ssn laia-nan? Pages 715-1060 ■ Year 2017, Vol. 64, No. 4 ActaChimicaSlc Acta Chimica Slc Slovenica ActaC w—^ M 64/2017 m % \ it- k / \ Dedicated to Professor and Academician Miha Tisler at the occasion of his 90th birthday Selected titles: Synthesis of Novel 3D-Rich a-Amino Acid-Derived 3-Pyrazolidinones ■ Synthesis and X-ray Structural Analysis of the Ruthenium(lIl) Complex Na[trans-RuCl4(DMSO)(PyrDiaz)], the Diazene Derivative of Antitumor NAMI-Pyr ■ Discrimination Between Synechocystis Members (Cyanobacteria) Based on Heterogeneity of Their 16S rRNA and ITS Regions ■ Geographical Origin Characterization of Slovenian Garlic Using Stable Isotope and Elemental Composition Analyses http://acta.chem-soc.si EDITOR-IN-CHIEF ALEKSANDER PAVKO Slovenian Chemical Society, Hajdrihova 19, SI-1000 Ljubljana, Slovenija, E-mail: ACSi@fkkt.uni-lj.si, Telephone: (+386)-1-476-0252; Fax: (+386)-1-1-476-0300 ASSOCIATE EDITORS Marija Bešter-Rogač, University of Ljubljana, Slovenia Janez Cerkovnik, University of Ljubljana, Slovenia Krištof Kranjc, University of Ljubljana, Slovenia Franc Perdih, University of Ljubljana, Slovenia Helena Prosen, University of Ljubljana, Slovenia Damjana Rozman, University of Ljubljana, Slovenia Melita Tramšek, Jožef Stefan Institute, Slovenia Irena Vovk, National Institute of Chemistry, Slovenia GUEST-EDITOR Janez Cerkovnik, University of Ljubljana, Slovenia ADMINISTRATIVE ASSISTANT Marjana Gantar Albreht, National Institute of Chemistry, Slovenia EDITORIAL BOARD Wolfgang Buchberger, Johannes Kepler University, Austria Alojz Demšar, University of Ljubljana, Slovenia Stanislav Gobec, University of Ljubljana, Slovenia Marko Goličnik, University of Ljubljana, Slovenia Günter Grampp, Graz University of Technology, Austria Wojciech Grochala, University of Warsaw, Poland Danijel Kikelj, Faculty of Pharmacy, Slovenia Ksenija Kogej, University of Ljubljana, Slovenia Janez Košmrlj, University of Ljubljana, Slovenia Blaž Likozar, National Institute of Chemistry, Slovenia Mahesh K. Iakshman, The City College and The City University of New York, USA Janez Mavri, National Institute of Chemistry, Slovenia Friedrich Srienc, University of Minnesota, USA Walter Steiner, Graz University of Technology, Austria Jurij Svete, University of Ljubljana, Slovenia Ivan Švancara, University of Pardubice, Czech Republic Jiri Pinkas, Masaryk University Brno, Czech Republic Gašper Tavčar, Jožef Stefan Institute, Slovenia Christine Wandrey, EPFL Lausanne, Switzerland Ennio Zangrando, University of Trieste, Italy Chairman Branko Stanovnik, Slovenia Members Josef Barthel, Germany Udo A. Th. Brinkman, The Netherlands Attilio Cesaro, Italy Dusan Hadzi, Slovenia Vida Hudnik, Slovenia Venceslav Kaucic, Slovenia ADVISORY EDITORIAL BOARD Željko Knez, Slovenia Radovan Komel, Slovenia Janez Levec, Slovenia Stane Pejovnik, Slovenia Anton Perdih, Slovenia Slavko Pečar, Slovenia Andrej Petrič, Slovenia Boris Pihlar, Slovenia Milan Randic, Des Moines, USA Jože Škerjanc, Slovenia Miha Tišler, Slovenia Durda Vasic-Rački, Croatia Marjan Veber, Slovenia Gorazd Vesnaver, Slovenia Jure Zupan, Slovenia Boris Žemva, Slovenia Majda Žigon, Slovenia Acta Chimica Slovenica is indexed in: Chemical Abstracts Plus, Current Contents (Physical, Chemical and Earth Sciences), PubMed, Science Citation Index Expanded and Scopus. Impact factor for 2016 is IF = 0.983. /-NCreative Articles in this journal are published under Creative Commons Attribution 3.0 License wCOl I IN lOnS http://creativecommons.org/licenses/by/3.0/ Izdaja - Published by: SLOVENSKO KEMIJSKO DRUŠTVO - SLOVENIAN CHEMICAL SOCIETY Naslov redakcije in uprave - Address of the Editorial Board and Administration Hajdrihova 19, SI-1000 Ljubljana, Slovenija Tel.: (+386)-1-476-0252; Fax: (+386)-1-476-0300; E-mail: chem.soc@ki.si Izdajanje sofinancirajo - Financially supported by: Slovenian Research Agency, Ljubljana, Slovenia National Institute of Chemistry, Ljubljana, Slovenia Jožef Stefan Institute, Ljubljana, Slovenia Faculty of Chemistry and Chemical Technology at University of Ljubljana, Slovenia Faculty of Chemistry and Chemical Engineering at University of Maribor, Slovenia Faculty of Pharmacy at University of Ljubljana, Slovenia University of Nova Gorica, Nova Gorica, Slovenia Chamber of Commerce and Industry of Slovenia - Chemical and Rubber Industry Association, Slovenia Članom je revija na voljo brezplačno. Za nečlane in pravne osebe znaša letna naročnina 50 EUR, za inozemstvo 110 EUR vključno s poštnino. Annual subscription: 110 EUR including postage. Transakcijski račun: 02053-0013322846 Bank Account No.: SI56020530013322846-Nova Ljubljanska banka d. d., Trg republike 2, SI-1520 Ljubljana, Slovenia, SWIFT Code: LJBA SI 2X Na podlagi Zakona o davku na dodano vrednost sodi revija Acta Chimica Slovenica med proizvode, od katerih se obračunava DDV po stopnji 9,5 %. Acta Chimica Slovenica izhaja štirikrat letno v 200 izvodih - Acta Chimica Slovenica appears quarterly in 200 copies Oblikovanje ovitka - Design cover: KULT, oblikovalski studio, Simon KAJTNA, s. p. Grafična priprava za tisk: Majanafin, d. o. o. Tisk - Printed by: Tiskarna Skušek, Ljubljana © Copyright by Slovenian Chemical Society ActaChimicaSlovenica Dedicated Issue Professor Miha Tišler Writing a tribute to Professor Emeritus Dr. Miha Tisler on the occasion of his 90th birthday fills me with great honor. He was born in Ljubljana on September 18th, 1926. He graduated in chemistry at the Faculty of Natural Sciences and at the Faculty of Chemistry and Chemical Engineering in Ljubljana, Slovenia. With a scholarship from the British Council he was a postgraduate researcher in the laboratories of Lord Todd in Cambridge, England (1953-1955). There he prepared his PhD which he then defended in Ljubljana to obtain subsequently a PhD in chemistry on the basis of his thesis entitled „Syntheses in the Cycloheptatrienone Series" (1955). In Cambridge he investigated 3-hydroxytropones and, among other compounds, he prepared at that time an unusual compound, the aromatic carboxycycloheptatrienylium (carboxytro-pylium) bromide. His first appointment was as assistant in the Laboratory of Organic Chemistry at the University of Ljubljana and with time he gradually advanced to the position of Lecturer (Docent), Associate Professor (1961) and Full Professor (1964) as a permanent position. During 19711984 he serverd as Head of the Laboratory of Organic Chemistry and in 1995 he retired. He was a British Council Visiting Scientist (1966), Visiting Professor at the Universities of Freiburg, Germany (1962), Trieste, Italy (1985, 1986), Brigham Young University, Provo, Utah (1986), and under an exchange program with the National Academy of Sciences, Washington, he lectured at several universities in the USA (1968, 1979). He also spent two months as Visit- ing Professor in Japan at the invitation of the Japan Society for Promotion of Science, Tokyo (1975), as well as in Australia (1982). He was plenary speaker at about 20 International Symposia or Congresses and at the Gordon Research Conference. Professor Tisler is author or co-author of over 50 books, textbooks of organic and heterocyclic chemistry, monographs or review articles and over 500 published scientific papers. Of the many organic chemists who obtained the PhD degree by accomplishing their thesis under his guidance, nine were later elected and appointed at several faculties at the universities of Ljubljana and Maribor. The Organic Chemistry laboratory in Ljubljana has become internationally known as the school for heterocy-clic chemistry. His research was mainly devoted to the field of heter-ocyclic chemistry, syntheses of new heterocyclic systems and their transformations, development of new reagents, structural studies, tautomerism, elucidation of reaction mechanisms, etc. New and interesting results were obtained from extensive investigation on azidotetrazolo isomerization. Target compounds were in the series of tetrazoloazines, and related tri- or polycyclic systems with an annelated tetra-zole ring, such as azasteroids. Heterocyclic diazo compounds, in contrast to the corresponding diazonium salts, were practically unknown. Their synthesis was developed and it was possible to obtain for the first time an X-ray structure of 3-diazo-3H-indazole. Graphical Contents Many years of research were devoted to investigations of azoloazines and azaindolizines, i.e. bicyclic 10 n-electron aromatic systems with bridgehead nitrogen atom which are, for example, isoelectronic with indole. Many new synthetic approaches towards bicyclic, tricyclic or polycyclic heterocyclic systems containing the azaindolizine unit (the six-membered ring being a pyridine, pyridazine, pyrazine or pyrimidine ring) were developed and their reactivity was investigated, such as the site of protonation and quaterniza-tion, hemolytic phenylation, etc. An enormous synthetic potential was opened up with the introduction of N,N-dimethylaminoformamide (or acetamide) dialkyl acetals for the construction of new heterocyclic rings. The formamidines themselves or hy-droxyiminomethyleneamino derivatives derived from them could be widely used for annelation reactions, for the formation of [2+2] cycloadducts with phenyl isocyanate, etc. Unsaturated N,N-dimethylamidine synthons, prepared from enamino amides, thioamides or esters and even activated methylene groups of pyridines, proved to be reactive with these acetals. A fruitful collaboration with research possibilities was provided in the form of an exchange programme with the participation of Professors Jerald S. Bradshaw (BYU University Provo, Utah), Ronald J. Pugmire and David M. Grant (University of Utah, Salt Lake City), Gordon B. Barlin and Desmond J. Brown (John Curtin School of Medical Research, Canberra, Australia). Last but not least one should mention and acknowledge that Professor Tisler had great pleasure in a long-standing friendship, collaboration and support from Professors Jerald S. Bradshaw and Alan R. Katritzky (University of Florida, Gainesville), dating from the time when the latter was professor at the University of East Anglia in Norwich. He has also many friends at universities and institutes throughout the world. Professor Tisler was Dean of the Faculty of Natural Sciences and Technology (1973-1976) and he was the first elected rector at the University of Ljubljana in the independent Slovenia (1991-1995). He served as Vice-President, President and Past-President of the International Society of Heterocyclic Chemistry (1973-1980). He is member of the ACS, MRSC, International Standing Committe of the International Association „Ius Primi Viri" in Rome, Italy, and member of the Governing Council of the European Science Foundation (Strasbourg). He was also a member of the Permanent Committee of the European Rectors Conference and the State Council of Republic of Slovenia (1992-1997). He served as the National Representative in Organic Chemistry Division, Commission on Physical Organic Chemistry of IUPAC (1986-1997). He was elected to membership of the Slovenian Academy of Sciences and Arts (1970), Serbian Academy of Sciences and Arts (1978), Croatian Academy of Sciences and Arts (1979), New York Academy of Sciences (1980) and Academia Scientiarum et Artium Europaea, Salzburg (1995). He was decorated as Knight of the Order of St. Gregorius the Great (1995). He received the Award for Science from Slovenia (1977), Plaque from the International Society of Heterocyclic Chemistry (1979), Honorary Medal and Diploma of the Slovak Technical University, Bratislava (1981), Diploma of the Tohoku University, Sendai (1975) and Hoshi University, Tokio (1986); he received documents of recognition from the Faculty of Pharmacy (1982) and Faculty of Natural Sciences and Technology, University of Ljubljana (1989) and the award Ambassador of the Republic of Slovenia (1995). He is an Inaugurated Honorary Member of the Florida Center for Heterocyclic Compounds, Gainesville, Florida. In 2000 he received the Honorary Degree (Doctor honoris causa) of the University of Ljubljana. He served or is still serving at the Editorial or Advisory Board of the following scientific journals: Journal of Heterocyclic Chemistry, Heterocycles, Advances in Heterocyclic Chemistry, Heterocyclic Communications, Organic Preparations and Procedures International, Duga (Turkish Journal of Chemistry), Croatica Chemica Acta, Acta Chimica Slovenica (formerly Vestnik Slovenskega kemijskega društva). Professor Tišler (alone or with colleagues) organized several National or International Symposia or Congresses: 5th International Congress of Heterocyclic Chemistry (1975), 5th International Symposium on the Chemistry of Organic Sulfur Compounds (1978), 3rd Yugoslav Symposium of Organic Chemistry (1983) and TRISOC Symposium (symposium of the universities of Graz, Trieste and Ljubljana)(1985). Since his retirement he has remained active in preparing some review articles (on pyridazines, which was first published in 1968, and thereafter in 1979, 1990, 2000, on heterocyclic quinonesin 1989, and on heterocyclic amino acids (1995), all in Advances in Heterocyclic Chemistry). Recently he published two books, Molecules and their Messages, and Reminiscences and Reflections, both in the Slovene language. Professor Tišler has always been an excellent supervisor to his students and friendly and helpful to his associates and acquaintances. I, as one of them, have been extremely fortunate in having him first as a supervisor during my PhD studies then later becoming one of his coworkers and colleagues; the result has been fruitful collaboration and friendship for almost sixty years. Please join me in this celebration of Professor Miha Tišler commemorating the 90th anniversary and a distinguished career in chemistry. We wish him good health in the years ahead. Branko Stanovnik Faculty of Chemistry and Chemical Technology University of Ljubljana Graphical Contents Graphical Contents s V i t /s ActaChimicaSlo ActaChimicaSlo SlovenicaActaC Year 2017, Vol. 64, No. 4 SCiENTIFiC PAPER 715-726 Orga nic chemistry Synthesis of Novel 3D-Rich «-Amino Acid-Derived 3-Pyrazolidinones Jaka Glavač, Georg Dahmann, Franc Požgan, Sebastijan Ričko, Bogdan Štefane, Jurij Svete and Uroš Grošelj 727-736 Orga nic chemistry Asymmetric Bio- and Chemoreduction of 2-Benzylidenecyclopentanone Derivatives Bogdan Štefane, Uroš Grošelj, Jurij Svete and Franc Požgan 737-746 Orga nic chemistry A 26-Membered Macrocycle Obtained by a Double Diels-Alder Cycloaddition Between Two 2H-Pyran-2-one Rings and Two 1,1'-(Hexane-1,6-diyl)bis (1H-pyrrole-2,5-dione)s Bor Lucijan Turek, Marijan Kočevar, Krištof Kranjc and Franc Perdih Graphical Contents 747-762 Orga nic chemistry Iodine-catalyzed Transformation of Aryl-substituted Alcohols Under Solvent-free and Highly Concentrated Reaction Conditions Marjan Jereb and Dejan Vražič 763-770 Orga nic chemistry Synthesis and X-ray Structural Analysis of the Ruthenium(III) Complex Na[frans-RuCl4(DMSO) (PyrDiaz)], the Diazene Derivative of Antitumor NAMI-Pyr Jure Vajs, Andrej Pevec, Martin Gazvoda, Damijana Urankar, Evgeny Goreshnik, Slovenko Polanc and Janez Košmrlj 771-781 Biomed ical applications Chlorocarbonylsulfenyl Chloride Cyclizations Towards Piperidin-3-yl-oxathiazol-2-ones as Potential Covalent Inhibitors of Threonine Proteases Marko Jukič, Katarina Grabrijan, Selmir Kadic, Fernando Juan de Lera Garrido, Izidor Sosič, Stanislav Gobec and Aleš Obreza 804-817 Biochemistry and molecular biology Discrimination Between Synechocystis Members (Cyanobacteria) Based on Heterogeneity of Their 16S ^ f" ff rRNA and ITS Regions ! LJL J Mojca Jutersek, Marina Klemencic and Marko Dolinar 818-824 General chemistry Reaction Monitoring by Means of Multivariate Data Analysis of Near-Infrared and Raman Spectra Dardan Hetemi and Steve Janagap 825-831 inorganic chemistry Synthesis, Crystal Structures, Molecular Docking and MAO-B Inhibitory Activity of Transition Metal Complexes Derived from 2-(4-(Pyridin-2-yl)piperazin-1-yl)acetic Acid Yan-Jie Ren, Jin-Long Zhu, Li-Xin Zhang, Yin-Xiang Xu and Shao-Song Qian 832-841 Orga nic chemistry A Novel One-pot Synthesis of Isothiocyanates and Cyanamides from Dithiocarbamate Salts Using Environmentally Benign Reagent Tetrapropylammonium Tribromide Neivotsonuo Bernadette Kuotsu, Latonglila Jamir, Tovishe Phucho and Upasana Bora Sinha 842-848 Phy sical chemistry Prediction of Physico-chemical Properties of Bacteriostatic N1-Substituted Sulfonamides: Theoretical and Experimental Studies Hossein Nikoofard, Mohsen Sargolzaei and Farnosh Faridbod n u ^ u 849-857 Analytical chemistry Methyl Salicylate-Based Vortex-Assisted Surfactant-Enhanced Emulsification Microextraction and HPLC for Determination of Fungicides in Honey Samples Yanawath Santaladchaiyakit, Jutamas Bunchamnan, Darunee Tongsa and Supalax Srijaranai 858-864 Applied chemistry A One-pot Multicomponent Reaction for the Synthesis of Oligoetherols with Azacyclic Rings Jacek Lubczak, Renata Lubczak and Iwona Zarzyka Graphical Contents 865-876 Orga nic chemistry Synthesis and Structure Activity Relationship of Some Indole Derivatives as Potential Anti-inflammatory Agents Samar S. Fatahala, Mohammed A. Khedr and Mossad S. Mohamed 877-887 Materials science Preparation and Investigation of the Thermal Stability of Phosphate-modified TiO2 Anatase Powders and Thin Films Uroš Prah and Irena Kozjek Škofic 888-894 Applied chemistry Recovery of Rutin from Labisia pumila Extract Using Solid Phase Extraction Lee Suan Chua, Nur Nabihah Ruzlan and Mohamad Roji Sarmidi 895-901 Orga nic chemistry Synthesis and Biological Evaluation of 1,2,4-Triazoles and 1,3,4-Oxadiazoles Derivatives Linked to 1,4-Dihydropyridines Scaffold Maghsoud Ziaie, Karim Akbari Dilmaghani and Amir Tukmechi 902-910 Orga nic chemistry Design, Synthesis and Anti-inflammatory Activity of Derivatives 10-R-3-Aryl-6,7-dihydro-2ff-[1,2,4]triazino [2,3-c]quinazolin-2-ones of Spiro-fused Cyclic Frameworks Oleksandra Kolomoets, Oleksii Voskoboynik, Oleksii Antypenko, Galyna Berest, Inna Nosulenko, Vitaliy Palchikov, Olexandr Karpenko and Sergiy Kovalenko 911-918 Orga nic chemistry Green One-pot Synthesis of Novel Polysubstituted Pyrazole Derivatives as Potential Antimicrobial Agents Hamid Beyzaei, Zahra Motraghi, Reza Aryan, Mohammad Mehdi Zahedi and Alireza Samzadeh-Kermani 919-930 Orga nic chemistry Synthesis and Biological Evaluation of Some Novel 1,8-Naphthyridine Derivatives Sraa Abu-Melha 931-937 Materials science Preparation and Characterization of Chromium Doped Ni-Cu-Zn Nano Ferrites Bajarang Laxman Shinde, Laxman Appa Dhale, Venkat S. Suryavanshi and Kishan Shankarrao Lohar 938-944 Materials The Impact of a Silane Pigment Treatment on the Properties of Thickness-sensitive Spectrally Selective Paints Miha Steinbücher, Peter Venturini, Jože Hafner, Matevž Zupančič, Peter Gregorčič and Iztok Golobič 945-958 Materials science Graphene Oxide/Co3O4 Nanocomposite: Synthesis, Characterization, and Its Adsorption Capacity for the Removal of Organic Dye Pollutants from Water Kolsoum Pourzare, Saeed Farhadi and Yaghoub Mansourpanah Graphical Contents 959-967 Chem ical education The Integration of Submicroscopic Representations Used in Chemistry Textbook Sets into Curriculum Topics Špela Hrast and Vesna Ferk Savec 968-979 Analytical chemistry Optimization of UPLC Method for Simultaneous Determination of Rosuvastatin and Rosuvastatin Degradation Products Jure Zakrajšek, Katarina Bevc-Černilec, Simona Bohanec and Uroš Urleb 980-987 Materials science Ordering Effects and Percolation in the Structure Formation Process of the Oriented Polyolefin Porous Films Galina Kazimirovna Elyashevich, Dmitrii Vladimirovich Novikov, Ivan Sergeevich Kuryndin, Andreja Jelen and Vili Bukošek SEM images of the PE and PP parous films surface 988-1004 Organic chemistry Microwave-assisted One-pot Efficient Synthesis of Functionalized 2-Oxo-2-phenylethylidenes-linked 2-Oxobenzo[1,4]oxazines and 2-Oxoquino[1,4]oxalines: Vashundhra Sharma, Pradeep K. Jaiswal, Dharmendra K. Yadav, Mukesh Saran, Jaroslav Prikhodko, Manas Mathur, Ajit K. Swami, Irina V. Mashevskaya and Sandeep Chaudhary 1005-1019 Materials science Magnetically Recyclable Fe3O4/GO-NH2/H3PMo12O40 Nanocomposite: Synthesis, Characterization, and Application in Selective Adsorption of Cationic Dyes from Water Saeed Farhadi, Mohammad Hakimi and Mansoureh Maleki F'lOj Mpwrtf* ■»¡E^ïtVn h.PUT, pi-' I ^^ni-i-^-^l j 1020-1029 Organic chemistry Eco-Friendly Multi-Component Synthesis of y-Spiroiminolactones in Water Hamid Reza Safaei and Mohsen Shekouhy 1030-1041 Organic chemistry Synthesis, Nematicidal and Antifungal Properties of Hybrid Heterocyclics Avula Srinivas, Malladi Sunitha, Pulluri Karthik and K. Vasumathi Reddy 1042-1047 i norganic chemistry Preparation, Structure, Photoluminescent and Semiconductive Properties, and Theoretical Calculation of a Novel Cadmium Complex with Mixed Ligands Xiu-Guang Yi, Wen-Tong Chen, Jian-Gen Huang, Ding-Wa Zhang and Yin-Feng Wang SHORT COMMUNICATION 782-789 Orga nic chemistry Synthesis of Novel 5-(N-Boc-N-Benzyl-2-aminoethyl)-7-oxo-4,7-dihydropyrazolo[1,5-fl]pyrimidin-3-carboxamides and Their Inhibition of Cathepsins B and K Branislav Lukic, Uroš Grošelj, Marko Novinec, and Jurij Svete 790-797 Orga nic chemistry Synthesis and Reduction of 10-Phthalimidocamphor Oxime Uroš Grošelj, Amalija Golobič, Jurij Svete and Sebastijan Ričko 798-803 Orga nic chemistry The Synthesis of 7-Substituted-2,3-dihydropyrido [4,3-d]pyridazine-1,4-diones and 1,4-Dioxo-7-substituted-1,2,3,4-tetrahydropyrido[4,3-d]pyridazine 6-Oxides from Methyl Ketones Benjamin Prek and Branko Stanovnik 1048-1055 Applied chemistry Geographical Origin Characterization of Slovenian Organic Garlic Using Stable Isotope and Elemental Composition Analyses Anja Mahne Opatic, Marijan Nečemer, David Kocman and Sonja Lojen i M V" £ \ _ y *jt * ** M i « » DOI: 10.17344/acsi.2017.3438 Acta Chim. Slov. 2017, 64, 715-726 ¿^creative tS1 commons Scientific paper Synthesis of Novel 3D-Rich a-Amino Acid-Derived 3-Pyrazolidinones Jaka Glavač,1 Georg Dahmann,2 Franc Požgan,1 Sebastijan Ričko,1 Bogdan Štefane,1 Jurij Svete,1 and Uroš Grošelj1* 1 Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, SI - 1000 Ljubljana, Slovenia. 2 Medicinal Chemistry, Boehringer-Ingelheim Pharma GmbH&Co. KG, 88397Biberach, Germany * Corresponding author: E-mail: uros.groselj@fkkt.uni-lj.si Received: 12-04-2017 Dedicated to Professor Emeritus Miha Tišler, University of Ljubljana, on the occasion of his 90th birthday. Abstract Synthetic approaches towards novel 3-pyrazolidinone derivatives functionalized at positions N(1) and/or C(5) were studied. 5-Aminoalkyl-3-pyrazolidinones were prepared in four steps from N-protected glycines via Masamune-Claisen homologation, reduction, O-mesylation, and cyclisation with a hydrazine derivative. The free amines were prepared by acidolytic deprotection. Title compound was also prepared by 'ring switching' transformation of N-Boc-pyrrolin-2(5H)-one with hydrazine hydrate. Hydrogenolytic deprotection of 5-(N-alkyl-N-Cbz-aminomethyl)pyrazolidine-3-ones followed by cyclisation with 1,1'-carbonyldiimidazole (CDI) gave two novel representatives of perhydroimidazo[1,5-fo] pyrazole, which is an almost unexplored heterocyclic system. Amidation of 3-oxopyrazolidine-5-carboxylic acid gave the corresponding carboxamides in moderate yields. Diastereomeric non-racemic carboxamides obtained from (S)-AlaOMe and (S)-ProOMe were separated by MPLC. Keywords: 3-Pyrazolidinones, amino acids, cyclization, heterocycles, synthesis 1. Introduction Hetero(bi)cycles are commonly used building blocks for applications in medicinal chemistry, catalysis, and materials science.1,2 In this context, 3-pyrazolidinones and their bicyclic analogues are attractive targets due to their easy availability from ^-unsaturated esters and because of their applicability and biological activity.3-6 Pyrazolidi-none derivatives have been employed as dyes and photographic developers3,5 and as inhibitors of cyclooxygenase, lipoxygenase,7 and y-aminobutyrate aminotransferase8 exhibiting analgesic, antipyretic, anti-inflammatory, and an-orectic activity. Among bicyclic analogues, perhydropyra-zolo[1,2-a]pyrazolones belong to azabicycloalkane amino acids, which are U-shaped conformationally constrained heterocyclic analogues of peptides that simulate ^-turn structures.9,10 Consequently, bicyclic pyrazolidinones are used as drugs to relieve Alzheimer's disease11 and as antibacterial (Eli-Lilly's Y-lactam antibiotics),12 and antitrypanosomal agents.13 Synthetic applications of 3-pyrazolidi- nones comprise their use as chiral auxiliaries,14-19 as templates in asymmetric Diels-Alder cycloadditions,20-22 and as a new scaffold in organocatalysis.23-30 Typical examples of important 3-pyrazolidinone derivatives are depicted in Figure 1. However, in spite of easy availability of simple pyrazolidinones from ^-unsaturated esters and hydrazine derivatives,3-6,31,32 the synthesis of functionalized polysub-stituted pyrazolidinones remains challenging. Consequently, a majority of saturated bi- and tricyclic 3-pyrazo-lidinones are either unknown or unexplored heterocyclic systems. In the context of our ongoing work on the synthesis of chiral heterocycles with emphasis on pyrazole33,34 and pyrazolidinone derivatives,31,32 we reported the synthesis of tetrahydropyrazolo[1,5-c]pyrimidine-2,7-diones as the first representatives of a novel saturated heterocyclic system,35,36 followed by preparation of closely related tetrahy-dropyrazolo[1,5-c]pyrimidine-3-carboxamides37 and tet-rahydro-1H-imidazo[1,5-fr]pyrazole-2,6-diones.38 In ex- Glavač et al.: Synthesis of novel 3D-rich a-amino acid-derived 716 Acta Chim. Slov. 2017, 64, 715-726 H R O Phenidone (photographic developer) (COX- and LOX-inhibitor) Ph i O"Bu Phenylbutazone (anti-inflammatory) 8 M N O BW357U (anorectic) co2h Yr° nh oU V=N A * ' N OMe nh2 LY73013 (R = C02Me) LY186826 (R = COMe) LY193239 (R = S02Me; LY255262 (R = CN) Eli-Lilly's y-lactams (antibiotics) Figure 1. Examples of important 3-pyrazolidinone derivatives. tension, the first representatives of octahydro-2H-2a,2a1-di-azacyclopenta[cd]inden-2-one as a novel tricyclic pyrazo-lidinone-based system were also prepared.39 Crucial for all of the above syntheses was the preparation of a pyrazolid-inone key-intermediate with suitably functionalized substituent at position 5 allowing for cyclization to position 1. The 5-substituted pyrazolidinone was obtained by cyclization of the corresponding ^-mesyloxy ester, which in turn was obtained in three steps from a suitably functionalized carboxylic acid.31 Pyrazolidinones with 2-hydroxyethyl36 and 2-aminoethyl35,37 functional groups at position 5 were used as key intermediates in the synthesis of novel saturated heterocyclic systems, while 5-[(S)-1-aminoalkyl] derivatives prepared from N-protected a-amino acids were used as scaffolds for potential organo catalysts38 and as key-intermediates in the synthesis of 3-pyrrolinones.40 In addition to previously published 5-aminoethyl and 5-hydroxymethyl-3-pyrazolidinones, we also tried to prepare the 5-aminomethyl analogues, because they could be useful intermediates in the synthesis of novel saturated heterocycles in the imidazo[1,5-fr]pyrazole and pyra-zolo[1,5-fl]pyrazine series. In this paper, we report the preparation and some follow-up transformations of 5-ami- Scheme 1. Synthesis of the 5-aminomethyl-3-pyrazolidinones 5a, 5b, 5'b, and 7-9. Reaction conditions: i) CDI, THF, r.t. 2 h, then MeO2CCH2CO2K, MgCl2, r.t.; ii) NaBH4, MeOH, 0-20 °C; iii) MsCl, pyridine, CH2Cl2, 0 °C; iv) N2H4-H2O, MeOH, r.t.; v) MeNHNH2, MeOH, r.t., then chromatograph-ic separation (MPLC); vi) HCl-EtOAc, MeOH, r.t. Glavac et al.: Synthesis of novel 3D-rich a-amino acid-derived ... 717 Acta Chim. Slov. 2017, 64, 715-726 nomethyl and 5-carboxy substituted 3-pyrazolidinones available from glycine derivatives and from dimethyl maleate, respectively. These novel pyrazolidinone derivatives are interesting intermediates in the synthesis of chiral saturated pyrazolidine-based heterocyclic systems. 2. Results and Discussion First, 5-tert-butoxycarbonylaminomethyl-3-pyrazo-lidinones 5a, 5b, and 5'b were prepared in four steps from commercially available N-Boc-glycine (1a) following a well-established literature protocol.35-39 Masamune-Clais-en condensation of amino acid 1a, i.e. activation of 1a with 1,1'-carbonyldiimidazole (CDI) followed by treatment of the intermediate imidazolide with a mixture of potassium monomethyl malonate and magnesium chloride gave the corresponding ^-keto ester 2a in 93% yield. Reduction of 2a with NaBH4 in methanol followed by O-mesylation of the so formed alcohol 3a afforded the ^-mesyloxy ester 4a in 71% yield over two steps. The mesylate 4a was then cy-clized with hydrazine hydrate or methylhydrazine to furnish the N(5')-protected 5-aminomethyl-3-pyrazolidi-nones 5a, 5b, and 5'b. Cyclisation of the mesylate 4a with methylhydrazine was regioselective to give a ~5:1 mixture of the major 1-methyl regioisomer 5b and the minor 2-methyl isomer 5'b. Upon chromatographic separation (MPLC), the pure regioisomers 5b and 5'b were obtained in 66% and 14% yields, respectively. To shorten the synthetic procedure for the preparation of 5a, commercially available fert-butyl 2-oxo-2,5-dihydro-1H-pyrrole-1-car-boxylate (6) was treated with hydrazine hydrate in methanol at room temperature to afford the pyrazolidinone 5a in 45% yield. However, in spite of its greater simplicity, the latter procedure was less effective in terms of product yield. Finally, the respective free amines 7-9 were prepared Scheme 2. Reaction conditions: i) TFA, CH2Cl2, r.t.; ii) 50% aq. glyoxal or (MeO)2CH2CHO, H2, Pd-C, MeOH, r.t.; iii) 50% aq. (MeO)2CH2CHO, NaB-H3CN, MeOH, r.t.; iv) aq. HCl, MeOH, H2, Pd-C, r.t.; v) CDI, THF, r.t. 2 h, then MeO2CCH2CO2K, MgCl2, r.t.; vi) NaBH4, MeOH, 0-20 °C; vii) MsCl, pyridine, CH2Cl2, 0 °C; viii) N2H4-H2O, MeOH, r.t.; ix) Boc2O, r.t.; x) Mel, DMF, K2CO3, r.t.; xi) TFA-CH2Cl2, r.t.; xii) H2, Pd-C, MeOH, r.t.; xiii) CDI, DMF, r.t.. 2 2 2 4 2 2 2 3 2 2 2 Glavac et al.: Synthesis of novel 3D-rich a-amino acid-derived ... 718 Acta Chim. Slov. 2017, 64, 715-726 by acidolytic N-deprotection of 5a, 5b, and 5'b. Quite unexpectedly, treatment of 5a with HCl-MeOH gave the open-chain diamine 7, which is explainable by acid-catalyzed ring-opening of the initially formed intermediate 7' with methanol (Scheme 1). Next, cyclisation of the pyrazolidinone 5a was studied. Our initial goal was to prepare hexahydropyra-zolo[1,5-fl]pyrazin-2(1H)-one (10) by concomitant N-de-protection and reductive alkylation of 5a with glyoxal or with dimethoxyacetaldehyde. Unfortunately, this approach did not work and furnished mixtures of products regardless of the variation of the reaction conditions. Nevertheless, we were able to detect the presence of the desired compound 10 in the crude reaction mixture by HRMS (m/z = 142.0974, MH+). Attempted isolation and purification of this highly polar compound 10 failed. On the other hand, reductive alkylation of 5a with dimethoxyacetaldehyde and NaBH3CN in methanol at room temperature gave the corresponding 1-(2,2-dimethoxyethyl) derivative 11a in 37% yield. In the same way, the Cbz-analogue 11b was prepared in five steps from N-Cbz-glycine (1b). Finally, two novel 1,5-dialkyltetrahydro-1H-imidazo[1,5-fr]pyrazole-2,6-di-ones 14a and 14b were synthesized. Following the established one-pot protocol (cf. Scheme 1), N-Cbz-sarcosine (1c) and N-benzyl-N-Cbz-glycine (1d) were transformed in four steps into the corresponding pyrazolidinones 5c and 5d. In a subsequent one-pot procedure,35 compounds 5c and 5d were Boc-protected at N(1), methylated at N(2), and Boc-deprotected to give the N(1)-unsubstituted intermediates 12a and 12b in good yields over seven steps. Somewhat expectedly,38 cyclizations of 12a,b into imida-zo[1,5-fr]pyrazole derivatives 14a,b proceeded well. Hy-drogenolytic deprotection of the pyrazolidinones 12a and 12b followed by cyclisation of the intermediate free amines 13 with CDI furnished the expected 1,5-dimethyltetrahy-dro-1H-imidazo[1,5-fr]pyrazole-2,6-diones 14a and 14b in 42% and 53% yield, respectively (Scheme 2). In continuation, the amidation of 5-oxo-1-phe-nylpyrazolidine-3-carboxylic acid (17) was studied. Compound 17 was obtained in three steps from dimethyl maleate (15) following the literature procedure.41 Activation of the carboxylic acid 17 with CDI followed by treatment with primary amines 18a-c gave the corresponding carboxamides 19a-c in moderate yields. Somewhat surprisingly, amidation proceeded equally well with secondary diethylamine (18d) to afford the tertiary carboxamide 19d in 49% yield. Attempted cyclisation of the glycine derivative 19a into 1-phenyltetrahydropyrazolo[1,5-fl]pyrazine-2,4,7(1H)-tri-one (20) in refluxing toluene failed (Scheme 3). Finally, amidation of racemic carboxylic acid was also performed with the non-racemic a-amino esters, (S)-AlaOMe (18e) and (S)-ProOMe (18f). These amidations afforded mixtures of non-racemic diastereomers 19e/19'e and 19f/19'f. Subsequent separation of diastereomeric mixtures by medium pressure liquid chromatography furnished the non-racemic diastereomerically pure carboxamides 18a, 19a CH2C02Me H 45 18b, 19b CH2CH2C02Me H 45 18c, 19c CH2CH2Ph H 50 18d, 19d Et Et 49 Scheme 3. Synthesis of 3-pyrazolidinone-5-carboxamides 19a-d. 19e, 19'e, 19f, and 19'f in 13-23% yields. Unfortunately, all products 19e, 19'e, 19f, and 19'f were obtained as oils and their absolute configuration could not be determined by X-ray diffraction. Therefore, the configurations of the products 19e, 19'e, 19f, and 19'f are arbitrary (Scheme 4). The structures of novel compounds 5a,b, 5'b, 7-9, 11a,b, 14a,b, 19a-f, and 19'e,f were determined by spectroscopic methods (1H NMR, 13C NMR, IR, MS, HRMS) and by elemental analyses for C, H, and N. Compounds 5b, 5'b, 8, 9, 11a,b, 14a,b, 19e,f, and 19'e,f were not obtained in analytically pure form. Their identities were confirmed by 13C NMR and HRMS. 3. Experimental 3. 1. General Methods Melting points were determined on a Stanford Research Systems MPA100 OptiMelt automated melting Glavac et al.: Synthesis of novel 3D-rich a-amino acid-derived ... 719 Acta Chim. Slov. 2017, 64, 715-726 Ph HN ft H (s)V- IH fS,N-/ H Me02Cf{^ ^ Me02C^ ^ 19e (5R,2'S)-lsomer 19'e {5S,2'S)-lsomer 19e; (-)-isomer: 23% 19'e; (+)-isomer: 21% 17 1. CD!, MeCN, r.t. 2. (S)-AiaOMe (18e), r.t. 3. MPLC 2. (S)-ProOMe (iSf>, r.t. 3. MPLC 19f; (-)-isomer: 13% ph 19'f; (+)-isomer: 14% ph A HN ¡in CQ2Me 19f ($R,2'S)-Isomer 19'f {5S,2'S)-lsomer Scheme 4. Synthesis of non-racemic 3-pyrazolidinones 19e,f and 19'e,f. point system. The NMR spectra were obtained on a Bruker Avance III UltraShield 500 plus at 500 MHz for 1H and 126 MHz for 13C, using CDCl3 and DMSO-d6 (with TMS as the internal standard) as solvents. Mass spectra were recorded on an Agilent 6224 Accurate Mass TOF LC/MS spectrometer, IR spectra on a Bruker FTIR Alpha Platinum ATR spectrophotometer. Microanalyses were performed on a Perkin-Elmer CHN analyser 2400 II. Column chromatography (CC) was performed on silica gel (Fluka, Silica gel 60, particle size 35-70 ^m). Medium performance liquid chromatography (MPLC) was performed on a Büchi Flash Chromatography System (Büchi Fraction Collector C-660, Büchi Pump Module C-605, Büchi Control Unit C-620) on silica gel (LiChroprep® Si 60, 15-25 ^m), column dimensions: 23 x 460 mm, backpressure: 10 Bar, detection: UV (254 nm). Catalytic hydrogenation was performed on a Parr Pressure Reaction Hydrogenation apparatus (500 mL). Optical rotation of chiral nonracemic compounds was measured on a Perkin-Elmer 241MC polarimeter. N-Boc-Glycine (1a), N-Cbz-glycine (1b), N-Cbz-sar-cosine (1c), N-benzyl-N-Cbz-glycine (1d), CDI, potassium monomethyl malonate, anhydrous magnesium chloride, sodium borohydride, mesyl chloride, ferf-butyl 2-oxo-2,5-dihydro-1H-pyrrole-1-carboxylate (6), glyoxal, dimeth-oxyacetaldehyde, sodium cyanoborohydride, sodium triacetoxyborohydride, tetrabutylammonium borohydride, trifluoroacetic acid (TFA), methyl glycinate hydrochloride (18a), methyl ß-alaninate (18b), 2-phenylethylamine (18c), diethylamine (18d), (S)-N-Boc-alaninate (18e), and (S)-N-Boc-prolinate (18f) are commercially available. Methyl 4-ferf-butoxycarbonylamino-3-oxobutanoate (2a),40 methyl 4-benzyloxycarbonylamino-3-oxobutanoate (2b),42 and 5-oxo-1-phenylpyrazolidine-3-carboxylic acid (17)41 were prepared following the literature procedures. 3. 2. General Procedure for the Synthesis of N-protected 5-aminomethyl-3-pyrazolidinones 5a, 5b, and 5'b Method A. Compounds 5a, 5b, and 5'b were prepared in a one-pot procedure following the combined slightly modified general literature procedures for the preparation of analogous compounds.35,38,39 3. 2. 1. Methyl 4-ferf-butoxycarbonylamino-3-oxobutanoate (2a)42 Under argon, CDI (1.94 g, 12 mmol) was added to a solution of Boc-glycine (1a) (1.75 g, 10 mmol) in anh. THF (20 mL) and the mixture was stirred at room temperature for 2 h. Then a solid mixture of anh. MgCl2 (0.893 g, 9.5 mmol) and potassium mono-methyl malonate (2.184 g, 14 mmol) was added under Ar in one portion via a powder funnel, which was rinsed with anh. THF (5 mL) and the mixture was stirred under Ar at r.t. for 20 h. Volatile components were evaporated in vacuo and the residue was triturated with EtOAc (80 mL). The resulting suspension was washed with 1 M aq. NaHSO4 (2 x 20 mL) and brine (20 mL). The organic phase was dried over anh. Na2SO4, filtered, and the filtrate was evaporated in vacuo to give 2a, which was used in the next step without purification. Yield: 2.15 g (93%) of yellow oil. Spectral data were in agreement with the literature data.42 3. 2. 2. Methyl 4-ieri-butoxycarbonylamino-3-hydroxybutanoate (3a)43 Finely powdered NaBH4 (650 mg, 17.2 mmol) was slowly added to a cooled (0 °C) stirred solution of ß-keto ester 2a (6.94 g, 30 mmol) in MeOH (100 mL) and the resulting mixture was stirred at 0 °C for 1 h and then quenched at 0 °C by the addition of H2O (150 mL) followed by the addition of 1 M aq. HCl (30 mL, 30 mmol). The product was extracted with dichloromethane (3 x 150 mL) and the combined organic phase was washed with brine (150 mL). The organic phase was dried over anh. Na2SO4, filtered, and the filtrate was evaporated in vacuo. Glavac et al.: Synthesis of novel 3D-rich a-amino acid-derived ... 720 Acta Chim. Slov. 2017, 64, 715-726 The residue was dissolved in anh. toluene (30 mL) and the solution was evaporated in vacuo at 40 °C/2 mbar to give anhydrous crude 3a, which was used in the next step without further purification. Yield: 5.93 g (84%) of yellowish oil. Spectral data were consistent with the literature data.43 3. 2. 3. Methyl 4-ieri-butoxycarbonylamino-3-mesyloxybutanoate (4a) MsCl (2.25 ml, 29 mmol) was added to a cooled (0 °C) solution of ^-hydroxy ester 3 (5.83 g, 25 mmol) in anh. pyridine (30 mL) and the resulting mixture was stirred at 0 °C for 1 h and then at room temperature for 2 h. The reaction mixture was poured into cooled (0 °C) toluene (350mL) and the toluene solution was washed thoroughly with 1 M aq. HCl (200 mL) and brine (2 x 200mL). The organic phase was dried over anhydrous Na2SO4, filtered, and volatile components evaporated in vacuo to give crude 4a, which was used in the next step without purification. Yield: 6.58 g (84%) of yellowish oil. 1H-NMR (500 MHz, DMSO-d6): 5 1.37 (s, f-Bu); 2.20 (dd, J = 9.0; 15.1 Hz, 1H of CH2); 2.44 (dd, J = 3.8; 15.1 Hz, 1H of CH2); 2.86-2.98 (m, CH2); 3.82-3.90 (m, CH); 4.94 (d, J = 5.6 Hz, OH); 6.77 (f, J = 5.8 Hz, NH). 13C-NMR (126 MHz, DMSO-d6): 5 28.2, 37.0, 37.9, 43.0, 51.7, 77.4, 78.2, 155.8, 169.8. ESI (m/z): [MH+] calcd for C9H18N3O3, 216.1343; found, 216.1339. Anal. Calcd for C9H17N3O3: C 50.22, H 7.96, N 19.52. Found: C 50.09, H 8.09, N 19.13. IR (ATR) v 3344, 2970, 1692, 1649, 1522, 1445, 1392, 1365, 1277, 1252, 1175, 1124, 1081, 1053, 1000, 964, 901, 874, 782, 732, 638 cm-1. Tert-Butyl [ (2-methyl-5-oxopyrazolidin-3-yl)methyl] carbamate (5b) and tert-butyl [(1-methyl-5-oxopyrazo-lidin-3-yl)methyl]carbamate (5'b). Prepared from 4a (1.38 g, 4.43 mmol) and methylhy-drazine (557 ^L, 10.37 mmol), stirring for 72 h. tert-Butyl [(2-methyl-5-oxopyrazolidin-3-yl)methyl]car-bamate (5b). Yield: 670 mg (66%) of yellow oil. 1H-NMR (500 MHz, DMSO-d6): 5 1.38 (9H, s, f-Bu), 1.95 (1H, dd, J = 4.2, 16.8 Hz, 4-Ha), 2.46 (3H, s, 2-Me), 2.70 (1H, dd, J = 8.1, 16.7 Hz, 4-Hb), 2.82-2.90 (1H, m, 3'-Ha), 2.99-3.08 (2H, m, 3'-Hb and 3-H), 6.89 (1H, t, J = 5.3 Hz, NHCH2), 9.30 (1H, s, 1-H). 13C-NMR (126 MHz, DMSO-d6): 5 28.2, 32.5, 42.7, 46.6, 63.5, 77.8, 155.8, 172.1. m/z (ESI) = 230 (MH+). HRMS-ESI (m/z): [MH+] calcd for C10H20N3O3, 230.1499; found, 230.1496. IR (ATR) v 3301, 2975, 20931, 1692, 1520, 1455, 1392, 1366, 1278, 1253, 1168, 1094, 1044 cm-1. 3. 2. 4. Preparation of 3-pyrazolidinones 5a, 5b, and 5'b Method A. Hydrazine monohydrate (0.75 mL, 15 mmol) or methylhydrazine (789 15 mmol) was added to a solution of the mesylate 4a (3 mmol) in CH2Cl2 (25 mL) and the mixture was stirred at room temperature for 24-72 h. Volatile components were evaporated in vacuo and the residue was purified by CC. First, the non-polar impurities and starting material 4a were eluted (EtOAc-hexane, 1:1), followed by elution of the products 5 and 5' (EtOAc-MeOH, 10:1). Fractions containing the product were combined and volatile components evaporated in vacuo to give 5a or 5b/5'b. A mixture of regioisomers 5b and 5'b was separated by MPLC (EtOAc-MeOH, 20:1). Fractions containing the products were combined and volatile components were evaporated in vacuo to give 5b and 5'b, respectively. tert-Butyl [(5-oxopyrazolidin-3-yl)methyl]carbamate (5a) Prepared from 4a (1.87 g, 6 mmol) and hydrazine hydrate (685 ^L, 13.8 mmol), stirring for 24 h. Yield: 1.12 g (86%) of white solid; m.p. 103-110 °C. 1H-NMR (500 MHz, DMSO-d6): 5 1.38 (9H, s, f-Bu), 2.00 (1H, dd, J = 4.9; 16.4 Hz, 4-Ha), 2.36 (1H, dd, J = 7.9; 16.1 Hz, 4-Hb), 2.88-3.06 (2H, m, 3'-CH2), 3.48 (1H, br s, 3-H), 5.29 (1H, br s, 2-H), 6.88 (1H, t, J = 5.8 Hz, NHCH2), 8.98 (1H, s, 1-H). 13C-NMR (126 MHz, DMSO-d6): 5 28.2, 35.0, 42.4, 56.4, 77.8, 155.9, 175.0. m/z (ESI) = 216 (MH+). HRMS- tert-Butyl [(1-methyl-5-oxopyrazolidin-3-yl)methyl]car-bamate (5'b). Yield: 150 mg (14%) of yellow oil. 'H-NMR (500 MHz, DMSO-d6): 5 1.38 (9H, s, f-Bu), 2.09 (1H, dd, J = 5.1, 16.3 Hz, 4-Ha), 2.45 (1H, dd, J = 8.2, 16.2 Hz, 4-Hb), 2.81 (3H, s, 1-Me), 2.86-2.94 (1H, m 3'-Ha); 2.96-3.03 (1H, m, 3'-Ha), 3.41 (1H, br 5, 3-H), 5.67 (1H, br s, 2-H), 6.89 (1H, t, J = 5.8 Hz, NHCH2). 13C-NMR (126 MHz, DMSO-d6): 5 28.2, 30.6, 35.2, 42.6, 53.0, 77.8, 155.8, 170.5. m/z (ESI) = 230 (MH+). HRMS-ESI (m/z): [MH+] calcd for C10H20N3O3, 230.1499; found, 230.1503. IR (ATR) v 3332, 29977, 2933, 1694, 1520, 1455, 1394, 1367, 1277, 1253, 1169, 1060, 957 cm-1. Method B. Hydrazine hydrate (729 ^L, 15 mmol) was added to a solution of 6 (0.916 g, 5 mmol) in methanol (15 mL) and the mixture was stirred at r.t. for 48 h. Volatile components were evaporated in vacuo and the residue was purified by CC (EtOAc-MeOH, 10:1). Fractions containing the products were combined and volatile components were evaporated in vacuo to give 5a. Yield: 481 mg (45%) of a yellow resin. Characterisation data for 5a are given above in Section 3.2.4.1. 3. 3. General Procedure for Acidolytic Deprotection of Compounds 5a, 5b, and 5'b. Synthesis of Free Amines 7-9 2 M HCl in ethyl acetate (10 mL, 20 mmol) was added to a stirred solution of 5a, 5b, or 5'b (4 mmol) in methanol (20 mL) and the mixture was stirred at r.t. for 72 h. Glavac et al.: Synthesis of novel 3D-rich a-amino acid-derived ... 721 Acta Chim. Slov. 2017, 64, 715-726 The precipitate was collected by filtration, washed with anh. Et2O (50 mL) and dried in vacuo to give 7-9. 3. 3. 1. 2-(1-Ammonio-4-methoxy-4-oxobutan-2-yl)hydrazin-1-ium chloride (7) Prepared from 5a (861 mg, 4 mmol). Yield: 778 mg (88%) of white solid; mp 187-191 °C. 1H-NMR (500 MHz, DMSO-d6): 8 2.75 (1H, dd, J = 6.9, 17.0 Hz, 3-Ha), 2.83 (1H, dd, J = 6.3, 17.0 Hz, 3-Hb), 3.06 (2H, br s, 1-CH2); 3.64 (4H, br s, CO2Me, 2-H), 5.75 (1H, br s, NH), 8.34 (3H, br s, NH3+), 9.65 (3H, br s, NH3+). 13C-NMR (126 MHz, DMSO-d6): 8 34.7, 39.3, 51.8, 52.8, 170.9. Anal. Calcd for C5H15Cl2N3O2: C 27.28, H 6.87, N 19.09. Found: C 27.58, H 6.691 N 18.84. IR (ATR) v 3437, 3198, 2987, 1727, 1598, 1523, 1471, 1442, 1376, 1301, 1232, 1189, 1054, 1000, 987, 942, 902, 869, 772 cm-1. 10:1). Fractions containing the product were combined and evaporated in vacuo to give 7a. Yield: 1.606 g (37%) of white foam. 1H-NMR (500 MHz, DMSO-d6): 5 1.37 (9H, s, f-Bu), 1.89 (1H, dd, J = 2.3, 16.4 Hz, 4'-Ha), 2.70 (1H, dd, J = 4.5, 12.7 Hz, 1H of NCH2), 2.75 (1H, dd, J = 8.4, 16.8 Hz, 4'-Hb), 2.78-2.87 (1H, m, 1H of NCH2), 2.89 (1H, dd, J = 5.9, 12.7 Hz, 1H of NCH2), 3.00 (1H, m, 1H of NCH2), 3.25 and 3.26 (6H, 2s, 1:1, 2 x OMe), 3.23-3.29 (1H, m, 3'-H overlapped by the signal for H2O), 4.40 (1H, dd, J = 4.5, 5.9 Hz, CH(OMe)2), 6.82 (1H, t, J = 5.9 Hz, NHCH2), 9.34 (1H, s, 1'-H). 13C-NMR (126 MHz, DMSO-d6): 5 28.2, 31.8, 43.0, 52.9, 53.2, 60.8, 61.9, 77.7, 101.8, 155.7, 172.2. m/z (ESI) = 304 (MH+). HRMS-ESI (m/z): [MH+] calcd for C13H25N3O5, 304.1867; found, 304.1866. IR (ATR) v 3395, 33055, 2982, 2936, 2836, 2360, 2340, 1699, 1507, 1452, 1423, 1392, 1367, 1266, 1169, 1132, 1074, 974, 896, 866, 741, 705, 668 cm-1. 3. 3. 2. 5-(Ammoniomethyl)-1-methyl-3-oxopyrazolidin-1-ium chloride (8) Prepared from 5b (688 mg, 3 mmol). Yield: 250 mg (41%) of white solid; mp 170-183 °C. 1H-NMR (500 MHz, DMSO-d6): 8 2.42 (1H, br d, J = 11.4 Hz, 4-Ha), 2.81 (3H, s, 1-Me), 2.98 (1H, br d, J = 13.3 Hz, 4-Hb), 3.09 (2H, br d, 3'-CH2), 3.89 (1H, br s, 3-H), 7.90 (2H, br s, 2-H and NH+), 8.50 (3H, br s, NH3+). 13C-NMR (126 MHz, DMSO-d6): 8 32.6, 39.2, 45.2, 62.3, 171.5. m/z (ESI) = 130 (MH+). HRMS-ESI (m/z): [MH+] calcd for C5H12N3O, 130.0975; found, 130.0974. IR (ATR) v 3438, 3004, 2484, 1750, 1493, 1456, 1443, 1425, 1385, 1322, 1302, 1261, 1223, 1166, 1118, 1104, 1053, 1013, 919 cm-1. 3. 3. 3. 5-(Ammoniomethyl)-2-methyl-3-oxopyrazolidin-1-ium chloride (9) Prepared from 5'e (85 mg, 0.37 mmol). Yield: 60 mg (80%) of very hygroscopic white semi-solid. 1H-NMR (500 MHz, DMSO-d6): 8 2.40 (1H, dd, J = 4.7, 16.8 Hz, 4-Ha), 2.75 (1H, dd, J = 8.7, 16.8 Hz, 4-Hb), 2.94 (3H, s, 2-Me), 2.97-3.07 (2H, m, 5'-CH2), 3.92-4.01 (1H, m, 5-H), 4.91 (2H, br s, NH2+), 8.33 (3H, br s, NH3+). 13C-NMR (126 MHz, DMSO-c26): 8 30.7, 33.9, 39.9, 51.2, 169.9. m/z (ESI) = 130 (MH+). HRMS-ESI (m/z): [MH+] calcd for C5H12N3O, 130.0975; found, 130.0972. 3. 5. Benzyl ((2-(2,2-dimethoxyethyl)-5-oxopyrazolidin-3-yl)methyl)carbamate (11b) The crude pyrazolidinone 5b was prepared in four steps from N-Cbz-glycine (1b) following a one-pot procedure for the preparation of its N-Boc analogue 5a (cf. Section 3.2. and Scheme 1). Reductive alkylation of the intermediate pyrazolidinone 5b (1.246 g, 5 mmol) was performed in the same way as described above for the preparation of 11a. The crude product 11b was additionally purified by MPLC (EtOAc-MeOH, 10:1). Yield: 700 mg (41%) of yellow oil. 1H-NMR (500 MHz, DMSO-d6): 8 1.91 (1H, dd, J = 2.5, 16.9 Hz, 4'-Ha), 2.71 (1H, dd, J = 4.5, 12.8 Hz, 1H of NCH2), 2.78 (1H, dd, J = 8.3, 16.8 Hz, 4'-Hb), 2.89 (1H, dd, J = 3.2, 5.9 Hz, 1H of NCH2), 2.90 (1H, m, 1H of NCH2), 3.08 (1H, m, 1H of NCH2), 3.24 and 3.25 (6H, 2s, 1:1, 2 >2 OMe), 3.29-3.36 (1H, m, 3'-H), 4.40 (1H, dd, J = 4.5, 5.9 Hz, CH(OMe)2), 5.02 (2H, d, J = 4.3 Hz, PhCH2), 7.29-7.39 (6H, m, Ph and NHCH2), 9.36 (1H, s, 1'-H). 13C-NMR (126 MHz, DMSO-d6): 8 31.8, 43.4, 53.0, 53.1, 60.8, 61.8, 65.3, 101.8, 127.7, 127.8, 128.3, 137.1, 156.3, 172.9. m/z (ESI) = 338 (MH+). HRMS-ESI (m/z): [MH+] calcd for C H N O , 338.1711; found, 338.1709. 16 23 3 5 IR (ATR) v 3336, 3058, 2938, 2836, 2360, 2342, 1698, 1519, 1455, 1266, 1134, 1073, 977, 918, 869, 830, 739, 701, 668 cm-1. 3. 4. Tert-Butyl ((2-(2,2-dimethoxyethyl)-5-oxopyrazolidin-3-yl)methyl)carbamate (11a) NaBH3CN (465 mg, 15 mmol) was added in small portions within 1 h to a stirred solution of 5 (3.23 g, 15 mmol) and dimethoxyacetaldehyde (50% in H2O, 4.5 mL, 30 mmol) in methanol (30 mL) and the mixture was stirred at r.t. for 48 h. Volatile components were evaporated in vacuo and the residue was purified by CC (EtOAc-MeOH, 3. 6. General Procedure for the Synthesis of 5-alkyl-1-methyltetrahydro-1H-imidazo[1,5-&]pyrazole-2,6-diones 14a,b Bicyclic compounds 14a and 14b were obtained in nine steps from N-Cbz-sarcosine (1c) and N-benzyl-N-Cbz-glycine (1d). First, 3-pyrazolidinones 5c and 5d were prepared following a one-pot procedure for the preparation of their N-Boc analogue 5a (cf. Section 3.2. and Scheme 1).35 Glavac et al.: Synthesis of novel 3D-rich a-amino acid-derived ... 722 Acta Chim. Slov. 2017, 64, 715-726 3. 6. 1. Preparation of the Free Diamines 12a,b Boc2O (2.4 g, 11 mmol) was added to a stirred solution of 5c,d (9 mmol) in a mixture of dioxane (12 mL), water (25 mL), and Na2CO3 (1.1 g, 10 mmol) and the mixture was stirred at r.t. for 24 h. Most of the dioxane was removed by evaporation in vacuo at 35 °C/50 mbar. EtOAc (50 mL) and brine (25 mL) were added to the aqueous residue, the biphasic system was transferred into a separatory funnel, shaken, and the phases were separated. The organic phase was washed with brine (2 x 20 mL), dried over anh. Na2SO4, filtered, and the filtrate was evaporated in vacuo. The residue was purified by CC (EtOAc/hexane, 1:1). Fractions containing the product were combined and evaporated in vacuo. Under argon, the residue was dissolved in anh. DMF (25 mL), K2CO3 (691 mg, 5 mmol) and methyl iodide (934 ^L, 15 mmol) were added and the mixture was stirred at r.t. for 72 h. Volatile components were evaporated in vacuo, EtOAc (100 mL) was added to the residue, and the mixture was washed with brine (3 x 30 mL). The organic phase was dried over anh. Na2SO4, filtered, and the filtrate was evaporated in vacuo. The residue was purified by CC (EtOAc/hexane, 1:1). Fractions containing the product were combined and evaporated in vacuo. The residue was dissolved in dichloromethane (20 mL), TFA (5 mL) was added and the mixture was stirred at r.t. for 24 h. Volatile components were evaporated in vacuo, EtOAc (150 mL) and brine (50 mL) were added, and the biphasic system was made alkaline by slow addition of solid K2CO3 until pH 8-9 was reached. The mixture was stirred vigorously at r.t. for 5 min and then stirring was stopped and the phases were allowed to separate. The organic phase was washed with brine (2 x 10 mL), dried over anh. Na2SO4, filtered, and the filtrate was evaporated in vacuo. The residue was purified by CC (EtOAc/MeOH, 10:1). Fractions containing the product were combined and evaporated in vacuo to give 12a,b, which were used in the next step without further purification. 3. 6. 2. Preparation of tetrahydro-1H- imidazo[1,5-fo]pyrazole-2,6-diones 14a,b A mixture of crude 12c,d (1.5 mmol), methanol (20 mL), and 10% Pd-C (80 mg) was hydrogenated under 3 bar of H2 at room temperature for 1.5 h. The catalyst was removed by filtration through a short pad of Celite®, washed with methanol (3 x 10 mL), and the combined filtrate was evaporated in vacuo. The residue was dissolved in toluene (20 mL) and the solution was evaporated in vacuo again to give anhydrous free diamine 13a,b. The crude diamine 13 (1.5 mmol) was dissolved in anh. DMF (5 mL), CDI (262 mg, 1.5 mmol) was added, and the mixture was stirred at room temperature for 12 h. Volatile components were evaporated in vacuo and the residue was purified by CC (EtOAc-MeOH, 10:1). Fractions containing the product were combined an evaporated in vacuo. The residue (a mixture of 14 and imidazole) was dissolved in EtOAc (1 mL), 2 M HCl-Et2O (1 mL), was added and the precipitate (imidazole hydrochloride) was removed by filtration and washed with anh. Et2O (2 x 2 mL). The filtrate was evaporated in vacuo to give 14a,b. 1,5-Dimethyltetrahydro-1H-imidazo[1,5-fo]pyrazole-2, 6-dione (14a). Prepared from 12a (222 mg, 1.55 mmol) and CDI (265 mg, 1.55 mmol). Yield: 110 mg (42%) of yellow oil. 1H-NMR (500 MHz, DMSO-d6): 5 2.41 (1H, dd, J = 7.8, 16.0 Hz, 3'-Ha), 2.70 (1H, dd, J = 11.3, 16.4 Hz, 3'-Hb), 2.75 (3H, s, 5'-Me), 3.13 (3H, s, 1'-Me), 3.35 (1H, dd, J = 1.2, 9.7 Hz, 4'-H), 3.58 (1H, dd, J = 7.35, 9.82 Hz, 4'-H), 4.30 (1H, dtd, J = 1.20, 7.65, 7.61, 10.97 Hz, CH). 13C-NMR (126 MHz, DMSO-d6): 8 30.1, 32.3, 35.3, 47.6, 53.4, 162.6, 170.1. m/z (ESI) = 130 (MH+). HRMS-ESI (m/z): [MH+] calcd for C7H11N3O2, 170.0924; found, 170.0926. IR (ATr) v 3486, 2926, 2798, 1685, 1496, 1436, 1410, 1384, 1360, 1292, 1253, 1219, 1173, 1146, 1085, 1063, 1037, 1020, 974, 926, 891, 838, 790, 737, 675 cm-1. 5-Benzyl-1-methyltetrahydro-1H-imidazo[1,5-fo]pyra-zole-2,6-dione (14b). Prepared from 5'c (340 mg, 1.55 mmol) and CDI (265 mg, 1.55 mmol). Yield: 201 mg (53%) of yellow oil. 1H-NMR (500 MHz, DMSO-d6): 8 2.44 (1H, dd, J = 16.2, 7.7 Hz, 3-Ha), 2.59 (1H, ddd, J = 16.3, 11.2, 1.1 Hz, 3-Hb), 3.16 (1H, dd, J = 9.8, 1.1 Hz, 4-Ha), 3.34 (3H, s, 1-Me), 3.50 (1H, dd, J = 9.7, 7.3 Hz, 4-Hb), 4.25 (1H, dtd, J = 11.3, 7.5, 1.1 Hz, 3'-H), 4.34 (1H, d, J = 14.8 Hz, 1H of CH2Ph), 4.46 (1H, d, J = 14.9 Hz, 1H of CH2Ph), 7.18-7.26 (2H, m, 2H of Ph), 7.277.43 (3H, m, 3H of Ph). 13C-NMR (126 MHz, DMSO-d6): 8 32.7, 36.0, 45.4, 47.6, 54.0, 128.1, 128.1, 129.0, 135.2, 162.8, 169.6. m/z (ESI) = 246 (MH+). HRMS-ESI (m/z): [MH+] calcd for C H, N O, 246.1237; found, 246.1237. 13 16 3 2 3. 7. General Procedure for the Synthesis of 5-oxopyrazolidine-3-carboxamides 19a-d Under argon, CDI (0.892 g, 5.5 mmol) was added to a stirred suspension of carboxylic acid 17 (1.031 g, 5 mmol) in anh. acetonitrile (20 mL), the mixture was stirred at r.t. for 1.5 h, followed by addition of amine 18 (5 mmol). When amine 18 hydrochloride was used, one equivalent of N-methylmorpholine (NMM, 600 ^L, 5 mmol) was added as well. The mixture was stirred at r.t. for 12 h and volatile components were evaporated in vacuo. The residue was taken up in dichloromethane (30 mL) and the solution was washed with 1 M aq. NaHSO4 (2 x 20 mL), saturated aq. NaHCO3 (2 x 20 mL), and brine (2 x 20 mL). The organic phase was dried over anh. Na2SO4, filtered, and the filtrate was evaporated in vacuo. Volatile components were evaporated in vacuo and the residue was purified by CC (EtOAc). Fractions containing the product were combined an evaporated in vacuo to give 19a-d. Glavac et al.: Synthesis of novel 3D-rich a-amino acid-derived ... 723 Acta Chim. Slov. 2017, 64, 715-726 3. 7. 1. Methyl rac-(5-oxo-1-phenylpyrazolidine-3-carbonyl)glycinate (19a) Prepared from 17 (1.031 g, 5 mmol), CDI (0.892 g, 5.5 mmol), methyl glycinate hydrochloride (18a) (628 mg, 5 mmol), and NMM (600 ^L, 5 mmol). Yield: 653 mg (45%) of red crystals; m.p. 148-152 °C. 1H-NMR (500 MHz, DMSO-d6): 5 2.79 (1H, dd, J = 1.3, 16.6 Hz, 4'-Ha), 3.06 (1H, dd, J = 9.3, 16.6 Hz, 4'-Hb), 3.60 (3H, s, OMe), 3.85-3.97 (2H, m, CH2CO2Me), 4.16-4.22 (1H, m, 5'-h), 6.86 (1H, d, J = 6.8 Hz, 1'-H), 7.12 (1H, t, J = 7.4 Hz, 1H of Ph), 7.37 (2H, dd, J = 7.3, 8.6 Hz, 2H of Ph), 7.86 (2H, td, J = 1.2, 7.5 Hz, 2H of Ph), 8.57 (1H, t, J = 6.1 Hz, NHCH2). 13C-NMR (126 MHz, DMSO-d6): 5 36.8, 40.7, 51.7, 54.6, 118.1, 123.7, 128.4, 138.8, 169.8, 170.0, 171.2. m/z (ESI) = 278 (MH+). HRMS-ESI (m/z): [MH+] calcd for C13H15N3O4, 278.1135; found, 278.1138. Anal. Calcd for C13H15N3O4: C 56.31, H 5.45, N 15.15. Found: C 56.05, H 5.53, N 14.87. IR (ATR) v 3359, 3219, 3005, 2959, 2930, 1754, 1695, 1655, 1593, 1525, 1489, 1461, 1440, 1403, 1358, 1338, 1312, 1281, 1242, 1205, 1160, 1127, 1096, 1075, 1031, 1013, 983, 968, 956, 932, 909, 828, 764, 718, 692, 659, 617 cm-1. 3. 7. 2. Methyl rac-3-(5-oxo-1-phenylpyrazolidine-3-carboxamido)propanoate (19b) Prepared from 17 (208 mg, 1 mmol), CDI (178 mg, 1.1 mmol), methyl ^-alaninate hydrochloride (18b) (140 mg, 1 mmol), and NMM (120 ^L, 1 mmol). Yield: 131 mg (45%) of pale yellowish crystals; m.p. 88-91 °C. 1H-NMR (500 MHz, DMSO-d6): 5 2.41-2.54 (2H, m, CH2NH), 3.07 (1H, dd, J = 17.2, 9.3 Hz, 4'-Ha), 3.13 (1H, dd, J = 17.2, 3.5 Hz, 4'-Hb), 3.47 (1H, ddt, J = 13.6, 7.3, 5.1 Hz, CH2CO2Me), 3.50-3.60 (4H, m, OMe and 1H of CH2CO2Me), 4.09 (1H, ddd, J = 9.7, 6.6, 3.4 Hz, 5'-H), 5.43 (1H, d, J = 6.6 Hz, 1'-H), 7.14 (1H, t, J = 7.4 Hz, 1H of Ph), 7.37 (2H, dd, J = 8.7, 7.3 Hz, 2H of Ph), 7.78 (1H, s, NHCO), 7.82 (2H, d, J = 7.6 Hz, 2H of Ph). 13C-NMR (126 MHz, DMSO-d6): 8 33.7, 34.8, 37.2, 51.8, 55.31, 118.0, 124.7, 128.9, 138.2, 169.5, 170.0, 172.6. m/z (ESI) = 292 (MH+). HRMS-ESI (m/z): [MH+] calcd for C14H17N3O4, 292.1292; found, 292.1295. Anal. Calcd for C14H17N3O4: C, 57.72; H, 5.88; N, 14.42. Found: C, 57.89; H, 5.61; N, 14.20. IR (ATR) v 3371, 3284, 3073, 3026, 2954, 2932, 2883, 2848, 1722, 1695, 1593, 1525, 1496, 1455, 1434, 1398, 1361, 1337, 1323, 1309, 1272, 1231, 1198, 1178, 1160, 1121, 1078, 1054, 1028, 1009, 967, 925, 894, 875, 817, 753, 713, 690, 667, 615 cm-1. 3. 7. 3. 5-Oxo-N-phenethyl-1-phenylpyrazolidine-3-carboxamide (19c) Prepared from 17 (208 mg, 1 mmol), CDI (178 mg, 1.1 mmol), and 3-phenylethylamine (18c) (126 ^L, 1 mmol). Yield: 154 mg (50%) of white crystals; m.p. 131-133 °C. 1H-NMR (500 MHz, DMSO-d6): 8 2.73 (2H, td, J = 4.5, 6.7, 6.9 Hz, NHCH2), 3.02 (1H, dd, J = 9.6, 17.7 Hz, 4'-Ha), 3.09 (1H, dd, J = 3.3,17.3 Hz, 4'-Hb), 3.45 (1H, qd, J = 6.4, 13.1 Hz, 1H of CH2Ph), 3.58 (1H, qd, J = 6.5, 13.1 Hz, 1H of CH2Ph), 4.02 (1H, ddd, J = 3.3, 6.6, 9.8 Hz, 5'-H), 5.31 (1H, d, J = 6.7 Hz, 1'-H), 7.01-7.08 (1H, m, 1H of Ph), 7.11-7.24 (5H, m, Ph), 7.30-7.38 (2H, m, 2H of Ph), 7.67 (2H, d, J = 7.8 Hz, 2H of Ph). 13C-NMR (126 MHz, DMSO-d6): 8 35.4, 37.3, 40.4, 55.2, 117.8, 124.7, 126.6, 128.6, 128.7, 129.0, 138.2, 138.3, 169.4, 169.9. m/z (ESI) = 310 (MH+). HRMS-ESI (m/z): [MH+] calcd for C18H20N3O2, 310.1550; found, 310.1555. Anal. Calcd for C18H19N3O2: C, 69.88; H, 6.19; N, 13.58. Found: C, 69.78; H, 6.13; N, 13.53. IR (ATR) v 3314, 3193, 3079, 3061, 3024, 2936, 2863, 1944, 1872, 1805, 1686, 1651, 1593, 1539, 1492, 1479, 1454, 1434, 1361, 1323, 1311, 1298, 1287, 1252, 1217, 1189, 1153, 1120, 1087, 1065, 1031, 1004, 982, 952, 932, 902, 868, 833, 747, 716, 688, 657, 614 cm-1. 3. 7. 4. N,N-Diethyl-5-oxo-1-phenylpyrazolidine-3-carboxamide (19d) Prepared from 17 (208 mg, 1 mmol), CDI (178 mg, 1.1 mmol), and diethylamine (18d) (104 ^L, 1 mmol). Yield: 128 mg (49%) of pale greyish crystals; m.p. 77-79 °C. 1H-NMR (500 MHz, DMSO-d6): 8 1.05 (3H, t, J = 7.1 Hz, Me), 1.18 (3H, t, J = 7.0 Hz, Me), 2.85 (2H, d, J = 15.7 Hz, 4'-CH2), 3.32 (2H, m, CH2Me), 3.42 (2H, m, CH2Me), 4.55 (1H, m, 5'-H), 6.39 (1H, d, J = 9.37 Hz, 1'-H), 7.07 (1H, t, J =7.4 Hz, 1H of Ph), 7.33 (2H, t, J = 7.85 Hz, 2H of Ph), 7.77 (2H, d, J = 8.10 Hz, 2H of Ph). 13C-NMR (126 MHz, DMSO-d6): 8 12.7, 14.4, 37.4, 39.6, 40.9, 53.2, 117.7, 123.4, 128.4, 139.1, 168.2, 170.5. m/z (ESI) = 262 (MH+). HRMS-ESI (m/z): [MH+] calcd for C14H20N3O2, 262.1550; found, 262.1551. Anal. Calcd for C14H19N3O2: C, 64.35; H, 7.33; N, 16.08. Found: C, 64.44; H, 7.20; N, 15.93. IR (ATR) v 3212, 3063, 2979, 2932, 2901, 2873, 2159, 1699, 1631, 1593, 1496, 1481, 1471, 1424, 1369, 1320, 1272, 1233, 1218, 1154, 1140, 1102, 1072, 1042, 1029, 997, 965, 938, 906, 878, 843, 815, 760, 720, 692, 671, 660 cm-1. 3. 8. General Procedure for the Synthesis of Non-racemic Carboxamides 19e,f and 19'e,f Mixtures of diastereomeric carboxamides 19e/19'e and 19e/19'f were prepared from racemic carboxylic acid 17 and (S)-amino esters 18e and 18f, respectively, following the general procedure for the preparation of racemic carboxamides 19a-d. Mixtures of diastereomers 19e/19'e and 19f/19'f were separated by MPLC (EtOAc-hexane). Fraction containing the products were combined and evaporated in vacuo to give the non-racemic diastere-omerically pure carboxamides 19e, 19'e, 19f, and 19'f. 3. 8. 1. Methyl (5R,2'S)-(3-oxo-2- phenylpyrazolidine-5-carbonyl)alaninate (19e) and its (5S,2'S)-isomer 19'e Prepared from 17 (0.208 g, 1 mmol), CDI (0.178 g, 1.1 mmol), methyl (S)-alaninate hydrochloride (18e) (140 Glavac et al.: Synthesis of novel 3D-rich a-amino acid-derived ... 724 Acta Chim. Slov. 2017, 64, 715-726 mg, 1 mmol), and NMM (120 ^L, 1 mmol); MPLC (EtOAc-hexane, 1:1). Data for the (-)-isomer 19e. Yield: 67 mg (23%) of yellow oil; [a]D22 -64.5 (c 0.365, CH2Cl2), MPLC: Rt = 67 min. 1H NMR (500 MHz, CDCl3): 5 1.33 (3H, d, J = 7.2 Hz, Me), 3.13 (2H, d, J = 6.4 Hz, 4'-CH2), 3.75 (3H, s, CO2Me), 4.16 (1H, q, J = 6.6 Hz, 2-H), 4.54 (1H, m, 5'-H), 5.48 (1H, d, J = 6.7 Hz, 3-H), 7.16 (1H, t, J = 7.4 Hz, 1H of Ph), 7.38 (2H, dd, J = 7.4, 8.7 Hz, 2H of Ph), 7.73 (1H, s, 1'-H), 7.83 (2H, dd, J = 1.2, 8.8 Hz, 2H of Ph). 13C NMR (126 MHz, CDCl3): 8 18.25, 37.22, 48.06, 52.63, 55.29, 117.97, 124.84, 129.04, 138.23, 169.39, 169.80, 173.12. m/z (ESI) = 292 (MH+). HRMS-ESI (m/z): [MH+] calcd for C14H17N3O4, 292.1292; found, 292.1292. IR (ATR) v 3469, 3367, 3227, 3068, 2992, 2952, 2848, 1739, 1664, 1595, 1518, 1495, 1454, 1352, 1323, 1310, 1210, 1154, 1112, 1056, 1030, 979, 932, 894, 847, 827, 754, 691, 670, 629 cm-1. Data for the (+)-isomer 19'e. Yield: 60 mg (21%) of yellow oil; [a]D22 +82.2 (c 0.39, CH2Cl2), MPLC: Rt = 78 min. 1H NMR (500 MHz, CDCl3): 8 1.43 (3H, d, J = 7.1 Hz, Me), 3.09 (1H, dd, J = 9.3, 17.2 Hz, 4'-Ha), 3.16 (1H, dd, J = 3.5, 17.2 Hz, 4'-Hb), 3.62 (3H, s, CO2Me), 4.14 (1H, ddd, J = 3.5, 6.6, 9.7 Hz, 2-H), 4.54 (1H, m, 5'-H), 5.44 (1H, d, J = 6.7 Hz, 3-H), 7.16 (1H, t, J = 7.4 Hz, 1H of Ph), 7.38 (2H, dd, J = 7.4, 8.7 Hz, 1H of Ph), 7.81-7.90 (3H, m, 1'-H and 2H of Ph). 13C NMR (126 MHz, CDCl3): 8 18.4, 37.0, 48.2, 52.5, 55.4, 118.42, 124.9, 129.0, 138.2, 169.3, 169.6, 172.6. m/z (ESI) = 292 (MH+). HRMS-ESI (m/z): [MH+] calcd for C,„H N O 292.1292; found, 292.1293. IR (ATR) v 3486, 14 17 3 4 3369, 3226, 3066, 2992, 2952, 2848, 1739, 1665, 1595, 1518, 1495, 1453, 1353, 1325, 1310, 1211, 1154, 1110, 1061, 1030, 1019, 980, 933, 899, 846, 827, 754, 691, 670, 617 cm-1. 3. 8. 2. Methyl (5R,2'S)-(3-oxo-2- phenylpyrazolidine-5-carbonyl)prolinate (19f) and its (5S,2'S)-isomer 19'f Prepared from 17 (0.208 g, 1 mmol), CDI (0.178 g, 1.1 mmol), methyl (S)-prolinate hydrochloride (18f) (166 mg, 1 mmol), and NMM (120 ^L, 1 mmol); MPLC (EtOAc-hexane, 2:1). Data for the (-)-isomer 19f. Yield: 40 mg (13%) of yellow oil; [a]D22 -98.1 (c 0.425, CH2Cl2), MPLC: Rt = 67 min. 1H NMR (500 MHz, CDCl3): 8 2.03-2.10 (2H, m, 4'-CH2), 2.09-2.18 (1H, m, 3-CH2), 2.23-2.31 (1H, m, 3-CH2), 2.95 (1H, dd, J = 8.2, 16.5 Hz, 4'-CH2), 3.07 (1H, dd, J = 11.3, 16.5 Hz, 4'-CH2), 3.54-3.63 (1H, m, 5-CH2), 3.65-3.71 (1H, m, 5-CH2), 3.76 (3H, s, CO2Me), 4.51 (1H, dt, J = 8.2, 11.0 Hz, 5-H), 4.60 (1H, dd, J = 3.9, 8.8 Hz, 2'-H), 5.67 (1H, d, J = 10.8 Hz, NH), 7.13 (1H, t, J = 7.4 Hz, 1H of Ph), 7.36 (2H, dd, J = 7.3, 8.7 Hz, 2H of Ph), 7.84 (2H, d, J = 7.3 Hz, 2H of Ph). 13C NMR (126 MHz, CDCl3): 8 24.7, 28.8, 37.5, 46.4, 52.5, 54.9, 59.0, 118.4, 124.5, 128.7, 138.4, 168.4, 168.6, 172.0. m/z (ESI) = 318 (MH+). HRMS-ESI (m/z): [MH+] calcd for C16H19N3O4, 318.1448; found, 318.1447. IR (ATR) v 3496, 32101 3066, 2953, 2881, 2248, 1740, 1695, 1645, 1595, 1496, 1456, 1434, 1418, 1357, 1323, 1311, 1280, 1196, 1173, 1094, 1030, 998, 984, 963, 910, 861, 834, 790, 755, 728, 691, 670, 647, 616 cm-1. Data for the (+)-isomer 19'f. Yield: 43 mg (14%) of yellow oil; [a]D22 +2.5 (c 0.43, CH2Cl2), MPLC: R = 84 min. 1H NMR (500 MHz, CDCl3): 5 2.02-2.13 (2H, m, 4-CH2), 2.11-2.20 (1H, m, 3-CH2), 2.23-2.29 (1H, m, 3-CH2), 2.89 (1H, dd, J = 8.1, 16.1 Hz, 4'-CH2), 3.02 (1H, dd, J = 11.3, 16.2 Hz, 4'-CH2), 3.47 (1H, td, J = 7.1, 9.6, 5-CH2), 3.76 (3H, s, CO2Me), 3.85 (1H, ddd, J = 4.3, 8.0, 10.0 Hz, 5-CH2) 4.51-4.54 (1H, m, 5-H), 4.55-4.58 (1H, m, 2'-H), 5.59 (1H, d, J = 10.7 Hz, NH), 7.13 (1H, t, J = 7.4 Hz, 1H of Ph), 7.36 (2H, dd, J = 7.3, 8.7 Hz, 2H of Ph), 7.86 (2H, d, J =7.6 Hz, 2H of Ph). 13C NMR (126 MHz, CDCl3): 5 24.6, 29.0, 37.6, 46.6, 52.5, 55.1, 59.2, 118.4, 124.5, 128.7, 138.4, 168.3, 168.6, 172.0. m/z (ESI) = 318 (MH+). HRMS-ESI (m/z): [MH+] calcd for C H,N O , 318.1448; found, 318.1447. L J 16 19 3 4 7 ' IR (ATR) v 3468, 3212, 3064, 2953, 2882, 2251, 1739, 1695, 1643, 1595, 1495, 1455, 1434, 1419, 1356, 1323, 1311, 1282, 1196, 1172, 1094, 1031, 996, 981, 911, 856, 836, 792, 755, 729, 691, 670, 646, 615 cm-1. 4. Conclusions 1,2-Unsubstituted 5-aminomethyl-3-pyrazolidinones are available in four steps from N-protected glycines and their N-alkylated analogues. Although the alternative one-step 'ring switching' synthesis of 5-aminomethyl-3-pyrazo-lidinones is definitely simpler and shorter, the high price or difficult availability of the starting N-protected pyrrolin-2(5H)-one, as well as lower yield and purity of the so obtained product are disadvantageous. Regioselective reductive alkylation 1,2-unsubstituted pyrazolidinones with di-methoxyacetaldehyde provided the 1-(2,2-dimethoxyethyl) substituted 3-pyrazolidinones, which, unfortunately could not be cyclized into the desired hexahydropyrazolo[1,5-a] pyrazin-2(1H)-ones. On the other hand, a three step selective methylation provided selectively the 2-methyl regioiso-mers as key-intermediates in the preparation of two novel 1,5-dialkyltetrahydro-1H-imidazo[1,5-b]pyrazole-2,6 -di-ones, as rare representatives of almost unexplored 3D-rich heterocyclic system. The number of synthetic steps in the above preparations may seem disadvantageous, nevertheless, this is compensated by performing up to five subsequent steps via a one-pot procedure. Amidation of easily available 3-oxopyrazolidine-5-carboxylic acid yielded the corresponding carboxamides in moderate yields. Diastere-omeric non-racemic carboxamides obtained from (S)-AlaOMe and (S)-ProOMe are separable by MPLC. 5. Acknowledgement The authors acknowledge the financial support from the Slovenian Research Agency (research core funding No. Glavac et al.: Synthesis of novel 3D-rich a-amino acid-derived ... 725 Acta Chim. Slov. 2017, 64, 715-726 P1-0179). The financial support from the Boehringer-In-gelheim Pharma, Biberach, Germany in gratefully acknowledged. 6. References 1. J. A. Joule, K. Mills, In: Heterocyclic Chemistry, 5th ed., Wiley-Blackwell, West Sussex, UK, 2010. 2. G. L. Patrick, In: An Introduction to Medicinal Chemistry, 4th ed., Oxford University Press: Oxford, UK, 2009. 3. G. Varvounis, Y. Fiamegos, G. Pilidis, Adv. Heterocycl. Chem. 2001, 80, 73-156. 4. J. Elguero, In: A. R. Katritzky, C. W. Rees, E. F. V. Scriven, (Eds.), Pyrazoles, Comprehensive Heterocyclic Chemistry II, Elsevier, Oxford, 1996, Vol. 3, pp. 1-75. 5. R. M. Claramunt, J. Elguero, Org. Proc. Prep. Int. 1991, 23, 273-320. 6. H. Dorn, Chem. Heterocycl. Compd. USSR 1981, 3-31. 7. C. Cucurou, J. P. Battioni, D. C. Thang, N. H. Nam, D. Mansuy, Biochemistry 1991, 30, 8964-8970. 8. H. L. White, J. L. Howard, B. R. Cooper, F. E. Soroko, J. D. McDermed, K. J. Ingold, R. A. Maxwell, J Neurochem. 1982, 39, 271-273. 9. S. Hanessian, L. Auzzas, Acc. Chem. Res. 2008, 41, 1241-1251. 10. J. Cluzeau, W. D. Lubell, Biopolymers 2005, 80, 98-150. 11. E. M. Kosower, E. Hershkowitz, IL patent 1990-94658 94658. 1994 19900607. 12. R. J. Ternansky, S. E. Draheim, A. J. Pike, F. T. Counter, J. A. Eudaly, J. S. Kasher. J. Med. Chem. 1993, 36, 3224-3229. 13. E. M. Kosower, A. E. Radkowsky, A. H. Fairlamb, S. L. Croft, R. A. Neal, Eur. J. Med. Chem. 1995, 30, 659-671. 14. S.-G. Wang, H. R. Tsai, K. Chen, Tetrahedron Lett. 2004, 45, 6183-6185. 15. C. L. Fan, W.-D. Lee, N.-W. Teng, Y.-C. Sun, K. Chen, J. Org. Chem. 2003, 68, 9816-9818. 16. C. H. Lin, K. S. Yang, J. F. Pan, K. Chen, Tetrahedron Lett. 2000, 41, 6815-6819. 17. K.-S. Yang, K. Chen, J. Org. Chem. 2001, 66, 1676-1679. 18. K.-S. Yang, J.-C. Lain, C.-H. Lin, K. Chen, Tetrahedron Lett. 2000, 41, 1453-1456. 19. K.-S. Yang, K. Chen, Org. Lett. 2000, 2, 729-731. 20. M. P. Sibi, L. M. Stanley, X. Nie, L. Venkatraman, M. Liu, C. P. Jasperse, J. Am. Chem. Soc. 2007, 129, 395-405. 21. M. P. Sibi, S. Manyem, H. Palencia, J. Am. Chem. Soc. 2006, 128, 13660-13661. 22. M. P. Sibi, L. Venkatraman, M. Liu, C. P. Jasperse, J. Am. Chem. Soc. 2001, 123, 8444-8445. 23. M. Lemay, L. Aumand, W. W. Ogilvie, Adv. Synth. Catal. 2007, 349, 441-447. 24. M. Lemay, J. Trant, W. W. Ogilvie, Tetrahedron 2007, 63, 11644-11655. 25. M. Lemay, W. W. Ogilvie, J. Org. Chem. 2006, 71, 4663-4666. 26. M. Lemay, W. W. Ogilvie, Org. Lett. 2005, 7, 4141-4144. 27. E. Gould, T. Lebl, A. M. Z. Slawin, M. Reid, A. D. Smith, Tetrahedron 2010, 66, 8992-9008. 28. J. B. Brazier, J. L. Cavill, R. L. Elliott, G. Evans, T. J. K. Gibbs, I. L. Jones, J. A. Platts, N. C. O. Tomkinson, Tetrahedron 2009, 65, 9961-9966. 29. G. J. S. Evans, K. White, J. A. Platts, N. C. O. Tomkinson, Org. Biomol. Chem. 2006, 4, 2616-2627. 30. J. L. Cavill, R. L. Elliott, G. Evans, I. L. Jones, J. A. Platts, A. M. Ruda, N. C. O. Tomkinson, Tetrahedron 2005, 62, 410-421. 31. U. Grošelj, J. Svete, ARKIVOC 2015, Part vi, 175-205. 32. J. Svete, In: (4R*,5R*)-4-Benzoylamino-5-phenyl-3-pyrazo-lidinone - A Useful Building Block in the Synthesis of Func-tionalized Pyrazoles in Stereochemistry Research Trends, M. A. Horvat, J. H. Golob, Eds.; Nova Science Publishers, Inc., New York. 2008, p. 129-182. 33. L. Šenica, N. Petek, U. Grošelj, J. Svete, Acta Chim. Slov. 2015, 62, 60-71. 34. L. Šenica, K. Stopar, M. Friedrich, U. Grošelj, J. Plavec, M. Počkaj, Č. Podlipnik, J. Svete, J. Org. Chem. 2016, 81, 146161. 35. U. Grošelj, A. Podlogar, A. Novak, G. Dahmann, A. Golobič, B. Stanovnik, J. Svete, Synthesis 2013, 45, 639-650. 36. J. Mirnik, U. Grošelj, A. Novak, G. Dahmann, A. Golobič, M. Kasunič, B. Stanovnik, J. Svete, Synthesis 2013, 45, 3404-3412. 37. K. Lombar, U. Grošelj, G. Dahmann, B. Stanovnik, J. Svete, Synthesis 2015, 47, 497-506. 38. U. Grošelj, A. Golobič, J. Svete, B. Stanovnik, Chirality 2013, 25, 541-555. 39. E. Pušavec Kirar, M. Drev, J. Mirnik, U. Grošelj, A. Golobič, G. Dahmann, F. Požgan, B. Štefane, J. Svete, J. Org. Chem. 2016, 81, 8920-8933. 40. U. Grošelj, M. Žorž, A. Golobič, B. Stanovnik, J. Svete, Tetrahedron 2013, 69, 11092-11108. 41. Y. Bandala, R. Melgar-Fernández, R. Guzmán-Mejía, J. L. Olivares-Romero, B. R. Díaz-Sánchez, R. González-Olvera, J. Vargas-Caporali, E. Juaristi, J. Mex. Chem. Soc. 2009, 53, 147154. 42. C. R. Theberge, C. K. Zercher, Tetrahedron 2003, 59, 15211527. 43. M. P. C. Mulder, F. El Oualid, J. ter Beek, H. Ovaa, ChemBio 2014, 15, 946-949. Glavac et al.: Synthesis of novel 3D-rich a-amino acid-derived ... 726 Acta Chim. Slov. 2017, 64, 715-726 Povzetek Študirali smo sinteztne pristope za pripravo novih 3-pirazolidinonskih derivatov funkcionaliziranih na položajih N(1) in/ali C(5). 5-aminoalkil-3-pirazolidinone smo pripravili v štirih korakih iz N-zaščitenih glicinov preko Masam-une-Claisenove homologacije, redukcije, O-meziliranja in ciklizacije z derivati hidrazina. Proste amine smo pripravili z odščito v kislem. Ciljno spojino smo pripravili tudi z 'ring switching' transformacijo N-Boc-pirolin-2(5H)-ona s hidraz-in hidratom. S katalitskim hidrogeniranjem smo odščitili 5-(N-alkil-N-Cbz-aminometil)pirazolidine-3-one in s sledečo ciklizacijo z 1,1'-karbonildiimidazolom (CDI) pripravili dva nova predstavnika perhidroimidazo[1,5-fo]pirazola, ki je skoraj popolnoma neraziskan heterociklični system. Pri amidiranju 3-oksopirazolidin-5-karboksilne kisline smo dobili ustrezne karboksamide s srednjimi izkoristki. Diastereomerne neracemne karboksamide, pripravljene iz (S)-AlaOMe in (S)-ProOMe, smo ločili s pomočjo MPLC kromatografske tehnike. Glavac et al.: Synthesis of novel 3D-rich a-amino acid-derived ... DOI: 10.17344/acsi.2017.3543 Acta Chim. Slov. 2017, 64, 727-736 ¿^creative tS1 commons Scientific paper Asymmetric Bio- and Chemoreduction of 2-Benzylidenecyclopentanone Derivatives Bogdan Štefane, Uroš Grošelj, Jurij Svete and Franc Požgan* Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, SI-1000 Ljubljana, Slovenia * Corresponding author: E-mail: franc.pozgan@fkkt.uni-lj.si Received: 16-05-2017 Dedicated to Professor Emeritus Miha Tišler, University of Ljubljana, on the occasion of his 90th birthday. Abstract Highly efficient asymmetric reduction of 2-benzylidenecyclopentanone derivatives to give the respective exocyclic allylic alcohols in ee's up to 96% was performed with chiral oxazaborolidine-based catalysts. Complete enantioselectivity furnishing (S)-configured alcohol product could be achieved by bioreduction of 2-(4-chlorobenzylidene)cyclopentanone with Daucus carota root. The synthesized compounds may be used as enantiomerically enriched standards for the monitoring of the enzyme-catalyzed redox processes. Keywords: Enantioselective reduction, cyclopentanone, AKR1C inhibitors, oxazaborolidine, bioreduction 1. Introduction Chiral alcohols are important building blocks and intermediates in the synthesis of pharmaceuticals, fine chemicals, agro chemicals, flavors and fragrances, as well as functional materials.1 Since ketones represent one of the most common families of unsaturated compounds, their asymmetric reduction represents the simplest and most powerful method for the preparation of enantiomerically enriched alcohols. The stereospecific reduction of carbon-yl groups to the corresponding alcohols is also a function-alization reaction involved in the metabolism of endoge-neous compounds and xenobiotics containing these groups. Thus, it is often catalyzed by enzymes belonging to either dehydrogenase/reductase superfamily or the al-do-keto reductase (AKR) superfamily.2 The human members of the AKR subfamily 1C are involved in the biosynthesis and inactivation of steroid hormones, and also in the biosynthesis of neurosteroids and prostaglandins.3 These enzymes reduce carbonyl containing substrates to alcohols and also function in vivo as ketosteroid reduc-tases, and thus regulate the activity of androgens, estrogens and progesterone in target tissues, and ligand occupancy and transactivation of their corresponding receptors.4 Aberrant expression and action of AKR1C enzymes may lead to an imbalance in the metabolism of steroid hormones, and to further development of different patho- physiological conditions.5 These enzymes thus represent promising therapeutic targets in the development of new drugs. In the literature, structurally different compounds have been evaluated as AKR1C inhibitors, for example dietary phytoestrogens,6 benzodiazepines,7 cinnamic acids,8 benzofurans, and phenolphthalein derivatives,9 Ru(II) complexes,10 salicylic and aminobenzoic acids derivatives, as well as some nonsteroidal anti-inflammatory drugs and their analogues.11,12 In spite of a plethora of potent inhibitors of steroid metabolizing enyzmes that have emerged, the search for new and more selective ones is an important field of investigation. Štefane et al.13 indentified compounds based on cyclopentane scaffold, which are AKR1C1 and AKR1C3 substrates active in the low micromolar range, and thus represent promising starting points in the development of potential agents for treatment of hormone-dependent forms of cancer and other diseases involving these enzymes. AKR1C inhibitors are not only interesting as potential agents for the treatment of diseases, but also as molecular tools in the study of the patho-physiological roles of these enzymes. In the recent study Beranič et al. introduced new enzymatic assays employing racemic 2-(4-chlorobenzylidene)cyclopentanol (CBCP-ol) and its ketone counterpart 2-(4-chlorobenzylidene)cyclo-pentanone that allow monitoring of AKR1C-catalyzed reactions in the reductive and oxidative directions.14 Since enzymes perform highly stereoselective reactions, it seems Štefane et al.: Asymmetric Bio- and Chemoreduction 728 Acta Chim. Slov. 2017, 64, 727-736 useful to know, which enantiomer of CBCP-ol is involved in the redox process. For this reason we present herein the synthesis of enantiomerically enriched cyclopentyl alcohols (CBCP-ol and its 4-methoxy analogue) via the asymmetric chemo- and bioreduction of substituted 2-ben-zylidenecyclopentanones, which can serve as standards in monitoring of AKRIC-catalyzed reactions. The reduction of the benzene-fused analogue, indanone-derived chal-cone, to the corresponding secondary allylic alcohol is also included. 2. Results and Discussion The starting compounds, a-arylmethylene cyclic ketones 3 and 6 were synthesized in a base-induced aldol condensation from cyclopentanone (1) or 1-indanone (5) and the corresponding p-substituted benzaldehydes 2 following slightly modified literature procedure15 (Scheme 1). The reaction of cyclopentanone with p-methoxybenzalde-hyde (2b) towards benzylidenecyclopentanone 3b proceeded smothly, while using p-chlorobenzaldehyde (2a), besides the desired product 3a, symmetrical abis(ben-zylidene) derivative 4a was isolated as the by-product. 1-Indanone reacted with p-chlorobenzaldehyde leading to the product 6 in a very low 9% isolated yield. We were not, however, interested in the optimization of these aldol condensation reactions. With a,^-unsaturated ketones 3 and 6 in hand, we investigated different methods for the selective carbonyl reduction to obtain the highest possible enantiomeric excess of the corresponding allylic alcohol products with exocyclic C=C double bond. The most elegant method for the asymmetric reduction of prochiral ketones is either homogeneous or heterogeneous hydrogenation or transfer hydrogenation catalyzed by chiral metal catalysts.16 Highly efficient asymmetric hydrogenation of a-arylmethylene cyclopentanones was realized by chiral tailor-made iridium-spiroamino-phosphine catalysts;17 for example, reduction of 3b gave 7b with 95% ee (enantiomeric excess). Unfortunately, in our case the use of some commercially available chiral rhodium and ruthenium catalysts C1-C4 (Figure 1) in hydrogenation of cyclopentanone 3a with molecular hydrogen (80 bars) led to very low yields and ee values of the secondary alcohol 7a; the best ee of 12% (31% isolated yield) was obtained with Noyori's bifunctional ruthenium catalyst C4. After report by Itsuno18 that chiral aminoalcohols together with BH3 effected the enantioselctive reduction of prochiral ketones, Corey19 isolated the primarily formed oxazaborolidine derivative, and developed a powerful catalytic version of an original stoichometric reduction. Con- Scheme 1. Synthesis of a-arylmethylene cyclic ketones 3, 4 and 6. Figure 1. Chiral catalysts employed in the asymmetric reduction of cyclic ketones 3 and 6. Štefane et al.: Asymmetric Bio- and Chemoreduction ... 729 Acta Chim. Slov. 2017, 64, 727-736 sequently, enantioselective reduction of prochiral ketones with borane (or its derivatives) catalyzed by chiral oxaz-aborolidines has emerged as an excellent route to alcohols of high enantiomerical purity.20 Since this method has many advantages such as predictable absolute configuration and high ee of chiral secondary alcohol products, it seemed logical to investigate whether oxazaborolidine-ca-talysed reduction of ketones 3 and 6 could afford the desired exocyclic allylic alcohols in high enantioselectivity. Indeed, the borane reduction of chlorobenzylidenecyclo-pentanone 3a in the presence of 10 mol% of oxazaboroli-dine catalyst (S)-C5 at room temperature afforded the desired alcohol (R)-7a in 77% ee (as juged by chiral HPLC) (Table 1, entry 1). By varying different solvents, reaction temperatures, amount of reductant, and catalyst loading (Table 1, entries 2-8), the highest ee of 96% in reduction of 3a was achieved with 1.88 equiv. BH3 x Me2S, 20 mol% (S)-C5 in toulene at 0 °C. Typically, reduction was carried out by slow addition of a toluene solution of the ketone to an ice-cooled toluene solution of BH3 x Me2S and catalyst (stirred for 10 min prior to adding the ketone). The same protocol was used in the reduction of the methoxy-substi-tuted analogue 3b giving (R)-7b but with significant loss of enantioselectivity (Table 1, entry 9). The opposite enantio- mers, (S)-7a and (S)-7b, were obtained by the borane reduction with the oxazaborolidine catalyst (R)-C5 (Table 1, entries 10 and 11). Interestingly, chloro-substituted alcohols (S)-7a and (R)-7a were obtained with practically identical ee values (~95%), while catalyst (R)-C5 reduced methoxy-benzylidenecyclopentanone 3b with increased enantioselectivity compared to catalyst (S)-C5 (90% vs. 82% ee). A dramatic drop in chemical yield and optical purity of the indanol alcohol 6 was observed in reduction of the indanone derivative 6 with either (S)-C5 or (R)-C5 catalyst. In spite of applying different reaction conditions (Table 1, entries 12-17), the corresponding alcohol 8 was not obtained in ee higher than 33%. Lower ee values associated with asymmetric reduction of indanone 6 as compared to cyclopentanone 3 may suggest that a fused benzene ring has a pronounced influence on the level of asymmetric induction with oxazaborolidine catalysts C5. Additionally, low isolated yield of indanol 8 might be due to its decomposition (or of parent ketone) under applied reaction conditions as was also established for reduction of analogous indanone-derived chalcones.21 The enantiomeric excess of the allylic alcohols 7 and 8 was determined by chiral stationary phase HPLC. The corresponding racemic alcohols were synthesized by che- Table 1: Asymmetric reduction of cyclic ketones 3a,b and 6 with oxazaborolidine catalysts C5. entry ketone catalyst (mol%) solvent BH3 x Me2S (equiv.) T (°C) product yield a (%) eeb (%) 1 3a (S)-C5, 10 THF 1.10 r. t. 7a n.d. 77 (R) 2 3a (S)-C5, 10 THF 2.74 -20 7a 88 81 (R) 3 3a (S)-C5, 10 THF 1.70 -20 7a 67 32 (R) 4 3a (S)-C5, 10 THF 3.42 -20 7a 91 75 (R) 5 3a (S)-C5, 20 DCM 1.70 -6 7a 84 90 (R) 6 3a (S)-C5, 10 THF 2.74 0 7a 82 88 (R) 7 3a (S)-C5, 10 THF 1.70 0 7a 80 90 (R) 8 3a (S)-C5, 20 toluene 1.88 0 7a 77 96 (R) 9 3b (S)-C5, 20 toluene 1.88 0 7b 67 82 (R) 10 3a (R)-C5, 20 toluene 1.88 0 7a 70 95 (S) 11 3b (R)-C5, 20 toluene 1.88 0 7b 59 90 (S) 12 6 (R)-C5, 20 toluene 1.88 0 8 15 23 13td 6 (R)-C5, 20 toluene/CH2Cl2 2.10 0 8 21 24 14d 6 (S)-C5, 20 toluene/CH2Cl2 1.27 0 8 20 33 15 6 (S)-C5, 20 CH2Cl2 1.27 0 8 19 21 16ef 6 (S)-C5, 20 THF 1.27 -30 - - - 17e 6 (S)-C5, 20 ch2CI2 1.27 -30 8 22 19 "Isolated yield is given. bDetermined by chiral HPLC. cFirst solution of 6 added to a solution of (R)-C5, CH2Cl2, and catalyst in toluene. 'Reaction quenched with MeOH. The desired alcohol was not isolated. then BH3 x Me2S. dKetone dissolved in Štefane et al.: Asymmetric Bio- and Chemoreduction ... 730 Acta Chim. Slov. 2017, 64, 727-736 Štefane et al.: Asymmetric Bio- and Chemoreduction 731 Acta Chim. Slov. 2017, 64, 727-736 f) 1 . I S \ & -^---- § LÏ£Î_| T- c6 A .1 V mAU7 16001400-1 1200-j 1000 -j 800-i 600 -, 400 200 0 2.5 7.5 I 10 12.5 15 17.5 20 22 5 g) I * s i <•> « A i I -Tî-iî^-a;- r-g rj -E-l- I l A * 1 ■ ■ i 1 ■ □ 5 10 15 20 25 30 35 mir mAU 40D 300 200' 10D' 0 Figure 2. Chromatograms of (a) racemic alcohol 7a, (b) racemic alcohol 7b, (c) racemic alcohol 8, (d) enantiomerically enriched (R)-7a obtained with (S)-C5, (e) enantiomerically enriched (S)-7a obtained with (R)-C5, (f) enatiomerically enriched (R)-7b obtained with (S)-C5, (g) alcohol 8 obtained with (R)-C5, (h) enantiomerically enriched (R)-7a obtained with Daucus carota root reduction. moselective reduction with NaBH4 in the presence of CeCl3 x 6H2O. They were used to find the optimal HPLC conditions for the separation of the pairs of the enatiomer-ic alcohols. Although Corey's (S)-proline-derived or stereo-chemically related oxazaborolidines in general delivered ^-configured allylic alcohols in reduction of enones,22 the R absolute configuration of chlorobenzylidenecyclopenta-nol 7a obtained from reduction with (S)-C5 was unambi-gously confirmed by X-ray crystallography (Figure 3). Additionally, this established also the configuration around the exocyclic C=C double bond as E. It should be made clear that stereochemical assignment for (R)-7a has not been previously made, although the absolute stereochemistry of related 2-benzylidenecyclopentanol obtained with Corey (S)-oxazaborolidine catalyst was determined to be R.23 Thus, formation of the alcohol (R)-7a from chloro-sub-stituted cyclopentanone 3a in the presence of oxazaboroli-dine catalyst (S)-C5 is also consistent with the sense of asymmetric induction predicted by the Corey mechanistic model.24 Consequently, we ascribed the R stereochemistry also to the methoxy-substituted alcohol 7b provided by oxazaborolidine catalyst (S)-C5, while for alcohols 7a,b arising from the borane reduction with catalyst (R)-C5 the S configuration was concluded. This was further supported by comparison of the sense of optical rotation and HPLC elution sequence of the enantiomeric forms of the alcohols 7a and 7b obtained with catalysts (S)-C5 and (R)-C5, respectively. Examination of the chromatogram (d) depicted in Figure 2 reveals, that for chloro-cyclopentanol 7a delivered with catalyst (S)-C5, the (+)-(R)-form of the enantiomers separated on chiral column is eluted second. Štefane et al.: Asymmetric Bio- and Chemoreduction ... 732 Acta Chim. Slov. 2017, 64, 727-736 Methoxy-cyclopentanol 7b obtained with catalyst (S)-C5 (Figure 2, chromatogram (f)) is also eluted second and returned a specific rotation of [a]^5 + 3.8, identical in sign to that of (R)-7a; consequently its configuration was proposed to be R. The opposite enantiomers of 7a and 7b, ob- Figure 3. X-ray crystal structure of (R)-7a; thermal ellipsoids are set at 40% probability. Table 2 Crystallographic data, structure refinement summary, selected bond lengths, bond angles, and torsion angles for compound (R)-7a. Property Compound (R)-7a Empirical formula C12H13Cl° Formula weight 208.67 Temperature 1 50(2) Crystal system orthorhombic Space group P2 2 2 111 a (A) 4.99305(10) b (A) 9.3428(2) c (A) 22.2446(4) a (°) 90.00 b (°) 90.00 g (°) 90.00 Volume (A3) 1037.69(4) Z 4 F (000) 440 Crystal size (mm3) 0.08 x 0.25 x 0.60 m (CuK\a) ( /mm) 0.079 Vref 6371 R 0.0327 wR2 0.0876 S 1.08 Bond length (A) C2(1) - Cl(1) 1.7440(16) C(8) - C(9) 1.336(2) O(1) - C(10) 1.4238(19) Bond angles (°) C(5) - C(7) - C(8) 125.0(1) C(8) - C(9) - C(11) 129.9(2) C(8) - C(9) - C(10) 122.4(1) C(9) - C(10) -O(1) 115.2(1) Torsion angles (°) C(5) - C(7) - C(8) - C(9) 10.0(1) C(7) - C(8) - C(9) - C(11) -4.5(1) tained with catalyst (R)-C5, both eluted on column first and the samples show levorotatory character. The dominant enantiomer of the indanol alcohol 8 obtained with (R)-C5, though in low excess, also eluted first on chiral column (Figure 2, chromatogram (g)), and the optical rotation of the sample was measured as [a]^5 - 13.4. On this basis it can be speculated that catalyst (R)-C5 preferentially delivers the (S)-indanol 8 in the reduction of indanone 6, while with catalyst (S)-C5 the (R)-alcohol 8 is obtained as the major enantiomer. Efficient asymmetric reduction of carbonyl compounds can also be achieved by means of bioreduction employing either isolated enzymes or whole cells system as mild and environmentally benign reduction systems. Fog-liato et al. used baker's yeast25 for the reduction of arylidene cyclopentanones and cyclohexanones reaching satisfactory enantioselectivity, while the secondary alcohols of excellent optical purity were obtained from Daucus carota26 root reduction of structurally different prochiral ketones (up to 100% ee). Similarly, an a,^-unsaturated ketone tra«s-4-phenylbut-3-en-2-one was regio- and stereoselec-tively reduced using carrot, celeriac, and beetroot enzyme systems to the corresponding (S)-allylic alcohol in ees 72-99%.27 In our case, the baker's yeast reduction of chlo-robenzylidenecyclopentanone 3a gave very low isolated yield (5%) and optical purity (ee = 9%) of the corresponding alcohol 7a even after incubating the reaction mixture at 38 oC for 10 days. On the contrary, the 24-hour-biore-duction with Daucus carota root (substrate/carrot, 1/134 (w/w)) delivered alcohol (S)-7a with >99% ee as determined in the crude product (Figure 2, chromatogram (h)), the amount of which was, however, very low after removal of the biomaterial (Scheme 2). Interestingly, asymmetric induction turned out to be time-dependent, namely ee value of (S)-7a reduced to 92% after incubating reaction mixture for four days at room temperature. It is noteworthy that isolation of the desired alcohol product from biore-duction is intrinsically messy, as the aqueous media contains the cellular mass, usual metabolites, nutrients, and the starting ketone. baker's yeast, D-glucose HsO H20, r. t., 24 h >99% ee Scheme 2. Bioreduction of benzylidenecyclopentanone 3a. 3. Experimental General. Toluene was dried with sodium and distilled. All other reagents and solvents were used as received from commercial suppliers. Melting points were deter- Štefane et al.: Asymmetric Bio- and Chemoreduction 733 Acta Chim. Slov. 2017, 64, 727-736 mined on a Kofler micro hot stage. The NMR spectra were recorded at 302 K either on a Bruker Avance DPX 300 or Avance III 500 MHz spectrometer operating at 300 MHz or 500 MHz and 75.5 MHz or 126 MHz for 'H and 13C nuclei. The 'H NMR spectra in CDCl3 are referenced with respect to TMS as the internal standard. The 13C NMR spectra are referenced against the central line of the solvent signal (CDCl3 triplet at 5 = 77.0 ppm). The coupling constants (J) are given in Hz. The multiplicities are indicated as follows: s (singlet), d (doublet), t (triplet), q (quartet), qn (quintet), m (multiplet) and br (broad). IR spectra were obtained on a Bruker FTIR Alpha Platinum ATR spectro-photometer. MS spectra were recorded with an Agilent 6224 Accurate Mass TOF LC/MS instrument. Elemental analyses (C, H, N) were performed with a Perkin-Elmer 2400 Series II CHNS/O Analyzer. TLC was carried out on Fluka silica gel TLC-cards. Column chromatography was performed on 230-400-mesh silica gel. Merck silica gel 60 PF254 containing gypsum was used to prepare chromato-tron plates. Radial chromatography was performed with Harrison Research, model 7924T chromatotron. HPLC analyses were performed with Agilent Technology 1260 Infinity HPLC instrument with UV detection. The known compounds were characterized by comparison of their physical or spectrosopic data with those in the literature. Synthesis of ketones 3a and 4a: Cyclopentanone (1) (5.0 g, 59.4 mmol) and p-chlorobenzaldehyde (2a) (4.22 g, 30.02 mmol) were added into a 0.2 M aqueous NaOH solution (210 mL), and stirred at room temperature for 72 h. The reaction was quenched with water (210 mL), and the reaction mixture was acidified with 3.6% aqueous HCl solution (60 mL) to pH ~ 4. The product was extracted with CH2Cl2 (3 x 150 mL), and combined organic layers were dried over anhydrous Na2SO4. The solvent was evaporated in vacuo, and the residue was purified by SiO2 column chromatography (petroleum ether : EtOAc = 20 : 1) to give 1.90 g (31%) of 3a and 4.00 g (40%) of 4a. 2-(4-Chlorobenzylidene)cyclopentanone (3a): mp 7476 oC (lit.28 77-79 °C). 2,5-Bis(4-chlorobenzylidene)cyclopentanone (4a): mp 225-227 oC (lit.29 224-226 °C). Synthesis of 2-(4-methoxybenzylidene)cyclopentanone (3b): Cyclopentanone (1) (1.0 g, 11.89 mmol) and p-me-thoxybenzaldehyde (2b) (896 mg, 6.58 mmol) were added into a 0.2 M aqueous NaOH solution (90 mL), and stirred at room temperature for 24 h, to which additional amount of cyclopentanone (298 mg, 3.54 mmol) was added, and stirred for further 12 h. The reaction was quenched with water (90 mL), and the reaction mixture was acidified with 3.6% aqueous HCl solution (20 mL) to pH ~ 4. The product was extracted with CH2Cl2 (3 x 100 mL), and the combined organic layers were dried over anhydrous Na2SO4. The solvent was evaporated in vacuo, and the residue was purified by SiO2 column chromatography (petroleum ether : EtOAc = 10 : 1) to give 843 mg (41%) of yellow crystalline product. Mp 65.4-66.3 °C (lit.28 68-69 °C). Synthesis of 2-(4-chlorobenzylidene)-2,3-dihydro-1H-inden-1-one (6): To a solution of 1-indanone (5) (4.0 g, 30.27 mmol) and p-chlorobenzaldehyde (2a) (5.32 g, 37.85 mmol) in MeOH (20 mL), a 0.2 M aqueous NaOH solution (250 mL) was added and the reaction mixture was stirred at room temperature for 48 h. The reaction was quenched with water (200 mL), and the reaction mixture was acidified with 3.6% aqueous HCl solution (60 mL) to pH ~ 1. The product was extracted with CH2Cl2 (3 x 150 mL) and combined organic layers were dried over anhydrous Na2SO4. The solvent was evaporated in vacuo and the residue was purified by SiO2 column chromatography (petroleum ether : EtOAc = 50 :1; 20 : 1; 10: 1; 5 : 1; 2 : 1) to give 732 mg (9%) of light yellow crystalline product. Mp 180.4-181.0 °C (lit.30 179 °C). Typical procedure for the synthesis of racemic alcohols - (±)-2-(4-chlorobenzylidene)cyclopentanol (±)-(7a): 2-(4-Chlorobenzylidene)cyclopentanone (3a) (400 mg, 1.94 mmol) and CeCl3 x 6H2O were dissolved in MeOH (20 mL), and stirred at room temperature for 30 min. Then solid NaBH4 (296 mg, 7.83 mmol) was added portion-wise. After 15 min additional amount of NaBH4 (140 mg, 3.70 mmol) was added, and stirred for further 1 h. The reaction was quenched with 1 M aqueous HCl solution (10 mL) and water (40 mL). The reaction mixture was stirred for 30 min and then extracted with EtOAc (3 x 50 mL). The combined organic layers were successively washed with 5% aqueous NaHCO3 solution (50 mL), water (30 mL), and brine (30 mL), and dried over anhydrous Na2SO4. The solvent was evaporated in a vacuo to give 360 mg (89%) of white crystalline product (±)-(7a), mp 76-77 °C. IR (ATR) v 3358, 2964, 1911, 1705, 1620, 1493, 1405, 1325, 1243, 1166, 1143, 1090, 1027, 1009, 980, 940, 887, 823, 730 cm-1. 1H NMR (500 MHz, CDCl3) 8 1.60 (br s, 1H, OH), 1.61-1.68 and 1.70-1.79 (2 x m, 2 x 1H, CH2), 1.93-2.02 (m, 2H, CH2), 2.51-2.59 and 2.65-2.74 (2 x m, 2 x 1H, CH2), 4.59 (m, 1H, OCH), 6.53 (br q, J 2.5 Hz, 1H, C=CH), 7.28 (AA'BB', J 8.8 Hz, 2H, Ar), 7.30 (AA'BB', J 8.8 Hz, 2H, Ar). 13C NMR (125 MHz, CDCl3) 8 22.3, 29.2, 34.7, 77.1, 122.4, 128.4, 129.5, 132.1, 136.2,148.3. ESI-HRMS (m/z): [M+H-H2O] + calcd for C12H12Cl, 191.0622; found, 191.0627. (±)-2-(4-Methoxybenzylidene)cyclopentanol (±)-(7b): Prepared by the above procedure from 3b (100 mg, 0.49 mmol). Yield 71%, mp 75.5-78.3 °C. IR (ATR) v 3252, 2998, 2955, 2931, 2833, 1604, 1510, 1463, 1420, 1294, 1242, 1178, 1112, 1034, 973, 938, 886, 831, 753 cm-1. 1H NMR (500 MHz, CDCl3) 8 1.50 (s, 1H, OH), 1.62-1.69 and 1.721.79 (2 x m, 2 x 1H, CH2), 1.89-2.01 (m, 2H, CH2), 2.502.59 and 2.67-2.75 (2 x m, 2 x 1H, CH2), 3.82 (s, 3H, Me), Štefane et al.: Asymmetric Bio- and Chemoreduction ... 734 Acta Chim. Slov. 2017, 64, 727-736 4.58 (m, 1H, OCH), 6.52 (br q, J 2.5 Hz, 1H, C=CH), 6.88 (AA'BB', J 8.8 Hz, 2H, Ar), 7.30 (AA'BB', J 8.8 Hz, 2H, Ar). 13C NMR (125 MHz, CDCl3) 5 22.7, 29.3, 34.9, 55.2, 77.5, 113.7, 123.2, 129.6, 130.5, 145.5, 158.2. ESI-HRMS (m/z): [M+H-H2O]+ calcd for C13H15O, 187.1117; found, 187.1114. Analytical data are in agreement with the literature data.17 (±)-2-(4-Chlorobenzylidene)-2,3-dihydro-1H-in-den-1-ol (±)-(8): Prepared by the above procedure from 6 (125 mg, 0.49 mmol). Yield 39%, mp 105.8-108.6 °C. IR (ATR) v 3319, 3069, 3025, 2887, 2321, 2155, 2107, 1904, 1692, 1677, 1608, 1586, 1490, 1461, 1405, 1354, 1312, 1296, 1255, 1212, 1186, 1176, 1133, 1092, 1009, 954, 895, 866, 844, 823, 806, 745, 732 cm-1. 1H NMR (500 MHz, CDCl3) 5 1.97 (br s, 1H, OH), 3.79-3.99 (m, 2H, CH2), 5.62 (br s, 1H, OCH), 6.86 (br q, J 2.5 Hz, 1H, C=CH), 7.27-7.32 (m, 3H, Ar), 7.34-7.37 (m, 4H, Ar), 7.51-7.55 (m, 1H, Ar). 13C NMR (125 MHz, CDCl3) 5 35.5, 78.3, 124.8, 124.9, 125.1, 127.3, 128.6, 128.8, 129.8, 132.7, 135.5, 140.7, 142.9, 145.5. ESI-HRMS (m/z): [M+H-H2O]+ calcd for C16H12Cl, 239.0622; found, 239.0612. Typical procedure for the asymmetric reduction with oxazaborolidines - synthesis of (R)-2-(4-chloroben-zylidene)cyclopentanol (R)-(7a). To the ice-cooled (0 °C) solution of (S)-C5 (0.1 mL, 0.1 mmol; 1 M in toluene) in dry toluene (1 mL), BH3:Me2S (470 ^L, 0.94 mmol; 2 M in toluene) was added dropwise, and the mixture was strirred for 10 min. A solution of ketone 3a (0.5 mmol) in dry toluene (1 mL) was slowly added to the previously prepared solution of reductant at 0 °C. After completion (jugded by TLC) of the reaction, the mixture was evaporated, and the residue purified by SiO2 radial chromatography (petroleum ether : EtOAc = 5 : 1) to give 80 mg (77%) of enantio-merically enriched product. 96% ee; t = 11.6 min (minor), 13.2 min (major), (chiracel OD-H chiral column, mobile phase: ¿-PrOH/hexane = 98/2, flow rate: 1.5 mL/min, wavelength: 240 nm), [a]D5 + 41.3 (1.13, CH2Cl2). (S)-2-(4-Chlorobenzylidene)cyclopentanol (S)-(7a): for the synthesis details see Table 1, entry 10. Yield 70%, 95% ee; tR = 11.2 min (major), 12.8 min (minor) (chiracel OD-H chiral column, mobile phase: ¿-PrOH/hexane = 98/2, flow rate: 1.5 mL/min, wavelength: 240 nm), [a]D5 -31.9 (1.17, CH2Cl2). (R)-2-(4-Methoxybenzylidene)cyclopentanol (R)-(7b): for the synthesis details see Table 1, entry 9. Yield 67%, 82% ee; tR = 16.1 min (minor), 18.4 min (major), (chiracel OD-H chiral column, mobile phase: ¿-PrOH/hexane = 98/2, flow rate: 1.5 mL/min, wavelength: 240 nm), [a]^ 3.8 (1.11, CH2Cl2). ]D5 ■ (S)-2-(4-Methoxybenzylidene)cyclopentanol (S)-(7b): for the synthesis details see Table 1, entry 11. Yield 59%, 90% ee; tR = 16.0 min (major), 18.7 min (minor) (chiralcel OD-H), ¿PrOH:heksan = 98:2, 1.5 ml/min, [a]D5 - 9.9 (1.08, CH2Cl2). 2-(4-Chlorobenzylidene)-2,3-dihydro-1H- inden-1-ol (8): for the synthesis details see Table 1, entry 13. Yield 21%, 24% ee; tR = 19.9 min (major), tR = 23.8 min (minor) (chiracel OD-H chiral column, mobile phase: ¿-PrOH/ hexane = 98/2, flow rate: 1.5 mL/min, wavelength: 240 nm), [a]D5 - 13.4 (0.96, CH2Cl2). 2-(4-Chlorobenzylidene)-2,3-dihydro-1H-inden-1-ol (6): for the synthesis details see Table 1, entry 14. Yield 20%, 33% ee; tR = 20.3 min (minor), tR = 24.1 min (major) (chiracel OD-H chiral column, mobile phase: ¿-PrOH/ hexane = 98/2, flow rate: 1.5 mL/min, wavelength: 240 nm). Hydrogenation of 3a with Noyori's catalyst C4: 2-(4- Chlorobenzylidene)cyclopentanone (3a) (103 mg, 0.498 mmol), catalyst C4 (6 mg, 4.9 ^mol) and isopropanol (2 mL) were added to hydrogenation vessel under nitrogen atmosphere. Then K2CO3 (10 mg, 0.072 mmol) was added, the autoclave was pressurized to 80 bars of H2, and the reaction mixture was stirred at room temperature. After 2 days additional amount of C4 (4.6 mg, 3.8 ^mol) was added (ketone 3a still present). Because the ketone 3a was still not consumed after 7 days, additional amount of K2CO3 (50 mg, 0.362 mmol) was added. After additional 5 days the reaction was still not complete, therefore K2CO3 (10 mg, 0.072 mmol), catalyst C4 (5 mg, 4.1 ^mol) and isopropanol (1 mL) were added, and hydrogenated for further 4 days. The solvent was evaporated and the residue was purified by SiO2 column chromatography (petroleum ether : EtOAc = 5 : 1) to afford 32 mg (31%) of the product 7a; ee = 12%. Reduction of 3a with baker's yeast: To a stirred solution of D-glucose (10.0 g, 55.5 mmol) and baker's yeast (56.0 g) in water (200 mL) at 38 °C, 2-(4-chlorobenzylidene)cyclo-pentanone (3a) (1.0 g, 4.84 mmol) dissolved in the minimum amount of EtOH (5 mL) was added; the reaction mixture was stirred for 10 days. Then EtOAc (100 mL) was added and the crude reaction mixture was filtered through a pad of Celite. The filtrate was extracted with EtOAc (3 x 100 mL), the organic phase was dried over anhydrous Na-2SO4, and the solvent was evaporated under reduced pressure. The residue was purified by SiO2 column chromatography (petroleum ether : EtOAc = 5 : 1) to afford 45 mg (5%) of the product 7a as a light yellow oil; ee = 9%. Reduction of 3a with Daucus carota root: An ethanolic (5 mL) solution of 2-(4-chlorobenzylidene)cyclopenta-none (3a) (100 mg, 0.484 mmol) was added to a suspension of freshly grated carrot root (13.4 g) in water (70 mL). The raction mixture was stirred at room temperature for Štefane et al.: Asymmetric Bio- and Chemoreduction 735 Acta Chim. Slov. 2017, 64, 727-736 24 h, then carrot root was filtered off and washed with water. Filtrate was extracted with EtOAc (3 x 50 mL). The organic phase was dried over anhydrous Na2SO4 and the solvent was evaporated under reduced pressure to give 10 mg (10%) of crude red oily product 7a; ee >99%. 4. Conclusion In summary, we synthesized enantiomerically enriched exocyclic allylic alcohols by asymmetric reduction of cyclic a-arylmethylene cyclic ketones. Highly enanti-oselective chemoreduction of 2-benzylidenecyclopenta-none derivatives was achieved by applying chiral oxaz-aborolidine-derived catalysts under mild reaction conditions. The sense of asymmetric induction was in accordance with Corey mechanistic model, thus (S)-catalyst delivered (_R)-alcohols, while (_R)-catalyst gave (S)-alcohol products with ee values of up to 96%. The indanone-de-rived chalcone was much less efficiently reduced regarding the chemical yield and optical purity (33% ee). Bioreduc-tion of 2-(4-chlorobenzylidene)cyclopentanone with baker's yeast gave very low ee of the corresponding allylic alcohol, while reduction with Daucus carota root turned out to be completely enantioselective. The synthesized allylic alcohols can serve as enantioenriched probes for the monitoring of oxidation-reduction processes catalyzed by AKR1C enzymes; these studies are currently under progress. 5. Acknowledgments We acknowledge with thanks the financial support from the Slovenian Research Agency through grant P1-0179. We thank EN-FIST Centre of Excellence, Trg Osvobodilne fronte 13, 1000 Ljubljana, Slovenia, for using SuperNova diffractometer and FTIR spectrophotometer. We also thank D. Potočnik for laboratory assistance. 6. References 1. H.-U. Blaser, C. Malan, B. Pugin, F. Spindler, H. Steiner, M. Studer, Adv. Synth. Catal. 2003, 345, 103-151. (a) T. M. Penning, Chem. Biol. Interact. 2015, 234, 236-246; (b) Y. Jin, T. M. Penning, Annu. Rev. Pharmacol. Toxicol. 2007, 47, 263-292; (c) U. Oppermann, Annu. Rev. Pharmacol. Toxicol. 2007, 47, 293-322. 3. (a) P. Brozic, S. Turk, T. L. Rizner, S. Gobec, Curr. Med. Chem. 2011, 18, 2554-2565; DOI:10.2174/092986711795933713 (b) T. L. Rizner, T. M. Penning, Steroids 2014, 79, 49-63. DOI:10.1016/j.steroids.2013.10.012 4. (a) T. M. Penning, M. E. Burczynski, J. M. Jez, C.-F. Hung, H.K. Lin, H. Ma, M. Moore, N. Palackal, K. Ratnam, Biochem. J. 2000, 351, 67-77; (b) S. Steckelbroeck, Y. Jin, S. Gopishet- ty, B. Oyesanmi, T. M. Penning, J. Biol. Chem. 2004, 279, 10784-10795; DOI:10.1074/jbc.M313308200 (c) O. A. Barski, S. M. Tipparaju, A. Bhatnagar,Drug.Metab.Rev., 2008, 40, 553-624. DOI:10.1080/03602530802431439 5. (a) T. M. Penning, J. E. Drury, Arch. Biochem. Biophys. 2007, 464, 241-250; DOI:10.1016/j.abb.2007.04.024 (b) T. M. Penning, M. C. Byrns, Ann. N. Y. Acad. Sci. 2009, 1155, 33-42; DOI:10.1111/j.1749-6632.2009.03700.x (c) B. E. Henderson, H. S. Feigelson, Carcinogenesis 2000, 21, 427-433; DOI:10.1093/carcin/21.3.427 (d) P. Brožič, S. Turk, T. L. Rižner, S. Gobec, Curr. Med. Chem. 2011, 18, 2554-2565. DOI:10.2174/092986711795933713 6. A. Krazeisen, R. Breitling, G. Möller, J. Adamski, Mol. Cell. Endocrinol. 2001, 171, 151-162. DOI: 10.1016/S0303-7207(00)00422-6 7. N. Usami, T. Yamamoto, S. Shintani, Y. Higaki, S. Ishikura, Y. Katagiri, A. Hara, Biol. Pharm. Bull. 2002, 25, 441-445. DOI: 10.1248/bpb.25.441 8. P. Brožič, B. Golob, N. Gomboc, T. L. Rižner, S. Gobec, Mol. Cell. Endocrinol. 2006, 248, 233-235. DOI: 10.1016/j.mce.2005.10.020 9. Y. Higaki, N. Usami, S. Shintani, S. Ishikura, O. El-Kabbani, A. Hara, Chem. Biol. Interact. 2003, 143, 503-513. DOI: 10.1016/S0009-2797(02)00206-5 10. K. Traven, M. Sinreih, J. Stojan, S. Seršen, J. Kljun, J. Bez-enšek, B. Stanovnik, I. Turel, T. L. Rižner, Chem. Biol. Interact. 2015, 234, 349-359. DOI:10.1016/j.cbi.2014.11.005 11. P. Brožič, S. Turk, A. O. Adeniji, J. Konc, D. Janežič, T. M. Penning, T. L. Rižner, S. Gobec, J. Med. Chem. 2012, 55, 74177424. DOI:10.1021/jm300841n 12. S. Gobec, P. Brožič, T. L. Rižner, Bioorg. Med. Chem. Lett. 2005, 15, 5170-5175. DOI:10.1016/j.bmcl.2005.08.063 13. B. Štefane, P. Brožič, M. Vehovc, T. L. Rižner, S. Gobec, Eur. J. Med. Chem. 2009, 44, 2563-2571. DOI:10.1016/j.ejmech.2009.01.028 14. N. Beranič, B. Štefane, B. Brus, S. Gobec, T. L. Rižner, Chem. Biol. Interact. 2013, 202, 204-209. DOI: 10.1016/j.cbi.2012.12.003 15. A. P. Phillips, J. Mentha, J. Am. Chem. Soc. 1956, 78, 140-145. DOI:10.1021/ja01582a040 16. (a) B. Štefane, F. Požgan, Top. Curr. Chem. 2016, 374, 1-67; DOI:10.1007/s41061-015-0002-2 (b) B. Štefane, F. Požgan, Cat. Rev. Sci. Eng. 2014, 56, 82-174; DOI: 10.1080/01614940.2013.869461 (c) F. Požgan, B. Štefane in: I. Karame (Ed.): Hydrogenation: Asymmetric hydrogenation and transfer hydrogenation of ketones, InTech, Rijeka, Croatia, 2012, pp. 31-68. 17. J.-B. Xie, J.-H. Xie, X.-Y. Liu, W.-L. Kong, S. Li, Q.-L. Zhou, J. Am. Chem. Soc. 2010, 132, 4538-4539. DOI: 10.1021/ja100652f 18. A. Hirao, S. Itsuno, N. Nakahama, N. Yamazaki, J. Chem. Soc., Chem. Commun 1981, 315-317. DOI:10.1039/c39810000315 19. E. J. Corey, R. K. Bakshi, S. Shibata, J. Am. Chem. Soc. 1987, 109, 5551-5553. DOI:10.1021/ja00252a056 20. E. J. Corey, C. J. Helal, Angew. Chem. Int. Ed. 1998, 37, Štefane et al.: Asymmetric Bio- and Chemoreduction ... 736 Acta Chim. Slov. 2017, 64, 727-736 1986-2012. D01:10.1002/(SICI)1521-3773(19980817)37:15 <1986::AID-ANIE1986>3.0.CO;2-Z 21. T. Yildiz, Tetrahedron: Asymmetry 2015, 26, 497-504. D0I:10.1016/j.tetasy.2015.03.008 22. (a) E. J. Corey, R. K. Bakshi, S. Shibata, C.-P. Chen, V K. Singh, J. Am. Chem Soc. 1987, 109, 7925-7926. DOI: 10.1021/ja00259a075 (b) E. J. Corey, A. V. Gavai, Tetrahedron Lett. 1988, 29, 3201-3204; D0I:10.1016/0040-4039(88)85121-9 (c) E. J. Corey, J. O. Link, Tetrahedron Lett. 1989, 30, 62756278; D0I:10.1016/S0040-4039(01)93871-7 ( d) E. J. Corey, R. K. Bakshi, Tetrahedron Lett. 1990, 31, 611-614; C. Y. Hong, N. Kado, L. E. Overman, J. Am. Chem. Soc. 1993, 115, 11028-11029. 23. A. F. Simpson, C. D. Bodkin, C. P. Butts, M. A. Armitage, T. Gallagher, J. Chem. Soc., Perkin Trans 1 2000, 3047-3054. DOI: 10.1039/b004540n 24. (a) D. A. Evans, Science 1988, 240, 420-426; D01:10.1126/science.3358127 (b) D. K. Jones, D. C. Liotta, J. Org. Chem. 1993, 58, 799-801; D0I:10.1021/jo00056a001 (c) L. P. Linney, C. R. Self, I. H. Williams, J. Chem. Soc., Chem. Commun. 1994, 1651-1652. D0I:10.1039/C39940001651 25. G. Fogliato, G. Fronza, C. Fuganti, S. Lanati, R. Rallo, R. Rig-oni, S. Servi, Tetrahedron 1995, 51, 10231-10240. D0I:10.1016/0040-4020(95)00578-V 26. J. S. Yadav, S. Nanda, P. T. Reddy, A. B. Rao, J. Org. Chem. 2002, 67, 3900-3903. D0I:10.1021/jo010399p 27. E. Majewska, M. Kozlowska, Tetrahedron Lett. 2013, 54, 6331-6332. D0I:10.1016/j.tetlet.2013.09.041 28. M Austin, O. J. Egan, R. Tully, A. C. Pratt, Org. Biomol. Chem. 2007, 5, 3778-3786. D0I:10.1039/b711620a 29. J.-X. Wang, L. Kang, Y. Hu, B. guo Wei, Synth. Commun. 2002, 32, 1691-1696. D0I:10.1081/SCC-120004261 30. N. P. Buu-Hoï, N. D. Xuong, J. Chem. Soc. 1952, 2225-2228. D0I:10.1039/JR9520002225 Povzetek Učinkovita asimetrična redukcija 2-benzilidenciklopentanonskih derivatov s kiralnimi oksazaborolidinskimi katalizatorji vodi selektivno do nastanka eksocikličnih alilnih alkoholov z enantiomernimi presežki do 96 %. Popolno enantiose-lektivnost lahko dosežemo z bioredukcijo 2-(4-klorobenziliden)ciklopentanona s korenjem, pri čemer nastane ustrezen alkohol z S konfiguracijo. Sintetizirane spojine lahko služijo kot enantiomerno obogateni standardi pri spremljanju encimsko kataliziranih Štefane et al.: Asymmetric Bio- and Chemoreduction DOI: 10.17344/acsi.2017.3566 Acta Chim. Slov. 2017, 64, 737-746 ¿^creative tS1 commons Scientific paper A 26-Membered Macrocycle Obtained by a Double Diels-Alder Cycloaddition Between Two 2H-Pyran-2-one Rings and Two 1,1'-(Hexane-1,6-diyl)bis (1H-pyrrole-2,5-dione)s Bor Lucijan Turek, Marijan Kočevar, Krištof Kranjc* and Franc Perdih* Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, SI-1000 Ljubljana, Slovenia * Corresponding author: E-mail: franc.perdih@fkkt.uni-lj.si; kristof.kranjc@fkkt.uni-lj.si Received: 19-05-2017 Dedicated to Professor Emeritus Miha Tišler, University of Ljubljana, on the occasion of his 90th birthday. Abstract With the application of a double dienophile 1,1'-(hexane-1,6-diyl)bis(1ff-pyrrole-2,5-dione) for a [4+2] cycloaddition with a substituted 2ff-pyran-2-one a novel 26-membered tetraaza heteromacrocyclic system 3 was prepared via a direct method under solvent-free conditions with microwave irradiation. The macrocycle prepared is composed of two units of the dienophile and two of the diene. The structure of the macrocycle was characterized on the basis of IR, 1H and 13C NMR and mass spectroscopy, as well as by the elemental analysis and melting point determination. With X-ray diffraction of a single crystal of the macrocycle we have determined that the two acetyl groups (attached to the bridging double bond of the bicyclo[2.2.2]octene fragments) are oriented towards each other (and also towards the inside of the cavity of the macrocycle), therefore, mostly filling it completely. Keywords: Macrocycles, 2H-Pyran-2-ones, [4+2] Cycloaddition, Crystal structure, Hydrogen bonds, Polymorphs 1. Introduction Macrocycles are privileged molecule structures that are of paramount importance in many areas of chemistry, including drug development,1 formation of coordination compounds and metal-organic frameworks.2 Generally they possess properties (structural, chemical, physical and biological) that set them apart from their linear or small-ring analogues, the reason being that they can often provide sufficient flexibility for interactions with other molecules (e.g. for binding to an enzyme's active site or for a coordination to a guest ion during phase catalysis) combined with the advantages brought by the fact that they often contain more than one binding motif. This means that all of the interactions between the host (or enzyme) and the macrocycle are taking place between two molecules only and consequently the enthropy of the interaction is not so unfavourable as would be in the case where more (smaller) ligands interact simultaneously with the host. Even though the synthesis of macrocycles has achieved some remarkable successes, there is still a lack of general approach towards them.3 There were many successful attempts towards the preparation of macrocycles, one of the most-often used being dilution techniques triggering the macrocyclization via lactonization, lactamiza-tion, metathesis reaction etc. (that were recently used for the first asymmetric total synthesis of aspergillide D4 or for the total synthesis of mandelalide A).5 Other options include the template-induced cyclization (around the host ion)6 and cyclization on a solid support (like Merri-field-based synthesis of cyclic peptides or such inspired by non-ribosomal peptide aldehydes).7 More contemporary approaches are based on multi multicomponent macrocy-clizations (MiBs)8 that include various bifunctional building blocks. However, neither of the above mentioned ap- Turek et al.: A 26-Membered Macrocycle Obtained by a Double 738 Acta Chim. Slov. 2017, 64, 737-746 proaches can be applied universally. So there is still place for new routes. Recently, a lot of effort was devoted to mul-ticomponent reactions that efficiently offer access to various macrocycles, including the possibility to incorporate points of diversity, which are, nevertheless, generally introduced before or after the key cyclization step.9 However, even this approach is generally applied just to obtain the requisite linear precursors that are latter assembled via a suitable ring-closing reaction into the final macrocyclic target.10-14 Of interest are also preparations of calix[4]arene systems linked with 1,2,4-triazole and 1,3,4-oxadiazole derivatives,15 as well as other tetraaza macrocycles applied as ligands in various coordination compounds.16 Herein we present another approach, where two double Diels-Alder cycloadditions between two molecules of the substituted 2H-pyran-2-ones (each acting as a "double" diene)17 and two molecules of the double dieno-phile provide a 26-membered tetraaza macrocyclic system. This strategy can be termed a multicomponent reaction (as four molecules react to form the macrocycle) with four individual [4+2] pericyclic reactions representing the crucial ring-closing steps. 2. Experimental 2. 1. Materials and Measurements Melting points were determined on a micro hot stage apparatus and are uncorrected. !H NMR spectra were recorded at 29 °C with a Bruker Avance III 500 spectrometer at 500 MHz using Me4Si as an internal standard. 13C NMR spectra were recorded at 29 °C with a Bruker Avance III 500 spectrometer at 125 MHz and were referenced against the central line of the solvent signal (CDCl3 triplet at 77.0 ppm or DMSO-d6 septet at 39.5 ppm). The coupling constants (J) are given in Hertz. IR spectra were obtained with a Bruker Alpha Platinum ATR FT-IR spectrometer on a solid support as microcrystalline powder. MS spectra were recorded with an Agilent 6624 Accurate Mass TOF LC/MS instrument (ESI ionization). Elemental analyses (C, H, N) were performed with a Perkin Elmer 2400 Series II CHNS/O Analyzer. TLC was carried out on Fluka silica-gel TLC-cards. The starting 2H-pyran-2-one 1 was prepared by the method devised by Kepe, Kocevar et al.18 as follows: from acetylacetone, N,N-dimethylformamide dimethyl acetal (DMFDMA) and hippuric acid by heating in acetic anhydride according to the published procedure 5-acetyl-3-ben-zoylamino-6-methyl-2H-pyran-2-one was obtained; followed by the removal of the benzoyl group (in concentrated H2SO4 upon heating) analogously as previously described19,20 and subsequent derivatization of the free 3-amino group with acetyl chloride the 2H-pyran-2-one 1 was obtained.21 Dienophile 2 was prepared by a modification of the procedures published by Cava et al.22 All other reagents and solvents were used as received from commercial suppliers. Microwave reactions were performed in air using a focused microwave unit (Discover by CEM Corporation, Matthews, NC, USA). The machine consists of a continuous, focused microwave power-delivery system with an operator-selectable power output ranging from 0 to 300 W. Reactions were conducted in darkness in glass vessels (capacity 10 mL) sealed with rubber septum. The pressure was controlled by a load cell connected to the vessel via the septum. The temperature of the reaction mixtures was monitored using a calibrated infrared temperature controller mounted below the reaction vessel and measuring the temperature of the outer surface of the reaction vessel. The mixtures were stirred with a Teflon-coated magnetic stirring bar in the vessel. Temperature, pressure, and power profiles were recorded using commercially available software provided by the manufacturer of the microwave unit. Synthesis of 1,1'-(Hexane-1,6-diyl)bis(1H-pyrrole-2,5-dione) (2)22 To a clear solution of maleic anhydride (2.03 g, 20 mmol) in diethyl ether (30 mL) a separately prepared mixture of hexane-1,6-diamine (2.07 g, 10 mmol) in diethyl ether (10 mL) is added dropwise at room temperature. The viscous suspension is further stirred at room temperature for 1 h and thereafter cooled on ice. Precipitated product is isolated by vacuum filtration and used in the next step without drying or additional purification. The entire obtained solid is slowly added to a mixture of sodium acetate (0.66 g, 8 mmol) and acetic anhydride (8 mL) in an Erlenmayer flask while vigorously stirring at room temperature. After the completion of the addition, the reaction mixture is heated on water bath (ap-prox. 100 °C) for 1 h, cooled to room temperature and poured onto ice-water mixture (30 g). The precipitated product is isolated by vacuum filtration, rinsed 3 times with distilled water and once with a few mL of petroleum ether yielding crude 2 (0.56 g, 20%) that is further crystallized from ethanol. M.p. 139-141 °C (EtOH), m.p. (lit.)23 139-141 °C (EtOH). IR (ATR) 3104, 3087, 2936, 2856, 1686, 1453, 1418, 1372, 1327, 1240, 1129 cm-1. 1H NMR (500 MHz, CDCl3): 5 1.29 (m, 4H, 2 x NCH2CH2CH2), 1.59 (m, 4H, 2 x NCH2CH2CH2), 3.51 (t, 4H, 2 x NCH2CH2CH2), 6.69 (s, 4H, 4 x CH). 13C NMR (125 MHz, CDCl3): 5 25.6, 27.8, 36.9, 124.4, 171.1. MS (ESI+) m/z 277 (MH+). HRMS (ESI+) calcd. for C14H17N2O4 (MH+): 277.1183. Found: 277.1181. Anal. calcd. for C14H16N2O4-0.1 H2O: C, 60.47; H, 5.87; N, 10.07. Found: C, 60.43; H, 5.89; N, 9.95. Synthesis of the Macrocycle 3 A 10 mL quartz microwave vessel is loaded with 2H-pyran-2-one 1 (105 mg, 0.5 mmol), dienophile 2 (152 mg, 0.55 mmol) and «-butanol (100 mg). A stirring bar is added and the vessel closed with the rubber septum. The reaction mixture is irradiated with microwaves (150 W) at Turek et al.: A 26-Membered Macrocycle Obtained by a Double ... Acta Chim. Slov. 2017, 64, 737-746 739 150 °C for 45 min. Thereafter, the reaction mixture is cooled to room temperature and diisopropyl ether is added (0.5 mL). The precipitated product is collected by vacuum filtration providing crude macrocycle 3 (150 mg, 34%) that is further crystallized from DMF. M.p. 255-257 °C (DMF). IR (ATR) 3368, 2940, 2860, 1766, 1698, 1548, 1437, 1399, 1367 cm-1. 1H NMR (500 MHz, DMSO-d6): 5 1.03 (m, 8H, 2 x NCH2CH2 CH2CH2CH2CH2N), 1.23 (m, 8H, 2 x NCH2CH2CH,, CH2CH2CH2N), 1.85 (s, 6H, 2 x Me), 1.95 (s, 6H, 2 x NHCOCH3), 2.02 (s, 6H, 2 x COMe), 3.00 (d, J = 7.5 Hz, 4H, 2 x 3a-H, 4a-H), 3.18 (m, 8H, 2 x NCH2CH2CH2CH2 CH2CH2N), 4.11 (d, J = 7.5 Hz, 4H, 2 x 7a-H, 8a-H), 6.82 (s, 2H, 2 x CH), 8.43 (s, 2H, 2 x NH). 13C NMR (125 MHz, DMSO-d6): S 18.2, 23.5, 25.8, 27.0, 27.4, 37.7, 41.2, 42.9, 48.8, 57.2, 138.2, 142.7, 170.3, 174.1, 175.3, 195.8. MS (ESI+) m/z 884 (MH+). HRMS (ESI+) calcd. for C^HNA, (MH+): 883.3872. Found: 883.3844. Anal. cal- 46 55 6 12 x ' cd. for C H NO,-0.8 H,O: C, 61.57; H, 6.24; N, 9.37. 46 54 6 12 2 Found: C, 61.57; H, 6.36; N, 9.27. 2. 2. Crystallography Single-crystal X-ray diffraction data were collected at room temperature on a Nonius Kappa CCD diffractom-eter using graphite monochromated Mo-Ka radiation (A = 0.71073 A). The data were processed using DENZO.24 Structures were solved by direct methods implemented in SIR9725 and refined by a full-matrix least-squares procedure based on F2 with SHELXL-2014.26 All non-hydrogen atoms were refined anisotropically. Hydrogen atoms were readily located in a difference Fourier maps and were subsequently treated as riding atoms in geometrically idealized positions, with C-H = 0.93 (aromatic), 0.98 (methine), 0.97 (methylene) or 0.96 A (CH3), N-H = 0.86 A and with Uiso(H) = kUeq(C or N), where k = 1.5 for methyl groups, which were permitted to rotate but not to tilt, and 1.2 for all other H atoms. To improve the refinement results, two reflections in the case of 2a, eleven reflections in the case of 2b and twenty eight reflections in the case of 3-2DMF with too high values of 5(F2)/e.s.d. and with Fo2 < Fc2 were deleted from the refinement. In 2b a proposed twin law has been applied according to Platon analysis and the R1 factor has improved from 8.13% to 7.75%, however, instead of estimated BASF 0.19 the refined BASF was found to be 0.00939. In the crystal structure of 3-2DMF a solvate DMF molecule is disorder over two positions with refined ratio 0.82:0.18 and ISOR instruction was used for the refinement of C25B atom in DMF. Crystallographic data are listed in Table 1. X-Ray powder diffraction data were collected at room temperature using a PANalytical X'Pert PRO MPD diffractometer with 9-29 reflection geometry, Table 1. Crystal data and refinement parameters for the compounds 2a, 2b and 3-2DMF. Compound 2a 2b 3-2DMF CCDC 1547701 1547702 1547703 Molecular formula C14H16N2O4 276.29 C14H16N2O4 276.29 C52H68N8O14 1029.14 Molecular weight Crystal system Triclinic Monoclinic Orthorhombic Space group P-1 P 21/a P c a n a (À) 4.5975(2) 8.4999(3) 10.4260(10) b (À) 5.5190(3) 6.6347(2) 17.5217(2) c (À) 14.1680(10) 12.6120(5) 27.8937(4) « (°) 93.956(3) 90 90 P (°) 97.222(4) 98.295(2) 90 y (°) 97.692(4) 90 90 V (À3) 352.08(4) 703.80(4) 5095.7(5) Z 1 2 4 Dcalc (g cm-3) 1.303 1.304 1.341 p (mm-1) 0. 097 0.097 0.098 F(000) 146 292 2192 Crystal dimensions (mm) 0.60 x 0.35 x 0.05 0.60 x 0.50 x 0.05 0.28 x 0.13 x 0.08 Reflections collected 2464 3070 11000 Data / restraints / parameters 1540 / 0/91 1566 / 0 / 93 5819 / 6 / 369 Rint 0.0227 0.0204 0.0320 R1, wR2 [I > 2a(I)]a 0.0444, 0.1231 0.0775, 0.2475 0.0486, 0.1157 R1, wR2 (all data)b 0.0580, 0.1343 0.0854, 0.2540 0.0855, 0.1335 Goodness of fit on F2, Sc 1.039 1.097 1.010 Extinction coefficient - 0.62(15) - Ap ,Ap . (e À-3) ' max ' min v ' 0.128, -0.150 0.226, -0.229 0.263, -0.306 0 R = X||FJ - |FJ|/X|FJ.b wR2 = (Z[w(Fo2 - Fc2)2]/X[w(Fo2)2]}1/2. c S = (Z[(Fo2 - Fc2)2]/(n/p)}1/2 where n is the number of reflections and p is the total number of parameters refined. Turek et al.: A 26-Membered Macrocycle Obtained by a Double 740 Acta Chim. Slov. 2017, 64, 737-746 primary side Johansson type monochromator and Cu-Ka1 radiation (A = 1.54059 â). 3. Results and Discussion 3. 1. Synthesis The strategy for the synthesis of the macro cycle 3 was based on our previous experiences with the [4+2] cycloadditions of variously substituted 2H-pyran-2-ones and appropriate dienophiles, including N-substituted maleim-ides.27-31 Namely, it was already observed that 2H-pyran-2-ones can act as "double" dienes, reacting in two consecutive Diels-Alder reactions with two distinctive molecules of the dienophiles, yielding bicyclo [2.2.2] octenes.30 The initial cycloaddition step leads to the formation of CO2-bridged oxabicyclo[2.2.2]octenes that in the next step eliminate a molecule of CO2 (via a retro-hetero-Diels-Al-der reaction) providing cyclohexadiene systems that act as new dienes for another molecule of dienophile finally providing the double cycloadducts. On the other hand, if the two molecules of the dienophile would be connected by a suitable tether, it would be possible to expect that the second cycloaddition step would take place intramolecularly. At least in theory, the smallest possible cyclic product would consist of just one bicyclo [2.2.2] octene fragment (formed out of one 2H-pyran-2-one ring) and one molecule of the double dienophile. Related examples were already described by the application of cycloocta-1,4-di-ene.32 Of course, it could be also possible that larger cycles would be obtained, for example such that contain two bi-cyclo[2.2.2]octene moieties and two molecules of the double dienophile. Here, we have focused our attention to a 3-acetyl-amino-6-methyl-2H-pyran-2-one (1) and 1,1'-(hexane- n-BuOH NHCOMe 150'Cst. -rViPi + f YTI+n conditions s a? K.J K J 0 0 ^ 10 11a 11Ö 11c Entry Catalyst Reaction time, rt Conversion (%)b 11a 11b 11c 1 3% I2/SFRCc 15 min 100 50 50 2 3% yMeOH 5 min 100 100 3 PMA/SFRCd 15 min 29e 7 7 4 PMA/MeOHf 5 min 100 8 8 84 5 3% HI (57%)« 15 min 100 50 50 6 3% HI (57%)/MeOHh 5 min 100 10 10 80 7 3% I2/3% H2O/MeOH' 5 min 100 100 8 3% y3% KI/SFRO 15 min 100 50 50 9 3% I2/3% KI/MeOHk 5 min 100 100 10 3% I2/3% Bu4NI/SFRC' 15 min 40m 2 2 11 3% 1/3% BuNI/MeOH" 24 5 min 95 100 759 a Reaction conditions: 10 (1 mmol, 198 mg) and various catalysts, rt. b Conversion and product distribution determined by 'H NMR spectroscopy. c Entries 1 and 2 from Table 6. d 10 (1 mmol, 198 mg) and phosphomolybdic acid hydrate (PMA, H3[P(Mo3O10)4] • xH2O), 60 mg. e 71% of unreacted 10 and 15% of a new, unidentified product presumably a ROR type dimer of 10. f 10 (1 mmol, 198 mg), methanol (3 mmol, 96 mg) and PMA (60 mg). g 10 (1 mmol, 198 mg) and HI (57 % aqueous solution, 0.03 mmol, 6.7 mg). h 10 (1 mmol, 198 mg), methanol (3 mmol, 96 mg) and HI (57% aqueous solution, 0.03 mmol, 6.7 mg). i 10 (1 mmol, 198 mg), H2O (0.03 mmol, 0.6 mg), methanol (3 mmol, 96 mg) and I2 (0.03 mmol, 7.6 mg). j KI (0.03 mmol, 5 mg) and I2 (0.03 mmol, 7.6 mg) were stirred for 20 minutes, 10 (1 mmol, 198 mg) was added. k KI (0.03 mmol, 5 mg), I2 (0.03 mmol, 7.6 mg) were stirred for 20 minutes, 10 (1 mmol, 198 mg) and methanol (3 mmol, 96 mg) were added. l I2 (0.03 mmol, 7.6 mg) and Bu4NI (0.03 mmol, 11.1 mg) were stirred for 20 minutes, 10 (1 mmol, 198 mg) was added. m 60% of unreacted 10 and 36% of a new product, presumably a ROR type dimer of 10. n I2 (0.03 mmol, 7.6 mg), Bu4NI (0.03 mmol, 11.1 mg) were stirred for 20 minutes, 10 (1 mmol, 198 mg) and methanol (3 mmol, 96 mg) were added. 10 in a mixture of 3% of I2, 3 % of water and 3 mmol of methanol furnished 11c as the only product (entry 7). The result indicates that iodine is relatively highly water tolerant76 and retains its catalytic activity in contrast to the numerous other Lewis acids. Iodine and KI were stirred together before 10 were added in order to establish the effect of iodine complexation. Disproportionation took place quantitatively, indicating at least two possible scenarios. Complexation of iodine and potassium iodide might be poor; on the other hand, triiodide could possibly catalyze the disproportionation (entry 8). Reaction of 10 with the system I2/KI/MeOH yielded the methoxy ether 11c as a (substitution) Scheme 1. A suggested role of iodine in transformation of alcohols under SFRC and HCRC. Jereb and Vražič: Iodine-catalyzed Transformation of Aryl-substituted 760 Acta Chim. Slov. 2017, 64, 747-762 sole product (entry 9). Additional complexation of I2 with Bu4NI almost completely suppressed disproportionation, suggesting that formation of triiodide was a key (entry 10). Reaction of 10 with I2/Bu4NI in the presence of methanol yielded the methoxy ether 11c only, while no disproportionation took place (entry 11). In reactions in entries 3 and 10 an unidentified product appeared, seemingly a di-meric ether of 10. There is often speculated, though not experimentally proven, that the in-situ formed HI is the actual catalyst in the iodine-catalyzed transformations.77 A potential formation of HI would probably result in a loss of the reaction selectivity (comparison of entries 2 and 6). The results indicate that iodine was the active catalyst, where complexation changed the reaction pathway considerably. Additionally, iodine was titrated with a standard solution of Na2S2O3 after the end of the disproportionation of 10. The entire amount of iodine was present at the end of the reaction. Similar observation was made in the case of dimerization of a secondary alcohol and substitution reaction with methanol, strongly indicating iodine as the active catalyst in these reactions. A tentative explanation of the reaction pathways is presented on Scheme 2. The driving force in all cases is presumably polarization of the reactants by iodine. We proposed such halogen bond78 activation in disproportionation of ethers under SFRC39, which is in agreement with recent computational37 and experimental studies.38 A simultaneous TS-1 or two separated activation processes TS-2, including carbenium ion TS-3 could be proposed as the key steps in the dimerization process. In the absence of a better nucleophile, the starting alcohol took over a role of an attacking nucleophile, affording the dimer 2. The dehydration process of the tertiary alcohols might be initiated by polarization of the starting alcohol as shown in TS-4. The substitution step is suggested as a concomitant activation TS-5 or a divided activation TS-6 or by carbenium ion TS-7. In the presence of added stronger nucleophile, substitution products 6 and 7 substantially prevailed over the dimerization products 2. ary alcohols. The difference in thermodynamic stability of the alkenes, derived from the tertiary and the secondary alcohols, is supposedly a driving force for the observed selectivity. Substitution was another process observed in the presence of the hydroxylic solvents; their acidity, nucleop-hilicity and hydrophobicity were important parameters for studying the reactivity of those alcohols. 4-Methoxyphe-nyl-substituted alcohols possessed higher reactivity than phenyl analogues; their pentafluorophenyl counterparts were unreactive under the studied conditions. The results indicated the electron-deficient intermediates to be likely involved in these processes, the geometries of the molecule and heteroatom share an important part in reactivity. 4-Methoxybenzyl alcohol yielded its dimeric ether and bis(4-methoxyphenyl)methane, a product derived via the I2-catalyzed ipso-substitution. 4-Methoxybenzyl alcohol exhibited higher reactivity in TFE and HFIP than in EtOH and in i-PrOH under HCRC, thus indicating stronger stabilization of the reaction intermediates in the fluorinated alcohols. A tertiary benzylic alcohol 1n was demonstrated to possess a special reactivity. It appears that upon its dehydration all three possible alkenes were obtained. The thermodynamically less stable alkene unexpectedly reacted with the initially formed intermediate, furnishing two dimeric alkenes. It is worth mentioning that certain processes take place under SFRC and HCRC, but not under the classical diluted conditions. This is an indication that reacting species have to be in close vicinity. Iodine catalyzed the disproportionation of 9H-xanthene-9-ol 10 under SFRC and HCRC, and in contrast, the substitution took place in the presence of MeOH. Iodine is a convenient catalyst for transetherification under mild conditions, it has a potential for interconversion of ether to ester. The Hammett correlation analysis of the I2-catalyzed dimerization of substituted 1-phenylethanols under SFRC (T = 55 °C) furnished straight-line p+ = -2.8 (r2 = 0.98). This fact strongly suggests the involvement of the electron-deficient intermediates with a certain degree of the developed charge in the transition state. 4. Conclusions To summarize, we have studied iodine-catalyzed transformations of aryl-substituted alcohols under SFRC and under HCRC, the concentration was proved to have an exceptional impact on the transformation. Achieving a pasty aggregate state of solid substrates in the presence of I2 was of vital importance for the reaction progress. Primary and secondary alcohols underwent two main transformations, depending on the reaction conditions. Dimerization took place in absence of the good nucleophiles under SFRC and HCRC, while substitution prevailed in presence of the good nucleophiles. The tertiary alcohols exhibited a strong tendency of dehydration into alkenes, which is in sharp contrast with the reactivity of primary and second- 5. Acknowledgement We thank Prof. Marko Zupan for his initiative part of the research. We are grateful the Slovenian Research Agency for the financial support (P1-0134 and P1-0230), to Dr. D. Žigon at the Mass Spectroscopy Centre at the 'Jožef Stefan' Institute in Ljubljana, and to Mrs. T. Stipanovič and Prof. J. Svete for elemental combustion analysis. 6. References 1. J. Clark, D. Macquarrie, Eds. Handbook of Green Chemistry & Technology, Blackwell: Oxford, U. K. 2002. DOI: 10.1002/9780470988305 Jereb and Vražič: Iodine-catalyzed Transformation of Aryl-substituted Acta Chim. Slov. 2017, 64, 747-762 761 2. M. Lancaster, Green Chemistry: An Introductory Text; Royal Society of Chemistry, Cambridge, U. K., 2002. 3. P. Tundo, V. Esposito, V., (Eds.): Green Chemical Reactions; Springer, Dordrecht, Netherlands, 2008. 4. K. Tanaka, (Ed.): Solvent-Free Organic Synthesis; Wiley-VCH: Weinheim, Germany, 2009. 5. P. J. Walsh, H. Li, C. A. de Parrodi, Chem. Rev. 2007, 107, 2503-2545. DOI:10.1021/cr0509556 6. M. B. Gawande, V. D. B. Bonifácio, R. Luque, P. S. Branco, R. S. Varma, ChemSusChem 2014, 7, 24-44. DOI:10.1002/cssc.201300485 7. M. A. P. Martins, C. P. Frizzo, D. N. Moreira, L. Buriol, P. Machado, Chem. Rev. 2009, 109, 4140-4182. DOI:10.1021/cr9001098 8. S. Nakamatsu, S. Toyota, W. Jones, F. Toda, Chem. Commun. 2005, 3808-3810. DOI:10.1039/b503922c 9. A. Orita, G. Uehara, K. Miwa, J. Otera, Chem. Commun. 2006, 4729-4731. DOI:10.1039/b609567d 10. M. Feller, A. Karton, G. Leitus, J. M. L. Martin, D. Milstein, J. Am. Chem. Soc. 2006, 128, 12400-12401. DOI:10.1021/ja0641352 11. Z. Tang, Z.-H. Yang, L.-F. Cun, L.-Z. Gong, A.-Q. Mi, Y.-Z. Jiang, Org. Lett. 2004, 6, 2285-2287; DOI:10.1021/ol049141m 12. I. Pravst, M. Zupan, S. Stavber, Tetrahedron Lett. 2006, 47, 4707-4710. DOI:10.1016/j.tetlet.2006.04.119 13. G. Angelici, R. J. Correa, S. J. Garden, C. Tomasini, Tetrahedron Lett. 2009, 50, 814-817. DOI:10.1016/j.tetlet.2008.12.007 14. Y. Hayashi, T. Sumiya, J. Takahashi, H. Gotoh, T. Urushima, M. Shoji, Angew. Chem. Int. Ed. 2006, 45, 958-961. DOI: 10.1002/anie.200502488 15. P. Dziedzic, W. Zou, J. Háfren, A. Córdova, Org. Biomol. Chem. 2006, 4, 38-40. DOI:10.1039/B515880J 16. S. Minegishi, S. Kobayashi, H. Mayr, J. Am. Chem. Soc. 2004, 126, 5174-5181. DOI:10.1021/ja031828z 17. T. Bug, H. Mayr, J. Am. Chem. Soc. 2003, 125, 12980-12986. DOI:10.1021/ja036838e 18. T. B. Phan, M. Breugst, H. Mayr, Angew. Chem. Int. Ed. 2006, 45, 3869-3874. DOI:10.1002/anie.200600542 19. H. Mayr, J. Ammer, M. Baidya, B. Maji, T. A. Nigst, A. R. Ofi-al, T. Singer, J. Am. Chem. Soc. 2015, 137, 2580-2599. DOI:10.1021/ja511639b 20. T. L. Amyes, I. W. Stevens, J. P. Richard, J. Org. Chem. 1993, 58, 6057-6066. DOI:10.1021/jo00074a036 21. H. F. Schaller, H. Mayr, Angew. Chem. Int. Ed. 2008, 47, 39583961. DOI:10.1002/anie.200800354 22. R. Breslow, Acc. Chem. Res. 2004, 37, 471-478. DOI:10.1021/ar040001m 23. R. Breslow, Acc. Chem. Res. 1991, 24, 159-164. DOI: 10.1021/ar00006a001 24. H. Y. Bae, C. E. Song, ACS Catal. 2015, 5, 3613-3619. DOI:10.1021/acscatal.5b00685 25. R. N. Butler, A. G. Coyne, W. J. Cunningham, E. M. Moloney, J. Org. Chem. 2013, 78, 3276-3291. DOI:10.1021/jo400055g 26. F. C. Küpper, M. C. Feiters, B. Olofsson, T. Kaiho, S. Yanagi-da, M. B. Zimmermann, L. J. Carpenter, G. W. Luther III, Z. Lu, M. Jonsson, L. Kloo, L. Angew. Chem. Int. Ed. 2011, 50, 11598-11620. 27. Z. Jinjin, G. Wenchao, C. Honghong, L. Xing, L. Qiang, W. Wenlong, Chin. J. Org. Chem. 2014, 34, 1941-1957. DOI:10.6023/cjoc201405003 28. M. Jereb, D. Vražič, M. Zupan, Tetrahedron 2011, 67, 13551387. DOI:10.1016/j.tet.2010.11.086 29. Y.-M. Ren, C. Cai, R.-C. Yang, RSC Adv. 2013, 3, 7182-7204. 30. H. Hibbert, J. Am. Chem. Soc. 1915, 37, 1748-1763. DOI:10.1021/ja02172a015 31. G. Stavber, M. Zupan, S. Stavber, Tetrahedron Lett. 2006, 47, 8463-8466. DOI:10.1016/j.tetlet.2006.09.154 32. M. Jereb, D. Vražič, M. Zupan, Tetrahedron Lett. 2009, 50, 2347-2352. DOI:10.1016/j.tetlet.2009.02.224 33. M. Jereb, D. Vražič, M. Zupan, Acta Chim. Slov. 2009, 56, 652-658. 34. Y. Kasashima, A. Uzawa, T. Nishida, T. Mino, M. Sakamoto, T. Fujita, J. Oleo Sci. 2009, 58, 421-427. DOI:10.5650/jos.58.421 35. Y. Ide, Y. Hori, S. Kobayashi, M. D. Hossain, T. Kitamura, Synthesis 2010, 3083-3086. 36. W. Rao, A. H. L. Tay, P. J. Goh, J. M. L. Choy, J. K. Ke, P. W. H. Chan, Tetrahedron Lett. 2008, 49, 122-126. DOI: 10.1016/j.tetlet.2007.11.005 37. M. Breugst, E. Detmar, D. von der Heiden, ACS Catal. 2016, 6, 3203-3212. DOI:10.1021/acscatal.6b00447 38. D. von der Heiden, S. Bozkus, M. Klussmann, M. Breugst, J. Org. Chem. 2017, 82, 4037-4043. DOI: 10.1021/acs.joc.7b00445 39. M. Jereb, D. Vražič, Org. Biomol. Chem. 2013, 11, 1978-1999. DOI: 10.1039/c3ob27267b 40. Vogels Textbook of Practical Organic Chemistry, Longman Group Limited: London, U. K., 1959, pp 252. 41. Y. Suh, J.-s. Lee, S. H. Kim, R. D. Rieke, J. Organomet. Chem. 2003, 684, 20-36. DOI:10.1016/S0022-328X(03)00500-X 42. E. D. Bergmann, A. M. Meyer, J. Org. Chem. 1965, 30, 28402841. DOI:10.1021/jo01019a510 43. G. V. M. Sharma, K. L. Reddy, P. S. Lakshmi, R. Ravi, A. C. Kunwar, J. Org. Chem. 2006, 71, 3967-3969. DOI: 10.1021/jo052418r 44. V. Dimitrov, S. Stanchev, B. Milenkov, T. Nikiforov, P. Demi-rev, Synthesis 1991, 228-232. 45. R. Stoermer, O. Kippe, Chem. Ber. 1903, 36, 3992-4013. DOI: 10.1002/cber.19030360459 46. C. F. Koelsch, J. Am. Chem. Soc. 1932, 54, 2487-2493. DOI:10.1021/ja01345a046 47. I. Ho, J. G. Smith, Tetrahedron 1970, 26, 4277-4286. DOI:10.1016/S0040-4020(01)93071-6 48. D. J. Cram, D. R. Wilson, J. Am. Chem. Soc. 1963, 85, 12491257. DOI:10.1021/ja00892a009 49. Y. Sprinzak, J. Am. Chem. Soc. 1958, 80, 5449-5455. DOI:10.1021/ja01553a033 50. W. Ried, J. Schönherr, Chem. Ber. 1960, 93, 1870-1877. DOI: 10.1002/cber.19600930826 Jereb and Vražič: Iodine-catalyzed Transformation of Aryl-substituted 762 Acta Chim. Slov. 2017, 64, 747-762 51. I. T. Badejo, R. Karaman, J. L. Fry, J. Org. Chem. 1989, 54, 4591-4596. DOI:10.1021/jo00280a026 52. G.-Q. Li, Z.-Y. Yan, Y.-N. Niu, L. Y. Wu, H.-L. Wei, Y.-M. Liang, Tetrahedron: Asymmetry 2008, 19, 816-821. DOI:10.1016/j.tetasy.2008.03.012 53. J. M. Khurana, A. Sehgal, A. Gogia, A. Manian, G. C. Maikap, J. Chem. Soc. Perkin Trans 1, 1996, 2213-2215. DOI:10.1039/P19960002213 54. A. F. Holleman, Org. Synth., Coll. Vol. 1, 1941, 554-555. 55. M. Zupan, P. Skulj, S. Stavber, Arkivoc, 2001, 2, 108-118. 56. M. Zupan, P. Skulj, S. Stavber, Tetrahedron 2001, 57, 1002710031. DOI:10.1016/S0040-4020(01)01031-6 57. S. J. Cristol, J. R. Douglass, J. S. Meek, J. Am. Chem. Soc. 1951, 73, 816-818. DOI:10.1021/ja01146a094 58. G. Stavber, M. Zupan, M. Jereb, S. Stavber, Org. Lett. 2004, 6, 4973-4976. DOI:10.1021/ol047867c 59. R. E. Pearson, J. C. Martin, J. Am. Chem. Soc. 1963, 85, 31423146. DOI:10.1021/ja00903a021 60. C. K. Ingold, F. R. Shaw, J. Chem. Soc. 1927, 2918-2926. DOI: 10.1039/JR9270002918 61. X. Zhang, W. Rao, P. W. H. Chan, Synlett 2008, 2204-2208. 62. J. S. Yadav, B. V. S. Reddy, N. Thrimurtulu, N. M. Reddy, A. R. Prasad, Tetrahedron Lett. 2008, 49, 2031-2033. DOI:10.1016/j.tetlet.2008.01.017 63. S. Ward, T. Messier, M. Lukeman, Can. J. Chem. 2010, 88, 493-499. DOI:10.1139/V10-032 64. X. Creary, A. Wolf, J. Phys. Org. Chem. 2000, 13, 337-343. DOI:10.1002/1099-1395(200006)13:6<337::AID-P0C249 >3.0.C0;2-T 65. S. M. Bonesi, M. Fagnoni, Chem. Eur. J. 2010, 16, 1357213589. DOI:10.1002/chem.201001478 66. G. G. Gurzadyan, S. Steenken, Chem. Eur. J. 2001, 7, 18081815. DOI:10.1002/1521-3765(20010417)7:8<1808::AID-CHEM18080>3.0.CO;2-8 67. A. Berkessel, M. R. M. Andreae, H. Schmickler. J. Lex, Angew. Chem. Int. Ed. 2002, 41, 4481-4484. DOI:10.1002/1521-3773(20021202)41:23<4481::AID-ANIE 4481>3.0.C0;2-7 68. J. L. Wahlstrom, R. C. Ronald, J. Org. Chem. 1998, 63, 60216022. DOI:10.1021/jo972222u 69. S. Das, R. Borah, R. R. Devi, A. J. Thakur, Synlett, 2008, 27412762. 70. Hammett, L. P. J. Am. Chem. Soc. 1938, 59, 96-103. DOI:10.1021/ja01280a022 71. Hammett, L. P. Chem. Rev. 1935, 17, 125-136. DOI:10.1021/cr60056a010 72. M. Jereb, M. Zupan, S. Stavber, Helv. Chim. Acta, 2009, 92, 555-566. DOI:10.1002/hlca.200800308 73. K. Zmitek, M. Zupan, S. Stavber, J. Iskra, J. Org. Chem. 2007, 72, 6534-6540. DOI:10.1021/jo0708745 74. F. Goronwy Kny-Jones, A. Miles Ward, J. Chem. Soc. 1930, 535-542. DOI:10.1039/JR9300000535 75. C. Waterlot, D. Couturier, M. De Backer, B. Rigo, B. Can. J. Chem. 2000, 78, 1242-1246. DOI:10.1139/v00-082 76. K. Ramalinga, P. Vijayalakshmi, T. N. B. Kaimal, Tetrahedron Lett. 2002, 43, 879-882. DOI:10.1016/S0040-4039(01)02235-3 77. K. M. Kim, D. J. Jeon, E. K. Ryu, Synthesis 1998, 835-836. 78. G. Cavallo, P. Metrangolo, R. Milani, T. Pilati, A. Priimagi, G. Resnati, G. Terraneo, Chem. Rev. 2016, 116, 2478-2601. DOI: 10.1021/acs.chemrev.5b00484 Povzetek Preučevali smo z jodom katalizirane pretvorbe alkoholov pri reakcijskih pogojih brez topil (RPBT) in pri visoko koncentriranih pogojih (VKP) v prisotnosti različnih topil, da bi dobili vpogled v obnašanje reakcijskih intermediatov pri takšnih pogojih. Ugotovili smo, da z benzilnimi alkoholi potečejo trije različni tipi pretvorb: dimerizacija, dehidratacija in substitucija. Dimerizacija in substitucija pretežno potečeta v primeru primarnih in sekundarnih alkoholov, medtem, ko dehidratacija prevladuje v primeru terciarnih alkoholov. Relativna reaktivnost substituiranih 1-feniletanolov pri z jodom katalizirani dimerizaciji pri RPBT daje dobro Hammettovo korelacijo p+ = -2.8 (r2 = 0.98), ki kaže na prisotnost elektronsko siromašnih intermediatov z določeno stopnjo razvitega naboja v prehodnem stanju. Jereb and Vražič: Iodine-catalyzed Transformation of Aryl-substituted DOI: 10.17344/acsi.2017.3844 Acta Chim. Slov. 2017, 64, 763-770 ¿^creative tS1 commons Scientific paper Synthesis and X-ray Structural Analysis of the Ruthenium(III) Complex Na[trans-RuCl4(DMSO) (PyrDiaz)], the Diazene Derivative of Antitumor NAMI-Pyr Jure Vajs,1 Andrej Pevec,1 Martin Gazvoda,1 Damijana Urankar,1 Evgeny Goreshnik,2 Slovenko Polanc1 and Janez Košmrlj1* 1 Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, SI-1000 Ljubljana, Slovenia 2 Department of Inorganic Chemistry and Technology, Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia * Corresponding author: E-mail: janez.kosmrlj@fkkt.uni-lj.si Received: 09-17-2017 Dedicated to Professor Emeritus Miha Tišler, University of Ljubljana, on the occasion of his 90th birthday. Abstract Novel tetrachloridoruthenium(III) complex Na[trans-RuCl4(DMSO)(PyrDiaz)] (3) with pyridine-tethered diazenedi-carboxamide PyrDiaz ligand (PyrDiaz = N1-(4-isopropylphenyl)-N2-(pyridin-2-ylmethyl)diazene-l,2-dicarboxamide) was synthesized by direct coupling of PyrDiaz with sodium trans-bis(dimethyl sulfoxide)tetrachloridoruthenate(III) (Na-[trans-Ru(DMSO)2Cl4]). Compound 3 is the analogue of the antimetastatic Ru(III) complex NAMI-A and NAMI-Pyr. Single crystal X-ray diffraction analysis revealed that the compound 3 is a polymeric complex with the ruthenium and sodium centres. Keywords: Diazene, pyridine, ruthenium(III), sodium, polymeric complex 1. Introduction Based on the WHO estimates, cancer is one of the leading causes of mortality worldwide accounting for 8.8 million deaths in 2015.1 In addition to surgical removal of tumours and radiation therapy, chemotherapy is one of the most commonly applied treatments of cancer. Burden associated with chemotherapy, however, is intrinsic and acquired resistance,2 and severe side-effects that are responsible for considerable morbidity, greatly reducing the effectiveness of the therapy. This, and the striking estimate that the global incidence of cancer continues to increase, urges for new strategies and chemical entities to be de-vised.3 Commenced with a group of platinum chemothera-peutic agents that has been in clinical use for half of a century,4-6 other precious-metal-based alternatives lately enticed an army of chemists worldwide.7 As a result, some ruthenium-based compounds of unique properties, sur- passing cisplatin in activity, particularly on resistant tumours, and with reduced host toxicity at active doses have been discovered.8,9 In the treatment of a complex disease like cancer it is unlikely for a single drug to be effective. To increase the chemotherapeutic success, a combination of two or more active agents having separate targets is commonly administered at the therapy.10 A promising alternative to these so-called cocktails are hybrid molecules that are composed of two or more covalently bound drugs; the compounds that can possess combined pharmacological properties of the individual drugs10,11 yet superior synergistic effects.12,13 Over the past years, we have investigated redox-ac-tive14 diazenecarboxamides as potential anti-cancer agents.15 The results of the in vitro experiments suggested that they likely target tumour cell redox mechanism by oxidation of glutathione into glutathione disulphide.16 A synergistic effect was noted by treating some tumour cell lines with the combination of cisplatin and selected diazenecar- Vajs et al.: Synthesis and X-ray Structural Analysis 764 Acta Chim. Slov. 2017, 64, 763-770 boxamides,16e prompting us to consider novel diazenecar-boxamide-platinum conjugates (Figure 1).6,17 This has led to complex A, possessing higher cytotoxicity against T24 bladder carcinoma cells as compared to the parent platinum precursor ([PtCl(DMSO)(en)]Cl; en = ethylenedi-amine) and organic ligand, for example.18 Redox-active diazenecarboxamides were also combined with organo-metallic [Ru(II)-Arene] to generate complexes with interesting coordination modes and chemical reactivity (Figure 2).19,20 In the context of our endeavour in the field of potential anti-cancer agents, complex B was identified as highly cytotoxic against tumour cell lines with IC50 values in the low micro-molar range.19,21 The activity of B was cell-type specific and comparable in both cancer cell lines and their drug-resistant subline. A tenfold increase in the sensitivity of tumour cervical carcinoma cell lines (HeLa) with depleted intracellular glutathione level in comparison to the untreated HeLa suggested glutathione as the molecular target of B.19 Encouraged by these results and inspired by rutheni-um(III) complexes NAMI-A (imidazolium trans-[tetra-chlorido(dimethyl sulfoxide)imidazole ruthenium(III)]),22 KP1019 (indazolium trans-[tetrachloridobis(1H-inda-zole)ruthenium(III)]) and its sodium salt KP133923 (Fig- H2 fY -K M r"\ ^ Pt W s A-n n H H>Cr y\ o +2 O r-O CI o n-n. Hj ,PI Íb Figure 1. Selected diazenecarboxamide-platinum conjugates. Figure 2. Selected organometallic [Arene-Run-Diazenecarboxamide] compounds. NAMI-Pyr I AziRu Figure 3. Selected Ru(III) complexes having anti-cancer activity. ure 3), we were prompted to examine the coordination ability of diazenedicarboxamide N!-(4-isopropylphenyl)-N2- (pyridin- 2-ylmethyl) diazene-1,2- dicarb oxamide ( 1 ) (Scheme 1). Compound 1 has been previously screened for anti-cancer activity.24 2. Experimental Starting materials and solvents for the synthesis of the examined compounds were used as obtained, and without further purification, from Aldrich, Fluka and Alfa Aesar. IR spectrum was obtained with a Bruker ALPHA Platinum ATR spectrometer on a solid sample support (ATR). NMR spectra were recorded in D2O with a Bruker Avance III 500 MHz instrument operating at 500 MHz, at 296 K, and referenced to the peak of HOD (5 = 4.63 ppm). An Agilent 6224 time-of-flight (TOF) mass spectrometer equipped with a double orthogonal electrospray source at atmospheric pressure ionization (ESI) coupled to an Agilent 1260 HLPC was used for recording HRMS spectra. Vajs et al.: Synthesis and X-ray Structural Analysis ... 765 Acta Chim. Slov. 2017, 64, 763-770 Mobile phase composed of two solvents: A was 0.1% formic acid in Milli-Q water, and B was 0.1% formic acid in acetonitrile mixed in the ratio of 1:1. Compound was prepared by dissolving the sample in acetonitrile and injected (0.1 ^L) into the LC-MS. Flow rate was 0.4 mL/min. Fragmentor voltage was 150 V. Capillary voltage 4000 V. Mass range 100-1100. Elemental analysis (C, H, N) was performed with Perkin Elmer 2400 Series II CHNS/O Analyser. Melting points were determined on the microscope hot stage. 2. 1. The Synthesis of Compound 3 N1-(4-Isopropylphenyl)-N2-(pyridin-2-ylmethyl)di-azene-1,2-dicarboxamide24 (1, 163 mg, 0.5 mmol) was added to a solution of sodium trans-bis(dimethyl sulfox-ide)tetrachloridoruthenate(III) (Na[trans-Ru(DMSO-S)2 Cl4])25 (2, 106 mg, 0.25 mmol) in acetone (10 mL) and stirred at room temperature for 2 h. The solvent was removed under reduced pressure. Dry residue was re-suspended in dichloromethane and filtered off to remove un-reacted ligand 1. The precipitate was dried in air to give compound 3 (165 mg, 99%). Crystal suitable for X-ray structure determination was found in the crude product. Purple solid; mp >250 °C dec.; IR v 3245, 3035, 2959, 1736, 1718, 1603, 1534, 1482, 1459, 1438 cm-1; HRMS (ESI-) m/z for C19H25Cl4N5O3RuS- [M - Na]-: calcd 646.9462, found 646.9467, m/z for C17H18Cl3N5O2Ru-[M-Na-DMSO -HCl]-: calcd 532.9560, found 532.9554; Anal. calcd for C19H25Cl4N5O3NaRuS ■ 0.1 H2O: C, 34.00; H, 3.78; N, 10.43; found: C, 34.48; H, 3.96; N, 10.60. 2. 2. X-ray Structure Determination Crystal data and refinement parameters of 3 ([NaRu-Cl41(DMSO)]n) are listed in Table 1. Single-crystal data were collected at 150 K on a Gemini A diffractometer equipped with an Atlas CCD detector, using graphite monochromated CuKa radiation (À = 1.54184 Â). The data were treated using the CrysAlisPro software suite program package.26 Analytical absorption correction was applied. Structure of compound 3 was solved using direct methods with SHELXS-9727 and refined using the least-squares method on F2 with SHELXL-201428 and using the graphical interface of OLEX2.29 Figures were prepared using Diamond software.30 CCDC 1569486 contains the supplementary crystallographic data for 3. These data can be obtained free of charge from The Cambridge Crystallo-graphic Data Center via www.ccdc.cam.ac.uk/data_re-quest/cif. 3. Results and Discussion The pyridine-tethered diazenedicarboxamide, N1-(4-isopropylphenyl)-N2-(pyridin-2-ylmethyl)diazene-1,2-dicarboxamide (1), was prepared in a three step reaction sequence starting from the commercial reactants as previously described by us (Scheme 1).24 Thus, the addition of carbazate to 4-isopropylphenyl isocyanate gave ethyl 2- ( (4-isopropylphenyl) carb amoyl) hydrazine-1 - carb ox-ylate, which was oxidized with N-bromosuccinimide (NBS) into ethyl 2-((4-isopropylphenyl)carbamoyl)dia-zene-1-carboxylate. Subsequent nucleophilic displacement with 3-picolylamine afforded the target compound 1. Sodium trans--bis(dimethyl sulfoxide)tetrachlori-doruthenate(III) (Na-[trans-Ru(DMSO-S)2ClJ, 2) was selected as the ruthenium(III) precursor. This compound was prepared by the literature procedure starting from the commercial hydrated RuCl3 via hydrogen trans-bis(di-methyl sulfoxide)tetrachloridoruthenate(III) ([(DMSO)2H] [trans-Ru(DMSO-S)2Cl4]) as shown in Scheme 2.25 The coordination of ligand 1 to the ruthenium of 2 was performed using procedure based on that reported for Scheme 1. Synthesis of pyridine-tethered diazenecarboxamide ligand 1 (PyrDiaz).24 Scheme 2. Preparation of Ru(III) precursor 2.25 Vajs et al.: Synthesis and X-ray Structural Analysis ... 766 Acta Chim. Slov. 2017, 64, 763-770 Scheme 3. Synthesis of Ru(III) complex 3 (left) and schematic presentation of sodium ion stabilized three-dimensional framework (right). NAMI-A.31 Combining complex 2 with an excess of ligand 1 in acetone solution resulted in displacement of one dimethyl sulfoxide ligand in the ruthenium coordination sphere and the formation of a new compound 3 shown in Scheme 3 (left). In contrast to our previous findings19,20 for arene-ruthenium(II) compounds from Figure 2, in complex 3 diazene moiety did not participate in coordination to the metal centre. Instead, the pyridine part of the molecule replaced one axial dimethyl sulfoxide ligand. The charge compensation was provided by sodium ion. In solid state, the sodium ion interconnects the anionic ruthenium units into a three-dimensional framework, schematically presented in Scheme 3 (right). The structure of the complex 3 was fully characterized by 1H NMR, elemental analysis and ESI-HRMS. Complex 3 was analysed by high-resolution (HRMS) elec-trospray ionization mass spectrometry in negative ion mode (ESI-). The spectrum, shown in Figure 4, was dominated by the parent [RuIIICl4(DMSO)(PyrDiaz)]- ion at m/z 646.9467 (calcd for C19H25Cl4N5O3RuS-: 646.9462). Another peak at m/z 532.9566 was interpreted as a result of in source collision-induced dissociation of the parent ion giving [RuIIICl3(PyrDiaz - H)]- ion at m/z 532.9554 (calcd for C17H18Cl3N5O2Ru- ([M - Na - HCl]-: 532.9560). In positive mode (ESI+), the spectrum of compound 3 was featureless. The paramagnetic ruthenium(III) ion severely broadened the NMR signals of coordinated ligands, not allowing the assignment procedure. The 1H NMR spectra of freshly prepared D2O solutions of complexes 2 and 3 are shown in Figure 5. Chemical shifts of the coordinated DMSO ligand, peaks at ca. -15 ppm to -18 ppm, are in agreement with published data for the NAMI-type complexes.31,32 Monitoring the D2O solution of 3 for 24 h at Figure 4. ESI- HRMS spectrum of compound 3. Peaks at m/z 112.9856 and m/z 1033.9881 are due to calibrants. Vajs et al.: Synthesis and X-ray Structural Analysis ... Acta Chim. Slov. 2017, 64, 763-770 767 Figure 5. 'H NMR spectrum of a) complex 3, and b) complex 2 as D2O solutions. Relevant insets are only shown (spectra were recorded with spectral width of 60 ppm, number of scans = 128). room temperature by 'H NMR indicated the formation of a complex mixture of products as seen by the appearance of several overlapping broad resonances in the spectra. The structures of these by-products could not be deduced from the spectra. The structure of compound 3 could unambiguously be determined by a single crystal X-ray structure determination and is displayed in Figure 6. Selected bond lengths and angles are given in Table 2. Compound 3 is a polymeric complex with the ruthenium and sodium metallic atoms. Each of the Ru111 atoms are hexacoordinated by sulphur atom from DMSO molecule and the pyridine nitrogen of 1 in apical positions and four chlorido ligands in an equatorial plane. The sulphur-bound DMSO and ligand 1 are trans in nearly octahedral geometry of ruthenium cen- tral atom. The Ru-N and Ru-S bond distances of 2.109(6) and 2.2924(17) A, respectively, are very similar to those found in the related structures containing trans-RuNCl4S donor set.33-35,37 Three of the chlorido ligands are terminal while Cl4 forms a bridge between the ruthenium and sodium atoms. The DMSO ligand also forms a bridge between a ruthenium centre (through its sulphur atom) and a sodium (through its oxygen) thus forming a five membered RuClNaOS ring in the crystal structure. The sodium has six atoms in its distorted octahedrally coordinated environment with the resulting ClN2O3 donor set. Sodium atom is coordinated by two ligands 1, each through one of the diazene nitrogen and one carbonyl oxygen atom. The coordination sphere of sodium atom in the structure of compound 3 is fulfilled by oxygen (bridging DMSO) and Figure 6. Part of the crystal structure of the compound 3, showing atom numbering scheme and hydrogen bonding interactions. The hydrogens on carbon atoms have been omitted for clarity. Vajs et al.: Synthesis and X-ray Structural Analysis ... 768 Acta Chim. Slov. 2017, 64, 763-770 Table 1. Crystal data and structure refinement details for 3. 3 formula Fw (g mol-1) 669.36 crystal size (mm) 0.59 x 0.12 x 0.01 crystal color Yellow crystal system Orthorhombic space group P n a 2l a (Â) 9.64914(19) b (Â) 21.4978(6) c (Â) 14.5013(3) V (Â3) 3008.07(12) Z 4 calcd density (g cm-3) 1.478 F(000) 1348 no. of collected reflns 9948 no. of independent reflns 4713 Rint 0.0443 no. of reflns observed 4388 no. parameters 323 Flack parameter -0.022(16) R[I > 2a (I)]a 0.0415 wR2 (all data)b 0.1113 Goof, Sc 1.067 maximum/minimum residual +0.909/-0.462 electron density (e Â-3) 0 R = £||Fo| - |Fc||/I|Fo|. b wR2 = {X[w(Fo2 - Fc2)2]/X[w(Fo2)2]}1/2. c S = {X[(F 2 - F 2)2]/(n/p)}1/2 where n is the number of reflections and p is the total number of parameters refined. Table 2. Selected bond lengths (Â) and angles (°) for 3. Ru1 - Cl1 2.3294(17) Cl1 - Ru1 - Cl2 92.44(7) Ru1 - Cl2 2.3624(18) Cl1 - Ru1 - Cl3 90.27(7) Ru1 - Cl3 2.3387(17) Cl1 - Ru1 - Cl4 175.82(7) Ru1 - Cl4 2.3797(17) Cl1 - Ru1 - S1 89.86(7) Na1 - O1 2.397(7) Cl1 - Ru1 - N5 89.57(18) Na1 - O2 2.290(6) N5 - Ru1 - S1 179.32(18) Na1 - O3 2.319(6) Cl2 - Ru1 - Cl3 177.29(7) Na1 - N2 2.558(6) O1 - Na1 - O2 104.3(3) Na1 - N3 2.542(7) O1 - Na1 - O3 141.3(2) Na1 - Cl4 2.683(3) O1 - Na1 - N2 77.2(2) N1 - ■ C10 1.321(11) O1 - Na1 - N3 64.0(2) N2 - N3 1.228(9) O1 - Na1 - Cl4 83.72(19) N2 - ■ C10 1.490(9) N2 - N3 - C11 109.5(6) N3 - ■ C11 1.474(9) N3 - C11 - N4 110.1(6) N4 - ■ C11 1.345(10) N3 - N2 - C10 111.3(6) O1 - ■ C10 1.198(9) C10 - N1 - C7 127.7(7) O2 - ■ C11 1.204(9) C11 - N4 - C12 119.4(6) bridging chlorido ligand. Both diazene nitrogen atoms and both carbonyl oxygen atoms of ligand 1 are involved in the coordination to sodium atoms in the polymeric structure of compound 3. A similar coordination environment around sodium ion has been found in related tetrachlori-doruthenium(III) complexes.33-36 The crystal structure of 3 is further stabilized by three intramolecular hydrogen bonds of the type N-H-O and N-H-F (Table 3). A pyridine-heterocycle unsubstituted analogue of 3, called NAMI-Pyr37 and AziRu38 (Figure 3), has been reported to show interesting reactivity profiles towards biologically relevant targets.9 Derivatives with functionalized pyridine ligand have also been investigated.32 Unfortunately, no biological studies of 3 were possible due to its instability and decomposition in protic solvents (vide supra). 4. Conclusions We have reported the synthesis of novel tetrachlori-doruthenium(III) complex Na[frans-RuCl4(DMSO) (PyrDiaz)] (PyrDiaz = N1-(4-isopropylphenyl)-N2-(pyri-din-2-ylmethyl)diazene-1,2-dicarboxamide), a pyridine-tethered derivative of NAMI-A that has a redox-active di-azencarobxamide ligand (PyrDiaz) in the structure. It has been designed to target tumour cell-lines synergistically by means of known antiproliferative activity of NAMI-A and glutathione oxidation ability enacted by the diazene part of the molecule. Although in this particular case the instability and decomposition of Na[frans-RuCl4(DMSO)(PyrDiaz)] in protic solvents disabled biological studies, work is in progress to improve the physicochemical properties of such PyrDiaz-ruthenium(III) complexes. 5. Acknowledgments The authors acknowledge the financial support from the Slovenian Research Agency (research core funding No. P1-0230, Young Researcher Grant to JV, and No. P1-0175). 6. References 1. http://www.who.int/mediacentre/factsheets/fs297/en/ (accessed: July 29, 2017) 2. V Brabec, J. Kasparkova, Drug Resist. Updates 2005, 8, 131146. D01:10.1016/j.drup.2005.04.006 Table 3. Hydrogen bonding geometry for 3. D - H ••• A d(D - H)/ A d(H ••• A)/ A d(D ••• A)/ A <(DHA)/ ° N1 - H1N1 ••• Cl4 0.88 2.72 3.455(5) 141.9 N1 - H1N1 ••• O1 0.88 2.53 3.236(7) 137.4 N4 - H1N4 ••• O3 0.88 2.05 2.922(6) 169.9 Vajs et al.: Synthesis and X-ray Structural Analysis ... Acta Chim. Slov. 2017, 64, 763-770 769 3. B. A. Chabner, T. G. Roberts, Jr., Nat. Rev. Cancer. 2005, 5, 65-72. D01:10.1038/nrc1529 4. B. Rosenberg, L. VanCamp, J. E. Trosko, V. H. Mansour, Nature 1969, 222, 385-386. D0I:10.1038/222385a0 5. For selected informative reading, see: (a) M. Galanski, M. A. Jakupec, B. K. Keppler, Curr. Med. Chem. 2005, 12, 20752094; DOI: 10.2174/0929867054637626 (b) A. V. Klein, T. W. Hambley, "Platinum-Based Anticancer Agents", in: T. Storr (Ed.): Ligand design in medicinal inorganic chemistry, John Wiley & Sons, 2014; D0I:10.1002/9781118697191.ch2 (c) T. C. Johnstone, K. Suntharalingam, S. J. Lippard, Chem. Rev. 2016, 116, 3436-3486; D0I:10.1021/acs.chemrev.5b00597 (d) D. M. Cheff, M. D. Hall, J. Med. Chem. 2017, 60, 45174532. DOI: 10.1021/acs.jmedchem.6b01351 6. J. D. White, M. M. Haley, V J. DeRose Acc. Chem. Res. 2016, 49, 56-66. D0I:10.1021/acs.accounts.5b00322 7. For selected informative reading, see: (a) S. P. Fricker, Dalton Trans. 2007, 4903-4917; (b) P. C. A. Bruijnincx, P. J. Sadler, Curr. Opin. Chem. Biol. 2008, 12, 197-206; (c) I. Romero-Canelon, P. J. Sadler, Inorg. Chem. 2013, 52, 12276-12291; (d) K. Dralle Mjos, C. Orvig, Chem. Rev. 2014, 114, 45404563; (e) R. D. Teo, J. Y. Hwang, J. Termini, Z. Gross, H. B. Gray, Chem. Rev. 2017, 117, 2711-2729; (f) T. Lazarevic, A. Rilak, 2. D. Bugarcic, Eur. J. Med. Chem. 2017. http://dx.doi.org/10.1016/j.ejmech.2017.04.007. 8. For selected informative reading, see: (a) C. S. Allardyce, P. J. Dyson, Platinum Metals Rev. 2001, 45, 62-69; (b) I. Kostova, Curr. Med. Chem. 2006, 13, 1085-1107; D01:10.2174/092986706776360941 (c) W. H. Ang, P. J. Dyson, Eur. J. Inorg. Chem. 2006, 40034018; D0I:10.1002/ejic.200600723 (d) E. S. Antonarakis, A. Emadi, Cancer Chemother Pharmacol. 2010, 66, 1-9; D0I:10.1007/s00280-010-1293-1 (e) A. Bergamo, G. Sava, Dalton Trans. 2011, 40, 7817-7823; D0I:10.1039/c0dt01816c (f) C. G. Hartinger, N. Metzler-Nolte, P. J. Dyson, Organome-tallics 2012, 31, 5677-5685; D0I:10.1021/om300373t (g) A. Bergamo, C. Gaiddon, J. H. M. Schellens, J. H. Beijnen, G. Sava, J. Inorg. Biochem. 2012, 106, 90-99; D0I:10.1016/j.jinorgbio.2011.09.030 (h) C. Mu, C. J. Walsby, "Ruthenium Anticancer Compounds with Biologically-Derived Ligands", in: T. Storr. (Ed.): Ligand design in medicinal inorganic chemistry, John Wiley & Sons, 2014; D0I:10.1002/9781118697191.ch15 (i) M. J. Chow, W. H. Ang, "Organoruthenium(II)-Arene Complexes: Structural Building Blocks for Anticancer Drug Discovery", in: K. Kam-Wing Lo (Ed.): Inorganic and Orga-nometallic Transition Metal Complexes with Biological Molecules and Living Cells, Academic Press, 2016. 9. C. Riccardi, D. Musumeci, C. Irace, L. Paduano, D. Montesar-chio, Eur. J. Org. Chem. 2017, 1100-1119. 10. L. K. Gediya, V. C. O. Njar, Expert Opinion on Drug Discovery 2009, 4, 1099-1111. D0I:10.1002/ejoc.201600943 11. B. Meunier, Acc. Chem. Res. 2008, 41, 69-77. DOI: 10.1021/ar7000843 12. A. Muller-Schiffmann, J. Marz-Berberich, A. Andreyeva, R. Ronicke, D. Bartnik, O. Brener, J. Kutzsche, A. H. C. Horn, M. Hellmert, J. Polkowska, K. Gottmann, K. G. Reymann, S. A. Funke, L. Nagel-Steger, C. Moriscot, G. Schoehn, H. Sticht, D. Willbold, T. Schrader, C. Korth, Angew. Chem. Int. Ed. 2010, 49, 8743-8746. D0I:10.1002/anie.201004437 13. S. Patyar, A. Prakash, B. Medhi, J. Pharm. Pharmacol. 2011, 63, 459-471. D0I:10.1111/j.2042-7158.2010.01236.x 14. J. Kosmrlj, M. Kocevar, S. Polanc, J. Chem. Soc., Perkin Trans.1 1998, 3917-3920. D0I:10.1039/a808381i 15. For reviews, see: (a) J. Kosmrlj, M. Kocevar, S. Polanc, Synlett 2009, 2217-2235; (b) H. Kaur, S. Yadav, B. Narasimhan, Anti-Cancer Agents in Medicinal Chemistry 2016, 16, 1240-1265. DOI: 10.2174/1871520616666160607012042 16. (a) L. Pieters, J. Kosmrlj, R. Lenarsic, M. Kocevar. S. Polanc, Arkivoc 2001, 42-50. (b) T. Cimbora-Zovko, S. Bombek, J. Kosmrlj, L. Kovacic, S. Polanc, A. Katalinic, M. Osmak, Drug Dev. Res. 2004, 61, 95-100. DOI: 10.1002/ddr. 10336 (c) I. Martin-Kleiner, S. Bombek, J. Kosmrlj, B. Cupic, T. Cimbora-Zovko, S. Jakopec, S. Polanc, M. Osmak, J. Ga-brilovac, Toxicol. In Vitro 2007, 21, 1453-1459. DOI: 10.1016/j.tiv.2007.06.005 (d) S. Jakopec, K. Dubravcic, S. Polanc, J. Kosmrlj, M. Osmak, Toxicol. In Vitro 2006, 20, 217-226. DOI: 10.1016/j.tiv.2005.06.008 (e) S. Jakopec, K. Dubravcic, A. Brozovic, S. Polanc, M. Osmak, Cell Biol. Toxicol. 2006, 22, 61-71. DOI: 10.1007/s10565-006-0023-2 17. (a) D. Urankar, A. Pevec, J. Kosmrlj, Eur. J. Inorg. Chem. 2011, 1921-1929; DOI:10.1002/ejic.201001051 (b) N. Stojanovic, D. Urankar, A. Brozovic, A. Ambrio-vic-Ristov, M. Osmak, J. Kosmrlj, Acta Chim. Slov. 2013, 60, 368-374. 18. S. Grabner, J. Kosmrlj, N. Bukovec, M. Cemazar, J. Inorg. Biochem. 2003, 95, 105-112. DOI:10.1016/S0162-0134(03)00092-8 19. M. G. Sommer, P. Kureljak, D. Urankar, D. Schweinfurth, N. Stojanovic, M. Bubrin, M. Gazvoda, M. Osmak, B. Sarkar, J. Kosmrlj, Chem. Eur. J. 2014, 20, 17296-17299. DOI: 10.1002/chem.201404448 20. M. G. Sommer, S. Marinova, M. J. Krafft, D. Urankar, D. Schweinfurth, M. Bubrin, J. Kosmrlj, B. Sarkar, Organometal-lics 2016, 35, 2840-2849. DOI:10.1021/acs.organomet.6b00424 21. For reviews on organometallic anticancer drugs, see refs. 8f,h,i. 22. J. M. Rademaker-Lakhai, D. van den Bongard, D. Pluim, J. H. Beijnen, J. H. M. Schellens, Clin. Cancer Res. 2004, 10, 37173727. DOI:10.1158/1078-0432.CCR-03-0746 23. C. G. Hartinger, M. A. Jakupec, S. Zorbas-Seifried, M. Groessl, A. Egger, W. Berger, H. Zorbas, P. J. Dyson, B. K. Keppler, Chem. Biodivers. 2008, 5, 2140-2155. DOI: 10.1002/cbdv.200890195 Vajs et al.: Synthesis and X-ray Structural Analysis ... 770 Acta Chim. Slov. 2017, 64, 763-770 24. J. Vajs, S. Sovicek, P. Kureljak, N. Stojanovic, I. Steiner, D. El-juga, D. Urankar, M. Kocevar, J. Kosmrlj, S. Polanc, M. Os-mak, Acta Chim. Slov. 2013, 60, 842-852. 25. E. Alessio, G. Balducci, M. Calligaris, G. Costa, W. M. Attia, G. Mestroni, Inorg. Chem. 1991, 30, 609-618. DOI: 10.1021/ic00004a005 26. Rigaku Oxford Diffraction, CrysAlisPro Software System, Version 1.171.38.41, Rigaku Corporation, Oxford, UK, 2015. 27. G. M. Sheldrick, Acta Crystallogr. 2008, A64, 112-122. DOI: 10.1107/S0108767307043930 28. G. Sheldrick, Acta Crystallogr. Sect. 2015, C71, 3-8. DOI: 10.1107/S2053273314026370 29. O. V. Dolomanov, L. J. Bourhis, R. J. Gildea, J. A. K. Howard, H. Puschmann, J. Appl. Crystallogr. 2009, 42, 339-341. DOI: 10.1107/S0021889808042726 30. Crystal Impact GbR, Bonn, Germany (2004-2005) DIAMOND v3.1. 31. E. Alessio, G. Balducci, A. Lutman, G. Mestroni, M. Calligaris, W. M. Attia, Inorg. Chim. Acta 1993, 203, 205-217. DOI:10.1016/S0020-1693(00)81659-X 32. For selected recent example, see: C. Mu, S. W. Chang, K. E. Prosser, A. W. Y. Leung, S. Santacruz, T. Jang, J. R. Thompson, D. T. T. Yapp, J. J. Warren, M. B. Bally, T. V. Beischlag, C. J. Walsby, Inorg. Chem. 2016, 55, 177-190. DOI:10.1021/acs.inorgchem.5b02109 33. C. M. Anderson, A. Herman, F. D. Rochon, Polyhedron 2007, 26, 3661-3668. DOI:10.1016/j.poly.2007.03.041 34. S. Ferrara, A. Kreider-Mueller, J. M. Tanski, C. M. Anderson, Acta Crystallogr. 2011, E67, m756-m757. 35. C. M. Anderson, S. S. Jain, L. Silber, K. Chen, S. Guha, W. Zhang, E. C. McLaughlin, Y. Hu, J. M. Tanski, J. Inorg. Bio-chem. 2015, 145, 41-50. DOI:10.1016/j.jinorgbio.2014.12.017 36. Z. Travnicek, M. Matikova-Malarova, Acta Crystallogr. 2010, E66, m348-m349. 37. M. I. Webb, R. A. Chard, Y. M. Al-Jobory, M. R. Jones, E. W. Y. Wong, C. J. Walsby, Inorg. Chem. 2012, 51, 954-966. DOI:10.1021/ic202029e 38. G. Mangiapia, G. D'Errico, L. Simeone, C. Irace, A. Radules-cu, A. Di Pascale, A. Colonna, D. Montesarchio, L. Paduano, Biomaterials 2012, 33, 3770-3782. DOI:10.1016/j.biomaterials.2012.01.057 Povzetek Z neposredno reakcijo med PyrDiaz ligandom (PyrDiaz = N1-(4-izopropilfenil)-N2-(piridin-2-ilmetil)diazen-1,2-dikar-boksamid)) in natrijevim trans-bis(dimetil sulfoksid)tetrakloridorutenatom(III) (Na-[trans-Ru(DMSO)2Cl4]) smo sin-tetizirali nov tetrakloridorutenijev(III) kompleks Na[trans-RuCl4(DMSO)(PyrDiaz)] (3) s piridin-funkcionaliziranim diazenkarboksamidnim ligandom PyrDiaz. Spojina 3 je analog antimetastatičnega Ru(III) kompleksa NAMI-A in NAMI-Pyr. Rentgenska difrakcijska analiza monokristala je pokazala, da je spojina 3 polimeren kompleks z rutenijevimi in natrijevimi kovinskimi atomi. Vajs et al.: Synthesis and X-ray Structural Analysis ... DOI: 10.17344/acsi.2017.3883 Acta Chim. Slov. 2017, 64, 771-781 ¿^creative tS1 commons Scientific paper Chlorocarbonylsulfenyl Chloride Cyclizations Towards Piperidin-3-yl-oxathiazol-2-ones as Potential Covalent Inhibitors of Threonine Proteases Marko Jukič,1 Katarina Grabrijan,1 Selmir Kadic,1 Fernando Juan de Lera Garrido,1,2 Izidor Sosič,1 Stanislav Gobec1 and Aleš Obreza1* 1 University of Ljubljana, Faculty of Pharmacy, Department of medicinal chemistry; Aškerčeva 7, SI-1000, Ljubljana, Slovenia 2 Universidad de Castilla-La Mancha (Albacete); Universidad de Castilla-La Mancha, Altagracia, 50 13071 Ciudad Real, Spain * Corresponding author: E-mail: ales.obreza@ffa.uni-lj.si phone: +386 1 47 69 677; fax: +386 1 42 58 031 Received: 29-09-2017 Dedicated to Professor Emeritus Miha Tišler, University of Ljubljana, on the occasion of his 90th birthday. Abstract Using rescaffolding approach, we designed piperidine compounds decorated with an electrophilic oxathiazol-2-one moiety that is known to confer selectivity towards threonine proteases. Our efforts to prepare products according to the published procedures were not successful. Furthermore we identified major side products containing nitrile functional group, resulting from carboxamide dehydration. We systematically optimized reaction conditions towards our desired products to identify heating of carboxamides with chlorocarbonylsulfenyl chloride and sodium carbonate as base in di-oxane at 100 °C. Our efforts culminated in the preparation of a small series of piperidin-3-yl-oxathiazol-2-ones that are suitable for further biological evaluation. Keywords: Cyclization, amide dehydration, oxathiazole-2-one, threonine protease, covalent inhibitors, irreversible inhibition 1. Introduction Proteases play key roles in complex biological systems and in multiple structural and signalling pathways. They constitute a historically important field in medicinal chemistry and continue to represent a source of potential drug targets. They are involved in the pathology of hypertension, autoimmune and inflammatory diseases, reperfusion injury, blood clotting disorders, HIV and other viral infections, parasitic and bacterial infections, and last but not least, cancer.1 Protease inhibitors are not valuable only as potential drugs but also as experimental tools for structural biology,2 as they can be used as molecular probes in the elucidation of protease structures and protease pathway mechanisms.3 Recently, databases of proteases (sometimes also termed peptidases, proteinases or proteolytic enzymes) have been established as a resource in this immense research field; namely the Merops database with over 4000 individual entries.4 Our research efforts are mainly focused on the N-ter-minal threonine proteases that form stable covalent acyl-enzyme complexes and are subsequently hydrolyzed to afford product peptides. Threonine proteases constitute 99 entries in the Merops database, where we specifically study the threonine-type endopeptidases, such as the pro-teasomes.5 The proteasomes consist of a central proteolytic unit, known as the 20S proteasome, and the 19S regulators, which together make up a 26S structure (Figure 1). The constitutive isoform of the proteasome is expressed in all eukaryotic cells while its immunomodulatory isoform, the immunoproteasome, is mainly expressed in cells associated with the immune system, such as lymphocytes and Jukič et al.: Chlorocarbonylsulfenyl Chloride Cyclizations 772 Acta Chim. Slov. 2017, 64, 771-781 monocytes.5,6 The constitutive proteasome contains three enzymatically active subunits, namely the blc (caspase-like), the b2c (trypsin-like), and the ^5c (chymotryp-sin-like) that are embedded into a barrel-shaped structure consisting of four rings of ^-subunits and a-subunits in an abba order. The immunoproteasome has essentially the same overall structure, only the catalytically active sub-units of cCP are replaced by their counterparts bli, b2i, and ^5i (Figure 1). The 20S proteasome core particle of both isoforms is a protease of 720 kDa and 28 individual subunits and is responsible for essential proteolytic degradation during cellular inflammatory and oxidative stress.7 Immunoproteasome is also important for the generation of peptides for antigen presentation; moreover, recent studies also suggest a pleiotropic role in cellular function of the immunoproteasome.8-10 There is an amounting body of research on the small-molecule inhibitors of proteasomes.5,11 Both marketed medicines, bortezomib and carfilzomib, equally inhibit the catalytically active ^5 subunits of the constitutive pro-teasome and the immunoproteasome. The combined inhibition of both isoforms leads to cytotoxicity that limits the clinical application of these broad spectrum proteasome inhibitors.6 In addition, many of the investigational compounds are peptide-like compounds and this represents a serious limitation to their metabolic stability and bioavail-ability.5 To overcome these problems, multiple approaches can be found in literature: design of reversible proteasome inhibitors,12 use of structural differences in the binding sites of both proteasomes in structure-based drug de-sign,13,14 design of highly selective and hydrolytically more stable peptidic compounds,15 design of highly selective non-peptidic compounds,16 use of non-catalytic residues or allosteric sites in inhibitor design,17 and the design of selective electrophilic warheads.18 The majority of these compounds are covalent irreversible inhibitors bearing an electrophilic warhead that is capable of reacting with the N-terminal threonine residue in the catalytic active site of the examined protease.5,11 Electrophilic warheads belong to structural classes of aldehydes, a'p-epoxyketones, a-keto aldehydes, ^-lactones, vinyl sulfones, Michael-acceptor systems, and boronates.19 The active interest in this field is clearly represented by a very recent publication,19 where a new mechanism for an existing warhead was reported, i.e. the formation of 1,4-oxazepane upon reaction of an a'P'-epoxyketone warhead with the N-terminal threonine rather than the previously reported morpholine ring.14,19 Such new developments provide invaluable data for the design of novel and selective irreversible inhibitors of threonine proteases. In order to design targeted covalent inhibitors of threonine protease, we sought to examine the available electrophilic warheads.20 We were in particular interested in compounds that could provide a suitable reactivity and selectivity towards threonine proteases. Recently, oxathi-azol-2-one moiety was identified in a high-throughput screening campaign as a promising candidate.21 The proposed mechanism of the covalent modification of N-termi-nal threonine induced by this electrophilic fragment is depicted in Figure 2 and proceeds through cyclocarbonyla-tion.18,21 In current paper we describe an optimized synthetic approach towards oxathiazol-2-one electrophilic war- Figure 1. Proteasome (immunoproteasome) assembly with P1i (caspase-like), P2i (trypsin-like) and P5i (chymotrypsin-like) N-terminal threonine proteases. Jukič et al.: Chlorocarbonylsulfenyl Chloride Cyclizations Acta Chim. Slov. 2017, 64, 771-781 773 Figure 2. Oxathiazol-2-one electrophilic warhead and its interaction mechanism with the N-terminal threonine in the active site head in compounds with basic nitrogen atom and the preparation of a focused library of piperidin-3-yl-oxathi-azol-2-ones that are suitable for further biological evaluation. 2. Results and Discussion We designed our compounds on the basis of their synthetic accessibility and their potential to be modified accordingly during further optimizations. Therefore, we selected a piperidine central core derivatized with an elec-trophilic oxathiazol-2-one warhead that could confer the selectivity towards threonine proteases as reported beforehand (Figure 3).18,21 We started the synthesis with the alkylation of nipecotamide employing a set of alkyl bromides in DMF as a solvent and Na2CO3 as a base to obtain compounds 2a, 2b and 2c-e. In the case of compound 2f, alkylation with p-nitrobenzylbromide was followed by hydrogenation in MeOH with final acylation using benzyl chloride. The key step in the synthesis was the cyclization of suitably substituted nipecotamides 2a-f into piperidin-3-yl-oxathiazol-2-ones 7-3e using chlorocarbonylsulfenyl chloride as a reagent (Figure 4). This synthetic approach was reported by Gryder et al. when they described the synthesis of the oxathiazol-2-one analogue of bortezomib. The penultimate carboxamide dipeptide was successfully transformed into the oxathiazol-2-one-bortezomib in high yield by using chlorocarbonylsulfenyl chloride in refluxing THF.22 Despite our numerous attempts to obtain the final oxathiazol-2-ones 3a-f by following the original procedure no product could be isolated. Initial experiments in refluxing THF resulted in a complex mixture of products.23 If the experiments were performed at lower temperature (0 °C, room temperature), no apparent conversion was observed. Our first modification of the original procedure was to use relatively nonpolar and system-inert toluene as a solvent that could provide an alternative reactant/intermediate stabilization pattern and would enable a broader temperature sweep. This system was also described by Gurjar et al. where they heated the mixture of amide and chlorocarbonylsulfenyl chloride in toluene from 60 to 90 °C until the settlement of HCl evolution, followed by 1 h of reflux; this yielded > 50% of isolated oxathiazol-2-one.23 No conversion was observed in our case at lower temperatures (0 °C, room temperature) with a formation of complex mixture of products at 60 °C and reflux conditions. Further experiments using pyridine as solvent afforded similar results. Nevertheless, a difference in reaction scope can be observed as besides previously mentioned report by Gryder et al.,22 literature only describes a relatively simple case of benzamide cyclization towards final 5-phenyl-1,3,4-ox-athiazol-2-one. In our case, the reaction incorporated a piperidin-3-yl central scaffold (compounds 2a-e) containing an additional basic centre. We also conducted a thor- Q = C, N n = 0, 1 Figure 3. Design of piperidin-3-yl-oxathiazol-2-ones as potential covalent inhibitors of threonine proteases. Jukič et al.: Chlorocarbonylsulfenyl Chloride Cyclizations 774 Acta Chim. Slov. 2017, 64, 771-781 Figure 4. Cyclization of piperidine carboxamides towards final piperidin-3-yl-oxathiazol-2-ones. ough separation of complex product mixtures in the case of cyclization of compound 2a and identified a dominant side product (> 30% yield) flanked by a myriad of other chemical species that could not be obtained at a significant quantity. The dominant side product was identified when examining its 13C NMR spectrum. Namely, the carbon atom of the carboxamide 2a can be found as expected at 178.3 ppm (400 MHz, DMSO-d6), whereas the carbon of the dominant side product species was found upfield at 121.8 ppm. When recording IR spectrum, a marked peak at 2240 cm-1 was found indicating the presence of a nitrile functionality; the formation of the side product 1-benzylpiperidine-3-car-bonitrile 4a (Figure 5) was then further confirmed by HRMS. The nature of this reaction outcome can be rationalized as presented in Figure 5. In our reaction system, the dehydration process is facilitated by the primary amide 2a (Figure 5) that readily couples with the chlorocarbonylsulfenyl chloride to form an active intermediate (Figure 5). The coupling is followed by rapid elimination that is catalyzed either with the starting substituted piperidine as a base or is assisted by other bases in the reaction system (such as pyridine) to form the corresponding nitrile 4a (Figure 5). Indeed, similar dehydrations of primary carboxamides using an acidic reagent such as POCl3, SOCl2 are well documented in literature.24,25 More recent, chemoselective and milder methods were Figure 5. The proposed mechanism of dexydration of primary amides to nitriles using chlorocarbonylsulfenyl chloride. Jukič et al.: Chlorocarbonylsulfenyl Chloride Cyclizations Acta Chim. Slov. 2017, 64, 771-781 775 Figure 6. Possible side reaction pathways in the synthesis of piperidin-3-yl-oxathiazol-2-ones. also reported, where ethyl dichlorophosphate/DBU system or methyl (carboxysulfamoyl)triethylammonium hydroxide (Burgess reagent) were used as the dehydrating reagents.26,27 In addition, Vilsmeier reagents,28 bromodi-methylsulfonium bromide (BDMS),29 PdCl2 in aqueous acetonitrile,30 Swern oxidation conditions and other catalytic or alternative methods using silanes, silazanes, chlo-rosilanes, alkoxysilanes, and aminosilanes were also de- scribed.31-33 The myriad of reaction side products that was observed is a consequence of multitude of side reactions that can occur during dehydration reactions, such as thermal decomposition of the formed oxathiazol-2-one and hydrolysis reactions (Figure 6). The formed oxathi-azol-2-one can also take part in the 1,3-dipolar nitrile sulphide cycloaddition reaction with available nitrile to obtain thiadiazoles as side products.34 The nitrile sulphide is Table 1. Synthesis of piperidin-3-yl-oxathiazol-2-ones 3a-f. Cpd. No Product Yield (n) (%)a 3e 43 3f 16 a Yield after purification using column chromatography (SiO2 support with n-hexane:EtOAc solvent system as an eluent). Jukič et al.: Chlorocarbonylsulfenyl Chloride Cyclizations 776 Acta Chim. Slov. 2017, 64, 771-781 generated in situ by thermal decomposition of oxathi-azol-2-one.35 Nitrile sulphides are short-lived species prone to fragmentation and can take part in further cyc-loadditions.36-38 After initial unsuccessful attempts to prepare the desired compounds 3a-f, we turned our attention to microwave-assisted report on flow-chemistry synthesis of ox-athiazol-2-one in dioxane at 200 °C and residence time of 1 min in a flow reactor reported by Ohrngren et al.39 On this basis, we modified the reaction procedure and dissolved the carboxamides 2a-f (Figure 4) in dry dioxane (27 mL/1 mmol carboxamide), used an excess of solid Na-2CO3 (5 eq) and chlorocarbonylsulfenyl chloride (2 eq), and stirred the reaction mixture at 100 °C for 16 h under argon to obtain the desired oxathiazol-2-ones 3a-f (Figure 4) in 16 to 68% yields (Table 1). 3. Experimental Chemicals from commercial sources were used without further purification. Anhydrous THF, DCM and Et3N were dried and purified by distillation over Na, K2CO3 and KOH, respectively. Analytical thin-layer chromatography (TLC) was performed on Merck silica gel (60F254) plates (0.25 mm). Column chromatography was performed on silica gel 60 (Merck, particle size 0.0400.063 mm). Melting points were determined on a Reichert hot stage microscope and are uncorrected. 'H-, COSY-, HMQC- and 13C-NMR spectra were recorded on a Bruker AVANCE DPX400 spectrometer in CDCl3 or DMSO-d6 solution with TMS as internal standard. Chemical shifts are reported in ppm (5) downfield from TMS. All the coupling constants (J) are in hertz. IR spectra were recorded on a PerkinElmer Spectrum BX System FT-IR spectrometer. Mass spectra were obtained with a VG-Analytical Autospec Q mass spectrometer with ESI ionization (MS Centre, Jožef Stefan Institute, Ljubljana). All reported yields are those of purified products. 1-Benzylpiperidine-3-carboxamide (2a) To a solution of piperidine-3-carboxamide (500 mg, 3.90 mmol) in 50 mL DMF, solid Na2CO3 (460 mg, 4.33 mmol) and benzyl bromide (1.44 g, 8.42 mmol) were added. The reaction was stirred at 100 °C overnight. DMF was removed under reduced pressure, the residue dissolved in EtOAc (30 mL) and extracted with 0,5 M HCl (2 x 15 mL). The pH of combined aqueous phases was adjusted to 8 with NaHCO3 and extracted with EtOAc (4 x 30 mL). Combined organic phases were washed with H2O (1 x 30 ml), brine (1 x 30 mL) and dried over Na2SO4. The volatiles were removed under reduced pressure to give compound 2a as white solid. Yield = 56 %; TLC (EtOAc:MeOH = 2:1), Rf = 0,50; m.p. 108-110 °C; 1H NMR (400 MHz, DMSO-d6) 5 1.34-1.48 (m, 2H, H-5 ), 1.58-1.74 (m, 2H, H-4), 1.85-1.99 (m, 2H, H-6), 2.27-2.32 (m, 1H, H-2), 2.68-2.78 (m, 2H, H-2 and H-3), 3.44 (dd, J 4.4, 13.2 Hz, 2H, CH2), 6.75 (br s, 1H, NH2), 7.22-7.34 (m, 6H, ArH and NH2); 13C NMR (400 MHz, CDCl3) 5 22.8 (C-5), 26.9 (C-4), 41.8 (C-3), 53.8 (C-6), 55.0 (c-2), 63.5 (CH2), 127.4 (C-4'), 128.4 (C-3' and C-5'), 129.2 (C-2' and C-6'), 137.6 (C-1'), 178.0 (OCNH2); IR (ATR) v 3333, 3148, 2932, 2758, 1632, 1429, 1368, 1356, 1337, 1253, 1100, 1070, 1002,858, 734,698, 666, 564, 518 cm-1; MS m/z (relative intensity): 240.95 (M+Na, 100), 219.03 (M+H, 30). 1-(4-Nitrobenzyl)piperidine-3-carboxamide (2b) To a solution of piperidine-3-carboxamide (500 mg, 3.90 mmol) in 50 mL DMF, solid Na2CO3 (460 mg, 4.33 mmol) and 4-nitrobenzyl bromide (1.69 g, 7.82 mmol) were added. The reaction was stirred at 100 °C overnight. DMF was removed under reduced pressure, the residue dissolved in EtOAc (30 mL) and extracted with 0.5 M HCl (2 x 15 mL). The pH of combined aqueous phases was adjusted to 8 with NaHCO3 and extracted with EtOAc (4 x 30 mL). Combined organic phases were washed with H2O (1 x 30 ml), brine (1 x 30 mL) and dried over Na2SO4. The volatiles were removed under reduced pressure to give compound 2b as pale orange solid. Yield = 93%; TLC (EtOAc:MeOH = 2:1), Rf = 0.63; m.p. 111-114 °C; 1H NMR (400 MHz, DMSO-d) 5 1.33-1.36 (m, 1H, H-5), 1.43-1.48 (m, 1H, H-4), 1.60-1.65 (m, 1H, H-5), 1.721.76 (m, 1H, H-4), 1.91-2.09 (m, 2H, H-6 and H-2), 2.30-2.33 (m, 1H, H-6), 2.69-2.78 (m, 2H, H-2 and H-3), 3.55-3.63 (m, 2H, CH2), 6.76 (br s, 1H, NH2), 7.27 (br s, 1H, NH2), 7.56-7.60 (m, 2H, H-2' and H-6'), 8.19 (dd, J 2.0, 4.8 Hz, 2H, H-3' and H-5'); 13C NMR (400 MHz, DMSO-d6) 5 24.4 (C-5), 27.0 (C-4), 42.3 (C-3), 53.2 (C-6), 55.8 (C-2), 61.4 (CH2), 123.3 (C-3' and C-5'), 129.6 (C-2' and C-6'), 146.5 (C-1'), 146.9 (C-4'), 175.4 (OCNH2); IR (ATR) v 3385, 3180, 2926, 2787, 1644, 1605, 1512, 1421, 1341, 1249, 1204, 1166, 1102, 1048, 989, 862, 797, 736,720 cm-1; MS m/z (relative intensity): 261.97 (M-H, 100). 1-(4-Chlorobenzyl)piperidine-3-carboxamide (2c) To a solution of piperidine-3-carboxamide (500 mg, 3.90 mmol) in 50 mL DMF, solid Na2CO3 (460 mg, 4.33 mmol) and 3-chlorobenzyl bromide (1.64 g, 7.99 mmol) were added. The reaction was stirred at 100 °C overnight. DMF was removed under reduced pressure, the residue dissolved in EtOAc (30 mL) and extracted with 0.5 M HCl (2 x 15 mL). The pH of combined aqueous phases was adjusted to 8 with NaHCO3 and extracted with EtOAc (4 x 30 mL). Combined organic phases were washed with H2O (1 x 30 ml), brine (1 x 30 mL) and dried over Na2SO4. The volatiles were removed under reduced pressure to give compound 2c as pale orange solid. Yield = 53%; TLC (EtOAc:MeOH = 2:1), Rf = 0.60; 1H NMR (400 MHz, CDCl3) 5 1.55-1.61 (m, 1H, H-5), 1.65-1.77 (m, 2H, H-4 and H-5), 1.82-1.84 (m, 1H, H-4), 2.26 (s, 1H, H-6), 2.44-2.53 (m, 2H, H-2 and H-6), 2.59 (s, 1H, H-2), 2.73 Jukič et al.: Chlorocarbonylsulfenyl Chloride Cyclizations Acta Chim. Slov. 2017, 64, 771-781 777 (s, 1H, H-3), 3.46 (s, 2H, CH2), 6.07 (br s, 1H, NH2), 7.147.16 (m, 1H, NH2), 7.24-7.26 (m, 4H, ArH); 13C NMR (400 MHz, CDCl3) 5 22.9 (C-5), 26.9 (C-4), 41.9 (C-3), 53.7 (C-6), 55.1 (c-2), 62.9 (CH2), 127.2, 127.6 (C-3' and C-5'), 129.1, 129.7 (C-2' and C-6'), 134.3 (C-4'), 139.8 (C-1'), 177.8 (CONH2); IR (ATR) v 3342, 3162, 2937, 2800, 2763, 1626, 1597,1573, 1426, 1371, 1342, 1300, 1256, 1207, 1157, 1106, 1075, 1044, 996, 985, 930, 892, 875, 863, 808, 786, 776, 717, 682, 655, 569, 516 cm-1; MS m/z (relative intensity): 275.29 (M+Na, 100), 253.29 (M+H, 20). Methyl 4-((3-carbamoylpiperidin-1-yl)methyl)benzo-ate (2d) To a solution of piperidine-3-carboxamide (200 mg, 1.56 mmol) in 50 mL DMF, solid Na2CO3 (320 mg, 3.02 mmol) and methyl 4-(bromomethyl)benzoate (540 mg, 2.36 mmol) were added. The reaction was stirred at 100 °C overnight. DMF was removed under reduced pressure, the residue dissolved in EtOAc (30 mL) and extracted with 0.5 M HCl (2 x 15 mL). The pH of combined aqueous phases was adjusted to 8 with NaHCO3 and extracted with EtOAc (4 x 30 mL). Combined organic phases were washed with H2O (1 x 30 ml), brine (1 x 30 mL) and dried over Na2SO4. The volatiles were removed under reduced pressure to give compound 2d as white solid. Yield = 91 %; TLC (EtOAc:MeOH = 2:1), Rf = 0.62; 1H NMR (400 MHz, DMSO-d.) 5 1.32-1.42 (m, 1H, H-5), 1.44-1.49 (m, 1H, H-4), 1.59-1.64 (m, 1H, H-5), 1.71-1.75 (m, 1H, H-4), 1.88-1.95 (m, 1H, H-6), 1.98-2.03 (m, 1H, H-2), 2.292.35 (m, 1H, H-6), 2.68-2.71 (m, 1H, H-2), 2.74-2.78 (m, 1H, H-3), 3.52 (d, J 2.8 Hz, 2H, CH2), 3.85 (s, 3H, CH3), 6.77 (br s, 1H, NH2), 7.27 (br s, 1H, NH2), 7.44 (d, J 8.4 Hz, 2H, H-2' and H-6'), 7.92 (dd, J 2.0, 4.8 Hz, 2H, H-3' and H-5'); IR (ATR) v 3403, 3183, 2936, 2797, 1715, 1647, 1434, 1415, 1273, 1239, 1199, 1165, 1112,1086, 1027, 995, 964, 860, 806, 760, 754, 706 cm-1. 1-(4-Cyano-2-fluorobenzyl)piperidine-3-carboxamide (2e) To a solution of piperidine-3-carboxamide (500 mg, 3.90 mmol) in 50 mL DMF, solid Na2CO3 (460 mg, 4.33 mmol) and 4-(bromomethyl)-3-fluorobenzonitrile (1.75 g, 8.17 mmol) were added. The reaction was stirred at 100 °C overnight. DMF was removed under reduced pressure, the residue dissolved in EtOAc (30 mL) and extracted with 0.5 M HCl (2 x 15 mL). The pH of combined aqueous phases was adjusted to 8 with NaHCO3 and extracted with EtOAc (4 x 30 mL). Combined organic phases were washed with H2O (1 x 30 ml), brine (1 x 30 mL) and dried over Na2SO4. The volatiles were removed under reduced pressure to give compound 2e as pale yellow solid. Yield = 71 %; TLC (EtOAc:MeOH = 2:1), Rf = 0.66; m.p. 126-128 °C; 1H NMR (400 MHz, DMSO-d.) 5 1.30-1.31 (m, 1H, H-5), 1.42-1.45 (m, 1H, H-4), 1.59-1.64 (m, 1H, H-5), 1.69-1.73 (m, 1H, H-4), 1.93-1.99 (m, 1H, H-6), 2.022.08 (m, 1H, H-2), 2.28-2.33 (m, 1H, H-6), 2.67-2.70 (m, 1H, H-2), 2.74-2.78 (m, 1H, H-3), 3.58 (s, 2H, CH2), 6.77 (br s, 1H, NH2), 7.27 (br s, 1H, NH2), 7.61 (t, J 7.6 Hz, 1H, H-5'), 7.68 (dd, J 1.6, 6.4 Hz, 1H, H-2'), 7.82 (dd, J 1.6, 8.4 Hz, 1H, H-3'); 13C NMR (400 MHz, CDCl3) 5 22.9 (C-5), 26.8 (C-4), 41.9 (C-3), 53.5 (C-6), 55.3 (C-2), 56.0 (CH2), 112.8(C-4'), 117.5 (CN), 119.2 (C-5'), 128.1 (C-3'), 130.8 (C-1'), 132.3 (C-2'), 162.1 (C-6'), 177.5 (OCNH2); IR (ATR) v 3364, 3187, 2929, 2811, 2227, 1646, 1611, 1569, 1486, 1411, 1350, 1298, 1246, 1201, 1166, 1089, 1002, 941, 849, 830, 731 cm-1; MS m/z (relative intensity): 283.71 (M+Na, 100), 261.78 (M+H, 20). 1-(4-Benzamidobenzyl)piperidine-3-carboxamide (2f) Argon was bubbled into a solution of 2b (5.811 g, 17.6 mmol) in MeOH (70 mL) for 15 minutes. 10% Pd/C, unreduced, was then added and H2 was bubbled into the stirred solution until the starting compound was no longer observed with TLC. Pd/C was filtered off and the solution concentrated in vacuo to yield crude product which was purified with column cromatography (EtOAc:MeOH = 2:1). Oily product was dissolved in DCM (30 mL). Et3N (250 mg, 2.47 mmol) and benzoyl chloride (265 mg, 2.36 mmol) were added and the reaction mixture was stirred at room temperature overnight. DCM was removed under reduced pressure, the residue dissolved in EtOAc (30 mL) and extracted with 0.5 M HCl (1 x 10 mL). The pH of combined aqueous phases was adjusted to 8 with NaHCO3 and extracted with EtOAc (2 x 15 mL). Combined organic phases were washed with H2O (1 x 20 ml), brine (1 x 20 mL) and dried over Na2SO4. The volatiles were removed under reduced pressure to give compound 2f as colourless oil. Yield = 16 %; TLC (EtOAc:MeOH = 2:1), Rf = 0.36; 1H NMR (400 MHz, DMSO-d.) d 1.06-1.63 (m, 1H, H-5), 1.64-1.70 (m, 3H, H-4 and H-5), 1.89-1.99 (m, 1H, H-6), 2.29-2.34 (m, 2H, H-2 and H-6), 2.67-2.76 (m, 1H, H-2), 2.78-2.99 (m, 1H, H-3), 3.01-3.51(m, 2H, CH2), 6.76 (br s, 1H, NH2), 7.24-7.29 (m, 3H, H-2' and H-6' and NH2), 7.52-7.62 (m, 3H, H-3''and H-4''and H-5''), 7.72-7.7 6 (m, 2H, H-3' and H-5'), 7.95-7.97 (m, 2H, H-2'' and H-6''), 10.26 (d, J 4.4 Hz, 1H, NH). 5-(1-Benzylpiperidin-3-yl)-1,3,4-oxathiazol-2-one (3a) To a solution of 1-benzylpiperidine-3-carboxamide (2a, 240 mg, 1.10 mmol) in dioxane (30 mL) in a three-necked flask, solid Na2CO3 (580 mg, 5.47 mmol) and chlo-rocarbonylsulfenyl chloride (288 mg, 2.20 mmol) were added under argon. The reaction mixture was stirred at 100 °C overnight, cooled to room temperature and after the addition of Et3N (0.75 mL) stirred for 15 minutes. The precipitate was filtered off and the residue concentrated in vacuo. Product was purified with column chromatography using hexane:EtOAc=3:1 as an eluent to give yellow oily product. Yield = 68%; TLC (hexane:EtOAc = 3:1), Rf = 0.56; 1H NMR (400 MHz, DMSO-d.) 5 1.50-1.56 (m, 2H, H-5 and H-4), 1.70-1.72 (m, 1H, H-5), 1.88-1.93 (m, 1H, H-4), 2.08-2.12 (m, 1H, H-6), 2.20-2.25 (m, 1H, H-2), Jukič et al.: Chlorocarbonylsulfenyl Chloride Cyclizations 778 Acta Chim. Slov. 2017, 64, 771-781 2.65-2.68 (m, 1H, H-6), 2.88-2.92 (m, 2H, H-2 and H-3), 3.50 (d, / 2.8 Hz, 2H, CH2), 7.23-7.34 (m, 5H, ArH); 13C NMR (400 MHz, CDCl3) 5 24.2 (C-5), 27.0 (C-4), 38.6 (C-3), 53.5 (C-6), 55.1 (C-2), 63.1 (CH2), 127.2 (C-4'), 128.3 (C-3' and C-5'), 128.9 (C-2' and C-6'), 137.8 (C-1'), 162.9 (NCO), 174.3 (SCO); IR (ATR) v 2942, 2800, 1758, 1666, 1599, 1493, 1467, 1450, 1350, 1320, 1287, 1188, 1152, 1097, 1071, 1049, 1025, 981, 926, 879, 791, 769, 738, 697, 664, 633, 599, 569, 522 cm-1; HRMS-ESI (m/z): [M+H]+ calcd for CH NO,S, 277.1015, found, 277.1011. L J 14 16 2 2 5-(1-(4-Nitrobenzyl)piperidin-3-yl)-1,3,4-oxathiazol-2-one (3b) To a solution of 1-(4-nitrobenzyl)piperidine-3-car-boxamide (2b, 100 mg, 0.380 mmol) in dioxane (20 mL) in a three-necked flask, solid Na2CO3 (200 mg, 1.87 mmol) and chlorocarbonylsulfenyl chloride (100 mg, 0.760 mmol) were added under argon. The reaction mixture was stirred at 100 °C overnight, cooled to room temperature and after the addition of Et3N (0.27 mL) stirred for 15 minutes. The precipitate was filtered off and the residue concentrated in vacuo. Product was purified with column chromatography using hexane:EtOAc = 2:1 as an eluent to give yellow oily product. Yield = 22%; TLC (hexane:EtOAc = 2:1), Rf = 0.38; 1H NMR (400 MHz, DMSO-dfi) 5 1.521.58 (m, 2H, H-4 and H-5), 1.71-1.74 (m, 1H, H-5), 1.91-1.94 (1H, H-4), 2.16 (s, 1H, H-6), 2.26 (d, / 9.6 Hz, H-2), 2.65-2.68 (m, 1H, H-6), 2.88-2.96 (m, 2H, H-2 and H-3), 3.65 (s, 2H, CH2), 7.58 (d, / 8.8 Hz, 2H, H-3' and H-5'), 8.18 (d, / 8.8 Hz, 2H, H-2' and H-6'); 13C NMR (400 MHz, DMSO-d6) 5 24.1 (C-5), 26.6 (C-4), 38.3 (c-3), 53.5 (C-6), 54.9 (C-2), 61.5 (CH2), 123.8 (C-2' and 6'), 130.1 (C-3' and C-5'), 147.0 (C-1' and C-4'), 162.9 (NCO), 174.7 (SCO); IR (ATR) v 2939, 2792, 1758, 1598, 1515, 1340, 1191, 1092, 926, 858, 802, 738, 650, 571 cm-1; HRMS-ESI (m/z): [M+H]+ calcd for C14H15N3O4S, 322.0867, found, 322.0862. 14 15 3 4 5-(1-(4-Chlorobenzyl)piperidin-3-yl)-1,3,4-oxathiazol-2-one (3c) To a solution of 1-(4-chlorobenzyl)piperidine-3-car-boxamide (2c, 300 mg, 1.19 mmol) in dioxane (30 mL) in a three-necked flask, solid Na2CO3 (630 mg, 5.94 mmol) and chlorocarbonylsulfenyl chloride (466 mg, 3.55 mmol) were added under argon. The reaction mixture was stirred at 100 °C overnight, cooled to room temperature and after the addition of Et3N (0.83 mL) stirred for 15 minutes. The precipitate was filtered off and the residue concentrated in vacuo. Product was purified with column chromatography using hexane:EtOAc = 3:1 as an eluent to give brown oily product. Yield = 33 %; TLC (H:EtOAc = 3:1), Rf = 0.42; 1H NMR (400 MHz, CDCl3) 5 1.57-1.69 (m, 2H, H-5 and H-4), 1.77-1.83 (m, 1H, H-5), 2.01-2.04 (m, 1H, H-4), 2.09-2.14 (m, H, H-6), 2.28-2.33 (m, 1H, H-2), 2.73-2.76 (m, 1H, H-6), 2.89-2.98 (m, 2H, H-2 and H-3), 3.50 (s, 2H, CH2), 7.16-7.31 (m, 4H, ArH); 13C NMR (400 MHz, CDCl3) 5 24.2 (C-5), 26.9 (C-4), 38.6 (C-3), 53.5 (C-6), 55.1 (C-2), 62.5 (CH2), 126.9, 127.4 (C-3' and C-5'), 128.9, 128.6 (C-2' and C-6'), 134.2 (C-4'), 140.2 (C-1'), 162.7 (NCO), 174.3 (SCO); IR (ATR) v 3339, 3160, 2938, 2801, 2764, 1762, 1626, 1598, 1573, 1463, 1427, 1371, 1342, 1300, 1257, 1207, 1157, 1105, 1075, 1046, 997, 930, 892, 876, 863, 808, 776, 717, 705, 682, 655, 569, 536, 516 cm-1; HRMS-ESI (m/z): [M+H]+ calcd for C14H15ClN2O2S, 311.0626, found, 311.0621. Methyl 4-((3-(2-oxo-1,3,4-oxathiazol-5-yl)piperidin-1-yl) methyl)benzoate (3d) To a solution of methyl 4-((3-carbamoylpiperi-din-1-yl)methyl)benzoate (2d, 300 mg, 1.09 mmol) in di-oxane (30 mL) in a three-necked flask, solid Na2CO3 (570 mg, 5.38 mmol) and chlorocarbonylsulfenyl chloride (285 mg, 2.16 mmol) were added under argon. The reaction mixture was stirred at 100 °C overnight, cooled to room temperature and after the addition of Et3N (0.75 mL) stirred for 15 minutes. The precipitate was filtered off and the residue concentrated in vacuo. Product was purified with column chromatography using hexane:EtOAc = 2:1 as an eluent to give yellow oily product. Yield = 43%; TLC (hexane:EtOAc = 2:1), Rf = 0.29; 1H NMR (400 MHz, DMSO-d6) 5 1.45-1.57 (m, 2H, H-5, H-4), 1.72-1.75 (m, 1H, H-5), 1.91-1.95 (m, 1H, H-4), 2.09-2.17 (m, 1H, H-6), 2.24-2.34 (m, 1H, H-2), 2.65-2.68 (m, 1H, H-6), 2.87-2.96 (m, 2H, H-2 and H-3), 3.59 (s, 2H, CH2), 3.85 (s, 3H, CH3), 7.45 (d, / 8.4 Hz, 2H, H-2' and H-6'), 7.93 (dd, J 2.0, 4.8 Hz, 2H, H-3' and H-5'); 13C NMR (400 MHz, DMSO-d6) 5 23.5 (C-5), 26.1 (C-4), 37.5 (CH2), 52.0 (C-6), 53.0 (C-2), 54.5 (CH3), 61.5 (CH2), 128.3, 128.7, 128.8, 129.1, 129.1 (C-2',3',4',5,,6'), 144.0 (C-1'), 162.4 (CO), 166.1 (NCO), 174.2 (SCO); IR (ATR) v 2946, 2801, 1759, 1717, 1609, 1434, 1415, 1395, 1349, 1309, 1275, 1190, 1173, 1106, 1049, 980, 928, 885, 801, 758, 731, 701, 650, 572, 538 cm-1; HRMS-ESI (m/z): [M+H]+ calcd for C16H18N2O4S, 335.1061, found, 335.1066. 3-Fluoro-4-((3-(2-oxo-1,3,4-oxathiazol-5-yl)piperi-din-1-yl)methyl)benzonitrile (3e) To a solution of 1-(4-cyano-2-fluorobenzyl)piperi-dine-3-carboxamide (2e, 300 mg, 1.15 mmol) in dioxane (30 mL) in a three-necked flask, solid Na2CO3 (608 mg, 5.74 mmol) and chlorocarbonylsulfenyl chloride (303 mg, 2.30 mmol) were added under argon. The reaction mixture was stirred at 100 °C overnight, cooled to room temperature and after the addition of Et3N (0.75 mL) stirred for 15 minutes. The precipitate was filtered off and the residue concentrated in vacuo. Product was purified with column chromatography using hexane:EtOAc = 3:1 as an eluent to give yellow oily product. Yield = 43%; TLC (hexane:EtOAc = 3:1), Rf = 0.29; 1H NMR (400 MHz, DMSO-dfi) 5 1.511.56 (m, 2H, H-5 and H-4), 1.70-1.72 (m, 1H, H-5), 1.91-1.92 (m, 1H, H-4), 2.14-2.19 (m, 1H, H-6), 2.302.35 (m, 1H, H-2), 2.64-2.67 (m, 1H, H-6), 2.91-2.95 (m, Jukič et al.: Chlorocarbonylsulfenyl Chloride Cyclizations Acta Chim. Slov. 2017, 64, 771-781 779 2H, H-3 and H-2), 3.65 (s, 2H, CH2), 7.62 (t, / 7.6 Hz, 1H, H-5'), 7.69 (dd, / 1.6, 6.4 Hz, 1H, H-2'), 7.83 (dd, / 1.2, 8.4 Hz, 1H, H-3'); 13C NMR (400 MHz, DMSO-d6) 5 23.5 (C-5), 26.0 (C-4), 37.5 (C-3), 52.7 (C-6), 54.2 (C-2), 54.3 (CH2), 111.3 (C-4'), 117.7 (CN), 119.3 (C-5'), 128.5 (C-3'), 131.2 (C-1'), 132.2 (C-2'), 158.8 (C-6), 162.4 (NCO), 174.2 (SCO); IR (ATR) v 2947, 2811, 2232, 1758, 1599, 1571, 1496, 1413, 1356, 1260, 1189, 1153, 1089, 1050, 1024, 981, 941, 925, 870, 835, 783, 711, 621, 569 cm-1; HRMS-ESI (m/z): [M+H]+ calcd for C15H14FN3O2S, 320.0873, found, 320.0869. N-(4-((3-(2-oxo-1,3,4-oxathiazol-5-yl)piperidin-1-yl) methyl)phenyl)benzamide (3f) To a solution of 1-(4-benzamidobenzyl)piperi-dine-3-carboxamide (2f, 83 mg, 0.250 mmol) in dioxane (30 mL) in a three-necked flask, solid Na2CO3 (130 mg, 1.23 mmol) and chlorocarbonylsulfenyl chloride (65 mg, 0.492 mmol) were added under argon. The reaction mixture was stirred at 100 °C overnight, cooled to room temperature and after the addition of Et3N (0.75 mL) stirred for 15 minutes. The precipitate was filtered off and the residue concentrated in vacuo. Product was purified with column chromatography using hexane:EtOAc = 3:1 as an elu-ent to give yellow oily product. Yield = 16%; TLC (hexane:EtOAc = 3:1), Rf = 0.07; 1H NMR (400 MHz, DMSO-d6) 5 1.02-1.24 (m, 2H, H-5 and H-4), 1.54-1.56 (m, 1H, H-5), 1.68 (s, 2H, H-4 and H-6), 2.28-2.30 (m, 1H, H-2), 2.46-2.47 (m, 1H, H-6), 2.58-2.60 (m, 1H, H-2), 3.02 (s, 1H, H-3), 3.44-3.55 (m, 2H, CH2), 7.28 (d, / 8.4 Hz, 2H, H-2' and H-6'), 7.52-7.60 (m, 3H, H-3'' and H-4'' and H-5''), 7.74 (d, / 8.4 Hz, 2H, H-3' and H-5'), 7.94-7.97 (m, 2H, H-2'' and H-6''), 10.26 (s, 1H, NH); 13C NMR (400 MHz, CDCl3) 5 23.4 (C-5), 27.6 (C-4), 52.9 (C-3), 54.7 (C-6), 62.1 (C-2), 76.5 (CH2), 120.2 (C-3' and C-5'), 127.1 (C-2'' and C-6''), 128.8 (C-2', C-6', C-3'' and C-5''), 129.8 (C-4''), 131.9 (C-1' and C-1''), 134.9 (C-4'), 165.8 (NHCO); IR (ATR) v 2950, 2916, 2868, 2837, 1655, 1601, 1523, 1458, 1410, 1376, 1319, 1258, 1167, 1098, 997, 973, 841, 809, 168, 694 cm-1; HRMS-ESI (m/z): [M+H] + calcd for C12H21N3O3S, 396.1377, found, 396.1382. 1-Benzylpiperidine-3-carbonitrile (4a) In a three-necked flask 1-benzyl piperidine-3-car-boxamide (2a, 100 mg, 0.458 mmol) was dissolved in pyr-idine (5 mL). Solution was cooled on ice and chlorocarbo-nylsulfenyl chloride (120 mg, 0.916 mmol) was added dropwise under argon. The reaction mixture was stirred at 80 °C overnight, cooled to room temperature and after the addition of Et3N (0,32 mL) stirred for 15 minutes. The precipitate was filtered off and the residue concentrated in vacuo. Product was purified with column chromatography using DCM: MeOH = 9:1 as an eluent to give a pure brownish solid. Yield = 33 %; TLC (DCM:MeOH = 9:1), Rf = 0.95; 1H NMR (400 MHz, DMSO-d6) 5 1.54 (s, 1H, H-4), 1.67 (s, 3H, H-4 and H-5), 2.27-2.28 (m, 1H, H-6), 2.46-2.55 (m, 2H, H-2 and H-3), 2.59 (s, 1H, H-6), 3.01 (s, 1H, H-2), 3.50-3.57 (m, 2H, CH2), 7.26-7.36 (m, 5H, ArH); 13C NMR (400 MHz, DMSO-d6) 5 22.8 (C-5), 26.6 (C-4), 42.3 (C), 53.1 (C-6), 55.8 (C-2), 61.5 (CH2), 121.8 (CN), 126.9 (C-4'), 128.1, 128.2, 128.4, 128.6 (C-2',3',5',6'), 137.8 (C-1'); IR (aTR) v 2943, 2804, 2766, 2240, 1493, 1452, 1393, 1349, 1309, 1257, 1205, 1151, 1099, 1072, 1011, 985, 959, 911, 868, 773, 604, 565, 542, 510 cm-1; MS m/z (relative intensity): 201.1 (M+Na, 100); HRMS-ESI (m/z): [m+H]+ calcd for CH NO,S, 201.1397, found, 201.1392. 14 16 2 2 1-(4-Nitrobenzyl)piperidine-3-carbonitrile (4b) In a three-necked flask 1-(4-nitrobenzyl)piperi-dine-3-carboxamide (2b, 100 mg, 0.380 mmol) was dissolved in pyridine (10 mL). The solution was cooled on ice and chlorocarbonylsulfenyl chloride (100 mg, 0.760 mmol) was added under argon. The reaction mixture was stirred at 80 °C overnight, cooled to room temperature and after the addition of Et3N (0.32 mL) stirred for 15 minutes. The precipitate was filtered off and the residue concentrated in vacuo. Product was purified with column chroma-tography using DCM:MeOH = 9:1 as an eluent to give a brownish solid. Yield = 34 %; TLC (DCM:MeOH = 9:1) Rf = 0.96; 1H NMR (400 MHz, CDCl3) 5 1.64-1.65 (m, 1H, H-5), 1.79-1.85 (m, 3H, H-5 and H-4), 2.46 (s, 2H, H-6), 2.63 (s, 2H, H-2), 2.81-2.84 (m, 1H, H-3), 3.63 (dd, / 6.8, 14.4 Hz, 2H, CH2), 7.53 (d, 2H, H-2' and H-6'), 8.19 (dd, / 2.0, 4.8 Hz, 2H, H-3' and H-5'); 13C NMR (400 MHz, CDCl3) 5 23.4 (C-5), 27.4 (C-4), 27.9 (C-3), 53.3 (C-6), 55.1 ((C-2), 61.7 (CH2), 121.0 (CN), 123.7 (C-3' and C-5'), 129.2 (C-2' and C-6'), 145.8 (C-1'), 147.3 (C-4'); IR (ATR) v 2945, 2802, 2240, 1599, 1467, 1439, 1154, 1093, 1033, 1012, 990, 955, 801, 773, 696, 651, 547 cm-1; MS m/z (relative intensity): 246.1 (M+Na, 100); HRMS-ESI (m/z): [m+H]+ calcd for C14H15N3O4S, 246.1242, found, 246.1243. 4. Conclusion Based on the previously reported oxathiazol-2-one-bearing and nonpeptidic inhibitors of the chymotryp-sin-like (^5i) subunit of the immunoproteasome, we designed a novel series of piperidin-3-yl-oxathiazol-2-ones as potential covalent inhibitors of threonine proteases. Compounds were designed with a synthetically accessible piperidine central core derivatized with an oxathi-azol-2-one electrophilic moiety. In lieu of previously reported synthetic approaches, we identified a synthetic protocol that enables the cyclization of carboxamides incorporating a basic centre into oxathiazol-2-ones. This straightforward protocol using chlorocarbonylsulfenyl chloride as a reagent in dioxane afforded the desired products in moderate to good yields. Thus, a vast chemical space of 5-substituted oxathiazol-2-ones can be explored and various chemical libraries of inhibitors of threonine proteases can be compiled. Jukič et al.: Chlorocarbonylsulfenyl Chloride Cyclizations 780 Acta Chim. Slov. 2017, 64, 771-781 Abbreviations ATR IR attenuated total reflectance infrared spectroscopy BDMS bromodimethylsulfonium bromide CDCl3 deuterated chloroform DBU 1,8-diazabicyclo(5.4.0)undec-7-ene D CM dichloromethane DMSO dimethyl sulfoxide DMF dimethylformamide EtOAc ethyl acetate MTB Mycobacterium tuberculosis Py pyridine THF tetrahydrofuran TLC thin-layer chromatography Conflict of interest The authors declare they have no conflict of interest. 5. Acknowledgments The authors acknowledge the financial support from the Slovenian Research Agency (research core funding No. P1-0208). We thank Dr. Dušan Žigon (Mass Spectrometry Center, Jožef Stefan Institute, Ljubljana, Slovenia) for mass spectra. 6. References 1. M. Drag, G. S. Salvesen, Nat. Rev. Drug Discov. 2010, 9, 690701. DOI:10.1038/nrd3053 2. D. H. Lee, A. L. Goldberg, Trends Cell Biol. 1998, 8, 397-403. DOI:10.1016/S0962-8924(98)01346-4 3. C. H Arrowsmith, J. E. Audia, C. Austin, J. Baell, J. Bennett, J. Blagg, C. Bountra, P. E. Brennan, P. J. Brown, M. E. Bunnage, C. Buser-Doepner, R. M. Campbell, A. J. Carter, P. Cohen, R. A. Copeland, B. Cravatt, J. L. Dahlin, D. Dhanak, A. M. Edwards, M. Frederiksen, S. V. Frye, N. Gray, C. E. Grimshaw, D. Hepworth, T. Howe, K. V. M. Huber, J. Jin, S. Knapp, J. D. Kotz, R. G. Kruger, D. Lowe, M. M. Mader, B. Marsden, A. Mueller-Fahrnow, S. Müller, R. C. O'Hagan, J. P. Overington, D. R. Owen, S. H. Rosenberg, R. Ross, B. Roth, M. Schapira, S. L. Schreiber, B. Shoichet, M. Sundström, G. Superti-Furga, J. Taunton, L. Toledo-Sherman, C. Walpole, M. A. Walters, T. M. Willson, P. Workman, R. N. Young, W. J. Zuercher, Nat. Chem. Biol. 2015, 11, 536-541. DOI:10.1038/nchembio.1867 4. N. D. Rawlings, E. O'Brien, A. J. Barrett, Nucleic Acids Res. 2002, 30, 343-346. DOI:10.1093/nar/30.1.343 5. E. M. Huber, M. Groll, Angew. Chem. Int. Ed. 2012, 51, 87088720. DOI:10.1002/anie.201201616 6. L. R. Dick, P. E. Fleming, Drug Discov. Today 2010, 15.5, 243249. DOI:10.1016/j.drudis.2010.01.008 7. D. A. Ferrington, D. S. Gregerson, Prog. Mol. Biol. Transl. Sci. 2012, 109, 75-112. DOI:10.1016/B978-0-12-397863-9.00003-1 8. A. Schweitzer, A. Aufderheide, T. Rudack, F. Beck, G. Pfeifer, J. M. Plitzko, E. Sakata, K. Schulten, F. Förster, W. Baumeister, Proc. Natl. Acad. Sci. 2016, 113, 7816-21. DOI:10.1073/pnas.1608050113 9. R. Raynes, L. C. D. Pomatto, K. Davies, J. A. Kelvin, Mol. Aspects Med. 2016, 50, 41-55. DOI:10.1016/j.mam.2016.05.001 10. M. Schmidt, D. Finley, Biochim. Biophys. Acta - Molecular Cell Research 2014, 1843, 13-25. 11. A. F. Kisselev, W. A. Van Der Linden, H. S. Overkleeft, Chem. Biol. 2012, 19, 99-115. DOI:10.1016/j.chembiol.2012.01.003 12. M. Groll, Y. Koguchi, R. Huber, J. Kohno, J. Mol. Biol. 2001, 311, 543-548. DOI:10.1006/jmbi.2001.4869 13. E. M. Huber, M. Basler, R. Schwab, W. Heinemeyer, C. J. Kirk, M. Groettrup, M. Groll, Cell 2012, 148, 727-738. DOI:10.1016/j.cell.2011.12.030 14. M. Groll, K. B. Kim, N. Kairies, R. Huber, C. M. Crews, J. Am. Chem. Soc. 2000, 122, 1237-1238. DOI:10.1021/ja993588m 15. H. W. B. Johnson, J. L. Anderi, E. K. Bradley, J. Bui, J. Jones, S. Arastu-Kapur, L. M. Kelly, E. Lowe, D. C. Moebius, T. Mu-chamuel, C. Kirk, Z. Wang, D. McMinn, Med. Chem. Lett. 2017, 8, 413-417. DOI:10.1021/acsmedchemlett.6b00496 16. I. Sosič, M. Gobec, B. Brus, D. Knez, M. Živec, J. Konc, S. Lešnik, M. Ogizek, A. Obreza, D. Žigon, D. Janežic, I. Mlinar-ič-Raščan, S. Gobec, Angew. Chem. Int. Ed. 2016, 55, 57455748. DOI:10.1002/anie.201600190 17. C. Dubiella, R. Baur, H. Cui, E. M. Huber, M. Groll, Angew. Chem. Int. Ed. 2015, 54, 15888-15891. DOI:10.1002/anie.201506631 18. G. Lin, D. Li, L. P. S. de Carvalho, H. Deng, H. Tao, G. Vogt, K. Wu, J. Schneider, T. Chidawanyika, J. D. Warren, H. Li, C. Nathan, Nature 2009, 461, 621-626. DOI:10.1038/nature08357 19. J. Schrader, F. Henneberg, R. A. Mata, K. Tittmann, T. R. Schneider, H. Stark, G. Bourenkov, A. Chari, Science 2016, 353, 594-598. DOI: 10.1126/science.aaf8993 20. J. Singh, R. C. Petter, T. A. Bailie, A. Whitty, Nat. Rev. Drug Discov. 2011, 10, 307-317. DOI:10.1038/nrd3410 21. H. Fan, N. G. Angelo, J. D. Warren, C. F. Nathan, G. Lin, Med. Chem. Lett. 2014, 5, 405-410. DOI:10.1021/ml400531d 22. B. E. Gryder, W. Guerrant, C. H. Chen, A. K. Oyelere, Med. Chem. Comm. 2011, 2, 1083-1086. DOI:10.1039/c1md00208b 23. C. Kuo, J. Zhu, J. Wu, C. Chu, C. Yao, K. Shia, Chem. Comm. 2007, 3, 301-303. DOI:10.1039/B614061K 24. A. S. Gurjar, V. Andrisano, A. D. Simone, V. S. Velingkar, Bi-oorg. Chem. 2014, 57, 90-98. DOI:10.1016/j.bioorg.2014.09.002 25. B. Rickborn, F. R. Jensen, J. Org. Chem. 1962, 27, 4608-4610. DOI:10.1021/jo01059a114 26. K. Mai, G. Patil, Tetrahedron Lett. 1986, 27, 2203-2206. DOI:10.1016/S0040-4039(00)84487-1 27. D. A. Claremon, B. T. Phillips, Tetrahedron Lett. 1988, 29, 2155-2158. DOI:10.1016/S0040-4039(00)86697-6 Jukič et al.: Chlorocarbonylsulfenyl Chloride Cyclizations Acta Chim. Slov. 2017, 64, 771- 781 781 28. T. M. Bargar, C. M. Riley, Syn. Comm. 1980, 10, 479-487. DOI: 10.1080/00397918008064272 29. L. D. S. Yadav, V. P. Srivastava, R. Patel., Tetrahedron Lett. 2009, 50, 5532-5535. D0I:10.1016/j.tetlet.2009.07.100 30. S. I. Maffioli, E. Marzorati, A. Marazzi, Org. Lett. 2005, 7, 5237-5239. D0I:10.1021/ol052100l 31. N. Nakajima, M. Ubukata, Tetrahedron Lett. 1997, 38, 20992102. D0I:10.1016/S0040-4039(97)00316-X 32. W. E. Dennis, J. Org. Chem. 1970, 35, 3253-3255. D0I:10.1021/jo00835a016 33. S. Zhou, K. Junge, D. Addis, S. Das, M. Beller, Org. Lett. 2009, 11, 2461-2464. D0I:10.1021/ol900716q 34. A. Aitha, S. Yennam, M. Behera, J. S. Anireddy, Tetrahedron Lett. 2016, 57, 1507-1510. D01:10.1016/j.tetlet.2016.02.082 35. P. C. Unangst, G. P. Shrum, D. T. Connor, J. Heterocycl. Chem. 1993, 30, 357-359. D0I:10.1002/jhet.5570300211 36. J. Crosby, M. C. McKie, M. Paton, J. F. Ross, Arkivoc 2000, 1, 720-734. 37. R. M. Paton, Chem. Soc. Rev. 1989, 18, 33-52. D0I:10.1039/cs9891800033 38. R. K. Howe, T. A. Gruner, L. G. Carter, L. L. Black, J. E. Franz. J. Org. Chem. 1978, 43, 3736-3742. D0I:10.1021/jo00413a024 39. P. Ohrngren, A. Fardost, F. Russo, J. S. Schanche, M. Fagrell, M. Larhed, Org. Process Res. Dev. 2012, 16, 1053-1063. Povzetek Z zamenjavo molekulskega skeleta smo načrtovali spojine s piperidinskim jedrom, derivatiziranim z oksatiazol-2-on-skim elektrofilnim centrom, ki omogoča selektivno zaviranje treoninskih proteaz. Sinteza produktov po postopkih, opisanih v literaturi, ni bila uspešna, poleg tega smo identificirali nitrile kot glavne stranske produkte, ki nastanejo pri dehidraciji karboksamidne funkcionalne skupine. S sistematično optimizacijo reakcijskih pogojev, smo s segrevanjem karboksamidov, klorokarbonilsulfenil klorida in natrijevega karbonata kot baze v dioksanu pri 100 °C pripravili serijo piperidin-3-il-oksatiazol-2-onov, primerno za nadaljnje biološko vrednotenje. Jukič et al.: Chlorocarbonylsulfenyl Chloride Cyclizations 782 DOI: 10.17344/acsi.2017.3421 Acta Chim. Slov. 2017, 64, 782-789 ^creative ty com mor Short communication Synthesis of Novel 5-(N-Boc-N-Benzyl-2-aminoethyl)-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-3-carboxamides and Their Inhibition of Cathepsins B and K Branislav Lukic, Uroš Grošelj, Marko Novinec* and Jurij Svete* Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, SI - 1000 Ljubljana, Slovenia. * Corresponding author: E-mail: marko.novinec@fkkt.uni-lj.si, jurij.svete@fkkt.uni-lj.si Received: 10-04-2017 Dedicated to Professor Emeritus Miha Tišler, University of Ljubljana, on the occasion of his 90th birthday. Abstract Eight novel 5-(N-Boc-N-benzyl-2-aminoethyl)-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-3-carboxamides were prepared in three steps from methyl 3-amino-1ff-pyrazole-4-carboxylate and methyl 5-(benzyl(ferf-butoxycarbonyl)ami-no)-3-oxopentanoate. The synthetic procedure comprises cyclocondensation of the above starting compounds, hydrolysis of the ester, and bis(pentafluorophenyl) carbonate (BPC)-mediated amidation. Title carboxamides were tested for inhibition of cathepsins K and B. The N-butylcarboxamide 5a exhibited appreciable inhibition of cathepsin K (IC ~ 25 |M), while the strongest inhibition of cathepsin B was achieved with N-(2-picolyl)carboxamide 5c (IC ~ 45 |M). Keywords: Pyrazolo[1,5-a]pyrimidines, cathepsin inhibition, cyclization, synthesis 1. Introduction Various 5-6 annulated heterocycles are important scaffolds for the preparation of compound libraries for medicinal and pharmaceutical applications.1,2 Due to biological activity of many of its derivatives, pyrazolo[1,5-a] pyrimidine is an important heterocycle among 5-6-fused systems.3,4 The importance of pyrazolo[1,5-a]pyrimidine is reflected in the results of a literature search5 showing around 150,000 known pyrazolo[1,5-a]pyrimidine derivatives within 6,500 references and with preparation, biological study, and uses as the predominant substance roles. For 2016 alone, 74 references can be found for a term "pyrazolo[1,5-fl]pyrimidines". Among bioactive pyra-zolo[1,5-fl]pyrimidines there are hepatitis C virus inhibitors,6 antagonists of serotonin 5-HT6 receptors,7 kinase inhibitors,8-10 PET tumor imaging agents,11 and inhibitors of amyloid ^-peptide aggregation.12 Sedative agents zale-plon and indiplon and the anxiolytic agent ocinaplon are approved drugs containing a pyrazolo[1,5-a]pyrimidine core (Figure 1). Cathepsin K, a cysteine protease that is selectively and abundantly expressed within osteoclasts, is believed to Ocinaplon Indiplon Zaleplon Figure 1. Approved drugs based on a pyrazolo[1,5-a]pyrimidine scaffold. be crucial for the resorption of bone matrix.13-17 The ability to degrade type I collagen allows cathepsin K to make a unique contribution to the balance between bone resorption and bone formation.18,19 Inhibitors of cathepsin K could prevent bone resorption and may provide a promising approach for the treatment of osteoporosis, therefore inhibition of cathepsin K has been proposed as a promising strategy for the treatment of osteoporosis, cancer, and other diseases.13-15 Several inhibitors have progressed into Lukic et al.: Synthesis of novel 5-(N-Boc-N-benzyl-2-aminoethyl)-7-oxo- Acta Chim. Slov. 2017, 64, 782-783 789 clinical trials but there are, as yet, no inhibitors on the market.20 Pyrazolo[1,5-fl]pyrimidines are commonly available by cyclo condensation of a 3-aminopyrazole derivative with a 1,3-dicarbonyl compound or its synthetic equivalent.3,21 Due to this ease of access, a plethora of known pyrazolo[1,5-fl]pyrimidine derivatives is not surprising. Nevertheless, a more detailed literature search also reveals that 5-(2-aminoethyl) substituted pyrazolo[1,5-a]pyrimi-dines are much less known - 135 examples can be found by SciFinder", however, without any literature reference available. Furthermore, the 5-(2-aminoethyl)pyrazolo Scheme 1. Synthesis of title carboxamides 5a-h. Lukic et al.: Synthesis of novel 5-(N-Boc-N-benzyl-2-aminoethyl)-7-oxo- ... 784 Acta Chim. Slov. 2017, 64, 782-789 [1,5-fl]pyrimidine-3-carboxamides are, to the best of our knowledge, unknown. Recently, a substantial part of our studies were focused on the synthesis of novel pyra-zolo[1,5-fl]pyridine and pyrazolo[1,5-c]pyridine derivatives. In this connection, we reported (parallel) syntheses of libraries of novel 7-heteroarylpyrazolo[1,5-a]pyridine-3-carb oxamides,22 7-oxopyrazolo[1,5-a]pyrimidine-3-car-b oxamides,23 7- (1 - aminoethyl) pyr azolo [ 1,2- a]pyrimi-dines,24 and tetrahydropyrazolo[1,5-c]pyrimidine-3-car-boxamides.25 In extension, we explored another synthetic approach based on direct cyclisation of methyl 5-ami-no-1H-pyrazole-4-carboxylate (1) with methyl 5-[ben-zyl(tert-butoxycarbonyl)amino]-3-oxopentanoate (2) to obtain a 5-(2-aminoethyl)pyrazolo[1,5-a]pyrimidine central building block for a late-stage derivatization at the car-boxy function. Herein we report the results, the synthesis of 5-(N-Boc-N-benzyl-2-aminoethyl)-7-oxo-4,7-dihy-dropyrazolo[1,5-fl]pyrimidin-3-carboxamides 5a-h and their evaluation for inhibition of cathepsins B and K. 2. Results and Discussion The starting ^-keto ester, methyl 5-[benzyl(tert-bu-toxycarbonyl)amino]-3-oxopentanoate (2) was prepared in four steps from benzylamine (6b) and methyl acrylate following the literature procedures.23,26 Subsequent cyclisa-tion of 2 with methyl 5-amino-1H-pyrazole-4-carboxylate (1)27 was performed in acetic acid at 80 °C for 24 h to afford methyl 5-(N-Boc-N-benzyl-2-aminoethyl)-7-oxo-4,7-dihydropyrazolo[1,5-fl]pyrimidin-3-carboxylate (3) in 95% yield. Notably, heating at temperatures above 80 °C shortened the reaction times at the expense of the product yield due to partial acidolytic removal of the Boc group and concomitant formation of undesired by-products. Somewhat expectedly,23,25 attempted hydrolysis of the ester function with aq. NaOH failed. Fortunately enough, hydrolysis of 3 into the desired carboxylic acid 4 could be performed upon prolonged treatment of the ester 3 with excess LiOH in aq. methanol to furnish the central intermediate 4 in 54% yield. For the final amidation step 1,1'-carbonyldiimidazole (CDI), 2-ethoxy-1-ethoxycar- bonyl-1,2-dihydroquinoline (EEDQ), and bis(pen-tafluorophenyl) carbonate (BPC) were tested as the reagents for the activation of the carboxy group of 4. As we already experienced previously in amidation of related hetarenecarboxylic acids,22-26 BPC proved to be the most suitable reagent, because it gave the corresponding car-boxamides 5 reproducibly and in good yields. Thus, upon activation of 4 with BPC to form the intermediate pen-tafluorophenyl ester 4', further treatment with 1:1 mixtures of amines and triethylamine for 12 h furnished the target carboxamides 5a-h in 55-87% yields upon chroma-tographic workup (Scheme 1). The structures of novel compounds 3, 4, and 5a-h were determined by spectroscopic methods (1H NMR, 13C NMR, IR, MS, HRMS). Spectral data for compounds 3, 4, and 5a-h were in agreement with the data of closely related pyrazolo[1,5-fl]pyrimidin-7(4H)-ones. 1>3>4'21-23 Some physicochemical properties were calculated to estimate the drug-likeness of compounds 3, 4, and 5a-h. The compounds have molecular weight (MW) between 412 and 503, number of atoms between 54 and 72, clogP between 1.3 and 3.6, number of hydrogen bond donors (HBD) < 2, number of hydrogen bond acceptors (HBA) < 5, and polar surface area (PSA) below 116 A2. These calculated physicochemical properties are compliant with Li-pinski's rule of five28-30 indicating promising drug-likeness of the synthesized compounds 3, 4, and 5a-h (Table 1). The biological activity of compounds 3, 4, and 5a-h was tested against the cysteine peptidases cathepsins B and K, which are both important drug targets.31 All compounds were initially tested for their activity at a concentration of 100 ^M. As shown in Table 2, compound 5a had the strongest inhibitory effect on cathepsin K, with an IC50 value of 25 ± 5 ^M under the experimental conditions used in the assay and complete (100%) inhibition was observed at concentrations of 600 ^M or higher. The effect of other compounds was significantly weaker and resulted in less than 50% inhibition. Cathepsin B was most strongly inhibited by compound 5c (IC50 value of 45 ± 15 ^M) and to a lesser extent by compounds 5a and 5d. Altogether these results identify three compounds, 5a, 5c and 5d, as potential lead compounds for further development (Table 2). Table 1. Calculated physicochemical properties of compounds 3, 4, and 5a-h. Compd. MW (g mol1) No. of atoms ClogP No. of HBD No. of HBA PSA (A2) 3 426.47 57 2.62 1 4 100.5 4 412.45 54 2.41 2 4 111.5 5a 467.57 67 3.19 2 4 103.3 5b 501.59 68 3.57 2 4 103.3 5c 502.57 67 2.07 2 5 115.7 5d 469.54 65 1.81 2 5 112.6 5e 496.6 72 2.29 2 5 106.6 5f 479.58 68 2.34 1 4 94.6 5g 481.55 66 1.31 1 5 103.8 5h 494.60 70 1.87 1 5 97.8 Lukic et al.: Synthesis of novel 5-(N-Boc-N-benzyl-2-aminoethyl)-7-oxo- Acta Chim. Slov. 2017, 64, 782-785 789 Table 2: Effect of compounds 3, 4 and 5a-h on the activity of cathepsins K and B.a Compound Cathepsin K RA (%)b IC50 (pM) RA (%) Cathepsin B b 1C50 (MM) control 100 100 3 89 89 4 84 84 5a 29 25 ± 5 36 110 ± 30 5bc - - 5c 94 20 45 ± 15 5d 95 23 150 ± 50 5e 69 104 5f 60 - 5g 74 61 5h 112 101 a) All experiments were performed in 50 mM sodium acetate buffer pH 5.5 containing 1 mM EDTA, 2.5 mM DTT and the fluorigenic substrate Z-Phe-Arg-AMC (5 |iM final concentration). Final enzyme concentrations were 1 nM. IC 50 values were determined from titration curves. b) Residual activity at saturation. c) Activity of 5b could not be determined fluorometrically due to strong absorption of the compound at the excitation wavelength. 3. Experimental 3. 1. General Methods Melting points were determined on a Stanford Research Systems MPA100 OptiMelt automated melting point system. The NMR spectra were obtained on a Bruker Avance III UltraShield 500 plus at 500 MHz for 1H and 126 MHz for 13C, using CDCl3 and DMSO-d6 (with TMS as the internal standard) as solvents. Mass spectra were recorded on an Agilent 6224 Accurate Mass TOF LC/MS spectrometer, IR spectra on a Bruker FTIR Alpha Platinum ATR spectrophotometer. Flash column chromatography (FC) was performed on silica gel (Fluka, Silica gel 60, particle size 35-70 pm). Amines 6a-h, bis(pentafluorophenyl) carbonate (BPC), triethylamine, and LiOH ■ H2O are commercially available. Methyl 5-amino-1H-pyrazole-4-carboxylate (1)27 and methyl 5-(benzyl(tert-butoxycarbonyl)amino)-3-oxopentanoate (2)26 were prepared following the literature procedures. 3. 2. Synthesis of methyl 5-(N-Boc-N-benzyl- 2-aminoethyl)-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-3-carboxylate (3) A mixture of 1 (1.413 g, 10 mmol), 2 (3.694 g, 10 mmol), and AcOH (20 mL) was stirred at 80 °C for 24 h. Volatile components were evaporated in vacuo and the residue was purified by FC (EtOAc). Fractions containing the product were combined and evaporated in vacuo to give 3. Yield: 4.059 g (95%) of pale beige solid; m.p. 161165 °C. 1H NMR (500 MHz, CDCl3): 5 1.30 (9H, s, t-Bu); 2.95 (2H, t, J = 10.0 Hz, CH2); 3.54 (2H, t, J = 10.0 Hz, CH2); 3.86 (3H, s, OMe); 4.45 (2H, s, CH2Ph); 5.72 (1H, s, 6-H); 7.29 (5H, m, Ph); 8.15 (1H, s, 2-H); 11.45 (1H, s, NH). 13C NMR (126 MHz, CDCl3): 8 27.6, 44.8, 48.3, 51.3, 59.7, 78.7, 96.5, 99.4, 127.1, 127.4, 128.3, 138.3, 143.0, 143.3, 154.4, 155.1, 162.0, 170.3. m/z (ESI) = 427 (MH+). HRMS-ESI (m/z): [MH+] calcd for C22H27N4O5, 427.1976; found, 427.1971. Anal. Calcd for CTL N„C> : C 61.96, H 22 26 4 5 6.15, N 13.14. Found: C 61.90, H 6.29, N 13.17. IR (ATR) v 3344, 2963, 1710, 1671, 1620, 1580, 1529, 1495, 1466, 1442, 1414, 1365, 1323, 1303, 1259, 1247, 1185, 1167, 1145, 1124, 1115, 1051, 1019, 963, 933, 887, 847, 791, 776, 729, 695, 683, 657, 632 cm-1. 3. 3. Synthesis of 5-(N-Boc-N-benzyl-2- aminoethyl)-7-oxo-4,7-dihydropyrazolo [1,5-a]pyrimidin-3-carboxylic acid (4) A mixture of the ester 3 (3.408 g, 8 mmol), LiOH ■ H2O (2.016 g, 48 mmol), and methanol (30 mL) was stirred at 50 °C for 48 h. The reaction mixture was cooled to room temperature, and acidified to pH ~ 4 by careful addition of 1 M aq. NaHSO4. The precipitate was collected by filtration and washed with cold (0 °C) water (5 mL) to give 4. Yield: 2.215 g (54%) of white solid; m.p. 166-172 °C. 1H NMR (500 MHz, CDCl3): 8 1.21 (9H, s, f-Bu); 2.90 (2H, t, J = 10.0 Hz, CH2); 3.36 (2H, t, J = 10.0 Hz, CH2); 4.45 (2H, s, CH2Ph); 5.68 (1H, s, 6-H); 7.29 (5H, m, PI2); 8.26 (1H, s, 2-H); 12.78 (1H, s, NH), CO2H exchanged. 13C NMR (126 MHz, CDCl3): 8 27.5, 31.3, 44.9, 48.2, 78.7, 97.5, 98.7, 127.0, 127.4, 128.4, 138.3, 143.2, 144.2, 153.7, 154.8, 155.4, 163.3. m/z (ESI) = 413 (MH+). HRMS-ESI (m/z): [MH+] calcd for C21H25N4O5, 413.1806; found, 413.1812. Anal. Calcd for C21H24N4O^H2O: C 58.60, H 6.09, N 13.02. Found: C 58.50, H 5.74, N 12.89. IR (ATR) v 3648, 3368, 2977, 1682, 1635, 1575, 1495, 1464, 1446, 1404, 1366, 1345, 1302, 1281, 1252, 1218, 1200, 1160, 1131, 1073, 1047, 1015, 963, 940, 858, 841, 812, 780, 758, 725, 695, 669, 653 cm-1. Lukic et al.: Synthesis of novel 5-(N-Boc-N-benzyl-2-aminoethyl)-7-oxo- ... 786 Acta Chim. Slov. 2017, 64, 782-789 3. 4. Synthesis of 5-(N-Boc-N-benzyl-2- aminoethyl)-7-oxo-4,7-dihydropyrazolo [1,5-a]pyrimidin-3-carboxamides 5a-h A mixture of carboxylic acid 4 (207 mg, 0.5 mmol), MeCN (5 mL), and Et3N (70 ^L, 0.5 mmol) was stirred at room temperature for 5 minutes. Then, BPC (197 mg, 0.5 mmol) was added and the reaction mixture was stirred at r.t. for 2 h (activation of carboxylic acid 4 via formation of the pentafluorophenyl ester 4'). Next, amine 6 (0.5 mmol) and Et3N (70 ^L, 0.5 mmol) were added and stirring at room temperature was continued for 24 h. The reaction mixture was evaporated in vacuo (60 °C/2 mbar) and the crude semi-solid carboxamide 5 was purified by FC on silica gel (first EtOAc to elute the non-polar impurities, then CH2Cl2-MeOH, 10:1, to elute the product). Fractions containing the product were combined and evaporated in vacuo to give carboxamides 5a-h. 3. 4. 1. tert-Butyl benzyl{2-[3-(butylcarbamoyl)-7-oxo-4,7-dihydropyrazolo[1,5-«]pyrimidin-5-yl]ethyl}carbamate (5a) Prepared from 4 (207 mg, 0.5 mmol) and butylamine (6a) (50 ^L, 0.5 mmol). Yield: 167 mg (72%) of yellowish resin. 1H NMR (500 MHz, CDCl3): 5 0.85 (3H, t, J = 7.0 Hz, CH2CH3); 1.27 (2H, m, CH2); 1.34 (9H, s, f-Bu); 1.42 (2H, m, CH2); 2.29 (2H, m, CH2); 3.23 (2H, m, CH2); 3.44 (2H, m, CH2); 4.38 (2H, s, CH2Ph); 5.41 (1H, s, 6-H); 7.28 (5H, m, Ph); 7.90 (1H, s, 2-H); 8.50 (1H, br s, NHBu); py-rimidone NH exchanged. 13C NMR (126 MHz, CDCl3): 5 13.0, 13.7, 19.7, 28.2, 31.8, 38.5, 45.8, 51.0, 80.0, 101.1, 126.0, 127.2, 127.5, 127.9, 128.4, 128.7, 138.1, 155.7, 156.0, 159.0, 164.0. m/z (ESI) = 468 (MH+). HRMS-ESI (m/z): [MH+] calcd for C25H34N5O5, 468.2605; found, 468.2601. IR (ATR) v 3300, 2930, 2175,2110, 1985, 1960, 1684, 1619, 1537, 1512, 1494, 1451, 1413, 1364, 1245, 1157, 1115, 1047, 980, 885, 808, 775, 733, 697 cm-1. 3. 4. 2. tert-Butyl benzyl{2-[3-(benzylcarbamoyl)-7-oxo-4,7-dihydropyrazolo[1,5-«] pyrimidin-5-yl]ethyl}carbamate (5b) Prepared from 4 (207 mg, 0.5 mmol) and benzyla-mine (6b) (54 ^L, 0.5 mmol). Yield: 137 mg (55%) of yellowish resin. 1H NMR (500 MHz, CDCl3): 5 1.30 (9H, br s, f-Bu); 2.56 (2H, br s, CH2); 3.35 (2H, br s, CH2); 4.29 and 4.43 (4H, 2 br s, 3:1, 2 >2 CH2Ph); 5.57 (1H, br s, 6-H); 6.84-7.34 (10H, m, 2xPh); 8.10 (1H, br s, 2-H); 8.76 (1H, br s, NH); pyrimidone NH exchanged. 13C NMR (126 MHz, CDCl3): 5 28.1, 28.3, 36.5, 42.8, 46.1, 51.3, 51.9, 81.0, 100.7, 125.1, 127.3, 127.4, 128.5, 128.6, 136.7, 138.2, 138.9, 140.6, 142.7, 156.0, 159.6, 163.9. m/z (ESI) = 502 (MH+). HRMS-ESI (m/z): [MH+] calcd for C28H32N5O4, 502.2449; found, 502.2444. IR (ATR) v 3278, 2975, 2114, 1618, 1535, 1494, 1451, 1413, 1364, 1244, 1207, 1156, 1115, 976, 884, 809, 774, 728, 696, 665, 630 cm-1. 3. 4. 3. tert-Butyl benzyl(2-{7-oxo-3-[(pyridin-2-ylmethyl)carbamoyl]-4,7-dihydro-pyrazolo[l,5-«]pyrimidin-5-yl}ethyl) carbamate (5c) Prepared from 4 (207 mg, 0.5 mmol) and 2-pico-lylamine (6c) (51 ^L, 0.5 mmol). Yield: 190 mg (72%) of yellowish resin. 1H NMR (500 MHz, DMSO-d6): 5 1.27 and 1.32 (9H, 2 br s, 2:1, f-Bu); 2.67-2.77 (2H, br s, CH2); 3.42-3.50 (2H, br s, CH2); 4.33 and 4.37 (2H, 2 br s, 1:2, CH2Ph); 4.60 (2H, d, J = 5.7 Hz, CH2Py); 5.48 and 5.50 (1H, 2 br s, 2:1, 6-H); 7.20-7.30 (5H, m, Ph); 7.30-7.37 (2H, m, 2H of Ph); 7.71 (1H, td, J = 7.7, 1.8 Hz, 1H of Py); 8.09 (1H, br s, 2-H); 8.47 (1H, br d, J = 4.2 Hz, 1H of Py); 9.16 (1H, br t, J = 6.0 Hz, NHCO); pyrimidone NH exchanged. 13C NMR (126 MHz, DMSO-d6): 5 27.8, 43.8, 45.6, 45.8, 49.0, 78.8, 100.2, 120.9, 122.0, 127.0, 127.2, 127.4, 128.4, 136.7, 138.1, 138.5, 140.0, 141.5, 148.8, 155.0, 157.2, 159.0, 162.9. m/z (ESI) = 503 (MH+). HRMS-ESI (m/z): [MH+] calcd for C27H31N6O4, 503.2397; found, 503.2394. IR (atr) v 3679, 36077, 2926, 1730, 1624, 1537, 1497, 1393, 1368 cm-1. 3. 4. 4. tert-Butyl benzyl(2-{3-[(2-methoxyethyl) carbamoyl]-7-oxo-4,7-dihydropyrazolo-[1,5-«]pyrimidin-5-yl}ethyl)carbamate (5d) Prepared from 4 (207 mg, 0.5 mmol) and 2-methox-yethylamine (6d) (63 ^L, 0.5 mmol). Yield: 143 mg (61%) of yellowish resin. 1H NMR (500 MHz, CDCl3): 5 1.45 (9H, s, f-Bu); 2.72-2.83 (2H, br s, CH2); 3.40 (3H, br s, OMe); 3.513.59 (4H, m, 2xCH2); 3.59-3.64 (2H, m, CH2); 4.44 (2H, br s, CH2Ph); 5.69 (1H, s, 6-H); 7.14-7.29 (6H, m, Ph and NHCO); 8.03 (1H, br s, 2-H); pyrimidone NH exchanged. 13C NMR (126 MHz, CDCl3): 5 28.4, 39.2, 46.2, 51.8, 59.0, 71.1, 81.3, 99.2, 126.0, 127.8, 128.8, 132.2, 137.6, 139.0, 143.5, 151.0, 154.1, 155.8, 156.3, 163.1. m/z (ESI) = 470 (MH+). HRMS-ESI (m/z): [MH+] calcd for C24H32N5O5, 470.2398; found, 470.2393. IR (ATR) v 3313, 2978, 2916, 1685, 1624, 1585, 1532, 1513, 1479, 1453, 1414, 1365, 1244, 1156, 1122, 1051, 1012, 993, 976, 858, 819, 774, 733, 698, 660 cm-1. 3. 4. 5. tert-Butyl benzyl[2-(3-{[3-(dimethylamino) propyl]carbamoyl}-7-oxo-4,7-dihydro-pyrazolo[1,5-«]pyrimidin-5-yl)ethyl] carbamate (5e) Prepared from 4 (207 mg, 0.5 mmol) and 3-dimeth-ylaminopropylamine (6e) (63 ^L, 0.5 mmol). Yield: 200 mg (81%) of yellowish resin. 1H NMR (500 MHz, CDCl3): 5 1.40 (9H, s, f-Bu); 1.96-2.05 (2H, m, CH2); 2.71 (6H, br s, NMe2); 2.67-2.81 (2H, m, CH2); 3.03-3.12 (2H, m, CH2); 3.43-3.51 and 3.55-3.63 (4H, 2m, 3:1, 2 x CH2); 4.37 (2H, br s, CH2Ph); 5.70 (1H, s, 6-H); 7.16-7.34 (5H, m, Ph); 8.14 (1H, br s, 2-H); 8.65 (1H, br s, NHCO); pyrimidone NH exchanged. 13C NMR (126 MHz, CDCl3): 5 25.9, 28.4, 28.5, 35.9, 43.4, 43.5, 45.7, 56.2, 79.7, 95.4, Lukic et al.: Synthesis of novel 5-(N-Boc-N-benzyl-2-aminoethyl)-7-oxo- Acta Chim. Slov. 2017, 64, 782-787 789 101.1, 127.3, 127.7, 128.6, 137.3, 138.1, 139.1, 141.0, 156.0, 159.3, 165.0. m/z (ESI) = 497 (MH+). HRMS-ESI (m/z): [MH+] calcd for C H„N O , 497.2857; found, 497.2863. L J 26 3/ 6 4 IR (ATR) v 3285, 2937, 1995, 1690, 1619, 1537, 1493, 1450, 1411, 1364, 1243, 1158, 1112, 1020, 886, 806, 776, 735, 698, 665, 631 cm-1. 3. 4. 6. tert-Butyl benzyl{2-[7-oxo-3-(piperidine-1-carbonyl)-4,7-dihydropyrazolo[1,5-«] pyrimidin-5-yl]ethyl}carbamate (5f) Prepared from 4 (207 mg, 0.5 mmol) and piperidine (6f) (37 ^L, 0.5 mmol). Yield: 128 mg (61%) of yellowish resin. 1H NMR (500 MHz, CDCl3): 5 1.46 (9H, s, f-Bu); 1.68 (4H, br s, 2 x CH2); 1.74 (2H, br s, CH2); 2.76 (2H, br s, CH2); 3.53 (2H, br s, CH2); 3.73 (4H, br s,2 x CH2); 4.41 (2H, br s, CH2Ph); 5.69 (1H, s, 6-H); 7.13-7.34 (5H, m, Ph); 7.96 (1H, br s, 2-H); pyrimidone NH exchanged. 13C NMR (126 MHz, CDCl3): 5 24.6, 26.2, 28.5, 33.0, 46.0, 50.9, 52.1, 80.9, 99.1, 127.4, 127.7, 128.8, 137.8, 141.1, 141.1, 145.1, 150.7, 155.6, 156.3, 162.8. m/z (ESI) = 480 (MH+). HRMS-ESI (m/z): [MH+] calcd for C26H34N5O4, 480.2605; found, 480.2599. IR (ATR) v 2931, 28849, 1687, 1617, 1578, 1578, 1495, 1438, 1410, 1364, 1258, 1159, 1122, 1002, 970, 875, 851, 814, 764, 731, 698, 672, 629 cm-1. 3. 4. 7. tert-Butyl benzyl{2-[3-(morpholine-4-carbonyl)-7-oxo-4,7-dihydropyrazolo [1,5-«]pyrimidin-5-yl]ethyl}carbamate (5g) Prepared from 4 (207 mg, 0.5 mmol) and morpholine (6g) (44 ^L, 0.5 mmol). Yield: 184 mg (76%) of yellowish resin. 1H NMR (500 MHz, CDCl3): 8 1.46 (9H, s, f-Bu); 2.76 (2H, br s, CH2); 3.50-3.59 (2H, m, CH2); 3.79 (4H, br s, 2 x CH2); 3.81 (4H, br s, 2 x CH2); 4.44 (2H, br s, CH2Ph); 5.71 (1H, s, 6-H); 7.16-7.35 (5H, m, Ph); 7.97 (1H, br s, 2-H); pyrimidone NH exchanged. 13C NMR (126 MHz, CDCl3): 8 28.5, 33.3, 45.9, 51.7, 60.6, 66.8, 81.4, 99.3, 127.8, 128.8,133.6, 137.7, 140.9, 143.7, 145.3, 151.1, 155.7, 156.2, 163.2. m/z (ESI) = 482 (MH+). HRMS-ESI (m/z): [MH+] calcd for C25H32N5O5, 482.2398; found, 482.2393. IR (ATr) v 2974, 2922, 2843, 1685, 1619, 1580, 1532, 1513, 1453, 1434, 1412, 1365, 1245, 1157, 1114, 1065, 1051, 1010, 978, 935, 884, 817, 765, 733, 699, 630 cm-1. 3. 4. 8. tert-Butyl benzyl{2-[3-(4-methylpiperazine -1-carbonyl)-7-oxo-4,7-dihydro-pyrazolo [1,5-«]pyrimidin-5-yl]ethyl}carbamate (5h) Prepared from 4 (207 mg, 0.5 mmol) and 4-meth-ylpiperazine (6h) (56 ^L, 0.5 mmol). Yield: 215 mg (87%) of yellowish resin. 1H NMR (500 MHz, CDCl3): 5 1.45 (9H, s, f-Bu); 2.40 (3H, br s, NCH3); 2.59 (4H, br t, J = 5.1 Hz, 2 x CH2); 2.70 and 2.76 (2H, 2br s, 1:1, CH2); 3.54 (2H, br s, CH2); 3.85 (4H, br s, 2 x CH2); 4.42 (2H, br s, CH2Ph); 5.70 (1H, s, 6-H); 7.16-7.31 (5H, m, Ph); 7.96 (1H, br s, 2-H); pyrimidone NH exchanged. 13C NMR (126 MHz, CDCl3): 5 28.5, 33.2, 43.8, 45.8, 46.1, 52.7, 54.8, 80.9, 98.9, 127.7, 128.8, 136.6, 137.2, 137.8, 138.9, 140.6, 141.3, 155.7, 156.6, 163.3. m/z (ESI) = 495 (MH+). HRMS-ESI (m/z): [MH+] calcd for C26H35N6O, 495.2714; found, 495.2707. IR (ATR) v 2977, 2958, 1685, 1621, 1583, 1531, 1495, 1414, 1364, 1243, 1155, 976, 879, 807, 767, 731, 698, 606 cm-1. 3. 5. Activity assays against cathepsins K and B The activity of all compounds was tested against recombinant human cathepsins K and B produced in-house according to the known protocol.32 All assays were performed in 50 mM sodium acetate buffer pH 5.5 containing 1 ^M EDTA and 2.5 mM DTT. The hydrolysis of the synthetic substrate Z-Phe-Arg-AMC (5 ^M final concentration) was followed fluorimetrically at an excitation wavelength of 370 nm and an emission wavelength of 455 nm. Final concentrations of the enzymes in the reaction mixtures were 1 nM. Experiments were first performed at a fixed compound concentration of 100 ^M. Compounds with significant inhibitory activity were re-tested by measuring residual enzyme activity in the presence of increasing concentrations of the compounds and IC50 values were calculated from these titration curves. 4. Conclusions Eight novel 5-(N-Boc-N-benzyl-2-aminoethyl)-7-oxo-4,7-dihydropyrazolo[1,5-fl]pyrimidin-3-carboxam-ides 5a-h were prepared in three synthetic steps from methyl 3-amino-1H-pyrazole-4-carboxylate (1) and methyl 5-(benzyl(ferf-butoxycarbonyl)amino)-3-oxopentanoate (2). The synthetic procedure comprises cyclocondensation of the above starting compounds, hydrolysis of the ester function, and BPC-mediated amidation. This method offers a quick access to various 5-(2-aminoethyl) substituted pyrazolo[1,5-fl]pyrimidin-3-carboxamides 5 from easily available starting materials. Testing of the intermediates 3 and 4 and title compounds 5a-h for inhibition of cathepsins B and K revealed that most of them were weak inhibitors at 100 mM concentration. Carboxamide 5a had the strongest inhibitory effect on cathepsin K, with an IC50 value of 25 ± 5 ^M. Cathepsin B was most strongly inhibited by compounds 5c and 5d with the respective IC50 values of 45 ± 15 ^M and 150 ± 50 ^M and to a lesser extent by compound 5a as well. Inhibitory activities of compounds 5a, 5c, and 5d against cysteine peptidases cathep-sins B and K identify them as potential leads for drug development. In summary, the synthetic method allows for a simple preparation of libraries of title compounds that could be useful for medicinal and pharmaceutical applications. Lukic et al.: Synthesis of novel 5-(N-Boc-N-benzyl-2-aminoethyl)-7-oxo- ... 788 Acta Chim. Slov. 2017, 64, 782-789 5. Acknowledgement The authors acknowledge the financial support from the Slovenian Research Agency (research core funding No. P1-0179 and P1-0140). We thank to EN-FIST Centre of Excellence, Ljubljana, Slovenia, for using FTIR spectro-photometer. 6. References 1. J. A. Joule, K. Mills, in: Heterocyclic Chemistry, 5th ed., Wiley-Blackwell, 2010. 2. G. L. Patrick, in: An Introduction to Medicinal Chemistry, 5th ed., Oxford University Press, Oxford, UK, 2013. 3. A. C. Regan, in: A. R. Katritzky, C. A. Ramsden, E. F. V. Scriv-en, R. J. K. Taylor (Ed.): Pyrazolo[1,5-c]pyrimidine (73) in Comprehensive heterocyclic chemistry III, Vol. 11, J. Cossy (Ed.), Elsevier Science Ltd., Oxford, 2008, pp. 577-577; and references cited therein. 4. M. H. Elnagdi, M. R. H. Elmoghayar, G. E. H. Elgemeie, Adv. Heterocycl. Chem. 1987, 41, 319-376. DOI:10.1016/S0065-2725(08)60164-6 5. SciFinder* Scholar substructure search performed on Febru-rary 25, 2017. 6. J. Y. Hwang, M. P. Windisch, S. Jo, H. C. Kim, S. Kim, H. Kim, M. E. Lee, D.-S. Park, E. Park, S. Ahn, J. Cechetto, J. Kim, M. Liuzzi, Z. No, J. Lee, Bioorg. Med. Chem. Lett. 2012, 22, 7297-7301. DOI:10.1016/j.bmcl.2012.10.123 7. A. V. Ivashchenko, E. S. Golovina, M. G. Kadieva, V. M. Kysil, O. D. Mitkin, I. M. Okun, Pharm. Chem. J. 2012, 46, 406-410. DOI:10.1007/s11094-012-0810-4 8. E. J. Hanan, A. van Abbema, K. Barrett, W. S. Blair, J. Blaney, C. Chang, C. Eigenbrot, S. Flynn, P. Gibbons, C. A. Hurley, J. R. Kenny, J. Kulagowski, L. Lee, S. R. Magnuson, C. Morris, J. Murray, R. M. Pastor, T. Rawson, M. Siu, M. Ultsch, A. Zhou, D. Sampath, J. P. Lyssikatos, J. Med. Chem. 2012, 55, 10090-10107. DOI:10.1021/jm3012239 9. T. Asano, H. Yamazaki, C. Kasahara, H. Kubota, T. Kontani, Y. Harayama, K. Ohno, H. Mizuhara, M. Yokomoto, K. Misumi, T. Kinoshita, M. Ohta, M. Takeuchi, J. Med. Chem. 2012, 55, 7772-7785. DOI:10.1021/jm3008008 10. T. Kosugi, D. R. Mitchell, A. Fujino, M. Imai, M. Kambe, S. Kobayashi, H. Makino, Y. Matsueda, Y. Oue, K. Komatsu, K. Imaizumi, Y. Sakai, S. Sugiura, O. Takenouchi, G. Unoki, Y. Yamakoshi, V. Cunliffe, J. Frearson, R. Gordon, C. J. Harris, H. Kalloo-Hosein, J. Le, G. Patel, D. J. Simpson, B. Sherborne, P. S. Thomas, N. Suzuki, M. Takimoto-Kamimura, J. Med. Chem. 2012, 55, 6700-6715. DOI:10.1021/jm300411k 11. J. Xu, H. Liu, G. Li, Y. He, R. Ding, X. Wang, M. Feng, S. Zhang, Y. Chen, S. Li, M. Zhao, Y. Li, C. Qi, Z. Naturforsch. 2012, 67B, 827-834. DOI:10.5560/ZNB.2012-004 12. L. C. Lopez, S. Dos-Reis, A. Espargaro, J. A. Carrodeguas, M.-L. Maddelein, S. Ventura, J. Sancho, J. Med. Chem. 2012, 55, 9521-9530. DOI:10.1021/jm301186p 13. T. Inaoka, G. Bilbe, O. Ishibashi, K. Tezuka, M. Kumegawa, T. Kokubo, Biochem. Biophys. Res. Commun. 1995, 206, 89-96. DOI:10.1006/bbrc.1995.1013 14. A. G. Dossetter, H. Beeley, J. Bowyer, C. R. Cook, J. J. Crawford, J. E. Finlayson, N. M. Heron, C. Heyes, A. J. Highton, J. A. Hudson, A. Jestel, P. W. Kenny, S. Krapp, S. Martin, P. A. MacFaul, T. M. McGuire, P. M. Gutierrez, A. D. Morley, J. J. Morris, K. M. Page, L. R. Ribeiro, H. Sawney, S. Steinbacher, C. Smith, M. Vickers, J. Med. Chem. 2012, 55, 6363-6374. DOI:10.1021/jm3007257 15. J. J. Crawford, P. W. Kenny, J. Bowyer, C. R. Cook, J. E. Finlay-son, C. Heyes, A. J. Highton, J. A. Hudson, A. Jestel, S. Krapp, S. Martin, P. A. Macfaul, B. P. McDermott, T. M. McGuire, A. D. Morley, J. J. Morris, K. M. Page, L. R. Ribeiro, H. Sawney, S. Steinbacher, C. Smith, A. G. Dossetter, J. Med. Chem. 2012, 55, 8827-8837. DOI:10.1021/jm301119s 16. J. Borišek, M. Vizovišek, P. Sosnowski, B. Turk, D. Turk, B. Mohar, M. Novič, J. Med. Chem. 2015, 58, 6928-6937. DOI: 10.1021/acs.jmedchem.5b00746 17. F. Lecaille, D. Bromme, G. Lalmanach, Biochimie 2008, 90, 208-226. DOI:10.1016/j.biochi.2007.08.011 18. P. Garnero, O. Borel, I. Byrjalsen, M. Ferreras, F. H. Drake, M. S. McQueney, N. T. Foged, P. D. Delmas, J. M. Delaisse, J. Biol. Chem. 1998, 273, 32347-32352. DOI:10.1074/jbc.273.48.32347 19. W. Kafienah, D. Bromme, D. J. Buttle, L. J. Croucher, A. P. Hollander, Biochem. J. 1998, 331, 727-732. DOI:10.1042/bj3310727 20. Y. Wang, R. Li, Z. Zheng, H. Yia, Z. Li. RSC Adv. 2016, 6, 82961-82968. DOI:10.1039/C6RA14251F 21. G. Hajos, Z. Riedl, in: R. Neier (Ed.): Aza analogues of pyra-zolo[1,5-a]pyridines containing additional nitrogen atoms in the sixmembered ring in Science of synthesis, Houben-Weyl: methods of molecular transformations, Vol. 12, Thieme, Stuttgart, 2006, pp. 667-678. 22. S. Ahmetaj, N. Velikanje, U. Grošelj, I. Šterbal, B. Prek, A. Golobič, D. Kočar, G. Dahmann, B. Stanovnik, J. Svete, Mol. Divers. 2013, 17, 731-743. DOI:10.1007/s11030-013-9469-3 23. M. Drev, U. Grošelj, Š. Mevec, E. Pušavec, J. Štrekelj, A. Golobič, G. Dahmann, B. Stanovnik, J. Svete Tetrahedron 2014, 70, 8267-8279. DOI:10.1016/J.tet.2014.09.020 24. L. Šenica, N. Petek, U. Grošelj, J. Svete, Acta Chim. Slov. 2015, 62, 60-71. DOI:10.17344/acsi.2014.712 25. K. Lombar, U. Grošelj, G. Dahmann, B. Stanovnik, J. Svete, Synthesis 2015, 47, 497-506. DOI:10.17344/acsi.2014.712 26. D. Zerovnik, U. Grošelj, D. Kralj, Č. Malavašič, J. Bezenšek, G. Dahmann, K. Stare, A. Meden, B. Stanovnik, J. Svete, Synthesis, 2010, 3363-3373. DOI:10.1055/s-0030-1257864 27. T. J. Nitz, K. Salzwedel, C. Finnegan, C. Wild, S. Brunton, S. Flanagan, C. Montalbetti, T. S. Coulter, M. Kimber, F. Maga-raci, D. Johnston, WO 2008134035 A1 20081106, date of patent November 8, 2008; Chem. Abstr. 2008, 149, 534234. 28. C. A. Lipinski, F. Lombardo, B. W. Dominy, P. J. Feeney, Adv. Drug. Del. Rev. 2001, 46, 3-26. DOI:10.1016/S0169-409X(00)00129-0 Lukic et al.: Synthesis of novel 5-(N-Boc-N-benzyl-2-aminoethyl)-7-oxo- Acta Chim. Slov. 2017, 64, 782-789 789 29. A. K. Ghose, V N. Viswandhan, J. J. Wendoloski, J. Comb. Chem. 1999, 1, 55-68. DOI:10.1021/cc9800071 31. M. Novinec, B. Lenarčič, BioMol. Concepts 2013, 4, 287-308. DOI:10.1515/bmc-2012-0054 32. M. Novinec, M. Pavšič, B. Lenarčič, Prot. Expr. Purif. 2012, 82, 1-5. DOI: 10.1016/j.pep.2011.11.002 30. A. Nadin, C. Hattotuwagama, I. Churcher, Angew. Chem. Int. Ed. 2012, 51, 1114-1122. DOI:10.1002/anie.201105840 Povzetek Izhajajoč iz metil 3-amino-1ff-pirazol-4-karboksilata in metil 5-(benzil(terc-butoksikarbonil)amino)-3-oksopentanoa-ta (2) smo v treh sinteznih stopnjah pripravili osem novih 5-(N-Boc-N-benzil-2-aminoetil)-7-okso-4,7-dihidropira-zolo[1,5-a]pirimidin-3-karboksamidov 5a-h. Sintezni postopek sestavljajo ciklokondenzacija izhodnih spojin, hidroliza estra in amidiranje tako nastale karboksilne kisline z uporabo bis(pentafluorofenil) karbonata (BPC) kot aktivacijskega reagenta. Karboksamide 5a-h smo testirali na inhibicijo katepsinov B in K. Najbolj aktiven inhibitor katepsina K (IC50 ~ 25 |M) je bil N-butilkarboksamid 5a, medtem ko smo najmočnejšo inhibicijo katepsina B izmerili z N-(2-pikolil) karboksamidom 5c (IC50 ~ 45 |M). Lukic et al.: Synthesis of novel 5-(N-Boc-N-benzyl-2-aminoethyl)-7-oxo- ... 790 DOI: 10.17344/acsi.2017.3474 Acta Chim. Slov. 2017, 64, 790-797 'commons Short communication Synthesis and Reduction of 10-Phthalimidocamphor Oxime Uroš Grošelj, Amalija Golobič, Jurij Svete and Sebastijan Ričko* Department of Organic Chemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Vecna pot 113, SI - 1000 Ljubljana, Slovenia * Corresponding author: E-mail: sebastijan.ricko@fkkt.uni-lj.si Tel.: +386 1 479 8606 Received: 24-04-2017 Dedicated to Professor Emeritus Miha Tisler, University of Ljubljana, on the occasion of his 90th anniversary. 10-Phthalimidocamphor oxime was prepared from easily available 10-iodocamphor in two steps. Reduction of the oxime functionality resulted in the formation of two novel polycyclic isoindolinone heterocycles, the attempted preparation of the primary amine failed. The structures of novel heterocycles were unambiguously confirmed by single crystal X-ray diffraction as well as NMR techniques. Keywords: 10-iodocamphor, 10-phthalimidocamphor oxime, camphor derived amines, reduction Abstract 1. Introduction Instead of the desired diamine 5, isoindolinone heterocycles 6 and 7 were isolated. Isoindolinone/isoindole derivatives can be found in numerous natural and pharmaceutical compounds shoving multiple biological activities (Figure 1).25 (1R)-(+)-Camphor and its enantiomer are renewable enantiomerically pure chiral pool starting materials. The unique reactivity of camphor enables its derivatization at positions 2, 3, 4, 5, 8-10, as well as selective cleavage of the C1-C2 and C2-C3 bonds (Figure 1).1,2 All of the above makes camphor a very desirable starting compound for the preparation of a wide variety of products3 ranging from natural products1,2 to chiral auxiliaries,4,5 ligands in asymmetric synthesis,6-10 organocatalysts,11 and NMR shift reagents.12 12 Within our continuing study on camphor-based diamines as potential organo catalyst scaffolds,13-15 we recently reported on the synthesis of a novel type of 1,3-di-amine-derived bifunctional squaramide organocatalysts A prepared from 10-iodocamphor and their application as highly efficient catalysts in Michael additions of 1,3-di-carbonyl nucleophiles to trans-^-nitrostyrenes.16 10-Io-docamphor17 has seen surprisingly limited application as the starting compound,18-24 although, it can easily be prepared in sufficient quantities from (1S)-(+)-10-camphor-sulfonic acid.16 Herein we report the results of the synthesis and reduction of 10-phthalimidocamphor oxime (4), which is a potential precursor for the preparation of mono-protected primary diamine camphor building block 5. 13-15 (1fl)-(+)-camphor RHN A \J o O 5 Grošelj et al.: Synthesis and reduction of 10-phthalimidocamphor Acta Chim. Slov. 2017, 64, 790-791 797 2. Results and Discussion Following the literature procedure, (1 S)-(+)-10-cam-phorsulfonic acid (1) was transformed into 10-iodocam-phor (2).16 The following reaction of 2 with potassium phthalimide gave the corresponding 10-phthalimidocam-phor (3) in 72% yield. Finally, condensation of 3 with NH2OH furnished in 92% yield the expected 10-phthalim-idocamphor oxime (4). Next, reduction of the oxime 4 was studied with the aim of preparing mono-protected primary diamine camphor building block 5 (Scheme 1). Thus, the results of the reduction of oxime 4 are summarized in Scheme 2 and Table 1. Catalytic hydrogena- tion of 4 using Pd-C in MeOH with or without HCl yielded only the recovered starting material (Entries 1 and 2). On the other hand, reduction of 4 with Na in n-PrOH, as expected, gave a complex mixture of products (Entry 3). Catalytic hydrogenation using Raney-Ni gave the polycy-clic secondary amine 6 in 37% isolated yield (Entry 4). Clearly, the reduction of oxime 4 was successful, though the reaction did not stop at the desired diamine level 5. Therefore, the reduction with Raney-Ni was repeated in the presence of AcOH (Entry 5) and aqueous formaldehyde (Entry 6) in order to obtain either the amine 5 or a tertiary dimethylamine derivative. The former reaction again delivered compound 6 in 20% yield, while the later Scheme 1. Attempted synthesis of monoprotected diamine 5. Scheme 2. Synthesis of amine 6 and imine 7 from oxime 4. Table 1. Reduction of oxime 4 under various reaction conditions. Entry Reducing agent Solvent T (°C) t (h) Product/Yield (%) 1 Pd-C MeOH r.t. 8 no reaction 2 Pd-C/HCl MeOH r.t. 8 no reaction 3 Na n-PrOH 90 2 complex mixture 4 Raney-Ni MeOH r.t. 8 6 (37) 5 Raney-Ni/AcOH MeOH r.t. 8 6 (20) 6 Raney-Ni/HCHO MeOH r.t. 8 complex mixture 7 Zn/HCl MeOH r.t. a) 7 (45) 8 Zn AcOH r.t. a) complex mixture 9 Zn/HCl AcOH r.t. a) complex mixture a) Till the disappearance of the starting material (TLC analysis). Groselj et al.: Synthesis and reduction of 10-phthalimidocamphor ... 792 Acta Chim. Slov. 2017, 64, 790-797 yielded a complex mixture of products. Next, reduction of oxime 4 with Zn in MeOH in the presence of excess aqueous HCl was performed, furnishing imine 7 in 45% yield (Entry 7). Repeating the reduction of 4 with Zn in AcOH with or without aqueous HCl yielded complex mixtures of products (Entries 8 and 9). The formation of the products 6 and 7 could be rationalized by the initial formation of the primary amine 5, followed by the condensation with the proximal carbonyl group of the phthalimide functionality to give intermediate 8. Isomerization of 8 to imine 7 is explained by a simple imine-imine tautomerisation, while reduction (or iso-merization/reduction) of 8 would lead to amine 6 (Scheme 3). The configuration of the newly formed stereogenic centers seems to be dictated by the reducing agent applied. 2. 1. Crystal Structures of Compounds 6 and 7 The asymmetric units of compounds 6 and 7 are depicted in Figures 2 and 3, respectively. In both structures there is one molecule in the asymmetric unit. Bond lengths are given in Table 2. Most of bond lengths are very similar both in 6 and 7, with the exception of bonds including atoms N2 and C9. This is in accordance with their structural chemical formulas (as shown in Scheme 2) which differ only in the closeness of these two atoms. Bond N2-C9 in 6, 1.463(3) Â, is significantly longer than 1.265(2)  in 7, which is in accordance with the fact that this is a single bond in 6 and a double bond in 7. The average C(sp3)-N(3) single bond and C(sp2) = N(2) double bond in the literature1 are 1.469(14) and 1.279(8) Â, respectively. Usually C(sp3)-C(sp3) bond distances are longer in comparison to C(sp3)-C(sp2). In accordance to this, C9-C10 and C9-C15 are longer in 6 than in 7. Scheme 3. Rationalization of the formation of products 6 and 7. Compound 6 was tested as a potential covalent or-ganocatalyst in the addition of 1-methylindole to cinnamal-dehyde.26 Amine 6 failed to catalyze the reaction (Scheme 4). The structures of novel compounds 3, 4, 6, and 7 were determined by spectroscopic methods (1H-NMR, 13C-NMR, IR, HRMS). Molecules of 6 and 7 are asymmetric. In both structures, chiral carbon centres are C8, C10, and C14; in 6 C9 atom is also chiral. C10 and C14 from camphor part of the molecule have in both compounds absolute configuration (S) and (R), respectively. The absolute configuration of C8 atom from phthalimde ring is (R) in 6 and (S) in 7, respec- Grošelj et al.: Synthesis and reduction of 10-phthalimidocamphor Acta Chim. Slov. 2017, 64, 790-793 797 tively. Consequently, the conformation of molecules of 6 and 7 is different in a way how a camphor part is bonded to the remaining part of molecule which is shown in Figure 4. In accordance with their optical activity, both compounds crystalize in chiral space group. Compound 6 crystalizes in orthorhombic crystal system in P212121 and 7 in tetragonal P43212, respectively. The packing of molecules is presented in Figures 5 and 6. In 6 molecules are connected via N2-H---O1 hydrogen bonds into chains parallel to b axis. Geometrical parameters of this H-bond are given in Table 3. The distance between the donor, N2, Table 2. Bond lengths in 6 and 7 (Á). bond 6 7 O1-C1 1.230(2) 1.222(1) N1-C1 1.347(3) 1.356(2) N1-C8 1.466(3) 1.460(2) N1-C11 1.453(3) 1.451(2) N2-C8 1.438(2) 1.464(2) N2-C9 1.463(3) 1.265(2) C1-C2 1.490(3) 1.493(2) C2-C7 1.381(3) 1.379(2) C2-C3 1.377(3) 1.384(2) C3-C4 1.375(4) 1.387(2) C4-C5 1.373(5) 1.385(2) C5-C6 1.389(4) 1.381(2) C6-C7 1.387(3) 1.383(2) C7-C8 1.504(3) 1.502(2) C9-C10 1.565(3) 1.519(2) C9-C15 1.546(3) 1.522(2) C10-C11 1.521(3) 1.519(1) C10-C12 1.551(3) 1.550(2) C10-C16 1.557(3) 1.553(2) C12-C13 1.540(3) 1.556(2) C13-C14 1.531(3) 1.523(2) C14-C15 1.532(3) 1.536(2) C14-C16 1.551(3) 1.554(2) C16-C17 1.538(3) 1.531(2) C16-C18 1.526(3) 1.528(2) Figure 2. Ortep28 drawing of asymmetric unit of compound 6. Displacement ellipsoids are drawn with 25% probability level and the hydrogen atoms are shown as small spheres of arbitrary radii. and acceptor, O1, is not short, which means that H-bond is weak. In 7 there are no N-H or O-H groups and consequently no classical intermolecular H-bonds. N and O atoms are acceptors of weak intermolecular H-bonds, donated by C-H moieties and presented in Table 3. In 6 and 7 Figure 3. Ortep28 drawing of asymmetric unit of compound 7. Displacement ellipsoids are drawn with 25% probability level and the hydrogen atoms are shown as small spheres of arbitrary radii. Groselj et al.: Synthesis and reduction of 10-phthalimidocamphor ... 794 Acta Chim. Slov. 2017, 64, 790-797 Figure 4. Mercury29 drawing of compounds 6 (on left) and 7 (on right) with labelling of chiral carbon centers of the phthalimide part of the molecule. Figure 5. Mercury29 drawing of molecular packing in 6. Light blue lines show intermolecular N-H.. .O hydrogen bonds. there are no or stacking interaction between aromatic rings. 3. Conclusion The title 10-phthalimidocamphor oxime (4) was prepared as a precursor for the preparation of monoprotected camphor derived 1,3-diamine building block 5. Reduction thereof under various reaction conditions could never be stopped at the diamine 5 level, instead polycyclic isoin-dolinone heterocycles 6 and 7 were isolated. The structures of 6 and 7 were confirmed by X-ray analysis of the corresponding monocrystals. 4. Experimental Section Solvents for extractions and chromatography were of technical grade and were distilled prior to use. Extracts were dried over technical grade Na2SO4. Melting points were determined on a Kofler micro hot stage and on SRS OptiMelt MPA100 - Automated Melting Point System (Stanford Research Systems, Sunnyvale, California, United States). The NMR spectra were obtained on a Bruker Ul-traShield 500 plus (Bruker, Billerica, Massachusetts, United States) at 500 MHz for 1H and 126 MHz for 13C nucleus, using DMSO-d6 and CDCl3 with TMS as the internal standard, as solvents. Mass spectra were recorded on an Agilent 6224 Accurate Mass TOF LC/MS (Agilent Technologies, Santa Clara, California, United States), IR spectra on a Perkin-Elmer Spectrum BX FTIR spectrophotometer (PerkinElmer, Waltham, Massachusetts, United States). Catalytic hydrogenation was performed on a Parr Pressure Reaction Hydrogenation Apparatus (Moline, IL, USA). Figure 6. Mercury29 drawing of molecular packing in 7. Light blue lines show weak intermolecular C-H.O and C-H.N hydrogen bonds. Table 3. Hydrogen-bond geometry in 6 and 7 (Á, °). D-H-A D-H H-A D-A D-H-A N2-H2'—O1i 0.91(2) 2.25(2) 3.127(2) 161(2) C8-H-O1ii 0.98 2.57 3.509(2) 162 C15-H-N2iii 0.97 2.69 3.654(2) 173 Symmetry codes: (i) -x, y+1/2, -z+1/2, (ii) 1/2+x,3/2-y,1/4-z, (iii) y,x,-z. Column chromatography (CC) was performed on silica gel (Silica gel 60, particle size: 0.035-0.070 mm (Sigma-Al-drich, St. Louis, Missouri, United States)). Synthesis of 2-(((1fl,4fl)-7,7-dimethyl-2-oxobicyclo[2.2.1] heptan-1-yl)methyl)isoindoline-1,3-dione (3). To a suspension of 10-iodocamphor (2) (420 mg, 1.51 mmol) in anhydrous DMSO (10 mL) under argon Grošelj et al.: Synthesis and reduction of 10-phthalimidocamphor Acta Chim. Slov. 2017, 64, 790-795 797 potassium phthalimide (524 mg, 2.83 mmol) was added and the resulting reaction mixture was heated at 100 °C under argon for 16 h. Volatile components were evaporated in vacuo. The residue was suspended in H2O (20 mL) and extracted with EtOAc (3 x 40 mL). The combined organic phase was washed with H2O (20 mL) and NaCl (aq. sat., 20 mL), dried over anhydrous Na2SO4, filtered, and volatile components evaporated in vacuo. The residue was purified by column chromatography (EtOAc:petroleum ether = 1:2). Fractions containing the pure product 3 were combined and volatile components evaporated in vacuo. Yield: 320 mg (1.076 mmol, 72%) of white solid; mp 123129 °C. [a]Drt = -2.4 (c = 0.25, CH2Cl2). EI-HRMS: m/z = 298.1437 (MH+); C18H20NO3 requires: m/z = 298.1438 (MH+); v 3469, 3189, 30(56, 2959, 2888, 1773, 1731, v ' max 1712, 1604, 1466, 1426, 1398, 1373, 1361, 1309, 1295, 1191, 1157, 1142, 1106, 1089, 1053, 1031, 1008, 935, 872, 763, 712, 642, 625 cm-1. 1H-NMR (500 MHz, CDCl3): 8 0.95 (5, 3H, Me); 1.10 (5, 3H, Me); 1.30 - 1.37 (m, 1H); 1.52 - 1.60 (m, 1H); 1.88 (d, J = 18.3 Hz, 1H); 1.84-1.99 (m, 2H); 2.01 (t, J = 4.5 Hz, 1H); 2.43 (ddd, J = 2.5; 4.9; 18.4 Hz, 1H); 3.77 (d, J = 14.9 Hz, 1H); 4.07 (d, J = 14.9 Hz, 1H); 7.72 (dd, J = 3.0; 5.5 Hz, 2H of Ar); 7.85 (dd, J = 3.1; 5.4 Hz, 2H of Ar). 13C-NMR (126 MHz, CDCl3): 8 19.5, 19.7, 26.7, 26.7, 34.7, 43.3, 43.5, 47.2, 61.1, 1233.4, 132.2, 134.1, 168.9, 216.7. Synthesis of 2-(((1.R,4.R)-2-(hydroxyimino)-7,7-dimet-hylbicydo[2.2.1]heptan-1-yl)methyl)isoindoline-1,3-di-one (4). To a solution of ketone 3 (2.76 g, 9.28 mmol) in EtOH (45 mL) NH2OH-HCl (1.30 g, 18.7 mmol) and pyridine (1.10 g, 13.9 mmol) were added and the resulting reaction mixture was heated under reflux for 16 h. Volatile components were evaporated in vacuo, followed by the addition of H2O (25 mL) and finely powdered NaOH till the pH ~ 10-12. The resulting mixture was extracted with Et2O (5 x 40 mL). The combined organic phase was washed with H2O (5 mL) and NaCl (aq. sat., 5 mL), dried over anhydrous Na2SO4, filtered, and volatile components evaporated in vacuo. The residue was purified by column chro-matography (EtOAc:petroleum ether = 1:2). Fractions containing the pure product 4 were combined and volatile components evaporated in vacuo. Yield: 2.67 g (8.54 mmol, 92%) of white solid; mp 151-155 °C. [a]Drt = -50.6 (c = 0.33, CH2Cl2). EI-HRMS: m/z = 313.1547 (MH+); C18H-21N2O3 requires: m/z = 313.1547 (MH+); vmax 3469, 32380, 2945, 2881, 1774, 1713, 1612, 1467, 1427, 1395, 1387, 1362, 1338, 1312, 1297, 1245, 1197, 1158, 1104, 1028, 1015, 987, 962, 927, 913, 875, 855, 821, 800, 717, 611 cm-1. 1H-NMR (500 MHz, CDCl3): 8 0.90 (5, 3H, Me); 1.07 (5, 3H, Me); 1.20-1.28 (m, 1H); 1.58-1.65 (m, 1H); 1.76-1.85 (m, 2h); 1.95- 2.03 (m, 1H); 2.08 (d, J = 17.8 Hz, 1H); 2.59 (dt, J = 3.8; 17.9 Hz, 1H); 3.88 (d, J = 14.7 Hz, 1H); 4.10 (d, J = 14.8 Hz, 1H); 7.63 (br 5, 1H); 7.72 (dd, J = 3.0; 5.5 Hz, 2H of Ar); 7.85 (dd, J = 3.1; 5.4 Hz, 2H of Ar). 13C-NMR (126 MHz, CDCl3): 8 19.2, 19.3, 27.0, 29.5, 32.9, 35.8, 44.6, 48.7, 55.5, 123.4, 132.2, 134.1, 168.5, 169.1. Synthesis of (4b.,5a.,7.,9aS)-13,13-dimethyl-5,5a,6, 7,8,9-hexahydro-10H-7,9a-methanoisoindolo[1,2-fo] quinazolin-12(4b.H)-one (6). A mixture of compound 4 (246 g, 0.788 mmol), MeOH (50 mL), and Raney-Ni (100 mg) was hydrogenat-ed (4 bar of H2) at room temperature for 8 h. The reaction mixture was filtered through a short pad of Celite®, washed with MeOH (20 mL), and the filtrate evaporated in vacuo. The residue was purified by column chromatography (1. n-hexane:Et2O = 1:3 to elute the nonpolar impurities; 2. Et3N:Et2O = 1:40 to elute the product 6). Fractions containing the pure product 6 were combined and volatile components evaporated in vacuo. Yield: 83 mg (0.294 mmol, 37%) of white solid; mp 154-158 °C. [a]Drt = -163.0 (c = 0.40, CH2Cl2). EI-HRMS: m/z = 283.1801 (MH+); C18H23N2O requires: m/z = 283.1805 (MH+); vmax 3326, 29141, 2881, 1672, 1485, 1460, 1431, 1388, 1368, 1356, 1331, 1300, 1276, 1263, 1243, 1192, 1153, 1130, 1112, 1087, 1053, 1013, 976, 948, 931, 898, 875, 846, 816, 793, 740, 708, 687, 675 cm-1. 1H-NMR (500 MHz, CDCl3): 8 0.87 (5, 3H, Me); 0.99 (5, 3H, Me); 1.07-1.19 (m, 2H); 1.19-1.24 (m, 1h); 1.51-1.58 (m, 1H); 1.59-1.67 (m, 1H); 1.72-1.81 (m, 2h); 1.95 (dd, J = 8.9; 13.5 Hz, 1h); 3.18 (d, J = 14.3 Hz, 1h); 3.25 (dd, J = 4.6; 8.9 Hz, 1H); 4.44 (d, J = 14.4 Hz, 1H); 5.12 (5, 1H); 7.47-7.60 (m, 3H, 3H of Ar); 7.81-7.86 (m, 1H, 1H of Ar). 13C-NMR (126 MHz, CDCl3): 8 21.2, 21.7, 26.9, 33.9, 37.9, 38.5, 44.1, 45.1, 46.4, 62.9, 70.6, 123.1, 123.8, 129.6, 131.7, 133.2, 142.8, 165.4. Synthesis of (4bS,7.,9aS)-13,13-dimethyl-6,7,8,9-tetra-hydro-10H-7,9a-methanoisoindolo[1,2-fo]qumazolin-12(4bH)-one (7). To a solution of 4 (113 mg, 0.362 mmol) in MeOH (10 mL) at room temperature HCl (aq. 12 M, 1 mL) was added. Next, at room temperature under vigorous stirring, Zn dust (100 mg, 1.53 mmol) was added. After the disappearance of the starting material (TLC analysis), the reaction mixture was filtered and the filtrate evaporated in vacuo. The residue was suspended in H2O (10 mL), finely powdered NaOH was added till the pH ~ 10-12 followed by extraction with Et2O (3 x 30 mL). The combined organic phase was washed with H2O (10 mL) and NaCl (aq. sat., 10 mL), dried over anhydrous Na2SO4, filtered, and volatile components evaporated in vacuo. The residue was purified by column chromatography (1. n-hexane:Et2O = 1:3 to elute the nonpolar impurities; 2. Et3N:Et2O = 1:25 to elute the product 7). Fractions containing the pure product 7 were combined and volatile components evaporated in vacuo. Yield: 46 mg (0.163 mmol, 45%) of white solid; mp 164-172 °C. [a]Drt = +102.5 (c = 0.33, CH2Cl2). EI-HRMS: m/z = 281.1646 (MH+); C18H21N2O requires: m/z = 281.1648 (MH+); v 2951, 2930, 2869,1677, 1615, 1468, 1447, 1412, max 1310, 1281, 1225, 1152, 1102, 1057, 1025, 975, 320, 795, Groselj et al.: Synthesis and reduction of 10-phthalimidocamphor ... 796 Acta Chim. Slov. 2017, 64, 790-797 747, 709, 691, 621 cm-1. 'H-NMR (500 MHz, CDCl3): 5 1.02 (5, 3H, Me); 1.04 (5, 3H, Me); 1.18-1.27 (m, 1H); 1.321.39 (m, 1H); 1.82-1.97 (m, 4H); 2.57-2.65 (m, 1H); 3.24 (d, J=13.3, 1H); 4.43 (d, J = 13.3 Hz, 1H); 5.83- 5.86 (m, 1H); 7.48-7.53 (m, 1H, 1H of Ar); 7.58-7.63 (m, 1H, 1H of Ar); 7.77-7.84 (m, 2H, 2H of Ar). 13C-NMR (126 MHz, CDCl3): 8 18.6, 20.0, 26.9, 30.0, 38.3, 39.6, 43.3, 47.1, 53.0, 73.8, 123.4, 123.5, 129.1, 131.5, 132.1, 143.4, 167.6, 180.4. 4. 1. Single Crystal X-ray Structure Analysis of Compounds 6 and 7 Single crystal X-ray diffraction data of compounds 6 and 7 have been collected on an Agilent SuperNova dual source diffractometer with an Atlas detector with CuKa radiation (1.54184 A) at room temperature. The diffraction data were processed using CrysAlis PRO software.30 Structure of both compounds was solved by direct methods, using SIR97.31 A full-matrix least-squares refinement on F2 was employed with anisotropic displacement parameters for all non-hydrogen atoms. H atoms were placed at calculated positions and treated as riding. For H atoms from methyl groups, torsion angles were calculated from electron density. Only H atom bonded to N2, was located from difference Fourier map and refined with isotropic displacement parameter. The absolute structure of both compounds was confirmed also by the refinement of Flack parameter. SHELXL97 software32 was used for structure refinement and interpretation. Drawings of the structures were produced using ORTEP-328 and Mercury29. Structural and other crystallographic details on data collection and refinement have been deposited with the Cambridge Crys-tallographic Data Centre as supplementary publication numbers CCDC 1539864-1539865, for 6 and 7, respectively. These data can be obtained free of charge via www.ccdc. cam.ac.uk/conts/retrieving.html (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; fax: 44 1223 336033; e-mail: deposit@ccdc.cam.ac.uk). 5. Acknowledgements The authors acknowledge the financial support from the Slovenian Research Agency(research core funding No. P1-0179). We also thank to EN-FIST Centre of Excellence (Ljubljana, Slovenia) for using the SuperNova diffractom-eter. 6. References 1. T. Money, Remote functionalization of camphor: application to natural product synthesis. Org. Synth.: Theory Appl. 1996, 3, 1-83. 2. T. Money, Nat. Prod. Rep. 1985, 2, 253-289. DOI: 10.1039/np9850200253 3. U. Grošelj, Targets Heterocycl. Syst. 2015, 19, 62-100. 4. W. Oppolzer, Tetrahedron 1987, 43, 1969-2004. DOI:10.1016/S0040-4020(01)86780-6 5. W. Oppolzer, Pure Appl. Chem. 1990, 62, 1241-1250. 6. S. A. Matlin, W. J. Lough, L. Chan, D. M. H. Abram, Z. Zhou, J. Chem. Soc., Chem. Commun. 1984, 1038-1040. D0I:10.1039/C39840001038 7. C. Chapuis, J. Jurczak, Helv. Chim. Acta 1987, 70, 436-440. DOI: 10.1002/hlca.19870700223 8. K. Tomioka, Synthesis 1990, 541-549. D0I:10.1055/s-1990-26935 9. R. Noyori, M. Kitamura, Angew. Chem. Int. Ed. Engl. 1991, 30, 49-69. D0I:10.1002/anie.199100491 10. H. C. Brown, P. V. Ramachandran, Pure Appl. Chem. 1991, 63, 307-16. DOI:10.1351/pac199163030307 11. U. Grošelj, Curr. Org. Chem. 2015, 19, 2048-2074. DOI: 10.2174/1385272819666150713180204 12. H. L. Goering, J. N. Eikenberry, G. S. Koermer, J. Chem. Soc. 1971, 93, 5913-5914. D0I:10.1021/ja00751a065 13. U. Grošelj, A. Golobič, K. Stare, J. Svete, B. Stanovnik, Chiral-ity 2012, 24, 307-317. D0I:10.1002/chir.21999 14. U. Grošelj, S. Ričko, J. Svete, B. Stanovnik, Chirality 2012, 24, 412-419. D0I:10.1002/chir.22035 15. S. Ričko, A. Golobič, J. Svete, B. Stanovnik, U. Grošelj, Chirality 2015, 27, 39-52. D0I:10.1002/chir.22386 16. S. Ričko, J. Svete, B. Štefane, A. Perdih, A. Golobič, A. Meden, U. Grošelj, Adv. Synth. Catal. 2016, 358, 3786-3796. D0I:10.1002/adsc.201600498 17. J. D. Loudon, J. Chem. Soc. 1933, 823-825. D0I:10.1039/jr9330000823 18. M. J. Spallek, G. Storch, O. Trapp, Eur. J. Org. Chem. 2012, 2012, 3929-3945. 19. K. Bica, G. Gmeiner, C. Reichel, B. Lendl, P. Gaertner, Synthesis 2007, 1333-1338. 20. S.-i. Watanabe, R. Hasebe, J. Ouchi, H. Nagasawa, T. Kataoka, Tetrahedron Lett. 2010, 51, 5778-5780. D0I:10.1016/j.tetlet.2010.08.082 21. J. A. Gladysz, J. L. Hornby, J. E. Garbe, J. Org. Chem. 1978, 43, 1204-1208. D0I:10.1021/jo00400a040 22. M. C. Galan, K. Jouvin, D. Alvarez-Dorta, Carbohydr. Res. 2010, 345, 45-49. D0I:10.1016/j.carres.2009.09.034 23. J. E. H. Buston, I. Coldham, K. R. Mulholland, J. Chem. Soc., Perkin Trans. 1 1999, 2327-2334. D0I:10.1039/a903050f 24. T. Sell, S. Laschat, I. Dix, P. G. Jones, Eur. J. Org. Chem. 2000, 4119-4124. D0I:10.1002/1099-0690(200012)2000:24<4119: :AID-EJ0C4119>3.0.C0;2-X 25. K. Speck, T. Magauer, Beilstein J. Org. Chem. 2013, 9, 20482078 and references cited therein. D0I:10.3762/bjoc.9.243 26. J. F. Austin, D. W. C. MacMillan, J. Am. Chem. Soc. 2002, 124, 1172-1173. D0I:10.1021/ja017255c 27. F. H. Allen, D. G. Watson, L. Brammer, A. G. Orpen, R. Taylor, International Tables for Crystallography (2006). Vol. C, chapter 9.5, 790-811. 28. L. J. Farrugia, J. Appl. Crystallogr. 1997, 30, 567-567. D01:10.1107/S0021889897003117 29. C. F. Macrae, P. R. Edgington, P. McCabe, E. Pidcock, G. P. Grošelj et al.: Synthesis and reduction of 10-phthalimidocamphor Acta Chim. Slov. 2017, 64, 790-797 797 Shields, R. Taylor, M. Towler, J. van de Streek, J. Appl. Cryst. 2006, 39, 453. DOI:10.1107/S002188980600731X 30. Agilent Technologies. CrysAlis PRO. Version 1.171.35.11; Agilent Technologies: Yarnton, Oxfordshire, England, (2011). 31. A. Altomare, M. C. Burla, M. Camalli, G. L. Cascarano, C. Giacovazzo, A. Guagliardi, A. G. G. Moliterni, G. Polidori, R. Spagna, J. Appl. Crystallogr. 1999, 32, 115-119. D01:10.1107/S0021889898007717 32. G. M. Sheldrick, Acta Crystallogr., Sect. A 2008, A64, 112-122. D0I:10.1107/S0108767307043930 Povzetek 10-Ftalimidokafra oksim smo pripravili iz enostavno dostopne 10-jodokafre v dveh korakih. Pri redukciji oksima ni prišlo do tvorbe primarnega amina ampak sta nastala dva nova policiklična izoindolidinska heterocikla. Njuni strukturo smo nedvoumno potrdili z rentgensko strukturo in NMR tehnikami. Groselj et al.: Synthesis and reduction of 10-phthalimidocamphor ... 798 DOI: 10.17344/acsi.2017.3695 Acta Chim. Slov. 2017, 64, 798-803 ^creative ty com mor Short communication The Synthesis of 7-Substituted-2,3-dihydropyrido [4,3-d]pyridazine-1,4-diones and 1,4-Dioxo-7-substituted-1,2,3,4-tetrahydropyrido[4,3-d]pyridazine 6-Oxides from Methyl Ketones Benjamin Prek and Branko Stanovnik* Faculty of Chemistry and Chemical Technology, University of Ljubljana, Vecna pot 113, P. O. Box 537, 1000 Ljubljana, Slovenia * Corresponding author: E-mail: branko.stanovnik@fkkt.uni-lj.si Received: 12-07-2017 Dedicated to Professor Emeritus Miha Tisler, University of Ljubljana, on the occasion of his 90th birthday. Abstract A general four-step transformation of alkyl, cycloalkyl, aryl, and heteroaryl methyl ketones via 3-(dimethylamino)-1-sub-stituted-prop-2-en-1-ones, followed by microwave [2+2] cycloaddition of dimethyl acetylenedicarboxylate, cyclization of (2E,3E)-2-[(dimethylamino)methylene]-3-(2-substituted)succinates with ammonia or hydroxylamine hydrochloride into 2-substituted-pyridine-4,5-dicarboxylates and their N-oxides and final cyclization with hydrazine hydrate into of 7-substituted-2,3-dihydropyrido[3,4-d]pyridazine- 1,4-diones and 1,4-dioxo-7-substituted- 1,2,3,4-tetrahydropyri-do[4,3-d]pyridazine 6-oxides is shown. Keywords: methyl ketones, 7-substituted-2,3-dihydropyrido[4,3-d]pyridazine- 1,4-diones, 1,4-dioxo-7-substituted-1,2,3,4-tetrahydropyrido [4,3-d]pyridazine 6-oxides 1. Introduction There are several methods for the preparation of 2,3-dihydro[4,3-d]pyridazine-1,4- dione derivatives. They have been prepared by treatment of diethyl or dimethyl pyridine-3.4-dicarboxylate with hydrate of hydrazine in refluxing ethanol, and 6-aryl- and 6-aryl-2-methyl derivatives with hydrate of hydrazine in refluxing ethanol, which allow the formation of the corresponding 7-substituted and 5,7-disubstituted-2,3-dihydropyridazine[3,4-d]pyri-dazine-1,4-diones.1-4 Cyclization of ethyl 3-cyanoisonico-tinate with hydrazine proceeds at room temperature to give 4-aminopyrido[3,4-d]pyridazine-1(2H)-one,5,6 while pyridine-3,4-dicarbonitriles give the corresponding pyr-ido[3,4-d]pyridazine-1,4-diones.7 Other methods include cycloamination of 4-carbofunctional-5-vinylpyridazi-nes,A9 condensation of 4,5-dicarbofunctional pyridazines with amines,9,10 condensation of 4-(iminomethyl)pyri-dazines with enolates,10 intramolecular cyclization of pyri-dinecarbohydrazides,1,11 intramolecular cyclization of 4-vinylpyridazine-5-carbonitriles,12,13 by ring enlargement of furo[3,4-c]pyridine-1,3-diones,2,3,5,14,15 1H-pyrrolo[3,4-c] pyridine-1,3(2H) -diones with hydrazine,1,4,16,17 by reaction of 5H-pyrano[3,4-d]pyridazines with amines,8 intramolecular [4+2]cycloaddition of 1,2,4,5-tetrazines,18 ring contraction of 2H-1,2,4-triazepines.14 For a review see.19 Enaminones are well known starting compounds in the synthesis of heterocyclic systems. Their reactivity enables various transformations and functionalizations. Their synthetic value and broad applicability has also been demonstrated in the preparation of natural products and their analogues, such as aplysinopsins,20 meridi-anines,21 and dipodazines.22 Besides the evident reactions with nucleophiles, they also exhibit reactivity with elec-trophiles as well, which only adds to their importance as building blocks in organic synthesis.23 Reactions with electrophiles have been demonstrated in the synthesis of polysubstituted butadienes by microwave-assisted formal [2 + 2] cycloadditions of enaminones to electron-poor acetylenes.24 Prek and Stanovnik: The Synthesis of 7-Substituted-2,3-dihydropyrido Acta Chim. Slov. 2017, 64, 798-803 799 The functionalized buta-1,3-dienes as the basis of the synthetic route presented in this paper are prepared from simple and commercially available compounds such as al-kyl, aryl, and heteroaryl methyl ketones. These are transformed by treatment with N,N-dimethylformamide dimethyl acetal (DMFDMA) or tert-butoxybis(dimethyl-amino)methane (Bredereck's reagent) into the corre- sp onding 3 - ( dimethylamino) -1 -substituted-prop- 2- en-ones, which are further transformed in a regiospecific microwave assisted [2 + 2] cycloaddition with dimethyl acetylenedicarboxylate (DMAD)25 to the before mentioned 1,3-butadienes. These highly functionalized buta-1,3-dienes proved to be useful and versatile reagents in the formation of highly substituted pyridine, pyridine N-oxides, Scheme 1. Preparation of 7-substituted-2,3-dihydropyrido[3,4-90% identity in the variable segment of the 16S rRNA-coding region. ITS regions were either very similar among strains or quite varied, e.g. S. minuscula and S. pevalekii display only 48% identity, while S. salina and S. minuscula share 78% identity in the ITS region (Table 5). This is a good basis for development of Jutersek et al.: Discrimination Between Synechocystis Members (Cyanobacteria) 816 Acta Chim. Slov. 2017, 64, 804-817 ITS-specific primers that could differentiate between species of the same genus. Including genomic regions outside the rRNA operon in the analysis could contribute to fine-positioning of genus members into a system, but it was not essential for discrimination between strains, as our results clearly show. Although our prime interest remains the development of a tool for easy determination of Synechocystis members in water bodies, our current results demonstrate the applicability of DNA-based approach in discriminating between species/strains belonging to the same cyano-bacterial genus. Moreover, they represent a solid basis for taxonomic reconsideration of Synechocystis and related cyanobacterial genera. 4. Conclusions ITS region sequences proved to discriminate among species and strains of Synechocystis members and thus represent a solid basis for DNA barcoding. The observed differences between genus members indicate the presence of several genetic clusters which might lead to a taxonomic reinvestigation of the genus. Interestingly, we observed that two out of 11 strains obtained from cell culture collection show morphological and genetic properties different from expected for Synechocystis genus members. Our results greatly expand the range of Synechocystis representatives with available genomic sequence data and demonstrate that Synechocystis genus currently consists of members that are genetically too different to form one single genus. The need for reconsideration of the genus, previously suggested by Komarek et al.24 is thus additionally substantiated. 5. Acknowledgements We wish to thank Dr. Bojan Sedmak from the National Institute of Biology for access to the epifluorescence microscope. This project has received funding from the European Union's Seventh Programme for research, technological development and demonstration under grant agreement No 308518, CyanoFactory. Parts of this work have also been supported by the Slovenian Research Agency within the research programme P1-0048a. 6. References 1. J. Komarek, Algae 2006, 21, 349-375. DOI:10.4490/ALGAE.2006.21.4.349 2. U. Nubel, F. Garcia-Pichel, G. Muyzer, Appl. Environ. Microbiol. 1997, 63, 3327-3332. 3. M. Gugger, C. Lyra, P. Henriksen, A. Coute, J. F. Humbert, K. Sivonen, Int. J. System. Evol. Microbiol. 2002, 52, 1867-1880. 4. N. Engene, R. C. Coates, W. H. Gerwick, J. Phycol. 2010, 46, 591-601. DOI:10.1111/j.1529-8817.2010.00840.x 5. E. M. Eckert, D. Fontaneto, M. Coci, C. Callieri, Life 2015, 5, 50-64. DOI:10.3390/life5010050 6. C. R. Woese, Microbiol. Rev. 1987, 51, 221-271. 7. S. Smit, J. Widmann, R. Knight, Nucl. Acids Res. 2007, 35, 3339-3354. DOI:10.1093/nar/gkm101 8. N. Engene, H. W. Gerwick, Fottea 2011, 1, 17-24. DOI:10.5507/fot.2011.003 9. G. E. Fox, J. D. Wisotzkey, P. Jurtshuk, Jr., Int. J. Syst. Evol. Microbiol. 1992, 42, 166-170. 10. N. Walworth, U. Pfreundt, W. C. Nelson, T. Mincer, J. F. Heidelberg, F. Fu, J. B. Waterbury, T. Glavina del Rio, L. Lynne Goodwin, N. C. Kyrpides, M. L. Land, T. Woyke, D. A. Hutchins, W. R. Hess, E. A. Webb, Proc. Natl. Acad. Sci. U.S.A. 2015, 112, 4251-4256. DOI:10.1073/pnas.1422332112 11. D. Honda, A. Yokota, J. Sugiyama, J. Mol. Evol. 1999, 48, 723739. DOI:10.1007/PL00006517 12. P. S. Seo, A. Yokota, J. Gen. Appl. Microbiol. 2003, 49, 191203. DOI:10.2323/jgam.49.191 13. V Piccin-Santos, M. Mendes Brandao, M. Do Carmo Bitten-court-Oliveira, J. Phycol. 2014, 4, 736-743. DOI:10.1111/jpy.12204 14. I. Iteman, R. Rippka, N. Tandeau de Marsac, M. Herdman, Microbiol. 2002, 148, 481-496. DOI: 10.1099/00221287-148-2-481 15. G. Rocap, D. L. Distel, J. B. Waterbury, S. W. Chisholm, Appl. Environ. Microbiol. 2002, 68, 1180-1191. DOI: 10.1128/AEM.68.3.1180-1191.2002 16. T. A. Sarma. Handbook of cyanobacteria. CRC Press, Boca Raton, USA, 2013. Also available from: http://www.crcnetbase. com/isbn/978-1-4665-5941-7, (accessed January 19, 2017) 17. W. Lu, E. H. Evans, S. M. McColl, V. A. Saunders, FEMS Microbiol. Lett. 1997, 153, 141-149. DOI: 10.1111/j.1574-6968.1997.tb10475.x 18. I. Janse, M. Meima, W. E. A. Kardinaal, G. Zwart, Appl. Environ. Microbiol. 2003, 69, 6634-6643. DOI:10.1128/AEM.69.11.6634-6643.2003 19. W. E. A. A. Kardinaal, I. Janse, M. Kamst-van Agterveld, M. Meima, J. Snoek, L. R. Mur, J. Huisman, G. Zwart, P. M. Visser, Aquat. Microb. Ecol. 2007, 48, 1-12. DOI:10.3354/ame048001 20. K. M. Orcutt, U. Rasmussen, E. A. Webb, J. B. Waterbury, K. Gundersen, B. Bergman, Appl. Environ. Microbiol. 2002, 68, 2236-2245. DOI:10.1128/AEM.68.5.2236-2245.2002 21. S. Becker, M. Fahrbach, P. Boger, A. Ernst, Appl. Environ. Microbiol. 2002, 68, 4486-4494. DOI: 10.1128/AEM.68.9.4486-4494.2002 22. A. A. Shibl, L. R. Thompson, D. K. Ngugi, U. Stingl, FEMS Microbiol. Lett. 2014, 356, 118-126. DOI: 10.1111/1574-6968.12490 23. N. D. Crosbie, M. Pockl, T. Weisse, Appl. Environ. Microbiol. 2003, 69, 5716-5721. DOI:10.1128/AEM.69.9.5716-5721.2003 24. J. Komarek, J. Kastovsky, J. Mares, J. R. Johansen, Preslia 2014, 86, 295-335. Jutersek et al.: Discrimination Between Synechocystis Members (Cyanobacteria) ... Acta Chim. Slov. 2017, 64, 804- 817 817 25. J. Komarek, Hydrobiol. 2016, 764, 259-270. DOI:10.1007/s10750-015-2242-0 26. I. Iteman, R. Rippka, N. Tandeau de Marsac, M. Herdman, Microbiol. 2000, 146, 1275-1286. DOI: 10.1099/00221287-146-6-1275 27. S. F. Altschul, W. Gish, W. Miller, E. W. Myers, D. J. Lipman, J. Mol. Biol. 1990, 215, 403-410. D0I:10.1016/S0022-2836(05)80360-2 28. W. Li, A. Cowley, M. Uludag, T. Gur, H. McWilliam, S. Squiz-zato, Y. M. Park, N. Buso, R. Lopez, Nucl. Acids Res. 2015, 43(W1), W580-584. D0I:10.1093/nar/gkv279 29. K. Tamura, G. Stecher, D. Peterson, A. Filipski, S. Kumar, Mol. Biol. Evol. 2013, 30, 2725-2729. D0I:10.1093/molbev/mst197 30. J. R. Cole, Q. Wang, J. A. Fish, B. Chai, D. M. McGarrell, Y. Sun, C. T. Brown, A. Porras-Alfaro, C. R. Kuske, J. M. Tiedje, Nucl. Acids Res. 2014, 42(D1), 633-642. D0I:10.1093/nar/gkt1244 31. P. Schattner, A. N. Brooks, T. M. Lowe, Nucl. Acids Res. 2005, 33, W686-9. D0I:10.1093/nar/gki366 32. J. Korelusova, J. Kastovsky, J. Komarek, J. Phycol. 2009, 45, 928-937. D0I:10.1111/j.1529-8817.2009.00701.x 33. J. Komarek, K. Anagnostidis, in: H. Ettl, G. Gärtner, H. Heynig, D. Mollenhauer (Eds.), Süsswasserflora von Mitteleuropa 19/1, Gustav Fischer, Jena, Germany, 1998. 34. I. Moro, N. Rascio, N. La Rocca, M. Di Bella, C. Andreoli, Algol. Stud. 2007, 123, 1-15. DOI: 10.1127/1864-1318/2007/0123-0001 35. R. W. Castenholz, J. B. Waterbury, in: J. T. Staley, M. P. Bryant, N. Pfenning, J. G. Holt (Eds.): Bergeys manual of systematic bacteriology, Williams and Wilkins, Baltimore, USA, 1989, pp. 1710-1789. 36. F. von Winztigerode, U. B. Göbel, E. Stackebrandt, FEMS Microbiol. Rev. 1997, 21, 213-29. DOI: 10.1111/j.1574-6976.1997.tb00351.x 37. W. Laloui, K. A. Palinska, R. Rippka, F. Partensky, N. Tandeau de Marsac, M. Herdman, I. Iteman, Microbiol. 2002, 148, 453-465. DOI: 10.1099/00221287-148-2-453 38. P. M. Shih, D. Wu, A. Latifi, S. D. Axen, D. P. Fewer, E. Talla, A. Calteau, F. Cai, N. Tandeau de Marsac, R. Rippka, M. Herdman, K. Sivonen, T. Coursin, T. Laurent, L. Goodwin, M. Nolan, K. W. Davenport, C. S. Han, E. M. Rubin, J. A. Eisen, T. Woyke, M. Gugger, C. A. Kerfeld, Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 1053-1058. DOI:10.1073/pnas.1217107110 39. M. Kopf, S. Klähn, B. Voss, K. Stuber, B. Huettel, R. Reinhardt, W. R. Hess. Genome Announc. 2014, 2, e00757-14. DOI: 10.1128/genomeA.00757-14 40. T. Kaneko, S. Sato, H. Kotani, A. Tanaka, E. Asamizu, Y. Na-kamura, N. Miyajima, M. Hirosawa, M. Sugiura, S. Sasamoto, T. Kimura, T. Hosouchi, A. Matsuno, A. Muraki, N. Nakazaki, K. Naruo, S. Okumura, S. Shimpo, C. Takeuchi, T. Wada, A. Watanabe, M. Yamada, M. Yasuda, S. Tabata, DNA Res. 1996, 3,109-136. DOI:10.1093/dnares/3.3.109 41. O. Zhaxybayeva, J. P. Gogarten, R. L. Charlebois, W. F. Doolit-tle, R. T. Papke, Genome Res. 2006, 16, 1099-1108. DOI: 10.1101/gr. 5322306 42. S. Yerrapraganda, J. L. Siefert, G. E. Fox, Methods Mol. Biol. 2009, 532, 339-366 DOI:10.1007/978-1-60327-853-9_20 43. P. Rajaniemi-Wacklin, A. Rantala, M. A. Mugnai, S. Turic-chia, S. Ventura, J. Komarkova, L. Lepistö, K. Sivonen, J. Phy-col. 2006, 42, 226-232. DOI: 10.1111/j.1529-8817.2006.00179.x 44. J. Komarek, K. Anagnostidis, Algol. Stud. 1989, 56, 247-345. 45. F. Garcia-Pichel, L. Prufert-Bebout, G. Muyzer, Appl. Environ. Microbiol. 1996, 62, 3284-3291. 46. S. Gkelis, P. Rajaniemi, E. Vardaka, M. Moustaka-Gouni, T. Tanaras, K. Sivonen, Microb. Ecol. 2005, 49, 176-182. DOI: 10.1007/s00248-003-2030-7 47. R. Hoshina, BMC Res. Notes 2014, 7, 592. DOI: 10.1186/1756-0500-7-592 48. M. Kopf, S. Klähn, N. Pade, C. Weingärtner, M. Hagemann, B. Voss, W. R. Hess, DNA Res. 2014, 21, 255-266. DOI: 10.1093/dnares/dst055 49. K. Zerulla, K. Ludt, J. Soppa, Microbiol. 2016, 162, 730-739. DOI: 10.1099/mic.0.000264 Povzetek Cianobakterije so pomembna skupina mikroorganizmov z zelo raznoliko morfologijo, na podlagi katere lahko fenotip-sko razlikujemo med taksonomskimi linijami cianobakterij. Vendar je morfološko razlikovanje zanesljivo predvsem na ravni rodu, na ravni vrste ali seva pa pogosto ne. Osredotočili smo se na rod enoceličnih cianobakterij Synechocystis, ki vključuje tudi modelni cianobakterijski sev PCC 6803. Določili smo zaporedja variabilnega dela genomske regije rRNA 16 S in regije ITS med zapisoma za rRNA 16 S in 23 S za 11 predstavnikov rodu Synechocystis. Zaporedja dveh od enajstih analiziranih sevov iz zbirk kultur so se pomembno razlikovala od zaporedij tipičnih predstavnikov rodu Synechocystis. Opaženo razlikovanje na molekularni ravni smo potrdili tudi z mikroskopijo. Za ostale seve smo ugotovili, da sta obe genomski regiji, ki se sicer že uporabljata v taksonomiji bakterij, ustrezni za razlikovanje med analiziranimi vrstami, pri čemer regija ITS omogoča tudi zanesljivo razlikovanje med sevi iz rodu Synechocystis. Jutersek et al.: Discrimination Between Synechocystis Members (Cyanobacteria) 818 DOI: 10.17344/acsi.2017.3264 Acta Chim. Slov. 2017, 64, 818-824 /^creative tycommor Scientific paper Reaction Monitoring by Means of Multivariate Data Analysis of Near-Infrared and Raman Spectra Dardan Hetemi12* and Steve Janagap1,3 1 Department of Chemistry, University of Bergen, Allégaten 41, N-5007 Bergen, Norway. 2 Pharmacy Department, Medical Faculty, University of Prishtina "Hasan Prishtina", Rr. "Dëshmorët e Kombit"p.n., 10000 Prishtina, Kosovo. 3 Department of Chemistry, College of Arts and Sciences, University of the Philippines Visayas, 5023 Miagao, Iloilo, Philippines. * Corresponding author: E-mail: dardan.hetemi@uni-pr.edu Received: 06-02-2017 Abstract An efficient, versatile and non-destructive in situ method in reaction monitoring using vibrational spectroscopy is described. A Suzuki cross-coupling reaction was monitored in which the substrate 1-iodo-2-nitrobenzene reacted with the electrophile phenylboronic acid to form the product 2-nitrobiphenyl. To hasten the reaction, palladium(II) acetate and potassium carbonate were added to serve as catalyst and to promote transmetalation, respectively. This reaction was monitored using near-infrared and Raman spectroscopy. The recorded data was subjected to multivariate analysis such as principal component analysis in order to detect spectral changes due to the formation of the product. To confirm the presence of the desired product, offline analyses were performed using gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy. The results demonstrate how Raman spectroscopy is able to detect the formation of the product in real time, whereas near-infrared spectroscopy fails to do so. Keywords: Raman spectroscopy, near-infrared spectroscopy, reaction monitoring, Suzuki cross-coupling, multivariate data analysis 1. Introduction Organic synthesis enables the creation of diverse chemical structures of complex architecture in the laboratory. New strategies to develop syntheses, optimize reaction outcomes, and investigate reaction mechanisms are important. One of the interesting reactions to synthesize substituted biphenyls1 involves the Suzuki cross-coupling reaction, a protocol of which has been disclosed elsewhere. 2 The carbon-carbon bond construction method3,4,5,6 in organic synthesis has allowed chemists to assemble complex molecular frameworks to prepare numerous natural products,7 drug precursors,8 biological compounds,9 organic products10 and herbicides.11 Lately, the conditions developed for the cross-coupling reaction have many desirable features for large scale syntheses and are persuasively used in the industrial synthesis of pharmaceutical and chemical products. In daily laboratory work, time together with accuracy plays a crucial role in monitoring techniques particularly in in situ reactions. Systems requiring long reaction times are frequently experienced in organic synthesis. For such reactions, in situ reaction monitoring techniques coupled with multi-variate analysis serve as an excellent approach for determining reaction progress without interruption. Thus, online vibrational spectroscopy has become an increasingly useful tool for research and process development.12,13,14 Near-infrared (NIR) and Raman spectroscopy are suitable instrumental techniques in monitoring the formation of a desired product in real time. In comparison to gas chromatography-mass spectrometry (GC-MS) analysis which requires considerable time to provide a result, the NIR and Raman spectroscopic techniques provide an immediate response that considerably shortens analysis time. By these techniques, it is possible to obtain a huge amount of information about the reaction with minimum effort and time.15 Another advantage of NIR and Raman spec-troscopy in reaction monitoring is that they can provide information on the appearance of reaction intermediates. Hetemi and Janagap: Reaction Monitoring by Means of Multivariate Acta Chim. Slov. 2017, 64, 818-824 819 They also give information about whether the reaction proceeds in the right direction. These advantages make NIR and Raman attractive techniques in pharmaceutical and industrial production. The usefulness of the above-mentioned vibrational spectroscopic techniques in monitoring in situ reactions is much improved if they are coupled with proper data analysis. Chemometrics involves processing chemical data using mathematical and statistical methods in order to extract useful information. Principal components analysis (PCA)16 is applied in order to extract chemical information from the spectra. Spectral data collected by NIR and Raman spectroscopy are complex and normally possesses broad overlapping absorption bands. A single NIR spectrum contains absorptions at many wavelengths that are measured at different times and the vast amount of spectrum generated by the instrument requires chemometric tools for analysis. Often, the measured absorbances at various wavelengths are highly correlated (they are not independent of each other) and this multicollinearity results to poor prediction. PCA is applied to significantly reduce the multidimensionality of the data while retaining most of the information where it decomposes the originally correlated variables to a new limited set of latent variables called principal components. These principal components represent the common variations in the data set where the first principal component accounts for most variations in the data; the second principal component explains the second greatest variations and so on. With PCA, data size is reduced and data overfitting is avoided. In this study, a Suzuki cross-coupling reaction was investigated1 and monitored using NIR and Raman spec-troscopy. The model reaction involved the palladium-catalyzed Suzuki cross-coupling of 1-iodo-2-nitrobenzene and phenylboronic acid to produce 2-nitrobiphenyl. The reaction protocol involves addition of a catalyst (palladium(II) Scheme 1. Synthesis of 2-nitrobiphenyl. acetate) and a base (potassium carbonate) to promote transmetalation. Scheme 1 shows the synthesis of 2-nitro-biphenyl. A review of literature revealed vast amount of materials concerning the Suzuki cross-coupling reaction4,5,17,18 but only few reports19,20 describe the use of NIR and Raman with the reaction. To the best of our knowledge, no report on combining NIR or Raman spectroscopy and multivariate analysis in monitoring Suzuki cross-coupling reactions have been published before. Thus, this study presents a novel monitoring of a Suzuki cross-coupling reaction through vibrational spectroscopic methods and multivariate data analysis. As the Suzuki cross-coupling reactions lead to the formation of by-products as the reaction proceeds, the reaction time and other experimental conditions need to be monitored and controlled. Additionally, the monitoring should provide an immediate estimate of the progress of the ongoing reactions. This is important because Suzuki cross-coupling reactions give very low yield. The reaction of 2-chlorophenylboronic acid with 1-iodo-2-nitroben-zene has been reported as giving as little as 2% product yield1 under some conditions. 2. Experimental Section 2. 1. Chemicals 1-Iodo-2-nitrobenzene (>97%), phenylboronic acid (>99%), methanol (>99.9%), dichloromethane (>99.9%) and potassium carbonate (>99%) were purchased from Sigma-Aldrich (Missouri, USA). Palladium(II) acetate (>98%) was purchased from Fluka (Buchs, Switzerland). All products were used as received without further purification. 2. 2. Reaction Procedure The first step of the boscalid synthesis was chosen as a model for the Suzuki cross-coupling reaction. Instead of using 1-chloro-2-nitrobenzene as substrate for the synthesis of boscalid, an organohalide compound 1-iodo-2-ni-trobenzene was used. This is due to the higher activity of ArI compared with ArCl in oxidative addition (ArI>>Ar- Table 1. Temperature, concentration and sampling times at each experiment run. No. exp. T [°C] C [mmol/mL] Sample 1 Samples were withdrawn Sample 2 Sample 3 in specific time [min] Sample 4 Sample 5 Sample 6 1 23 0.167 60 2 50 0.167 60 3 60 0.167 5 10 15 30 45 60 4 50 0.250 30 60 1 60 0.250 5 10 15 30 45 60 2 65 0.250 5 10 15 30 45 60 Hetemi and Janagap: Reaction Monitoring by Means of Multivariate ... 820 Acta Chim. Slov. 2017, 64, 818-824 Br>>ArCl).21,22 The procedure for the synthesis of 2-nitro-biphenyl has been published elsewhere.1 Briefly, methanol (5 mL) and water (1 mL) were mixed in a round-bottom flask. Potassium carbonate (0.414 g, 3 mmol), palladium^) acetate (0.006 g, 0.03 mmol), 1-iodo-2-nitroben-zene (0.362 g, 1.5 mmol) and phenylboronic acid (0.177 g, 1.5 mmol) were then added to make the concentration 0.250 mmol/mL with respect to substrate (1-iodo-2-nitro-benzene). The solution was flashed with N2 gas and covered with aluminum foil. The reaction mixture was heated at 50 or 60 °C under stirring. The reaction was monitored online with NIR and Raman methods. Samples were withdrawn at specific times without interrupting the reaction (as shown in Table 1), filtered through cotton and silica gel with dichloromethane and analyzed by GC-MS. 2. 3. Workup Procedure of Product Water (30 mL) was added to a crude product obtained after evaporation of methanol under reduced pressure. The solution was extracted three times with dichloro-methane using 30 mL every extraction. The resulting organic solution was washed with saturated aqueous solution of sodium chloride (30 mL). The organic solution was evaporated under reduced pressure and the target product 2-nitrobiphenyl was obtained as dark yellowish oil and analyzed by NMR. 2. 4. Instrumentation A Perstorp NIR Systems 6500 (Maryland, USA) was used for recording the NIR spectra. The NIR spectra of the reaction mixture were obtained at specific time intervals using the fiber optic probe and were recorded from 1100-2500 nm range at 2 nm data interval and using 32 scans per spectrum. The path length was set to 0.5 mm using a 0.25 mm feeler gauge. An average of about 20 s was required to obtain one spectrum. Background and sample spectra were collected using the same parameter settings. The instrument control software used was Vision (FOSS NIRSystems, Inc., MD). The spectrum data was exported as an ASCII file. MATLAB 7.4 (The MathWorks Inc., Natick, MA) was used for multivariate analysis. The software was run on a Microsoft Windows 7 Professional 2009 operating system (Microsoft Corporation, WA, USA). Raman scattering spectra were recorded on a Ra-manRxn1 analyzer (Kaiser Optical Systems, Inc., USA). Measurements were made with an immersion BallProbe®23 connected to the instrument via a fiber optic bundle. The laser wavelength and power were set to 785 nm and 100 mW, respectively. The spectral range was from 0-3800 cm-1 and spectral acquisition setting parameters were set (time of exposure was fixed to 2 s; number of accumulations was set to two). HoloGram 4.1 (Kaiser Optical Systems Inc., Michigan, USA) was used as the instrument control software. The recorded spectra were exported using HoloReact 2.0 (Kaiser Optical Systems Inc., Michigan, USA) to MAT-LAB. The Raman spectra were combined into one data matrix (D.j with dimensions i x j, where (i) represents spectra and (j) represents frequencies. The GC-MS used was HP 5890 II gas chromatograph coupled with HP 5971 mass spectrometer. The capillary column used was fused silica (30 m x 0.2 mm I.D.) coated with Chrompack, CP-Sil 8 CB low bleed/ MS (0.25 ^m film thickness). The GC oven temperature was programed as follows: start temperature was 50 °C ramped to 70 °C at 5 °C/min then increased up to 300 °C at 25 °C/min. Direct on-column injection was used, and the injector port temperature was 250 °C. Helium was used as carrier gas. Xcal-ibur 1.2 (Thermo Fisher Scientific Inc.) was used for instrument control and data analysis. NMR spectra were recorded on a Bruker BioSpin DPX400 (1H NMR: 400.13 MHz, 13C NMR: 100.61 MHz) spectrometer. Deuterated chloroform (CDCl3) was used as solvent for the preparation of the samples and the internal standard. The chemical shifts were expressed in ppm values relative to tetramethylsilane (TMS). Multiplicities were reported using the following abbreviations: s (singlet), d (doublet), t (triplet), q (quartet), dd (double doublet) and m (multiplet). The spectra were recorded at room temperature and the data were processed with MestReNova 5.2 (Santiago de Compostela, Spain). 3. Results and Discussion The reaction being monitored in this study is the synthesis of 2-nitrobiphenyl from 1-iodo-2-nitrobenzene and phenylboronic acid in the presence of palladium(II) acetate and potassium carbonate (see Scheme 1) via the Suzuki cross-coupling mechanism. To determine the best conditions that would give a high product yield, several factors were tested: amount of substrate (1-iodo-2-nitro-benzene), reaction temperature and time. It is therefore important to perform an optimization experiment by varying the levels of the variables and checking the yield at different time intervals as the reaction proceeds. In situ monitoring of the reaction was carried out by using NIR and Raman spectroscopy. 3. 1. Reaction Optimization Similar results were obtained in the synthesis of 2-ni-trobiphenyl following the protocol described in a previous study,1 where 99% yield was achieved after 90 min reaction time at 20 °C. With the aim to shorten the reaction time (to 60 min) but still obtaining a high yield, temperatures higher than 20 oC were tested. Since reaction temperature and substrate concentration (1-iodo-2-nitrobenzene) are Hetemi and Janagap: Reaction Monitoring by Means of Multivariate Acta Chim. Slov. 2017, 64, 818-824 821 Table 2. Experimental set-up employed in the optimization procedure. Exp. T C Time Yield by no. [oC] [mmol/mL] [min] GC-MS [%] 1 23 0.167 60 4.0 2 50 0.167 60 35.3 3 60 0.167 60 71.1 4 50 0.250 60 49.2 5 60 0.250 60 90.0 6 65 0.250 60 99.7 critical factors affecting the yield, several experiments were conducted by varying these factors, assigning low and high values. A summary of the optimization experiments is presented in Table 2. Results show consistently that the yield of the reaction increases as temperature is increased. The higher the temperature, the higher is the yield. When the concentration of the substrate is increased from 0.167 to 0.250 mmol/mL, the same trend is observed: the greater the concentration of the substrate, the higher is the yield. This suggests that the substrate is stoichiometri-cally converted to the product, 2-nitrobiphenyl. The reaction carried out at 65 oC and 0.250 mmol/mL substrate concentration, afforded the highest reaction yield (99.7%). This optimized reaction was monitored by means of fiber optical NIR and Raman spectroscopy (Figures 1 and 2). 3. 2. In Situ Vibrational Spectroscopy Monitoring Visual inspection of the NIR spectra (Figure 1) of the samples monitored from the start of the reaction until the end shows no noticeable spectrum profile changes to distinguish the appearance of the product 2-nitrobiphenyl. WVavalength (cm"1) Figure 1. A series of NIR spectra recorded of a Suzuki cross-coupling reaction that was conducted at reaction temperature of 65 °C. 8000 7000 £000 ™ 5000 500 1000 1500 2000 2500 3000 3500 Wavelength [cm1) Figure 2. A series of Raman spectra recorded of a Suzuki cross-coupling reaction that was conducted at reaction temperature of 65 °C. Hetemi and Janagap: Reaction Monitoring by Means of Multivariate ... fies 1210 1240 1270 1300 1335 1186 1210 1240 1270 1300 1336 Wavelength (cm1) Wavelength (cm1) Figure 3. A) Magnified view of a specific region in the Raman spectra; B) The black and red spectra correspond to the first and last recorded spectra, respectively, as reaction proceeds. By using principal component analysis (PCA), the data were pre-processed by mean-centering and first derivative method using 4th order polynomial fit.24 Mean-centering and use of derivatives are pretreatment techniques to correct systematic variations in the NIR raw data between samples. The variations may be due to nonhomogeneous reaction mixing, sample density gradient, sample viscosity, and other factors that could affect the integrity of the samples during in situ sampling. These lead to light scattering effects and result in baseline shifts and scaling variations. Mean-centering and first derivative methods were selected as optimal pretreatments as they give the lowest statistical errors in preliminary data analysis. PCA was performed in the pretreated spectra to reduce the dimension of NIR spectra matrix for processing and handling multicollinear-ity. Emerged peak were analysed for the substrates and product (data not shown) and there were no significant profile changes in the spectra as reaction proceeds. This shows that NIR method is not sensitive in detecting formation of the product. On the other hand, the Raman spectra (Figure 2) show characteristic stretching vibrations of the biphenyl C-C bridge in the region 1200-1320 cm-1.25,26,27 Spectral changes in this region signal conversion of the reactants (1-iodo-2-nitrobenzene and phenyl-boronic acid) into the desired product (2-nitrobiphenyl). To see more clearly, the region is magnified and presented in Figure 3A. In Figure 3B, the black spectrum corresponds to the initial condition when the substrate was present in the reaction, while the red spectrum represents the final recorded spectrum, which shows the presence of the target molecule in the reaction mixture. The emergence of the peaks at the specific region (1200-1320 cm-1) in the last spectrum shows the emergence of the desired product (C-C bridge stretch of biphenyl). Further offline analysis by GC-MS and NMR confirmed that the spectral changes could be attributed to the formation of the product 2-nitrobiphe-nyl via the Suzuki cross-coupling reaction. The GC-MS chromatogram and NMR spectrum are presented in Supporting Information. 3. 3. Multivariate Data Analysis Principal component analysis (PCA) is an exploratory technique that highlights the differences in the spectra collected and gives a simple visual idea as to the relationship between the spectra. By using PCA, we have analysed the emerged peak for the substrates and product (data not shown). No significant change that could be attributed to the formation of the product was observed. In the contrary, good results were obtained for the specific region (1200-1320 cm-1) in the Raman spectra treated with PCA where the data matrix (120 x 120) was pre-processed using the same techniques as for NIR data. The first principal component (PC1) explains 87.91% of the total variance and from the sample and score plot (Figure 4), it is possible to establish a time-concentration profile. Figure 4 shows that the concentration of the product increases proportionally with time. This increase continues until the reaction is complete. During the course of the reaction, a series of spectra were recorded every 30 s so the sample 120 corresponds to the time of 60 min, the end of reaction monitoring (99.7% yield by GC-MS). The curve shows that the reaction has almost finished after 30 min (corresponding to sample 60). This is in full agreement with the offline measurements of GC-MS that shows the yield of 98.3% for 30 min (Table 3). Also, in the beginning of the reaction (blue circle in Figure 4) we do not have the product yet so the black curve is not increasing. All offline measurements for GC-MS can be seen in the Table 3. As mentioned earlier, PCA was also performed in the NIR data and results show that no significant findings can be established from the collected data. NIR instrumentation is inadequate for monitoring the reaction due to low sensitivity of detection. This study shows that Ra- Hetemi and Janagap: Reaction Monitoring by Means of Multivariate Acta Chim. Slov. 2017, 64, 818-824 823 Figure 4. Samples and scores plot of the data in 1200-1320 cm 1 region. The red line shows the 4th degree polynomial fit. Table 3. The time of sample withdrawn and the yield measured by GC-MS. Time Yield by [min] GC-MS [%] 0 0 5 59.5 10 79.8 15 87.9 30 98.3 45 99.6 60 99.7 man spectroscopy is more sensitive and is the appropriate technique over NIR in reaction monitoring. 4. Conclusions Monitoring of reactions using vibrational spectros-copy coupled with multivariate data analysis finds very important applications nowadays in pharmaceutical and chemical industries. The possibility of online detection of a desired product in an ongoing chemical reaction without system interruption is the main advantage why these techniques in combination with PCA have wide use in the industry. Monitoring of reactions in industry commonly uses the NIR method but this present paper demonstrated that the Raman method is more sensitive and effective and thus, suitable for reaction monitoring. The power of multivariate technique such as PCA in combination with the Raman instrumentation has been shown. The Raman results have been validated by offline techniques such as GC-MS and NMR. Results of online and offline techniques confirm the detection of the desired product making feasible the monitoring of a Suzuki cross-coupling reaction by real time Raman spectroscopy. 5. Acknowledgments European Commission (Erasmus Mundus Scholarships) and University of Bergen are acknowledged for funding the project. The former is acknowledged for financial support. 6. Supporting Information GC-MS of 2-nitrobiphenyl; H NMR of 2-nitrobiphe- nyl. 7. References 1. R. R. Gonzale, L. Liguori, A. M. Carrillo, H.-R. Bj0rsvik, J. Org Chem. 2005, 70, 9591-9594. DOI:10.1021/jo051589t 2. N. Miyura, K. Yamada, A. Suzuki, Tetrahedron Lett. 1979, 20, 3437-3440. DOI:10.1016/S0040-4039(01)95429-2 3. E. Negishi, Acc. Chem. Res. 1982, 15, 340-348. DOI:10.1021/ar00083a001 4. A. Suzuki, J. Organomet. Chem, 1999, 576, 147-168. DOI:10.1016/S0022-328X(98)01055-9 5. N. Miyaura, Chem. Rev. 1995, 95, 2457-2483. DOI: 10.1021/cr00039a007 Hetemi and Janagap: Reaction Monitoring by Means of Multivariate ... 824 Acta Chim. Slov. 2017, 64, 818-824 6. L. Zhao, F. Derridj, S. Djebbar, Ch. Bruneau, H. Doucet, Tetrahedron Lett. 2015, 56, 4354-4358. D01:10.1016/j.tetlet.2015.05.082 7. S. J. Danishefsky, J. J. Masters, W. B. Yound, J. T. Link, L. B. Snyder, Th. V. Magee, D. K. Jung, R. C. A. Isaacs, W. G. Bornmann, Ch. A. Alaimo, C. A. Coburn, M. J. Di Grandi, J. Am. Chem. Soc. 1996, 118, 2843-2859. DOI: 10.1021/ja952692a 8. H. A. Saader, I. M. Mosleh, M. M. El-Abadelah, Molecules, 2009, 14, 2758-1767. D0I:10.3390/molecules14082758 9. X. Zheng, W. Meng, F. Qing, Tetrahedron Lett. 2004, 45, 80838085. D0I:10.1016/j.tetlet.2004.08.180 10. A. Ganesan, Drug Discovery Today, 2002, 7, 47-55. D0I:10.1016/S1359-6446(01)02087-6 11. H. H. Szmant, Organic building blocks of the chemical industry. Wiley: New York,1989, Chapter 4. 12. I. Marziano, D. C. A. Sharp, P. J. Dunn, P. A. Hailey, Org. Process Res. Dev. 2000, 4, 357-361. D0I:10.1021/op000030m 13. K. Hossain, H. Y. Cho, K. J. Kim, J. W. Choi, Biosens. Bioe-lectron. 2015, 71, 300-305. D0I:10.1016/j.bios.2015.04.053 14 . Y. Wu, Y. Jin, Y. Li, D. Sun, X. Liu, Y. Chen, Vib. Spectrosc. 2012, 58, 109-118. D0I:10.1016/j.vibspec.2011.10.006 15. W. L. Guo, Y. P. Du, Y. C. Zhou, S. Yang, J. H. Lu, H. Y. Zhao, Y. Wang, L. R. Teng, World J. Microbiol. Biotechnol. 2012, 28, 993-1002. D0I:10.1007/s11274-011-0897-x 16. I. T. Jollife, Principal Component Analysis; Springer. New York, 2002. 17. S. D. Dreher, S.-E. Lim, D. L. Sandrock, G. A. Molander, J. Org. Chem., 2009, 74, 3626-3631. D01:10.1021/jo900152n 18. M. P. Lorenzo, J. Phys. Chem. Lett. 2012, 3, 167-174. D0I:10.1021/jz2013984 19. G.; Rothenberg, S. C. Cruz, G. P. F. van Strijdonck, H. C. J. H. Hoefsloot, Adv. Synth. Catal. 2004, 346, 347- 473. 20. N. E. Leadbeater, R. J. Smith, Org. Lett. 2006, 8 , 4589-4591. D0I:10.1021/ol061803f 21. A. F. Littke, Ch. Dai, G. C. Fu, J. Am. Chem. Soc. 2000, 122, 4020-4028. D0I:10.1021/ja0002058 22. H. Kurosawa, A. Yamamoto, (Ed.): Fundamentals of Molecular Catalysis, Current Method in Inorganic Chemistry; Vol.3; Elsevier Science, Tokyo, Japan 2003, pp.115-123. 23.ihttp://ballprobe.com/index.html (accessed: March 14, 2016). 24. A. Savitzky, M. J. E. Golay, Anal. Chem., 1964, 38, 1627-1639. DOI: 10.1021/ac60214a047 25. J. S. Gromiuk, H. Gluchowska, B. Tarsiuk, L. Mazur, Z. Rzaczynska, J. Mol. Struct. 2014, 1070, 110-116. DOI:10.1016/j.molstruc.2014.04.030 25. K. C. Bantz, Ch. L. Haynes, Vib. Spectrosc. 2009, 50, 29-35. DOI:10.1016/j.vibspec.2008.07.006 27. G. Socrates, Infrared and Raman Characteristic Group Frequencies: Tables and Charts, 3rd ed., Wiley: New York, 2001. Povzetek Opisujemo učinkovito, vsestransko in neporušno »in situ« metodo za spremljanje reakcij z uporabo vibracijske spektroskopije. Spremljali smo Suzukijevo reakcijo, pri kateri je substrat 1-jodo-2-nitrobenzen reagiral z elektrofilom fenil-borovo kislino in tvoril produkt 2-nitrobifenil. Da bi pospešili reakcijo, smo dodali paladijev(II) acetat kot katalizator in kalijev karbonat, ki promovira reakcijo transmetalacije. To reakcijo smo spremljali z bližnjo infrardečo in z Ramansko spektroskopijo. Podatke smo obdelali z multivariantno analizo, kot je metoda glavnih osi (PCA), da bi ugotovili, do katerih sprememb v spektrih pride zaradi nastanka produkta. Za potrditev prisotnosti želenega produkta smo izvajali tudi »off-line« analizo s plinsko kromatografijo-masno spektrometrijo in z jedrsko magnetno resonančno spektroskopijo. Rezultati pokažejo, da z Ramansko spektroskopijo lahko zaznamo tvorbo produkta v realnem času, medtem ko z bližnjo infrardečo spektroskopijo to ni možno. Hetemi and Janagap: Reaction Monitoring by Means of Multivariate DOI: 10.17344/acsi.2017.3315 Acta Chim. Slov. 2017, 64, 825-831 ¿^creative tS1 commons Scientific paper Synthesis, Crystal Structures, Molecular Docking and MAO-B Inhibitory Activity of Transition Metal Complexes Derived from 2-(4-(Pyridin-2-yl)piperazin-1-yl)acetic Acid Yan-Jie Ren, Jin-Long Zhu, Li-Xin Zhang, Yin-Xiang Xu and Shao-Song Qian* School of Life Sciences, Shandong University of Technology, Zibo 255049, P. R. China * Corresponding author: E-mail: sdutqianss@163.com Tel.: 0086-533-2780271; Fax: 0086-533-2781329. Received: 22-02-2017 Abstract Three new complexes derived from 2-(4-(pyridin-2-yl)piperazin-1-yl)acetic acid (HL), [M(L)2(H2O)2] where M = Cu11 (1), Zn11 (2) and Cd11 (3), have been synthesized and characterized by IR spectroscopy, elemental analysis and X-ray crystallography. The inhibitory activity of these three complexes against MAO-B was tested in vitro, and the molecular docking experiments were also carried out to rationalize their binding models. Both the experimental and docking simulation results indicated that complex 1 has the best inhibitory activity with IC50 value being 6.5 ± 0.31 |M. Keywords: Metal complexes, Crystal Structure, MAO-B inhibitor; Molecular Docking 1. Introduction Monoamine oxidases (MAOs, EC 1.4.3.4) are well known enzymes bound to the outer membrane of mitochondria through a C-terminal transmembrane helix and catalyze the oxidative deamination of monoamine neurotransmitters in the central nervous system.1-3 MAOs occur in two forms designated as MAO-A and MAO-B, which have different substrate preference and inhibitor specificity.4,5 An increase of the dopamine levels as well as a neuroprotective effect can be observed following the inhibition of MAO-B.6 Therefore, MAO-B inhibitors can be used to treat the neurodegenerative disorders such as Aizheimer's disease (AD) and Parkinson's disease (PD).7-9 Kinds of heterocyclic scaffolds such as chal-cone,10 coumarin,11 pyrazoline12 and oxadiazole13 derivatives have been demonstrated as MAO-B inhibitors. Recently, pyridoxine-resveratrol hybrids Mannich base derivatives have been reported as MAO-B inhibitors by Yang et al.14 As mentioned above, most reported MAO-B inhibitors are organic heterocyclic molecules while MAO-B inhibitors based on metal complexes are seldom discussed. In our earlier work, we investigated the urease inhibitors and the MAO-B inhibitory activity of metal complexes.15,16 The inhibitory activities of these bio active complexes are affected by the central metal and the coordination modes of the ligand. As a continuation of our study, in this paper, three new complexes [M(L)2(H2O)2] where M = Cu11 (1), Zn11 (2) and Cd11 (3), were obtained derived from 2-(4-(pyr-idin-2-yl)piperazin-1-yl)acetic acid (HL) and corresponding metal nitrates. Herein, the synthesis, characterization, MAO-B inhibitory activity and molecular docking are presented. 2. Experimental Section 2. 1. Materials and Methods All chemicals and solvents were analytical reagent grade and purchased from Aladdin Industrial Corporation (China). They were used without purification. Elemental analyses for C, H, and N were conducted using the Pregl-Dumas technique on a Thermo Fischer Flash EA1112. FT-IR spectra were recorded from 400-4000 cm-1 on a Nicolet 750 Magna IR spectrometer using KBr pellets. The enzyme inhibitory activity was measured on a Bio-Tek Synergy™ HT Microplate reader. Ren et al.: Synthesis, Crystal Structures, Molecular Docking 826 Acta Chim. Slov. 2017, 64, 825-831 2. 2. Synthesis of 2-(4-(pyridin-2-yl)piperazin-1-yl)acetic Acid (HL) HL was prepared according to the method reported with suitable modification.17,18 l-(pyridin-2-yl)piperazine (1.00 g, 0.0060 mol), bromoacetic acid (2.50 g, 0.0179 mol), and potassium hydroxide (1.50 g, 0.267 mol) were dissolved in 30 mL absolute ethanol. The mixture was refluxed for 10 h. When the reaction was finished, the solution was cooled to room temperature and neutralized with 1 M HCl. White precipitate was filtered to give HL (1.01 g, 74%). 2. 3. General Procedure for the Synthesis of Complexes 1-3 Nitrate (0.040 mmol) was dissolved in 4 mL of methanol solution which was carefully layered on the top of 4 mL water solution of HL (8.84 mg, 0.040 mmol) and KOH (2.24 mg, 0.040 mmol). The solutions were left for a few days at room temperature and then the crystals were obtained. [Cu(L)2(H2O)2] (1) Yield: 0.0372 g, (43% on the basis of HL). IR (KBr, cm-1): 3365; 2970; 1596; 1467; 1384; 845; 681. Anal. Calcd. for C22H32N6O6Cu: C, 48.97; H, 5.97; N, 15.56. Found: C, 49.09; H, 5.95; N, 15.56%. [Zn(L)2(H2O)2] (2) Yield: 0.0442 g, (51% on the basis of HL). IR (KBr, cm-1): 3338; 2943; 1595; 1454; 1395; 831; 664. Anal. Calcd. for C22H32N6O6Zn: C, 48.76; H, 5.95; N, 15.51; Found: C, 48.91; H, 5.92; N, 15.56.%. [Cd(L)2(H2O)2] (3) Yield: 0.0433 g, (46% on the basis of HL). IR (KBr, cm-1): 3359; 2995; 1594; 1458; 1394; 836; 671. Anal. Calcd. for C22H32N6O6Cd: C, 44.87; H, 5.48; N, 14.27; Found: C, 45.02; H, 5.46; N, 14.29%. 2. 4. X-ray Crystallography Diffraction intensities for the complexes were collected at 298(2) K using a Bruker D8 VENTURE PHOTON diffractometer with Mo-Ka radiation (X = 0.71073 A). The structures were solved by direct methods and refined against F2 by full-matrix least-squares methods using the SHELXTL-97.19,20 All non-hydrogen atoms were refined anisotropically, the water H atoms in the complexes were located in difference Fourier maps and refined isotropically with O-H distances restrained to 0.85(1). All other H atoms were placed in idealized positions and constrained to ride on their parent atoms. Details of the crystal parameters, data collection, and refinement are listed in Table 1. The selected bond length and angle data are given in Table 2. The hydrogen bonding data are summarized in Table 3. 2. 5. Rat Brain MAO-B Inhibition Assay MAO-B was obtained from wista mice with the method that was reported earlier. 21 The content of MAO-B was detected by MU30905 ELISA Kit (details in supplementary CH3CH2OH Scheme 1 Synthesis of 2-(4-(pyridin-2-yl)piperazin-1-yl)acetic acid Table 1. Crystallographic data for complex 1-3. 1 2 3 Empirical formula C22H32CuN606 C22H32N606Zn C22H32CdN6O6 Mr 540.09 541.93 588.95 Crystal system triclinic monoclinic monoclinic Space group P-1 P 2/c P 2/c a (A) 6.1916(6) 15.0279(13) 15.0305(14) b (A) 8.1504(8) 7.1943(6) 7.2772(6) c (A) 12.7804(13) 11.7348(9) 11.8887(10) « (°) 95.079(3) 90 90 P (°) 103.019(3) 101.273(3) 101.986(3) Y (°) 106.241(3) 90 90 V (A3) 595.33(10) 1244.23(18) 1272.04(19) Z 1 2 2 pc (g cm-3) 1.507 1.446 1.538 F(000) 283.0 568.0 604.0 Data / param. / restr. 2187 / 148 / 0 2202 / 161/4 2929 / 157 / 12 ^(Mo-Ka)/ mm-1 0.968 1.036 0.907 GOF 1.108 1.110 1.090 R*, wR2h (I > 2o(I)) 0.0663, 0.1684 0.0660, 0.2290 0.0370 , 0.1085 R1 = Z||F| - |Fc||/Z|Fo|.b wR2 = [Ew(Fo2 - Fc2)2/Ew(Fo2)]1'2 Ren et al.: Synthesis, Crystal Structures, Molecular Docking 827 Acta Chim. Slov. 2017, 64, 825-831 materials).22 The MAO-B inhibitory properties of complex 1-3 were carried out as it was reported previously.23 2. 6. Molecular Docking Molecular docking of complexes 1-3 with the active site of human MAO-B (3LA4) was performed by the AUTODOCK 4.2 program suite. The graphical user interface AutoDockTools (ADT) was performed to setup every inhibitor enzyme interaction, where all hydrogen atoms were added, Gasteiger charges were calculated and non-polar hydrogen atoms were merged to carbon atoms. The result file was saved as pdbqt file. The 3D structures of ligand molecules were saved in Mol2 format with the aid of the program Mercury 3.0. The partial charges of Mol2 file were further modified by using the ADT package (version 1.5.4) so that the charges of the non-polar hydrogen atoms would be assigned to the atom to which the hydrogen is attached. The choice of the flexible bonds in the ligands was in accordance with SP3 hybridization. The nitrogen atoms of ligands in complexes 1-3 were assembled as non-protonated. The resulting file was saved as pdbqt file. The AutoDock Vina docking procedure was used to generate the docking output files.24 In all docking, a grid box size of 60 x 60 x 60 pointing in x, y and z directions was built, the maps were centered on N5 atom of FAD6 00.25,26 A grid spacing of 0.375 A and a distances-dependent function of the dielectric constant were used for the calculation of the energetic map. Default parameters were used except num-modes, which was set to 10. The results of the most favorable free energy of binding were selected as the resultant complex structures. At the end of the docking, the result was analyzed using Pymol 2.5 program. 3. Results and Discussion 3. 1. IR Spectroscopy The IR spectra of these complexes were similar. They all show broad band ranging from 3500 cm-1 to 3300 cm-1 indicating the O-H stretching of the water and methanol molecules. The asymmetric stretching mode vas(COO-) was located around 1595 cm-1 (1596 cm-1 in 1, 1595 cm-1 in 2 and 1594 cm-1 in 3), while the strong symmetric stretching mode v (COO-) for complexes 1-3 was clearly visible around 1391 cm-1 (1384 cm-1 in 1, 1395 cm-1 in 2 and 1394 cm-1 in 3). The separation value Av [v (COO-) - vs(COO-)] of the carboxylic based complexes could be used to discriminate the coordination mode of the carboxyl group. Av < 200 cm-1 indicated the bidentate mode, whereas Av > 200 cm-1 indicated the monodentate mode.27,28 Therefore, the Av values [v (COO ) - v(COO )] for complexes 1-3 is around 204 cm-1, which means that the coordination mode of the carboxyl group in these complexes is monodentate. Figure 1. Molecular structure showing the atom-labelling scheme. Displacement spheres are drawn at the 50% probability level. The molecular diagram of (a) 1, (b) 2, (c) 3. Ren et al.: Synthesis, Crystal Structures, Molecular Docking 828 Acta Chim. Slov. 2017, 64, 825-831 3. 2. Description of Crystal Structures Complex 1 crystallizes in triclinic space group P-1, while complexes 2 and 3 crystallize in monoclinic space group P21/c. As shown in Figure 1, these three complexes possess very similar mononuclear structure, so only the structure of 1 is described here in detail. As shown in Figure 1(a), one asymmetric unit contained half of the complex molecule [Cu(L)2(H2O)2]. Every mononuclear complex molecule included one copper ion, two ligand molecules and two coordinated water molecules. Ligand L behaves as a bidentate ligand resulting in the forming of a novel distorted five-membered heterocyclic ring around copper ion. These two five-membered rings are coplanar. The equatorial plane is surrounded by two O-atom donors (O2 and O2a) and two N-atom donors (N1 and N1a) from two L ligands, while the axial positions are occupied by O-atom donors (O3 and O3a) from two coordinated water molecules. In addition, the sum of the equatorial angles N1-Cu1-O2, O2-Cu1-N1a, N1a-Cu1-O2a and O2a-Cu-N1 for complex 1 (= 360.00°) is equal to the ideal value (360.00°), which ensures the planarity of equatorial plane. The axial Cu-O average distance (2.866 A) is longer than the equatorial Cu-O average distance (2.048 A) and Cu-N average distance (1.922 A), showing the stretched tetrahedroid surrounding the Cu(II) center. Compared with the other piperazine-Cu(II) complexes, the Cu-O Table 2. Selected bond lengths (Â) and angles (°) for 1-3 Cu1-O2 1.922(4) Cu1-O3 2.866(4) Cu1-N1 2.048(3) O2-Cu1-O3 98.30(13) O2-Cu1-N1 84.33(12) O2-Cu1-O3a 81.70(13) O2-Cu1-N1a 95.67(12) O3-Cu1-N1 93.97(11) O3-Cu1-N1a 86.03(11) 2 Zn1-O2 2.006(4) Zn1-O3 2.107(4) Zn1-N1 2.362(4) O2-Zn1-O3 94.02(14) O2-Zn1-N1 78.76(13) O2-Zn1-O3a 85.98(14) O2-Zn1-N1a 101.25(13) O3-Zn1-N1 87.95(15) O3-Zn1-N1a 92.05(15) 3 Cd1-O1 2.213(2) Cd1-O1W 2.3055(19) Cd1-N3 2.456(3) O1-Cd1-O1W 94.92(7) O1-Cd1-N3 74.32(8) O1-Cd1-O1Wa 85.08(7) O1-Cd1-N3a 105.68(8) O1W-Cd1-N3 87.69(8) O1W-Cd1-N3a 92.31(8) Figure 2. 1-D zig-zag chain of complex 1. Dashed lines denote hydrogen bonds. Symmetry code: (i) -x, 1 - y, -z. Symmetry codea: (a) 1 - x, 1 -1 - x, -y, 1 - z for 3. y, -z for 1; 1 - x, 2 - y, 2 - z for 2 and Figure 3. The hydrogen-bond-driven 2D sheet of 1 extended in bc plane. Symmetry code: (ii) 1 + x, y, z Ren et al.: Synthesis, Crystal Structures, Molecular Docking 1 Acta Chim. Slov. 2017, 64, 825-831 829 Table 3. Geometrical parameters for hydrogen bonds for 1 Hydrogen bonds D-H (A) H-A (A) D-A (A) D-H-A(°) O3-H3D •O1i 0.85 1.96 2.788(5) 164 C3-H3A—O1" 0.97 2.53 3.476(6) 166 Symmetry codes: (i) -x, 1 - y, -z; (ii) 1 + x, y, z. carboxyl bond (1.938 A) is similar to the Cu-O carbonyl bond length (1.923 A), and Cu-N bond length (2.033 A) in complex 1 is also similar to the other piperazine-Cu(II) complexes. The bond distances and bond angles are normal compared to other reported Cu (II) complexes.29 Water acts a hydrogen bond donor. As shown in Figure 2, complex 1 presents enhanced hydrogen-bonding framework in the solid state (Table 3). Two coordinated water molecules (O3 and O3a) are involved in a chain formation through O3-H3D1—O1' hydrogen bonding (symmetry code: (i) -x, 1 - y, -z). As shown in Figure 3, these chains stack in a interleaved fashion in bc plane, the hydrogen bonds exist between the carboxyl group of ligand L and the carbon atom of other ligand L form intermolecular C3-H3A—O1ii (symmetry code: (ii) 1 + x, y, z) 3. 3. Inhibitory Activity Against MAO-B Statistical analyses of data were performed using SPSS 19.0 program. Data reported as means ± SEM for three independent samples in duplicate. Statistical differences between the groups were considered significant if the p value was < 0.05. Specific results please see Table 4. It was found that compared with iproniazid phosphate as the positive control (IP, IC50 = 7.59 ± 1.17 ^M), 30 complex 1 (IC50 = 6.52 Table 4. Inhibition of rat brain MAO-B for HL, complexes 1-3, and IP Test material ic50 (MM) 1 6.52 ± 0.31 2 >100 3 >100 HL 76.1 ± 2.51 Cu(NO3)2 12.31 ± 1.06 Zn(NO3)2 >100 Cd(NO3)2 >100 IP* 7.59 ± 1.17 ± 0.31 ^M) had good inhibitory activity on MA0-B, while complexes 2 and 3 (IC50 > 100^M) showed no inhibitory activity. Both Cu(NO3)2 and ligands have a certain inhibitory capacity for monoamine oxidase, after the two combine to form a complex 1, complex 1 showed good inhibitory activity, therefore, they show certain synergistic effects. 3. 4. Molecular Docking The binding models of complexes 1, 2, 3 with MAO-B (1S3E) were simulated using the AutoDock Vina docking program to validate their structure-activity relationships. The docking results show that only the complex 1 has good binding with the active site of MAO-B (1S3E). The binding model of complex 1 and 1S3E is shown in Figure 4. All amino acid residues around complex 1 are shown. In the binding model, the main stabilizing factors that stabilize the Cu(L)2-1S3E complex are the hydropho-bic contacts and hydrogen bonding interactions. The O1 atom in complex 1 serves as a hydrogen bond acceptor re- Figure 4. Binding mode of complex 1 with human monoamineoxidase-B. The complex molecule and MAO-B were shown as stick, the hydrogen bonds were shown as yellow dash lines Ren et al.: Synthesis, Crystal Structures, Molecular Docking 830 Acta Chim. Slov. 2017, 64, 825-831 ceived one strong hydrogen bonding interaction from Tyr393. The hydrogen-bonding distance of Tyr393 N-H—O2 is 2.8 A. The results of the molecular docking indicate that the complex 1 could be well fitted in the active pocket of MAO-B. 4. Conclusion This paper reports that synthesis, crystal structure, molecular docking, and monoamine oxidase B inhibitory activities of three transition metal complexes with 2-(4-(pyridin-2-yl)piperazin-1-yl)acetic acid ligand. Molecular docking assay showed the potential binding model of complex 1 with MAO-B. The complex 1 exhibits MAO-B inhibiting activity in vitro at micromole concentrations (IC = 6.5 ± 0.31 ^M), whereas complex 2 and 3 exhibits no ¡MAO-B inhibiting activity (IC50 > 100 ^M). Now, we are synthesizing specific compounds that inhibit the MAO-B based on the complex 1 structure. 5. Supplementary Information CCDC files 1515965 (1), 1515778 (2) and 1515777(3) contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc. cam.ac.uk/data_request/cif. 6. Referances 1. M. B. Youdim, G. G. Gollins, M. Sandler, J. Biochem. 1971, 121, 34-36. DOI:10.1042/bj1210034P 2. J. C. Shih, Neuropsychopharmacol. 1991, 4, 1-7. 3. J. W. Greenawalt, C. Schnatiman, J. Cell. Biol. 1970, 46, 173179. DOI:10.1083/jcb.46.1.173 4. C. W. Abell, S. W. Kwan, Prog. Nucleic. Acid. Res. 2001, 65, 129-156. http://dx.doi.org/10.1016/S0079-6603(00)65004-3 DOI:10.1016/S0079-6603(00)65004-3 5. O. Nakagawasai , Y. Arai , E. H. Satoh, N. Satoh, M. Neda, M. Hozumi, R. Oka, H. Hiraga, T. Tadano, Neurotoxicology 2004, 25, 223-232. DOI:10.1016/S0161-813X(03)00101-3 6. H. P. Volz, C. H. Gleiter, Drug. Aging 1998, 13, 341-355. DOI: 10.2165/00002512-199813050-00002 7. J. C. Jean, Neurotoxicology 2004, 25, 21-30. DOI:10.1016/S0161-813X(03)00112-8 8. M. B. Youdim, D. Edmondson, K. F. Tipton, Nat. Rev. Neuro-sci. 2006, 7, 295-309. DOI:10.1038/nrn1883 9. S. S. Jossan, P. G. Gillberg, C. G. Gottfries, I. Karlsson, L. Oreland, Nat. Rev. Neurosci. 1997, 45, 1-12. http://dx.doi. org/10.1016/0306-4522(91)90098-9 DOI: 10.1016/0306-4522(91)90098-9 10. T. Shigeo, Y. Kuwai, M. Tabata, Planta Med. 1987, 53, 5-8. DOI:10.1055/s-2006-962604 11. J. Joubert, G. B. Foka, B. P. Repsold, D. W. Oliver, E. Kapp, S. F. Malan, Eur. J. Med. Chem. 2017, 125, 853-864. DOI:10.1016/j.ejmech.2016.09.041 12. B. Evranos-Aksoz, S. Yabanoglu-^ift^i, G. U^ar, K. Yelek^i, R. Ertan, Bioorg. Med. Chem. Lett. 2014, 24, 3278-3284. DOI:10.1016/j.bmcl.2014.06.015 13. S. Distinto, R. Meleddu, M. Yanez, R. Cirilli, G.Bianco, M. L. Sanna, A. Arridu, P. Cossu, F. Cottiglia, C. Faggi, F. Ortuso, S. Alcaro, E. Maccioni, Eur. J. Med. Chem. 2016, 108, 542-552. DOI:10.1016/j.ejmech.2015.12.026 14. X. Yang, X. M. Qiang, Y. Li, L. Luo, R. Xu, Y. X. Z. Zheng, Z. C. Cao, Z. H. Tan, Y. Deng, Bioorg. Chem. 2017, 71, 305-314. DOI:10.1016/j.bioorg.2017.02.016 15. J, Qin, Q. Yin, S. S. Zhao, J. Z. Wang, S. S. Qian, Acta Chim. Slov. 2016, 63, 55-61. 16. D. D. Yang, R. Wang, J. L. Zhu, Q. Y. Cao, J. Qin, H. L. Zhu, S. S. Qian, J. Mol. Struct. 2017, 1128, 493-498. DOI:10.1016/j.molstruc.2016.08.037 17. C. T. Sadashiva, S. C. J. N. Narendra, K. C. Ponnappa, G. T. Veerabasappa, K. S. Rangappa, Bioorg. Med. Chem Lett. 2016, 16, 3932-3936. DOI:10.1016/j.bmcl.2006.05.030 18. Z. J. Chen, C. N. Xu, J. L. Zhu, D. D. Yang, S. S. Zhao, Y. N. Chen, S. S. Qian, Acta Chim. Slov. 2016, 63, 165-172. DOI:10.17344/acsi.2015.2109 19. G. M. Sheldrick, Acta Crystallogr. A 2008, 64, 112-122. DOI:10.1107/S0108767307043930 20. Bruker, SMART (Version 5.63), SAINT (Version 6.02), SADABS (Version 2.03), Bruker AXS Inc. 2002, Madi-son,Wisconsin, USA. 21. B. E. Yoon, J. Woo, Y. E. Chun, H. Chun, S. Jo, J. Y. Bae, H. An, J. O. Min, S. J. Oh, K. S. Han, H. Y. Kim, T. Kim, Y. S. Kim, Y. C. Bae , C. J. Lee, J Physiol. 2014, 592, 4951-4968. DOI: 10.1113/jphysiol.2014.278754 22. A. Holt, D. F. Sharman, G. B. Baker, M. M. Palcic, Anal. Biochem. 1977, 244, 384-392. DOI:10.1006/abio.1996.9911 23. M. O. Ogunrombi, S. F. Malan, G. T. Blanche, N. Castagnoli, J. J. Bergh, J. P. Petzer, Bioorg. Med. Chem. 2008, 16, 2463-2472. http://dx.doi.org/10.1016/j.bmc.2007.11.059 DOI:10.1016/j.bmc.2007.11.059 24. O. Trott, A. J. Olson, J. Comput. Chem. 2010, 31, 455-461. DOI:10.1002/jcc.21334 25. M. B. Nunez, F. P. Maguna, N. B. Okulik, E. A. Castro, Bioorg. Med. Chem. Lett. 2004, 14, 5611-5617. DOI:10.1016/j.bmcl.2004.08.066 26. L. Santana, H. Gonzalez-Diaz, E. Quezada, J. Med. Chem. 2008, 51, 6740-6751. DOI:10.1021/jm800656v 27. C. T. Sadashiva, J. N. N. S. Chandra, K. C. Ponnappa, G. T. Veerabasappa, K. S. Rangappa, J. Bioorg. Med. Chem. Lett. 2006, 16, 3932-3936. DOI:10.1016/j.bmcl.2006.05.030 28. I. Turel, J. Kljun, J. Curr. Top. Med. Chem. 2011, 11, 26612687. DOI:10.2174/156802611798040787 29. H. Y. Luo, J. M. Lo, P. E. Fanwick, J. G. Stowell, M. A. Green, J. Inorg. Chem. 1999, 38, 2071-2078. DOI:10.1021/ic981324f 30. S. S. Xie, X. Wang, N. Jiang, Y. W. Yu, D. G. K. Wang, J. S. Lan, Z. R. Li, L. Y. Kong, Eur. J. Med. Chem. 2015, 95, 153-165. DOI:10.1016/j.ejmech.2015.03.040 Ren et al.: Synthesis, Crystal Structures, Molecular Docking Acta Chim. Slov. 2017, 64, 825- 831 831 Povzetek Sintetizirali smo tri nove komplekse z 2-(4-(piridin-2-il)piperazin-1-il)ocetno kislino (HL), [M(L)2(H2O)2], kjer je M = Cu11 (1), Zn11 (2) in Cd11 (3), ter jih okarakterizirali z IR spektroskopijo, elementno analizo in rentgensko kristalografijo. In vitro smo določili inhibitorno aktivnost pripravljenih treh kompleksov proti MAO-B ter izvedli simulacijo molekularnega dockinga za razumevanje načina vezave. Eksperimentalni podatki in računalniške simulacije kažejo, da ima kompleks 1 največjo inhibitorno aktivnost z IC50 vrednostjo 6.5 ± 0.31 |M. Ren et al.: Synthesis, Crystal Structures, Molecular Docking 832 DOI: 10.17344/acsi.2017.3342 Acta Chim. Slov. 2017, 64, 832-841 ^creative tycommor Scientific paper A Novel One-pot Synthesis of Isothiocyanates and Cyanamides from Dithiocarbamate Salts Using Environmentally Benign Reagent Tetrapropylammonium Tribromide Neivotsonuo Bernadette Kuotsu,1,3 Latonglila Jamir,2 Tovishe Phucho3 and Upasana Bora Sinha3* 1 Department of chemistry, Kohima Science College (Autonomous), Jotsoma-797002, Nagaland, India 2 Department of Environmental Science, Nagaland University, Lumami-798627, Nagaland, India 3 Department of Chemistry, Nagaland University, Lumami-798627, Nagaland, India * Corresponding author: E-mail: upasanabsinha@gmail.com Received: 06-03-2017 Abstract A highly efficient and simple protocol for the synthesis of isothiocyanates and cyanamides from their respective amines in the presence of a mild, efficient, and non-toxic reagent tetrapropylammonium tribromide is described. High environmental acceptability of the reagents, cost effectiveness and high yields are the important attributes of this methodology. Keywords: TPATB, desulfurization, oxidation, isothiocyanates, sodium bicarbonate, cyanamide. 1. Introduction Isothiocyanates are one of the most important synthetic intermediates for the preparation of both sulphur and nitrogen containing organic compounds especially for heterocycles.1 The isothiocyanate functionality is frequently encountered in natural products, including sesquiterpenes.2 Additionally, synthetic isothiocyanates have proven to have some biological activity, such as antiproliferative3 and enzyme inhibitory for the HIV virus.4 Numerous methods for the preparation of isothiocyanates have been reported, starting from amines,5 dithiocarbamates,6 organic halides,7 olefins8 and aldoximes.9 Among the literature methods, the most widely used procedure is the synthesis by the decomposition of dithiocarbamates using heavy metals,10 thiophosgene, iodine, ethylchlorocarbamate and claycop.11 Although many synthetic methods for the preparation of isothiocyanates have been reported to date,12-32 most methods suffer from the employment of highly toxic reagents. Thus, there is still need for a commercially viable and environmentally acceptable protocol for the synthesis of isothiocyanates. We describe herein a modified, environmentally benign and cost-effective met- hod for the synthesis of isothiocyanates via TPATB-medi-ated decomposition of dithiocarbamate salt in the presence of sodium bicarbonate in water/ethyl acetate biphasic solvent system at room temperature (Scheme 1). Similarly, in continuation of our work we have syn-thesised cyanamides from the dithiocarbamate salt. Cyan-amides have attracted considerable attention as a class of versatile organic molecules. They have a wide range of uses in organic synthetic chemistry33 and coordination chemis-try34 as they can be used as building blocks for the construction of not only diverse N-containing compounds but also as metal ligands with their unique reactivity and structure of the cyanamide unit. Additionally, it has been found that some cyanamide based compounds show a diversity of interesting bioactivities, inhibition of spontaneous myogenic, and peptide activator activities.35 It has also been proved that cyanamide is a natural product present in higher plants even though its distribution is limit-ed.36 The broad applications of cyanamides have resulted in the development of several methods for their synthesis over the years. The most common among these is the reaction of cyanogens chloride or bromide with amines or with imide salts.37 However, this method involves the use of po- Kuotsu et al.: A Novel One-pot Synthesis of Isothiocyanates Acta Chim. Slov. 2017, 64, 832-833 841 tassium or sodium cyanide and bromine for the preparation of cyanogen halide (which is again highly toxic), making the protocol environmentally unacceptable. Literature reports on various other methods for the preparation of cyanamide using different synthetic strategies, such as cy-anation of amines using CN+ equivalents as synthons,38-44 Tiemann rearrangement of amidoximes,45 coupling reactions involving Pd isocyanides, allyl carbonates and trimethylsilyl azide,46 and sodium bis(trimethylsilyl)amide as deoxygenating or desulfurizing agents.47 Yet another method for the preparation of cyanamides involves the reaction of hypervalent iodine(V) species with N.N'-disub-stituted glycylamide.48 However, all the procedures reported so far seem to have certain environmental concerns as they involve direct or indirect use of toxic and corrosive reagents, strong alkaline conditions, expensive reagents and catalysts, high reaction temperatures and tedious purification procedures. We have been interested to an extent in the synthesis of isothiocyanates and cyanamides, therefore, in this context we develop an alternative method involving the use of alkyl or aryl dithiocarbamate using tet-rapropylammonium tribromide as a double desulfurizing agent. 2. Experimental 2. 1. General Procedure for the Synthesis of Phenyl Isothiocyanate To a stirred and ice cooled suspension of phenyl dithiocarbamate salt (Table 1, compound 1) (540 mg, 2 mmol) in ethylacetate (5 mL), and water (5 mL) was added NaHCO3 (336 mg, 4 mmol). To this was added TPATB (0.852 g, 2 mmol) pinch wise over a period of 10-15 minutes to yield phenylisothiocyante (Table 1, compound 1a). During this period, precipitation of elemental sulfur was observed. Completion of the reaction was confirmed by TLC. Ethyl acetate (10 mL) was further added to the reaction mixture. The organic layer was washed with water (2-5 mL), dried over anhydrous Na2SO4, concentrated under reduced pressure and purified over a short column of silica gel while eluting it with hexane-ethyl acetate (97:3) to give the pure product (Table 1, compound 1a) (216 mg, 80%). 2. 2. General Procedure for the Synthesis of Phenylcyanamide To a stirred and ice cooled suspension of dithiocar-bamate (Table 2, compound 1) (540 mg, 2 mmol) in ethyl acetate (5 mL), was added NaHCO3 (336 mg, 4 mmol). To this was then added TPATB (0.852 g, 2 mmol) pinch wise over a period of 10-15 minutes to yield phenylisothiocya-nate. During this period precipitation of elemental sulfur was observed. After complete addition of TPATB, 25% aqueous NH3 (2.5 mL) was added drop wise to the stirred reaction mixture to give 1-phenylthiourea. After stirring for 10 minutes at room temperature, the excess of NH3 was removed in a rotary evaporator whereby the solvent ethyl acetate was also simultaneously removed leaving behind the aqueous layer. To the crude reaction mixture was then further added ethyl acetate (5 mL) and NaHCO3 (336 mg, 4 mmol). To the resultant solution, TPATB (0.852 g, 2 mmol) was added in small pinches, during which further precipitation of elemental sulfur was observed. The conversion of 1-phenylthiourea to phenylcyanamide (Table 2, compound 1b) was observed within 5 minutes of the complete addition of TPATB. Completion of the reaction was confirmed by TLC. The precipitated sulfur was filtered, washed with ethyl acetate (2 x 5 mL). The organic layer was washed with water (2 x 5 mL) and dried over anhydrous Na2SO4, concentrated under reduced pressure and purified over a short column of silica gel eluting it with hexane-ethyl acetate (97:3) to give the pure product (Table 2, compound 1b) (188 mg, 80%) as an oily liquid. 3. Results and Discussion The dithiocarbamic acid salt is readily converted into the corresponding isothiocyanate (Table 1, compound 1a), simply by treating it with TPATB in the presence of sodium bicarbonate in water/ethyl acetate biphasic medium in good to excellent yields in shorter time (15 minutes) as shown in Scheme 1. N A, S"-+NHEt, TPATB NaHC03 H-.O/EtOAc R—N=C—S 3r + Br-+NHEtj + H,CO., + S| Scheme 1. Preparation of isothiocyanate from dithiocarbamate salt The use of water/ethyl acetate biphasic solvent system has several potential advantages. In addition to the benign character of both water and ethyl acetate, the coexistence of water with ethyl acetate helps in extracting the isothiocyanate to the organic layer leaving behind the impurities in the aqueous layer which in turn facilitates an easy workup. TPATB is soluble in ethyl acetate and on stirring dissolves, thus getting delivered to the water-ethyl acetate interphase for desulfurization. Moreover, the water phase dissolves the base sodium bicarbonate and retains the dithiocarbamic acid salt in aqueous layer. The use of sodium bicarbonate over organic bases offers a mild and effective green approach towards the synthesis of isothio-cyanates. The mechanism of the present transformation is given below in Scheme 2.48 Thus, the method provides an ecologically and economically viable process for the preparation of isothiocyanates. The preparation of isothiocyanates was performed Kuotsu et al.: A Novel One-pot Synthesis of Isothiocyanates 834 Acta Chim. Slov. 2017, 64, 832-841 Table 1. Preparation of Isothiocyanates from Dithiocarbamate Salt and TPATBa Substrate Productb Yield (%)c a Reactions were monitored by TLC; b Confirmed by IR,1 HNMR and 13CNMR; c Isolated yield. Kuotsu et al.: A Novel One-pot Synthesis of Isothiocyanates ... Acta Chim. Slov. 2017, 64, 832-835 841 Scheme 2. Mechanism of the formation of isothiocyanate from dithiocarbamate salt on freshly prepared dithiocarbamate salts synthesized from a variety of structurally different alkyl and aryl amines. The results are summarized in Table 1. Substrates containing activating substituents (Table 1, compounds 2-3) gave the expected products efficiently as also did substrates containing deactivating substituents (Table 1, compounds 4-7). Trisubstituted substrates (Table 1, compounds 8-10) as well as highly hindered substrates (Table 1, compound 11) gave the corresponding isothiocyanates in high yields. Benzylic substrate (Table 1, compound 12) and aliphatic substrates (Table 1, compounds 13-15) also gave their expected products (Table 1, compound 12a) and (Table 1, compounds 13a-15a) respectively in excellent yields. Similarly, for the synthesis of cyanamides the methodology was based on: (i) formation of isothiocyanate from alkyl/aryl dithiocarbamate salt by desulfurization with TPATB in the presence of triethylamine as the base in ethyl acetate solvent, (ii) treating the in situ generated isothiocyanate with aqueous NH3 to afford alkyl/aryl thioam-ides and (iii) further oxidative desulfurization of thioam-ides to cyanamide with TPATB in the presence of triethylamine (Scheme 2). The mechanism of the present transformation is given below.49 Based on these findings, we thus report herein a practical, environmentally benign, high yielding and one pot preparation of cyanamides from dith-iacarbamate salts using cheap and non-toxic reagent TPATB (Scheme 3) in an innocuous solvent ethyl acetate. Implementing this one pot method, a wide variety of aromatic and aliphatic cyanamides have been prepared from their parent dithiocarbamate salts. Phenyl cyanamide (Table 2, compound 1b) was obtained in excellent yield from its dithiocarbamate (Table 2, compound 1). Monosubstituted substrates bearing deactivating substituents (Table 2, compounds 2-6) readily underwent this reaction to produce the desired cyanamides (Table 2, compounds 2b-6b) in high yields. The method worked smoothly for substrates with electron donating substitu-ents such as (Table 2, compounds 7-8) to afford the corre- sponding cyanamides (Table 2, compounds 7b-8b) in high yields. A noteworthy aspect is that the present methodology can be applied to substrates containing sensitive substituents such as keto functionality (Table 2, compound 9b) as well as trisubstituted ones (Table 2, compound 10b). Cyclic and open chain aliphatic cyanamides (Table 2, compounds 11b-12b) were obtained from their corresponding dithiocarbamate salts (Table 2, compounds 11-12) under the same reaction conditions. We further investigated the efficacy of this method towards benzylic dithiocarbamate (Table 2, compounds 13-14) and dithiocarbamate salt of homoveratryl amine (Table 2, compound 15) which underwent the reaction smoothly to yield their respective corresponding cyanamide products (Table 2, compounds 13b-14b) and (Table 2, compound 15a) in good yields under the similar reaction conditions. 4. Spectroscopic and Analytical Data 1-Isothiocyanato-benzene (1a). Oily; 1H NMR (400 MHz, CDCl3) 5 7.21-7.37 (m, 5H, ArH). 13C NMR (100 MHz, CDCl3) 5 125.8, 127.4, 129.6, 131.3, 135.3. IR (KBr) 3064, 2164, 2063, 1591, 1489, 1474, 1451, 1070, 927, 905, 749, 684 cm-1. Anal. Calcd for C7H5NS (135.19): C, 62.19; H, 3.73; N, 10.36; S, 23.72. Found: C, 62.22; H, 3.71; N, 10.35; S, 23.73. 1-Isothiocyanato-4-methyl-benzene (2a). Oily; 1H NMR (400 MHz, CDCl3) 5 2.33 (s, 3H, CH3), 7.06-7.13 (m, 4H, ArH). 13C NMR 3100 MHz, CDCl3) 5 21.2, 125.4, 128.2, 130.1, 134.4, 137.4. IR (KBr) 2920, 2094, 1503, 929, 812, 790, 497 cm-1. Anal. Calcd for C8H7NS (149.15): C, 64.36; H, 4.73; N, 9.39; S, 21.51. Found: C, 64.32; H, 4.75; N, 9.41; S, 21.56. 1-Isothiocyanato-4-methoxy-benzene (3a). Oily; 1H NMR (400 MHz, CDCl3) 5 3.80 (s, 3H, CH3), 6.85 (d, 2H, J = 8.8 Hz, ArH), 7.16 (d, 2H, J = 8.8 Hz, ArH). 13C NMR R. S •"^e-.n TPATB H I R—N—C—N y S - NHEt3 NaHC03, Aq, NH3 H H2O/Et0Ac NaBr + Br" +NHEt3 + H2C03" + si Scheme 3. Mechanism for the formation of cyanamide Kuotsu et al.: A Novel One-pot Synthesis of Isothiocyanates 836 Acta Chim. Slov. 2017, 64, 832-841 Table 2. Preparation of cyanamides from dithiocarbamates and TPATBa Substrate Productb Yield (%)c 72 70 70 75 71 77 70 68 65 78 60 62 Kuotsu et al.: A Novel One-pot Synthesis of Isothiocyanates Acta Chim. Slov. 2017, 64, 832-837 841 Substrate Productb Yield (%)c " Reactions were monitored by TLC; b Confirmed by IR,1 HNMR and 13CNMR; c isolated yieldw (100 MHz, CDCl3) 5 55.4, 114.6, 123.2, 126.8, 133.7, 158.4. IR (KBr) 3000, 2956, 2835, 2170, 2098, 1580, 1599, 1503, 1459, 1440, 1292, 1251, 1179, 1166, 1028, 927, 824, 614, 513 cm-1. Anal. Calcd for C8H7NOS (165.22): C, 58.16; H, 4.27; N, 8.48; S, 19.40. Found: C, 58.08; H, 4.23; N, 8.34; S, 19.34. 8.11-8.14 (m, 1H, ArH). 13C NMR (100 MHz, CDCl3) 5 120.7, 121.9, 130.6, 131.6, 133.3, 139.6, 148.8. IR (KBr) 3091, 3074, 2227, 2161, 2106, 1526, 1470, 1348, 1302, 892, 809, 736, 665 cm-1. Anal. Calcd for C7H4N2O2S (180.13): C, 46.63; H, 2.23; N, 15.55; S, 17.81. Found: C, 46.65; H 2.26; N, 15.51; S, 17.78. 1-Bromo-4-isothiocyanato-benzene (4a). White solid, m.p. 58 °C (lit.1a 58 °C), 1H NMR (400 MHz, CDCl3) 5 7.09 (d, 2H, J = 8.8 Hz, ArH), 7.47 (d, 2H, J = 8.8 Hz, ArH). 13C NMR (100 MHz, CDCl3) 5 120.8, 127.2, 130.5, 132.8, 136.9. IR (KBr) 3074, 2925, 2171, 2071, 1578, 1478, 1474, 1399, 1067, 1011, 923, 818, 490, 438 cm-1. Anal. Calcd for C7H4BrNS (214.03): C, 39.24; H, 1.88; N, 6.54; S, 14.99. Found: C, 39.21; H, 1.93; N, 6.50; S, 15.04. 4-Isothiocyanato-benzonitrile (5a). White solid; m.p. 119-120 °C (lit.21121 °C), 1H NMR (CDCl3, 400 MHz) 5 7.31 (d, J = 8.8 Hz, 2H), 7.66 (d, J = 8.8 Hz, 2H); 13C NMR (CDCl3, 100 MHz) 5 110.6, 117.9, 126.5, 133.6, 135.9, 139.4; IR (KBr) 3435, 2197, 2124, 2104, 1591, 1492, 1277, 933, 836, 544 cm-1. Anal. Calcd for C8H4N2S (166.13): C, 59.95; H, 2.51; N, 17.49; S, 20.03. Found: C, 59.98; H, 2.49; N, 17.45; S, 19.98. 1-Isothiocyanato-4-trifluoromethyl-benzene (6a). White solid; m.p. 43 °C (lit.22 bp 205-207 °C); 1H NMR (CDCl3, 400 MHz) 5 7.32 (d, J = 8.4 Hz, 2H, ArH), 7.61 (d, J = 8.4 Hz, 2H, ArH); 13C NMR (CDCl3, 100 MHz) 5 122.4, 125.1, 126.1, 126.9, 127.0, 129.0, 129.4, 135.15, 138.4; IR (KBr) 3427, 2081, 1613, 1413, 1325, 1137, 1106, 1066, 839, 590 cm-1. Anal. Calcd for C8H4F3NS (203.12): C, 47.26; H, 1.98; N, 6.89; S, 15.79. Found: C, 47.22; H, 1.96; N, 6.91; S, 15.75. 1-Isothiocyanato-2,4-dimethyl-benzene (8a). Oily; 1H NMR (400 MHz, CDCl3) 5 2.30 (s, 3H, CH3), 2.33 (s, 3H, CH3), 6.96 (d, 1H, J = 9.2 Hz, ArH), 7.01 (s, 1H, ArH), 7.07 (d, 1H, J = 8.0 Hz, ArH). 13C NMR (100 MHz, CDCl3) 5 18.2, 21.1, 125.6, 127.4, 131.2, 134.6, 137.4. IR (KBr) 2920, 2131, 2085, 1490, 1455, 1379, 1229, 1036, 947, 901, 875, 812 cm-1. Anal. Calcd for C9H9NS (163.17): C, 66.19; H, 5.55; N, 8.58; S, 19.66. Found: C, 66.15; H, 5.52; N, 8.63; S, 19.64. 2-Iodo-1-isothiocyanato-4-methylbenzene (9a). White solid; m.p. 62-65 °C; 1H NMR (400 MHz, CDCl3) 5 2.30 (s, 3H, CH3), 7.13 (m, 2H, ArH), 7.62 (s, 1H, ArH). 13C NMR (100 MHz, CDCl3) 5 20.9, 94.2, 126.7, 130.1, 132.3, 136.1, 139.1, 139.9. IR (KBr) 2916, 2134, 1633, 1474, 1042, 929, 811 cm-1. 2-Bromo-1-isothiocyanato-4-methoxy-benzene (10a). White solid; m.p. 77 °C; 1H NMR (400 MHz, CDCl3) 5 3.80 (s, 3H, CH3), 6.82 (m, 1H, ArH), 7.11 (m, 1H, ArH), 7.18 (d, 1H, J = 8.0 Hz, ArH); 13C NMR (100 MHz, CDCl3) 5 56.0, 114.4, 118.5, 121.6, 124.1, 127.7, 136.7, 158.8; IR (KBr) 2972, 2125, 1594, 1560, 1483, 1296, 1263, 1220, 1039, 807, 617 cm-1. Anal. Calcd for C8H6BrNOS (244.05): C, 39.33; H, 2.47; N, 5.73; S, 3.14. Found: C, 39.29; H, 2.52; N, 5.70; S, 3.11. 1-Isothiocyanato-3-nitro-benzene (7a). Oily; 1H NMR (400 MHz, CDCl3) 5 7.54 (s, 2H, ArH), 8.06 (s, 1H, ArH), 1-Iodo-2-isothiocyanato-4,5-dimethyl-benzene (11a). White solid; m.p. 54 °C; 1H NMR (400 MHz, CDCl3) 5 2.18 Kuotsu et al.: A Novel One-pot Synthesis of Isothiocyanates 838 Acta Chim. Slov. 2017, 64, 832-841 (s, 3H, CH3), 2.22 (s, 3H, CH3), 6.99 (s, 1H, ArH), 7.30 (s, 1H, ArH); 13C NMR (100 MHz, CDCl3) 5 19.4, 19.5, 117.2, 124.1, 127.8, 129.3, 133.8, 137.4, 137.9; IR (KBr) 2922, 2853, 2192, 2118, 1472, 1372, 1273, 1076, 1048, 933, 882, 824, 708 cm-1. Anal. Calcd for C9H8INS: C, 37.39; H, 2.79; N, 4.84; S, 11.09. Found: C, 37.42; H, 2.81; N, 4.81; S, 11.06. MS (AP+) Calcd for C9H8INS: 288.94. Found: 288.97 (M+). 5-(Isothiocyanatomethyl)benzo[d][1,3]dioxole (12a). Reddish oil. 1H NMR (400 MHz, CDCl3) 5 4.59 (s, 2H, CH2), 5.98 (s, 2H, CH2), 6.74-6.80 (m, 3H, ArH). 13C NMR (100 MHz, CDCl3) 5 48.7, 101.5, 107.7, 108.6, 120.7, 128.0, 132.1, 147.8, 148.2. IR (KBr) 2895, 2087, 1503, 1445, 1369, 1322, 1251, 1101, 1028, 924 cm-1. Isothiocyanato-cyclohexane (13a). 1H NMR (400 MHz, CDCl3) 5 1.28-1.96 (m, 10H, CH2), 3.67 (m, 1H, CH). 13C NMR (100 MHz, CDCl3) 5 23.0, 24.9, 33.0, 55.2, 129.6. IR (KBr) 2937, 2858, 2175, 2102, 2060, 1450, 1361, 1320, 986, 891, 720, 702 cm-1. Anal. Calcd for C7H11NS (141.23): C, 59.53; H, 7.85; N, 9.92; S, 22.70. Found: C, 59.50; H, 7.81; N, 9.88; S, 22.74. 1-Isothiocyanato-n-butane (14a). Oily liquid. 1H NMR (400 MHz, CDCl3) 5 0.92 (t, J = 7.4 Hz, 3H, CH3), 1.371.47 (m, 2H, CH2), 1.61-1.69 (m, 2H, CH,), 3.42 (t, J = 6.6 Hz, 2H, CH2). 13C NMR (100 MHz, CDCl3) 5 13.2, 19.7, 31.9, 44.7, 129.4. IR (KBr) 2925, 2088. 1597, 1401, 1218, 1116, 753 cm-1. 1-Isothiocyanato-octadecane (15a). Gummy, 1H NMR (CDCl3, 400 MHz) 5 0.879 (t, J = 6.8 Hz, 3H, CH3), 1.25 (m, 28H, CH2), 1.71-1.72 (m, 4H, CH2), 3.50 (t, J = 6.4 Hz, 2H, CH2); 13C NMR (CDCl3, 100 MHz) 5 14.3, 22.8, 26.7, 29.0, 29.5, 29.7, 29.8, 30.1, 32.1, 45.2; IR (KBr) 2923, 2853, 2185, 2096, 1463, 1455, 1346, 721 cm-1 Anal. Calcd for C19H37NS: C, 73.24; H, 11.97; N, 4.50; S, 10.29. Found: C, 73.27; H, 12.01; N, 4.48; S, 10.25. MS (ES-) Calcd for C19H37NS: 311.26. Found: 311.19 (M+). Phenyl cyanamide (1b). Gummy; 1H NMR (CDCl3, 400 MHz) 5 7.02-7.07 (m, 3H, ArH), 7.28-7.33 (m, 2H, ArH), 7.64 (brs, 1H, NH). 13C NMR (100 MHz, CDCl3) 5 112.2, 115.5, 123.6, 129.8, 137.4. IR (KBr) 3175, 2919, 2227, 1600, 1501, 1249, 748, 689 cm-1. Anal. Calcd for C7H6N2 (118.13): C, 71.17; H, 5.12; N, 23.71. Found: C, 71.27; H, 5.09; N, 23.67. 2-Fluorophenyl cyanamide (2b). White solid; m.p. 95 °C; 1H NMR (CDCl3, 400 MHz) 5 6.87 (brs, 1H, NH), 6.907.45 (m, 4H, ArH). 13C NMR (100 MHz, CDCl3) 5 110.9, 115.7, 115.9, 116.8, 124.1, 124.1 125.09, 125.12, 125.6, 125.8, 150.1, 152.5. IR (KBr) 3068, 2037, 1606, 1587, 1495, 1265, 1212, 1104, 941, 808, 752 cm-1. Anal. Calcd for C7H5FN2 (136.13): C, 61.76; H, 3.70; N, 20.58. Found: C, 61.80; H, 3.73; N, 23.53. 2-Chlorophenyl cyanamide (3b). White solid; m.p. 101103 °C; 1H NMR (CDCl3, 400 MHz) 5 6.56 (brs,1H, NH), 7.05 (m, 1H, ArH), 7.31 3m, 2H, ArH), 7.35 (m, 1H, ArH). 13C NMR (100 MHz, CDCl3) 5 110.0, 116.2, 120.4, 124.5, 128.6, 129.9, 134.3. IR (KBr) 3163, 2921, 2243, 1598, 1500, 1426, 1295, 1049 cm-1. Anal. Calcd for C7H5ClN2 (152.58): C, 55.10; H, 3.30; N, 18.36. Found: C, 55.11; H, 3.32; N, 18.29. 3-Chlorophenyl cyanamide (4b). White solid; m.p. 9395 °C; 1H NMR (CDCl3, 400 MHz) 5 6.92 (m, 1H, ArH), 7.03 (m, 2H, ArH), 7.26 (t, J = 8.0 Hz, 1H, ArH). 13C NMR (100 MHz, CDCl3) 5 111.1, 113.8, 115.9, 124.0, 130.9, 135.7, 138.7. IR (KBr) 3154, 2910, 2237, 1602, 1513, 1423, 1256 cm-1. Anal. Calcd for C7H5ClN2 (152.58): C, 55.10; H, 3.30; N, 18.36. Found: C, 55.10; H, 3.29; N, 18.29. MS (ESI): 152 (M+). 3-Nitrophenyl cyanamide (5b). Yellow solid; m.p. 133135 °C; 1H NMR (CDCl3 + DMSO, 400 MHz) 5 7.38 (d, J = 8.4 Hz, 1H, ArH), 7.52 (t, J = 8.4 Hz, 1H, ArH), 7.85 (m, 2H, ArH). 13C NMR (100 MHz, CDCl3 + DMSO) 5 109.6, 110.7, 116.8, 120.8, 130.1, 139.9, 148.4. IR (KBr) 3147, 2919, 2241, 1621, 1531, 1354, 1260, 1071, 937, 871 cm-1. Anal. Calcd for C7H5N3O2 (163.14): C, 51.54; H, 3.09; N, 25.76. Found: C, 51.58; H, 3.12; N, 25.71; MS (ESI): 163 (M+). 4-Chlorophenyl cyanamide (6b). White solid; m.p. 95 °C; 1H NMR (CDCl3, 400 MHz) 5 6.91 (d, J = 8.0 Hz, 2H, ArH), 7.28 (d, J = 8.0 Hz, 2H, ArH). 13C NMR (100 MHz, CDCl3) 5 111.4, 116.9, 128.9, 129.9, 136.2. IR (KBr) 3166, 2954, 2234, 1600, 1494, 1251, 1091 cm-1. Anal. Calcd for C7H5ClN2 (152.58): C, 55.10; H, 3.30; N, 18.36. Found: C, 557.09; H, 3.33; N, 18.32. 4-Methoxyphenyl cyanamide (7b). White solid; m.p. 8689 °C; 1H NMR (CDCl3, 400 MHz) 5 3.78 (s, 3H, CH3), 6.87 (d, J = 8.8 Hz, 2H, ArH), 6.95 (d, J = 8.8 Hz, 2H, ArH). 13C NMR (100 MHz, CDCl3) 5 55.8, 112.8, 115.2, 117.0, 130.6, 156.1. IR (KBr) 3180, 2926, 2218, 1456, 1295, 1238, 1105, 1037, 826 cm-1. Anal. Calcd for C7H6N2O (148.17): C, 64.85; H, 5.44; N, 18.91. Found: C, 64.91; H, 5.40; N, 18.93. 4-Hydroxyphenyl cyanamide (8b). White solid; m.p. 259261 °C; 1H NMR (CDCl3 + DMSO, 400 MHz) 5 5.67 (brs, 1H, NH), 6.77 (d, J = 8.8 Hz, 2H, ArH), 6.83 (d, J = 8.8 Hz, 2H, ArH), 8.98 (brs, 1H, OH). 13C NMR (100 MHz, CDCl3 + DMSO) 5 112.8, 115.6, 115.8, 129.5, 152.2. IR (KBr) 3213, 2992, 2230, 1613, 1519, 1444, 1258, 1224 cm-1. Anal. Calcd for C7H6N2O (134.14): C, 62.68; H, 4.51; N, 20.88. Found: C, 62.72; H, 4.55; N, 20.83. 4-Acetylphenyl cyanamide (9b). White solid; m.p. 153157 °C; 1H NMR (CDCl3 + DMSO, 400 MHz) 5 2.56 (s, Kuotsu et al.: A Novel One-pot Synthesis of Isothiocyanates ... Acta Chim. Slov. 2017, 64, 832-839 841 3H, CH3), 7.08 (d, J = 8.8 Hz, 2H, ArH), 7.91 (d, J = 8.8 Hz, 2H, ArH). 13C NMR (100 MHz, CDCl3 + DMSO) 5 25.9, 110.9, 114.5, 129.8, 131.2, 142.9, 196.2. IR (KBr) 3188, 2966, 2228, 1666, 1599, 1585, 1411, 1362, 1278, 1176, 962 cm-1. Anal. Calcd for C9H8N2O (160.18): C, 67.49; H, 5.03; N, 17.48. Found: C, 67.53; H, 5.08; N, 17.44. MS (ESI): 160 (M+). 2-Iodo-4-methylphenyl cyanamide (10b). White solid; m.p. 144 °C; 1H NMR (CDCl3, 400 MHz) 5 2.29 (s, 3H, CH3), 6.17 (brs, 1H, NH), 7.17 (dd, J = 8.2 Hz, 2H, ArH), 7.56 (s, 1H, ArH). 13C NMR (100 MHz, CDCl3) 5 20.4, 84.2, 110.7, 115.4, 130.9, 135.4, 139.6. IR (KBr) 3229, 2919, 2217, 1603, 1573, 1502, 1420, 1383, 1283, 1032, 866, 805 cm-1. Anal. Calcd for C8H7N2 (258.06): C, 37.23; H, 2.73; N, 10.86. Found: C, 37.27; H, 2.75; N, 10.84. «-Butyl cyanamide (11b). Gummy; 1H NMR (CDCl3, 400 MHz) 5 0.94 (t, J = 7.6 Hz, 3H, CH3), 1.40 (m, 2H, CH2), 1.58 (m, 2H, CH2), 3.06 (m, 2H, CH2), 4.61 (brs, 1H). 13C NMR (100 MHz, CDCl3) 5 13.6, 19.5, 31.7, 45.7, 117.2. IR (KBr) 3207, 2961, 2875, 2221, 1614, 1463, 1373, 1171 cm-1. Anal. Calcd for C5H10N2 (98.15): C, 61.19; H, 10.27; N, 28.54. Found: C, 61.21!; if, 10.23; N, 28.48. Cyclohexyl cyanamide (12b). Gummy; 1H NMR (CDCl3, 400 MHz) 5 1.31 (m, 5H, CH2), 1.61 (m, 1H, CH2), 1.78 (m, 2H, CH2), 1.95 (m, 2H, CH2), 3.09 (m, 1H, CH2), 3.91 (brs, 1H, NH). 13C NMR (100 MHz, CDCl3) 5 24.3, 25.1, 32.6, 54.3, 115.9. IR (KBr) 3196, 2933, 2857, 2217, 1453, 1367, 1167 cm-1. Anal. Calcd for C7H12N2 (124.19): C, 67.70; H, 9.74; N, 22.56. Found: C, 67.(57; H, 9.70; N, 22.50. Benzyl cyanamide (13b). Gummy; 1H NMR (CDCl3, 400 MHz) 5 4.11 (d, J = 5.2 Hz, 2H, CH2 CH2), 4.66 (brs, 1H, NH), 7.27-7.37 (m, 5H, ArH). 13C NMR (100 MHz, CDCl3) 5 49.9, 116.7, 127.9, 128.4, 128.9, 136.4. IR (KBr) 3207, 2925, 2220, 1455, 1359, 1155, 1014 cm-1. Anal. Calcd for C8H8N2 (132.17): C, 72.70; H, 6.10; N, 21.19. Found: C, 72.66; H, 6.13; N, 21.11. Benzo[1,3]dioxol-5-ylmethyl cyanamide (14b). White solid; m.p. 82-84 °C; 1H NMR (CDCl3, 400 MHz) 5 4.05 (d, J = 5.2 Hz, 2H, CH2), 4.57 (brs, 1H, NH), 5.94 (s, 2H, OCH2), 6.77 (m, 3H). 13C NMR (100 MHz, CDCl3) 5 49.9, 101.42 108.46, 108.54, 116.5, 121.7, 130.1, 147.8, 148.2. IR (KBr) 3233, 2952, 2897, 2220, 1500, 1445, 1038, 925, 809 cm-1. Anal. Calcd for C9H8N2O2 (176.18): C, 61.36; H, 4.58; N, 15.90. Found: C, 61.41; H, 4.61; N, 15.85. 3,4-Dimethoxyphenylethyl cyanamide (15b). Gummy; 1H NMR (CDCl3, 400 MHz) 5 2.84 (t, 2H, CH2), 3.28 (q, J = 7.2 Hz, 2H, CH2), 3.83 (s, 3H, CH3), 3.84 (s, 3H, CH3), 4.37 (brs, 1H, NH), 6.76 (m, 3H, ArH). 13C NMR (100 MHz, CDCl3) 5 35.5, 47.5, 55.92, 55.95, 111.4, 111.9, 116.5, 120.9, 130.0,147.8, 148.9. IR (KBr) 3274, 2937, 2219, 1592, 1517, 1464, 1262, 1236, 1156, 1142, 1026, 913 cm-1. Anal. Calcd for C11H14N2O2(206.24): C, 64.06; H, 6.84; N, 13.58. Found: C, 64.12; H, 6.80; N, 13.54. 5. Conclusion In conclusion, we have developed a general, economical and environmentally benign method for the preparation of isothiocyanates and cyanamides from their corresponding dithiocarbamic acid salts. The use of non-toxic and eco-friendly reagents and solvents without the formation of any side products makes this methodology potentially useful. The yield could in fact be considered as very good if not excellent. Acknowledgements UBS acknowledges the financial support received from UGC (UGC/MRP 43-192/ 2014). N. B. Kuotsu acknowledges the support of this research from UGC (F.5.77/2014-15/MRP/NERO) Thanks are due to CIF/IIT Guwahati for NMR spectra 6. References 1. (a) A. K. Mukerjee, R. Ashare, Chem. Rev. 1991, 91, 1-24. DOI: 10.1021/cr00001a001 (b) M. Dobosz, M. Wujec, Heterocycles, 2002, 57, 1135-1141. DOI: 10.3987/C0M-02-9461 2. (a) N. Kuhnert, G. Williamson, B. Holst, J. Labl. Comp. Radi-opharm. 2001, 44, 347-355. DOI:10.1002/jlcr.462 (b) N. Kuhnett, Y. Lu, Labl. Comp. Radiopharm. 2004, 47, 501-507. DOI:10.1002/jlcr.833 3. C. Nastruzzi, R. Cotesi, E. Eposito, E. Menegatti, O. Leoni, R. Ironi, S. J. Palmier, Agric. Food. Chem. 2000, 48, 3572-3575. DOI:10.1021/jf000191p 4. K. Xu, P. Thornalley, J. Biochem. Pharmacol. 2000, 6, 221-231. DOI:10.1016/S0006-2952(00)00319-1 5. C. X. Zhang, K. Y. Lee, A. J. Kelly, R. T. Burke, J. Org. Chem. 2000, 65, 6237-6240. DOI:10.1021/jo000139s 6. (a) J. E. Hodgkins, W. P. Reeves, J. Org. Chem. 1956, 21, 404405. DOI:10.1021/jo01110a006 (b) J. E. Hodgkins, W. P. Reeves, J. Org. Chem. 1964, 29, 30983099. DOI:10.1021/jo01033a524 7. C. G. Cho, G. H. Posne, Tetrahedron Lett. 1992, 33, 35993602. DOI:10.1016/S0040-4039(00)92512-7 8. T. Kitamura, S. Kobayashi, H. Taniguchi, J. Org. Chem. 1990, 55, 1801-1805. DOI:10.1021/jo00293a025 9. J. N. Kim, K. S. Jung, J. H. Lee, J. S. Son, Tetrahedron Lett. 1997, 38, 1597-1598. DOI:10.1016/S0040-4039(97)00121-4 10. J. Goerdeler, C. Ho, Chem. Ber. 1984, 117, 1636-1639. DOI: 10.1002/cber.19841170431 11. H. M. Mesheram, S. Dale, J. S. Yadav, Tetrahedron Lett. 1997, 38, 8743-8744. DOI:10.1016/S0040-4039(97)10158-7 12. A. Toshimitsu, S. Uemura, M. Okano, N. Watanabe, J. Org. Chem. 1983, 48, 5246-5251. DOI:10.1021/jo00174a018 13. D. Albanese, M. Penso, Synthesis 1991, 1001-1002. Kuotsu et al.: A Novel One-pot Synthesis of Isothiocyanates 840 Acta Chim. Slov. 2017, 64, 832-841 DOI:10.1055/s-1991-26629 14. W. G. Shan, G. F. Bian, W. K. Su, X. R. Liang, Org. Prep. Pro-ced. Int. 2004, 36, 283-286. DOI: 10.1080/00304940409355967 15. J. N. Kim, E. K. Ryu, Tetrahedron Lett. 1993, 34, 8283-8284. D0I:10.1016/S0040-4039(00)61411-9 16. J. N. Kim, K. S. Jung, H. J. Lee, J. S. Son, Tetrahedron Lett. 1997, 38, 1597-1598. D0I:10.1016/S0040-4039(97)00121-4 17. W. Adam, R. M. Bargon, S. G. Bosio, W. A. Schenk, D. Stalke, J. Org. Chem. 2002, 67, 7037-7041. D0I:10.1021/jo026042i 18. M. Arisawa, M. Ashikawa, A. Suwa, M. Yamaguchi, Tetrahedron Lett. 2005, 46, 1727-1729. D0I:10.1016/j.tetlet.2005.01.069 19. L. Valette, S. Poulain, X. Fernandez, L. Lizzani-Cuvelier, J. Sulfur Chem. 2005, 26, 155-161. DOI: 10.1080/17415990500070144 20. T. Isoda, K. Hayashi, S. Tamai, T. Kumagai, Y. Nagao, Chem. Pharm. Bull. 2006, 54, 1616-1619. D0I:10.1248/cpb.54.1616 21. B. Zhong, R. S. Al-Awar, C. Shih, J. H. Grimes Jr., M. Vieth, C. Hamdouchi, Tetrahedron Lett. 2006, 47, 2161-2164. D0I:10.1016/j.tetlet.2006.01.119 22. W. J. Neely, Aust. J. Chem. 1960, 13, 341-346. D0I:10.1071/CH9600341 23. M. Bollini, J. J. Casal, D. E. Alvarez, L. Boiani, M. González, H. Cerecetto, A. M. Bruno, Bioorg. Med. Chem. 2009, 17, 1437-1444. D0I:10.1016/j.bmc.2009.01.011 24. E. Dyer, T. B. Johnson, J. Am. Chem. Soc. 1932, 54, 777-787. D0I:10.1021/ja01341a048 25. G. M. Dyson, T. Harrington, J. Chem. Soc. 1942, 374-375. D01:10.1039/JR9420000374 26. J. C. Jochims, A. Seeliger, Tetrahedron 1965, 21, 2611-2616. D0I:10.1016/S0040-4020(01)93917-1 27. R. Gottfried, Angew. Chem., Int. Ed. Engl. 1966, 5, 963-964. D01:10.1002/anie.196609632 28. C. Larsen, K. Steliou, D. N. Harpp, J. Org. Chem. 1978, 43, 337-339. D0I:10.1021/jo00396a035 29. C. Larsen, D. N. Harpp, J. Org. Chem. 1981, 46, 2465-2466. D01:10.1021/jo00325a007 30. S. Kim, K. Y. Yi, J. Org. Chem. 1986, 51, 2613-2615. D01:10.1021/jo00363a046 31. S. Kim, K. Y. Yi, Tetrahedron Lett. 1985, 26, 1661-1664. D01:10.1016/S0040-4039(00)98578-2 32. J. Grayson, I. Org. Process Res. Dev. 1997, 1, 240-246. D0I:10.1021/op970002c 33. (a) A. Servais, M. Azzouz, D. Lopes, C. Courillon, M. Malac-ria, Angew. Chem. Int. Ed. 2007, 46, 576-579. D0I:10.1002/anie.200602940 (b) M. H. Larraufie, C. Ollivier, L. Fensterbank, M. Malacria, E. Laocte, Angew. Chem. Int. Ed. 2010, 49, 2178-2181. D01:10.1002/anie.200907237 (c) Z. Pan, S. M. Pound, N. R. Rondla, C. J. Douglas, Angew. Chem., Int. Ed. 2014, 53, 5170-5174. (d) H. Basavaprabhu, V. V. Sureshbabu, Org. Biomol. Chem. 2012, 10, 2528-2533. D0I:10.1039/c2ob06916d (e) V. Panduranga, H. Basavaprabhu, V. V. Sureshbabu, Tetrahedron Lett. 2013, 54, 975-979. D01:10.1016/j.tetlet.2012.12.027 (f) S. Kamijo, T. Jin, Y. Yamamoto, Angew. Chem. Int. Ed. 2002, 41, 1780-1782. D0I:10.1002/1521-3773(20020517) 41:10<1780::AID-ANIE1780>3.0.C0;2-# (g) L. V. R. Bonaga, H. C. Zhang, B. E. Maryanoff, Chem. Commun. 2004, 2394-2395. D0I:10.1039/B410012C (h) K. Fukumoto, T. Oya, M. Itazaki, H. Nakazawa, J. Am. Chem. Soc. 2009, 131, 38-39. D0I:10.1021/ja807896b (i) R. L. Giles, J. D. Sullivan, A. M. Steiner, R. E. Looper, Angew. Chem., Int. Ed. 2009, 48, 3116-3120. D0I:10.1002/anie.200900160 (j) S. Guin, S. K. Rout, A. Gogoi, W. Ali, B. K. Patel, Adv. Synth. Catal. 2014, 356, 2559-2565. D0I:10.1002/adsc.201400011 (k) T. K. Lane, B. R. D'Souza, J. Louie, J. Org. Chem. 2012, 77, 7555-7563. (l) V. Kumar, M. P. Kaushik, A. Mazumdar, Eur. J. Org. Chem. 2008, 1910-1916. D0I:10.1021/jo3012418 34. (a) C. J. Adams, J. Chem. Soc., Dalton Trans. 1999, 2059-2064. D0I:10.1039/a901959f (b) A. S. Smimov, E. S. Butukhanova, N. A. Bokach, G. L. Starova, V. V. Gurzhiy, M. L. Kuznetsov, V. Y. Kukushkin, Dalton Trans. 2014, 43, 15798-15811. D0I:10.1039/C4DT01812E (c) M. Yuan, S. Gao, H. L. Sun, G. Su, Inorg. Chem. 2004, 43, 8221-8223. D0I:10.1021/ic048547s 35. (a) D. G. Barret, D. N. Deaton, A. M. Hassell, R. B. McFadyen, A. B. Miller, L. R. Miller, J. A. Payne, L. M. Shewchuk, D. H. Willard, L. L. Wright, Bioorg. Med. Chem. Lett. 2005, 15, 3039-3043. D0I:10.1016/j.bmcl.2005.04.032 (b) H. W. K. Bischofsheim, H. J. L. Hogheim, J. R. S. Kelkheim, A. W. Egelsbach, S. F. Idstein, H. W. J. Niedernhausen, S. P. Frankfurt, US 6369069B1, 2002. (c) K. S. Atwal, G. J. Grover, S. Z. Ahmed, P. G. Sleph, S. Dzwonczyk, A. J. Baird, D. E. Normandin, J. Med. Chem. 1995, 38, 3236-3245. D0I:10.1021/jm00017a007 (d) P. W. Manley, U. Quast, J. Med. Chem. 1992, 35, 23272340. D0I:10.1021/jm00090a025 (e) G. Danger, A. Michaut, M. Bucchi, L. Boiteau, J. Canal, R. Plascal, Angew. Chem. Int. Ed. 2013, 52, 611-614. D0I:10.1002/anie.201207730 36. T. Kamo, M. Endo, M. Sato, R. Kasahara, H. Yamaya, S. Hi-radate, Y. Fujii, N. Hirai, M. Hirota, Phytochemistry 2008, 69, 1166-1172. D01:10.1016/j.phytochem.2007.11.004 37. (a) B. J. Von, Ber. Dtsch. Chem. Ges. 1900, 33, 1438-1452. D0I:10.1002/cber.19000330208 (b) L. Y. Hu, J. Guo, S. S. Mager, J. B. Fischer, K. J. Burke-How-ie, G. J. Durant, Ber. Dtsch. Chem. Ges. 1900, 33, 1438-1452. D0I:10.1002/cber. 19000330208 (c) G. Kaupp, J. Schmeyers, J. Boy, Chem.-Eur. J. 1998, 4, 24672474. D0I:10.1002/(SICI)1521-3765(19981204)4:12<2467: :AID-CHEM2467>3.0.C0;2-D 38. (a) W. A. Davis, M. P. Cava, J. Org. Chem. 1983, 48, 27742775. D0I:10.1021/jo00164a030 (b) D. Kahne, D. Collum, Tetrahedron Lett. 1981, 22, 50115014. D0I:10.1016/S0040-4039(01)92406-2 39. K. H. Boltz, H. D. Dell, Justus Liebigs Ann. Chem. 1967, 709, Kuotsu et al.: A Novel One-pot Synthesis of Isothiocyanates ... Acta Chim. Slov. 2017, 64, 832-841 841 63-67. D01:10.1002/jlac.19677090107 40. M. E. Hermes, F. D. Marsh, J. Org. Chem. 1972, 37, 29692979. DOI: W.1021/jo00984a013 41. T. V. Hughes, S. D. Hammond, M. P. Cava, J. Org. Chem. 1998, 63, 401-402. D0I:10.1021/jo9717548 42. R. C. Wheland, E. L. Martin, J. Org. Chem. 1975, 40, 31013109. D0I:10.1021/jo00909a019 43. Y. Q. Wu, D. C. Limburg, D. E. Wilkinson, G. S. Hamilton, Org. Lett. 2000, 2, 795-297. D0I:10.1021/ol0055263 44. J. J. Kim, D. J. Kweon, S. D. Cho, K. H. Kim, E.Y. Jung, S. G. Lee, J. R. Falck, Y. J. Yoon, Tetrahedron 2005, 61, 5889-5894. D0I:10.1016/j.tet.2005.03.138 45. S. A. Bakunov, A. V. Rukavishnikov, A. V. Tkachev, Synthesis 2000, 1148-1159. D0I:10.1055/s-2000-6317 46. K. Shin, J. Tienan, Y. Yoshinori, J. Am. Chem. Soc. 2001, 123, 9453-9454. D01:10.1021/ja016355f 47. (a) F. F. Wong, C. Y. Chen, M. Y. Yeh, Synlett 2006, 559-562. DOI: 10.1055/s-2006-932470 (b) C. Y. Chen, F. F. Wong, J. J. Haung, S. K. Lin, M. Y. Yeh, Tetrahedron Lett. 2008, 49, 6505-6507. D0I:10.1016/j.tetlet.2008.08.106 48. K. H. Chaudhuri, U. S. Mahajan, D. S. Bhalerao, K. G. Akamanchi, Synlett 2007, 2815-2818. 49. J. Nath, B. Patel, L. Jamir, U. B. Sinha, K. V. V. V. Satyanaraya-na, Green Chem. 2009, 11, 1503-1506. D0I:10.1039/b914283p Povzetek Opisujemo zelo učinkovit in enostaven pristop k sintezi izotiocianatov in cianamidov iz ustreznih aminov ob prisotnosti blagega, učinkovitega in nestrupenega reagenta tetrapropilamonijevega tribromida. Dobra okoljska sprejemljivost, stroškovna učinkovitost in visoki izkoristki so glavne odlike te metodologije. Kuotsu et al.: A Novel One-pot Synthesis of Isothiocyanates 842 DOI: 10.17344/acsi.2017.3357 Acta Chim. Slov. 2017, 64, 842-848 /^creative ty com mor Scientific paper Prediction of Physico-chemical Properties of Bacteriostatic ^-Substituted Sulfonamides: Theoretical and Experimental Studies Hossein Nikoofard,1* Mohsen Sargolzaei1 and Farnosh Faridbod2 1 Faculty of Chemistry, Shahrood University of Technology, Shahrood 63199-95161, Iran. 2 Center of Excellence in Electrochemistry, Faculty of Chemistry, University of Tehran, Tehran, Iran * Corresponding author: E-mail: hnikoofard@shahroodut.ac.ir Received: 13-03-2017 Abstract A computational study at the density functional theory (DFT) as well as electrochemical methods, was carried out on the structural and physico-chemical properties of a series of sulfonamide derivatives (SAs) as WHO essential medications in the treatment of basic health system. The B3LYP/6-311++G(d,p) level of theory carried out on sulfadiazine (SDZ), sul-fathiazole (STZ), sulfaquinoxaline (SQX), sulfacetamide (SAA), and the reference unsubstituted sulfonamide (SA) was discussed and rationalized in term of the N1-sulfonamide substituent. The geometric structures and the electronic properties related to the bacteriostatic reactivity were revealed to be affected by the steric and "push-pull" characteristics of the substituents. Electrochemical experiments on oxidation of SAs, using cyclic voltammetry are presented. The results obtained showed that the calculated ionization potentials (IPs) could be correlated linearly with the electro-oxidation potentials. From the molecules studied it is evident that SDZ act as the most electro-active agent, possessing the highest biological activity. DFT computations carried out using the standard molar enthalpies of formation in the gas phase predicted improvements in the thermodynamic stabilization of the SDZ, SQX, and SAA molecules and an unstabilization of STZ with respect to the parent molecule SA. Keywords: Sulfonamide, Substituent, Density functional theory, Electrochemical behavior 1. Introduction Sulfa drugs (sulfonamides), characterized by a p-aminophenyl group and a sulfonamido one (-SO2N-) in their molecular structures, are the basis of some important bacteriostatic agents. These molecules represent a substantial class of pharmaceutical compounds, which are extensively employed as chemotherapeutic agents,1 and anti-tu-mor,2 anti-thyroid,3 anti-carbonic anhydrase,4 antiinflammatory,5 hypoglycemic,6 diuretic,7 COX-inhibitors, and anti-impotence drugs,8 and also have been used as azo dyes to achieve improved light stability, water solubility, and fixation to fiber. Sulfonamides act as competitive inhibitors of the enzyme dihydropteroate synthase in bacteria, and catalyze changing p-aminobenzoic acid into a nutrient necessary for some bacteria.9 Some sulfonamide derivatives are still extensively used for the treatment of numerous bacterial, fungal infections, protozoal, and the first effective chemotherapeutic agents used in safe therapeutic dosage ranges.10 Due to their biological and pharmaceuti- cal ingredients, sulfonamide derivatives find a lot of importance in the literature related to the synthesis of new classes of compounds.11-15 Molecular structure of the sulfa drug is analogous to that for p-aminobenzoic acid (PABA) (Scheme 1). The similarity between them has been shown by the Wood-fielder theory.16 According to Bell and Robin,17 a structure that is comparable to the molecular structure of PABA may interfere within its biological function. The sul-fonamide mechanism has been recognized at the enzyme level. In bacteria, anti-bacterial sulfonamides act as the competitive inhibitors of dihydropteroate synthetase. Hence, sulfonamide interferes with the enzyme folic acid synthetase, which is involved in changing PABA to folic acid, which results in the deficiency of folic acid, causing injury to the bacterial cell. Most bacteriostatic SAs have been derivatized basically by variation in the R-substitu-tion linked to the N1 atom of the sulfonamido group (Scheme 1). Substitution with a heterocyclic structure has Nikoofard et al.: Prediction of Physico-chemical Properties Acta Chim. Slov. 2017, 64, 842-848 843 Scheme 1. Sketch map of PABA structure and SA structure with N atom numbering. produced compounds more active than the parent molecule SA (R = H). More widespread experimental studies carried out by Bell,17 Kumler,18 Seydel,19 and others have been devoted to the analysis of the structure-electronic effects that could be related to the pharmaceutical activities, paying special interest to the role of the acidity of the sul-fonamido group.20-22 Sulfonamides are weak acids compared to carboxylic acid amides. Their acidic nature results from the ability of the SO2 moiety to stabilize the nitrogen anion via resonance. It has been found that their pharmaceutical activity is favored directly by the increased sulfonamide acidity (related to the lower pKa values). The relationship between the chemical structure and pharmaceutical activity of the SA derivatives has prompted the current experimental and theoretical investigations for new sulfa compounds that would possess a greater pharmacological activity.23-26 In this way, sulfadiazine is one of the substantial sulfonamide antibiotics that are listed as WHO crucial medications in the cure of basic health system.15 In comparison to the SA derivatives, it has been found that the original SA is at the lowest end of the activity spectrum. Although these drugs are clinically effective in the treatment of various medical disorders, they cause some negative side-effects, which may lead to hepatitis and arthritis. Through relocation, these drugs reach the environment and cause acute toxicity and serious public health hazards.14 Now we wish to report a theoretical investigation carried out on the SA derivatives including SAA, SDZ, SQX, STZ, and the original sulfonamide SA as the reference molecule, which can be used to evaluate the relevance of the physico-chemical and structural properties toward the steric and electronic influences of the N1-substituents. The R groups were chosen by taking into account the structures of some sulfonamide-based drugs. The sketch map of the substituent structures for all the studied SAs is depicted in Scheme 2. Investigations have been carried out to identify the geometric structure, energy disparity be- tween the lowest unoccupied molecular orbital (LUMO) and the highest occupied molecular orbital (HOMO), atomic and group Mulliken charge distribution, and some appropriate quantum descriptors. Since electrochemical methods are accurate, simple and economical in terms of both time and cost for drug residue monitoring as well as control processes, electro-oxidation behavior of SAs was initially studied on a carbon paste electrode (CPE) using cyclic voltammetry technique. 2. Methodology 2. 1. Computation Method The ground-state geometry of each sulfonamide was fully optimized using the gradient procedure at the restricted DFT technique, as implemented in the Gaussian 09 program package.27 A preliminary basis set test carried out for calculations on the electronic ground state for the unsubstituted reference molecule (SA), showed that that 6-311++G(d,p) was the best basis set that can be used within our available hardware/software facilities within a reasonable time. The reports by others28,29 have also shown that the B3LYP/6-311+G(d,p) level of theory appeared notably adapted to describe sulfonamides to obtain experimental data. The fully-optimized structures by the DFT-B3LYP level30,31 were confirmed to be the real minima through the zero imaginary frequencies. For all cases, both the radical cation and radical anion were treated as open-shell systems by UB3LYP/6-311++G(d,p). In particular, the values for the electronic chemical potential (^), global hardness (n), electrophilicity index (w), and maximal flow of electrons (ANMax) were determined using the Koopman's theorem eigenvalues.32,33 Furthermore, the standard molar enthalpies of formation in the gas phase at 298.15 K for all the studied species were estimated by the atomization energy route. The detailed description of this calculation procedure has been reported in the literature.34 Scheme 2. Sketch map of R group structure for all studied SAs. Nikoofard et al.: Prediction of Physico-chemical Properties ... 844 Acta Chim. Slov. 2017, 64, 842-848 2. 2. Experimental All chemical material used for this project were reagent grade from Merck and used as received. A na-no-composite modified carbon past electrode consisted of 5% reduced graphene oxide (RGO) decorated CeO2 na-no-particle was employed to study the electro-oxidation of SAs solution using cyclic voltammetry. A homemade ul-tra-voltammetry system designed in Center of Excellence Electrochemistry (CEE) of University of Tehran was used for the electrochemical measurements at 25.0 ± 0.1 °C. A stock solution (1mM) for each SA derivatives was prepared and kept in refrigerator. Standard solution for measurements was prepared by dilution of stock solution by phosphate buffer (pH 7.4). The external electrode surface was smoothed with a clean and soft paper. A new surface was produced by scraping out the old surface and replacing the carbon paste. 3. Results and Discussion 3. 1. Geometric Structures of SAs Full-optimized geometrical structure of each studied sulfonamide obtained at the B3LYP/6-311 ++G(d,p) level of theory are given in Fig. S1 in the Supporting Information. In Table 1, some selected dihedral (D) and bond (A) angles of the SA derivatives are tabulated. According to this table (column 2), for the dihedral angle DN4ph, defining the torsion between the amino group (NH2) and phe-nyl ring (ph), a value of 178o is an evidence of complete planarity, and our calculated results indicated that all SAs presented values for DN4-ph close to 180o. It is interesting that introduction of the selected R groups did not affect the planarity of the amino group and phenyl ring present in the parent molecule (SA). It has been found that this planarity in the SA drugs is a necessary condition for a pharmaceutical activity.23 The dihedral angle between the SO2 group and the phenyl ring, Dph SO2, was also close to 180° in all SAs (Table 1, column 3).FH[owever, for the SAA, SDZ, and SQX species, the C-S-N1 bond angle (A C ) displayed a value of nearby 105°, although it was found to be 100o for the STZ molecule. This may be attributed to the different steric effects resulting from the proximity of the substituent R to the sulfonamido group. Our calculated results for all SAs revealed that the bond angle for O-S-O Table 1. Dihedral and bond angles (in degrees) for SA species in neutral state at the B3LYP/6-311++G(d,p) level of theory. Species DN4-ph Dph.-S A CSN1 A OSO SA 178 -180 104 122 SAA 178 179 106 122 SDZ 178 179 105 121 SQX 178 179 105 122 STZ 178 180 100 120 was near 120°, indicating that introduction of the R group did not affect it considerably. In the case of the optimized structures in the ground state, some selected bond lengths (d) of the studied molecules are given in Table 2. As we can see in this table, for all SAs, the bond length for N4-ph is 1.38 Â, which is in the order of the C-C bond length in the resonance structure of phenyl ring (on average, 1.39 Â). This means that the amino group is well-conjugated with the phenyl ring, and is not affected by the substituent type. In this way, the N4-ph bond distances for both the radical anion and radical cation species, tabulated in Table S1 of the Supporting Information, indicated that the N4-ph bond length varied in the following order: anionic (1.40 Â) > neutral (1.38 Â) > cat-ionic (1.34 Â). It is interesting that the ph-S and S-N1 bond lengths were not varied considerably by the R groups, where the distance between the S atom and R group changed due to the substituent steric hindrance. It has been established23 that the sulfonamide activity is accompanied by a small distance between the N1 atom and R group corresponding to the large bond order for dN1 R. According to Table 2 (column 5), the SDZ molecule with a shorter N1-R distance possesses a more bacteriostatic activity with respect to the other substituted SAs. It was concluded that the electronic and structural properties of the substituent could contribute to the bacteriostatic activity of a sulfa drug. Table 2. Some selected bond lengths (A) for SA species in neutral state at the B3LYP/6-311++G(dip) level of theory. Species dN4-ph dph-S dS-N1 dN1-R SA 1.3851 1.7835 1.7001 - SAA 1.3803 1.7815 1.7102 1.4022 SDZ 1.3842 1.7773 1.7152 1.3846 SQX 1.3829 1.7758 1.7201 1.3916 STZ 1.3810 1.7787 1.7247 1.4127 3. 2. Electronic Properties of SAs One of the important parameters involved in the bacteriostatic activity of a sulfa drug is the charge distribution of an atom and a group over their molecular structures. We investigated qualitatively the "push-pull" effect of the R substituents on the SA molecules by the Mulliken population analysis. Some atomic and group charge distributions obtained for the neutral and both the radical anion (-) and radical cation (+) of SAs are summarized in Table 3. In all the neutral species, linkage of the R group on the parent molecule SA did not affect the charge on the N4 atom (Table 3, column 2). This is expected because R group is far from it (see Scheme 1). The calculated results show that the influence of substituent introduction is manifested in the increased negative charge on the N1 atom in the SAA, SDZ, and SQX species, and an increased positive Nikoofard et al.: Prediction of Physico-chemical Properties Acta Chim. Slov. 2017, 64, 842-848 845 charge on it in STZ with respect to the reference molecule SA. These are referred to the electron-withdrawing effect of the R group in the SAA, ADZ, and SQX molecules, and the electron-donating effect of the R group in the STZ molecule, respectively. The same trend was observed for the negative charge on the phenyl ring. Depending on the substituent type, the calculated charge at the SO2 group shows a large variation. In both the experimental and theoretical works carried out by Bell et al.17 and Soriano-Cor-rea et al.,23 respectively, the increase in the acidity of SAs (which is equivalent to an increase in the bacteriostatic activity) was found to be related to a reduction in the negative charge of the SO2 group. In other words, the substituent electronegativity is an important parameter that controls the bacteriostatic activity in sulfa drug. Our calculated results showed that SDZ with a more positive charge (or a less negative charge) of the SO2 group can have a higher reactivity (Table 3, column 5). For the case of radical cation species, the main influence of the injection of one positive charge is manifested in the increased positive charge on the N4 atom with respect to the N1 atom. Consequently, the N4 atom in the _p-amino group, which is far from the substitution position, was found to be a more reactive zone to the protonation processes than the N1 atom that is closer to the substituent (see Scheme 1). An inverse trend was observed for the negative charge on the radical anion species. In this regards, the negative charge was distributed mainly on the N1 atom, which provides that the deproton-ation processes were take place most probability at the N1 atom position with respect to the N4 atom. tion systems and improved stabilizations. The extended aromatic structure can correspond to the narrow HO-MO-LUMO (H-L) gap energy, which provides a reasonable qualitative indication of the excitation properties and of the ability of electron or hole transport [35-37]. Table 4 displays the H-L gaps for all the SA molecules. According to this data, reduction in the H-L gap values for both the radical anion and radical cation species becomes more considerable with respect to the ones in the neutral state. Thus we may predict that the SA derivatives have the most reactivity in their ionic forms. It is interesting that the H-L gaps for the R substituted species are lower with respect to the reference SA molecule, which is in good agreement with the less bacteriostatic reactivity of the original SA.23 Among the compounds studied, SDZ and SQX have the lower H-L gaps, indicating that they can be show the higher reactivity. The results obtained revealed that the HO-MO-LUMO electronic transitions could be attributed to the tendency of the considered R groups to contribute the ^-electrons with the molecular system. Table 4. Calculated values for HOMO-LUMO gaps (eV) for studied species in neutral and ionic states. Species Neutral Cationic Anionic SA 0.197 0.074 0.034 SAA 0.192 0.052 0.034 SDZ 0.174 0.050 0.025 SQX 0.166 0.049 0.034 STZ 0.193 0.054 0.034 Table 3. Some atomic and group charge distributions of SAs in neutral and both radical cation (+) and radical anion (-) states. Species N4 N1 ph SO2 SA -0.29 -0.34 -0.14 -0.34 SAA -0.29 -0.40 -0.22 0.03 SDZ -0.29 -0.49 -0.34 0.36 SQX -0.31 -0.36 -0.24 0.23 STZ -0.29 -0.05 -0.07 -0.20 SA(+) -0.18 -0.38 0.30 -0.08 SAA(+) -0.18 -0.33 0.27 0.07 SDZ(+) -0.20 -0.50 -0.01 0.57 SQX(+) -0.26 -0.41 -0.11 0.53 STZ(+) -0.25 -0.23 0.16 0.07 SA(-) -0.23 -0.35 0.12 -0.54 SAA(-) -0.24 -0.39 -0.22 -0.08 SDZ(-) -0.25 -0.41 -0.30 -0.07 SQX(-) -0.30 -0.31 0.00 0.00 STZ(-) -0.25 -0.33 -0.34 -0.42 It is expected that the presence of a desired R substituent on the sulfonamido group could improve the electron delocalization along the molecular structure. For the case of the SA derivatives, delocalization of the ^-electrons onto the molecular backbone led to satisfactory conjuga- According to density functional theory, the energy E can be expressed as a function of the electron number N and as a functional of the external potential v(r). Derivatives of E[N; v(r)] with respect to N and v(r) produce a set of global and local quantities that allow to quantify the concept of reactivity and site selectivity, respectively. The electronic chemical potential the molecular hardness q, the electrophilicity index w, and the maximal flow of electrons that a system may accept ANM are defined as:32,33 ON 2 1 ,d2E 1 (i) (2) (3) (4) where EH and EL are the energies of HOMO and LUMO. These electronic that characterize the charge injection and Nikoofard et al.: Prediction of Physico-chemical Properties ... 846 Acta Chim. Slov. 2017, 64, 842-848 charge transport properties of such materials g are given in Table 5. It was found that the electronic descriptors of SAs are influenced by the electronic and steric properties of the substituent. The values for u, w, and AM. are related with r ' ' Max the escaping tendency of electrons and stabilization energy of the system, and increase with presenting the R group to the parent SA molecule. This indicates that the escaping tendency of electrons in the structures of the SA derivatives, in particular for the deprotonation process, is stabilized by the electron-withdrawing character of the substituent. An inverse trend was observed for the molecular hardness, which points out an obvious substitution effect on the reactivity of the molecules, in particular for the SDZ, SQX, and SAA species. The less change in the electronic properties of the STZ molecule may be referred to the less contribution of its substituent to the conjugated system. Table 5. Calculated values for electronic descriptors u, n, w, and Species ? (eV) n (eV) u> (eV) Max SA -0.136 0.098 0.187 1.377 SAA -0.149 0.097 0.230 1.545 SDZ -0.146 0.087 0.246 1.679 SQX -0.151 0.084 0.271 1.798 STZ -0.141 0.097 0.209 1.411 In order to elucidate the electrochemical behavior of the titled SA derivatives, their voltammetric responses obtained at a carbon past electrode. Owning to non-solubility of SAs in acidic rezones, a phosphate buffer solution with pH = 7.4 was used in the cyclic voltammetry measurements. These compounds can be electrochemical oxidized at the amino group (NH2). Figure 1 shows the cyclic voltammograms of each SAs containing 1.0 mM of sulfacetamide, sulfaquinoxaline, sulfadiazine, and sulfathiazole at the CPE. In all voltammograms, just one oxidation peak was observed which could be expressed as a two-electron, two-proton process via the following reaction: (5) As it can be seen in Figure 1, the anodic peak current and potential at which the oxidation reaction occurs (I and Epa, respectively) are strongly dependent on the characteristic of R group (Scheme 2). Among the SAs, sulfadiazine shows a significant increase in the response current with respect to the other SAs. Under these conditions, it Figure 1. Electrochemical behavior of sulfacetamide (SAA), sulfadiazine (SDZ), sulfaquinoxaline (SQX), and sulfathiazole (STZ) at a CPE. All solutions were studied in concentration of 1 mM in a phosphate buffer solution (pH = 7.4) with scan rate of 0.1 V s-1. can be concluded that SDZ possesses a higher electrochemical reactivity, and so it can be considered as a desired pharmaceutical application. Since the anodic peak potential E , where the oxidation current is maximum, is related pa' to the ionization potential, we calculated the adiabatic IP values for SAs at B3LYP/6-311++G(d,p) level defined as: SAs - SAs+° + e- (6) where SAs and SAs+° stand for the neutral and radical cation states of the sulfonamide derivatives, respectively. In the case of sulfonamide derivatives, the calculated IP is an important parameter for use to estimate the energy barrier for their electro-oxidation reaction. In Figure 2, the calculated IP values are plotted against the experimental values for Epa. According to this figure, one can observe a good correlation between the IP and Epa values (the correlation coefficient r2 > 0.90). The observed correlation indicates that a highly delocalized Figure 2. Correlation plot between the calculated IP (eV) and experimental Epa (V) for SA derivatives. Nikoofard et al.: Prediction of Physico-chemical Properties Acta Chim. Slov. 2017, 64, 842-848 847 system corresponding to a low Epa value may accept a less barrier energy than a relatively more localized system (with higher E ). This trend is reasonable because a higher electron-conjugation character of the substituent stabilizes the oxidation product (Eq. 2). It can also be observed in Figure 2 that the SAA molecule with a non-cyclic group (R = COCH3) has a large positive shift for Epa. 3. 3. Thermodynamic Stability of SAs Full-optimized geometrical structure of SAs were used to calculate the vibrational frequencies by means of the B3LYP/6-311 ++G(d,p) level of theory. All vibrational frequency values together some thermochemical quantities of the sulfonamide derivatives including the total energy (E), zero-point energy (ZPE), enthalpy (H), and thermal corrected energy (Hcorr) at 298.15 K were tabulated in Tables S2 and S3. As mentioned in section 2, the gas-phase standard molar enthalpies of formation at 298 K, AH°f 298(g), for SAs were calculated through the atomization energy route, and the results obtained were displayed in Figure 3. As it can be seen in this figure, in a comparative study in the gas phase, the improvement in stability (corresponding to AH°f298(g) < 0) was obtained for the SAA, SDZ, and SQX species with respect to the reference molecule SA. Indeed, attachment of an electron-attracting substituent to the sul-fonamido group leads to an evident decrease in the standard molar enthalpies of formation and followed by an increase in the thermodynamic stabilization. In agreement with the electronic results obtained in section 3.2, we observed that the thermodynamic stability of the STZ molecule decreased with respect to the unsubstituted parent SA. Since the thermal stability of compounds is an important factor to be considered for the standardization of drugs and pharmaceuticals, it may be concluded that the considered processor helps us to predict the relative thermody-namic stability of new SA derivatives for which the respective experimental determination has not been reported. 4. Conclusion In the current work, the B3LYP/6-311++G(d,p) level of theory was employed to investigate the influence of the ^-sulfonamide substituent on the geometrical structure and electronic properties of the SAA, SDZ, SQX, and STZ molecules. Substituting the hydrogen atom of the sulfana-mido group by four different substituents played a fine-tune effect on the physico-chemical properties and ther-modynamic stabilities of the SA derivatives. In the case of the R substituted species, improvements were obtained in the HOMO-LUMO gap, charge density, and some electronic descriptors with respect to the ones in the reference SA molecule, which were in good agreement with the higher bacteriostatic reactivity of these molecules. Comparison of the cyclic voltammetry experiments for the oxidation potential of SAs obtained in this work, with the calculated ionization potential values shows a linear correlation, which corresponds to the conjugational character of the substituent. The calculations carried out on the neutral and ionic SAs as well as electrochemical measurements showed that the SDZ molecule had a more satisfactory structural and electronic characteristic for the bacterio-static reactivity. Besides, the calculated results for the standard molar enthalpies of formation in the gas phase revealed an improvement in the thermodynamic stabilization of the SDZ, SQX, and SAA molecules with respect to the unsubstituted parent molecule. Generally, the theoretical data obtained for the efficient injection and transport of the carrier charges involving holes and electrons can be applied for the rational design of a sulfa drug of desired properties. 5. Acknowledgment The authors wish to thank the Shahrood University of Technology for the financial support of this research work. 300 250 ZOO o Ü 150 CO u £ ™ S J 50 <1 •60 • - -100 -1-1-1-1-1- sa saa s 01 sqx stz SA derivatives Figure 3. Gas-phase standard molar enthalpy of formation values for SA derivatives at 298 K at the B3LYP/6-311++G(d,p) level of theory. 6. References 1. S. Alyar, N. Karacan, J. Enzyme, Inhib. Med. Chem. 2009, 24, 986-992. DOI: 10.1080/14756360802561220 2. T. Owa, T. Nagasu, Exp. Opin. Ther. Pat. 2000, 10, 1725-1740. 3. R. C. Ogden, C. W. Flexner, Protease inhibitors in AIDS therapy, New York, Marcel Dekker, 2001. 4. I. Nishimori, D. Vullo, A. Innocenti, A. Scozzafava, A. Mas-trolorenz, C. T. Supuran, Bioorg. Med. Chem. Lett. 2005, 15, 3828-3833. D0I:10.1016/j.bmcl.2005.06.055 5. J. J. Li, G. D. Anderson, E. G. Burton, J. N. Cogburn, J. T. Collins, D. J. Garland, S. A. Gregory, H-C. Huang, P. C. Isakson, J. Med. Chem. 1995, 38, 4570-4578. DOI: 10.1021/jm00022a023 6. C. W. Thornbe, Chem. Soc. Rev. 1979, 8, 563-580. DOI: 10.1039/cs9790800563 Nikoofard et al.: Prediction of Physico-chemical Properties ... 848 Acta Chim. Slov. 2017, 64, 842-848 7. A. E. Boyd, Diabetes 1988, 37, 847-850. DOI:10.2337/diab.37.7.847 8. C. T. Supuran, A. Cosini, A. Scozzafave, Med. Res. Rev. 2003, 23, 535-558. DOI:10.1002/med.10047 9. S. Roland, R. Ferone, R. J. Harvey, V. L. Styles, R.W. Morrison, J. Biol. Chem. 1979, 254, 10337-10345. 10. U. Kalidhar, A. Kaur, Res. J. Pharm., Biol. Chem. Sci. 2011, 2, 1116-1135. 11. R. J. Henry, Bacteriol. Rev. 1943, 7, 175-262. 12. S-Y. Won, P. Chandra, T. S. Hee, Y-B. Shim, Biosens. Bioelec-tron. 2013, 39, 204-209. DOI:10.1016/j.bios.2012.07.043 13. G. Stoev, A. Michailova, Chromatography 2000, 25, 37-42. DOI:10.1016/S0021-9673(99)00904-8 14. K. H. Lu, C.Y. Chen, M. R. Lee, Talanta 2007, 72, 1082-1087. DOI:10.1016/j.talanta.2007.01.022 15. http://www.who.int/medicines/publications/essentialmedi-cines/en/index.html. 16. W. G. Harter, H. Albrect, K. Brady, B. Caprathe, J. Dunbar, J. Gilmore, S. Hays, C. R. Kostlan, B. Lunney, N. Walker, Bioorg. Med. Chem. Lett. 2004, 14, 809-813. DOI: 10.1016/j.bmcl.2003.10.065 17. P. H. Bell, R. O. Roblin, J. Am. Chem. Soc. 1942, 64, 29052917. DOI:10.1021/ja01264a055 18. W. D. Kumler, T.C. Daniels, J. Am. Chem. Soc. 1943, 65, 21902196. DOI:10.1021/ja01251a053 19. J. K. Seydel, Pharm. Sci. 1968, 57, 1455-1478. DOI: 10.1002/jps.2600570902 20. Z. H. Chohan, H. A. Shad, L. Toupet, T. B. Hadda, M. Akkurt, J. Chem. Crystallogr. 2011, 41, 159-162. DOI:10.1007/s10870-010-9856-x 21. A. M. Mansour, J. Mol. Struct. 2013, 1035, 114-123. DOI:10.1016/j.molstruc.2012.09.048 22. A. M. Mansour, N. T. Abdel Ghani, J. Mol. Struct. 2013, 1040, 226-237. DOI:10.1016/j.molstruc.2013.02.028 23. C. Soriano-Correa, R. O. Esquivel, R. P. Sagar, Int. J. Quant. Chem. 2003, 94, 165-172. DOI:10.1002/qua.10597 24. A. Chandran, H.T. Varghese, Y. Sheena Mary, C. Yohannan Panicker, T. K. Manojkumar, C. V. Alsenoy, G. Rajendran, Spectrochim. Acta A 2012, 87, 29-39. DOI:10.1016/j.saa.2011.10.073 25. S. Alyar, Ü. Özdemir Özmen, N. Karacan, O. §. §entürk, K. A. Udachin, J. Mol. Struct. 2008, 889, 144-149. DOI:10.1016/j.molstruc.2008.01.048 26. H. A. Dabbagh, A. Teimouri, R. Shiasi, A. Najafi Chermahini, J. Iran. Chem. Soc. 2008, 5, 74-82. DOI:10.1007/BF03245818 27. M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, V. G. Zakrzewski, J. A. Montgomery Jr., R. E. Stratmann, J. C. Burant, S. Dapprich, J. M. Millam, A. D. Daniels, K. N. Kudin, M. C. Strain, O. Farkas, J. Toma-si, V. Barone, M. Cossi, R. Cammi, B. Mennucci, C. Pomel-li, C. Adamo, S. Clifford, J. Ochterski, G. A. Petersson, P. Y. Ayala, Q. Cui, K. Morokuma, D. K. Malick, A. D. Rabuck, K. Raghavachari, J. B. Foresman, J. Cioslowski, J. V. Ortiz, B. B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaromi, R. Gomperts, R. L. Martin, D. J. Fox, T. Keith, M. A. Al-Laham, C. Y. Peng, A. Nanayakkara, C. Gonzalez, M. Challacombe, P. M.W. Gill, B. Johnson, W. Chen, M. W. Wong, J. L. Andres, C. Gonzalez, M. Head-Gordon, E. S. Replogle, J. A. Pople, Gaussian 09W, Gaussian Inc., Pittsburgh PA, 2009. 28. M. Karabacak, M. Cinar, M. Kurt, J. Mol. Struct. 2010, 968, 108-114. DOI:10.1016/j.molstruc.2010.01.033 29. J. R. B. Gomes, P. Gomes, Tetrahedron 2005, 61, 27015-2712. DOI:10.1016/j.tet.2005.01.034 30. A. D. Becke, J. Chem. Phys. 1993, 98, 5648-5652. DOI: 10.1063/1.464913 31. C. T. Lee, W. T. Yang, R.G. Parr, Phys. Rev. B 1998, 37, 785789. DOI: 10.1103/PhysRevB.37.785 32. R. G. Parr, L. V. Szentpaly, S. Liu, J. Am. Chem. Soc. 2009, 121, 1922-1924. DOI:10.1021/ja983494x 33. J. L. Moncada, A. Toro-Labbe, Chem. Phys. Lett. 2006, 429, 161-165. DOI:10.1016/j.cplett.2006.06.087 34. J. W. Ochterski, Gaussian white paper, Thermochemistry in Gaussian 2000, http://www.gaussian.com/g_whitepap/ther-mo.htm. 35. H. Cao, J. Ma, G. Zhang, Y. Jiang, Macromolecules 2005, 38, 1123-1130. DOI:10.1021/ma048534y 36. G. Zhang, J. Ma, Y. Jiang, J. Phys. Chem. B 2005, 109, 1349913509 DOI:10.1021/jp051259c 37. M. A. De Oliveira, H. Duarte, J. Pernaut, W. B. De Almeida, J. Phys. Chem. A 2000, 104, 8256-8265. DOI:10.1021/jp001252p Povzetek S teorijo gostotne funkcije (DFT) in z uporabo elektrokemijskih metod smo proučevali strukturne in fizikalno-kemijske lastnostih serije derivatov sulfonamida (SA), ki imajo vlogo nujnih zdravil WHO pri osnovnem zdravljenju. Izračune za sulfadiazin (SDZ), sulfatiazol (STZ), sulfakvinoksalin (SQX), sulfacetamid (SAA) in referenčni nesubstituirani sulfo-namid (SA), smo izvedli na B3LYP / 6-311 ++ G (d, p) nivoju. Ugotovili smo, da geometrijske strukture in elektronske lastnosti, povezane z bakteriostatično aktivnostjo, vplivajo na sterične in „push-pull" značilnosti substituent. Predstavili smo tudi elektrokemijske eksperimente oksidacije SA z uporabo ciklične voltametrije. Izkazalo se je, da med izračunani ionizacijski potenciali (IP) in elektrooksidacijskimi potenciali lahko obstaja linearna zveza. Izmed proučevanih molekul je očitno SDZ najbolj elektroaktiven in izkazuje tudi največjo biološko aktivnost. Izračuni DFT, izvedeni s uporabo standardnih molskih tvorbenih entalpij za tvorbo v plinski fazi, so predvideli možne izboljšave pri termodinamski stabilnosti molekul SDZ, SQX in SAA ter relativno nestabilnost STZ glede na molekulo SA. Nikoofard et al.: Prediction of Physico-chemical Properties DOI: 10.17344/acsi.2017.3413 Acta Chim. Slov. 2017, 64, 849-857 ¿^creative ty commons Scientific paper Methyl Salicylate-Based Vortex-Assisted Surfactant-Enhanced Emulsification Microextraction and HPLC for Determination of Fungicides in Honey Samples Yanawath Santaladchaiyakit,1* Jutamas Bunchamnan,1 Darunee Tongsa1 and Supalax Srijaranai2 1 Department of Chemistry, Faculty of Engineering, Rajamangala University of Technology Isan, Khon Kaen Campus, Khon Kaen 40000, Thailand. 2 Materials Chemistry Research Center, Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand * Corresponding author: E-mail: sanyanawa@gmail.com, yanawath.sa@rmuti.ac.th; Tel.: (+66)-4333-8869-70, Fax: (+66)-4333-8869-70 Received: 26-06-2017 Abstract Methyl salicylate based vortex-assisted surfactant-enhanced emulsification microextraction (MeSA-VASEME) has been developed and applied for rapid preconcentration of fungicides (i.e., carbendazim, thiabendazole, and fluberidazole) in honey samples followed by high performance liquid chromatographic analysis. MeSA was used as an extraction solvent, while surfactant was used to enhance the extraction performance under the dispersion by vortex agitation. The optimum MeSA-VASEME conditions were 100 |L MeSA, 2.0 mmol L"1 sodium dodecyl sulfate, and vortex agitation at 1200 rpm for 90 s. Preconcentration factors were obtained in the range of 32-40. The limit of detection in the studied honey samples was 0.5 |g L"1. The recovery of the spiked target fungicides at 20, 50, and 100 |g L"1 were 81.5-116.8 % with the relative standard deviation below 11%. The proposed method is simple, sensitive, less organic solvent consuming, inexpensive, and a rapid procedure for the residue analysis of fungicides in honey samples. Keywords: Methyl salicylate, VASEME, HPLC, Fungicide, Honey 1. Introduction Bee products including honey are natural products that are rich in minerals, antioxidants, and simple sugars.1 Honey is found to be used as enzymatic and nonenzymatic antioxidant to prevent deteriorative oxidation reactions in foods such as the browning of fruit and vegetables, lipid oxidation in meat, and to inhibit the growth of food borne pathogens and microorganisms leading to food spoilage.1 In addition, honey has potential therapeutic properties in infections, wound healing, and cancer.1 However, bee products can also be a source of toxic substances, such as heavy metals, radioactive isotopes, organic pollutants, and pesticides (e.g., insecticides, fungicides, herbicides, and bactericides) due to environmental pollution and misuse of beekeeping practices.1,2 Indirect contamination of hon- ey by pesticides can be found during the pesticide applications in agriculture through soil, water, air, and flowers and then bees come into contact with the pesticides and collect nectar to produce the honey.3 Pesticide residues (e.g., organohalogens, organophosphates, organonitrogen, pyrethroids, and carbamates) in honey samples have been reported in the range of 0.05-4310 ^g kg-1 and were found in many countries.2 According to the legislations of maximum residue limits (MRLs) set by the European Union (EU) and Official of Brazil, it should be below 50 ^g kg-1 for most pesticides.3 Benzimidazole fungicides are widely used in agriculture for pre- and post-harvest treatment to control and kill fungi or fungal spores in order to prevent the spoilage of crops.4-6 The active benzimidazole fungicides include benomyl (BN), carbendazim (CBZ), thiabendazole Santaladchaiyakit et al.: Methyl Salicylate-Based Vortex-Assisted 850 Acta Chim. Slov. 2017, 64, 849-857 (TBZ), fuberidazole (FuBZ), thiophanate (TP), and thio-phanate-methyl (TPM). Most of the fungicides are normally used to control various diseases in various fruits and vegetables. They are directly applied to soil or sprayed over the crop fields.4,7 Hence, the studied fungicides may contaminate natural honey after bees come into contact with contaminated plants. From the literature, it was found that CBZ at the level of 1.62 ^g kg-1 was detected in the honey sample.8 There are several toxic effects from this fungicide exposure including teratogenicity, congenital malformations, polyploidy, diarrhea, anemia, pulmonary edemas, or necrotic lymphoadenopathy.5,9 Therefore, the development of highly sensitive techniques for trace residue analyses of fungicides in various sample matrices (e.g. food and enverionmental samples) has been increasingly important for the environment and health protection. The simultaneous residue determinations of ben-zimidazole fungicides using micellar electrokinetic chro-matography (MEKC),10 and high performance liquid chromatography (HPLC)11-14 have been popularly employed. Recently, the solvent-microextraction technique based on the application of vortex agitation, namely vortex-assisted liquid-liquid microextraction (VALLME) has been re-viewed.15 Furthermore, surfactants (as emulsifiers) are used instead of disperser solvents (used in DLLME). This technique is named vortex-assisted surfactant-enhanced emulsification microextraction (VASEME). It was found that VALLME overcomes the disadvantage of DLLME (required disperser solvents), while surfactants used in VASEME assist extraction solvents to better disperse into a sample solution.16 The combination of vortex agitation and surfactant has also been widely applied to improve the extraction performance and used for the analysis of various compounds.16-21 In VASEME, extraction solvents/surfactants such as carbon tetrachloride/Triton X-100, tolu-ene/CTAB, 1-octanol/SDS+CTAB, trichloromethane/am-monium perfluorooctanoate, 1-undecanol/Triton X-100, and methyl benzene/Tween 20 can be used.16-21 As mentioned above,16-21 it was found that a toxic extraction solvent (e.g. carbon tetrachloride) was used. Meanwhile, the use of lighter density solvents (e.g. toluene, octanol, unde-canol) proved difficult to separate and collect the upper extract phase and normally needed special devices to accomplish the phase separation. To overcome these limitations, an alternative extraction solvent such as methyl sa-licylate (MeSA) seems to be interesting for the extraction and preconcentration of organic compounds, such as fungicides. MeSA has some important characteristics such as (1) high density (1.17 g mL-1), (2) clear liquid solution at room temperature, (3) low water solubility (700 mg L-1), and (4) low cost.22 As our previous work demonstrated, MeSA was used in quite a large volume (250 ^L) and extraction was carried out in the presence of salt in the extraction solution.22 However, it seems to be suitable for non-polar compounds, except CBZ. Thus, the further de- velopment of preconcentration based on MeSA is of interest. The use of MeSA and surfactant (as emulsifier) instead disperser solvent and salt could maybe improve the performance of extraction of target fungicides, especially CBZ. The application of the proposed VASEME using MeSA as an extraction solvent has not been used for the analysis of fungicides in honey samples. This work is aimed at the development and extension of our previous work using a method named methyl salicylate based vortex-assisted surfactant-enhanced emulsification microextraction (MeSA-VASEME) coupled with HPLC for the simultaneous analysis of target benzimida-zole fungicides (e.g. CBZ, TBZ, and FuBZ) in honey samples. The variables affecting MeSA-VASEME procedure were investigated, and analytical performances as well as method validation were also evaluated. 2. Experimental 2. 1. Chemicals and Reagents The chemicals and reagents used in this study are of AR grade or higher. The analytical standards of fungicides were purchased from Sigma-Aldrich including CBZ (Munich, Germany), TBZ (Milan, Italy), and FuBZ (Munich, Germany). The stock solutions of each fungicide were prepared at 1,000 mg L1 by dissolving an appropriate amount in a small volume (~500 ^L) of formic acid and further dilution with methanol (MeOH). Methyl salicylate was obtained from Sigma-Aldrich (Shanghai, China). MeOH, ethanol (EtOH), formic acid, and acetonitrile (ACN) were purchased from Merck (Darmstadt, Germany). Sodium dodecyl sulfate (SDS) was purchased from BDH Prolab (Leuven, Belgium). Triton X-100 was purchased from Sig-ma-Aldrich (MO, USA), while Tergitol® TMN-10 (Sig-ma-Aldrich, MO, USA) and cetyl trimethylammonium bromide (CTAB) (Sigma-Aldrich, Bangalore, India) were also purchased. The solutions of SDS (100 mmol L1), CTAB (100 mmol L1), Triton X-100 (25%, w/v), and Tergi-tol TMN-10 (25%, w/v) were prepared in deionized water before use. Sodium chloride (NaCl) (Ajax Finechem, Auckland, New Zealand), anhydrous sodium sulfate (anh. Na2SO4) (Carlo Erba, Val de Reuil, France), sodium car-bon2ate4 (Na2CO3) (RFCL Limited, New Delhi, India), and anhydrous sodium acetate (anh. NaOAc) (Carlo Erba, Val de Reuil, France) were used. All aqueous solutions were prepared in deionized water with resistivity of 18.2 MO cm from RiOsTM Type I Simplicity 185 (Millipore water, MA, USA). 2. 2. Instruments The HPLC coupled with a photo-diode array detector (PDA) (Shimadzu Corporation, Kyoto, Japan) was used. Data analysis and acquisition of the system were controlled using LCsolution software (Shimadzu). An In- Santaladchaiyakit et al.: Methyl Salicylate-Based Vortex-Assisted ... Acta Chim. Slov. 2017, 64, 849-857 851 ertsil C8 column (4.6 x 150 mm, 5.0 ^m) connected to a guard C8 column (4.0x10 mm, 5.0 ^m) (GL Science, Tokyo, Japan) was used as the separation column for target fungicides. Centrifuge NF200 model (Nuve Inc., Ankara, Turkey) and a vortex mixer Genie-2 model (Scientific Industries Inc., NY, USA) were also used. 2. 3. HPLC Separation Conditions The reversed-phase HPLC was used for complete separation of the studied fungicides under the gradient elution of ACN and 0.1% (v/v) formic acid as an optimal mobile phase. A flow rate of 1.0 mL/min was performed throughout the separation process. The chromatographic separation was performed at 25 °C. The detection of the target analytes was performed at 280 nm for CBZ, and at 311 nm for TBZ and FuBZ. The column gradient program12-22 consisted of 0-2.0 min 15% ACN, 2.0-4.0 min ramped linearly from 15 to 45% ACN, and then 4.0-6.0 min ramped linearly to 75% ACN. After the composition was further kept constant at 75% ACN for 3 min, ACN was linearly decreased to 45% and 15%, respectively. When the pressure reached its initial value, the next separation process could be performed. 2. 4. Sample Analysis Honey samples were purchased from a supermarket in Khon Kaen province, Thailand. Accurate weight (1.00 g) of sample was dissolved in 10 mL water. Then, the 10 mL sample solution was extracted using the Me-SA-VASEME procedure and analyzed by HPLC. To evaluate the accuracy, the studied honey samples were fortified with the standard fungicides at various concentration levels of 20, 50, and 100 ^g L-1 prior to the preconcentration. 2. 5. MeSA-VASEME Procedure Methyl salicylate (100 ^L) and SDS (2 mmol L-1) were injected into the 15 mL conical tube containing a standard or sample solution (10.00 mL). Then, the solution was manually shaken for 15 s before vortex agitation at 1200 rpm for 90 s. After centrifugation at 3000 rpm for 1 min, the extract phase was obtained (at the bottom of the tube). The aqueous phase was then removed by microsy-ringe. Subsequently, the extract rich phase was mixed with MeOH (100 ^L) before subjecting it (20 ^L) to HPLC for the analysis. 2. 6. Calculation of Preconcentration Factor and Extraction Recovery Preconcentration factor (PF) and extraction recovery (ER) were used to evaluate the performance of the extraction method and were calculated using the following equations: where Cext is defined as target compound concentration in the collected phase, while C0 is the initial analyte concentration. The calculation of Cext was conducted from the standard calibration curves obtained from the direct analysis (without preconcentration). Vext and V0 are the volume of the collected phase and initial aqueous sample solution (10 mL), respectively. 3. Results and Discussion The parameters affecting extraction recovery of target fungicides were investigated including surfactant, extraction solvent, salt additive, solution pH, vortex agitation and centrifugation. One-parameter-at-a-time was used for optimizing extraction conditions, while the other remaining factors were kept constant. 3. 1. Effect of Surfactants By trial and error, three different extraction compositions including (a) MeSA in the presence of sodium acetate, (b) MeSA containing MeOH (as disperser solvent) and sodium acetate, and (c) MeSA in the presence of SDS, were studied. As the results shown in Figure S1 indicate, the chromatograms obtained from the conditions (a) and (b) are similar. This means that the extraction performance of the methods for three studied compounds is insignificantly different under the presence of disperser solvent and/or salt. Meanwhile, MeSA in the presence of SDS gave the highest peak height especially for CBZ. This behavior indicated that SDS can be used as a good emulsifier for improving the extraction of polar analytes. Therefore, the effect of SDS in comparison with other surfactants on the extraction performance of the target fungicides was further investigated. Theoretically, surfactant was used as an emulsifier in various microextraction methods to accelerate the emulsi-fication of water-immiscible solvent in the aqueous sample solution.23-25 It has been proven that the addition of surfactant can improve the penetration of different target hy-drophobic compounds due to its hydrophobic and hydro-philic groups within the molecule.26 In this study, surfactants included SDS (at 2.00 mmol L-1), CTAB (at 0.50 mmol L-1), Tergitol TMN-10 (at 2.71 mmol L-1), and Triton X-100 (0.12 mmol L-1), while the concentration tested was lower than the critical micelle concentration (CMC) for each surfactant. The CMCs of SDS, CTAB, Tergitol TMN-10, and Triton X-100 were 8, 0.92, 5.7, and 0.24 mmol L-1, respectively. The results (Figure 1a) show that SDS (anionic surfactant) provided the highest extraction Santaladchaiyakit et al.: Methyl Salicylate-Based Vortex-Assisted ... 852 Acta Chim. Slov. 2017, 64, 849-857 recovery in comparison to no surfactant addition, cationic (e.g. CTAB), and non-ionic (e.g. Tergitol TMN-10 and Triton X-100) surfactants. Is may be assumed that the target fungicides (p^a~5-6) were in the positive charge5 under the acidic conditions studied (pH 4) and consequently favorably penetrated and were strongly attracted to SDS molecules. Meanwhile, less interaction between positively charged analytes and cationic or non-ionic surfactant was expected. In addition, it has been reported that good emul-sification process was obtained when the concentration of surfactant was lower than CMC.16 Thus, SDS was then selected for further investigation. SDS(mmol/L) Figure 1: Effects of (a) surfactant and (b) SDS on the extraction recovery of the target analytes The influence of SDS concentration on the extraction recovery of target fungicides was evaluated in the range of 0-4 mmol L-1 (see Figure lb). Increase in the extraction recovery for most target fungicides when concentration of SDS increased was observed up to 2 mmol L-1. High concentrations of SDS did not promote better extraction recovery of the target analytes. This behavior may be because of strong self interaction of SDS molecules and the analytes more favorably dissolve in the aqueous phase resulting in decreased extraction recoveries. In this study, 2 mmol L-1 SDS was chosen. 3. 2. Selection of Extraction Solvent and its Volume Methyl salicylate was used as an extraction solvent in this study. Based on the log^ow (related to hydrophobic-ity of the analytes) of target fungicides (1.5-2.7) and MeSA (2.55), MeSA seems to be good for extraction of the hydrophobic target fungicides, especially for TBZ and FuBZ. For CZB, the combination of MeSA and SDS could be used for improving the extraction performance of the method. Good characteristics of MeSA as an extraction solvent include (i) the presence of the extract phase at the bottom of the bulk solution (due to its density >1.0 g mL-1), (ii) highly solubility in the organic mobile phase, and (iii) no interference of the excess MeSA with the target fungicide peaks. In this study, the volume of MeSA on the extraction recovery was further studied by varying the volume in the a) I-1-1-1-1-1-1-■-1-■-1— 0 100 200 300 400 S00 Methyl salicylate {]nL) Figure 2: Effects of (a) volume of methyl salicylate and (b) salt addition on the extraction recovery of the target analytes (250 |ig L-1 each). Santaladchaiyakit et al.: Methyl Salicylate-Based Vortex-Assisted ... Acta Chim. Slov. 2017, 64, 849-857 853 range of 50-500 ^L (Figure 2a). The highest extraction recovery was observed at the MeSA volume of 100 ^L and decrease in the extraction performance afterwards. It may be due to the dilution of the target fungicides in higher volume of extract MeSA phase. Thus, 100 ^L MeSA was chosen as the optimal value. 3. 3. Effect of Salt Addition In general, the addition of salt into the aqueous sample solution can enhance the extraction recovery of target analytes by decreasing the solubility of the analytes in the aqueous phase and increasing mass transfer toward the organic phase.15 The decrease in water solubility of target compounds in bulk aqueous phase was expected, resulting in increasing mass transfer of target compounds towards the extract phase. In this investigation, CH3COONa, NaCl, Na2SO4, and Na2CO3 were studied at the equal molar concentration (~1.71 mmol L-1) and compared to with and without salt addition (Figure 2b). It is clearly seen that the addition of salt could not help to improve the extraction recovery of target analytes when compared to the process without salt addition. This may be explained by (i) charge interaction between SDS and counter ions of salts resulting in decreased capability of SDS in the solution, and (ii) salt addition leading to increased viscosity of the bulk aqueous phase. In this study, salt addition was not required throughout the experiments. 3. 4. Effect of Solution pH The effect of sample pH on the extraction recovery of the target fungicides was investigated in the range of 2.08.0 (data not shown). It can be found that the pH value strongly affected the extraction efficiency of MsSA-VASEME for fungicide analytes. The highest extraction recovery was found at the pH 4.0 (as the original pH value, without pH adjustment), while higher pHs decreased the extraction efficiency for most analytes. The reason may be the fact that the analytes (pKa1 ~4-5) are in the cationic form (i.e. positive charge) under the acidic pHs and favorably attract SDS molecules (negative charge). On the other hand, the analytes present in the neutral form or negatively charged form result in less interaction between the analytes and SDS. From the investigation, the original pH of the solution of about 4.0 was chosen for the extraction of target fungicides. 3. 5. Effect of Vortex Agitation (Extraction Time) Vortex agitation (speed and time) is one of the important factors in vortex-based microextraction method because it affects the extraction equilibrium (e.g. emulsifi-cation and distribution process) of target analytes, and consequently influences the extraction efficiency12,17,27 The vortex agitation time was investigated between 30 and 150 s at speed of 1200 rpm, while the agitation speed was studied in the range of 600-2100 rpm. The results are shown in Figure S2 (a & b), which reveals that appropriate speed and time for the vortex agitation can improve extraction efficiency of the method. In this study, the highest extraction recoveries were found at 90 s at 1200 rpm. A higher speed rate (> 1200 rpm) and longer time (> 90 s) decreased the extraction recovery of the target fungicides. Thus, the vortex agitation was chosen at 1200 rpm for 90 s for further evaluation. 3. 6. Effect of Centrifugation Time and Speed Centrifugation speed and time were also investigated in this study because they affect the phase separation of a sample solution. In our previous work,22 it was reported that a low centrifugation speed (e.g. < 2500 rpm) could not cause complete phase separation, while the decreased extraction recoveries were observed at high centrifugation speed (i.e. 4000 rpm). In this study, the centrifugation speed (2000-3500 rpm) and time (0-5 min) were investigated (see Figure S3 (a & b)). It is clearly seen that the best extraction recoveries were obtained at a speed of 3000 rpm as the optimum speed for obtaining complete phase separation, and there was a decrease in extraction recoveries afterwards. Meanwhile, the highest extraction recoveries were also observed at the appropriate centrifugation time of 1 min. Therefore, centrifugation at 3000 rpm for 1 min was selected. 3. 7. Analytical Performance of the Method The analytical performance and method validation of the proposed MeSA-VASEME were investigated in two sample matrices (i.e. ultrapure water and honey). The studied parameters were linear dynamic range, coefficient of determination (R2), limits of detection (LODs), limits of quantitation (LOQs) and precision (intra-day and inter-day measurements). LODs were defined as the concentration of the target analytes giving the signal-to-noise ratio of 3 (S/N = 3), while LOQs were defined as the S/N = 10. In ultrapure water medium, the linearity was found in the range of 0.1-100 ^g L-1 with R2 greater than 0.995. LODs were obtained between 0.01 and 0.05 ^g L-1, while LOQs were in the range of 0.1-0.2 ^g L-1. On the other hand, the LODs obtained from the method without pre-concentration were found to be 3 ^g L-1 for the studied analytes. The intra-day (n = 6) and inter-day (n = 6 x 3 days) precisions were also investigated by replicate injections of the certain concentration of 100 ^g L-1 in a day and over several days. The relative standard deviations (RSDs) in terms of peak area and retention time were calculated. It was found that the RSDs below 8.3% for peak area and retention time were obtained. Under the optimal Santaladchaiyakit et al.: Methyl Salicylate-Based Vortex-Assisted ... 854 Acta Chim. Slov. 2017, 64, 849-857 conditions, preconcentration factors and extraction recoveries were obtained in the range of 32-40, and 6479%, respectively. For the investigation in honey samples, the analytical features and method validations were studied in real honey samples. Matrix-matched calibration was performed in this study. The results are summarized in Table 1. The linear dynamic range was in the range of 2-200 ^g L-1 with R2 higher than 0.995. The calibrations obtained in each sample are listed in Table 2. LODs and LOQs in honey sample (Brand#1 as a representative sample) were 0.5 and 2 ^g L-1, respectively. Precisions in terms of intra-day (n = 6) and inter-day (n = 3 x 3 days) were also studied and expressed as the relative standard deviations (RSDs) of the studied target fungicides at a certain concentration each. High precisions with RSDs below 12% were accepted. where Cdetect is the detected concentration of analytes after the addition of known amount of standard to real sample, Creal is the concentration of the target analytes found in real sample, and Cadd is the concentration of the spiked known amount of standard solution in the real sample. The chromatograms obtained from the spiked samples (see Figure 3) and recovery results (Table 3) are shown. Good relative recoveries of the target fungicides in honey samples were found in the range of 81.5-116.8% with the relative standard deviation below 11%. Intra-day precision (n = 6) and intermediate precision (n = 3 x 3 days) of the proposed method were also studied in the spiked honey sample (Brand#1 as a representative sample) at 100 ^g L-1 of each fungicide. The studied precisions provided the RSD below 12%. The obtained recoveries and %RSD were in good agreement with the acceptable values of 70-120% Table 1: Figures of merit of the proposed method for the determination of the benzimidazole fungicides in honey samples Analyte Linearity (^g L-1) LOD (^g L-1) LOQ (^g L-1) Intra-day precision" (n = 6), %RSD Inter-day precisiona (n = 3 x 3 days), %RSD CBZ 2-200 0.5 2 7.7 11.7 TBZ 2-200 0.5 2 8.4 10.0 FuBZ 2-200 0.5 2 8.6 7.2 a Precisions were evaluated at the concentration of 100 |ig/L for each fungicide spiked in honey brand # 1 (as a representative sample). Table 2: Calibrations obtained by the proposed method for the determination of the benzimidazole fungicides in honey samples . Honey Brand#1 Honey Brand#2 Honey Brand#3 Honey Brand#4 Linear equation R2 Linear equation R2 Linear equation R2 Linear equation R2 CBZ y = 532x + 3385 0.996 y = 805x - 3714 0.996 y = 508x - 1717 0.998 y = 438x + 1506 0.997 TBZ y = 708x + 8048 0.995 y = 1069x - 4201 0.997 y = 791x - 537 0.999 y = 573x + 3776 0.999 FuBZ y = 1068x + 1600 0.996 y = 1865x - 8732 0.997 y = 1249x + 796 0.997 y = 940x + 6855 0.997 3. 8. Application to Real Samples The proposed method was then evaluated in different commercial brands of honey samples (4 brands). The matrix-matched calibration was used in this study to avoid endogenous interferences effect on the analysis. The identification and confirmation of the target peaks of the ana-lytes were performed using comparison of retention time of the standard analytes and their absorption spectra data obtained from PDA. An example of honey sample blank is demonstrated in Figure 3. It was found that contamination by the studied fungicides in the studied honey samples was not detected. Accuracy in terms of relative recovery (RR) test at different concentrations spiked (e.g. 20, 50, and 100 ^g L-1) in real honey samples was also investigated. The RR(%) was used for the evaluation of real honey sample analyses. The calculation of RR(%) is as follows: RR(%) = Cietec' ~Cred x 100 (3) C add with RSD less than of 20%, at the concentrations spiked in the range of 10-100 ^g L-1.28 According to the results obtained, the proposed method was effective and reliable for the determination of target fungicides in honey samples. 3. 9. Comparison of the Proposed Method to Other Relevant Strategies The developed MeSA-VASEME method and other related strategies coupled to HPLC for the analysis of benzimidazole fungicides in various samples (e.g. water) are compared and summarized in Table 4. The SPME4 and MISPE6 are promising methods but SPME is expensive and requires a long incubation time (~40 min), high temperature, and high electrolyte salt. Meanwhile, MISPE is a tedious procedure and requires a long synthesis time for the sorbent. Micellar extractions (or cloud-point extractions) using surfactants are normally performed at high incubation temperature (e.g. 90 °C) for a long time.29 The Santaladchaiyakit et al.: Methyl Salicylate-Based Vortex-Assisted ... Acta Chim. Slov. 2017, 64, 849-857 855 Table 3: Recovery obtained from the determination of benzimidazole fungicides in honey samples (n = 3) Spiked (^g L1) Honey brand #1 Honey brand #2 Honey brand #3 Honey brand #4 Analyte Detected (^g L1) RR (%) RSD (%) Detected (^g L1) RR (%) RSD (%) Detected (^g L1) RSD (%) RR (%) Detected (^g L1) RR (%) RSD (%) CBZ 0 ND - - ND - - ND - - ND - - 20 22.97 114.8 3.7 17.47 87.3 4.8 21.15 105.8 5.7 17.99 90.0 1.8 50 52.81 105.6 1.4 57.55 115.1 7.1 47.60 95.2 3.2 52.88 105.8 9.2 100 111.62 111.6 10.8 115.05 115.0 3.3 85.56 85.6 4.4 109.89 109.9 9.1 TBZ 0 ND - - ND - - ND - - ND - - 20 16.77 83.8 3.9 17.77 88.9 4.1 19.10 95.5 8.1 16.55 82.7 2.4 50 46.82 93.6 6.9 53.41 106.8 6.9 50.99 102.0 5.9 44.05 88.1 9.4 100 89.27 89.3 6.7 115.22 115.2 1.6 90.30 90.3 5.9 111.12 111.1 6.9 FuBZ 0 ND - - ND - - ND - - ND - - 20 19.90 99.5 8.0 16.34 81.7 4.3 20.97 104.9 6.0 16.58 82.9 2.4 50 50.70 101.4 0.6 55.18 110.4 6.8 47.27 94.5 5.9 40.76 81.5 10.0 100 103.05 103.0 5.7 116.81 116.8 2.1 90.19 90.2 8.7 107.20 107.2 8.5 ND: Not detected RR: Relative recovery developed method is useful for the quantification and qualification of the target fungicides at trace levels in the studied samples. The advantages of the method are a simple procedure, short extraction time, short analysis time, and low cost. The proposed MeSA-VASEME can also be used as an alternative powerful method to the other our previous works demonstrated.22 4. Conclusions A simple and fast procedure for preconcentration and analysis using MeSA-VASEME and HPLC-PDA has been successfully developed for target fungicides in honey samples. The preconcentration based MeSA in the presence of surfactant (e.g. SDS) has also been proven to improve the extraction efficiency of target compounds, especially polar analytes. Good extraction efficiency, recovery, and reproducibility were achieved. Low limits of detection at 0.5 ^g L-1 in honey samples were also obtained. Less consumption of solvents used for the preconcentration step (< 500 ^L), short extraction time (< 10 min), and short separation time (< 5 min) are the advantages of the developed method. The proposed method can be used as an alternative method for trace residue analysis of target fungicides in the studied sample and other related matrices. 5. Acknowledgements This research was financially supported by the Thailand Research Fund (TRF) and Rajamangala University of Technology Isan, through the research grant for new scholars under grant no. TRG5780038. The authors would like to acknowledge Department of Chemistry, Faculty of Science, Khon Kaen University for providing deionized water. Santaladchaiyakit et al.: Methyl Salicylate-Based Vortex-Assisted ... Figure 3: Overlaid chromatograms of honey samples (Brand#1) with (a) honey sample blank and (b-d) spiked at various concentrations of target fungicides (20, 50, and 100 |ig L-1, respectively), evaluated at 280 nm and 311 nm. conventional DLLME uses toxic chlorinated solvent (e.g. chloroform) and needs disperser solvent for emulsifica-tion.30 It was difficult to withdraw the upper extract rich phase and remove the aqueous lower phase in SALLME.11 It is clearly seen that the proposed MeSA-VASEME provides comparable results such as LODs and recovery. The 856 Acta Chim. Slov. 2017, 64, 849-857 Table 4: Comparison of the HPLC technique with different preconcentration methods for the determination of benzimidazole fungicides in various sample matrices Analyte Extraction/ clean-up Extraction condition Sample matrix LOD (^g L-1) Recovery f (%) Ret' BN, CBZ, TBZ, FuBZ SPME • Carboxen-polydimethylsiloxane 75 |im fiber • Heated at 60 °C for 40 min and stirred at 600 rpm • Desorbed by MeOH for 10 min Water 0.03-1.30 80.9-119.6 4 BN, CBZ, Micellar • Genapol X-080 or POLE (4%, v/v), 4% (w/v) Water 0.008-6.4 74-92 29 TBZ, FuBZ extraction NaCl, 20-min at 90 °C (Genapol (Genapol X-080), X-080), 0.004-5.9 72-91 (POLE) (POLE) CBZ, TBZ DLLME • Chloroform (extraction solvent) + tetrahydrofuran (disperser solvent) + 10% (w/v) NaCl Water 0.5-1.0 84.0-94.0 30 BN, CBZ, TBZ, FuBZ, FluBZ, FBZ, ABZ MISPE • Molecularly imprinted polymer-divinylbenzene • Eluted by MeOH/acetic acid (50/50, v/v) • Evaporated and re-dissolved in ACN Water 0.0020.012 90-106 6 CBZ, FuBZ, TPM, TP SALLE • ACN (2 mL) + NaH2PO4 (0.1 M) + NaCl (5.0 M) • Dried extract phase and re-dissolved with ACN (70%, v/v) Water 0.14-0.38 60.4-99.1 11 CBZ, TBZ, FuBZ VA-DLLME • 250 |L methyl benzoate + 300 |L EtOH + NaOAc (1.0%, w/v) Water 0.01-0.05 77.4-110.9 12 CBZ, TBZ, FuBZ MeSA-DLLME • 250 |L methyl salicylate + NaOAc (1.0%, w/v) Water 0.03-0.05 74.1-118.4 22 CBZ, TBZ, FuBZ MeSA-VASEME • 100 |L methyl salicylate + 0.2 mM SDS Honey 0.5 81.5-116.8 Proposed method 6. References 1. N. Al-Waili, K. Salom, A. Al-Ghamdi, and M. J. Ansari, Sci. World J., 2012, 2012, 1-9. D01:10.1100/2012/930849 2. D. R. Lopez, D. A. Ahumada, A. C. Diaz, and J. A. Guerrero, Food Control, 2014, 37, 33-40. D0I:10.1016/j.foodcont.2013.09.011 3. P. A. S. Tette, L. R. Guidi, and M. B. de A. Gloria, Talanta, 2016, 149, 124-141. D0I:10.1016./j.talanta.2015.11.045 4. A. L. Monzón, D. V. Moreno, M. E. T. Padrón, Z. S. Ferrera, and J. J. S. Rodríguez, Anal. Bioanal. Chem., 2007, 387, 19571963. D0I:10.1007/s00216-006-1083-0 5. O. Zamora, E. E. Paniagua, C. Cacho, L. E. Vera-Avila, and C. Perez-Conde, Anal. Bioanal. Chem., 2009, 393, 1745-1753. D0I:10.1007/s00216-009-2631-1 6. C. Cacho, E. Turiel, and C. Pérez-Conde, Talanta, 2009, 78, 1029-1035. D0I:10.1016/j.talanta.2009.01.007 7. D. P. Zamora, M. M. Galera, A. G. Frenich, J. L. M. Vidal, Analyst, 2000, 125, 1167-1174. D0I:10.1039/A909886K 8. K. M. Kasiotis, C. Anagnostopoulos, P. Anastasiadou, and K. Machera, Sci. TotalEnviron., 2014, 485-486, 633-642. D0I:10.1016/j.scitotenv.2014.03.042 9. M. Danaher, H. D. Ruyck, S. R. H. Crooks, G. Dowling, and M. O'Keeffe, J. Chromatogr. B, 2007, 845, 1-37. D0I:10.1016/j.jchromb.2006.07.046 10. R. Rodríguez, Y. Picó, G. Font, and J. Mañes, J. Chromatogr. A, 2001, 924, 387-396. DOI:10.1016/S0021-9673(01)00716-6 11. Y. Wen, J. Li, F. Yang, W. Zhang, W. Li, C. Liao, and L. Chen, Talanta, 2013, 106, 119-126. D0I:10.1016/j.talanta.2012.12.011 12. Y. Santaladchaiyakit, and S. Srijaranai, J. Sep. Sci., 2014, 37, 3354-3361. D0I:10.1002/jssc.201400699 13. X. Deng, X. Chen, K. Lin, G. Ding, and P. Yao, Food Anal. Methods, 2013, 6, 1576-1582. D0I:10.1007/s12161-013-9572-1 14. M. Asensio-Ramos, J. Hernández-Borges, T. M. Borges-Mi-quel, and M. Á. Rodríguez-Delgado, J. Chromatogr. A, 2011, 1218, 4808-4816. D0I:10.1016/j.chroma.2010.11.030 15. V Andruch, M. Burdel, L. Kocurova, J. Sandrejova, and I. S. Balogh, Trends Anal. Chem., 2013, 49, 1-19. D0I:10.1016/j.trac.2013.02.006 16. G. Leng, W. Chen, F. Huang, and Q. Cao, J. Sep. Sci., 2014, 37, 684-690. D0I:10.1002/jssc.201301033 17. Y. Zhang, and H. K. Lee, J. Chromatogr. A, 2013, 1274, 28-35. D0I:10.1016/j.chroma.2012.12.017 18. J. Vichapong, Y. Santaladchaiyakit, R. Burakham, W. Kan-chanamayoon, and S. Srijaranai, J. Food Compos. Anal., 2015, 37, 30-37. D0I:10.1016/j.jfca.2014.08.006 19. D. Moreno-Gonzalez, J. F. Huertas-Perez, A. M. Garcia-Campana, and L. Gami-Gracia, Talanta, 2015, 139, 174-180. D0I:10.1016/j.talanta.2015.02.057 Santaladchaiyakit et al.: Methyl Salicylate-Based Vortex-Assisted ... Acta Chim. Slov. 2017, 64, 849- 857 857 20. M. Asadi, A. M. H. Shabani, S. Dadfarnia, and B. Abbasi, J. Chromatogr. A, 2015, 1425, 17-24. D01:10.1016/j.chroma.2015.11.005 21. X. Chem, X. You, F. Liu, and X. Zhang, Anal. Methods, 2015, 7, 9513-9519. D0I:10.1039/c5ay02565f 22. Y. Santaladchaiyakit, N. Phiroonsoontorn, C. Sillapatiwat, K. Kotchalee, and S. Srijaranai, J. Braz. Chem. Soc., 2015, 26, 2014-2021. DOI: 10.5935/0103-5053.20150181 23. K. Seebunrueng, Y. Santaladchaiyakit, and S. Srijaranai, Anal. Methods, 2013, 5, 6009-6016. D0I:10.1039/C3AY40096D 24. C. Wu, N. Liu, Q. Wu, C. Wang, and Z. Wang, Anal. Chim. Acta, 2010, 679, 56-62. D0I:10.1016/j.aca.2010.09.009 25. Z.-H. Yang, P. Wang, W.-T. Zhao, Z.-Q. Zhou, and D.-H. Liu, J. Chromatogr. A, 2013, 1300, 58-63. D0I:10.1016/j.chroma.2013.02.054 26. Y. Santaladchaiyakit, and S. Srijaranai, Food Anal. Methods, 2014, 7, 1238-1246. DOI:10.1007/s12161-013-9738-x 27. D. Moreno-González, J. F. Huertas-Pérez, A. M. García-Campaña, and L. Gámiz-Gracia, Talanta, 2015, 139, 174-180. D0I:10.1016/j.talanta.2015.02.057 28. A. Ambrus, in: J. L. Tadeo (Ed.), Analysis of pesticides in food and environmental samples, 2008, CRC Press, Taylor & Francis Group, New York, 125-152. 29. R. Halko, P. Sanz, S. Ferrera, and J. J. S. Rodríguez, Chroma-tographia , 2004, 60, 151-156. D0I:10.1365/s10337-004-0364-z 30. Q. Wu, Y. Li, C. Wang, Z. Liu, X. Zang, X. Zhou, and Z. Wang, Anal. Chim. Acta, 2009, 638, 139-145. D0I:10.1016/j.aca.2009.02.017 Povzetek Za hitro predkoncentracijo fungicidov (karbendazim, tiabendazol, fluberidazol) iz vzorcev medu smo uporabili emulzi-fikacijsko ekstrakcijo s pomočjo surfaktanta in z uporabo vorteksa s topilom metil salicilatom (MeSA-VASEME), ki ji je sledila analiza z visokozmogljivo tekočinsko kromatografijo. Metil salicilat (MeSA) smo uporabili kot ekstrakcijsko topilo, medtem ko je surfaktant izboljšal ekstrakcijo ob disperziji z vorteks mešanjem. Optimalni MeSA-VASEME pogoji so bili: 100 |L MeSA, 2,0 mmol L-1 natrijevega dodecil sulfata in mešanje z vorteksom pri 1200 rpm za 90 s. Dobili smo predkoncentracijske faktorje v območju 32-40. Meja zaznave v preiskovanih vzorcih medu je bila 0,5 |g L-1. Izkoristek dodanih tarčnih fungicidov pri 20, 50 in 100 |g L"1 je bil 81,5-116,8 % z relativnim standardnim odklonom pod 11 %. Predlagana metoda je preprosta, občutljiva, porabi manj organskega topila, ni draga, je hiter postopek za analizo preostankov fungicidov v vzorcih medu. Santaladchaiyakit et al.: Methyl Salicylate-Based Vortex-Assisted ... 858 DOI: 10.17344/acsi.2017.3495 Acta Chim. Slov. 2017, 64, 858-864 ^creative tycommor Scientific paper A One-pot Multicomponent Reaction for the Synthesis of Oligoetherols with Azacyclic Rings Jacek Lubczak, Renata Lubczak and Iwona Zarzyka Rzeszow University of Technology, Department of Organic Chemistry, Al. Powstancow Warszawy 6, 35-959 Rzeszow, Poland * Corresponding author: E-mail: jml@prz.edu.pl Received: 04-05-2017 Abstract The one-pot multicomponent synthesis of oligoetherols containing azacycles is described. They were obtained by reaction of isocyanuric, barbituric, or uric acid or melamine with glycidol and alkylene carbonates. The isolated products were characterized by physical methods and their properties were compared with the same compounds obtained in two-step protocol. The oligoetherols with 1,3,5-triazine ring obtained by both methods were then used to form polyurethane foams and their properties were compared. Keywords: Azacyclic compounds, glycidol, alkylene carbonates, oligoetherols, one-pot multicomponent reactions, polyurethane foams 1. Introduction The methods of obtaining oligoetherols with azacycles like perhydro-1,3,5-triazine, 1,3,5-triazine, purine or pyrimidine rings, which are present in the structure of iso-cyanuric acid (IA, I), uric acid (UA, II), barbituric acid (BA, III), and melamine (MEL, IV) (Scheme 1) are described in the literature.1,2 These oligoetherols can be applied to form rigid polyurethane foams (PUFs) of enhanced thermal resistance due to the presence of above mentioned rings. Oli-goetherols can be obtained from IA, MEL or adenine and excess oxiranes like ethylene oxide (EO) or propylene oxide (PO)3-5 or from the reaction of alkylene carbonates (AC) with the mentioned azacylic compounds.2 General reaction pathway is shown in Scheme 2. Scheme 2. Reaction NH groups with oxiranes or alkylene carbonates where R = -H, CH3 The non-catalytic reactions of IA, UA, BA and MEL with glycidol (GL),6 and later with AC7-9 also led to the mentioned oligoetherols. Those syntheses were conducted in two steps; in the first step hydroxyalkyl derivatives of azacycles were obtained as semi-solid resins in reaction of Scheme 1. Azacyclic compounds used to obtain oligoetherols Lubczak et al.: A One-pot Multicomponent Reaction for the Synthesis Acta Chim. Slov. 2017, 64, 858-864 859 azacycles with GL, and then the semiproducts were hy-droxyalkylated with excess of ethylene (EC) or propylene (PC) carbonates in the presence of potassium carbonate catalyst according to the Scheme 3. Scheme 3. Obtaining of oligoetherols in two steps: reaction NH groups in azacycles compounds with glycidol and then with alky-lene carbonates where R= -H, CH3, p + q = n Those methods have some disadvantages. The deri-vatization of azacycles with oxiranes is difficult due to low solubility of azacycles in organic solvents suitable for synthesis. Moreover, high boiling solvents like dimethylsulf-oxide (DMSO) have to be vaccum distilled from post-reaction mixtures. The DMSO is also unstable at elevated temperatures and its odor originates from the products of its decomposition.2 On the other hand, the oxiranes are low boiling reagents, therefore the pressure reactors must be used. Also, the oxiranes are toxic, cancerogenic, and form explosive mixtures with air.10 When AC are used instead oxiranes, the structures of the products are very similar. The reactions with excess AC can be performed at temperatures between 160 °C and 200 °C in the presence of catalysts, like potassium carbonate or diaza[2.2.2]bicy-clooctane (DABCO). This protocol results in formation of products with limited functionality and partial decomposition of oligoetherols at synthesis temperature, especially the dehydration of products resulting in the formation of double C=C bonds. That side-reaction also diminishes the functionality of products and finally the PUFs obtained from them have diminished mechanical properties. Considering the two step synthesis by reaction of azacycle with GL, followed by AC, the exothermic effect observed during the first step causes the temperature increase even above 200 °C (Table 1) which may result in thermal decomposition of oligoetherols. Therefore, the scale-up of this process is technically demanding. Furthermore, the obtained products are semi-solid resins; the high viscosity of the oligoetherols very difficult proper homog-enization with isocyanates. Therefore the application of AC for further hydroxyalkylation is necessary to obtain a product of lower viscosity. Here we describe the results on multicomponent reaction and one-pot reaction (multicomponent one-pot reaction, MOPR) which were optimized in a way to eliminate above mentioned disadvantages. Thus we have ob- tained one product from at least three substrates introduced at the beginning of the process. In MOPR method the reagents were added consecutively, after the previous step was completed. The products obtained by both methods were isolated and purified in the same way and their properties were compared to those of the products obtained in the two-step protocol. The oligoetherols obtained from MEL were also tested as substrates for polyurethane foams (PUFs). The PUFs obtained here were also compared to those PUFs which were obtained from the oligoetherols synthesized in two-step protocol. 2. Experimental Section 2. 1. Synthesis To a three-neck round bottom flask equipped with mechanical stirrer, reflux condenser, and thermometer the following reagents were introduced: - 0.6 mol EC (pure, Fluka, Schwitzeland) or PC (pure, Fluka, Schwitzeland), 0.6 mol GL (pure, Sig-ma-Aldrich, Germany), and 0.1 mol IA (pure, Fluka, Switzerland) or - 0.7 mol EC or PC, 0.6 mol GL, and 0.1 mol UA (pure, Avocado, Germany) or - 0.8 mol EC or PC, 0.4 mol GL, and 0.1 mol BA (pure, BDH, Laboratory Supplies, UK) or - 1.6 mol EC or 1.2 mole PC, 0.8 mol GL and 0.1 mol MEL (pure, Fluka, Switzerland). The mixture was heated at 150 °C until azacyclic compound was dissolved and afterwards the system was kept as such for 1 hour. In the case the system containing MEL the temperature was raised to 120 °C; at that moment the exothermic effect resulted in increase of temperature to 160 °C. The reaction mixture was cooled down to maintain 150 °C until dissolution of MEL was completed and further for 1 hour longer. Then the potassium carbonate (pure, POCH, Poland) as catalyst for the reaction with AC was introduced. The amount of catalyst was the same as previously used for the two-step method;6-9 the reactions were performed with IA, BA and UA or MEL and EC at 150 °C or with PC at 160 °C until the completed reaction of AC. The progress of reaction was monitored by epoxide number and concentration of AC. 2. 2. Analytical Methods The course of the reaction between azacyclic compounds and GL was followed by measuring the content of epoxide groups.11 The progress of reaction between semi-product and AC was monitored using barium hydroxide method described in the literature.12 The sample was then treated with 2.5 mL of 0.15 M barium hydroxide, vigorously shaken and the excess of barium hydroxide titrated off with 0.1 M HCl solution. Hydroxyl number of Lubczak et al.: A One-pot Multicomponent Reaction for the Synthesis 860 Acta Chim. Slov. 2017, 64, 858-864 the obtained oligoetherols was determined with the use of acetic anhydride.13 Elemental analysis for C, H, N, were done with EA 1108, Carlo-Erba analyzer. IR spectra were registered on PARAGON 1000 FT IR Perkin Elmer spectrometer using ATR technique. 2. 3. Properties of Oligoetherols Density, viscosity, and surface tension of oligoetherols were determined with pycnometer, Hoppler viscometer (typ BHZ, prod. Prufgeratewerk, Germany) and by the detaching ring method, respectively.14,15 2. 4. Foam Preparation Into a 500 mL cup 10 g oligoeterol, then 2% water, 2.3% silicon L-6900 (pure, Houdry Hulls, USA) as surfactant, and 2.2% or 3.9% triethylamine (TEA, pure, POCH, Poland) as catalyst were introduced. The mixture was vigorously stirred and then the polymeric methylene diphe-nyl 4,4'-diisocyanate (pMDI containing 30 mass% of three-functional isocyanates, Merck, Darmstadt, Germany) was added. The mixture was vigorously stirred (1800 rpm) until creaming started. 2. 5. Studies of Foams The apparent density,16 water uptake,17 dimensional stability in temperature 150 °C18 heat conductance coefficient, heat capacity, and compressive strength19 of polyure-thanes foams with flame retardants were measured. Thermal resistance of modified foams was determined both by static methods. In static method the foams were heated at 150, 175 and 200 °C with continuous measurement of mass loss and determination of mechanical properties before and after heat exposure. 3. Results and Discussion 3. 1. Synthesis of Oligoetherols The synthesis of oligoetherols was performed as stepwise process in the mixture of all necessary reagents, i.e. azacycle, Gl, and AC. This can be considered as multi-components reaction. The first observed reaction was between azacycle and GL. The second step (the reaction with AC) needed addition of catalyst to the reaction mixture. Therefore, the process can also be considered as a one-pot Table 1. Exothermic effects accompanying the reaction of azacycles with GL in two-steps synthesis of oligoetherols (the starting amount of azacycle was 0.05 mole)6-9 GL-reaction fill (TAi>thpI*Al Reagents accompanied Reaction VyllgUClllClUl molar ratio exothermic effect time [h] temp. [°C] IA:GL:EC7 1:6:6 150 1,5 IA:GL:PC7 1:6:6 150 1,5 UA: GL:EC8 1:6:7 180 0.5 UA: GL:PC8 1:6: 7 180 0.5 BA:GL:EC9 1:4:8 130 3.0 BA:GL:PC9 1:4:7 130 3.0 MEL:GL:EC6 1:8:16 230 0.5 MEL:GL:PC6 1:8:12 220 0.5 reaction. After heating the reaction mixture up to 150 °C the reaction between azacycle and GL took place. At this stage the AC played the role of a solvent. No exothermic effect was observed in contrary to the two-step method, where after heating of azacycle and GL to 120-130 °C it was found (Table 1). In the case of MEL as azacycle the exothermic effect was observed at 120 °C leading to an increase of temperature to 160 °C (MEL:GL:AC = 1:8:8 system). The temperature of this system was kept at 150 °C by cooling. The AC are non-flammable, non-toxic, and very polar10 and they are good solvents for azacycles, therefore in the reaction mixture they are useful as solvents, which are not necessary to be removed after completion of the reaction with GL. They react further after introducing the catalyst (potassium carbonate) at 150 °C or 160 °C to give oligoetherols. On the other hand, the GL enables to obtain oligoetherols with higher functionality in relations to azacycle. The increase of functionality allows to obtain highly crosslinked, rigid PUFs. Moreover, the hydroxyal-kyl derivatives obtained from azacycle and GL are better soluble in AC than azacycles, thus homogenization of the mixtures with AC can be reached at lower temperatures than in the case of the azacycle-AC system (temperature needed is 180-230 °C). This allows to avoid thermal decomposition of the semiproducts. We have obtained oligoetherols in the reaction of azacycles with GL and AC at the same molar ratio of substrates as it has been used earlier in the two-step protocols.6-9 Analysis of the products obtained from IA, UA, or BA with GL and AC indicated that amount of AC in the Scheme 4. Reaction of alkylene carbonate with hydroxy! group Lubczak et al.: A One-pot Multicomponent Reaction for the Synthesis ... Acta Chim. Slov. 2017, 64, 858-864 861 reaction mixture did not change upon reaction of azacycle in the latter system, MEL is hydroxylated also with AC. with GL, while in the case of MEL, the AC was also con- Generally AC releases CO2 during the condensation with sumed at the level of up to 5% of the initial amount. Then, hydroxyl groups, according to the Scheme 4. Table 2. Elemental analysis and hydroxyl number of oligoetherols obtained in MOPR and two-steps reactions6-9 Oligoetherol Reagents molar ratio Method of synthesis of oligo-etherol C Elemental analysis calculated H N [% mas] C found H N Hydroxyl number [mgKOH/g] found calc. IA:GL:EC 1:6:6 MOPR two-step7 47. 31 7.53 5.02 47.21 47.51 7.72 7.24 4.95 5.04 592 603 IA:GL:PC 1:6:6 MOPR two-step7 50. 81 8.14 4.56 50.88 50.69 8.25 8.44 4.48 4.84 534 548 UA: GL:EC 1:6:7 MOPR two-step8 48. 26 7.39 6.09 48.13 48.51 7.12 7.03 6.25 6.21 571 595 610 UA: GL:PC 1:6: 7 1:6: 8 MOPR two-step8 51.87 52.42 8.06 8.18 5.50 5.20 52.52 51.95 8.25 8.40 5.38 5.83 504 525 551 521 BA:GL:EC 1:4:8 MOPR two-step9 49. 48 7.73 3.60 49. 79 8.03 3.58 560 558 578 BA:GL:PC 1:4:7 MOPR two-step9 53. 49 8.43 3.37 53. 41 8.46 3.51 535 527 540 MEL:GL:EC 1:8:16 MOPR two-step6 49. 79 8.30 5.90 50.07 49.60 8.21 8.43 5.53 5.44 517 544 552 MEL:GL:PC 1:8:12 MOPR two-step6 53.46 8.91 5.94 53.87 53.02 8.81 8.75 6.23 5.70 525 548 476 Table 3. The physical properties of oligoetherols at 20 °C obtained various methods in the reaction of azacycles with GL and AC Oligoetherol Reagents molar ratio Method of synthesis of oligo-etherol Density [g/cm3] Viscosity [mPa x s] Surface tension [N/m] x 103 IA:GL:EC 1:6:6 MOPR two-step7 1.28 1.28 17200 17220 50.8 52.6 IA:GL:PC 1:6:6 MOPR two-step7 1.24 1.20 16920 16970 48.2 46.9 UA:GL:EC 1:6:7 MOPR two-step8 1.27 1.28 30200 (30 °C) 35000 (30 °C) 52.4 57.0 UA:GL:PC 1:6:7 (8) MOPR two-step8 1.21 1.21 42800 47500 45.2 47.0 BA:GL:EC 1:4:8 MOPR two-step9 1.26 1.26 93.2 95.6 45.8 46.5 BA:GL:PC 1:4:7 MOPR two-step9 1.17 1.18 86.8 86.3 46.8 46.5 MEL:GL:EC 1:8:16 MOPR two-step6 1.28 1.26 8445 34260 54.2 46.2 MEL:GL:PC 1:8:12 MOPR two-step6 1.20 1.19 25640 65430 46.3 43.5 Lubczak et al.: A One-pot Multicomponent Reaction for the Synthesis 862 Acta Chim. Slov. 201?, 64, 858-864 Elemental analytical data and hydroxyl number measurements indicated that the composition of the obtained oligoetherols is comparable with those of oligoeth-erols obtained from the two-step reactions (Table 2). Exceptionally the oligoetherol obtained from UA:GL:PC = 1:6:8 system has one equivalent of PC-derived oxyal-kylene group more than the oligoetherol obtained in the two-step process. This difference is due to the higher temperature of reaction applied in the two-step protocol, which caused partial decomposition of PC to PO and CO2 volatiles. a) 3000 2000 1500 1000 500 b) 3000 2000 1500 1000 650 Fig. 1. IR spectrum of oligoetherols obtained in MOPR (a) and in the two-step reaction (b) in molar ratio MEL:GL:EC = 1:8:16 The density, viscosity, surface tension and hydroxyl number of oligoetherols obtained from azacycles, GL, and AC with MOPR method and two-steps method6-9 are collected in Table 3. The parameters of both series of oligoeth-erols are comparable except the oligoetherols obtained from MEL. Generally, the oligoetherols obtained in the two-step protocol had higher viscosity, which can be explained by condensation of hydroxyalkyl groups at high temperature and longer reaction time than in the MOPR method. The IR spectra of oligoetherols obtained from MEL in MOPR method are similar to those obtained in the two-steps method (Fig. 1a versus 1b). In both cases the broad valence hydroxyl group band is centered at 3324 cm-1. The ring C=N valence band was observed within 1660-1470 cm-1 region, while C-OH vibrations were found in the 1350-1450 cm-1 region. The product of the reaction between MEL and GL and EC had a characteristic band at 2870 cm-1 attributed to the methylene and methine groups. The band at 813 cm-1 was related to the 1,3,5-triazine ring, while ether bonds led to the appearance of the band at 1055 cm-1. The spectra of the products obtained from PC instead of EC are similar; the additional low-intensity band at 1372 cm-1 was attributed to the deformation vibration of methyl group. IR spectra of oligoetherols obtained from IA, BA and UA using MOPR and the two-stage method are very similar. 3. 2. Obtaining of Polyurethane Foams The substantial difference of physical properties of the oligoetherols obtained from MEL in MOPR method in comparison with the analogues obtained in the two-steps synthesis prompted us to further study these compounds as substrates to form PUFs. It has been found that amount of isocyanate necessary to form the PUF is independent of the method used (Table 4). However, the optimized amount of catalyst and surfactants are lower in the syntheses by MOPR protocol. Cream times were similar, while rise times are longer and drying times are shorter, which Table 4. Foaming process Oligoetherol Method of synthesis of oligo-etherol Amount of co-substrate [g/100g oligoetherol] pMDI TEA silicone Cream time[ s ] Foaming Process Rise time [s] Tack free time [s] MEL:GL:EC 1:8:16 MOPR two-step6 160 2.2 160 3.9 2.3 3.2 20 22 30 13 immediately 35 MEL: GL: P C 1:8:12 MOPR two-step6 152 3.9 152 6.2 2.3 3.2 24 24 24 11 immediately 24 2 mass % of water related to the mass of oligoetherol was used in both methods Cream time: the time elapsed from the moment of mixing to the start of volume expansion; Rise time: the time from the start of expansion to the moment of reaching the sample final volume; Tack free time: the time from reaching by the sample its final volume to the moment of losing its surface adhesion Lubczak et al.: A One-pot Multicomponent Reaction for the Synthesis 863 Acta Chim. Slov. 201?, 64, 858-864 Table 5. Some properties of polyurethane foams Foam Method of obtained synthesis Density Absorb. of water from of oligo- [kg/m3] [wt %] after oligoetherol etherol S min 3 h 24 h MEL:GL:EC MOPR 63.0 1.0 13 1.8 1:8:16 two-step6 67.2 4.4 6.5 8.5 MEL:GL:PC MOPR 42.1 1.2 1.6 1.9 1:8:12 two-step6 45.6 3.9 6.0 9.0 Dimensional stability [%] in temperature 150 °C Length Width Hight change [%] change [%] change [%] 20 h 40 h 20 h 40 h 20 h 40 h Heat conductance coefficient -0.44 -0.59 -0.69 -0.74 -0.46 -0.48 0.0322 ± 0.0005 0.01 0.01 0.01 0.02 0.01 0.02 0,0667 -0.39 -0.41 -0.61 -0.68 -0.80 -0.81 0,0344 ± 0.0006 0.01 0.02 0.01 0.02 0.01 0.02 0.0483 Table 6. Thermal stability and compressive strength of polyurethane foams Method of synthesis Mass loss in % wt. after Compressive strength [MPa] Oligoeterol of oligo- exposure in month before exposure after exposure etherol in temperature [oC] in temperature [°C] 150 175 200 1S0 17S 200 MEL:GL:EC MOPR 12.0 25.7 37.9 0.106 0.260 0.108 0.030 two-step6 15.0 23.9 36.8 0.134 0.208 0.245 0.229 MEL:GL:PC MOPR two-step6 11.8 13.5 30.0 23.4 45.6 35.1 0.152 0.146 0.404 0.246 0.316 0.205 0.034 0.166 Compression strength measured at 10% deformation could be the result of different reactivity of oligoetherols obtained by the MOPR method. The PUFs obtained from oligoetherols synthesized in MOPR are rigid and have similar apparent density as those obtained from the two-steps derived oligoetherols. They have water uptake value 1.9% in 24 hour test, which is related to domination of closed pores in the structure of PUF (Table 5). They also showed low polymerization shrinkage. It is noteworthy that generally water uptake of PUFs obtained both from EC and PC in the MOPR is similarly low, while that of PUFs obtained from oligoetherols synthesized in the two-steps method is considerably higher. Also the latter had higher heat conductance coefficient. The thermal resistance PUFs was studied by static method, i.e. by measuring mass loss within one month heat exposure at 150, 175 and 200 °C with concomitant measurements of compression strength (Table 6). Generally, the PUFs obtained from EC show higher thermal resistance than those obtained from PC (Table 6). For comparison the mass losses of PUFs obtained from MEL, GL, and AC in the two-step method are also collected in the Table 6. The mass loss of PUFs obtained from oligoetherols synthesized by MOPR with EC involved are comparable with the analogues obtained in the two-step procedure, while mass loss of PUFs obtained by MOPR and PC is considerably larger than that of analogous PUFs obtained from corresponding oligoetherols formed in two-step method. Compression strength of PUFs before thermal exposure does not depend on oligoetherol origin. However, the PUFs obtained from oligoetherols synthesized in MOPR showed considerable increase of compression strength upon annealing at 150 °C, presumable due to additional crosslinking. However annealing at 175 and 200 °C resulted in an important decrease of compression strength compared to the PUFs obtained from oligoetherols synthesized in the two-step processes. 4. Conclusions The one-pot multicomponent reaction (MOPR) of synthesis the oligoetherols with azacyclic rings was established. The substrates were azacycles, glycidol, and al-kylene carbonates. The oligoetherols obtained by this method showed similar properties as those obtained from the same reagents used consecutively, with isolation of semiproduct, except for oligoetherols obtained from melamine. The oligoetherols obtained in MOPR are useful substrates to obtain polyurethane foams of enhanced thermal resistance. 5. References 1. J. Lubczak, Polimery, 2011, 56, 360-368. 2. J. Lubczak, Polimery, 2011, 56, 452-460. 3. K. Frisch, D. Tummers, A. Nijenhuis, Preparation oftris(b-hy-droxypropyl)isocyanurate, US Patent Number 4,198,505, date of patent January 18, 1979. 4. T. Sniezek, E. Andrysiak, W. Montewski, H. Gniadowska, J. Lubczak et al.: A One-pot Multicomponent Reaction for the Synthesis 864 Acta Chim. Slov. 2017, 64, 858-864 Wojciechowski, Method of production of branched polyeth-ers, Polish Patent Number 69168, date of patent December 20, 1973. 5. R. Lubczak, J. Appl. Polym. Sci. 2002, 86, 489-497. D01:10.1002/app.11021 6. K. Cyzio, J. Lubczak, Polymer Int., 2013, 62, 1735-1743. DOI: 10.1002/pi.4476 7. K. Cyzio, J. Lubczak, J. Appl. Polym. Sci., 2011, 122, 417-426. D0I:10.1002/app.34131 1 8. K. Cyzio, J. Lubczak, Polym. Plast. Technol. Eng., 2017, 56, 13-21. DOI: 10.1080/03602559.2016.1211693 9. E. Kania, J. Lubczak, Polimery, 2014, 59, 851-854. D0I:10.14314/polimery.2014.851 10. D. Kijowska, Przemyst chemiczny, 2005, 84, 678-683. 11. Z. Brojer, P. Penczek, Epoxide resins, WNT, Warsaw, Poland, 1972, pp. 451-452. 12. D. Kijowska, S. Wolowiec, J. Lubczak, J. Appl. Polym Sci., 2004, 93, 294-300. D0I:10.1002/app.20453 13. B. Czuprynski, Questions of chemistry and technology of polyurethanes, The Publishing House of the Academy of Bydgoszcz, Poland, 2004, pp.195-196. 14. T. Broniewski, A. Iwasiewicz, J. Kapko, W. Placzek, Testing and evaluation of properties of plastics, WNT, Warsaw, Poland 1967, pp. 129-357. 15. T. Drynski, Laboratory of Physics, PWN, Warsaw, Poland 1967, pp. 125-132. 16. Cellular Plastics and Rubbers. Determination of apparent (bulk) Density, Polish (European) Standards PN-EN ISO 845-2000, Ed. Polish Committee for Standardization. 17. Cellular Plastics, rigid. Determination of Water Absorption, Polish (European) Standards PN-EN ISO 2896-1986, Ed. Polish Committee for Standardization. 18. Cellular Plastics, rigid. Test of dimensional Stability, Polish (European) Standards PN-EN ISO 2796-1986, Ed. Polish Committee for Standardization. 19. Cellular Plastics, Compression Test for rigid Materials, Polish (European) Standards PN- EN ISO 844-1978, Ed. Polish Committee for Standardization. Povzetek Opisujemo enolončno multikomponentno sintezo oligoeterolov, ki vsebujejo azaciklične sisteme. Pripravili smo jih z reakcijami izocianurne, barbiturne ali sečne kisline oz. melamina z glicidolom in alkil karbonati. Izolirane produkte smo karakterizirali s pomočjo fizikalnih metod in pripravljene spojine primerjali s spojinami, ki smo jih pridobili z dvostopenjskim protokolom. Oligoeterole z 1,3,5-triazinskim obročem, dobljene z obema metodama, smo uporabili za pripravo poliuretanskih pen; katerih lastnosti smo tudi primerjali. Lubczak et al.: A One-pot Multicomponent Reaction for the Synthesis ... DOI: 10.17344/acsi.2017.3481 Acta Chim. Slov. 2017, 64, 865-876 ^creative tS1 commons Scientific paper Synthesis and Structure Activity Relationship of Some Indole Derivatives as Potential Anti-inflammatory Agents Samar S. Fatahala,1* Mohammed A. Khedr 2 and Mossad S. Mohamed1 1 Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Helwan University, Ain-Helwan 2 Pharmaceutical Chemistry Department, Faculty of Pharmacy, Helwan University, Ain-Helwan Postal code: 11795, Helwan, Cairo, Egypt * Corresponding author: E-mail: ssfathatllah@yahoo.com, samarradwan1@yahoo.com Received: 25-04-2017 Abstract A series of fused pyrroles were synthesized and tested for their in vivo anti-inflammatory activity. Among 14 examined derivatives, 5 derivatives (1b-e, g and 5b), showed a promising anti-inflammatory activity equivalent to reference anti-inflammatory drugs (indomethacin and ibuprofen). A molecular docking study was conducted to interpret the biological activities of the tested compounds. The docking results were complementary with the phase of the biological survey and confirmed the biological effects. Keywords: Fused pyrroles, tetrahydroindoles, Molecular Docking, anti-inflammatory assay 1. Introduction Non-steroid anti-inflammatory drugs (NSAIDs) have been amongst the most widely developed drugs.1-5 They have provided an alternative to steroid therapy, which has revealed many problems related to parallel endocrine and metabolic activity, induced osteoporosis and hypercalcemia, as shown by Lessigiarska et al.6 The postponement in treatment causes severe side effects including rhinnorrhoea, rheumatoid arthritis, and atherosclerosis.7 With their anti-pyretic and analgesic activities, they represent a choice treatment in various inflammatory diseases such as arthritis and rheumatisms.8-105-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl The NSAIDs have exerted their anti-inflammatory activities through cycloo-xygenase (COX) inhibition.11-13 Structural variation of the heterocyclic rings through the manipulation of the heterocyclic core influences the activity of the resulting fused systemes, among these of pyrroles and their fused deivatives.14-16 Due to their pharmaceutical importance,17-22 attention was paid to develop a new synthetic route for pyrroles and their fused forms.23-28 Pyrrolylacetic acid derivatives such as tolmetin (Rumatol®) and zomepirac (Zomax®) were proved to be NSAIDs6 with strong anti-inflammatory activity.29,30 Other pyrrole and fused pyrrole compounds have been recently reported as potent COX-1 and COX-2 inhibitors:31,324-benzodioxine or pyrrole nucleus are described. All the newly synthesized compounds were examined for their in vitro and in vivo anti-inflammatory activity. Several derivatives, including (S indometha-cin (Indacin®), acemetacin (Emflex®) and etodolac (Eto-dine®) as indole derivatives, and ketorolac (Ketolac®) as a pyrrole derivative.33-36 These compounds blocked pros-taglandin synthesis by non-selective inhibition of COX-1 and COX-2 (indomethacin, acemetacin, tolmetin and ketorolac) or by selective inhibition of COX-2 (etodolac) (Fig. 1). Due to the importance of this ring system,39 we prepared some fused o-aminocyano tetrahydrobenzo[fo]pyr-role derivatives as an essential propagation step in our search for new pyrrole and pyrrolopyrimidine deriva-tives,40-43 and evaluated them for the anti-inflammatory activities. In addition, a molecular docking study has been done to explain the activity of the biologically active compounds. Radwan et al.: Synthesis and Structure Activity Relationship ... 866 Acta Chim. Slov. 2017, 64, 865-876 NS\llis contain Benzoj^jpyrrole jludole moietyj Rutaecarpinealkaloid |COX-2 Inhibitor«] Eiodolat I NSAIOs tin] !:iin Pyrrole ni n iit v Figure 1: Pyrroles and Indoles derivatives as NSAIDs37,38their mechanism of action at the molecular level such as cyclooxygenase (COX 2. Experimental 2. 1. Chemistry General Information for Chemicals All melting points were uncorrected and measured using Electro-thermal IA 9100 apparatus (Shimadzu, Japan). IR spectra were recorded as potassium bromide pellets on a Perkin-Elmer 1650 spectrophotometer (USA), Faculty of Science, Cairo University, Cairo, Egypt. 1H-NMR and 13C-NMR spectra were performed on Jeol NMR FXQ-300 MHz and Jeol NMR FXQ-500 MHz spectrometers; chemical shifts are expressed as ppm against TMS as the internal reference (Faculty of Science, Cairo University, Cairo, Egypt). Mass spectra were recorded at 70 eV EI Ms-QP 1000 EX (Shimadzu, Japan), Faculty of Science, Cairo University, Cairo, Egypt. Microanalyses were operated using Vario, Elemental apparatus (Shimad-zu, Japan), Organic Microanalysis Unit, Faculty of Science, Cairo University, Cairo, Egypt. Column Chromatography was performed on (Merck) Silica gel 60 (particle size 0.060.20 mm). Compounds 1a,b, 3a,b and 5a were prepared as reported in the literature.44 All new compounds yielded spectral data consistent with the proposed structures and microanalysis within ±0.4% of the theoretical values. General methods for the preparation of 2-oxo-substituded malononitriles I and II A mixture of 2-chloroketone (0.01 mol) and malo-nonitrile (1 g, 0.016 mol) in CHCl3 (50 mL), was cooled with stirring to 0-5 °C for 30 min. A cold solution of NaOH (2.5 g in 10 mL of water) was added to the mixture dropwise for 30 min. The stirring was continued for 50 min under ice and the reaction mixture then left for 72 h at room temperature. The precipitate formed was filtered off, recrystallized from appropriate solvent. 2-(2-Oxocyclohexyl)malononitrile (I) It was obtained as a brownish red solid [EtOH/H2O]. Yield 64%, m.p. 142-145 °C, FT-IR (KBr) v 2345, 1690 1 v ' max cm-1, EIMS m/z (%) 162 [M+] (7.5), 106 (100). Anal. Calcd for C9H10N2O: C, 66.65; H, 6.21; N, 17.27. Found: C, 66.98; H, 6.54; N, 17.03. 2-(1-Oxo-2,3-dihydro-1H-inden-2-yl)malononitrile (II) It was obtained as a reddish brown solid [EtOH/ HO]. Yield 65%, m.p. 142-147 °C, FT-IR (KBr) v 2360, 2 max 1705 cm-1, EIMS m/z (%) 196 [M+] (4), 144 (100). Anal. Calcd for C12H8N2O: C, 73.46; H, 4.11; N, 14.28. Found: C, 73.78; H, 4.423; N, 14.60. 2-Amino-1-(aryl)-4,5,6,7-tetrahydro-1H-indole-3-carboni-trile 1a-g Method A: A solution of I (1 g, 0.016 mol) in ethanol (30 mL), the appropriate aromatic amine (0.01 mol) and conc HCl (4 mL) were added. The reaction mixture was refluxed for 72 h (TLC monitored), cooled to room temperature, poured onto crushed ice (25 g.), and neutralized with NaOH. The precipitate formed was filtered off, dried and recrystallized from appropriate solvent to give compound 1. Radwan et al.: Synthesis and Structure Activity Relationship ... Acta Chim. Slov. 2017, 64, 865-876 867 Method B: A solution of I (1 g, 0.016 mol) in isopro-panol (30 mL), the appropriate aromatic amine (0.01 mol) was added. The reaction mixture was refluxed for 72 h (TLC monitored), then cooled to room temperature, and the solvent was removed under reduce pressure. The residue was dissolved in methanol (10 mL), poured into crushed ice (25 g). The precipitate formed was filtered off, dried and recrystallized from appropriate solvent to give compound 1 2-Amino-1-(4-chlorophenyl)-4,5,6,7-tetrahydro-1H-indole- 3-carbonitrile (1c) It was obtained as a brown solid [EtOH]. Yield for method A) 73% and for B) 52%, m.p. 184-188 °C. FT-IR (KBr) v 3380-3250, 2315 cm-1. 1H-NMR (DMSO-d, v ' max v 6 300 MHz) 8 = 1.63-1.88 (m, 4H, 2 x CH2), 2.5-2.78 (m, 4H, 2 x CH2), 5.78 (br.s, 2H, NH2, D2O exchangeable), 7.2-7.8 (m, 4H, Ar-H). 13C NMR (DMSO-d6): 8 34.1(CH2, C-4), 34.9 (CH2, C-7), 35.1 (CH2, C-5), 36.2 (CH2, C-6), 116.1 (N-*C=C, C-4a), 116.9 (N-C=C*, C-7a), 119.8 (C'N), 124.1 (C-2), 125.6 (C-3), 130.9 (CH aromatic, C-2'), 132.2 (CH aromatic, C-3'), 132.8 (CH aromatic, C-5'), 133.4 (CH aromatic, C-6'), 134.1(C-Cl, C-4'), 145.2 (C-N, C-1') ppm; EIMS m/z (%) 271 [M+] (25), 273 [M++2, 37Cl] (8.5), 149 (100). Anal. Calcd for C15H14ClN3; C, 66.30; H, 5.19; N, 15.46. Found: C, 66.68; H, 5.56; N, 15.30. 2-Amino-1-o-tolyl-4,5,6,7-tetrahydro-1H-indole-3-carbo-nitrile (1d) It was obtained as a brownish red solid [EtOH/H2O]. Yield for method A) 80%, B) 52%, m.p. 195-197 °C. FT-IR (KBr) v 3380-3280, 2285 cm-1. 1H-NMR (DMSO-d, max 6 300 MHz) 8 = 1.53-1.78 (m, 4H, 2 x CH2), 2.12 (s, 3H, CH3), 2.5-2.78 (m, 4H, 2 x CH2), 6.18 (br.s, 2H, NH2, D2O excliangeable), 7.2-7.6 (m, 4H, Ar-H) ppm. EIMS m/z (%) 251 [M+] (31) , 149 (100). Anal. Calcd for C16H17N3: C, 76.46; H, 6.82; N, 16.72. Found: C, 76.86; H, 6.41; N, 16.77. 2.2-2.45 (m, 4H, 2 x CH2), 2.33 (s, 3H, CH3), 3.12 (s, 3H, N-CH3), 5.28 (br.s, 2H, NH2, D2O exchangeable), 7.2-7.6 (m, 5H, Ar-H) ppm. EIMS m/z (%) 347 [M+] (38), 173 (100). Anal. Calcd for C20H21N5O: C, 69.14; H, 6.09; N, 20.16; O, 4.61. Found: C, 69.28; H, 6.16; N, 20.30; O, 4.30. 2-Amino-1-(pyridin-2-yl)-4,5,6,7-tetrahydro-1H-indole-3-carbonitrile (1g) It was obtained as a brownish black solid [EtOH]. Yield for method A) 65%, B) 48%, m.p. 194-196 °C. FT-IR (KBr) v 3370-3290, 2345 cm-1. 1H-NMR (DMSO-d, max 6 300 MHz) 8 = 1.7-1.75 (m,4H, 2 x CH2), 2.6-2.8 (m,4H, 2 x CH2), 5.28 (br.s, 2H, NH2, D2O exchangeable), 7.6-8.4 (m, 4H, Ar-H) ppm. EIMS m/z (%) 238 [M+] (100). Anal. Calcd for C14H14N4: C, 70.57; H, 5.92; N, 23.51. Found: C,70.71; H, 5.83;N, 23.46. 2-Amino-1-(aryl)-4,5,6,7-tetrahydro-1H-indole-3-carboni-trile 2a,b A solution of II (1 g, 0.016 mol) in ethanol (30 mL), the appropriate aromatic amine (0.01 mol) and conc HCl (4 mL) were added. The reaction mixture was refluxed for 72 h (TLC monitored), then cooled to room temperature, poured into crushed ice (25 g), and neutralized with NaOH. The precipitate formed was filtered off, dried and recrystallized from appropriate solvent to give compound 2a,b 2-Amino-1-phenyl-1,4-dihydroindeno[1,2-b]pyrrole-3-car-bonitrile (2a) It was obtained as a brownish black solid [EtOH/ HO]. Yield 60%, m.p. 218-220 °C. FT-IR (KBr) v 34102 max 3240, 2335 cm-1. 1H-NMR(DMSO-d6, 300 MHz) 8 = 3.4 (s, 2H, CH2), 6.18 (br.s, 2H, NH2, D2O exchangeable), 6.8-7.4 (m, 9H, Ar-H) ppm. EIMS m/z (%) 271 [M+] (100). Anal. Calcd for C18H13N3; C, 79.68; H, 4.83; N, 15.49. Found: C, 79.89; H, 4.97; N, 15.80. 2-Amino-1-p-tolyl-4,5,6,7-tetrahydro-1H-indole-3-carbo-nitrile (1e) It was obtained as a brown solid [EtOH/H2O]. Yield for method A) 80%, B) 54%, m.p. 168-172 °C. FT-IR (KBr) v 3390-3270, 2260 cm-1. 1H-NMR (DMSO-d, 300 max 6 MHz) 8= 1.53-1.78 (m, 4H, 2 x CH2), 1.98 (s, 3H, CH3), 2.4-2.78 (m, 4H, 2 x CH2), 5.58 (br.s, 2H, NH2, D2O exchangeable), 7.2-7.8 (m, 4H, Ar-H) ppm. EIMS m/z (%) 251 [M+] (61), 92 (100). Anal. Calcd for C16H17N3: C, 76.46; H, 6.82; N, 16.72. Found: C, 76.43; H, 6.91; N, 16.97. 2-Amino-1-(4-methoxyphenyl)-1,4-dihydroindeno[1,2-b] pyrrole-3-carbonitrile (2b) It was obtained as a brown solid [EtOH/H2O]. Yield 80%, m.p. 200-205 °C. FT-IR (KBr) v 3425-3220, 2340 max cm-1. 1H-NMR (DMSO-d6, 300 MHz) 8 = 3.3 (s, 2H, CH2), 3.7 (s, 3H, OCH3), 6.23 (br.s, 2H, NH2, D2O exchangeable2), 6.8-7.51 (m, 8H, Ar-H) ppm. EIMS m/z (%) 301 [M+] (7.6), 211 (100). Anal. Calcd for C19H15N3O: C, 75.73; H, 5.02; N, 13.94; O, 5.31. Found: C, 75.61; H, 5.39; N, 14.31; O, 5.41. 2-Amino-1-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H -pyrazol-4-yl)-4,5,6,7-tetrahydro-1H-indole-3-carbonitrile (1f) It was obtained as a yellowish brown solid [EtOH/ H2O]. Yield for method A) 76%, B) 55%, m.p. 234-238 °C. FT-IR (KBr) v 3350-3270, 2270, 1680 cm-1. 1H-NMR max (DMSO-d6, 300 MHz) 8 = 1.43-1.68 (m, 4H, 2 x CH2), N-(3-Cyano-1-(aryl)-pyrrol-2-yl) acetamide 3 and 4 A suspension of the appropriate compound 1 or 2 (0.01 mol) in acetic anhydride (40 mL) was refluxed for 2 h, cooled, poured onto ice-water, neutralized with ammonia to give a precipitate which was filtered off, dried, and recrystallized from appropriate solvent, to give compounds 3 and 4. Radwan et al.: Synthesis and Structure Activity Relationship ... 868 Acta Chim. Slov. 2017, 64, 865-876 N-(1-(4-Chlorophenyl)-3-cyano-4,5,6,7-tetrahydro-1H-in-dol-2-yl) acetamide (3c) It was obtained as a brownish red solid [MeOH/ HO]. Yield 70%, m.p. 184-188 °C. FT-IR (KBr) v 3300, 2 J * v ' max 2300, 1730 cm-1. 1H-NMR (DMSO-d6, 300 MHz) 8 = 1.631.88 (m, 4H, 2 x CH2), 2.5-2.78 (7H, (4H) 2 x CH2, 3H, CH3)), 7.2-7.8 (m, 4H, Ar-H), 10.15 (s, 1H, NH, D2O exchangeable) ppm. EIMS m/z (%) 313 [M+] (19.7), 315 [M++2, 37Cl] (6), 149 (100). Anal. Calcd for C17H16ClN3O: C, 65.07; H, 5.14; Cl, 11.30; N, 13.39. Found: C, (55.07; H, 5.14; Cl, 11.30; N, 13.39. N-(3-Cyano-1-o-tolyl-4,5,6,7-tetrahydro-1H-indol-2-yl) acetamide (3d) It was obtained as a yellowish brown solid [MtOH/ HO]. Yield 80%, m.p. 200-205 °C. FT-IR (KBr) v 3280, 2 max 2223, 1703 cm-1. 1H-NMR (DMSO-d6, 300 MHz) 8 = 1.531.78 (m, 4H, 2 x CH2), 2.12 (s, 3H, CH3), 2.2-2.58 (m, 7H, (4H) 2 x CH2, 3H, CH3), 7.2-7.6 (m, 4H, Ar-H), 9.85 (s, 1H, NH, D2O exchangeable) ppm. EIMS m/z (%) 293 [M+] (19.7) 92 (100). Anal. Calcd for C18H19N3O; C, 73.69; H, 6.53; N, 14.32. Found: C, 73.99; H, 6.73; N, 14.67. N-(3-Cyano-1-p-tolyl-4,5,6,7-tetrahydro-1H-indol-2-yl) acetamide (3e) It was obtained as a reddish black solid [EtOH/H2O]. Yield 70%, m.p. 186-190 °C. FT-IR (KBr) v 3330, 2210, max 1690 cm-1. 1H-NMR (DMSO-d6, 300 MHz) 8 = 1.53-1.78 (m, 4H, 2 x CH2), 1.98 (s, 3H, CH3), 2.2-2.68 (m, 7H, (4H) 2 x CH2, 3H, CH3), 7.2-7.8 (m, 4H, Ar-H), 9.5 (s, 1H, NH, D2O exchangeable) ppm. EIMS m/z (%) 293 [M+] (17.7) , 91 (100). Anal. Calcd for C18H19N3O: C, 73.69; H, 6.53; N, 14.32. Found: C, 73.79; H, 65.41; N, 14.39. N-(3-Cyano-1-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-4,5,6,7-tetrahydro-1H-indol-2-yl)aceta-mide (3f) It was obtained as a brownish red solid [MeOH/ HO]. m.p. 215-218 °C. FT-IR (KBr) v 3330, 2230, 1720, 2 max 1703 cm-1. 1H-NMR (DMSO-d6, 300 MHz) 8 = 1.43-1.68 (m, 4H, 2 x CH2), 2.2-2.45 ^ 7H, (4H) 2 x CH2), 2.332.4 (m, 6H, 2 x CH3), 3.12 (s, 3H, N-CH3), 7.2-7.6 (m, 5H, Ar-H). 10.3 (s, 1H, NH, D2O exchangeable) ppm. EIMS m/z (%) 389 [M+] (24.1), 159 (100). Anal. Calcd for C22H23N5O2; C, 67.85; H, 5.95; N, 17.98. Found: C, 67.78; H, 6.16; N, 17.91. N-(3-Cyano-1-(pyridin-2-yl)-4,5,6,7-tetrahydro-1H-indol-2-yl) acetamide (3g) It was obtained as a brown solid [EtOH/H2O]. Yield 62%, m.p. 168-170 °C. FT-IR (KBr) v 3300, 2300, 1730 max cm-1. 1H-NMR (DMSO-d6, 300 MHz) 8 = 1.7-1.75 (m, 4H, 2 x CH2), 2.4-2.6 (m, 7H, (4H) 2 x CH2, 3H, CH3), 7.6-8.4 (m, 4H, Ar-H), 9.9 (s, 1H, NH, D2O exchangeable) ppm. EIMS m/z (%) 280 [M+] (100). Anal. Calcd for C16H16N4O; C, 68.55; H, 5.75; N, 19.99. Found: C, 68.54; H, 5.42; N, 19.88. N-(3-Cyano-1-phenyl-1,4-dihydroindeno[1,2-b]pyrrol-2-yl)acetamide (4a) It was obtained as a brown solid [MeOH/H2O]. Yield 64%, m.p. 215-219 °C. FT-IR (KBr) vmax 3450 (NH), 2365 (CN), 1710 (C=O) cm-1. 1H-NMR (DMSO-d6, 300 MHz) 8 = 2.3 (s, 3H, CH3), 3.58 (s, 2H, CH2), 7.1-7.67 (m, 9H, Ar-H), 10.47 (br.s, 1H, NH, D2O exchangeable) ppm. EIMS m/z (%) 313 [M+] (8), 146 (100). Anal. Calcd for C20H15N3O: C, 76.66; H, 4.82; N, 13.41. Found: C, 76.69; H, 4.98; N, 13.57. General methods for the preparation ofaryl-pyrrolo [2,3-d]pyrimidin-4-ones 5 and 6 A suspension of the appropriate compound 1 or 2 (0.01 mol) in formic acid (20 mL, 85%) was refluxed for 3 h, cooled, poured onto ice-water to give a precipitate which was filtered off, dried, and recrystallized from appropriate solvent to afford 5 and 6. 9-(4-Methoxyphenyl)-5,6,7,8-tetrahydro-3H-pyrimido [4,5-b]indol-4(9H)-one (5b) It was obtained as a brown solid [EtOH]. Yield 65%, m.p. 272-276 °C. FT-IR (KBr) v 3230, 1690, 1560 cm-1. max 1H-NMR (DMSO-d6, 300 MHz) 8 = 1.33-1.58 (m, 4H, 2 x CH2), 2.2-2.48 (m, 4H, 2 x CH2), 3.52 (s, 3H, O-CH3), 6.9-7.5 (m, 4H, Ar-H), 9.3 (s, 1H, C2-H), 12.40 (s, 1H, NH, D2O exchangeable) ppm. EIMS m/z (%) 295 [M+] (18.7), 279 (100). Anal. Calcd for C17H17N3O2: C,69.14; H, 5.80; N, 14.23. Found: C, 69.19; H, 5.98; N, 14.53. 9-o-Tolyl-5,6,7,8-tetrahydro-3H-pyrimido[4,5-b]indol-4 (9H)-one (5d) It was obtained as a yellowish brown solid [MeOH]. Yield 79%, m.p. 228-231 °C. FT-IR (KBr) v 3230, 1690, max 1560 cm-1. 1H-NMR (DMSO-d6, 300 MHz) 8 = 1.53-1.78 (m, 4H, 2 x CH2), 2.12 (s, 3H, CH3), 2.2-2.58 (m, 4H, 2 x CH2), 7.2-7.6 (m, 4H, Ar-H), 9.3 (s, 1H, C2-H), 12.18 (s, 1H, NH, D2O exchangeable) ppm. EIMS m/z (%) 279 [M+] (17), 118 (100). Anal. Calcd for C17H17N3O: C, 73.10; H, 6.13; N, 15.04; O, 5.73. Found: C, 73.39; H, 6.23; N, 15.34; O, 5.94. 9-p-Tolyl-5,6,7,8-tetrahydro-3H-pyrimido[4,5-b]indol-4 (9H)-one (5e) It was obtained as a brown solid [EtOH]. Yield 71%, m.p. 206-210 °C. FT-IR (KBr) v 3430, 3330, 1720, 1690, max 1560 cm-1. 1H-NMR (DMSO-d6, 300 MHz) 8 = 1.53-1.78 (m, 4H, 2 x CH2), 1.98 (s, 3H, CH3), 2.2-2.68 (m, 4H, 2 x CH2), 7.2-7.8 (m, 4H, Ar-H), 9.4 (s, 1H, C2-H), 12.15 (s, 1H, NH, D2O exchangeable) ppm. EIMS m/z (%) 279 [M+] (19.3), 188 (100). Anal. Calcd for C17H17N3O: C,73.69; H, 6.53; N, 14.32. Found: C, 73.79; H, 6.61; N, 14.19. 9-(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-5,6,7,8-tetrahydro-3H-pyrimido[4,5-b]indol-4(9H)-one (5f) It was obtained as a yellowish brown solid [EtOH]. Yield 83%, m.p. 260-265 °C. FT-IR (KBr) v 3430, 3330, max Radwan et al.: Synthesis and Structure Activity Relationship ... Acta Chim. Slov. 2017, 64, 865-876 869 1720, 1690, 1560 cm-VH-NMR (DMSO-d6, 300 MHz) 8 = 1.43-1.68 (m, 4H, 2 x CH2), 2.2-2.45 (m, 4H, 2 x CH2), 2.33 (s, 3H, CH3), 3.12 (s, 3H, N-CH3), 7.2-7.6 (m, 5H, Ar-H), 9.1 (s, 1H, C2-H), 12.3 (s, 1H, NH, D2O exchangeable) ppm. EIMS m/z (%) 375 [M+] (24.6), 187 (100). Anal. Calcd for C21H21N5O2: C, 67.18; H, 5.64; N, 18.65. Found: C, 67.28; H, 5.3(5; N, 18.36. 9-(Pyridin-2-yl)-5,6,7,8-tetrahydro-3H-pyrimido[4,5-b] indol-4(9H)-one (5g) It was obtained as a brownish red solid [EtOH]. Yield 68%, m.p. 168-170 °C. FT-IR (KBr) v 3310, 1682, 1587 1 v ' max cm-1. 1H-NMR (DMSO-d6, 300 MHz) 8 = 1.7-1.75 (m, 4H, 2 x CH2), 2.4-2.6 (m, 4H, 2 x CH2), 7.6-8.4 (m, 4H, Ar-H), 9.3 (s, 1H, C2-H), 12.21 (s, 1H, NH, D2O exchangeable) ppm. EIMS m/z (%) 266 [M+] (16.4), 132 (100). Anal. Calcd for C15H14N4: C, 67.65; H, 5.30; N, 21.04. Found: C, 67.54; H, 5.62; IN, 21.88. 10-Phenyl-5,10-dihydro-3H-indeno[2',1':4,5]pyrrolo[2,3-d] pyrimidin-4-one (6a) It was obtained as an orange solid [MeOH]. Yield 58%, m.p. 215-218 °C. FT-IR (KBr) v 3330, 1705, 1590 max cm-1. 1H-NMR (DMSO-d6, 300 MHz) 8 = 3.51 (s, 2H, CH2), 7.3-7.8 (m, 10H, Ar-H + pym h), 12.21 (s, 1H, NH, D2O exchangeable) ppm. EIMS m/z (%) 299 [M+] (18), 221(100). C19H13N3O (299.33). Anal. Calcd for C19H13N3O: C, 76.24; H, 4.38; N, 14.04. Found: C, 76.57; H, 4.61; N, 14.53. 3. Biological Assay 3. 1. Anti-inflammatory Activity Animals Ninety adult male Sprague-Dawley rats (5 rats per group for 14 tested compounds, control (injected with 1 mL DMSO only, 2 standard drugs), weighing 120-150 g, were housed in cages in a temperature-controlled (25 ± 1 °C) environment and provided free access to pelleted food and purified drinking water ad libitum. The protocol of the study was approved by the animal ethics committee of the Faculty of Pharmacy, Helwan University on 10-01-2012. The study was conducted in accordance with the EC. DFT -IRective 86/609/EEC for animal experiments. Assessment of Anti-inflammatory Activity Rat paw oedema assay was carried out according to Winter et al.45 Prepared compounds (equimolar to active dose of the reference drug), control and 2 standard drugs were dissolved in 1mL DMSO and administrated subcuta-neously. One hour later, paw oedema was induced by sub -plantar injection of 0.1 mL of 1% carrageenan (Sigma-Al-drich, St. Louis, USA) into the right paw. Paw volume was measured using a water plethysmometer (Basile, Comerio, Italy). The difference between the right and left paw volu- me was measured at 1, 2, 3 and 4 h after induction of inflammation. Control group received 1 mL DMSO (as to evaluate the interference of DMSO itself in biological test) subcutaneously and carrageenan in sub-plantar region. Results were expressed as percentage inhibition of inflammation. Ibuprofen (70 mg/kg) and indomethacin (20 mg/kg) were used as the reference drugs. Statistical Analysis Results were expressed as the mean ± SEM, and different groups were compared using one way analysis of variance (ANOVA) followed by Tukey-Kramer test for multiple comparisons, using Graph Pad Instant (version 3.05) as the statistical software. Calculation: equimolar doses of tested compounds were calculated in relation to these of reference drug: swel = mean difference in rat paw volume between right and left paw ± SE. % inhibition = (1 - rt/rc) x 100 [rt = swel of tested group; rc = swel of control group]. 3. 2. Molecular Docking Study MOE 2013.08 Docking The molecular docking studies were done using MOE 2013.08 and Leadit 2.1.2. All compounds were built and saved as MOE. Rigid receptor was used as a docking protocol. Both receptor-solvent were kept as a »receptor«. Triangle matcher was used as a placement method. Two rescoring were computed, rescoring 1 was selected as London dG. Rescoring 2 was selected as affinity. The force field was used as a refinement. Leadit 2.1.2 Docking All compounds were built and saved as Mol2. The crystal structure of COX-2 enzyme complexes with indomethacin was downloaded from protein data bank (PDB: 4COX). The protein was loaded into Leadit 2.1.2 and the receptor components were chosen by selection of chain A as the main chain when complexes with indomethacin. Binding site was defined by choosing indomethacin as the reference ligand to which all coordinates were computed. Amino acids within radius 6.5  were selected in the binding site. All chemical ambiguities of residues were left as default. Ligand binding was driven by enthalpy (classic Triangle matching). For scoring, all default settings were restored. Intra-ligand clashes were computed by using clash factor = 0.6. Maximum number of solutions per iteration = 200. Maximum of solution per fragmentation = 200. The base placement method was used as the docking strategy. 4. Result and Discussion 4. 1. Chemistry The availability of a-amino ketones is key to the preparation of o-amino- cyanopyrroles.46-48 Research in- Radwan et al.: Synthesis and Structure Activity Relationship ... 870 Acta Chim. Slov. 2017, 64, 865-876 dicated that a-amino ketones used for the preparation of o-amino-cyanopyrroles were usually obtained in situ49-51 via the reaction of a-hydroxy ketones with amines in acid medium,52-55 or via the reaction of a-halo ketones with either amines and/or a-amino acids.56 As previously mentioned, a-hydroxy ketones and a-halo ketones, malononitriles or suitable substituted alkylidenemaloni-trile and primary amines constituted essential components for the synthesis of o-amino-cyanopyrrole deriva-tives.44,57,58 Regarding fused pyrrole and fused pyrrolopyrimi-dine derivatives there are few studies reporting the synthesis of o-aminocyano-tetrahydrobenzo[b]pyrrole and o-aminocyano-octahydroindeno[2,1-b]pyrrole derivatives. Literature also revealed that the reaction of a-hy-droxycyclohexanone (in place of the a-chloro analogue) with certain amines and malononitrile successfully afforded some o-aminocyanotetrahydrobenzo[b]pyrroles but in poor yields (20-30%).44,58,59 These results also indicated that some side reactions happened (Fig. 2a). Figure 2a. Typical synthesis of tetrahydrobenzo[b]pyrroles Attempts made to develop such compounds using the regular reactants (a-chlorocyclohexanone and/or 2-chloroindenone, with certain aromatic amines and malononitrile) were unfruitful.60 We also found that a-chlorocyclohexanone under the reaction conditions EtOH/NaOH (polar basic medium) might undergo what is called Favorskii rearrangement,61 rearrangement of an a-halo ketone upon treatment with a base; the reaction continues through cyclopropanone intermediate formed by nucleophilic attack [EtO-]. In addition to the possibility of condensation with amine to give the anil (Fig. 2b).62-63 Taking the previous results into consideration, we tried to diminish the potential of such side reactions. Our plan was to prepare the novel intermediate 2-(2-oxo-cyclohexyl) malononitrile (I) from the reaction of 2-chlo-rocyclohexanone with malononitrile in a nonpolar solvent, followed by condensation with the appropriate aromatic amines. By applying this method, we successfully set up the required tetrahydrobenzo[b]pyrroles 1 with a fair yield. Using the same conditions with 2-chloroindenone it produced 2 (Scheme 1). On the other hand, aminocyano pyrrole derivatives 1 and 2 were converted to the corresponding acetylated derivatives 3 and 4 via condensation with acetic anhydyri-de.64-663-d]pyrimidines were reported to act as potent anticancer agents, in this work, a series of novel 2-substituted- Figure 2b. Favoriskii rearrangement (polar basic medium rearrangement),57 and anil formation58,5 Radwan et al.: Synthesis and Structure Activity Relationship ... Acta Chim. Slov. 2017, 64, 865-876 871 Scheme 1: Synthesis of tetrahydroindoles 1a-g and 2a Scheme 2: Synthesis of acetylated pyrroles 3a-g, 4a,b and pyrrolopyrimidines 5a-g, 6a 3-cyano-4-phenyl-pyrrole 5, 6, 11-18, and 5-phenyl-pyr-rolo[2,3-d]pyrimidine derivatives 7-10, 19-24 bearing either sulfathiazole or sulfapyridine were synthesized. The structures of these compounds were confirmed by elemental analysis, IR, (1 Compounds 1 and 2 were converted to pyrrolo[2,3-d]pyrimidine-4-ones 5 and 6 via condensation with formic acid (Scheme 2).64 4. 2. Biological Results and Discussion Fourteen of the synthesized compounds were evaluated for their anti-inflammatory activity, using a method established by Harrk et a/.314-benzodioxine or pyrrole nucleus are described. All the newly synthesized compounds were examined for their in vitro and in vivo antiinflammatory activity. Several derivatives, including (S Five of the tested compounds induced significant anti-inflammatory activity, compared with that of ibuprofen and indomethacin. Compound 1b exerted significant activities compared to standard drugs at all time intervals post-car-rageenan (= 78%, = 80%, = 84% and = 85% inhibition at 1st, 2nd, 3rd and 4th hour interval post-carrageenan). The activity profile was the same as for standard drugs (response increasing with time). Compound 1c exerted noticeable acti- vities compared to standard drugs at the 1st and 2 nd hour post-carrageenan (= 81% and = 82% inhibition at 1st and 2nd hour interval post-carrageenan). The activity profile was the same as standard drugs (response increasing with time), yet the activity showed weak, yet significant activities, decreasing at 3rd and 4th hour post-carrageenan (= 79% and = 75% inhibition at 3rd and 4th hour interval post-car-rageenan). Compounds 1d and 5b showed a marked anti -inflammatory effect than standard drugs, from the 1st hour to 4th hour post-carrageenan. Compound 5b showed a moderate inhibitory action at the 4th hour interval: 76% inhibition. Yet, compound 1d showed the unusual profile compared to standard drugs: it showed = 79% inhibition at 2nd hour post-carrageenan and = 70% inhibition at 3rd hour post-carrageenan and then decreased to 66% inhibition at 4th hour post-carrageenan. Compound 1g exerted a moderate activity compared to standard drugs at the 3rd and 4th hour post-carrageenan (% inhibition s48 at 3rd hour and 61% at 4th hour post-carrageenan), it showed no activity at 1st and 2nd hour intervals post-carrageenan injection. Compounds 1f, 3c, 3d, 3e, 3f and 5d,e were all inactive over all tested periods, showing % inhibition < 12, 21, 25 and 38 at 1st to 4th hours, respectively, and were indicated as inactive in Table 1. Radwan et al.: Synthesis and Structure Activity Relationship ... 872 Acta Chim. Slov. 2017, 64, 865-876 Table 1. In vivo anti-inflammatory activity results for active compounds. Compounds Oedema induced by carrageenan (% Oedema inhibition relative to control) // (Ar=) 1 Hr 2 Hr 3 Hr 4 Hr Swel ± SE Swel ± SE % inh Swel ± SE % inh Swel ± SE % inh % inh 0.048 ± 0.024a 78.7 0.052 ± 0.02a 80 0.086 ± 0.048a 84.3 0.098 ± 0.04a 84.69 0.042 ± 0.008a 81.37 0.046 ± 0.009a 82.3 0.116 ± 0.048a 79.6 0.159 ± 0.029a 75.06 0.19 ± 0.036 15.9 0.076 ± 0.038a 70.76 0.022 ± 0.005a 95.98 0.048 ± 0.007a 92 0.2137 ± 0.03 6.35 0.054 ± 0.01a 78.16 0.086 ± 0.018a 69.43 0.083 ± 0.068a 66.73 Indomethacin Ibuprofen Control 0.2242 ± 0.031 0.79 0.25 ± 0.007 3.65 0.284 ± 0.04 48.17 0.251 ± 0.031a 60.74 0.152 ± 0.016 32.74 0.224 ± 0.004 0.88 0.216 ± 0.033 4.42 0.228 ± 0.027 0.15 ± 0.024 42.3 0.764 ± 0.009 20.7 0.158 ± 0.04 39.23 0.26 ± 0.037 0.234 ± 0.04a 57.29 0.286 ± 0.004 44.65 0.286 ± 0.008 48.175 0.548 ± 0.08 0.152 ± 0.057a 76.25 0.084 ± 0.01 a78.58 0.193 ± 0.007a 69.84 0.64 ± 0.038 swel = mean difference in rat paw volume between right and left paw. ± SE a: significantly different from control at the same time interval at p<0.05 % inhibition = (1-rt/rc) x 100 [rt = swel of tested group; rc = swel of control group] swel = swelling SE = standard error %inh = % inhibition 4. 3. Molecular Docking Results Molecular modeling has become very important in the discovery and design of new agents.67-703D-QSAR and docking studies were carried out on 23 pyrrole derivatives, to model their HIV-1 gp41 inhibitory activities. The 2D, 3D-QSAR studies were performed using CODESSA software package and comparative molecular field analysis a) AlaSÎ? TyrtSS SerJ53 Val523 Figure 3a. Binding modes of both A) indomethacin and B) ibuprofen. This was computed with Leadit 2.1.2 Radwan et al.: Synthesis and Structure Activity Relationship ... Acta Chim. Slov. 2017, 64, 865-876 873 (CoMFA It also helps in the interpretation and explanation of the biological results. Molecular docking is one of these approaches and is used to predict the binding mode of organic compounds.68 A molecular docking study had been done using both MOE 2013.0871 and Leadit 2.1.2 software.72,73 Possible binding modes of the active compounds inside the active site of COX-2 were estimated. Indomet-hacin and ibuprofen were also docked. The presence of a carboxylic group in both indomethacin and ibuprofen was important for the carboxylate anion to form an electrostatic interaction with the cationic guanidine moiety of Arg 120 residue found in the active site of the COX-2 enzyme. The oxygen atom of carbonyl group found in this carboxy-lic moiety participated with the formation of a hydrogen bond with the hydrogen atom of -OH group of Tyr 355 residue. The binding affinity of indomethacin and ibuprofen was found to be -30.24 kcal/mol and -19.09 kcal/mol, respectively (Fig. 3a). The clash score was computed with Leadit 2.1.2 software indicating that both drugs have a low clash score (Table 2). The presence of the p-chloro group in the derivative 1c with higher lipophilic contribution value (-10.90) improving the binding modes and interactions, compared with other tested compounds. All compounds with 2-ami-no-(substituted)-2H-indole-3-carbonitrile shared a hydrogen bond formed between their nitrile groups and the -OH group of Tyr 355. Compound 1g showed a mode of binding in which three hydrogen bonds with Tyr 355, Arg 120 and Ser 530 were formed. Finally, compound 5b showed three hydrogen bonds as well with His 90, Tyr 355 and Ser 530 (Fig. 3b). Compounds 1d and 1g had the highest clash penalty score (11.54) which affected their fitting in the binding site and resulted in the lowest affinity values in both MOE 2013.08 and Leadit 2.1.2 docking results. That could expla- Table 2. The clash score for active compounds and standard drugs using Leadit 2.1.2 software. Compound/ Standard drugs % of inhibition MOE docking score Docking score (kcal/mol) Leadit docking Lipo score Clash 1b Indomethacin 5b 1c Ibuprofen 1d 1g 84.69 78.58 76.25 75.06 69.84 66.73 60.73 -11.68 -15.25 -10.38 -10.52 -13.35 -10.24 -10.04 -17.03 -21.24 -15.47 -17.23 -19.09 -16.02 -12.54 -13.84 -12.93 -12.97 -10.90 -10.41 -11.42 -14.63 7.11 6.06 6.94 7.02 5.87 10.32 11.54 b) M' "v, Sor530 Figure 3b. A) Compound 1g possible binding mode inside COX-2. B) Compound 5b predicted interactions. Radwan et al.: Synthesis and Structure Activity Relationship ... 874 Acta Chim. Slov. 2017, 64, 865-876 in their lower inhibition activity compared to the other compounds. 4. 4. Structure-activity Relationships (SAR) To investigate SAR of any NSAIDs, pathophysiology of inflammation and its treatment have to be well understood. Inflammation represents the response to injury.74 Many processes are involved in the promotion of the inflammatory process, one of them is the secretion of ara-chidonic acid (AA) from damaged cells membranes. AA is metabolized by enzyme COX into prostanoids (as prostaglandins) and lipoxygenase (leukotrienes).75 The major mechanism of action of NSAIDs was found to be the inhibition of PG synthesis, through inhibition of COXs, that is to say preventing the AA from forming PG.76-78it has a very short half-live in blood, its oxidization to 15-keto-prostaglandins is catalyzed by 15-hydroxyprostaglandin dehydrogenase (15-PGDH In order to design any structure with pyrrole moiety or its fused form indole, vital considerations must be taken to ensure its anti-inflammatory activity.32,74,79,80 First, the structure should consist of an acidic moiety (carboxylic acid, enols, ester etc.) attached to a planar, aromatic functional group (appears to correlate with the double bond of AA),314-benzodioxine or pyrrole nucleus are described. All the newly synthesized compounds were examined for their in vitro and in vivo anti-inflammatory activity. Several derivatives, including (S and a polar linking group (which attaches the aromatic ring to a lipophilic group in AA).30 Addition of a second hydrophobic ring, not coplanar with the original aromatic ring, was found to enhance activity,35 this second heteroaromatic ring or he-terocyclic ring was believed to provide the necessary geometry to attach to AA.81 Taking indomethacin (benzo[foj pyrrole) as an example, it was found that N-benzoyl moiety seems to play an important role for the COX-1 activity of indomethacin.32 If the latter moiety is replaced with any other bulkier N-substituents, efficient binding to COX-1 pocket is prevented.12,82-84 Taking all this into consideration, and to analyze our SAR, two structural components were considered: the nature of the heterocycle nucleus and the character of the side chain (N-substitution). First, the influence of the nature of the aromatic heterocyclic system: fused pyrrole 1a, 1c, and 1d showed the highest activity over fused pyr-rolopyrimidine 5b. Regarding the side chain type, addition of bulky heterocyclic ring (anti-pyrine) in compound 1g causes the activity to decrease over the substituted phenyl in compounds 1a, 1c, and 1d. Also methoxy group at para-position in compound 1a has conferred significantly higher activity during all time intervals than the methyl goup at ortho-position in compound 1d. Replacing the antipyrine moiety in compound 1g with the halogen group at para-position in compound 1c has conferred significantly higher activity during 1st and 2nd hour time intervals, with decreased activity in 3rd and 4th hour post-carrageenan. 5. Conclusion A new strategy was developed to prepare a series of 2-amino-1-(aryl)-4,5,6,7-tetrahydro-1H-indole-3-carbo-nitriles 1a-g and 2-amino-1-(aryl)-4,5,6,7-tetrahydro-1H -indole-3-carbonitriles 2a,b as potential antiinflammatory agents. Based on their structure, we can conclude that the best aromatic nucleus was the pyrrole with a para substituted phenyl and cyclization to prepare pyrrolopyri-midine derivatives, added some anti-inflammatory activity in the heterocyclic system. The molecular docking study provided the interpretation of the biological activities of the active compounds compared to the two reference drugs indomethacin and ibuprofen. 6. Acknowledgements On the occasion of this, we would like to specify Dr. Rehab Kamel, Assistance professor of pharmacology, Faculty of Pharmacy, Helwan University; and Professor Dr. Aymen Goudah. Professor of Pharmacology, Faculty of Veterinary Medicine, Cairo University; with our sincerest gratitude in appreciation of their great assistance, sincere help, valuable advice and all facilities offered during this study. Our Profuse Thanks and Respects. 7. References 1. D.-L. Chen, G.-X. Ma, M.-J. He, Y.-Y. Liu, X.-B. Wang, X.-Q. Yang, Helv. Chim. Acta 2016, 99, 742-746. D01:10.1002/hlca.201600159 2. H. Li, S. Fan, J. Cheng, P. Zhang, B. Zhong, W. Shi, Molecules 2016, 21, 1-8. 3. S. S. Fatahala, S. Hasabelnaby, A. Goudah, G. I. Mahmoud, Molecules 2017, 22, 1-18. 4. M. Shroff, S. J. Daharwal, Br. Biomed. Bull. 2017, 5, art. no. 299, 7 pp. 5. M. A. Kale, A. S. Narute, T. M. Kalyankar, Clin. Anti-inflamm. Anti-Allergy Drugs 2014, No. 1, 39-44. 6. I. Lessigiarska, A. Nankov, A. Bocheva, I. Pajeva, A. Bijev, Farmaco 2005, 60, 209-218. D0I:10.1016/j.farmac.2004.11.008 7. A. Abbas, M. M. Naseer, Acta Chim. Slov. 2014, 61, 792-802. 8. A. A. Fadda, K. M. Elattar, J. Biosci. Med. 2015, 3, 114-123. 9. G. M. Cole, S. A. Frautschy, CNS Neurol. Disord. Drug Targets 2010, 9, 140-148. DOI: 10.2174/187152710791011991 10. B. O. Villoutreix, G. Laconde, D. Lagorce, P. Martineau, M. A. Miteva, P. Dariavach, PLoS One 2011, 6, e21117. D0I:10.1371/journal.pone.0021117 11. J. S. Bang, D. H. Oh, H. M. Choi, B.-J. Sur, S.-J. Lim, J. Y. Kim, Radwan et al.: Synthesis and Structure Activity Relationship ... Acta Chim. Slov. 2017, 64, 865-876 875 H.-I. Yang, M. C. Yoo, D.-H. Hahm, K. S. Kim, Arthritis Res. Thier. 2009, 11, R49. D01:10.1186/ar2662 12. K. D. Rainsford, Inflamm. Pathog. Chronic Dis. 2007, 42, 3-27. D0I:10.1007/1-4020-5688-5_1 13. A. O. H. El-Nezhawy, S. T. Gaballah, M. A. A. Radwan, A. R. B. Abdel-Salam, Med. Chem. (Los Angeles). 2009, No. 202, 558-569. 14. R. Mohareb, F. Al-Omran, M. Abdelaziz, R. Ibrahim, Acta Chim. Slov. 2017, 64, 349-364. D0I:10.17344/acsi.2017.3200 15. S. S. Fatahala, M. S. Mohamed, M. Youns, R. H. A.-E. Hameed, Anticancer. Agents Med. Chem. 2017, 17, 1-12. DOI: 10.2174/1871520617666170102152928 16. S. Fatahala, E. Shalaby, S. Kassab, M. Mohamed, Anticancer. Agents Med. Chem. 2015, 15, 517-526. DOI: 10.2174/1871520615666150105113946 17. S. D. Joshi, U. A. More, S. R. Dixit, H. H. Korat, T. M. Amina-bhavi, A. M. Badiger, Med. Chem. Res. 2013, 23, 1123-1147. D0I:10.1007/s00044-013-0709-y 18. J. A. Pfefferkorn, Y. Song, K.-L. Sun, S. R. Miller, B. K. Trivedi, C. Choi, R. J. Sorenson, L. D. Bratton, P. C. Unangst, S. D. Larsen, T.-J. Poel, X.-M. Cheng, C. Lee, N. Erasga, B. Auerbach, V. Askew, L. Dillon, J. C. Hanselman, Z. Lin, G. Lu, A. Robertson, K. Olsen, T. Mertz, C. Sekerke, A. Pavlovsky, M. S. Harris, G. Bainbridge, N. Caspers, H. Chen, M. Eberstadt, Bioorg. Med. Chem. Lett. 2007, 17, 4538-4544. D0I:10.1016/j.bmcl.2007.05.096 19. S. Y. Kang, E.-J. Park, W.-K. Park, H. J. Kim, D. Jeong, M. E. Jung, K.-S. Song, S. H. Lee, H. J. Seo, M. J. Kim, M. Lee, H.-K. Han, E.-J. Son, A. N. Pae, J. Kim, J. Lee, Bioorg. Med. Chem. Lett. 2010, 20, 1705-1711. D0I:10.1016/j.bmcl.2010.01.093 20. X. Teng, H. Keys, J. Yuan, A. Degterev, G. D. Cuny, Bioorg. Med. Chem. Lett. 2008, 18, 3219-3223. D0I:10.1016/j.bmcl.2008.04.048 21. N. Danchev, A. Bijev, D. Yaneva, S. Vladimirova, I. Nikolova, Arch. Pharm. (Weinheim) 2006, 339, 670-674. D0I:10.1002/ardp.200600116 22. N. Amishiro, A. Okamoto, C. Murakata, T. Tamaoki, M. Oka-be, H. Saito, J. Med. Chem. 1999, 42, 2946-2960. D0I:10.1021/jm990094r 23. S. B. Almasaudi, N. A. El-Shitany, A. T. Abbas, U. A. Ab-del-Dayem, S. S. Ali, S. K. Al Jaouni, S. Harakeh, Oxid. Med. Cell. Longev. 2016, 2016, 1-10. 24. S. Yang, Y. Kim, D. Jeong, J. H. Kim, S. Kim, Y. Son, B. C. Yoo, E. J. Jeong, T. W. Kim, I. H. Lee, J. Y. Cho, Biomol. Ther. 2016, 24, 595-603. D0I:10.4062/biomolther.2016.027 25. W. B. Han, A. H. Zhang, X. Z. Deng, X. Lei, R. X. Tan, Org. Lett. 2016, 18, 1816-1819. D0I:10.1021/acs.orglett.6b00549 26. X. T. Xu, X.-Q. Mou, Q.-M. Xi, W.-T. Liu, W.-F. Liu, Z.-J. Sheng, X. Zheng, K. Zhang, Z.-Y. Du, S.-Q. Zhao, S.-H. Wang, Bioorg. Med. Chem. Lett. 2016, 26, 5334-5339. D0I:10.1016/j.bmcl.2016.09.034 27. J. L. Amorim, D. L. R. Simas, M. M. G. Pinheiro, D. S. A. Moreno, C. S. Alviano, A. J. R. Da Silva, P. D. Fernandes, PLoS One 2016, 11, 1-18. D0I:10.1371/journal.pone.0153643 28. M. Malmsten, Curr. Top. Med. Chem. 2016, 16, 16-24. D01:10.2174/1568026615666150703121518 29. A. Bocheva, A. Bijev, A. Nankov, Arch. Pharm. (Weinheim) 2006, 339, 141-144. D0I:10.1002/ardp.200500191 30. M. T. Sarg, M. M. Koraa, A. H. Bayoumi, S. M. Abd El Gilil, Open J. Med. Chem. 2015, 5, 49-96. D0I:10.4236/ojmc.2015.54005 31. Y. Harrak, G. Rosell, G. Daidone, S. Plescia, D. Schillaci, M. D. Pujol, Bioorg. Med. Chem. 2007, 15, 4876-4890. D01:10.1016/j.bmc.2007.04.050 32. G. Dannhardt, W. Kiefer, G. Krämer, S. Maehrlein, U. Nowe, B. Fiebich, Eur. J. Med. Chem. 2000, 35, 499-510. D0I:10.1016/S0223-5234(00)00150-1 33. S. Ushiyama, T. Yamada, Y. Murakami, S. Kumakura, S. In-oue, K. Suzuki, A. Nakao, A. Kawara, T. Kimura, Eur. J. Pharmacol. 2008, 578, 76-86. D0I:10.1016/j.ejphar.2007.08.034 34. G. A. Elmegeed, A. R. Baiuomy, O. M. E. Abdel-Salam, Eur. J. Med. Chem. 2007, 42, 1285-1292. D0I:10.1016/j.ejmech.2007.01.027 35. J. T. Moon, J. Y. Jeon, H. A. Park, Y.-S. Noh, K.-T. Lee, J. Kim, D. J. Choo, J. Y. Lee, Bioorg. Med. Chem. Lett. 2010, 20, 734737. D0I:10.1016/j.bmcl.2009.11.067 36. M. S. Mohamed, R. Kamel, R. H. Abd El-hameed, Med. Chem. Res. 2012, 22, 2244-2252. D0I:10.1007/s00044-012-0217-5 37. P. N. Praveen Rao, E. E. Knaus, J. Pharm. Pharm. Sci. 2008, 11, 81-110. D0I:10.18433/J3T886 38. L. Gasparini, E. Ongini, G. Wenk, J. Neurochem. 2004, 91, 521-536. D0I:10.1111/j.1471-4159.2004.02743.x 39. A. Carbone, B. Parrino, P. Barraja, V. Spano, G. Cirrincione, P. Diana, A. Maier, G. Kelter, H.-H. Fiebig, Mar. Drugs 2013, 11, 643-654. D0I:10.3390/md11030643 40. M. S. Mohamed, A. E. Rashad, M. E. A. Zaki, S. S. Fatahala, Acta Pharm. 2005, 55, 237-249. 41. W. M. Hussein, S. S. Fatahala, Z. M. Mohamed, R. P. McGeary, G. Schenk, D. L. Ollis, M. S. Mohamed, Chem. Biol. Drug Des. 2012, 80, 500-515. D0I:10.1111/j.1747-0285.2012.01440.x 42. M. S. Mohamed, R. Kamel, S. S. Fathallah, Arch. Pharm. (Weinheim) 2011, 344, 830-839. D0I:10.1002/ardp.201100056 43. M. S. Mohamed, S. Ali, D. H. A. Abdelaziz, S. S. Fathallah, Biomed. Res. Int. 2014, 2014, 1-13. 44. K. M. H. Hilmy, E. B. Pedersen. Liebigs Ann. Chem. 1989, 1989, 1145-1146. 45. C. A. Winter, E. A. Risley, G. W. Nuss, J. Pharmacol. Exp. Ther. 1963, 141, 369-376. 46. E. C. Taylor, R. O. Kan, W. W. Paudler, J. Am. Chem. Soc. 1961, 83, 4484-4485. D0I:10.1021/ja01482a056 47. E. C. Taylor, R. W. Hendess, J. Am. Chem. Soc. 1965, 87, 19952003. D0I:10.1021/ja01087a025 48. E. C. Taylor, B. Liu, J. Org. Chem. 2001, 66, 3726-3738. D0I:10.1021/jo001580l 49. E. Fanghänel, K. Gewald, K. Pütsch, K. Wagner, J. Prakt. Chem. 1969, 311, 388-394. D01:10.1002/prac.19693110307 50. T. D. Duffy, D. G. Wibberley, J. Chem. Soc., Perkin I 1974, 1921-1929. D0I:10.1039/p19740001921 Radwan et al.: Synthesis and Structure Activity Relationship ... 876 Acta Chim. Slov. 2017, 64, 865-876 51. Z. Puterova, A. Krutošikova, D. Végh, Arkivoc 2010, (i), 209246. 52. Y. Ding, H. An, Z. Hong, J.-L. Girardet, Bioorg. Med. Chem. Lett. 2005, 15, 725-727. DOI:10.1016/j.bmcl.2004.11.019 53. T.-C. Chien, E. A. Meade, J. M. Hinkley, L. B. Townsend, Org. Lett. 2004, 6, 2857-2859. DOI:10.1021/ol049207d 54. M. Qian, R. Glaser, J. Am. Chem. Soc. 2004, 126, 2274-2275. DOI:10.1021/ja0389523 55. J. U. Jeong, X. Chen, A. Rahman, D. S. Yamashita, J. I. Luengo, Org. Lett. 2004, 6, 1013-1016. DOI:10.1021/ol049921v 56. R. W. Fischer, M. Misun, Org. Process Res. Dev. 2001, 5, 581586. DOI:10.1021/op010041v 57. C. G. Dave, N. D. Desai, J. Heterocycl. Chem. 1999, 36, 729733. DOI:10.1002/jhet.5570360325 58. A. O. Abdelhamid, A. M. Negm, I. M. Abbas, J. Prakt. Chemie 1989, 331, 31-36. DOI:10.1002/prac.19893310106 59. K. M. H. Hilmy, Arch. Pharm. (Weinheim) 2004, 337, 15-19. DOI: 10.1002/ardp.200300773 60. M. S. Mohamed, S. S. Fathallah, Mini. Rev. Org. Chem. 2014, 6, 477-507. 61. D. W. Goheen, W. R. Vaughan, Org. Synth. 1959, 39, 37-39. DOI: 10.15227/orgsyn.039.0037 62. McNelis, E. kon and E. J. Chem. Soc., Chem. Comm. 1973, 562-563. 63. J. Oh, C. Ziani-Cherif, J.-R. Choi, J. K. Cha, Org. Synth. 2002, 78, 212-219. DOI:10.15227/orgsyn.078.0212 64. P. M. Traxler, P. Furet, H. Mett, E. Buchdunger, T. Meyer, N. Lydon, J. Med. Chem. 1996, 39, 2285-2292. DOI:10.1021/jm960118j 65. M. M. Ghorab, F. A. Ragab, H. I. Heiba, H. A. Youssef, M. G. El-Gazzar, Bioorg. Med. Chem. Lett. 2010, 20, 6316-6320. DOI:10.1016/j.bmcl.2010.08.005 66. C. G. Dave, R. D. Shah, Molecules 2002, 7, 554-565. DOI: 10.3390/70700554 67. C. Teixeira, F. Barbault, J. Rebehmed, K. Liu, L. Xie, H. Lu, S. Jiang, B. Fan, F. Maurel, Bioorg. Med. Chem. 2008, 16, 30393048. DOI:10.1016/j.bmc.2007.12.034 68. M. Martin-Martinez, A. Marty, M. Jourdan, C. Escrieut, E. Archer, R. Gonzalez-Muniz, M. T. Garcia-Lopez, B. Maigret, R. Herranz, D. Fourmy, M. Marti, J. Med. Chem. 2005, 48, 4842-4850. DOI:10.1021/jm0501127 69. G. Cristalli, S. Costanzi, C. Lambertucci, S. Taffi, S. Vittori, R. Volpini, Farmaco 2003, 58, 193-204. DOI:10.1016/S0014-827X(03)00019-3 70. Y.-D. Gao, D. Feng, R. P. Sheridan, G. Scapin, S. B. Patel, J. K. Wu, X. Zhang, R. Sinha-Roy, N. A. Thornberry, A. E. Weber, T. Biftu, Bioorg. Med. Chem. Lett. 2007, 17, 3877-3879. DOI:10.1016/j.bmcl.2007.04.106 71. Molecular Operating Environment (MOE), 2013.08; Chemical Computing Group Inc.: 1010 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2014. 2014. 72. M. Rarey, B. Kramer, T. Lengauer, G. Klebe, J. Mol. Biol. 1996, 261, 470-489. DOI:10.1006/jmbi.1996.0477 73. D. M. Lorber, B. K. Shoichet, Protein Sci. 1998, 7, 938-950. DOI: 10.1002/pro.5560070411 74. A. Zarghi, S. Arfaei, Iran. J. Pharm. Res. 2011, 10, 655-683. 75. J. DeRuiter, In Principles of Drug Action; 2002; pp 1-26. 76. D. Choi, Y. L. Piao, Y. Wu, H. Cho, Bioorg. Med. Chem. 2013, 21, 4477-4484. DOI:10.1016/j.bmc.2013.05.049 77. T. H. Page, J. J. O. Turner, A. C. Brown, E. M. Timms, J. J. In-glis, F. M. Brennan, B. M. J. Foxwell, K. P. Ray, M. Feldmann, J. Immunol. 2010, 185, 3694-3701. DOI: 10.4049/jimmunol.1000906 78. R. Danesi, G. Pasqualetti, E. Giovannetti, F. Crea, G. Altavil-la, M. Del Tacca, R. Rosell, Adv. Drug Deliv Rev. 2009, 61, 408-417. DOI:10.1016/j.addr.2009.03.001 79. P. P. N. Rao, S. N. Kabir, T. Mohamed, Pharmaceuticals 2010, 3, 1530-1549. DOI:10.3390/ph3051530 80. S. Ushiyama, T. Yamada, Y. Murakami, S. I. Kumakura, S. I. Inoue, K. Suzuki, A. Nakao, A. Kawara, T. Kimura, Eur. J. Pharmacol. 2008, 578, 76-86. DOI:10.1016/j.ejphar.2007.08.034 81. W. O. Foye, In Williams & Wilkins; 2008. 82. M. R. Shaaban, T. S. Saleh, A. S. Mayhoub, A. Mansour, A. M. Farag, Bioorg. Med. Chem. 2008, 16, 6344-6352. DOI:10.1016/j.bmc.2008.05.011 83. H. N. Hafez, O. K. Al-duaij, A. B. A. El-gazzar, International J. Org. Chem. 2013, 3, 110-118. DOI:10.4236/ijoc.2013.32012 84. C. Limban, A. Missir, K. M. S. Fahelelbom, M. M. Al-Tab-akha, M. T. Caproiu, B. Sadek, Drug Des. Devel. Ther. 2013, 7, 883-892. Povzetek Sintetizirali smo serijo pripojenih pirolov in jih in vivo testirali za njihovo aktivnost proti vnetjem. Med 14 preiskovanimi derivati smo ugotovili, da 5 derivatov (1b-e,g and 5b) kaže obetavno aktivnost proti vnetjem. Njihova aktivnost je primerljiva z aktivnostjo referenčnih proti-vnetnih zdravil (indometacin in ibuprofen). Da bi interpretirali rezultate bioloških študij preiskovanih spojin, smo izvedli tudi študije molekulskega sidranja. Rezultati so bili komplementarni tistim, dobljenim z biološkimi testiranji; poleg tega so potrdili biološke učinke preiskovanih spojin. Radwan et al.: Synthesis and Structure Activity Relationship ... DOI: 10.17344/acsi.2017.3497 Acta Chim. Slov. 2017, 64, 877-887 ©commons 877 Scientific paper Preparation and Investigation of the Thermal Stability of Phosphate-modified TiO2 Anatase Powders and Thin Films Uros Prah* and Irena Kozjek Skofic Faculty of Chemistry and Chemical Technology, University of Ljubljana, Vecna pot 113, SI-Ljubljana, Slovenia * Corresponding author: E-mail: prah.uros@gmail.com Received: 04-05-2017 Abstract The temperature dependence of the anatase-to-rutile phase transition of TiO2 powders and thin films was studied. In order to shift the phase transition to higher temperature, samples were doped with a different amount of phosphate ions and their influence on the structure and thermal stability of the anatase phase was investigated. In addition, the effect of the catalyst form (powders or thin films) on the temperature of the anatase-to-rutile phase transition was observed. TiO2 thin films and powders were prepared using a simple sol-gel method with an alkoxide precursor and citric acid. The thin films were deposited on silicon and aluminum substrates using the dip-coating technique. The content of the anatase phase and the crystallite size at different annealing temperatures were monitored using X-ray diffraction. The course of the thermal decomposition was followed using thermal analyses. The morphology, particle size, shape and elemental makeup of the samples were investigated using scanning electron microscopy and energy-dispersive X-ray spectroscopy. The results showed that the phosphate ions successfully inhibited the growth of the anatase nanoparticles and delayed the phase transition to the rutile phase. Keywords: Anatase, phosphate, sol-gel, thermal stability, thin films 1. Introduction During the past few decades titanium dioxide has been one of the most intensively studied semiconductor materials. It has numerous useful characteristics, such as the unique positions of the valence and conduction bands, a relatively narrow band-gap, chemical and physical stability, favorable electronic and optical properties, non-toxici-ty and a low price.1-7 Furthermore, in nanocrystalline form it shows good catalytic and photocatalytic properties. Photons with sufficient energy excite electrons into the conduction band, which leads to the generation of free electrons in the conduction band and positive holes in the valence band. The energy required for the photogeneration of the electron-hole pairs in TiO2 nanocrystals is 3.03.2 eV, which is equivalent to the energy of light in the near-UV region.8 Some of these pairs react with electron-donor and electron-acceptor species on the semiconductor surface to form reactive radicals, which can be used for the degradation of environmental pollutants, self-cleaning, antifogging and the sterilization of surfaces.4,5 TiO2 naturally occurs in three polymorph crystal modifications: rutile, anatase and brookite.5,6,9 Of these, the anatase and rutile phases are the most frequently used, while brookite is less interesting for practical applications due to its lower thermal stability and difficult preparation. Although the band-gap of the anatase phase is wider (3.2 eV) in comparison to rutile (3.0 eV), anatase is considered to exhibit better photocatalytic activity due to its larger surface area and the slower recombination process for the charge carriers.6,10,11 The anatase is thermodynami-cally metastable and irreversibly converts to rutile at higher temperatures. This phase transition results in a reduction of the photocatalytic activity (formation of the less-active rutile form) and causes undesirable dimensional changes of the material.12 Improving the thermal stability of the anatase phase, by increasing the temperature of the anatase-to-rutile phase transition, is particularly important when using TiO2 in high-temperature applications, such as the degradation of toxic NOx and SOx, which are usually produced at high temperatures.13,14 To achieve a better thermal stability of the anatase phase and thereby inhibit the anatase-to-rutile phase transformation, different ion dopants (F-, Si4+, Fe3+, Al3+, etc.) were added to pure TiO2. These dopants can occupy both interstitial and substitutional positions in the TiO2 Prah and Škofic: Preparation and Investigation of the Thermal 878 Acta Chim. Slov. 2017, 64, 877-887 crystal lattice or act like a steric barrier (form a layer on the particles' surface) and thus shift the phase transformation to higher temperatures and therefore enhance the thermal stability of the anatase phase.1,3,6,10,13-16 Phosphate ions react with uncondensed hydroxyl groups on the surface of TiO2 particles and act as a steric barrier. Thereby phosphate ions effectively hold the anatase particles at certain distance (inhibit the contacts among the particles) and consequently decelerate their growth, because the rutile phase, which is responsible for a drastic increase in the particle size, begins to form at the interface between the anatase particles in the TiO2 agglomerates.1,17,18 By keeping the anatase particles separated at a certain distance, the phase transformation can be restricted and at the same time the small particle size can be maintained.1,3,6,16 The sol-gel technique is one of the most frequently used methods for the preparation of TiO2. The particle size and the morphology of the product can be easily controlled by changing the synthesis parameters. The variety of the prepared products, such as thin films, fibers, xe-rogels, aerogels, powders and dense ceramics, allows very diverse applications. Different types and amounts of dopants or additives can be easily added during the synthesis. A high degree of homogeneity for the prepared materials can be achieved in a single or even in multicomponent sys-tems.7,19,20 Using a powdered catalyst is not favorable for heterogeneous photocatalysis. The problem is its mobility in air and removal from aqueous systems. To avoid these problems, powders are often immobilized on various substrates, for example, thin films can be prepared.4 The advantages of using thin films are their easy removal from the liquid media and the low consumption of raw materials. In addition, very thin and transparent thin films can be prepared and used for different applications, such as self-cleaning windows and anti-fogging mirrors. Thin films often exhibit different properties compared to powders, such as phase composition, microstructure, reactivity, etc. Therefore, apart from the influence of phosphate ion addition, the influence of the TiO2 catalyst form (powder or thin film) and the impact of immobilization on the course of the phase conversion of anatase into rutile were studied. 2. Experimental 2. 1. Chemicals and Materials Titanium(IV) butoxide (97%), citric acid (> 99.5%) and absolute ethanol (> 99.8%) were purchased from Sigma Aldrich. Phosphoric acid (85%) was procured from Alfa Aesar. All chemicals were used without further purification. Aluminum foil (thickness 0.01 mm) and pure silicon wafers (1-0-0 single crystal, prepared by Czochralski method, MEMC Elect. Materials Sdn. Bhd.) were used as the substrates for the thin films. 2. 2. Synthesis Sols of TiO2 and TiO2 doped with phosphate ions were prepared by dissolving 0.01 mol of citric acid in 20 mL of absolute ethanol. The mixture was stirred on a magnetic stirrer until all the acid was dissolved and then 0.01 mol of titanium butoxide was slowly added to the solution. The beaker with the colloidal solution was closed with parafilm and the stirring was continued for approximately 12 hours. All the sols were stored in a refrigerator (5 °C) until further use. For the doped sols, the only difference was the addition of a different quantity of phosphoric acid to the homogenous solution of citric acid in ethanol before the addition of the Ti-precursor. Relative to the titanium ions, 5 mol%, 10 mol% and 15 mol% of phosphate ions were added to the solutions. 2. 3. Preparation of Powders and Thin Films For the preparation of the powders, the sols were dried in air at room temperature to produce the xerogels. The films were deposited using the dip-coating technique on aluminum foil and silicon plates, which were first cut to appropriate dimensions and cleaned in an ultrasonic bath in deionized water, followed by absolute ethanol, and then dried. The film thicknesses and their homogeneities were controlled using a constant pulling velocity (20 cm min-1). The thin films were dried in air at room temperature. The thin films had good adhesive properties (layers could not be removed by rubbing and cutting) and therefore no additional surfactant was needed. All the prepared xerogels and the dried thin films were then calcined for 1 hour at 400, 500, 600, 700, 800, 900 and 1000 °C. After the calcinations, the powders were thoroughly milled in an agate mortar. 2. 4. Characterization The thermal analyses of the xerogels and thin films were carried out in a dynamic air atmosphere with a flow rate of 100 mL min-1 on a Mettler Toledo TGA/DSC 1 thermo analyzer, coupled with a Balzers Thermostar quad-rupole mass spectrometer. Aluminum foil was used as the supporting material for the thin films. The thin films were cut into small pieces (~2 mm x 2 mm) and analyzed in the temperature range from room temperature up to 600 °C, while the xerogels were measured up to 800 °C. For all the measurements 150-^L platinum crucibles were used. Firstly, the samples were purged with air at 25 °C for 20 min and then heated at 5 K min-1. The gas products were transferred to the mass spectrometer through the quartz capillary heated to 190 °C. The baseline was subtracted for all the samples. The X-ray diffraction (XRD) patterns were recorded on an X PANalytical X'Pert PRO diffractometer using monochromatic Cu-Ka radiation. Measurements of the Prah and Škofic: Preparation and Investigation of the Thermal Acta Chim. Slov. 2017, 64, 877-879 887 heat-treated powders were recorded from 20 = 15° to 60° with a step of 0.034 degrees per second and an integration time of 100 s. Thin films were recorded from 20 = 23° to 30° with a step of 0.034 degrees per second and an integration time of 500 s. For the XRD analysis, silicon plates were used as a support for the thin films since silicon does not have any peaks in the 20 measuring range. Scanning electron microscope (SEM) images of the samples were taken with a Zeiss Ultra Plus field-emission scanning electron microscope. A small amount of powders and appropriately cut thin films on silicon plates (~5 mm x 5 mm) were attached to carbon tape on the metal holders. The electrical conductivity of titanium dioxide is sufficient; therefore, sputtering with conductive material was not needed. The elemental composition and the distribution of the elements in the samples were determined using energy-dispersive X-ray spectroscopy (EDS) coupled to SEM. 3. Results and Discussion 3. 1. Thermal Analysis The thermal decomposition of the xerogels and the thin films was investigated using thermal analysis. The mass losses, exothermic and endothermic changes of the samples during the thermal treatment were measured with thermogravimetric analysis (TG) and differential scanning calorimetry (DSC). For a qualitative analysis of the released gases and for better understanding of the thermal decomposition, mass spectrometry (MS) was also employed. DSC analysis was used to determine the temperature where the anatase-to-rutile phase transition took place. This phenomenon is very hard to detect for at least two reasons. One reason is a very small exothermic effect that accompanies this phase transformation and the other is its position, which is highly dependent on the selected synthesis method and the experimental parameters.21 Fig. 1 shows the results of the thermal decomposition of the undoped xerogel sample. Three distinct steps of mass loss were observed. In the first step between room temperature and 150 °C, the weight loss was 7.5%, corresponding to water and ethanol evaporation (m/z 18 and 46). Water evaporation also took place at the beginning of the following step of the mass loss, which is evident from the endo-thermic minimum on the DSC curve. In the second step, from 150 °C to around 370 °C, the mass decreases by approximately 46% and the third step continued up to 530 °C, with a mass loss of approximately 18%. The last two steps of the mass loss are associated with the decomposition and oxidation of the organic compounds (residues of the citric acid and butoxide groups), which is also supported by the peaks of the alkyl fragments, carbon dioxide and water from the MS signals. The total mass loss of the sample is around 72%. No exothermic peak that could represent the anatase-to-rutile phase transition is observed. The thermal decomposition of the doped samples is comparable to the undoped sample (Fig. 2). The first and second steps of the thermal decomposition of all the samples occur in the same temperature range and show almost ..... miz IB ■ miz 44 \ mfe 15 |TG \ ■ / 1 O a DSC t u / mfe 4 J 100 200 300 400 500 600 700 600 Temperature (°C) Figure 1. TG, DSC curves and signals from MS of undoped xerogel. Temperature (°C) Figure 2. A comparison of (a) TG and (b) DSC curves of doped and undoped xerogels. Prah and Škofic: Preparation and Investigation of the Thermal ... 880 Acta Chim. Slov. 2017, 64, 877-887 identical mass losses, regardless of the quantity of phosphate ions added (overlapping TG curves). The only observed difference is in the last stage of the thermal decomposition, where the temperature of the oxidation of the organic compounds moves to higher temperatures with an increasing amount of added dopant. Therefore, the mass is stabilized at higher values, which have an impact on the selection of the lowest annealing temperature. The same observations were made in the comparison of the DSC curves, wherein the addition of phosphate ions moved the exothermic peak of the last stage of thermal decomposition to higher temperatures, where also instead of one exothermic peak, two smaller one were observed. Thermal analyses of the thin films deposited on the aluminium foil were also investigated. Due to the much heavier aluminium substrate in comparison to the thin layer, all the effects were much harder to detect. Weight changes during the thermal treatment and also exothermic and endothermic effects were very low and their interpretation was easier in comparison to the results of the xerogels (Fig. 3).22,23 The total mass loss in the thin films was around 3.5%. However, it should be taken into consideration that the thermal decomposition of the thin films is often carried out differently than in the case of xerogels, because of the a) 100 200 300 4q0 500 600 700 800 Temperature (°C) b) 0 100 200 300 100 500 600 700 bod Temperature (°C) Figure 3. A comparison of (a) TG and (b) DSC curves for the un-doped xerogel and the thin film. suppressed diffusion of gases on the substrate side, the decomposition steps are not so clear.24 Despite this difference, the positions of the more intense exothermic and endo-thermic peaks and the temperature of the total mass loss are positioned in the same temperature ranges. Based on the results of the thermal analysis, a range of annealing temperatures was selected. Because we could not determine the exact temperature where the ana-tase-to-rutile phase transition occurred, we used a wider range of annealing temperatures. The used temperatures were 400, 500, 600, 700, 800, 900 and 1000 °C. 3. 2. XRD Analysis The content of the anatase phase and the particle size were determined by XRD analysis and calculated using the Rietveld analysis and Scherrer formula. The results were calculated from all the peaks in the measuring range and not only from the peaks 20 = 25.28° (101) and 27.40° (110), which are often taken as the characteristic peaks of the anatase and rutile phases.1,25,26 Firstly, the influence of adding phosphate ions on the average particle size and the thermal stability of the anatase phase in the powders were monitored (Fig. 4). In the un-doped samples, the content of the anatase phase quickly dropped with an increasing annealing temperature. At 400 °C amorphous and partially crystallized anatase phase was present, but the content of the anatase phase dropped rapidly to 6.4 wt%, when it was annealed at 600 °C. The particle size increased with the increasing temperature and it was 30 nm at 600 °C (Table 1). When the anatase particles are sufficiently large, they start to interact with each other and the phase transformation occurs at the interfaces between them. With an increasing annealing temperature, more ana-tase particles were converted to rutile and the phase transformation gradually extends over the entire TiO2 agglomerates. In doped TiO2, the phosphate ions can easily react with the surface hydroxyl groups and form a layer on the surfaces of the anatase nanoparticles. The phosphates act like a steric barrier that prevents any direct contact between the particles, inhibits their growth and the interactions among them. By inhibiting the particle growth and preventing any interaction between the particles, the phase transformation occurs at higher temperatures.1 The doped powders (with 5, 10 and 15 mol%) showed better thermal stability for the anatase with an increasing proportion of added dopant. The best results were shown by the sample with 15 mol% of added dopant, where only the anatase phase was present up to 700 °C. At 800 °C the anatase content decreased to 74.7% and at 900 °C to 6.8%. At higher dopant ratios (10 and 15 mol%) and high annealing temperatures (900 and 1000 °C) the formation of the new crystal phase, titanyl phosphate, was observed (3-10 wt%), otherwise only the anatase and rutile phases were present (Fig. 5b). All the samples annealed at 1000 °C contained, besides titanyl phosphate, only the rutile phase. The phos- Prah and Škofic: Preparation and Investigation of the Thermal Acta Chim. Slov. 201?, 64, 877-887 881 ■ 0% PC 5% PC 10% PC 15% PC i —i 1 i 400 500 600 700 aoo Annealing temperature (°C) 900 1000 Figure 4. Content of anatase phase depending on the dopant ratio at different annealing temperatures in the powders. Table 1. Average size of the anatase particles in powders with different annealing temperatures and dopant ratios. Annealing Dopant ratio (mol%) temperature 0 5 10 15 (°C) Average size of the anatase particles (nm) 400 ~8c 0a 0a 0a S00 i6.6 ~7c ~6c ~10c 600 30.0 8.4 6.6 6.0 700 0b 11.7 8.1 7.8 800 0b 22.9 2S.9 28.7 900 0b 26.4 38.8 43.6 i000 0b 0b 0b ob a - amorphous phase b - all anatase has been converted to rutile c - estimated value based on a partially crystallized anatase phate ions improved the thermal stability of the anatase phase and raised the temperature of the present anatase 2Theta (degrees) 2Theta (degrees) Figure 5. Evolution of the recorded powder diffraction patterns with an increasing annealing temperature (a: undoped TiO2 nanoparticles, b: TiO, nanoparticles doped with 15 mol% of phosphate ions). Prah and Škofic: Preparation and Investigation of the Thermal 882 Acta Chim. Slov. 2017, 64, 877-887 phase to 900 °C. The average size of the anatase particles was successfully inhibited by the phosphate ions up to 700 °C, where the average particles size remained under 12 nm (Table 1). At temperatures above 800 °C, the steric barrier is no longer able to prevent the particle growth and the phase transformation starts to take place. On the other hand, the addition of phosphate ions also increased the crystallization temperature of the anatase phase, because in doped samples, annealed at temperatures below 500 °C, no crystalline phase was observed (Table 1). The formation of all the crystalline phases and the particle growth are clear from the series of diffractograms (Fig. 5) that were recorded after the heat treatment at different temperatures for the undoped and (15 mol%) doped powder samples. In the undoped sample, in comparison to the doped sample, the anatase peaks are narrower and have a greater intensity, which is indicative of a larger particle size. 100 ,1—°,J J— 0v OH OH m/z 609>301 m/z 609>151 b) 1 09e>7 1 .OOH7 9.ÜOB6 ii.OOetj 7-OOee e.OOçe 5.00a6 4.00L-6 ï.Mce a.OOee 1 Mee OH \ 151.0 17a-»1fiB4 2S3.1 loo 1 50 2CÜ 25 563.5 591.3 300 350 400 450 rWz, tifa 500 55Ü BOO 6 5Ü 7QO 7 SO 8ÜÜ Figure 2. (a) Two transition ions of rutin peaks in multiple reaction monitoring and (b) mass fragmentation of rutin at the negative ion mode to be the second highest, which was about 52.27 ^g rutin in a gram of fraction in a milliliter of eluent. The content of rutin was increased from 0.85 ± 0.16 mg/g plant leaves (0.08%w/w) or 1.41 ± 0.54 mg/g crude extract (o.14%w/w) after reflux extraction, to 3.96 ± 0.39 mg/g fraction (0.40%w/w) after fractionation using 80% methanol as the eluent. The increment was about 2-fold after extraction and 4-fold after fractionation. The quantitation of rutin was measured by UPLC-MS/MS using the multiple reaction monitoring of two transition ions such as m/z 609>301 and m/z 609>151 at negative ion mode as shown in Figure 2. 3. 3. Scavenging Activity of Rutin Fraction The quality of rutin fraction was evaluated based on its scavenging activity using DPPH assay compared to standard chemicals such as rutin and ascorbic acid. The free radicals generated from DPPH were scavenged by antioxi-dants in a concentration dependent manner. This method measured the colour change based on the reduction of purple-coloured free radical DPPH' to yellow-coloured 2,2-di-phenyl-1-picrylhydrazine (DPPH-H). The colour density was recorded by a UV-Vis spectrophotometer at 517 nm. The results showed that IC50 for standard rutin and ascorbic acid was 122 and 84 ppm, respectively. The lower IC50 value indicates the lower amount of sample required to inhibit 50% of free radicals which means the sample has higher scavenging activity. The antioxidant capacity of standard rutin is comparable to ascorbic acid which is a well-known antioxidant compound. The IC50 of rutin fraction from the 80% methanol eluent system was 800 ppm which was 6 times lower scavenging activity than standard rutin, but almost 2 times higher scavenging activity than its crude extract (Table 2). Therefore, reversed phase fractionation increased the content of rutin in the plant sample, as well as improved its antioxidant capacity. The observation also explains that rutin could be the major radical scavenger. The increase of rutin content in the fractionated sample was found to increase its scavenging activity significantly. Table 2. Scavenging activity of standard chemicals and plant samples at 50% inhibition Sample IC50 (PPm) Standard ascorbic acid 84 Standard rutin 122 Crude extract 1500 80% methanol rutin fraction 800 4. Conclusion The detection of rutin has been reported by many investigators in L. pumila recently. It could be the prominent flavonol glycoside in the herbal plant which contributes to the significant pharmacological activities. Therefore, this study investigated the effects of methanol con- Chua et al.: Recovery of Rutin from Labisia pumila Acta Chim. Slov. 2017, 64, 888-894 893 centration for rutin recovery in SPE. A reserved phase SPE system could recover about 70% of rutin from the crude extract of L. pumila var. Alata using 80% methanol as the eluent. The rutin rich fraction was found to exhibit higher scavenging activity than crude extract, but the value was still lower than standard rutin. 5. Acknowledgement The authors would like to thank Universiti Teknologi Malaysia for internal grant (GUP 05J84), and Ministry of Higher Education Malaysia for HiCoE grant (4J263) to carry out the research activities. We also highly appreciate our internship student, Ms. Lee Si Ning to replicate some experimental works. 6. References 1. L. S. Chua, J. Ethnopharmacol. 2013, 150, 805-817. DOI:10.1016/j.jep.2013.10.036 2. G. Chen, H. Zhang, J. Ye, Anal. Chim. Acta 2000, 423, 69-76. DOI:10.1016/S0003-2670(00)01099-0 3. Y. Yang, F. Zhang, Ultrason. Sonochem. 2008, 15, 308-313. DOI:10.1016/j.ultsonch.2007.05.001 4. J. E. F. Reynolds, Martindale-The Extra Pharmacopoeia, 31st ed., The Royal Pharmaceutical Society, Council of the Royal Pharmaceutical Society of Great Britain, London, 1996, pp. 1679-1680. 5. W. Q. Sun, J. F. Sheng, Handbook of Natural Active Constituents, Chinese Medicinal Science and Technology Press, Beijing, 1998, pp. 2240-2316. 6. I. Erlund, T. Kosonen, G. Alfthan, J. Maenpaa, K. Perttunen, J. Kenraali, J. Parantainen, A. Aro, Eur. J. Clin. Pharmacol. 2000, 56, 545-553. DOI:10.1007/s002280000197 7. S. Y. Yoon, W. J. Choi, J. M. Park, J. W. Yang, Biotechnol. Tech. 1997, 11, 553-556. DOI:10.1023/A:1018434704902 8. E. Aehle, S. R. L. Grandic, R. Ralainirina, S. Baltora-Rosset, F. Mesnard, C. Prouillet, J. C. Maziere, M. A. Fliniaux, Food Chem. 2004, 86, 579-585. DOI:10.1016/j.foodchem.2003.10.006 9. J. Wang, F. A. Wu, H. Zhao, L. Liu, Q. S. Wu, Afr. J. Biotechnol. 2008, 7, 2147-2155. 10. B. Buszewski, S. Kawka, Z. Suprynowicz, T. Wolski, J. Pharm. Biomed. Anal. 1993, 11, 211-215. DOI:10.1016/0731-7085(93)80199-B 11. C. E. Sando, J. U. Lloyd, J. Biol. Chem. 1924, 737-745. 12. H. F. Koones, N. J. Clifton, Extraction of rutin, United States Patent Office: 2,450,555, 5 Oct 1948. 13. F. Fathiazad, A. Delazar, R. Amiri, S. D. Sarker, Iranian J. Pharm. Res. 2006, 5, 222-227. 14. S. Kreft, M. Knapp, I. Kreft, J. Agr. Food Chem. 1999, 47, 4649-4652. DOI:10.1021/jf990186p 15. K. H. Kim, K. W. Lee, D. Y. Kim, H. H. Park, I. B. Kwon, H. J. Lee, Bioresour. Technol. 2005, 96, 1709-1712. DOI:10.1016/j.biortech.2004.12.025 16. E. Altiok, D. Baycin, O. Bayraktar, S. Ulku, Sep. Purif. Technol. 2008, 62, 342-348. DOI:10.1016/j.seppur.2008.01.022 17. J. Dai, R. J. Mumper, Molecules, 2010, 15, 7313-7352. DOI:10.3390/molecules15107313 18. Z. Legnerova, D. Satinsky, P. Solich, Anal. Chim. Acta 2003, 497, 165-174. DOI:10.1016/j.aca.2003.07.007 19. B. Buszewski, S. Kawka, T. Wolski, Chromatographia 1993, 35, 311-316. DOI:10.1007/BF02277516 20. H. Zeng, Y. Wang, J. Kong, C. Nie, Y. Yuan, Talanta 2010, 83, 582-590. DOI: 10.1016/j.talanta.2010.10.006 21. M. Grilc, B. Likozar, J. Levec, ChemCatChem. 2016, 8, 180191. DOI: 10.1002/cctc.201500840 22. S. Machmudah, Wahyudiono, H. Kanda, M. Goto, ARPN J. Eng. App. Sci. 2016, 11, 9509-9518. 23. L. S. Chua, N. A. Latiff, S. Y. Lee, C. T. Lee, M. R. Sarmidi, R. A. Aziz, Food Chem. 2011, 127, 1186-1192. DOI:10.1016/j.foodchem.2011.01.122 24. A. Pandey, S. Tripathi, J. Pharmacog. Phytochem. 2014, 2, 115-119. 25. G. X. Wang, Vet. Parasitol. 2010, 171, 305-313. DOI:10.1016/j.vetpar.2010.03.032 26. P. Tiwari, B. Kumar, M. Kaur, G. Kaur, H. Kaur, Int. Pharm. Sci. 2011, 1, 98-106. 27. D. Xiao, P. M. Davidson, D. H. D'Souza, J. Lin, Q. Zhong, J. Food Eng. 2010, 100, 194-200. DOI:10.1016/j.jfoodeng.2010.03.044 28. M. Bimakr, R. A. Rahman, F. S. Taip, A. Ganjloo, L. M. Salleh, J. Selamat, A. Hamid, I. S. M. Zaidul, Food Bioprod. Process. 2011, 89, 67-72. DOI:10.1016/j.fbp.2010.03.002 29. I. Ammar, M. Ennouri, H. Attia, Ind. Crop. Prod. 2015, 64, 97-104. DOI: 10.1016/j.indcrop.2014.11.030 30. A. M. Azrie, A. Luqman Chuah, K. Y. Pin, J. Chem. Pharm. Res. 2014, 6, 172-176. 31. M. H. Ibrahim, H. Z. E. Jaafar, E. Karimi, A. Ghasemzadeh, Sci. World J. 2014, 2014, 360290. 32. Z. Ismail, H. K. Beh, M. S. R. Hamil, G. Ghafar, M. A. A. Saeed, A. H. Memon, S. Hashim, Publication number: W02016093692 A1. 16 June 2016. 33. E. Karimi, H. Z. E. Jaafar, S. Ahmad, 2011 Molecules 2011, 16, 4438-4450. 34. E. Karimi, H. Z. E. Jaafar, A. Ghasemzadeh, M. H. Ibrahim, Aus. J. Crop Sci. 2013, 7, 1016-1023. 35. E. Karimi, H. Z. E. Jaafar, Molecules 2011, 16, 6791-6805. DOI:10.3390/molecules16086791 36. R. Gevrenova, N. Denkov, D. Zheleva-Dimitrova, Pharmacia 2014, 61, 17-23. 37. The Official Hansen Solubility Parameter Site, Http://www. Hansen-Solubility.com (Accessed: 20 September 2015). 38. C. M. Hansen, Hansen Solubility Parameters, in A User's Handbook, 2nd ed. CRC Press, Boca Raton, 2007. DOI: 10.1201/9781420006834 39. J. Gao, Using Hansen solubility parameters (HSPs) to develop antioxidant-packing film to achieve controlled release. Michigan State University, Thesis for Master of Science, 2014. Chua et al.: Recovery of Rutin from Labisia pumila 894 Acta Chim. Slov. 2017, 64, 888-894 Povzetek Ekstrakcija z refluksom je bila uporabljena za pripravo osnovnega ekstrakta iz listov Labisia pumila var. Alata. Za izolacijo rutina z visokim izkoristkom je bil ta v nadaljevanju obdelan s C18 ekstrakcijo v trdni fazi z 20-80 % metanolom. Pri tem je bil z naraščajočo vsebnostjo metanola potreben manjši volumen eluenta, prav tako je naraščal tudi izkoristek, vendar je ta v čistem metanolu rahlo padel. Z 80 % metanolom je bilo izoliranega približno 70 % rutina. Bogata frakcija izolata z rutinom je dosegla 3.96 mg rutina /g frakcije, kar je 4-krat več kot v osnovnem ekstraktu. Tudi antioksidacijska aktivnost se je 2-krat povečala v primerjavi z osnovnim ekstraktom. Na boljši izkoristek rutina lahko vpliva voda (20 %) v 80 % sistemu z metanolom. Rutin je namreč glikoziliran flavonol in majhen delež vode lahko pospeši elucijo v primerjavi s 100 % metanolom. Chua et al.: Recovery of Rutin from Labisia pumila DOI: 10.17344/acsi.2017.3506 Acta Chim. Slov. 2017, 64, 895-901 ¿^creative tS1 commons Scientific paper Synthesis and Biological Evaluation of 1,2,4-Triazoles and 1,3,4-Oxadiazoles Derivatives Linked to 1,4-Dihydropyridines Scaffold Maghsoud Ziaie,1 Karim Akbari Dilmaghani1* and Amir Tukmechi2 1 Department of Organic Chemistry, Faculty of Chemistry, Urmia University, 57159, Urmia, Iran 2 Department of Microbiology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran * Corresponding author: E-mail: kadilmaghani@yahoo.com Received: 06-05-2017 Abstract A series of diethyl-2,6-dimethyl-4-phenyl-1,4-dihydropyridine-3,5-dicarboxylate derivative coupled to 1,3,4-oxadi-azole-5-thiones and 1,2,4-triazole-5-thiones moieties at C2,C6 positions of 1,4-dihydropyridine ring system was prepared. This linkage was carried out by the reaction of 1,3,4-oxadiazole-5-thiones and 1,2,4-triazole-5-thiones with 2,6-dibromomethyl-3,5-diethoxycarbonyl-4-phenyl-1,4-dihydropyridine in the presence of potassium carbonate as a weak base and dry acetone as the solvent. The newly synthesized compounds were characterized by FT-IR, 1H NMR, 13C NMR spectral data, elemental analysis and FAB-MS. The synthesized compounds were tested for their antimicrobial and antifungal activity against Escherichia coli and Aspergillus fumigatus in vitro in comparison with Enrofloxacin and Amphotericin as the reference drugs which are normally used for treating such infections. The synthetic compounds showed different inhibition zones against tested bacteria and fungi. Compound 8d showed more antagonistic activity against E. coli and A. fumigatus. Key words: 1,4-dihydropyridines, 1,3,4-oxadiazoles, 1,2,4-triazoles, antimicrobial activity 1. Introduction Antibiotics are drugs used for treating infection caused by microorganism such as bacteria or fungi and antibiotic resistance is the ability of microorganism to withstand the effect of antibiotics. The resistance of infective bacteria to present antibiotics remains a clinical obstacle in the chemotherapy of many cancers and demands research focused on the discovery of new drugs in the antibiotic drug field. The 1,4-dihydropyridines (1,4-DHPs) were found to be highly effective calcium antagonists and are used in treatment of various cardiovascular activities.1-4 Apart from CVS activities (1,4-DHPs), they posses a variety of biological activities such as anti-tubercular,5 antimicrobial,6-7 anti-inflammatory,8 anti-tumor,9-10 analgesic.11 A substance which has no calcium antagonistic activity but has antibiotic activity would be of value in cancer chemotherapy and has strong ability in overcoming anticancer drug resistance. Among the possible drug resistance modifiers the 1,4-dihydropyridines calcium antagonists such as Verapamil have been extensively studied.12-13 These examples clearly demonstrate the remarkable potential of novel DHP derivatives as a source of valuable drug candidates. In recent years 1,3,4-oxadiazole-5-thiones and 1,2,4-triazole-5-thiones derivatives which belong to an important group of heterocyclic compounds containing five membered ring have received significant attention and numerous reports have highlighted their diverse range of biological activities, such as antimicrobial,14-15 antifungal,16-17 antimycobacterial,18-19 anticancer,20-21 anti-inflammato-ry22-23 and anti convulsant.24-25 The connection of 1,3,4-oxa-diazoles or 1,2,4-triazoles to the 1,4-DHPs core has produced a combination scaffold. 1,4-DHPs can be selectively functionalized at several positions. Synthesis and antibacterial activities of bis(1,3,4-oxadiazole-2-thiol) and bis(4-amino-1,2,4-triazole-3-thiole) derivatives of 1,4-dihdropy-ridines derivatized at C3, C5 was reported.26 Synthesis and biological activities of 1,3,4-oxadiazole derivatives linked to N1 of 1,4-dihydropyridines ring system was reported.27 In spite of the widely developed chemistry of the 1,4-DHPs, much less is known about the synthesis of 1,4-DHPs bearing- substituents other than hydrogen atoms or alkyl groups Ziaie et al.: Synthesis and Biological Evaluation of 1,2,4-Triazoles 896 Acta Chim. Slov. 2017, 64, 895-901 at C2 and C6. These multicompound 1,4-DHP derivatives are useful in probing biological activity and might be used as antibiotics and inhibitors of the multidrug-resistant transporters. 1,4-DHP derivatives were prepared by Hantzsch method.28 Hantzsh dihydropyridine synthesis is a one-pot multicomponent condensation reaction where an aldehyde ^-ketoester and a nitrogen donor, such as ammonia or ammonium acetate, under reflux in ethanol froms the final product.29 In our previous works we reported the synthesis and antibacterial properties of a new series of thioglycoside heterocyclic derivatives of 1,2,4-triazole-5 -thiones and 1,3,4-oxadiazole-5-thiones.30-32 Following our studies on the design of potent antibacterial agents with 1,3,4-oxadiazoles and 1,2,4-triazole moieties, we have reported the connection of 1,2,4-triazole-5-thiones and 1,3,4-oxadiazole-5-thiones moieties to the 2,6-position of 1,4-DHP ring system. These newly synthesized multicom-pounds 1,4-DHPs are useful in probing biological activity, as they could exhibit antimicrobial effects and could act as inhibitors of the multidrug-resistant transporters. 2. Materials and Methods 2. 1. Bacterial Strain The antibacterial and antifungal activity of compounds was assayed according to our perviously published method.33 The antibacterial and antifungal activity of compounds was tested against Escherichia coli and Aspergillus fumigatus. 2. 2 Bacterium and Fungi Culture The following microorganisms were used in this study to test antimicrobial activity of compounds: E. coli (PTCC 1399) and Aspergillus fumigatus (PTCC 5009). All microorganisms were provided by Persian Culture Collections of Microorganism, Iran. Bacteria were cultured for 24 h at 37 °C in brain heart infusion broth (Merck, Darmstadt, Germany) and A. fumigatus was cultured for 72 h at 5 °C in Saburo Dextrose broth (Merck, Darmstadt, Germany) and were used as inoculums. 2. 3. Susceptibility Tests The following methods were used to evaluate the activity of the compounds. All tests were repeated three times, using distilled water without compounds as a control to test the inhibitory effect of the solvent. Minimum inhibition concentrations (MIC) of compounds against the tested pathological microorganisms were determined using micro broth dilution method.34 Briefly, serial twofold dilutions of each compounds (10% w/v) were prepared in 96-well micro titer plate ((from 1:2 to 1:8192) containing cation-adjusted Mueller-Hinton broth (Merck, Darmstadt, Germany). Control micro-titer plates containing medium and distilled water at the same dilutions were also made. Bacteria and fungi suspensions were adjusted to the 0.5 McFarland standards (approximately 1 to 2 x 108 CFU/mL). A constant amount of microorganisms were added to all wells and the plates were incubated at 37 and 25 °C for 24-72 hour for E. coli and A. fumigatus, respectively (final inoculates were adjusted to the 105 CFU per each well). Each well was examined for growth, comparing each well to the control. The MIC was defined as the lowest concentration of compounds at which there was no visible growth of the organisms. For each test enroflox-acin and amphotericin were used as the control antimicrobial agents. The minimal bactericidal concentration (MBC; the lowest concentration of compounds that resulted in a 99.9% reduction in CFU of the initial inoculums) was determined by plating count the contents of wells that showed no visible growth of bacteria onto Mueller-Hinton agar and Saburo Dextrose agar plates and incubating at 37 and 25 °C for 24-72 h for E. coli and A. fumigatus, respectively. The MBC was considered the lowest concentration of compounds that prevented any colony formation. 3. Result and Discussion The main synthetic route for substituted 1,3,4-oxadi-azole-5-thiones 2a-d involves an initial reaction between carboxylic acid hydrazides 1a-d and carbon disulfide in basic ethanol solution, followed by acidification with di- Scheme 1. Synthesis of 1,3,4-oxadiazole-5-thione and 1,2,4-triazole-5-thione derivatives Ziaie et al.: Synthesis and Biological Evaluation of 1,2,4-Triazoles ... Acta Chim. Slov. 2017, 64, 895-901 897 lute hydrochloric acid which resulted in the precipitation of oxadiazole. These compounds were synthesized according to the literature method (Scheme 1).35-36 1-Substitut-ed-4-phenylthiosemicarbazides 3a-d were prepared by the condensation of carboxylic acid hydrazides 1a-d with phenylisothiocyanate under reflux in absolute ethanol. The cyclodehydration of 1-substituted-4-phenylthiosemi-carbazides 3a-d were carried out in alkaline medium followed by acidification with dilute hydrochloric acid to give 1,2,4-triazole-5-thiones 4a-d according to the literature method (Scheme 1).37-39 The existence of thiol-thione tau-tomerism is known for the compounds 2a-d and 4a-d and generally one form is predominant. In the present study, the thione structure was dominated in the solid state. These constitutional isomers were distinguished by IR and 1H NMR. The appearance of a C=S absorption peak in the region 1248-1278 cm-1 indicated that the oxadiazoles and triazoles are in their thione form. The 1H NMR spectra of compounds 2a-d and 4a-d exhibited the NH signals (NH function of the oxadiazole and triazole ring) as a singlet in the range 12-14 ppm which also supports the proposed thione structure.35-39 4-Phenyl-1,4-dihydro-2,6-dimethyl-3,5-pyridine di-carboxylic acid ester (diethyl 2,6-dimethyl-4-phenyl-1,4-dihydropyridine-3,5-dicarboxylate)derivative (compound 5) was synthesized by condensation of benzaldehyde with two equivalents of ^-ketoester in the presence of a nitrogen donor such as ammonia or ammonium acetate according to the procedure reported in the literature. 40 The 1H NMR spectrum of compound 5 shows a characteristic singlet in the range of 5-7 ppm which was due to the NH proton of the 1,4-DHP ring and another important singlet at 5 ppm which was attributed to the CH at C4 of the 1,4-DHP ring. Allylic bromination is the replacement of a hydrogen on a carbon adjacent to a double bond and N-bromosuccinimide (NBS) is a brominating agent that is used as the source of bromine in radical reactions that are used for allylic bromination. The synthesis of 2,6-dibro-momethyl-3,5-diethoxycarbonyl-1,4-dihydropyridine (compound 6) was carried out by bromination of corresponding 2,6-dimethyl-1,4-dihydropyridine (compound 5) with NBS in methanol according to the procedure reported in the literature.41 Replacement of the bromines of compound 6 with 1,2,4-triazole-5-thiones 4a-d or 1,3,4-oxadiazole-5-thiones 2a-d was carried out in the presence of potassium carbonate as a weak base in dry acetone to afford the corresponding coupled 1,4-dihydropyridines (7a-d and 8a-d) (Scheme 2). The structures identification for compounds 7a-d and 8a-d was based on spectroscop-ic methods. In the IR spectra the disappearance of the C=S absorption peak in the region 1248-1278 cm-1 and furthermore the absence of NH peak at 12-14 ppm support the connection of oxadiazole and triazoles to 1,4-DHP ring. The CH2X protons at positions C2 and C6 of symmetrically substituted 1,4-dihydropyridine ring become diastereotopic and provide an AB system in the corresponding 1H NMR spectra. The extent of the observed anisochrony of the methylene protons should be influenced by the spatial conformation of ester groups and the formation of a CH—O=C intramolecular hydrogen bond- ing.41 The in vitro antibacterial and antifungal activities of the synthesized compounds against E. coli and A. fumiga-tus are shown in Tables 1 and 2. The minimal inhibition concentration for enrofloxacin as the reference antibacterial drug is 36 ^g/^L concentration and for amphotericin was 28 ^g/^L of drug in the same test conditions. Scheme 2. Synthesis of 1,4-dihydropyridine compounds bearing 1,3,4-oxadiazole-5-thiones and 1,2,4-triazole-5-thiones Ziaie et al.: Synthesis and Biological Evaluation of 1,2,4-Triazoles ... 898 Acta Chim. Slov. 2017, 64, 895-901 Table 1. In vitro antibacterial activity of synthetic compounds against E. coli. Compound (gS) MBC (g/mL) Compound MIC (g/mL) MBC (g/mL) 8a 250 500 7a 500 500 8b 250 500 7b 125 125 8c 125 250 7c 62.5 125 8d 62.5 62.5 7d 62.5 125 Table 2. In vitro antifungal activity of synthetic compounds against A. fumigatus. Compound (g/mL) MFC (g/mL) Compound MIC (g/mL) MFC (g/mL) 8a 500 500 7a 1000 2000 8b 500 500 7b 1000 2000 8c 500 500 7c 1000 2000 8d 250 500 7d 500 1000 From the data presented in Tables 1 and 2 it is clear that compounds 8a-d showed better antifungal activities than compounds 7a-d and the compounds 7c,7d,8d exhibited better and equipotent activity against E. coli. The compound 8d is highly active against E. coli and A. fumig-atus. 4. Experimental 4. 1. General The melting points of all compounds were recorded on a Philip Harris C4954718 apparatus without calibration. IR spectra were recorded on Thermo Nicolet Nexus 670 FT-IR spectrometer and 1H and 13C NMR spectra measured with Bruker Avance 300 MHz spectrometer. Mass spectra were recorded on a JEOL-JMS 600 (FAB MS) instrument. Thin layer chromatography (TLC) analyses were carried out on silica gel plates. All chemicals were purchased from Merck (Tehran, Iran) and used as received by standard procedures. All of the instruments, chemicals and solvents were dried according to standard methods. Freshly distilled solvents were used throughout, and anhydrous solvents were dried according to the method reported by Perrin and Armarego. Microanalyses were performed on a Leco Analyzer 932. 4. 2. General Procedure for the Synthesis of 2a-d Compounds 2a-d were synthesized by the reaction of CS2 (6.7 mL, 0.1 mol) with a suspension of carboxylic acid hydrazides 1a-d (0.1 mol) in chloroform (100 mL) in the presence of Et3N (15 mL, 0.1 mol). After refluxing the reaction mixture for 3 h, the resulting solution was filtered and acidified with acetic acid (20 mL, 20% v/v). On evaporation of the solvent, the solid obtained was washed twice with cold water and finally dried in vacuo, then recrystal-lized from ethanol (yield 80%).35-36 4. 3. General Procedure for the Synthesis of 4a-d Equimolar quantities of carboxylic acid hydrazides 1a-d (0.01 mol) and phenylisothiocyanate (1.35 g, 0.01 mol) in appropriate amount of absolute ethanol were re-fluxed for 6-8 h. The formed precipitate of 1-substitut-ed-4-phenylthiosemicarbazides 3a-d was filtered, and then a suspension of 0.04 g thiosemicarbazides 3a-d in sodium hydroxide 0.4 g (0.01 mol, as a 2N solution) was refluxed for 6 h. The reaction mixture was allowed to cool to the room temperature and was then adjusted to pH 6 with diluted hydrochloric acid. The formed precipitate was then filtered, dried and recrystallized from ethanol, to give 1,2,4-triazole-5-thiones 4a-d in 75% yields. The following compounds were prepared by an analogous procedure.37-39 Diethyl 2,6-Dimethyl-4-phenyl-1,4-dihydropyridine-3, 5-dicarboxylate (5)40 White crystals; 77% (2.535 g), mp 160-162 °C, 1H NMR (300 MHz, CDCl3) 5 1.25 (t, J = 7.2 Hz, 6H, -COOCH2CH3), 2.35 (s, 6H, 2CH3), 4.10 (q, 4H, -COOCH2CH3), 5.02 (s, 1H, DHP-C4-H), 5.72 (br s,1H, N-H), 7.13-7.32 (m, 5H, Ar-H), 13C NMR (75 MHz, CDCl3) S 14.27, 19.12, 39.66, 59.75, 103.59, 126.11, 127.85, 127.92, 144.76, 147.96, 168.03. IR (KBr) v 3340, 3078, 2976, 1651, 1483, 1378, 1100, 1030, 766, 695 cm-1. Anal. calcd for C19H23NO4: C, 69.28; H, 7.04; N, 4.25. Found: C, 69.18; H, 6.998; N, 4.29. Diethyl 2,6-Bis(bromomethyl)-4-phenyl-1,4-dihydro-pyridine-3,5-dicarboxylate (6)41 Lemon crystals; 72% (3.506 g), mp 135 °C, 1H NMR (300 MHz, CDCl3) S 1.25 (t, J = 7.2 Hz, 6H, -COOCH2CH3), 4.15 (q, J = 7.2 Hz, 4H, -COOCH2CH3), 4.6, 4.9 (dd, J1 = 4.65, J2 = 4.65 Hz, 4H, CH2-Br), 5.03 (s, 1H, DHP-C4-H), 6.53 (br s,1H, N-H), 7.12-7.31 (m, 5H, Ar-H), 13C NMR (75 MHz, CDCl3) S 14.11, 27.15, 40.08, 60.65, 105.79, 126.84, 128.06, 128.19, 142.19, 145.85, 166.31. IR (KBr) v 3334, 3084, 2979, 1686, 1508, 1372, 1095, 760, 704, 558 cm-1. Anal. calcd for C19H21Br2NO4: C, 46.84; H, 4.34; N, 2.88. Found: C, 46.76; H, 431; N, 2.95. 4. 4. General Procedure for the Synthesis of Compounds 7a-d and 8a-d A mixture of 2a-d or 4a-d (2 mmol), K2CO3 (0.276 g, 2 mmol) and acetone (20 mL) were stirred at room temperature for 30 minute, and then 0.487 g (1 mmol) com- Ziaie et al.: Synthesis and Biological Evaluation of 1,2,4-Triazoles ... Acta Chim. Slov. 2017, 64, 895-901 899 pound 6 and KI (cat.) were added and refluxed for 12 h and then evaporated. The residue was extracted with CH2Cl2 (30 mL) and the organic layer washed with water, dried over Na2SO4 and evaporated. Recrystallization of crude products from 2-propanol furnished 7a-d or 8a-d. Diethyl 4-Phenyl-2, 6-bis(((5-phenyl-1,3,4-oxadiazol-2-yl)thio)methyl)-1,4-dihydropyridine-3,5-dicarboxylate (7a) Pale yellow solid; 73% (0.498 g), m.p. 156-158 °C, 1H NMR (300 MHz, CDCl3): 5 25 (t, J = 7.2 Hz, 6H, -COOCH2CH3), 4.14 (q, J = 7.2 Hz, 4H,-COOCH2CH3), 4.62 (s, 4H, S-CH2), 5.05 (s, 1H, DHP-C4-H), 7.11-7.31 (m, 5H, Ar-H), 7.40-7.52 (m, 6H, Ar-H), 7.90 (d, J = 8.1Hz, 4H, Ar-H), 8.78 (br s, 1H, N-H), 13C NMR (75 MHz, CDCl3) 8 14.13, 32.32, 39.66, 60.37, 104.99, 123.54, 126.62, 126.66, 126.71, 128.11, 128.94, 131.59, 144.17, 147.76, 162.32, 165.38, 166.88. IR(KBr) v 3330, 3073, 2979, 1688, 1636, 1483, 1375, 1090, 764, 695, 595 cm-1. Anal. cal-cd for C35H31N5O6S2: C 61.66, H 4.58, N 10.27, S 9.40. Found: C, 61.69; H, 4.53; N, 10.24; S, 9.47. HRMS-FAB m/z [M+H]+calcd for QHNnS; 681.17. Found: 35 31 5 6 2 681.11. 1692, 1620, 1486, 1100, 753, 703 cm-1. HRMS-FAB m/z [M+H]+calcd for C35H31N2O8S2: 713.16. Found: 713.23. Anal. calcd for C^H^O^: C, 58.90; H, 4.38; N, 9.81; S, 8.98. Found: C, 58.86; H, 4.33; N, 9.84; S, 9.02. Diethyl 2,6-Bis((5-(3-nitrophenyl)-1,3,4-oxadiazol-2-ylthio)methyl) -4-phenyl-1,4-dihydropyridine-3,5-di-carboxylate (7d) Yellow crystals; 64% (0.493 g), m.p. 150-151 °C, 1H NMR (300 MHz, CDCl3) 8 1.26 (t, J = 7.2 Hz, 6H, -COOCH2 CH3), 4.15 (q, J = 7.2 Hz, 4H, -COOCH2CH3), 4.65 (s, 4H, S-CH2), 5.04 (s, 1H, DHP-C4-H), 7.10-7.30 (m, 5H, Ar-H, 1H N-H), 7.69 (t, J = 7.8 Hz, 2H, Ar-H), 8.28 (d, J = 7.8 Hz, 2H, Ar-H), 8.36 (d, J = 8.1 Hz, 2H, Ar-H), 8.72 (d, J = 17.1 Hz, 2H, Ar-H). 13C NMR (75 MHz, CDCl3) 8 14.12, 32.35, 39.65, 60.47, 105.25, 121.49, 125.13, 126.04, 126.66, 128.06, 128.09, 130.38, 132.04, 143.81, 146.65, 148.60, 162.32, 164.28, 166.85. IR (KBr) v 3333, 3087, 2978, 1693, 1633, 1525, 1479, 1353, 1097, 710 cm-1. HRMS-FAB m/z [M+H]+ calcd for C35H29N7O10S2:7 71.14. Found: 771.11. Anal. calcd for C35H29N7O10S2: C, 54.47; H, 3.79; N, 12.70; S, 8.31. Found: C, 54.51; H, 3.76; N, 12.67; S, 8.36. Diethyl 2,6-Bis((5-(2-chlorophenyl)-1,3,4-oxadiazol-2-ylthio)methyl) -4-phenyl-1,4-dihydropyridine-3,5-di-carboxylate (7b) Pale yellow solid; 62% (0.465 g), m.p. 156-158 °C, 1H NMR (300 MHz, DMSO-d6) 8 1.25 (t, J = 7.2 Hz, 6H, -COOCH2CH3), 4.15 (q, J = 7.2 Hz, 4H, -COOCH2CH3), 4.65 (dd, J1 = 14.1 Hz, J2 = 14.1 Hz, 4H, S-CH2), 5.05 (s, 1H, DHP-C4-H), 7.10-7.32 (m, 6H, Ar-H), 7.32-7.51 (m, 5H, Ar-H), 7.86 (d, J = 6.6 Hz, 2H, Ar-H), 8.72 (br s, 1H, N-H); 13C NMR (75 MHz, DMSO-d6) 8 14.32, 32.83, 39.60, 60.31, 104.53, 122.53, 126.94, 127.82, 128.19, 128.49, 131.48, 131.58, 132.22, 133.70, 44.16, 146.99, 162.32, 164.22, 166.41. IR (KBr) v 3282, 3080, 2978, 1678, 1630, 1488, 1097, 739 cm-1. HRMS-FAB m/z [M+H]+calcd for C H,0Cl N O^S,: 749.09. Found: 749.14. Anal. calcd for 35 29 2 5 6 2 C H,0Cl N O^S,: C, 56.00; H, 3.89; N, 9.33; S, 8.54. Found: 35 29 2 5 6 2 C, 56.04; H, 3.84; N, 9.35; S, 8.57. Diethyl 2,6-Bis((5-(2-hydroxyphenyl)-1,3,4-oxadiazol-2-ylthio)methyl)-4-phenyl-1,4-dihydropyridine-3,5-di-carboxylate (7c) Pale yellow solid; 69% (0.492 g), m.p. 155-157 °C, 1H NMR (300 MHz, CDCl3) 8 1.26 (t, J = 7.2 Hz, 6H, -COOCH2CH3), 4.14 (q, J = 7.2 Hz, 4H, -COOCH2CH3), 4.63 (dd, J1 = 14.1 Hz, J2 = 14.1 Hz, 4H, S-CH2), 5.03 2s, 1H, DHP-C4-H), 6.95 (t, J = 7.5 Hz, 2H, Ar-H), 7.05 (d, J = 8.4 Hz, 2H, Ar-H), 7.12-7.32 (m, 5H, Ar-H), 7.42 (t, J = 7.5 Hz, 2H, Ar-H), 7.65 (d, J = 7.8 Hz, 2H, Ar-H), 8.59 (s, 1H, N-H), 9.73 (s, 2H, OH). 13C NMR (75 MHz, CDCl3) 8 14.12, 32.43, 39.63, 60.47, 105.41, 107.66, 117.47, 119.92, 126.45, 126.73, 128.02, 128.12, 133.66, 143.66, 146.47, 157.16, 164.75, 165.64, 166.78. IR (KBr) v 3302, 2879, Diethyl 2,6-Bis((4,5-diphenyl-4H-1,2,4-triazol-3-ylt-hio)methyl)-4-phenyl-1,4-dihydropyridine-3,5-dicar-boxylate (8a) White solid; 76% (0.632 g), m.p. 189-191 °C, 1H NMR (300 MHz, CDCl3) 8 1.22 (t, J = 7.2 Hz, 6H, -COOCH2CH3), 4.08(q, J = 7.2 Hz, 4H, -COOCH2CH3), 4.49, 4.66 (dd, J = 14.1 Hz, J2 = 14.1 Hz, 4H, S-CH2), 5.04 (s, 1H, DHP-C4-H), 7.10-7.50 (m, 25H, Ar-H), 9.69 (s, 1H, N-H). 13C NMR (75 MHz, CDCl3) 8 14.17, 32.35, 39.82, 60.03, 104.42, 126.30, 126.68, 127.49, 127.94, 128.20, 128.33, 129.78, 129.84, 134.25, 145.36, 147.32, 153.61, 155.05, 167.11. IR (KBr) v 3292, 3050, 2976, 1688, 1635, 1493, 1095, 766, 695 cm-1. HRMS-FAB m/z [M+H]+ calcd for C47H41N7O4S2: 831.27. Found: 831.22. Anal. calcd for C47H41N7O4S2: C 67.85; H, 4.97; N, 11.78; S, 7.71. Found: C 67.88; H, 4.94; N, 11.79; S, 7.73. Diethyl 2,6-Bis((5-(2-chlorophenyl)-4-phenyl-4H-1,2,4 -triazol-3-ylthio)methyl)-4-phenyl-1,4-dihydropyri-dine-3,5-dicarboxylate (8b) White solid; 61% (0.549 g), m.p. 164-165 °C, 1H NMR (300 MHz, CDCl3) 8 1.22 (t, J = 7.2 Hz, 6H, -COOCH2CH3), 4.09 (q, J = 7.2 Hz, 4H, -COOCH2CH3), 4.52, 4.66 (dd, J = 14.1 Hz, J2 = 14.1 Hz, 4H, S-CH2), 5.04 (s, 1H, DHP-C4-H), 7.11-7.36 (m, 21H, Ar-H), 7.48 (d, J = 7.2 Hz, 2H, Ar-H), 9.70 (s, 1H, N-H). 13C NMR (75 MHz, CDCl3) 8 14.17, 32.35, 39.83, 60.08, 104.12, 126.32, 126.61, 126.78, 127.95, 128.23, 129.33, 129.62, 131.42, 132.79, 133.34, 134.28, 145.34, 147.25, 152.95, 153.53, 162.32, 167.12. IR (KBr) v 3277, 3068, 2978, 1685, 1637, 1499, 1094, 763, 697, 604 cm-1; HRMS-FAB m/z [M+H]+ calcd for C47H39Cl2N7O4S2: 899.19. Found: 899.25. Anal. calcd Ziaie et al.: Synthesis and Biological Evaluation of 1,2,4-Triazoles ... 900 Acta Chim. Slov. 2017, 64, 895-901 for C47H39Cl2N7O4S2: C, 62.66; H, 4.36; N, 10.88; S, 7.12. Found: C, 62.677; H, 4.35; N, 10.90; S, 7.15. Diethyl 2,6-Bis((5-(2-hydroxyphenyl)-4-phenyl-4H-1,2,4-triazol-3-ylthio)methyl)-4-phenyl-1,4-dihydropy-ridine-3,5-dicarboxylate (8c) Pale yellow solid; 71% (0.613 g), m.p. 226-228 °C, 1H NMR (300 MHz, CDCl3) 5 1.22 (t, J = 7.2 Hz, 6H, -COOCH2CH3), 4.08 (q, J = 7.2 Hz, 4H, -COOCH2CH3), 4.52, 4.70 (dd, J1 = 14.1 Hz, J2 = 14.1 Hz, 4H, S-CH2), 5.05 (s, 1H, DHP-C4-H), 6.40-6.54 (m, 5H, Ar-H), 6.97 (d, J = 8.4 Hz, 2H, Ar-H), 7.12 (t, J = 6.9 Hz, 2H, Ar-H), 7.22 (t, J = 7.2 Hz, 2H, Ar-H), 7.27-7.37 (m, 6H, Ar-H), 7.52-7.63 (m, 6H, Ar-H), 9.25 (s, 1H, N-H), 11.60 (br s, 2H, OH). 13C NMR (75 MHz, CDCl3) 8 14.12, 32.13, 39.74, 60.15, 104.04, 110.04, 117.78, 118.42, 125.48, 126.46, 127.65, 127.97, 128.16, 130.43,130.67, 131.27, 134.23, 144.61, 147.00, 153.68, 153.83, 157.86, 166.97. IR (KBr) v 3303, 2978, 1691, 1618, 1100, 752, 701 cm-1. HRMS-FAB m/z [M+H]+ calcd for Q,H,NOS,: 863.26. Found: 863.30. 47 41 7 6 2 Anal. calcd for Q,H,NOS,: C, 65.34; H, 4.78; N, 11.35; S, 47 41 7 6 2 7.42. Found: C, 65.33; H, 4.81; N, 11.32; S, 7.40. Diethyl 2,6-Bis((5-(3-nitrophenyl)-4-phenyl-4H-1,2,4-triazol-3-ylthio)methyl)-4-phenyl-1,4-dihydropyri-dine-3,5-dicarboxylate (8d) Pale yellow crystals; 68% (0.627 g), m.p. 189-190 °C, 1H NMR (300 MHz, CDCl3) 8 1.25 (t, J = 7.2 Hz, 6H, -COOCH2CH3), 4.08 (q, J = 7.2 Hz, 4H, -COOCH2CH3), 4.56, 4.66 (dd, J1 = 14.1 Hz, J2 = 14.1 Hz, 4H, S-CH2), 5.04 3s, 1H, DHP-C4-H), 7.17-7.58 (m, 19H, Ar-H), 7.68 (d, J = 7.8 Hz, 2H, Ar-H), 8.11 (d, J = 9.3 Hz, 2H, Ar-H), 9.58 (s, 1H, N-H). 13C NMR (75 MHz, CDCl3) 8 14.15, 32.31, 39.76, 60.09, 104.25, 126.39, 127,36, 127.46, 127.95, 128.23, 128.27, 129.52, 130.38, 130.61, 133.37, 133.53, 145.20, 147.28, 147.96, 152.93, 155.05, 162.34, 167.11. IR (KBr) v 3304, 3081, 2980, 1687, 1634, 1505, 1353, 1096, 909, 770, 701 cm-1. HRMS-FAB m/z [M+H]+ calcd for C47H39N9O8S2: 921.24. Found: 921.17. Anal. calcd for C47H39N9O8S2: C, (21.23; H, 4.26; N, 13.67; S, 6.95. Found: C, 61.2-49; H, 4.28; N, 13.65; S, 6.94. 5. Conclusion In this study we demonstrated the synthesis, antibacterial and antifungal activity of new derivatives of 1,4-di-hydropyridines bearing 1,2,4-triazole and 1,3,4-oxadiazole moieties at C2 and C6 of 1,4-DHP ring system. It should be noted that compound 8d exhibited the most potent activity against E. coli and A. fumigates. The structure-activity relationship of the compounds showed that substitution at the position 2, 6 of the pyridine ring enhances biological activity. Our results will have an impact on further investigation in this field in search of 1,4-dihydropyridine compounds connected with 1,2,4-triazole and 1,3,4-oxadiazole moieties as antibacterial and antifungal agents. Acknowledgments. The authors are grateful to Urmia University for providing a fellowship for the present work and thanks are also given to Prof. Dr. Joachim Thiem from Hamburg University for the ESI-MS measurements. 6. References 1. D. J. Triggle, Biochem. Pharmacol. 2007, 74, 1-9. DOI:10.1016/j.bcp.2007.01.016 2. D. A. Sica, J. Hypertens. 2006, 8, 53-56. 3. F. Bossert, H. Meyer, E. Wehinger, Angew. Chem. Int. Ed. Engl. 1981, 20, 762-769. DOI:10.1002/anie.198107621 4. J. G. Breitenbucher, G. Figliozz, Tetrahedron Lett. 2000, 41, 4311-4315. DOI:10.1016/S0040-4039(00)00660-2 5. G. A. Wachter, M. C. Davis, J. Med. Chem. 1998, 41, 24362438. DOI:10.1021/jm9708745 6. T. Akbarzadeh, S. Motagian, A. Fallah Tafti, A. Shafiee, A. R. Shahverdi, Res. Pharm. Sci. 2008, 3, 87-93. 7. S. Kalam, B. Darna, A. Garlapati, M. R. Vanga, Eur. J. Med. Chem. 2011, 46, 1564-71. DOI:10.1016/j.ejmech.2011.02.003 8. V M. Briukhanov, Exp. Clin. Pharmocol, 1994, 57, 47-49. 9. R. Boer, V. Gekeler, Drugs Future, 1995, 20, 499-509. 10. O. Firuzi, K. Javidnia, E. Mansourabadi, L. Saso, A. R. Meh-dipour, R. Miri, Arch. Pharm. Sci. Res. 2013, 36, 1392-1402. DOI:10.1007/s12272-013-0149-8 11. S. Ulloora, S. Kumar, R. Shabaraya, A.V. Adhikari, Med. Chem. Res, 2013, 22, 1549-1562. DOI:10.1007/s00044-012-0156-1 12. G. Hamilton, E. P. Cosentini, B. Teleky, T. Koperna, J. Zach-eri, M. Riegler, W. Feil, R. Schiessel, E. Wenzi, Anticancer Res. 1993, 13, 2059-2063. 13. H. W. van Veen, R. Callaghan, L. Soceneantu, A. Sardini, W. N. Konings, C. F. Higgins, Nature 1998, 391, 291-295. DOI: 10.1038/34669 14. N. B. Patel, J. C. Patel, Sci. Pharm. 2010, 78, 171-193. DOI:10.3797/scipharm.0912-16 15. T. Plech, M. Wujec, A. Siwek, U. Kosikowska, A. Malm, Eur. J. Med. Chem. 2011, 46, 241-248. DOI:10.1016/j.ejmech.2010.11.010 16. O. Prakash, M. Kumar, R. Kumar, C. Sharma, K. R. Aneja, Eur. J. Med. Chem. 2010, 45, 4252-4257. DOI:10.1016/j.ejmech.2010.06.023 17. T. Tsukuda, Y. Shiratori, M. Watanabe, H. Ontsuka, K. Hat-tori, M. Shirai, Bioorg. Med. Chem. Lett. 1998, 8, 1819-1824. DOI:10.1016/S0960-894X(98)00316-3 18. M. A. Alia, M. Shaharyar, Bioorg. Med. Chem. Lett. 2007, 17, 3314-3316. DOI:10.1016/j.bmcl.2007.04.004 19. T. A. Kaplancikli, G. Turan-Zitouni, P. Chevallet, J.Enzyme. Inhib.Med.Chem. 2005, 20, 179-182. DOI: 10.1080/14756360500043471 20. K. Liu, X. Lu, H. J. Zhang, J. Sun, H. L. Zhu, Eur. J. Med. Chem. 2012, 47, 473-478. DOI:10.1016/j.ejmech.2011.11.015 21. A. Kamal, N. Shankaraiah, V. Devaiah, K. L. Reddy, A. Ju- Ziaie et al.: Synthesis and Biological Evaluation of 1,2,4-Triazoles ... Acta Chim. Slov. 2017, 64, 895- 901 901 vekar S. Sen, N. Kurian, S. Zingde, Bioorg. Med. Chem. Lett. 2008, 18, 1468-1473. DOI:10.1016/j.bmcl.2007.12.063 22. A. Kumar, C. S. Rajput, Eur. J. Med. Chem. 2009, 44, 83-90. DOI:10.1016/j.ejmech.2008.03.018 23. H. Kumar, S. A. Javed, S. A. Khan. Eur. J. Med. Chem. 2008, 43, 2688-2698. DOI:10.1016/j.ejmech.2008.01.039 24. R. R. Somani, G. Kadam, R. Vohra, S. Vijayaraghavan, P. Y. Shirodkar, Int. J. Pharmacol. 2010, 6, 696-704. DOI:10.3923/ijp.2010.696.704 25. N. Siddiqui, W. Ahsan, Eur. J. Med. Chem. 2010, 45, 15361543. DOI: 10.1016/j.ejmech.2009.12.062 26. R. Surendrakumar, A. Manilal, A. J. Abdul Nasser, B. Merdeki-os, X. Chen, A. Idhayadhulla, J. Pharmacol. Toxicol. 2014, 9, 119-128. DOI:10.3923/jpt.2014.119.128 27. A. B. Archana, D. R. Dinesh, S. G. Paraag, Y. Shirodkar Prab-hakar, Int. J. Pharm. Chem. 2014, 4, 63-67. 28. A. Hantzsch, Ber. 1881, 14, 1637-1638. DOI: 10.1002/cber. 18810140214 29. A. Hantzsch, Ann. Chem. 1892, 215, 1-81. DOI:10.1002/jlac.18822150102 30. K. Akbari Dilmaghani, F. Nasuhi Pur, N. Hoseini Jazani, A. Alavi, Z. Niknam, F. Mirfakahraee, Phosphorus, Sulfur Silicon Relat. Elem.2014,189, 81-87. DOI: 10.1080/10426507.2013.789877 31. K. Akbari Dilmaghani, F. Nasuhi Pur, M. Hatami Nezhad, Iran. J. Pharm. Res. 2015, 14, 693-699. 32. K. Akbari Dilmaghani, F. Nasuhi Pur, M. Mahammadpour, J. Mahammadnejad, Iran. J. Pharm. Res. 2016, 15,777-782. 33. A. Tukmechi A. M. Ownagh, Braz. J. Microbiol. 2010, 41, 1086-1092. DOI:10.1590/S1517-83822010000400030 34. H. Katiroglu, Y. Beyatli, B. Aslim, Z. Yiiksekdag, T. Atici, Internet J. Microbiol.2006,2, 520-524. 35. N. K. Singh, R. J. Butcher, P. Tripathi, A. K. Srivastava. M. Kumar Bharty, Acta Cryst. 2007, E63, 0782- 0784. 36. C. H. Lee, H. I. Cho, K. J. Lee, Bull. Korean Chem. Soc. 2001, 22, 1153-1155. 37. M. Mhasalkar, M. Shah, S. Nikam, K. Anantanarayanan, Deli-wala, J. Med. Chem. 1970, 13, 672-674. DOI:10.1021/jm00298a021 38. I. Khan, S. Ali, S. Hameed, N. Rama, M. Hussain, A. Wadood, R. Uddin, Z. Ul-Haq, A. Khan, S. Ali, M. Choudhary, Eur. J. Med. Chem. 2010, 45, 5200-5207. DOI:10.1016/j.ejmech.2010.08.034 39. (a) B. V. I. Cohen, J. Heterocycl. Chem. 1978, 15, 237-240. DOI: 10.1002/jhet.5570150211 (b) K. Sung, A. R. Lee, J. Heterocycl. Chem. 1992, 29, 11011109. DOI:10.1002/jhet.5570290512 40. M. S. Bai, Y. Y. Chen, D. L. Niu, L. Peng, Acta Cryst. 2009, E65, 0799. 41. M. Petrova, R. Muhamadejev, B. Vigante, B. Cekavicus, A. Plotniece, G. Duburs, E. Liepinsh, Molecules 2011, 16, 80418052. DOI:10.3390/molecules16098041 Povzetek Pripravili smo serijo dietil-2,6-dimetil-4-fenil-1,4-dihidropiridin-3,5-dikarboksilatnih derivatov spojenih z 1,3,4-oksadi-azol-5-tionskimi in 1,2,4-triazol-5-tionskimi ostanki na položajih C2,C6 v 1,4-dihidropiridinskem obročnem sistemu. To povezavo smo izvedli z reakcijo med 1,3,4-oksadiazol-5-tioni in 1,2,4-triazol-5-tioni z 2,6-dibromometil-3,5-die-toksikarbonil-4-fenil-1,4-dihidropiridinom ob prisotnosti kalijevega karbonata kot šibke baze in v suhem acetonu kot topilu. Nove pripravljene spojine smo karakterizirali z FT-IR, 'H NMR in 13C NMR spektroskopskimi podatki, kot tudi z elementno analizo in FAB-MS. Za sintetizirane spojine smo in vitro preverili delovanje proti mikrobom in glivam (proti Escherichia coli in Aspergillus fumigatus) ter rezultate primerjali z vrednostmi za enrofloksacin in amfotericin kot referenčnima zdraviloma, ki se običajno uporabljata za zdravljenje tovrstnih infekcij. Pripravljene spojine so izkazale različne inhibicijske sposobnosti proti testiranim bakterijam in glivam. Spojina 8d je pokazala največje antagonistične lastnosti proti E. coli and A. fumigatus. Ziaie et al.: Synthesis and Biological Evaluation of 1,2,4-Triazoles ... 902 DOI: 10.17344/acsi.2017.3575 Acta Chim. Slov. 2017, 64, 902-910 ^creative tycommor Scientific paper Design, Synthesis and Anti-inflammatory Activity of Derivatives 10-R-3-Aryl-6,7-dihydro-2H-[1,2,4] triazino[2,3-c]quinazolin-2-ones of Spiro-fused Cyclic Frameworks Oleksandra Kolomoets,1 Oleksii Voskoboynik,1 Oleksii Antypenko,1 Galyna Berest,1 Inna Nosulenko,1 Vitaliy Palchikov,2 Olexandr Karpenko3 and Sergiy Kovalenko1* 1 Organic and Bioorganic Chemistry Department, Zaporizhzhya State Medical University, 26, Mayakovsky Ave., Zaporizhzhya, 69035, Ukraine 2 Chemistry Department, Oles Honchar Dnipropetrovsk National University, 72, Gagarina Ave., Dnipropetrovsk 49050, Ukraine 3 Enamine Ltd., Oleksandra Matrosova 23, Kyiv, 01103, Ukraine * Corresponding author: E-mail: kovalenkosergiy@gmail.com Received: 24-05-2017 Abstract Present work is devoted to the purposeful search of novel promising anti-inflammatory agents among the insufficiently known 3'-R-10'-R1-spiro[hetaryl-3(4),6'-[1,2,4]triazino[2,3-c]quinazolin]-2'(7'H)-ones. The virtual combinatorial library of previously unknown spiro-condensed derivatives of [1,2,4]triazino[2,3-c]quinazolines was formed and promising COX-2 inhibitors were identified by molecular docking method. Potential anti-inflammatory agents were synthesized by [5+1]-cyclocondensation of substituted 3-(2-aminophenyl)-6-R-1,2,4-triazin-5(2H)-ones with heterocyclic ketones. The structures of synthsized compounds were verified by complex of physicochemical methods and spectral characteristics features were discussed. Obtained compounds were studied for anti-inflammatory activity using formalin induced paw edema model and highly active compounds were identified. Conducted SAR-analysis showed that combination of triazino[2,3-c] quinazoline moiety with spiro-condensed fragments is a reasonable approach for creating novel anti-inflammatory agents. Keywords: 6,7-dihydro-2H-[1,2,4]triazino[2,3-c]quinazolin-2-ones, spiro-fused cycle, synthesis, molecular docking, anti-inflammatory activity 1. Introduction Inflammation is a chain of complex metabolic and morphological changes, aimed to restore the functions of the damaged tissues or organ in general. Despite the fact that the mentioned process is a natural response to a variety of factors, its role in the pathological states requires the development of drugs for pharmacotherapeutic correction. Thus, the use of drugs such as NSAIDs, may correct as certain stages of inflammation, so exclude the process in general. At the first stages of the antiphlogistics chemistry formation, the carboxylic acids of various nature (aspirin, diclofenac, ibuprofen and others) were considered as privileged objects of studies. Whereas recently, the majority of studies focus on substances with a heterocyclic fragment.1 This signifies considerable side effects of NSAIDs of first generation (COX-1), namely their negative impact on the gastrointestinal tract (gastrotoxicity). Nowadays, new classes of NSAIDs are found, which to some extent do not have mentioned side effect: selective COX-2 inhibitors (nimesulide, meloxicam, piroxicam, lornoxicam) and highly selective (specific) COX-2 inhibitors (celecoxib, ro-fecoxib, parecoxib, etoricoxib etc.).2 The current strategy of creating anti-inflammatory drugs is inextricably associated with further study of Kolomoets et al.: Design, Synthesis and Anti-inflammatory Activity Acta Chim. Slov. 2017, 64, 902-910 903 mechanism of inflammation. Advances in molecular biology in the last decades allowed to characterize every stage of this process and made it possible to form a number of approaches of the creation of this drug group.3,4 Thus, the main trends in the creation of innovational drugs include the development of C5a receptor antagonists, inhibitors of interleukin converting enzyme and tumor necrosis factor inhibitors, p38 MAP kinase inhibitors, inhibitors of matrix metalloproteinase, etc. Undoubtedly, the mentioned strategy with the use of de novo methodology (molecular docking) and X-ray analysis of the macromolecules active-site has significantly changed the direction of the synthetic work aimed at creating the drugs for correcting the inflammation. In particular, innovative anti-inflammatory drug of dual inhibition of COX-2/5-LOX - 2-(2,2-dimeth-yl-6,7-diphenyl-2,3-dihydro-1H-pyrrolizin-5-yl)acetic acid (Licofelone)5 was found. Moreover, new classes of biologically active substances with the mentioned type of activity were found among triazoles, imidazoles, thiazoli-dines, 2H-benzo[e][1,2]thiazine-1,1-dioxide, quinolines, quinazolines and other.1,6-11 Therefore, rational design based on structural similarity to innovative new structures of NSAIDs using the de novo methodology and traditional pharmacological screening is important and justified. So, purposeful search of selective anti-inflammatory agents among original derivatives of 10-R-3-aryl-6,7-dihy-dro-2H-[1,2,4]triazino[2,3-c]quinazolin-2-ones of spiro-fused cyclic frameworks was the aim of this work. That is based on rational design, namely structural similarity to a number of innovative and well-known drugs and forecasting the likely biological effects (COX inhibitors) using methods of computer modeling and in vivo tests. 2. Experimental Section 2. 1. Chemistry General Methods Melting points were determined in open capillary tubes in a «Stuart SMP30» apparatus and were uncorrected. Elemental analyses (C, H, N) were performed at the ELEMENTAR vario EL Cube analyzer (USA) and were within ± 0.3% from the theoretical values. IR spectra (4000-600 cm-1) were recorded on a Bruker ALPHA FT-IR spectrometer (Bruker Bioscience, Germany) using a module ATR eco ZnSe. 1H NMR spectra (400 MHz) and 13C NMR spectra (100 MHz) were recorded on a Vari-an-Mercury 400 (Varian Inc., Palo Alto, CA, USA) spectrometer with TMS as internal standard in DMSO-d 6 solution. LC-MS were recorded using chromatography/ mass spectrometric system which consists of a high performance liquid chromatograph «Agilent 1100 Series» (Agilent, Palo Alto, CA, USA) equipped with diode-matrix and mass-selective detector «Agilent LC/MSD SL» (atmospheric pressure chemical ionization - APCI). Electron impact mass spectra (EI-MS) were recorded on a Varian 1200 L instrument at 70 eV (Varian Inc., Palo Alto, CA, USA). Substances 1.1-1.5 were synthesized according to the reported procedures.12 Other starting materials and solvents were obtained from commercially available sources and were used without additional purification. 2. 2. Molecular Docking Research was conducted by flexible molecular docking, as an approach of finding molecules with affinity to a specific biological target. Macromolecules from Protein Data Bank (PDB) were used as biological targets, namely COX-1 enzyme in complex with diclofenac (PDB ID -3N8Y) and COX-2 in association with celecoxib (PDB ID - 3LN1).13 The choice of biological targets was due to the literature on the mechanism of action of antiinflammatory drugs.1 Ligand preparation. Substances were drawn using MarvinSketch 6.3.0 and were saved in mol format.14 After, they were optimized by program Chem3D using molecular mechanical MM2 algorithm and saved as pdb files. Molecular mechanics has been used to produce more realistic geometry values for the majority of organic molecules owing to the fact of being highly parameterized. Using Aut-oDockTools-1.5.6 pdb files were converted to PDBQT, number of active torsions was set as default.15 Protein preparation. Pdb files were downloaded from the protein data bank. Discovery Studio 4.0 was used to delete water molecules and ligand from crystal. Proteins were saved as pdb files. In AutoDockTools-1.5.6 polar hydrogens were added and saved as PDBQT. Grid box was set as following: center_x = 18.37, center_y = -52.296, cen-ter_z = 53.949, size_x = 18, size_y = 16, size_z = 16 for COX-2 (3LN1); center_x = 32.978, center_y = -44.488, center_z = -3.76, size_x = 16, size_y = 16, size_z = 16 for COX-1 (3N8Y). Vina was used to carry docking.15 For visualization Discovery Studio 4.0 was used. 2. 3. Pharmacology 2. 3. 1. Anti-inflammatory Activity Evaluation of anti-inflammatory activity of the synthesized compounds was performed on 84 Wistar white rats of 150-160 g, obtained from the nursery «Institute of Pharmacology and Toxicology of Ukraine» (Kyiv). All experimental procedures and treatments were carried out according to the European Convention and «Regulations on the use of animals in biomedical research».16 Screening of synthesized compounds with estimated antiinflammatory activity began with the study of their effect on exudative phase of acute aseptic inflammation (the formalin test). Phlogogen (1% aqueous solution of formaline)17 was subplantally administered at a dose of 0.1 mL in the back right paw of the rat, the left served as control. The studied compounds stabilized by Tween-80 were intragastric ad- Kolomoets et al.: Design, Synthesis and Anti-inflammatory Activity 904 Acta Chim. Slov. 2017, 64, 902-910 ministered in a dose of 10 mg/kg 1 h before the administration of phlogogen. Reference drug diclofenac sodium was intragastrically administered to rats at a recommended dose for pre-clinical studies of 8 mg/kg. Measuring paws volume was conducted before the experiment and 3 h after the administration of phlogogen using the described18 methods. The activity of these substances was determined by their ability to reduce the extension of swelling compared with control and expressed as a percentage showing how the substance inhibited formalin swelling in relation to control swelling where the value was taken as 100%. The activity of the studied compounds was calculated as follows: where A - antiexudative activity, %; Vx - the volume of swollen paw in the experiment; Vhe - the volume of healthy paw in the experiment; Vsc - the volume of swollen paw in control; Vhc - the volume of healthy paw in control. Statistical data processing was performed using a license program «STATISTICA® for Windows 6.0» (Stat-Softlnc., N AXXR712D833214FAN5) and «SPSS 16.0», «Microsoft Office Excell 2003». The results are presented as mean ± standard error of the mean. Arithmetic mean and standard error of the mean were calculated for each of the studied parameters. During verification of statistical hypothesis, null hypothesis was declined if statistical criterion is p < 0.05.19 General Procedure for the Synthesis of 3'-R-10'-R'-Spiro[hetaryl-3(4),6'-[1,2,4]triazino[2,3-c]quinazolin]-2'(7'H)-ones 2.1-2.11 To a solution of 0.01 M of compounds 1.1-1.5 in 20 mL of glacial acid was added 0.01 M of appropriate heterocyclic carbonyl compound (tetrahydro-4H-pyran-4-one, dihydrothiophen-3(2H)-one, dihydro-2H-thiopy-ran-3(4H) -one, 1-methylpiperidin-4-one, dihydrothio-phen-3(2H) -one 1,1-dioxide, dihydro-2H-thiopyran-3 (4H)-one 1,1 -dioxide). The reaction mixture was refluxed for 3 h. The solvent was removed by vacuum distillation, the residue was triturated in methanol, the precipitate was filtered and dried. If necessary, crystallized from dioxane. 3'-Phenyl-2,3,5,6-tetrahydrospiro[pyran-4,6'-[1,2,4]tria zino[2,3-c]quinazolin]-2'(7'H)-one (2.1). Yield: 3.8 g (92.4%); pale yellow crystals; m.p. >300; IR: 3256, 2952, 2856, 1638, 1624, 1611, 1592, 1551, 1514, 1498, 1479, 1435, 1417, 1394, 1334, 1299, 1261, 1214, 1177, 1160, 1150, 1136, 1107, 1097, 1080, 1041, 1027, 1004, 975, 951, 927, 874, 858, 836, 815, 778, 757, 709, 697, 675, 634, 620 cm-1; 1H NMR (DMSO-d6) 5 8.18 (d, J = 8.4 Hz, 2H, H-2, 6 Ph), 8.02 (d, J = 7.8 Hz, 1H, H-11), 7.55-7.29 (m, 5H, H-9, H-3, 4, 5 Ph, NH), 7.04 (d, J = 8.1 Hz, 1H, H-8), 6.88 (t, J = 7.5 Hz, 1H, H-10), 3.90-3.80 (m, 2H, H-2, 2',6, 6' pyrane), 2.56-2.51 (m, 2H, H-3, 5 pyrane), 1.99-1.99 (m, 2H, H-3', 5' pyrane); 13C NMR (DMSO-d6) S 34.19, 63.26, 75.98, 113.70, 116.81, 118.37, 119.81, 127.36, 128.62, 129.10, 130.27, 130.69, 133.37, 135.46, 144.56, 146.29, 147.28, 161.00. LC-MS: m/z = 347 [M+1]. Anal. Calcd. for C20H18N4O2: C, 69.35; H, 5.24; N, 16.17. Found: C, 69.38; H, 5.289; N, 16.28. 1-Methyl-3'-phenylspiro[piperidine-4,6'-[1,2,4]triazino [2,3-c]quinazolin]-2'(7'H)-one (2.2). Yield: 2.68 g (74.7%); pale yellow crystals; m.p. 269-271 °C; IR: 3274, 1700, 1637, 1623, 1611, 1594, 1550, 1516, 1498, 1484, 1457, 1436, 1416, 1365, 1336, 1266, 1222, 1202, 1186, 1157, 1139, 1108, 1080, 1064, 1031, 1016, 995, 951, 929, 882, 853, 816, 769, 748, 710, 694, 665, 636 cm-1; 1H NMR (DMSO-d6) S 8.21 (d, J = 8.0 Hz, 2H, H-2,6 Ph), 8.02 (d, J = 7.7 Hz, 1H, H-11), 7.547.37 (m, 3H, H-9; H-3, 5 Ph), 7.28 (s, 1H, NH), 7.07 (d, J = 7.8 Hz, 1H, H-8), 6.89 (t, J = 7.8 Hz, 1H, H-10), 2.71 (br.s, 2H, H-2, 6 piperidine), 2.51 (m, 2H, H-2', 6' piperidine), 2.29 (s, 3H, CH3), 2.08 (d, J = 7.3 Hz, 2H, H-3, 5 piperi-dine), 1.92 (m, 2H, H-3', 5' piperidine); 13C NMR (DMSO-d6) S 33.26, 45.79, 50.84, 76.54, 113.61, 116.77, 119.58, 127.28, 128.59, 129.03, 130.66, 133.36, 135.37, 144.57, 147.12, 152.35, 154.20, 156.50, 161.11. LC-MS: m/z = 360 [M+1]. Anal. Calcd. for C21H21N5O: C, 70.17; H, 5.89; N, 19.48. Found: C, 70.21; H, 5.93; N, 19.53. 1-Methyl-3'-(4-tert-butylphenyl)-spiro[piperidine-4,6'-[1,2,4]triazino[2,3-c]quinazolin]-2'(7'H)-one (2.3). Yield: 3.30 g (79.4%); pale yellow crystals; m.p. 255-257 °C; 1H NMR (DMSO-d6) S 8.13 (d, 2H, H-2, 6 Ph), 8.01 (d, 1H, H-11), 7.53-7.356 (m, 3H, H-9, H-3, 5 Ph), 7.30 (s, 1H, NH), 7.08 (d, 1H, H-8), 6.89 (t, 1H, H-10), 2.85-2.68 (m, 2H, H-2, 6 piperidine), 2.61-2.42 (m, 2H, H-2', 6' piperidine), 2.33 (s, 3H, -CH3), 2.21-2.02 (m, 2H, H-3, 5 piperidine), 2.00-1.81 (m, 2H, H-3', 5' piperidine), 1.37 (s, 9H, C(CH3)3); LC-MS: m/z = 416 [M+1]. Anal. Calcd. for C25H29N5O: C, 72.26; H, 7.03; N, 16.85. Found: C, 72.30; H, 7.11; N, 16.89. 3'-(4-Fluorophenyl)-1-methylspiro[piperidine-4,6'-[1,2, 4]triazino[2,3-c]quinazolin]-2'(7'H)-one (2.4). Yield: 3.3 g (88.3%); pale yellow crystals; m.p. 260-262 °C; 1H NMR (DMSO-d6) S 8.32 (t, J = 5.8 Hz, 2H, H-2, 6 Ph), 8.03 (d, J = 7.7 Hz, 1H, H-11), 7.42 (t, J = 7.4 Hz, 1H, H-9), 7.29-7.16 (m, 3H, NH, H-3, 5 Ph), 7.09 (d, J = 8.0 Hz, 1H, H-8), 6.90 (t, J = 7.3 Hz, 1H, H-10), 2.74 (m, 2H, H-2, 6 piperidine), 2.55 (m, 2H, H-2', 6' piperidine), 2.32 (s, 3H, -CH3), 2.09 (m, 1H, H-3, 5 piperidine), 1.92 (m, 2H, H-3', 5' piperidine); 13C NMR (DMSO-d6) S 33.15, 45.70, 50.78, 76.55, 113.57, 115.60 (d, J = 21.5 Hz), 116.78, 119.59, 127.30, 129.80 (d, J = 2.8 Hz), 131.49 (d, J = 8.6 Hz), 135.40, 144.56, 146.06, 152.36, 152.49, 161.10, 163.74 (d, J = 248.3 Hz), 172.54. LC-MS: m/z = 378 [M+1]; Anal. Calcd. for C21H20FN5O: C, 66.83; H, 5.34; N, 18.56; Found: C, 66.89; H, 5.41; N, 18.64. Kolomoets et al.: Design, Synthesis and Anti-inflammatory Activity Acta Chim. Slov. 2017, 64, 902-910 905 3'-(4-Methoxyphenyl)-1-methylspiro[piperidine-4,6'-[1,2,4]triazino[2,3-c]quinazolin]-2'(7'H)-one (2.5). Yield: 3.68 g (94.6%); pale yellow crystals; m.p. 270-272 °C; 'H NMR (DMSO-d6) 5 8.25 (d, J = 8.6 Hz, 2H, H-2, 6 Ph), 8.01 (d, J = 7.4 Hz, 1H, H-11), 7.40 (t, J = 6.9 Hz, 1H, H-9), 7.23 (s, 1H, NH), 7.08 (d, J = 8.0 Hz, 2H, H-8), 6.97 (d, J = 8.7 Hz, 2H, H-3, 5 Ph), 6.89 (t, J = 7.3 Hz, 1H, H-10), 3.86 (s, 3H, CH3), 2.73 (br.s, 2H, H-2, 6 piperidine), 2.51 (brs, 2H, H-2', 6' piperidine), 2.31 (s, 3H, -CH3), 2.09 (m, 1H, H-3, 5 piperidine), 1.92 (brs, 2H, H-3', 5' piperidine); 13C NMR (DMSO-d6) 8 33.24, 45.78, 50.84, 55.76, 76.43, 113.70, 114.06, 114.27, 116.72, 119.53, 125.65, 127.18, 130.69, 131.65, 144.46, 146.56, 151.98, 161.21, 161.42; LC-MS: m/z = 390 [M+1]. Anal. Calcd. for C22H23N5O2: C, 67.85; H, 5.95; N, 17.98. Found: C, 67.91; H, 6.02; N, 18.02. 10'-Bromo-1-methyl-3'-phenylspiro[piperidine-4,6'-[1,2,4]triazino[2,3-c]quinazolin]-2'(7'H)-one (2.6). Yield: 3.8 g (86.6%); pale yellow crystals; m.p. 270-272 °C; 1H NMR (DMSO-d6) 8 8.21 (d, J = 7.8 Hz, 2H, H-2, 6 Ph), 8.11 (s, 1H, H-11), 7.56-7.33 (m, 5H, H-9; NH; H-3, 4, 5 Ph), 7.05 (d, J = 8.4 Hz, 1H, H-8), 2.73 (m, 2H, H-2, 6 piperidine), 2.51 (m, 2H, H-2', 6' piperidine), 2.30 (s, 3H, -CH3), 2.14-1.99 (m, 2H, H-3, 5 piperidine), 1.92 (m, 2H, H-3', 5' piperidine); EI-MS, m/z (Irel, %) = 292 (6.2), 290 (5.9), 278 (7.5), 276 (6.5), 266 (5), 265 (18), 264 (6.3), 263 (19), 223 (12.1), 155 (5.4), 116 (8.9), 115 (5.3), 109 (11.1), 104 (20.3), 103 (71.9), 102 (6.4), 77 (14.8), 76 (22.6), 75 (5.8), 72 (32.2), 71 (32.5), 70 (100), 69 (6.1), 68 (8.1), 63 (12.3), 58 (17), 57 (41.7), 56 (20.8), 54 (10.3), 53 (6.8), 45 (11.2); LC-MS: m/z = 440 [M+2]. Anal. Calcd. for C21H20BrN5O: C, 57.54; H, 4.60; N, 15.98. Found: C, 57.50; H, 4.57; N, 15.93. 3'-Phenyl-2,3,5,6-tetrahydrospiro[thiopyran-4,6'-[1,2,4] triazino[2,3-c]quinazolin]-2'(7'H)-one (2.7). Yield: 3.5 g (89.0%); pale yellow crystals; mp >300 °C; 1H NMR (DM-SO-d6) 8 8.19 (d, J = 7.6 Hz, 1H, H-2, 6 Ph), 8.02 (d, J = 8.0 Hz, 1H, H-11), 7.51-7.43 (m, 3H, H-3, 4, 5 Ph), 7.40 (t, 1H, H-9), 7.26 (s, 1H, NH), 7.10 (d, J = 7.9 Hz, 1H, H-8), 6.89 (t, J = 7.3 Hz, 1H, H-10), 3.25-3.12 (m, 2H, H-2, 6 thiopyran), 2.52 (m, 6H, H-2', 3, 3', 5, 5', 6' thiopyran); 13C NMR (DMSO-^) 8 23.71, 35.25, 77.54, 113.41, 116.63, 119.74, 127.24, 128.61, 129.08, 130.71, 133.32, 135.51, 144.36, 147.29, 152.07, 161.05; LC-MS: m/z = 363[M+1]. Anal. Calcd. for C20H18N4OS: C, 66.28; H, 5.01; N, 15.46. Found: C, 66.31; H, 5.09; N, 15.49. 3'-Phenyl-4,5-dihydro-2H-spiro[thiophene-3,6'-[1,2,4] triazino[2,3-c]quinazolin]-2'(7'H)-one (2.8). Yield: 2.8 g (81.6%); m.p. 263-265 °C; pale yellow crystals; IR: 3299, 3076, 2926, 1714, 1640, 1626, 1615, 1598, 1551, 1503, 1486, 1440, 1411, 1338, 1312, 1271, 1249, 1221, 1191, 1170, 1154, 1107, 1080, 1028, 992, 948, 926, 867, 815, 776, 750, 692 cm-1; 1H NMR (DMSO-d6) 8 8.17 (d, J = 4.9 Hz, 2H, H-2,6 Ph), 8.03 (d, J = 7.6 Hz, 1H, H-11), 7.66 (s, 1H, NH), 7.44- 7.34 (m, 3H, H-9, H-3,5 Ph), 6.95 (d, J = 7.9 Hz, 1H, H-8), 6.88 (t, J = 7.9 Hz, 1H, H-10), 3.51 (d, J = 11.6 Hz, 1H, H-2 thiophene), 3.13 (d, J = 11.2 Hz, 1H, H-2' thiophene), 3.10-2.92 (m, 2H, H-5, 5' thiophene), 2.62-2.41 (m, 2H, H-4, 4' thiophene); 13C NMR (DMSO-d6) 8 27.85, 39.14, 39.41, 87.60, 113.17, 116.05, 119.72, 127.49, 128.61, 129.16, 130.69, 133.29, 135.48, 145.53, 147.02, 152.34, 161.22. LC-MS: m/z = 349 [M+1]. Anal. Calcd. for C,0H,N,OS: C, L J 19 16 4 65.50; H, 4.63; N, 16.08. Found: C, 65.61; H, 4.75; N, 16.12. 3'-Phenyl-5,6-dihydro-2H,4H-spiro[thiopyran-3,6'-[1,2, 4]triazino[2,3-c]qmnazoUn]-2'(7'H)-one (2.9). Yield: 3.3 g (91.8%); pale yellow crystals; m.p. 274-276 °C; IR: 3272, 2891, 1641, 1626, 1609, 1594, 1553, 1515, 1499, 1483, 1442, 1414, 1333, 1312, 1298, 1273, 1249, 1188, 1153, 1103, 1076, 1030, 1012, 980, 950, 923, 872, 859, 825, 814, 787, 774, 749, 694, 670 cm-1; 1H NMR (DMSO-d6) 8 8.18 (d, J = 5.3 Hz, 2H, H-2, 6 Ph), 8.00 (d, J = 7.7 Hz, 1H, H-11), 7.52-7.29 (m, 5H, H-9, H-3, 4, 5 Ph, NH), 7.17 (d, J = 8.0 Hz, 1H, H-8), 6.86 (t, J = 7.2 Hz, 1H, H-10), 3.38 (d, J = 13.4 Hz, 1H, H-2 thiopyran), 2.92 (d, J = 13.2 Hz, 1H, H-2' thiopyran), 2.78-2.59 (m, 1H, H-6 thiopyran), 2.55-2.05 (m, 5H, H-4, 4', 5, 5', 6' thiopyran); 13C NMR (DMSO-d6) 8 24.37, 27.02, 33.55, 34.76, 75.81, 112.99, 116.56, 116.62, 119.51, 127.27, 128.55, 129.20, 130.68, 133.32, 135.46, 144.45, 144.51, 152.17, 161.04. LC-MS: m/z = 363 [M+1]. Anal. Calcd. for C20H18N4OS: C, 66.28; H, 5.01; N, 15.46. Found: C, 66.30; H, 5.03; N, 15.49. 3'-Phenyl-4,5-dihydro-2H-spiro[thiophene-3,6'-[1,2,4] triazino[2,3-c]quinazolin]-2'(7'H)-one 1,1-dioxide (2.10). Yield: 2.1 g (56.1%); pale yellow crystals; m.p. 266-269 °C; 1H NMR (DMSO-d6) 8 8.25 (d, J = 5.3 Hz, 1H, H-2, 6 Ph), 8.04 (d, J = 7.5 Hz, 1H, H-11), 7.97 (s, 1H, NH), 7.49-7.41 (m, 4H, H-9, H-3, 4, 5 Ph), 7.04-6.84 (m, J = 7.7 Hz, 2H, H-8, 10), 4.24 (d, J = 14.9 Hz, 1H, H-2 thiophene), 3.50 (d, J = 15.0 Hz, 1H, H-2' thiophene), 3.47-3.34 (m, 2H, H-5, 5' thiophene), 3.30-3.03 (m, 1H, H-4 thiophene), 2.742.55 (m, 1H, H-4' thiophene); LC-MS: m/z = 381 [M+1]. Anal. Calcd. for C H NO S: C, 59.99; H, 4.24; N, 14.73. 19 16 4 3 Found: C, 60.03; H, 4.27; N, 14.77. 3'-Phenyl-5,6-dihydro-2H,4H-spiro[thiopyran-3,6'-[1,2,4] triazino[2,3-c]quinazolin]-2'(7'H)-one1,1-dioxide (2.11). Yield: 4.5 g (91.8%); pale yellow crystals; m.p. 296-299 °C; 1H NMR (DMSO-d6) 8 8.25 (d, J = 5.1 Hz, 2H, H-2, 6 Ph), 8.05 (d, J = 7.7 Hz, m, H-11), 7.46 (m, 4H, H-9, H-3, 4, 5 Ph), 7.14 (s, 1H, NH), 7.06 (d, J = 8.0 Hz, 1H, H-8), 6.96 (t, J = 7.3 Hz, 1H, H-10), 3.97 (d, J = 13.9 Hz, 1H, H-2 thiopyran), 3.53 (d, J = 15.4 Hz, 1H,H-2' thiopyran), 3.50-3.35 (m, 1H, H-6 thiopyran), 3.23-2.91 (m, 1H, H-6' thiopyran), 2.90-2.63 (m, 1H, H-4 thiopyran), 2.34 (m, 3H, H-4', 5, 5' thiopyran); 13C NMR (DMSO-d6) 8 18.09, 31.64, 50.06, 54.57, 77.78, 113.38, 116.95, 120.21, 127.32, 128.50, 129.38, 130.80, 133.18, 135.61, 143.78, 147.26, 151.84, 161.03; LC-MS: m/z = 395 [M+1]. Anal. Calcd. for Kolomoets et al.: Design, Synthesis and Anti-inflammatory Activity 906 Acta Chim. Slov. 2017, 64, 902-910 C20H18N4O3S: C, 60.90; H, 4.60; N, 14.20. Found: C, 60.93; H, 4.64; N, 14.26. 2. Results and Discussion Considering the roles of cyclooxygenases (COX-1 and COX-2) as important pharmacological targets and their inhibitors are the basis for the developing of antiinflammatory drugs, in the first phase of the study the virtual base (80 compounds) of 3'-R-10'-R1-spiro [het-aryl-3(4),6'-[1,2,4]triazino[2,3-c]quinazolin]-2'(7'H)-ones was analyzed using molecular docking. Virtual structural modification of studied compounds, which have heterocyclic fragments at spiro position (position 6) was held by the 3d position and at positions 8, 9, 10 and 11 of the aromatic ring. Complexes of COX-1 and COX-2 were downloaded from the Protein Data Bank, to determine the affinity (Table 1), http://www.rcsb.org/pdb/home/home. do. As a reference known selective COX-1 and COX-2 inhibitors were used, such as diclofenac sodium and celecox-ib. Results of the studies showed that the structures analyzed have a higher affinity to COX-2 and much lower to COX-1. Among 80 tested compound, 7 with the highest affinity, according to the docking, are presented in the Table 1. Compound 2.10 revealed the highest affinity, yet still lower than celecoxib. The visualization of the interaction of the structures with the active site of COX-2 (Fig. 1) showed, that compound 2.10 revealed the highest affinity (11.5 kcal/mol). There were four hydrogen bonds with the following amino acid residues D:TYR341 (3.08 A), D:HIS75 (3.37 A), D:GLY512 (3.33 Â), D:SER516 (3.63 Â), and besides n-cat-ion electrostatic interaction with D:ARG106 (4.86 Â), n-sigma hydrophobic interactions with D:VAL335 (3.76 Â), D:VAL509 (3.40 Â, 3.65 Â), D:ALA513 (3.82 Â), n-sul-fur interaction with D:TRP373 (5.93 Â), n-n stacked hydrophobic interaction with D:HIS75 (5.26 Â) and n-alkyl hydrophobic interactions with D:LEU517 (5.14 Â), D:ARG499 (5.27 Â), D:ALA502 (4.31 Â). Analyzing the complex of celecoxib and compound 2.10 with COX-2, similar interactions can be traced, namely with such ami-no acids D:ARG106, D:ARG499, D:VAL335, D:VAL509, D:LEU517, D:TRP373, D:ALA513. This may indicate that stated class of compound might have the ability to inhibit the COX-2 as celecoxib does. The second stage, namely the synthesis of the corresponding spiro-derivatives 2 provided the interaction of substituted 6-R1-3-(2-aminophenyl)-1,2,4-triazine-5(2H)-ones 1 and the corresponding heterocyclic ketones (Scheme 1). The heterocyclization was carried out by known method,20 namely the refluxing in glacial acid. Synthesized spiro-derivatives 2 are pale yellow crystalline substances, soluble in DMF, slightly soluble in diox-ane, insoluble in alcohols and water. The compounds' structures were established with elemental analysis, LC-MS-data, IR and NMR spectra. Thus, triazino[2,3-c]quinazoline fragment of compounds 2 in 1H NMR spectra has the appropriate chemical shifts and multiplicity: 8.11-8.00 ppm (H-11, d), 7.42-7.40 ppm (H-9, t), 7.17-6.95 ppm (H-8, d), and 6.96-6.86 ppm (H-10, t). It is important to note, that H-9 in most cases resonated together with the aromatic protons of substituent at the position 3 and NH proton of position 7 as multiplets. Whereas, protons H-11, H-8 and H-10 have Table 1. Results of molecular docking of the most active structures Affinity Affinity Comp. (kcal/mol) to COX-1 (kcal/mol) to COX-2 Types of interactions with amino acid residues of COX-2 2.1 -7.4 -10.1 D:ARG106i>, D:VAL509c, D:ALA513c, D:HIS75c, D:VAL335c, D:LEU517c, D:ARG499c, D:ALA502c. 2.2 -3.6 -10.3 D:ARG106i>, D:VAL509c, D:ALA513c, D:HIS75c, D:VAL335c, D:LEU517c, D:ARG499c, D:ALA502c. 2.7 -5.2 -9.0 D:ARG106i>, D:VAL509c, D:ALA513c, D:TRP373'i, D:HIS75c, D:LEU338c, D:PHE504c, D:VAL335c, D:LEU517c, D:ALA502c. 2.8 -3.3 -10.4 D:HIS75", D:VAL335c, D:VAL509c, D:ALA513c, D:TRP373'i, D:HIS75c, D:LEU338c, D:PHE504c, D:LEU517c, D:ARG499c, D:ALA502c. 2.9 -1.3 -10.6 D:TYR341", D:HIS75", D:VAL509c, D:ALA513c, D:SER339c, D:VAL335c, D:LEU338c, D:LEU517c, D:ALA502c. 2.10 -1.4 -11.5 D:TYR341", D:HIS75", D:GLY512", D:SER516", D:ARG1066, D:VAL335c, D:VAL509C, D:ALA513C, D:TRP373d, D:HIS75C, D:LEU517C, D:ARG499C, D:ALA502C. 2.11 -0.4 -10.9 D:TYR341", D:HIS75", D:SER516C, D:VAL509C, D:ALA513C, D:LEU338C, D:SER339C, D:VAL335C, D:LEU517C, D:ALA502C. Celecoxib -12.1 D:ARG106", D:ARG499", D:GLN178", D:LEU338", D:SER339", D:VAL335C, D:CEL682C, D:VAL509c, D:LEU370c, D:VAL335c, D:LEU345c, D:LEU517c, D:TYR371c, D:TRP373c, D:ALA513c. a - hydrogen, b - electrostatic, c - hydrophobic, d - others. Kolomoets et al.: Design, Synthesis and Anti-inflammatory Activity Acta Chim. Slov. 2017, 64, 902-910 907 a) b) c) ^I d) Figure 1. Interaction of compound 2.2 (a), 2.10 (b), 2.11 (c), cel-ecoxib (d) with COX-2, PDB ID: 3LN1 classical multiplicity and /-coupling (/ = 8.05-7.3 Hz).21 Phenyl moiety in position 3 of 1H NMR spectrum resonated as two proton singlets, H-2,6 at 8.25-8.17 ppm and three proton multiplets of H-3,4,5 at 7.56-7.35 ppm. Signals of p-substituted phenyl derivatives 2 were registered as A2B2-systems, as two doublets H-2,6 and H-3,5. The characteristic singlets of NH protons (position 7) in 1H NMR spectra of compounds 2 were observed at 7.97-7.14 ppm and their chemical shift depended on the electronic effects of spiro moiety at position 6. Thus, the signal of NH proton of compound 2.1 with spiropyran cycle were detected at 7.30 ppm, with spiropiperidine (2.22.6) at 7.33-7.23 ppm, with spirothiopyran (2.7, 2.9) at 7.29-7.26 ppm and with spirothiophene (2.8) cycle at 7.66 ppm. In 1H NMR spectra of compound 2.11 (oxidized analogue of compound 2.9) signal of NH proton were shifted to a higher field (7.14 ppm). Whereas, the stated proton of compound 2.10 resonated at a lower field, namely at 7.97 ppm, compared to compound 2.8, what is likely due to the formation of hydrogen bond between nearby structural fragments. In the 1H NMR spectra of compounds 2.1-2.11 proton signals of position 6 of spiro moiety were recorded as a complex set of multiplets in aliphatic part of spectrum due to the presence of magnetic nonequivalent axial and equatorial protons.21 Whereas, 1H NMR spectra of compounds 2.8, 2.9 have characteristic features, which were associated with the presence in the molecule of a center of asymmetry. Thus, in the spectra of mentioned compounds protons of methylene group of thiophene at position 2 or thiopy-ran were registered as doublets at 3.13 ppm H (/ = 11.2 Hz), 2.92 ppm (/ = 13.2 Hz) and H at 3.51 pprn (/ = 11.6 Hz), 3.38 ppm (/ = 13.4 Hz), respectively. A similar pattern of signals was characteristic for their dioxo analogues (compounds 2.10, 2.11). In the 13C NMR spectra of compounds 2 characteristic signal of ^-hybridized C in spiro position (position 6) was observed at 87.60-75.81 ppm, and its chemical shift was determined by electron withdrawing effect of the spiro cycle. In addition, further structure confirmation was made by the corresponding 13C signals of hydrogenated pyran cycle (compound 2.1) at 34.19 and 63.26 ppm, N-meth-ylpiperidine cycle (2.2, 2.4, 2.5) at 33.26-33.15 ppm, 45.7945.70 ppm and 50.84-50.78 ppm and hydrogenated thiopyrane cycle (2.7) at 23.71 ppm and 35.28 ppm at 4,6'-spiro position. While other electronic environments of carbon atoms of hydrogenated thiophene (2.8) and thiopy-ran (2.9) cycle at 3,6'-spiro position caused a series of four signals at a high field. A similar pattern of signals was characteristic for dioxo analogue compound 2.11. Mass spectrometric investigation showed, that compound 2.6 was characterized by a low-intensity molecular ion, that formed high-intensity fragmentation ions F1 [C6H5CH=N]+ (m/z 103, 71.9%), F2 [CH2N(Me) CH2=CH2]+ (m/z 70, 100%) and F3 [C10H6BrN3O]+ (m/z 265/263, 18.0/19.0%). The last fraagmented ion described Kolomoets et al.: Design, Synthesis and Anti-inflammatory Activity 908 Acta Chim. Slov. 2017, 64, 902-910 Scheme 1. The heterocyclization of 6-R1-3-(2-aminophenyl)-l,2J4-triazine-5(2fl)-ones the presence of bromine in the molecule and its isotopic profile. Further carbon-carbon bond breaking and hydrogen rearrangements in F2 created intense ions [CH2NHCH2=CH2]+ (m/z 57, 41.7%) and [CH2NCH3] + (m/z 45, 11.2%), indicating the presence ofN-methylpiper-idine fragment in the molecule.22 3. Pharmacology Formalin acute inflammation is characterized by a powerful inflammatory response, which in 3 h of the ex- periment can be verified by significant swelling of the paw in the control group of animals (average increase in volume of the paw is 47.38%). Administration of compounds 2 to the animals with experimental pathology led to a decrease of exudative reactions and most of compounds exhibited anti-inflammatory action comparable (compounds 2.2, 2.6, 2.7, 2.10) or higher (2.3-2.5, 2.8, 2.11) than the effect of the reference diclofenac sodium (Table 2). SAR-analysis (influence of substituents at positions 3 and 6) showed, that 3'-phenyl-2,3,5,6-tetrahydrospiro[pyran-4,6'-[1,2,4]triazino[2,3-c]quinazolin]-2'(7'H)-one (2.1) showed moderate anti-inflammatory effect (higher in the Table 2. Anti-inflammatory activity of synthesized compounds under formalin induced inflammation model Comp. Dosage, mg/kg Increase of the paw volume in 3 h, % Anti-inflammatory activity, % Experimental pathology - 47.38 0 2.1 10.0 24.22 39.39 2.2 10.0 26.28 44.44 2.3 10.0 15.43 69.19 2.4 10.0 20.47 56.57 2.5 10.0 6.74 85.86 2.6 10.0 24.75 48.48 2.7 10.0 22.60 44.44 2.8 10.0 21.61 55.56 2.9 10.0 33.45 29.29 2.10 10.0 23.91 49.49 2.11 10.0 20.04 60.61 Diclofenac sodium 8.0 26.58 45.45 Kolomoets et al.: Design, Synthesis and Anti-inflammatory Activity Acta Chim. Slov. 2017, 64, 902-910 909 control group to 39.4%). Replacing the hydrogenated spiropyran (2.1) by 1-methyl-piperidine (2.2) fragment at the spiro position 6 increased activity to 44.44% compared with the control. The intensity of anti-inflammatory activity of compound 2.2 is comparable with diclofenac sodium. The position of sulfur in spirocycle of the isomer hydrogenated spirothiopyranes (2.7, 2.9) determined the anti-inflammatory activity. Thus, the compound 2.7 with 4,6'-spiro position of thiopyran towards to triazinoquina-zoline cycle exhibited anti-inflammatory activity at the level of diclofenac sodium. Relocation of sulfur in the thiopyran cycle (3,6'-spiro position, compound 2.9) led to a significant decrease of activity, whereas contraction of thiopyran cycle by a homologous unit (2.8) in contrast increased activity to 11.11% (compared with diclofenac). The structural similarity of compounds 2.10 and 2.11 to the celecoxib-like drugs, as we consider led to high antiinflammatory activity. Modification of position 3 substituent of triazino-quinazoline cycle via replacing of phenyl substituent by the 4-tert-butylphenyl (2.3), 4-fluorophenyl (2.4) or 4-me-thoxyphenyl (2.5) was substantiated and led to increasing of anti-inflammatory activity at 11.12-40.21%, compared with the reference drug (Tab. 2). While, the introduction of additional bromine (2.6) at the 10th position of compound 2.2 led to a loss of activity compared to the compounds 2.3-2.5. Such, this way of modification of the molecule was not promising. 4. Conclusions Based on the methodology of purposeful search of NSAIDs an effective method for the synthesis of 6-spiro-fused 10-R-3-aryl-6,7-dihydro-2H-[1,2,4]triazino[2,3-c] quinazolin-2-ones (a promising class of anti-inflammatory agents) was proposed. Structural features of the synthesized compounds, as well as their 1H, 13C NMR spectrosco-py and mass spectrometry data were discussed. It was established that compounds 2.3-2.5, 2.8 and 2.10 under formaline-induced paw edema model revealed the activity higher comparing to the reference drug - diclofenac sodium. SAR analysis showed that combination of tri-azine[2,3-c]quinazoline cycle with other heterocyclic fragments is reasonable in scope of novel NSAIDs creation and calls for further research. 5. References 1. J. I. Levin, S. Laufer, Anti-Inflammatory drug discovery. Royal Society of Chemistry Series No. 26, Cambridge, 2012, 528 p. DOI: 10.1039/9781849735346. 2. G. A. FitzGerald, C. Patrono, N. Engl. J. Med. 2001, 345(6), 433-442. D01:10.1056/NEJM200108093450607. 3. R. G. Kulkarni, G. Achaiah, G. N. R. Sastry, Curr. Pharm. Des. 2006, 12, 2437-2454. D01:10.2174/138161206777698945. 4. B. Botz, K. Bölcskei, Z. Helyes, Challenges to develop novel anti-inflammatory and analgesic drugs, Wiley Interdiscip. Rev.: Nanomed. Nanobiotechnol. 2017, 9(3), 1427. DOI: 10.1002/wnan.1427. 5. S. K. Kulkarni, V. P. Singh, Curr. Top. Med. Chem. 2007, 7, 251-263. DOI: 10.2174/156802607779941305. 6. R. Paprocka, M. Wiese, A. Eljaszewicz, A. Helmin-Basa, A. Gzella, B. Modzelewska-Banachiewicz, J. Michalkiewicz, Bioorg. Med. Chem. Lett. 2015, 25, 2664-2667. D0I:10.1016/j.bmcl.2015.04.079. 7. A. Puratchikody, M. Doble, Bioorg. Med. Chem.2007, 15, 1083-1090. D0I:10.1016/j.bmc.2006.10.025. 8. C. D. Barros, A. A. Amato, T. B. de Oliveira, K. B. R. Iannini, A. L. da Silva, T. G. da Silva, E. S. Leite, M. Z. Hernandes, M. do C. A. de Lima, S. L. Galdino, F. de A. R. Neves, I. da R. Pitta, Bioorg. Med. Chem.2010,18, 3805-3811. D0I:https://doi.org/10.1016/j.bmc.2010.04.045. 9. Y.-L. Chen, Y.-L. Zhao, C.-M. Lu, C.-C. Tzeng, J.-P. Wang, Bioorg. Med. Chem.2006,14, 4373-4378. D0I:10.1016/j.bmc.2006.02.039. 10. X. Wen, S.-B. Wang, D.-C. Liu, G.-H. Gong, Z.-S. Quan, Med. Chem. Res. 2015, 24, 2591-2603. D0I:10.1007/s00044-015-1323-y. 11. M. A. Hussein, Med. Chem. Res. 2013, 22, 4641-4653. D0I:10.1007/s00044-013-0468-9. 12. T. Yu. Sergeieva, O. Yu. Voskoboynik, S. I. Okovytyy, S. I. Kovalenko, S. V. Shishkina, O. V. Shishkin, J. Leszczynski, J. Phys. Chem. A 2014, 118, 1895-1905. D0I:10.1021/jp4052616. 13. Protein Data Bank, pdb. Retrieved from http://www.pdb.org 14. MarvinSketch version: 6.3.0, 2015, ChemAxon (http://www. chemaxon.com). 15. O. Trott, A. J. Olson, J. Comput. Chem. 2010, 31, 455-461. D0I:10.1002/jcc.21334. 16. European convention for the protection of vertebrate animal used for experimental and other scientific purposes. Council of Europe, Strasbourg, 1986. 17. J. D. J. Liégeois, Naunyn-Schmiedeberg's Arch. Pharmacol. 1999, 359, 220-227. D0I:10.1007/PL00005345. 18. N. Shejawal, S. Menon, S. Shailajan, Hum. Exp. Toxicol. 2014, 33, 123-129. D01:10.1177/0960327113482594. 19. M. Pagano, K. Gauvreau, Principles of Biostatistics, Kimber-lee Gauvreau Statistics Series, Duxbury Press, 1994, 524 p. 20. S. V. Kholodnyak, K. P. Schabelnyk, O. Yu. Voskoboynik, O. M. Antypenko, S. I. Kovalenko, V. O. Palchykov, S. I. Okovyty, S. V. Shishkina, J. Org. Pharm. Chem. 2016, 14,3(55), 24-31. 21. E. Breitmaier. Structure Elucidation by NMR in Organic Chemistry: A Practical Guide, Third Edition, 2002, Wiley, 270 p. 22. C. Dass. Fundamentals of contemporary mass spectrometry, 2007, John Wiley & Sons, Inc., 608 p. Kolomoets et al.: Design, Synthesis and Anti-inflammatory Activity 910 Acta Chim. Slov. 2017, 64, 902-910 Povzetek Naše raziskave smo usmerili v načrtno iskanje novih, obetavnih antiinflamatornih spojin, zlasti med relativno neznanimi 3'-R-10'-R1-spiro[heteroarn-3(4),6'-[1,2,4]triazino[2,3-c]kinazolin]-2'(7'H)-oni. Pripravili smo virtualno kombi-natorialno knjižnico doslej neznanih spiro-pripojenih derivatov [1,2,4]triazino[2,3-c]kinazolinov ter s pomočjo metod molekulskega sidranja identificirali najbolj obetavne inhibitorje COX-2. Nato smo te potencialne spojine, ki bi lahko imele protivnetni učinek, sintetizirali s pomočjo [5+1] ciklokondenzacij substituiranih 3-(2-aminofenil)-6-R-1,2,4-tri-azin-5(2H)-onov s heterocikličnimi ketoni. Strukture pripravljenih spojin smo določili na osnovi fizikalno-kemijskih metod in spektroskopskih značilnosti. Za dobljene spojine smo določili antiinflamatorno aktivnost z uporabo modela s formalinom induciranega edema šape testnih živali. Na ta način smo identificirali najbolj aktivne spojine. Izvedena analiza primerjave aktivnosti od strukture spojin (SAR analiza) je pokazala, da je kombinacija triazino[2,3-c]kinazolinskega in spiro-pripojenega fragmetna smiselen pristop k pripravi še novih antiinflamatornih zdravil. Kolomoets et al.: Design, Synthesis and Anti-inflammatory Activity DOI: 10.17344/acsi.2017.3609 Acta Chim. Slov. 2017, 64, 911-918 ¿^creative tS1 commons Scientific paper Green One-pot Synthesis of Novel Polysubstituted Pyrazole Derivatives as Potential Antimicrobial Agents Hamid Beyzaei,1* Zahra Motraghi,1 Reza Aryan,1 Mohammad Mehdi Zahedi2 and Alireza Samzadeh-Kermani1 1 Department of Chemistry, Faculty of Science, University of Zabol, Zabol, Iran 2 Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, SK S7N 5C9, Canada * Corresponding author: E-mail: hbeyzaei@yahoo.com and hbeyzaei@uoz.ac.ir Tel: +98 5431232186 / Fax: +98 5431232180 Received: 05-06-2017 Abstract Various biological properties of natural and synthetic pyrazole derivatives such as anti-inflammatory, antimicrobial, neuroprotective, anticonvulsant, antidepressant and anticancer activities encouraged us to propose a new, fast, green and eco-friendly procedure for the preparation of some novel 5-amino-3-(aryl substituted)-1-(2,4-dinitrophenyl)-1H-pyra-zole-4-carbonitriles. They were efficiently synthesized via one-pot two-step process reaction of malononitrile, 2,4-dini-trophenylhydrazine and different benzaldehydes in deep eutectic solvent (DES) glycerol/potassium carbonate. The products yield and reaction times were considerably improved in the presence of applied DES. Antibacterial effects of all newly synthesized pyrazoles in comparison with several common antibiotics were evaluated against a variety of Gram-positive and Gram-negative pathogenic bacteria. In addition to, their inhibitory activities on three fungi were compared to some current antifungal agents. The moderate to good antimicrobial potentials particularly against fungi were observed in the major heterocyclic compounds according to the IZD, MIC, MBC and MFC results. Keywords: Green synthesis; deep eutectic solvent; glycerol/potassium carbonate; polysubstituted pyrazoles; antibacterial and antifungal activities 1. Introduction Pyrazoles are an important class of azoles containing two adjacent nitrogen atoms, which are found as major or minor scaffolds in various medicinal compounds and natural products. L-a-Amino-8-(pyrazolyl-N)-propanoic acid and withasomnine, which were isolated from Citrul-lus vulgaris (watermelon) juice and from the roots of With-ania somnifera Dun (Solanaceae), in fact, are two of the few naturally occurring pyrazoles that have found potential use as anti-diabetic and depressant agents in medicinal chemistry.1,2 Pyrazofurin and formycin are natural C-nu-cleoside antibiotics that are used to treat viral infections as well as inhibition of tumor cells growth. Stanozolol is a synthetic anabolic steroid that can be applied for treatment of anaemia and hereditary angioedema. In addition to, the pyrazole ring as a part of the chemical structure of drugs such as antipyrine, celecoxib and betazole, plays an essential role in the relief of ear pain and swelling, improvement of osteoarthritis signs, and treatment of bacterial and fungal infections (Figure 1). Compounds containing pyrazole moiety exhibit a wide variety of biological and pharmacological activities including analgesic, neuroprotective, anticonvulsant, an-giotensin converting enzyme (ACE) inhibitory, anti-an-giogenesis, antioxidant and antiviral activities.3-9 Numerous studies have also focused on antibacterial and antifungal properties of pyrazole derivatives.10-12 In a research project, inhibitory activities of some heterocyclic Schiff bases derived from thiocarbohydrazide were assessed against various pathogenic bacterial and fungal strains via measurement of their inhibition zone diameters. One of the synthesized 1,2,4-triazines could block the growth of all selected microorganism.13 Various methods were proposed for the synthesis of pyrazole and their analogues.14-17 In this regard, a solution of the appropriate triethylamine in 1,4-dioxane efficiently catalysed synthesis of pyridine, thiophene and 4H-pyrane Beyzaei et al.: Green One-pot Synthesis of Novel Polysubstituted 912 Acta Chim. Slov. 2017, 64, 911-918 Figure 1. Natural products and drugs containing pyrazole moiety. derivatives via one-pot or multicomponent protocols.18 Similar procedures were designed to prepare pyrazole derivatives.19-24 Most of these methods include simultaneous or multistep reaction of aldehyde, hydrazine and active methylene compounds under different conditions.25-29 Recently, deep eutectic solvents (DESs) were widely applied as eco-friendly media or efficient catalysts in organic synthesis especially for the preparation of pyrazoles.30-32 Glyc-erol/potassium carbonate is a new class of DES having its physical properties, such as surface tension, viscosity, density and refractive index, carefully measured.33 In order to apply glycerol/K2CO3 system in organic synthesis, some novel 5- amino -1 - (2,4- dinitrophenyl) -1 H-pyrazole-4-car-bonitrile derivatives were prepared via the reaction of malononitrile, 2,4-dinitrophenylhydrazine and various benzaldehydes. The in vitro antimicrobial activities of synthesized derivatives were studied against a variety of pathogenic bacteria and fungi, as well as structure-activity relationships were expanded. 2. Experimental 2. 1. Chemicals All reagents, solvents, antibiotics and antifungal agents were purchased from commercial sources (Merck, Sigma and Aldrich), and used without further purification. The bacterial and fungal culture media were obtained from HiMedia. Melting points were determined with Kru-ss type KSP1N melting point meter and are uncorrected. Reaction progress was monitored by aluminium TLC plates pre-coated by silica gel with fluorescent indicator F254 using CH2Cl2/CH3OH (9:1, v/v) as the mobile phase, being visualized under UV radiation (254 nm). FT-IR spectra of the products were collected using Bruker Ten-sor-27 FT-IR spectrometer. 1H and 13C NMR spectra were recorded at 400 and 100 MHz, respectively, on a Bruker FT-NMR Ultra Shield-400 spectrometer. Elemental analyses were performed for C, H and N on a Thermo Finnigan Flash EA microanalyzer. DESs were prepared in various ratios of glycerol/K2CO3 according to the procedures reported by Naser et a/.33 as follows: the mixture of different molar ratios of potassium carbonate and glycerol were vigorously stirred at 80 oC for 2 h to gain homogenous transparent colorless liquids. 2. 1. 1. General Procedure for the Synthesis of Pyrazoles 4a-f A mixture of K2CO3 (0.140 g, 0.001 mol) and glycerol (0.360 g, 0.004 mol) was stirred at 80 oC for 2 h to form a homogenous colorless liquid as DES1. Under the same conditions, the distilled water (0.25 mL), malononitrile (1) (0.660 g, 0.001 mol) and benzaldehydes 2a-f (2a: 0.163 g, 2b: 0.136 g, 2c: 0.151 g, 2d: 0.152 g, 2e: 0.175 g, 2f: 0.175 g; 0.001 mol) were respectively added to it. The intermediate benzylidene malononitriles 6a-f were produced in 2 min. 2,4-Dinitrophenylhydrazine (3) (0.198 g, 0.001 mol) was added to the mixture. The reaction continued for another 18-28 min. The reaction mixture was cooled to room temperature, and neutralized with glacial acetic acid (0.120 g, 0.002 mol). After adding 1 mL of ethanol, the mixture was poured into ice-cold saturated aqueous NaCl (5 mL). The resulting precipitates were collected by filtration, washed respectively with distilled water (5 mL) and ethanol (5 mL), and recrystallized from methanol to afford pure pyr-azoles 4a-f as colored crystals. 2. 1. 1. 1. AT-(4-(5-Amino-4-cyano-1-(2,4-dinitrophenyl)-1 H-pyrazol-3-yl)phenyl)acetamide (4a). Orange crystals; yield: 0.37 g (91%); m.p. 274-275 °C; IR (KBr) v 3444, 3281 (NH2, NH), 2231 (C=N), 1540, 1326 (NO2) cm-1; 1H NMR (400 MHz, DMSO-dfi) 5 2.06 (s, 3H, CH3), 7.75 (d, J = 8.7 Hz, 2H, H-3" and H-5"), 7.87 (d, J = 8.7 Hz, 2H, H-2" and H-6"), 7.98 (d, J = 8.5 Hz, 1H, H-6'), 8.30 (d, J = 8.5 Hz, 1H, H-5'), 8.55 (s, 2H, NH2), 8.79 (s, 1H, H-3'), 10.48 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) 5 24.67 (CH3), 78.00 (C-4), 114.21 (C=N), Beyzaei et al.: Green One-pot Synthesis of Novel Polysubstituted Acta Chim. Slov. 2017, 64, 911-918 913 119.20 (C-3" and C-5"), 123.43 (C-6'), 126.15 (C-1"), 128.60 (c-5'), 130.08 (C-3'), 132.78 (C-2'' and C-6''), 137.17 (C-1'), 141.91 (C-2'), 144.82 (C-4'), 145.38 (C-4''), 149.71 (C-3), 160.67 (C-5), 169.77 (C=O). Anal. Calcd. for C18H13N7O5: C, 53.07; H, 3.22; N, 24.07. Found: C, 53.01; H, 3.18; N, 24.12. 2. 1. 1. 2. 5-Amino-1-(2,4-dinitrophenyl)-3-(4- methoxyphenyl)-1H-pyrazole-4-carbonitrile (4b). Orange crystals; yield: 0.32 g (85%); m.p. 217-219 °C; IR (KBr) v 3456, 3326 (NH2), 2224 (C=N), 1538, 1319 (NO2) cm-1; 1H NMR (400 MHz, DMSO-dfi) 5 3.88 (s, 3H, CH3), 7.03 (d, J = 8.7 Hz, 2H, H-3'' and H-5''), 7.71 (d, J = 8.7 Hz, 2H, H-2'' and H-6''), 8.01 (d, J = 8.4 Hz, 1H, H-6'), 8.38 (d, J = 8.4 Hz, 1H, H-5'), 8.62 (s, 2H, NH2), 8.82 (s, IH, H-3'); 13C NMR (100 MHz, DMSO-dfi) 5 56.39 (CH3), 77.29 (C-4), 114.40 (C=N), 115.67 (C-3'' an d C-5''), 123.54 (C-6'), 124.59 (C-1''), 127.97 (C-5'), 130.04 (C-3'), 133.84 (c-2'' and C-6''), 137.13 (C-1'), 141.07 (C-2'), 144.93 (c-4'), 149.62 (C-3), 160.91 (C-5), 164.82 (C-4''). Anal. Calcd. for C17H12N6O5: C, 53.69; H, 3.18; N, 22.10. Found: C, 53.64; H, 3.17; N, 22.10. 2. 1. 1. 3. 5-Amino-1-(2,4-dinitrophenyl)-3-(4- nitrophenyl)-1H-pyrazole-4-carbomtrile (4c). Yellow crystals; yield: 0.35 g (89%); m.p. 295-296 °C; IR (KBr) v 3445, 3325 (NH2), 2228 (C=N), 1543, 1318 (NO2) cm-1; 1H NMR (400 MHz, DMSO-dfi) 5 8.06 (d, J = 9.3 Hz, 1H, H-6'), 8.15 (d, J = 7.9 Hz, 2H, H-2'' and H-6''), 8.24 (d, J = 7.9 Hz, 2H, H-3'' and H-5''), 8.38 (d, J = 9.3 Hz, 1H, H-5'), 8.48 (s, 2H, NH2), 8.81 (s, 1H, H-3'); 13C NMR (100 MHz, DMSO-dfi) 5 89.02 (C-4), 117.37 (C=N), 121.69 (C-2'' and C-6"), 123.34 (C-6'), 125.10 (c-3'' and C-5''), 130.35 (C-5'), 131.62 (C-3'), 136.32 (C-1'), 138.05 (C-1''), 142.82 (C-2'), 144.70 (C-4'), 147.29 (c-4''), 148.80 (C-3), 159.75 (C-5). Anal. Calcd. for CH.NO: C, 48.62; H, 2.30; N, 24.80. Found: C, 48.68; 16 9 / 6 H, 2.25; N, 24.84. 2. 1. 1. 4. 5-Amino-1-(2,4-dinitrophenyl)-3-(2-hydroxy-3-methoxyphenyl)-1H-pyrazole-4-carbonitrile (4d). Brown crystals; yield: 0.33 g (84%); m.p. 198-199 °C; IR (KBr) v 3537 (OH), 3428, 3287 (NH2), 2206 (C=N), 1517, 1331 (NO2) cm-1; 1H NMR (400 MHz, DMSO-dfi) 5 3.77 (s, 3H, CH3), 6.76 (m, 1H, H-4''), 6.93 (m, 1H, H-5''), 7.31 (m, 1H, H-6''), 7.91 (d, J = 8.1 Hz, 1H, H-6'), 8.24 (d, J = 8.1 Hz, 1H, H-5'), 8.87 (s, 1H, H-3'), 9.43 (s, 2H, NH2), II.63 (s, 1H, OH); 13C NMR (100 MHz, DMSO-dfi) 5 56.23 (CH3), 84.75 (C-4), 113.67 (C=N), 116.95 (C-4''), 118.12 (C-1''), 119.58 (C-5''), 120.71 (C-6''), 123.31 (C-6'), 129.44 (c-5'), 129.99 (C-3'), 137.05 (C-1'), 140.90 (C-2'), 142.15 (c-2''), 144.64 (C-4'), 146.43 (C-3), 148.43 (C-3''), 162.71 (C-5). Anal. Calcd. for C17H12N6O6: C, 51.52; H, 3.05; N, 21.21. Found: C, 51.45; H, 3.11; N, 21.18. 2. 1. 1. 5. 5-Ammo-3-(2,4-dichlorophenyl)-1-(2,4- dinitrophenyl)-1.H-pyrazole-4-carbomtrile (4e). Yellow crystals; yield: 0.36 g (86%); m.p. 184-186 °C; IR (KBr) v 3443, 3287 (NH2), 2227 (C=N), 1514, 1330 (NO2) cm-1; 1H NMR (400 MHz, DMSO-d6) 5 7.46 (d, J = 5.8 Hz, 1H, H-5"), 7.66 (s, 1H, H-3"), 7.86 (d, J = 5.8 Hz, 1H, H-6"), 8.01 (d, J = 8.0 Hz, 1H, H-6'), 8.29 (d, J = 8.0 Hz, 1H, H-5'), 8.77 (s, 1H, H-3'), 8.98 (s, 2H, NH2); 13C NMR (100 MHz, DMSO-d6) 5 87.52 (C-4), 113.76 (C=N), 123.21 (C-6'), 128.39 (C-5'), 128.77 (C-5''), 129.04 (c-1''), 130.06 (C-3'), 130.48 (C-6''), 131.40 (c-3''), 134.55 (c-2''), 135.81 (c-4''), 137.95 (C-1'), 139.16 (C-2'), 144.37 (C-4'), 144.65 (C-3), 157.17 (C-5). Anal. Calcd. for C16H8Cl2N6O4: C, 45.85; H, 1.92; N, 20.05. Found: C, 45.901 H, 1.89; N, 19.98. 2. 1. 1. 6. 5-Ammo-3-(2,6-dichlorophenyl)-1-(2,4- dinitrophenyl)-1H-pyrazole-4-carbonitrile (4f). Orange crystals; yield: 0.37 g (88%); m.p. 256-257 °C; IR (KBr) v 3443, 3287 (NH2), 2227 (C=N), 1514, 1330 (NO2) cm-1; 1H NMR (400 MHz, DMSO-d6) 5 7.44 (d, J = 6.1 Hz, 1H, H-4''), 7.58 (t, J = 6.1 Hz, 2H, H-3''), 7.96 (d, J = 8.4 Hz, 1H, H-6'), 8.40 (d, J = 8.4 Hz, 1H, H-5'), 8.83 (s, 1H, H-3'), 9.01 (s, 2H, NH2); 13C NMR (100 MHz, DM-SO-d6) 5 84.86 (C-4), 117.3(6 (C=N), 123.28 (C-6'), 126.36 (C-1'6), 130.48 (C-5'), 130.06 (C-3'' and C-5''), 130.61 (c-4''), 131.41 (c-3'), 131.64 (c-2'' and C-6''), 138.21 (C-1'), 140.90 (C-2'), 144.35 (C-4'), 144.89 (C-3), 158.66 (C-5). Anal. Calcd. for C16H8Cl2N6O4: C, 45.85; H, 1.92; N, 20.05. Found: C, 45.81; H, 1.87; N, 20.10. 2. 2. In vitro Antimicrobial Assay Gram-negative bacterial strains including Pseudomonas aeruginosa (PTCC 1310), Shigella flexneri (PTCC 1234), Shigella dysenteriae (PTCC 1188), Proteus mirabilis (PTCC 1776), Proteus vulgaris (PTCC 1079), Salmonella enterica subsp. enterica (PTCC 1709) and Salmonella typhi (PTCC 1609); Gram-positive bacterial strains including Streptococcus pyogenes (PTCC 1447), Streptococcus agalac-tiae (PTCC 1768), Streptococcus pneumoniae (PTCC 1240), Staphylococcus epidermidis (PTCC 1435) and Rhodococcus equi (PTCC 1633); and fungi including Aspergillus fumigatus (PTCC 5009), Candida albicans (PTCC 5027) and Fusarium oxysporum (PTCC 5115) were prepared from the Persian Type Culture Collection (PTCC), Tehran, Iran. Initial concentrations of 17.6 (ig/mL of positive controls were prepared in double-distilled water. Accordingly, heterocyclic compounds were dissolved in 10% DMSO to produce final concentrations of 10240 ^g/mL. All the antibiogram assays were repeated at least three times. The results were reported as the mean of three independent experiments. Antibacterial and antifungal activities were determined using both broth microdilution and Beyzaei et al.: Green One-pot Synthesis of Novel Polysubstituted ... 914 Acta Chim. Slov. 2017, 64, 911-918 disk diffusion methods, according to Clinical and Laboratory Standards Institute (CLSI) guidelines M07-A9, M26-A, M02-A11, M44-A and M27-A2 with a slight modification.34,35 3. Results and Discussion 3. 1. Chemistry In this project, 5-amino-1H-pyrazole-4-carboni-triles were synthesized via an efficient, environmentally friendly, cost-effective and fast process. One-pot two-step reaction of malononitrile (1), mono or disubstituted benz-aldehydes 2a-f and 2,4-dinitrophenylhydrazine (3) produced polyfunctional pyrazoles 4a-f in high yields (Scheme 1). The best results were obtained with glycerol/K2CO3 as the reaction media and catalyst. The reaction conditions were optimized in terms of solvent, presence or absence of the catalyst and temperature. 1 mmol each of malononitrile (1), 4-acetamido-benzaldehyde (2a) and 2,4-dinitrophenylhydrazine (3) were reacted under different conditions (Table 1). Glycerol as a green, cheap, non-toxic, inflammable and readily available solvent was the component present in all reactions. No target products were obtained when the reaction mixture was stirred at room temperature. The solubility of reagents was improved as the viscosity of glycerol largely decreased with increasing temperature to 80 °C. All efforts to perform three-component reaction in media containing glycerol alone were unsuccessful (Entries 1, 2). The formation of Schiff bases as major products in glycerol showed that the presence of K2CO3 catalyst is required for the synthesis of pyrazoles (Entries 3-6). There are two possible mechanisms to form the products, but it seems that only route b will afford the final compounds 4a-f (Scheme 2). Schiff-base condensation reaction was observed in route a under some of the applied conditions. Colorless solutions of various molar ratios of potassium carbonate to glycerol (DES1, 1:4; DES2, 1:5; DES3, 1:6) were selected because their physical properties, including conductivity, surface tension, viscosity, refractive index, density and pH have been evaluated very well in the temperature range of 10-80 °C.33 Three-component reaction in 0.5 g of each of these three DESs at room temperature have resulted in the Schiff bases and benzylidenemalono-nitriles as the major products (Entries 7, 9, 11), pyrazoles were obtained in 35-40% yields due to the increase of temperature to 80 °C (Entries 8, 10, 12). One-pot two-step process was screened according to route b. Two-step procedure was carried out in all DESs, the increase in molar ratios of glycerol reduced the product yield (Entries 13-18). This can be caused by the higher pH of DES1. The first stage reaction did not proceed completely at room temperature or at higher temperature even after 8 h, due to the lack of appearance of the intermediary benzylidenemalononitriles in the reaction media. Therefore, the next stage reaction of unconsumed reagents especially aldehydes with hydrazine was inevitable under these conditions (Entries 13, 15, 17). Adding water to achieve the final DES1/H2O ratios of 1:2, 1:1 and 3:1 (w:w) has improved reaction time and product yield, the condensation reaction of malononitrile with aldehyde Scheme 1. Total synthesis of polysubstituted pyrazoles 4a-f. Scheme 2. Proposed mechanisms for the formation of pyrazole derivatives 4a-f. Beyzaei et al.: Green One-pot Synthesis of Novel Polysubstituted Acta Chim. Slov. 2017, 64, 911-918 915 Table 1. Optimization of the model reaction conditions. Entry Solvent Condition Time (min) Yield (%) 1 Gly® Three-component, rt 240 - 2 Gly Three-component, 80°C 120 Schiff base 3 Gly One-pot two-step process (route a), rt 240 - 4 Gly One-pot two-step process (route a), 80 °C 180 Schiff base 5 Gly One-pot two-step process (route b), rt 240 - 6 Gly One-pot two-step process (route b), 80 °C 180 Schiff base 7 DES1 Three-component, rt 210 Benzylidene, Schiff base 8 DES1 Three-component, 80 °C 90 40 9 DES2 Three-component, rt 180 Benzylidene, Schiff base 10 DES2 Three-component, 80 °C 90 37 11 DES3 Three-component, rt 150 Benzylidene, Schiff base 12 DES3 Three-component, 80 °C 90 35 13 DES1 One-pot two-step process (route b), rt 180 Benzylidene, Schiff base 14 DES1 One-pot two-step process (route b), 80 °C 120 53 15 DES2 One-pot two-step process (route b), rt 150 Benzylidene, Schiff base 16 DES2 One-pot two-step process (route b), 80 °C 150 47 17 DES3 One-pot two-step process (route b), rt 120 Benzylidene, Schiff base 18 DES3 One-pot two-step process (route b), 80 °C 180 42 19 DES1/H2O, 1:2b One-pot two-step process (route b), 80 °C 50 60 20 DES1/H2O, 1:1b One-pot two-step process (route b), 80 °C 35 75 21 DES1/H2O, 2:1b One-pot two-step process (route b), 80 °C 20 91 22 DES1/H2O, 3:1b One-pot two-step process (route b), 80 °C 40 68 a Gly as glycerol; b Ratios as w:w; The amount 0.5 g of solvents containing glycerol was used. Under the optimized conditions, mono and disubstituted benzaldehydes 2b-f were also reacted with malononitrile (1) and 2,4-dinitrophenylhydrazine (3) to afford pyrazoles 4b-f. The results are presented in Table 2. Table 2. Synthesis of polysubstituted pyrazoles 4a-f under optimized conditions. Entry R Product Time Yield" (min) (%) 1 4-CH3CONH-C6H4 4a 20 91 2 4-CH3O-C6H4 4b 25 85 3 4-O2N-C6H4 4c 20 89 4 2-HO-3-CH3O-C6H3 4d 30 84 5 2.4-CVC6H3 4e 25 86 6 2,6-Cl2-C6H3 4f 25 88 a All yields refer to isolated products was thus completed within 2 min (Entries 19, 20, 22). The best results were obtained with DES1/H2O ratio of 2:1 (w:w), and this was considered as the optimized conditions (Entry 21). The molecular structures and purity of the newly synthesized compounds were identified by NMR (1H and 13C), FT-IR and elemental analysis (CHN). In FT-IR spectra, absorption bands attributed to symmetric and asymmetric stretching vibrations of amino groups appeared within v = 3428-3456 and 3281-3326 cm-1, as well as stretching vibrations of nitro groups were recorded within v = 1514-1543 and 1318-1331 cm-1. The presence of ni-trile groups was deduced both from IR bonds and 13C NMR signals appearing at v = 2206-2228 cm-1 and 8 113.67-117.37 ppm. In addition to these, 'H NMR spectra and microanalytical data are in agreement with the chemical structures. 3. 2. Antimicrobial Evaluation The in vitro inhibitory activities of the newly synthesized derivatives were evaluated against a variety of pathogenic bacteria and fungi. Amikacin, ceftriaxone and penicillin belonging to aminoglycoside, cephalosporin and penicillin antibiotics, respectively, were used as positive antibacterial controls, as well as antifungal agents including terbinafine, fluconazole and nystatin. The antimicrobial effects were presented as IZD, MIC, MBC and MFC values in Tables 3 and 4. According to the data reported in Table 3, the derivatives were ordered based on the spread of inhibitory properties and the MIC values as follows: 4b > 4e > 4d > 4c > 4f > 4a. The 3-phenyl ring in pyrazole derivative 4b was substituted by a methoxy group at para position, it was the only compound synthesized effective against Streptococcus pyogenes and Proteus vulgaris. The pyarazole 4a containing p-acetamidophenyl substituent was effective only against Gram-negative Salmonella typhi. The inhibitory effects of derivative 4e including 2,4-dichlorophenyl substituent were more significant than those of the derivative 4f with 2,6-dichlorophenyl substituent. Among pyrazoles 4a-f, the antibacterial properties against Proteus mirabilis and Beyzaei et al.: Green One-pot Synthesis of Novel Polysubstituted ... 916 Acta Chim. Slov. 2017, 64, 911-918 Table 3. Antibacterial effects of synthesized pyrazoles and antibiotics. Products Bacteria 4a 4b 4c 4d 4e 4f AMKa CROb PENc 1768 IZDd MICe MBCf - 9.23 512 1024 8.53 512 1024 - - - 17.20 2 8 - - 1447 IZD MIC MBC - - - - - 15.46 64 128 20.17 1 4 25.88 0.5 1 22.61 0.25 0.5 1709 IZD MIC MBC - 10.11 256 512 - 11.50 256 512 10.10 512 1024 11.51 256 512 10.10 0.5 1 32.64 2 8 14.03 4 16 1188 IZD MIC MBC - 15.87 256 128 - - - - 20.98 0.063 0.125 - - 1234 IZD MIC MBC - - - 13.26 1024 2048 - 15.01 512 1024 7.66 0.5 4 34.08 2 4 18.28 8 16 1776 IZD MIC MBC - - - 10.74 128 256 - - 14.65 0.25 4 33.91 0.063 1 20.73 8 32 1609 IZD MIC MBC 12.21 64 128 13.89 64 128 - 11.67 64 128 14.78 32 64 - 19.31 0.063 0.25 30.43 0.063 0.125 10.95 4 16 1435 IZD MIC MBC - 17.76 16 32 12.14 64 128 - 13.82 64 256 - 20.71 0.25 4 18.54 0.5 2 23.58 0.5 1 1310 IZD MIC MBC - 12.10 128 256 13.67 16 32 - 14.10 16 32 - 19.07 0.063 0.063 16.21 0.5 1 - 1240 IZD MIC MBC - 12.16 256 512 - 11.55 256 512 11.34 256 512 - 17.44 1 1 - 12.20 8 16 1079 IZD MIC MBC - 11.78 16 64 - - - - 22.42 4 4 - 12.82 8 32 1633 IZD MIC MBC - 19.45 32 64 11.92 32 128 11.08 256 512 15.20 128 256 9.52 128 256 19.47 1 2 21.51 2 2 17.29 8 16 -: No noticeable antibacterial effects at selected highest concentration. a Amikacin, b Ceftriaxone, c Penicillin, d Inhibition zone diameter in mm, ' Minimum inhibitory concentration in ^g/mL, f Minimum bactericidal concentration in ^g/mL. Shigella dysenteriae were observed for the compounds 4d and 4f, respectively. Amikacin in comparison with two other antibiotics could block the growth of all bacteria. The in vitro antifungal activities of prepared pyrazoles were also evaluated and the results were promising. No inhibitory effect was observed with derivative 4d containing 2-hydroxy-3-methoxyphenyl substituent at the 3-position of the pyrazole ring. The dichloro compounds 4e and 4f had the same antifungal properties despite their different stereochemistry. Data gathered in Table 4 show that terbinafine has more remarkable effects than the others. 4. Conclusions An efficient, one-pot two-step procedure was proposed and the synthesis of polysubstituted pyrazoles has been carried out. Some deep eutectic solvents including different molar ratios of potassium carbonate to glycerol were prepared and applied as reaction media and catalyst in this synthesis. The best results in terms of product yields and reaction times were achieved in molar ratios 1:4:14 of K2CO3/ glycerol/H2O. Efficiency of DES K2CO3/glycerol in organic synthesis is currently under our investigation, and will be in focus of our future research. Furthermore, antimicrobial ac- Beyzaei et al.: Green One-pot Synthesis of Novel Polysubstituted Acta Chim. Slov. 2017, 64, 911-918 917 Table 4. Antifungal effects of synthesized pyrazoles and drugs. Products Fungi 4a 4b 4c 4d 4e 4f TRBa FLCb NYT IZDd 11.56 - - - 15.25 15.29 23.94 15.23 20.45 5115 MICe 64 - - - 32 32 32 128 64 MBCf 128 - - - 64 64 64 256 128 IZD 21.87 14.77 14.71 - - - 36.24 14.81 - 5027 MIC 64 32 32 - - - 32 256 - MBC 128 64 64 - - - 64 512 - IZD - 23.57 - - - - 29.18 21.13 20.52 5009 MIC - 512 - - - - 32 32 32 MBC - 1024 - - - - 32 64 128 -: No noticeable antibacterial effects at selected highest concentration. a Terbinafine, b Fluconazole, c Nystatin, d Inhibition zone diameter in mm, e Minimum inhibitory concentration in ^g/mL, f Minimum fungicidal concentration in ^g/mL. tivities of all synthesized derivatives were evaluated against a broad range of pathogenic bacteria and fungi. Based on the broad-spectrum inhibitory effects of the pyrazole 4b, including 4-methoxy group on 3-aryl ring, it is suggested that benzaldehydes with small para electron donating substitu-ents should be used to synthesize future active analogues. 5. Acknowledgements This work was supported by the University of Zabol under Grant number UOZ-GR-9517-15. 6. References 1. F. F. Noe, L. Fowden, Nature 1959, 184, 69-70. DOI:10.1038/184069a0 2. A. A. Wube, E. M. Wenzig, S. Gibbsons, K. Asres, R. Bauer, F. Bucar, Phytochemistry 2008, 69, 982-987. DOI:10.1016/j.phytochem.2007.11.001 3. S. Domiati, A. El-Mallah, A. Ghoneim, A. Bekhit, H. A. El Razik, Inflammopharmacology 2016, 24, 163-172. DOI:10.1007/s10787-016-0270-7 4. G. Cocconcelli, E. Diodato, A. Caricasole, G. Gaviraghi, E. Genesio, C. Ghiron, L. Magnoni, E. Pecchioli, P. V. Plazzi, G. C. Terstappen, Bioorg. Med. Chem. 2008, 16, 2043-2052. DOI:10.1016/j.bmc.2007.10.090 5. M. J. Ahsan, H. Khalilullah, J. P. Stables, J. Govindasamy, J. Enzyme Inhib. Med. Chem. 2013, 28, 644-650. DOI:10.3109/14756366.2012.663364 6. M. Bonesi, M. R. Loizzo, G. A. Statti, S. Michel, F. Tillequin, F. Menichini, Bioorg. Med. Chem. Lett. 2010, 20, 1990-1993. DOI:10.1016/j.bmcl.2010.01.113 7. K. M. Kasiotis, E. N. Tzanetou, S. A. Haroutounian, Front. Chem. 2014, 2, 78 (7 pp). DOI:10.3389/fchem.2014.00078 8. Y.-F. Li, Z.-Q. Liu, Free Radic. Biol. Med. 2011, 52, 103-108. DOI:10.1016/j.freeradbiomed.2011.09.032 9. D. Swarnkar, R. Ameta, R. Vyas, European J. Biomed. Pharm. Sci. 2016, 3, 427-431. 10. R. E. El-Mekawy, J. Heterocycl. Chem. 2017, 54, 2367-2374. DOI: 10.1002/jhet.2828 11. S. Y. Hassan, Molecules 2013, 18, 2683-2711. DOI: 10.3390/molecules18032683 12. R. Surendra Kumar, I. A. Arif, A. Ahamed, A. Idhayadhull, Saudi J. Biol. Sci. 2016, 23, 614-620. DOI:10.1016/j.sjbs.2015.07.005 13. K. M. El-Mahdy, A. M. El-Kazak, M. Abdel-Megid, M. Seada, O. Farouk, Acta Chim. Slov. 2016, 63, 18-25. DOI: 10.17344/acsi.2015.1555 14. F. Gosselin, P. D. O'Shea, R. A. Webster, R. A. Reamer, R. D. Tillyer, E. J. J. Grabowski, Synlett 2006, 2006, 3267-3270. DOI: 10.1055/s-2006-956487 15. Y. Kong, M. Tang, Y. Wang, Org. Lett. 2014, 16, 576-579. DOI:10.1021/ol403447g 16. D. C. Schmitt, A. P. Taylor, A. C. Flick, R. E. Kyne Jr., Org. Lett. 2015, 17, 1405-1408. DOI:10.1021/acs.orglett.5b00266 17. Q. Zhang, L.-G. Meng, K. Wang, L. Wang, Org. Lett. 2015, 17, 872-875. DOI:10.1021/ol503735c 18. R. M. Mohareb, N. Y. M. Abdo, F. O. Al-farouk, Acta Chim. Slov. 2017, 64, 117-128. DOI:10.17344/acsi.2016.2920 19. J.-A. Jiang, C.-Y. Du, C.-H. Gu, Y.-F. Ji, Synlett 2012, 23, 29652968. DOI:10.1055/s-0032-1317668 20. A. Kamal, K. N. V. Sastry, D. Chandrasekhar, G. S. Mani, P. R. Adiyala, J. B. Nanubolu, K. J. Singarapu, R. A. Maurya, J. Org. Chem. 2015, 80, 4325-4335. DOI:10.1021/jo502946g 21. N. Panda, A. K. Jena, J. Org. Chem. 2012, 77, 9401-9406. DOI:10.1021/jo301770k 22. G. C. Senadi, W.-P. Hu, T.-Y. Lu, A. M. Garkhedkar, J. K. Van-davasi, J.-J. Wang, Org. Lett. 2015, 17, 1521-1524. DOI: 10.1021/acs.orglett.5b00398 23. S. Specklin, E. Decuypere, L. Plougastel, S. Aliani, F. Taran, J. Org. Chem. 2014, 79, 7772-7777. DOI:10.1021/jo501420r 24. L.-L. Wu, Y.-C. Ge, T. He, L. Zhang, X.-L. Fu, H.-Y. Fu, H. Chen, R.-X. Li, Synthesis 2012, 44, 1577-1583. DOI:10.1055/s-0031-1290772 25. A. Hasaninejad, S. Firoozi, Mol. Divers. 2013, 17, 459-469. DOI:10.1007/s11030-013-9445-y 26. S. Kumari, A. Shekhar, D. D. Pathak, New J. Chem. 2016, 40, 5053-5060. DOI:10.1039/C5NJ03380B Beyzaei et al.: Green One-pot Synthesis of Novel Polysubstituted ... 918 Acta Chim. Slov. 2017, 64, 911-918 27. M. O. M'hamed, O. K. Alduaij, Asian J. Chem. 2016, 28, 543547. D01:10.14233/ajchem.2016.19397 28. F. Nemati, S. H. Nikkhah, A. Elhampour, Chin. Chem. Lett. 2015, 26, 1397-1399. D0I:10.1016/j.cclet.2015.07.009 29. S. Yadav, P. Rai, M. Srivastava, J. Singh, K. P. Tiwari, J. Singh, Tetrahedron Lett. 2015, 56, 5831-5835. D0I:10.1016/j.tetlet.2015.07.039 30. M. Capua, S. Perrone, F. M. Perna, P. Vitale, L. Troisi, A. Salomone, V. Capriati, Molecules 2016, 21, 924 (11 pp). D0I:10.3390/molecules21070924 31. S. B. Katariya, Asian J. Pharm. Sci. Tech. 2015, 5, 199-201. 32. A. Moshtaghi Zonouz, D. Moghani, Synth. Commun. 2016, 46, 220-225. D0I:10.1080/00397911.2015.1129668 33. J. Naser, F. Mjalli, B. Jibril, S. Al-Hatmi, Z. Gano, Int. J. Chem. Eng. Appl. 2013, 4, 114-118. D0I:10.7763/IJCEA.2013.V4.275 34. H. Beyzaei, M. Moghaddam-Manesh, R. Aryan, B. Ghasemi, A. Samzadeh-Kermani, Chem. Pap. 2017, 71, 1685-1691. D0I:10.1007/s11696-017-0163-2 35. S. Arikan, Med. Mycol. 2007, 45, 569-587. D01:10.1080/13693780701436794 Povzetek Različne biološke lastnosti naravnih in sintetičnih pirazolskih derivatov, kot so npr. delovanja proti vnetjem in mikrobom, nevrozaščitni učinki, antiepileptični ter antidepresivni učinki in aktivnosti proti rakom, so nas spodbudili, da smo predlagali novo, hitro, zeleno in ekološko sprejemljivo pot za pripravo nekaterih novih 5-amino-3-(aril substitu-iranih)-1-(2,4-dinitrofenil)-1H-pirazol-4-karbonitrilov. Učinkovito smo jih pripravili z enolončno dvostopenjsko reakcijo med malononitrilom, 2,4-dinitrofenilhidrazinom in različnimi benzaldehidi v globoko evtektičnem topilu (DES) glicerol/kalijev karbonat. Uporaba tovrstnega topilnega sistema je opazno povečala izkoristke produktov in skrajšala reakcijske čase. Raziskali smo antibakterijsko delovanje novopripravljenih pirazolov in rezultate primerjali z učinki več običajnih antibiotikov na izbrane Gram-pozitivne in Gram-negativne patogene bakterije. Raziskali smo tudi inhibitorno aktivnost proti trem glivam in jo primerjali z nekaterimi običajnimi učinkovinami proti glivam. Zmerno do dobro anti-mikrobno delovanje, predvsem pa delovanje proti glivam, smo opazili v nekaterih primerih naših heterocikličnih spojin, kot je bilo razvidno iz izmerjenih IZD, MIC, MBC in MFC vrednosti. Beyzaei et al.: Green One-pot Synthesis of Novel Polysubstituted DOI: 10.17344/acsi.2017.3617 Acta Chim. Slov. 2017, 64, 919-930 ¿^creative tS1 commons Scientific paper Synthesis and Biological Evaluation of Some Novel 1,8-Naphthyridine Derivatives Sraa Abu-Melha* *Department of Chemistry, Faculty of Science of Girls, King Khaled University, Abha, Saudi Arabia * Corresponding author: E-mail: sraa201313@yahoo.com Tel: +966504757797 Received: 06-06-2017 Abstract A series of substituted 1,8-naphthyridine derivatives was synthesized to be used as cytotoxic and antioxidant agents by applying 1,4-dihydro-4-oxo-1,8-naphthyridine-3-carbohydrazide (1) as the starting material. Compound 1 was reacted with different reagents to afford the corresponding 3-heterarylcarbonyl-1,8-naphthyridine derivatives 3-19 which were tested for their in vitro cytotoxicity against Ehrlich Ascites Carcinoma, and antioxidant activity. Compound 15 showed the best cytotoxicity and antioxidant activity. Keywords: 1,8-naphthyridine; quinazolone; pyrazole; cytotoxicity; antioxidant activity 1. Introduction Studies on the synthesis of 1,8-naphthyridines have served as a fertile field of research in the perusal for antibacterial agents.1-3 Nalidixic acid (1-ethyl-3-carboxy-7-methyl-1,8-naphthyridine-4-one) has been found to be effective particularly against gram negative bacteria found in chronic urinary tract infections.4 1,8-Naphthyridine derivatives were found to display moderate cytotoxic activity against murine p388 leukemia, when changes were carried out at N-1 and N-7 positions.5,6 It has been reported that C-3 carboxamide derivatives with a spacer have shown good cytotoxicity along with anti-inflammatory activity.7 Pharmacologically, pyrazole and its derivatives represent one of the most important classes of organic heterocyclic compounds, possessing anti-bacterial, anti-fungal,8 herbicidal9 and anti-viral activities.10 Moreover, the chemistry of carbohydrazoles has gained increased interest in both synthetic organic chemistry and biological fields and has considerable value in many useful applications, such as the assessment process of the three dimensional ultra structure examination techniques of interphase nuclei and tissues, besides their therapeutic importance.11 2. Experimental 2. 1. Materials and Methods 2. 1. 1. Chemicals and Reagents All the chemicals and solvents used in this study were obtained from Merck (Germany) and Sigma-Aldrich chemical company (Germany). 2. 1. 2. Instruments All melting points were recorded on Gallenkamp electric melting point apparatus and are uncorrected. The IR spectra v cm-1 (KBr) were recorded on Perkin-Elmer Infrared Spectrophotometer Model 157, Grating. The 1H and 13C NMR spectra were run on Varian Spectrophotometer at 400 MHz and 100 MHz using TMS as the internal reference and DMSO-d as the solvent. Chemical 6 shifts (S) are given in ppm. The mass spectra (EI) were recorded on 70 eV with Kratos MS equipment and/or Varian MAT 311 A Spectrometer at Cairo University, Giza, Egypt, and at Assuit University Central Laboratory. Elemental analyses (C, H, and N) were carried out at the Microanalytical Center of Cairo University, Giza, Egypt (automatic analyzer CHNS, Vario ELIII-elementar, Ger- Abu-Melha : Synthesis and Biological Evaluation 920 Acta Chim. Slov. 2017, 64, 919-930 many). The results were found to be in good agreement with the calculated values. 2. 2. Synthesis 2. 2. 1. Synthesis of 1,4-Dihydro-N-(2-methyl-4-oxoquinazolin-3(4H)-yl)-4-oxo-1,8-naphthyridine-3-carboxamide (3) A mixture of separated or freshly prepared benzox-azine 212 (1.16 g, 10 mmol) and 4-oxo-1,4-dihy-dro-l,8-naphthyridine-3-carbohydrazide (1) (2.04 g, 10 mmol) in ethanol (25 mL) containing glacial acetic acid (5 mL) was refluxed for 4 h. The formed precipitate was filtered off, dried and recrystallized from ethanol to give compound 3. Yield 63%; White crystal; m.p. 289 °C; IR (KBr): vmax 3230-3238 (br, 2 NH), 1668, 1671 (C=O, amid-ic), mil^ß-unsaturated C=O),13 1569 (C=N) cm-1; 1H NMR (DMSO-d6) 5 1.93 (s, 3H, CH3), 7.62-8.24 (m, 7H, Ar-H), 9.01 (s, 1H, C2-H of naphthyridine ring), 9.21 (s, 1H, NH), 10.66 (s, 1H, NH); 13C NMR (DMSO-d6) 5 21.6, 113.8, 114, 121.1, 122.3, 123.9, 126.6, 127.8, 134.2, 138.1, 141.7, 148.2, 151.4, 152.6, 162.1, 164.4, 165.9, 176.8; MS (EI, 70 eV) m/z (%) 347 (M+, 4.03), 272 (6.05), 259 (5.26), 230 (10.31), 173 (61.90), 138 (8.82), 123 (8.09), 104 (62.02), 89 (9.44), 77 (100), 75 (17.03), 63 (24.58), 51 (67.43), 49 (13.12); Anal. calcd. for C18H13N5O3 (347.33): C, 62.24; H, 3.77; N, 20.16. Found: C, 62.3(5; H, 3.59; N, 20.31. 2. 2. 2. Synthesis of Ethyl 3-(2-(4-Oxo-1,4-dihydro-1,8-naphthyridine-3-carbonyl)hydrazono) butanoate (4) A mixture of compound 1 (2.04 g, 10 mmol) and ethyl acetoacetate (1.3 g, 10 mmol) was refluxed in ethanol (25 mL) containing drops of glacial acetic acid for 2 h. The reaction mixture was cooled to room temperature. The solid product that formed was filtered off, dried, and re-crystallized from ethanol to give compound 4. Yield 95%; pale yellow crystal; m.p. 209 °C; IR (KBr): vmax 3237-3255 (br, NH), 1724 (C=O of ester group), 1689 (C=O, amidic), 1625 («.^-unsaturated C=O), 1567 (C=N) cm-1; 1H NMR (DMSO-d6) 5 0.91 (s, 3H, CH3), 1.38 (t, 3H, CH3), 2.35 (s, 2H, CH2), 4.25 (q, 2H, CH2), 7.60-8.24 (m, 3H, CH-pyri-dine ring), 8.61 (s, 1H, C2-H of naphthyridine ring), 10.81 (s, 1H, NH), 11.01 (s, 1H, NH); MS (EI, 70 eV) m/z (%) 316 (M+, 17.5), 271 (2.8), 245 (0.2), 229 (69.0), 204 (0.5), 202 (1.4), 189 (1.6), 173 (100), 145 (2.3), 104 (12.3), 76 (10.6); Anal. calcd. for C15H16N4O4 (316.31): C, 56.96; H, 5.10; N, 17.71. Found: C, 56.96; H, 5.12; N, 17.93. 2. 2. 3. Synthesis of 3-(3-Methyl-5-oxo-4,5-dihydro-1H-pyrazole-1-carbonyl)-1,8-naphthyridin-4(1H)-one (5) Compound 4 (3.16 g, 10 mmol) was refluxed in ethanol (30 mL) containing sodium metal (0.23 g, 10 mmol) for 5 h, cooled to room temperature, and poured into ice cold water. The separated solid was filtered, dried, and re-crystallized from ethanol to give 5. Yield 59%; pale brown powder; m.p. >300 °C; IR (KBr): vmax 3134 (NH), 1669, 1674 (C=O, amidic), 1636 (a^-unsaturated C=O), 1606 (C=N) cm-1; 1H NMR (DMSO-d6) 5 1.22 (s, 3H, CH3), 2.07 (s, 2H, CH2), 6.76-8.63 (m, 3H, CH-pyridine ring), 8.41 (s, 1H, C22H of naphthyridine ring), 9.83 (s, 1H, NH); 13C NMR (DMSO-d6) 5 26.5, 43.2, 113.8, 114, 122.3, 138, 141.7, 151.4, 152.6, 158.2, 161.4, 167.1, 176.8; MS (EI, 70 eV) m/z (%) 270 (M+, 0.26), 173 (0.4), 168 (2.07), 125 (0.64), 111 (8.76), 97 (0.63), 84 (13.67), 77 (1.54), 68 (4.23), 52 (100); Anal. calcd. for C13H10N4O3 (270.24): C, 57.78; H, 3.73; N, 20.73. Found: C, 57.77; H, 3.64; N, 20.71. 2. 2. 4. Synthesis of 4-Oxo-N'-(4-oxopentan-2- ylidene)-1,4-dihydro-1,8-naphthyridine-3-carbohydrazide (6) A mixture of 1 (2.04 g, 10 mmol) and acetylacetone (1.0 g, 10 mmol) was refluxed in ethanol (25 mL) containing a few drops of glacial acetic acid for 2 h. The reaction mixture was left to cool, and then poured into ice cold water. The solid product was filtered off, dried, and recrystallized from ethanol to give compound 6. Yield 41%; dark yellow powder; m.p. >300 °C; IR (KBr): vmax 3251 (br, NH), 1743 (C=O, ketonic), 1681 (C=O, amidic)1, 1627 («,ß-un-saturated C=O), 1569 (C=N), 1548 (C=C) cm-1; 1H NMR (DMSO-d6) 5 1.20 (s, 3H, CH3), 2.11 (s, 3H, CH3), 2.54 (s, 2H, CH2), 8.48 (s, 1H, C2-H of naphthyridine ring), 7.628.76 (m, 3H, CH-pyridine ring), 10.49 (s, 1H, NH), 11.20 (s, 1H, NH); MS (EI, 70 eV) m/z (%) 286 (M+, 11.0), 285 (41.57), 148 (38.57), 134 (53.00), 117 (36.02), 77 (78.89), 64 (88.82), 50 (100), 49 (63.92), 45 (64.72); Anal. calcd. for C14H14N4O3 (286.29): C, 58.73; H, 4.93; N, 19.57. Found: C, 581.72; H, 4.83; N, 19.69. 2. 2. 5. Synthesis of 3-(3,5-Dimethyl-1H-pyrazole-1-carbonyl)-1,8-naphthyridin-4(1H)-one (7) A mixture of 6 (2.86 g, 10 mmol) and acetylacetone (1.0 g, 10 mmol) was refluxed in ethanol (30 mL) containing sodium metal (0.23 g, 10 mmol) for 4 h. The reaction mixture was cooled to room temperature, the separated solid filtered off, washed with a little cold ethanol and re-crystallized from ethanol to give 7. Yield 85%; pale yellow powder; m.p. sharing at 230 0C; IR (KBr): vmax 3205 (NH), 1706 (C=O, amidic), 1625 (a,ß-unsaturatecTC=O), 1567 (C=N), 1548 (C=C) cm-1; 1H NMR (DMSO-d6) 5 1.74, 1.79 (2s, 6H, 2 CH3), 6.08 (s, 1H, CH), 7.45-8.21 (m, 3H, CH-pyridine ring), 8.47 (s, 1H, C2-H of naphthyridine ring), 10.12 (s, 1H, NH); 13C NMR (DMSO-d6) 5 14.2, 19.6, 106.5, 114, 122.3, 125.5, 138, 140.3, 141.7, 151.4, 152.6, 153.1, 176.8, 193.9; MS (EI, 70 eV) m/z (%) 271 (M++3, Abu-Melha : Synthesis and Biological Evaluation Acta Chim. Slov. 2017, 64, 919-930 921 0.8), 270 (M++2, 0.5), 204 (9.8), 174 (12.9), 173 (100), 145 (3.3), 120 (0.4), 105 (56.2), 95 (0.2), 90 (4.1), 78 (76.5); Anal. calcd. for C14H12N4O2 (268.27): C, 62.68; H, 4.51; N, 20.88. Found: C, (52.81; H, 4.60; N, 20.73. 2. 2. 6. Synthesis of N'-((2-Hydroxynaphthalen-1-yl)methylene)-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carbohydrazide (8) A mixture of 1 (2.04 g, 10 mmol) and 2-hy-droxy-1-naphthaldehyde (1.72 g, 10 mmol) was refluxed in ethanol containing a catalytic amount of glacial acetic acid (5 drops) for 4 h. The reaction mixture was cooled to room temperature, the separated product filtered off, dried, and recrystallized from ethanol to give the hydrazone derivative 8. Yield 92%; pale yellow sheets; m.p. 176 °C; IR (KBr): vmax 3450 (OH), 3330 (NH), 1702 (C=O, amidic), 1626 (a^-unsaturated C=O), 1574 (C=N) cm-1; 1H NMR (DMSO-d6) 5 1.89 (s, 1H, CH), 6.97-8.61 (m, 9H, Ar-H), 9.09 (s, 1H, C2-H of naphthyridine ring), 9.21 (s, 1H, NH), 9.68 (s, 1H, NH), 12.24 (s, 1H, NH), 12.95 (s, 1H, OH); Anal. calcd. for C20H14N4O3 (358.35): C, 67.03; H, 3.94; N, 15.63. Found: C, 67.291; H, 3.78; N, 15.57. 2. 2. 7. Synthesis of 3-(3H-Benzo[e]indazole-3- carbonyl)-1,8-naphthyridin-4(1H)-one (9) Pathway 1 Compound 8 (3.58 g, 10 mmol) was refluxed in ethanol containing a few drops of piperidine or triethylamine for 4 h. The reaction mixture was cooled to room temperature, the separated product filtered off, dried, and recrys-tallized from ethanol to give compound 9. Pathway 2 An equimolar amount of 1 (2.04 g, 10 mmol) and 2-hydroxynaphthaldehyde (1.72 g, 10 mmol) in ethanol (25 mL) in the presence of a catalytic amount of piperidine or triethylamine (4 drops) or sodium ethoxide (10 mmol) was refluxed for 3 h. The reaction mixture was left to cool at room temperature overnight. The formed precipitate was filtered off, dried and recrystallized from ethanol to give compound 9. Yield 83%; yellow powder; m.p. 291 °C; IR (KBr): vmax 3340 (NH), 1710 (C=O, amidic), 1622 («^-unsaturated C=O), 1579 (C=N), 1549 (C=C) cm-1; 1H NMR (DMSO-d6) 8 8.08 (s, 1H, C2-H of naphthyridine ring), 8.20 (s, 1H, CH-pyrazole ring), 7.72-8.31 (m, 9H, Ar-H), 11.24 (s, 1H, NH); 13C NMR(DMSO-d6) 8 113.5, 118.0, 121.6, 122.8, 123.6, 124.4, 124.7, 124.9, 125.7, 127.2, 127.3, 127.8, 134.6, 141.9, 151.2, 152.5, 161.7, 164.3, 177.5; MS (EI, 70 eV) m/z (%)340 (M+, 6.95), 208 (3.42), 170 (26.02), 152 (13.20), 128 (19.71), 115 (72.86), 89 (13.88), 77 (8.22), 62 (13.54), 51 (17.97), 45 (100); Anal. calcd. for C20H12N4O2 (340.33): C, 70.58; H, 3.55; N, 16.46. Found: C, 70.67; H, 3.34; N, 16.62. 2. 2. 8. Synthesis of 3-(1H-Indazole-1-carbonyl)-1,8-naphthyridin-4(1H)-one(10) An equimolar amounts of 1 (2.04 g, 10 mmol) and salicyaldehyde (1.22 g, 10 mmol) in ethanol (25 mL) in the presence of a catalytic amount of piperidine (4 drops) was refluxed for 3 h. The reaction mixture was left to cool at room temperature overnight. The formed precipitate was filtered off, dried and recrystallized from ethanol to give compound 10. Yield 88%; white powder; m.p. 286 °C; IR (KBr): vmax 3073 (NH), 1667 (C=O, amidic), 1622 (a,ß-un-saturatecHC=O), 1569 (C=N), 1541 (C=C) cm-1; 1H NMR (DMSO-d6) 8 8.70 (s, 1H, CH-pyrazole ring), 9.08 (s, 1H, C2-H of naphthyridine ring), 6.90-9.24 (m, 5H, Ar-H), 11.28 (s, 1H, NH); 13C NMR(DMSO-d6) 8 114, 115.4, 121.5, 121.9, 122.3, 123.9, 125.5, 127.6, 128.3, 138, 143.2, 151.4, 152.6, 153.1, 176.8, 193.9; Anal. calcd. for C.,HlnN„O, (290.28): C, 66.20; H, 3.47; N, 19.30. Found: C, 16 1U 4 2 x ' 66.15; H, 3.56; N, 19.35. 2. 2. 9. Synthesis of 3-(3,4,6-Triamino-2H- pyrazolo[3,4-b]pyridine-2-carbonyl)-1,8-naphthyridin-4(1H)-one (13) A mixture of 1 (2.04 g, 10 mmol) and 2-amino-prop-1-ene-1,1,3-tricarbonitrile (1.32 g, 10 mmol) in ethanol (25 mL) containing a few drops of glacial acetic acid (5 drops) was refluxed for 4 h. The reaction mixture was then cooled to room temperature and the obtained solid material filtered off, dried and recrystallized from ethanol to give pyrazolopyridine 13. Yield 51%; pale brown powder; m.p. 288 °C; IR (KBr): vmax 3411, 3382 (NH2), 3228 (br, NH groups), 1671 (C=O, amidic), 1628 (a,ß-unsaturated C=O), 1608 (C=N), 1569 (C=C) cm-1; 1H NMR (DM-SO-d6) 8 4.18 (s, 4H, NH2), 5.90 (s, 1H, CH), 8.08 (s, 1H, C2-H of naphthyridine ring), 6.72-8.31 (m, 3H, CH-pyri-dine ring), 9.81 (s, 1H, NH), 11.60 (s, 2H, 2 C=NH); 13C NMR (DMSO-d6) 8 89.1, 95.1, 118.0, 121.6, 123.6, 134.6, 147.0, 151.2, 152.5, 153.7, 159.8, 161.7, 162.4, 164.3, 177.5; MS (EI, 70 eV) m/z (%) 336 (M+, 38.85), 104 (37.62), 90 (35.08), 84 (33.29), 78 (82.65), 64 (46.06), 54 (40.92), 53 (100), 50 (38.25), 48 (42.52); Anal. calcd. for C15H12N8O2 (336.31): C, 53.57; H, 3.60; N, 33.32. Found: C, 53.34; H, 3.53; N, 33.54. 2. 2. 10. Synthesis of 3-Amino-5-(methylthio)-1-(4-oxo-1,4-dihydro-1,8-naphthyridine-3-carbonyl)-1H-pyrazole-4-carbonitrile (15) A mixture of 1 (2.04 g, 10 mmol) and 2-(bis(meth-ylthio)methylene) malononitrile (1.7 g, 10 mmol) in ethanol (25 mL) containing a few drops of glacial acetic acid was refluxed for 4 h. The reaction mixture was then cooled to room temperature and the obtained solid material filtered off, dried and recrystallized from ethanol to give compound 15. Yield 90%; yellow sheets; m.p. 254 0C; IR (KBr): vmax 3413, 3382 (NH2), 3226 (br, NH groups), 2200 Abu-Melha : Synthesis and Biological Evaluation 922 Acta Chim. Slov. 2017, 64, 919-930 (CN), 1670 (C=O, amidic), 1627 («.^-unsaturated C=O), 1608 (C=N), 1569 (C=C) cm-1; 1H NMR (DMSO-d6) 5 1.96 (s, 3H, SCH3), 4.52 (s, 2H, NH2), 9.05 (s, 1H, C2-H of naphthyridine ring), 7.60-8.24 (m, 3H, CH-pyridine ring), 9.22 (s, 1H, NH), 10.66 (s, 1H, NH); 13C NMR (DMSO-d6) 8 16.9, 62.2, 113.8, 115.8, 118.0, 121.6, 134.6, 142.5, 152.6, 160.5, 161.7, 165.0, 177.5; MS (EI, 70 eV) m/z (%) 327 (M++1, 23.92), 294 (31.62), 253 (15.05), 230 (24.34), 173 (31.75), 147 (18.32), 111 (39.36), 105 (28.04), 97 (18.55), 88 (15.66), 71 (29.92), 65 (24.72), 61 (34.46), 56 (100), 50 (26.79); Anal. calcd. for C14H10N6O2S (326.33): C, 51.53; H, 3.09; N, 25.75. Found: C, 541.775; H, 3.13; N, 25.66. 2. 2. 11. Synthesis of 3-Imino-5-(4- methoxyphenylamino)-1-(4-oxo-1,4-dihydro-1,8-naphthyridine-3-carbon yl)-2,3-dihydro-1H-pyrazole-4-carbonitrile (16) An equimolar amount of compound 15 (3.26 g, 10mmol) and p-anisidine (1.23 g, 10 mmol) in ethanol (25 mL) in the presence of a few drops of piperidine was re-fluxed for 4 h. The reaction mixture was left to cool at room temperature overnight. The formed precipitate was filtered off, dried and recrystallized from ethanol to give compound 16. Yield 65%; dark yellow powder; m.p. 282 0C; IR (KBr): vmax 3227 (br, NH), 2200 (CN), 1669 (C=O, amidic), 1625 («^-unsaturated C=O), 1600 (C=N), 1571 (C=C) cm-1; 1H NMR (DMSO-d6) 5 3.52 (s, 2H, OCH3), 9.05 (s, 1H, C2-H of naphthyrid6ne ring), 7.60-8.24 (m, 3H, CH-pyridine ring), 10.66 (s, 1H, NH), 10.82 (s, 1H, C=NH); 13C NMR (DMSO-d6) 5 48, 54.1, 113.5 (2C), 113.6, 113.8, 114, 115.4 (2C), 122.3, 134.9, 138, 141.7, 149.5, 151.4, 152.6, 159.6, 160, 162, 176.8; MS (EI, 70 eV) m/z (%)401 (M+, 9.49), 383 (5.77), 318 (20.98), 308 (15.70), 245 (6.26), 227 (8.00), 211 (5.27), 207 (30.15), 201 (16.45), 186 (14.92), 173 (30.74), 120 (8.80), 108 (13.52), 77 (43.49), 68 (27.50), 52 (100); Anal. calcd. for C20H15N7O3 (401.38): C, 59.85; H, 3.77; N, 24.43. Found: C, 59.95; H, 3.86; N, 24.61. 2. 2. 12. Synthesis of 5-Amino-3-(4-nitrophenyl)-1-(4-oxo-1,4-dihydro-1,8-naphthyridine-3-carbonyl)-2,3-dihydro-1H-pyrazole-4-carbonitrile (17) A mixture of 1 (2.04 g, 10 mmol) in ethanol (25 mL) containing a few drops of glacial acetic acid (5 drops) and 2-(4-nitrobenzylidene)malononitrile (1.99 g, 10 mmol) was refluxed for 3 h, then left to stand at room temperature overnight. The separated solid product was filtered off, dried and recrystallized from ethanol to give 17. Yield 85%; dark yellow powder; m.p. 350 0C; IR (KBr): vmax 3468-3335 (NH2), 1685 (C=O, amidic), 1629 («^-unsaturated C=O), 1582 (C=n), 1550 (C=C), 1552, 1336 (NO2) cm-1; 1H NMR (DMSO-d,) 5 2.71 (s, 2H, NH2), 5.29 (s, 1H, CH), 9.11 (s, 1H, C2-H of naphthyridine ring), 7.57-9.22 (m, 7H, Ar-H), 10.81 (s, 1H, NH), 12.28 (s, 1H, NH); 13C NMR (DMSO-d6) 5 59.7, 63.4, 113.8, 117.3, 118.0, 121.6, 125.1, 128.3, 134.6, 142.5, 145.9, 149.4, 152.5, 158.1, 160.5, 161.7, 177.5; MS (EI, 70 eV) m/z (%)403 (M+, 100), 319 (34.34), 266 (44.02), 218 (42.66), 212 (20.30), 204 (21.45), 146 (40.11), 141 (36.01), 105 (28.76), 97 (33.65), 60 (63.03); Anal. calcd. for C19H13N7O4 (403.35): C, 56.58; H, 3.25; N, 24.31. Found: C, 516.71; H, 3.13; N, 24.26. 2. 2. 13. Synthesis of 5-Amino-3-(4-chlorophenyl)-1-(4-oxo-1,4-dihydro-1,8-naphthyridine-3-carbonyl)-2,3-dihydro-1H-pyrazole-4-carbonitrile (18) Equimolar quantities of the starting material 1 (2.04 g, 10 mmol) in ethanol (25 mL) containing a few drops of glacial acetic acid and 2-(4-chlorobenzylidene)malononi-trile (1.88 g, 10 mmol) were refluxed for 3 h, then left to stand at room temperature overnight. The separated solid product was filtered off, dried and recrystallized from eth-anol to give 18. Yield 93%; yellow powder; m.p. 307 0C; IR (KBr): vmax 3399-3341 (NH2), 3260 (NH), 1679 (C=O, amidic), 1625 («.^-unsaturated C=O), 765 (C-Cl) cm-1; 1H NMR (DMSO-d6) 5 2.83 (s, 2H, NH2), 5.69 (s, 1H, CH), 9.09 (s, 1H, C2-H of naphthyridine ring), 6.72-8.31 (m, 7H, Ar-H), 10.83 (s, 1H, NH), 11.40 (s, 1H, NH); 13C NMR (DMSO-d6) 5 113.8, 114, 118, 122.4, 125.8 (2C), 126.8 (2C), 131.2, 131.5, 135.5, 138, 141.6, 150, 151.4, 153.1, 161.5, 166.4, 176.2; MS (EI, 70 eV) m/z (%) 392 (M+-1, 0.01), 205 (0.11), 189 (77.30), 173 (100), 144 (21.49), 117 (10.94), 104 (45.37), 77 (47.32), 62 (16.20), 50 (31.49); Anal. calcd. for CloH, ClNA (392.8): C, 58.10; H, 3.34; N, 19 13 6 2 21.40. Found: C, 58.06; H, 3.12; N, 21.38. 2. 2. 14. Synthesis of 3-(3,5-Diamino-1H-pyrazole-1-carbonyl)-1,8-naphthyridin-4(1H)-one (19) Pathway 1 To a solution of 1 (2.04 g, 10 mmol) in ethanol (25 mL) containing a few drops of glacial acetic acid, cyanoac-etamide (0.84 g, 10 mmol) was added and the reaction mixture was refluxed for 5 h. The solution was cooled to room temperature, and the formed precipitate was filtered off, dried and recrystallized from ethanol to give pyrazole derivative 19. Pathway 2 A mixture of 1 (2.04 g, 10 mmol) in ethanol (25 mL) containing a few drops of glacial acetic acid and malononi-trile (0.66 g, 10 mmol) were refluxed for 3 h, then left to stand at room temperature overnight. The separated solid product was filtered off, dried and recrystallized from eth-anol to give 19. Yield 71%; gray powder; m.p. 255 0C; IR (KBr): vmax 3441 (NH2), 3232 (br, NH groups), 1668 (amid- Abu-Melha : Synthesis and Biological Evaluation Acta Chim. Slov. 2017, 64, 919-930 923 ic C=O), 1634 («.^-unsaturated C=O), 1612 (C=N), 1570 (C=C) cm-1; 1H NMR (DMSO-d6) 5 4.01 (s, 4H, 2 NH2), 6.37 (s, 1H, CH-pyrazole ring), 8.07 (s, 1H, C2-H of naph-thyridine ring), 7.72-8.31 (m, 3H, CH-pyridine ring), 10.20 (s, 1H, NH); 13C NMR (DMSO-d6) 5 78.6, 118, 121.6, 123.6, 134.6, 148.2, 151.2, 152.5, 161.7, 164.3, 177.5; MS (EI, 70 eV) m/z (%) 270 (M+, 10), 173 (28.63), 105 (76.12), 95 (0.40), 77 (100), 67 (20.41), 52 (38.72), and 50 (42.40); Anal. calcd. for C H N O (270.25): C, 53.33; H, 3.73; N, 12 1U 6 2 x ' 31.10. Found: C, 53.46; H, 3.64; N, 31.29. 2. 3. Antitumor Activity (or Cytotoxicity) Using In Vitro Ehrlich Ascites Assay The isolated compounds were screened for their antitumor activity. The viability of the cells used in control experiments exceeded 95%. Different concentrations of the tested compounds were prepared (100, 50 and 25 ^L from 1 mg/mL in DMSO (< 00.05%, v/v) and complete to 1 mL using RPMI-1640 medium). 5-Fluorouracil (25 ^g/ mL) was prepared in 100 ^L DMSO and complete to 1 mL using RPMI-1640 medium. Ehrlich ascites Carcinoma (EAC) were derived from ascetic fluid from diseased mouse (purchased from National Cancer Institute, by National Medical Research Ethics Committee). Ascites fluid from the peritoneal cavity of the diseased mouse (containing Ehrlich cells) was aseptically aspirated. The cells were grown partly floating and partly attached in a suspension culture in RPMI 1640 medium, supplemented with 10% fetal bovine serum. They were maintained at 37 °C in a humidified atmosphere with 5% CO2 for 2 h. The viability of the cells was determined by the microscopical examination using a hemocytometer and using trypan blue stain (that stains only the dead cells).14 2. 4. Antioxidant Activity Screening Assay 2,2'-Azino-bis-3-ethylbenzthiazoline-6-sulfonic Acid Method For each of the investigated compounds, 2 mL of ABTS solution (60 ^M) was added to 3 mL MnO2 solution (25 mhg/mL), all prepared in 5 mL aqueous phosphate buffer solution (pH 7; 0.1 M). The mixture was shaken, centrifuged, filtered, and the absorbance of the resulting green-blue solution (ABTS radical solution) at X 734 nm was adjusted to approximately 0.5. Then, 50 ^L of 2 Mm solution of the tested compound in spectroscopic grade MeOH/phosphate buffer (1:1) was added. The absorbance was measured and the reduction in color intensity was expressed as inhibition percentage. L-Ascorbic acid was used as the standard antioxidant (positive control). Blank sample was run without ABTS and using MeOH/phosphate buffer (1:1) instead of the tested compounds. Negative control was run with ABTS and MeOH/phosphate buffer (1: 1) only.15,16 The inhibition ratio (%) was calculated using the following formula: (1) 3. Results and Discussion 3. 1. Chemistry In view of these observations and in continuation of our previous work in quinazoline chemistry,17,18 we synthesized some new heterocyclic compounds containing quinazoline moiety to evaluate their biological activities. The synthetic procedures adopted to obtain the target compounds are depicted in Schemes 1-10. The starting material, 1,4-dihydro-4-oxo-1,8-naphthyridine-3-carbo-hydrazide (1)19 when heated with benzoxazine derivative 212 in refluxing ethanol containing a catalytic amount of glacial acetic acid afforded 1,4-dihydro-N-(2-methyl-4-oxo quinazolin-3(4H)-yl)-4-oxo-1,8-naphthyridine-3-car-boxamide (3) (Scheme 1). The IR spectrum showed absorption bands at 32303238, 1668, 1671, 1614, and 1569 cm-1 corresponding to stretching vibrations of two NH, amidic carbonyl groups, ^-unsaturated ketone, and C=N groups. 1H NMR spectrum revealed singlet signal at 8 1.93 ppm due to methyl protons, in addition to the classical pattern of 1,8-naph-thyridine protons. The mass spectrum provided more evidence for the correct structure, which showed the molecular ion peak at m/z 347 (M+). Compound 1 when reacted with ethyl acetoacetate in refluxing ethanol containing a catalytic amount of glacial acetic acid afforded the acyclic intermediate ethyl 3-(2-(4-oxo-1,4-dihydro-1,8-naphthyridine-3-carbonyl) hydrazono)butanoate (4). Heating compound 4 in boiling ethanol containing sodium ethoxide leads to cyclization with the formation of the pyrazolone derivative 3-(3-meth-yl-5-oxo-4,5-dihydro-1H-pyrazole-1-carbonyl)-1,8-naph-thyridin-4(1H)-one (5) (Scheme 2). Abu-Melha : Synthesis and Biological Evaluation 924 Acta Chim. Slov. 2017, 64, 919-930 Scheme 2 Similarly, the hydrazide 1 when subjected to react with acetyl acetone in refluxing ethanol containing a catalytic amount of glacial acetic acid afforded the acyclic hydrazone derivative 4-oxo-N'-(4-oxopentan-2-ylidene)-1,4-dihydro-1,8-naphthyridine-3-carbohydrazide (6) which when refluxed with ethanol containing sodium ethoxide afforded the pyrazole derivative 3-(3,5-dimeth-yl-1H-pyrazole-1-carbonyl)-1,8-naphthyridin-4(1H)-one (7) (Scheme 3). Structures 4-7 were proved by elemental and spectral analyses. The IR spectra of compounds 4 and 6 in general showed absorption frequencies at 1567-1569, 1724, and 1689 cm-1 corresponding to C=N and two C=O due to ketonic (ester), and amidic carbonyl functional groups, while the 1H NMR spectrum of compound 4 showed a characteristic signal at S 0.91 ppm as a singlet signal for CH3 protons, S 1.38 ppm as a triplet signal for CH3 protons, S 2.35 ppm as a singlet signal for CH2 protons and at S 4.25 ppm as a quartet signal for CH2 protons besides the aromatic protons of pyridine ring at S 7.60-8.24 ppm, and a singlet signal at 8.61 ppm due to C2-H of naphthyridine ring. On the other hand, the 1H NMR spectrum of compound 6 showed three singlet signals at S 1.20, 2.11, 2.54 ppm corresponding to two methyl protons and CH2 protons, respectively. In addition, the mass spectrometry measurement gave m/z 316 (M+) and 286 (M+) corresponding to the molecular ion peaks of compounds 4 and 6, respectively. For the pyrazole derivative 5 the IR spectrum showed a new absorption band at 1669 cm-1 corresponding to a new amidic carbonyl and absorption frequency at 1606 cm-1 corresponding to C=N stretching frequency while the IR spectrum of compound 7 showed stretching frequency at 1567 cm-1 due to C=N functional group. 1H NMR of compound 5 showed two new singlet signals at S 1.22 and 2.07 ppm corresponding to methyl and methylene protons of pyrazolone moiety. On the other hand, the 1H NMR spectrum of compound 7 revealed three singlet signals at S 1.74, 1.79, and 6.08 ppm attribut- Scheme 3 Abu-Melha : Synthesis and Biological Evaluation Acta Chim. Slov. 2017, 64, 919-930 925 able to two methyl groups and methine protons of pyra-zole moiety. The mass spectra of compounds 5 and 7 showed their molecular ion peak at m/z 270 (M+) and 270 (M++2), respectively. In a similar manner, it was found that 2-hy-droxy-1-naphthaldehyde when reacted with hydrazide 1 in ethanol containing a catalytic amount of glacial acetic acid afforded the hydrazone derivative 8. Hydrazone 8 cy-clized to the corresponding pyrazole derivative 3-(3H-ben-zo[e]indazole-3-carbonyl)-1,8-naphthyridin-4-(1H)-one (9) when heated in ethanol containing a catalytic amount of piperidine or triethylamine (Scheme 4). Structures 8 and 9 were proved based on the correct analytical and spectral data. The IR of compound 8 showed bands at 3450 and 1574 cm-1 corresponding to the hydrox-yl group and stretching vibration of C=N function. Compound 9 was confirmed by analytical and spectral data, beside it was confirmed chemically by an alternative synthesis. Thus, when hydrazide 1 reacted with 2-hy-droxy-1-naphthaldehyde in refluxing ethanol containing a catalytic amount of pipridine or triethylamine or sodium ethoxide afforded directly the corresponding pyrazole derivative 9. The IR spectrum of compound 9 showed bands at 1579 cm-1 corresponding to C=N function, and the disappearance of the band around 3450 cm-1 corresponding to hydroxyl group which indicates that the hydroxyl group was involved in the cyclization process. The 1H NMR spec- trum showed a characteristic singlet signal at 5 8.20 ppm due to the pyrazole CH proton plus the classical 1H NMR pattern of the rest of 1,8-naphthyridine protons. The mass spectroscopic measurement gives an additional confirmation for compound 9 which showed the molecular ion peak at m/z 340 (M+). In addition, salicylaldehyde when heated directly with the hydrazide 1 in refluxing ethanol containing a catalytic amount of piperidine or triethylamine afforded the corresponding pyrazole derivative 3-(1H-indazole-1-car-bonyl)-1,8-naphthyridin-4(1H)-one (10) (Scheme 4). The IR spectrum of pyrazole derivative 10 showed a characteristic absorption band at 1569 cm-1 corresponding to C=N function and the disappearance of any band around the region 3400 cm-1 corresponding to hydroxyl group. 1H NMR spectrum showed singlet signal at 8 8.70 ppm attributable to the pyrazole proton. The mass spectro-scopic measurement gives an additional confirmation for compound 10 which showed the molecular ion peak at m/z 290 (M+). An interesting reaction was observed when hydrazide 1 was heated with malononitrile dimmer (11) in re-fluxing ethanol containing a catalytic amount of glacial acetic acid affording the pyrazolopyridine 3-(3,4,6-tri-amino-2H-pyrazolo[3,4-fo]pyridine-2-carbonyl)-1,8-naphthyridin-4(1H)-one (13) via the non isolable intermediate 12. The reaction may be preceded first by addi- Scheme 8 Abu-Melha : Synthesis and Biological Evaluation tion of NHNH2 moiety to the two cyano groups with the formation of a pyrazole ring. The reaction followed by the attack of the lone pair of electrons of NH2 of pyrazole ring to the cyano group as shown in the following mechanism affording the pyrazolopyridine 13 and not the other possibility 12a because the exocyclic double bond will be more stable in the Z-form due to less steric hindrance. This fact confirms that nucleophilic addition occurred from NH2 group and not imino group. (Scheme 5). The IR spectrum of compound 13 showed absorption bands at 3411, 3382 cm-1 due to NH2 functions, besides a broad band at 3228 cm-1 for NH groups, 1671 and 1608 cm-1 corresponding to amidic C=O, C=N and the disappearance of any band due to cyano functions at 2220 cm-1 which indicate that the cyano group was involved in the cyclization reaction. The 1H NMR showed four singlet exchangeable signals at 6 4.18, 9.81, and 11.60 ppm attributable to one amino group and three NH protons, respectively. The mass spectrum showed the molecular ion peak at m/z 336 (M+). The hydrazide 1 when heated with dimethylth-iomethylene malonate (14) in the presence of ethanol containing a few drops of glacial acetic acid afforded the pyrazole derivative 15 (Scheme 6). The reaction proceeds according to the proposed following mechanism. The IR spectrum of compound 15 showed absorption bands at 3413, 3382 cm-1 corresponding to NH2 group, 3226 cm-1 due to NH function, 1670 and 1608 cm-1 corresponding to amidic C=O and C=N and a characteristic absorption band at 2220 cm-1 corresponding to CN group. The 1H NMR revealed a characteristic singlet signal of thiomethyl group at 8 1.96 ppm and three exchangeable Scheme 6 Abu-Melha : Synthesis and Biological Evaluation Acta Chim. Slov. 2017, 64, 919-930 927 Scheme 7 singlet signals at 8 4.52, 9.22, and 10.66 ppm attributable to NH2 and NH protons. The mass spectrum showed the molecular ion peak at m/z 327 (M++1). In addition, when compound 15 was subjected to react with p-anisidine in ethanol containing a catalytic amount of triethlylamine afforded 3-imino-5-(4-methoxy-phenylamino)-1-(4-oxo-1,4-dihydro-1,8-naphthyri-dine-3-carbonyl)-2,3-dihydro-1H-pyrazole-4-carbonitrile (16) (Scheme 7). Also, it has been found that when hydrazide 1 reacted with p-nitrobenzylidene malononitrile and p-chloro-benzylidene malononitrile in ethanol containing a few drops of glacial acetic acid afforded the pyrazole derivatives 5-amino-3-(4-nitrophenyl)-1-(4-oxo-1,4-dihydro-1,8-naphthyridine-3-carbonyl)-2,3-dihydro-1H-pyra-zole-4-carbonitrile (17) or 5-amino-3-(4-chlorophenyl)-1-(4-oxo-1,4-dihydro-1,8-naphthyridine-3-carbon-yl)-2,3-dihydro-1H-pyrazole-4-carbonitrile (18), respectively (Scheme 8). Structures 17 and 18 were proved based on the analytical and spectral data. The IR spectrum of both compounds showed, in general, characteristic absorption bands at 1582 and 1550 cm-1 corresponding to stretching frequencies of C=N and C=C functions, respectively. The 1H NMR of both compounds showed a characteristic sin- glet signal at 8 5.29 and 5.69 ppm attributable to methyne protons. The mass spectra of compounds 17 and 18 showed the molecular ion peak at m/z 403 (M+) and 392 (M+-1), respectively. Moreover, when hydrazide 1 reacted with cyanoacet-amide in refluxing ethanol in the presence of a catalytic amount of glacial acetic acid afforded the diaminopyrazole derivative 3-(3,5-diamino-1H-pyrazole-1-carbonyl)-1,8-naphthyridin-4(1H)-one (19). The same compound was obtained when the hydrazide 1 was subjected to react with malononitrile under the same reaction conditions (Scheme 9). Structure 19 was established based on both analytical and spectral data. The IR spectrum showed the classical pattern for carbonyl groups which appear at 1668 and 1634 cm-1, and showed the stretching vibration of C=N and C=C functions at 1612 and 1570 cm-1, respectively. On the other hand, the amino groups appeared as a tautomeric equilibrium with amino-imino groups and appeared at 3441 and 3232 cm-1, respectively. 1H NMR of compound 19 revealed two exchangeable signals at 8 4.01 and 10.20 ppm due to two NH2 and NH protons, in addition to two singlet signals at 8 6.37 and 8.07 ppm for CH-pyrazole and C2-H of naphthyridine ring, besides aromatic protons of pyridine ring at 8 7.72-8.31 ppm. The mass spectrum gave Scheme 8 Abu-Melha : Synthesis and Biological Evaluation 928 Acta Chim. Slov. 2017, 64, 919-930 additional evidence for structure 19 which showed its molecular ion peak at m/z 270 (M+). 3. 2. Pharmacology 3. 2. 1. Cytotoxicity Against Ehrlich Ascites Carcinoma Effect of Drugs on the Viability of Ehrlich Ascites Cells In Vitro The synthesis, antitumor evaluation and QSAR studies of novel pyrazol derivatives employed against Ehrlich Ascites Carcinoma (EAC, in vitro) cells are described. These novel analogues were molecularly designed with the goal of having significant potent cytotoxic effect against EAC cells. Pyrazoles and related analogues were tested for cyto-toxicity against EAC in vitro. EAC cells were used because they have a very well known established model of activi-ty.20 Results for the ED25 value of the active compounds are summarized in Table 1. The data showed clearly that compound 15 showed moderate activity (~45%) compar- ing with the drug reference (5-FU, 98% activity). The rest of compounds showed weak activity. Thus, it would appear that introducing thiomethyl tautomeric moiety enhanced the cytotoxic properties. Comparing the obtained cytotoxic activity of tested compounds in this study, the following structure-activity relationships (SAR) were postulated: 1) Compound 15 showed a mild cytotoxic activity (~45%), this may be due to the presence of thiomethyl and cyano groups which have toxic activity in nature. 2) All pyrazole derivatives showed weak activity at ED25. Thus the position and nature of substituents in the structure of pyrazole derivatives seems to modulate cyto-toxic activity. 3. 2. 2. ABTS Antioxidant Activity Screening The antioxidant activity assay employed here is one of the several assays that depends on measuring the consumption of stable free radicals, i.e. evaluates the free rad- Table 1. In vitro cytotoxicity of pyrazol against Ehrlich Ascites Carcinoma Compound No. EAC Assay Dead cells (%) ED25 ^L (1 mg/mL) Control 0 5-FU 98 Compound No. EAC Assay Dead cells (%) ED25 ^L (1 mg/mL) Control 0 5-FU 98 3 0 15 45 7 28 16 19 8 20 17 21 9 14 18 17 10 11 19 14 13 23 Where ED25 is the effective dose at 25 |iL of the compounds used. The dead % refers to the % of the dead tumor cells and 5-Flu is 5-fluorouracil as a well known cytotoxic agent. Abu-Melha : Synthesis and Biological Evaluation Acta Chim. Slov. 2017, 64, 919-930 929 ical scavenging activity of the investigated component. The methodology assumes that the consumption of the stable free radical (X') will be determined by the reaction as follows: (2) The rate and/or the extent of the process measured in terms of the decrease in X' concentration, would be related to the ability of the added compounds to trap free radicals. The decrease in color intensity of the free radical solution due to scavenging of the free radical by the antioxidant material is measured colorimetrically at a specific wavelength. The assay employs the radical cation derived from 2,2'-azino-bis(3-ethyl benzthiazoline-6-sulfonic acid) (ABTS) as a stable free radical to assess antioxidant potential of the isolated compounds and extracts. The advantage of ABTS-derived free radical method over other methods is that the produced color remains stable for more than one hour and the reaction is stoichiometric. The inhibition ratio (%) was calculated using the following formula: Inhibition = ¿(control) - ¿(test) ¿(control) x 100 (3) The antioxidant activity of some newly synthesized compounds was evaluated by ABTS method.21 The data in Table 2 showed clearly that compound 15 demonstrated moderate antioxidant activities. Thus, it would appear that introducing sulfur atoms and the presence of thiomethyl tautomeric equilibrium enhances the antioxidant properties of 1,8-naphthyridine derivatives. By comparing the results obtained of antioxidant of the compound reported in this study to their structures, the following SAR was postulated: compound 15 was nearly inpotent to vitamin C which may be attributed to the presence of amino and imino groups which trap the free radical X. 4. Conclusion This work aimed to synthesize a new series of pyrazole derivatives containing 1,8-naphthyridine ring via carbox-amide linkage. All the structures of the synthesized compounds were confirmed by different spectroscopic data and screened for their in vitro cytotoxicity against EAC and an-tioxidant activity. Results obtained show that compound 15 displayed the best cytotoxicity and antioxidant activity. 5. References 1. H. Egawa, T. Miyamota, A. Minamida, Y. Nishimura, H. Oka-da, H. Uno, T. Motosumota, J. Med. Chem. 1984, 27, 15431548. D01:10.1021/jm00378a004 2. K. Kohima, M. Motoyoshi, Japan Kotai Tokyo JP 1988, 01,100,603; Chem. Abstr. 1988, 109, 189591. 3. C. S. Cooper, P. L. Klock, D. T. W. Chu, D. J. Hardy, R. N. Swanson, J. J. Plattner, J. Med. Chem. 1992, 35, 1392-1398. DOI: 10.1021/jm00086a007 4. J. Nezval, K. Halocka, Experientia 1967, 23, 1043-1044. D0I:0.1007/BF02136439 5. K. Tomita, Y. Tsuzuki, K. Shibamori, M. Tshima, F. Kajikawa, Y. Sato, S. Kashimoto, K. Chiba, K. Hino, J. Med. Chem. 2002, 45, 5564-5575. D0I:10.1021/jm010057b 6. Y. Tsuzuki, K. Tomita, K. Shibamori, Y. Sato, S. Kashimoto, K. Chiba, J. Med. Chem. 2004, 47, 2097-2109. DOI: 10.1021/jm0304966 7. S. K. Srivastava, M. Jaggi, A. T. Singh, A. Madaan, N. Rani, M. Vishnoi, S. K. Agarwal, R. Mukherjee, A. C. Burman, Bioorg. Med. Chem. Lett. 2007, 17, 6660-6664. DOI: 10.1016/j.bmcl.2007.08.006 8. M. Lovu, C. Zalaru, F. Dumitrascu, C. Draghici, M. Moraru, E. Criste, Farmaco., 2003, 58, 301-307. http://www.sciencedirect.com/science/article/pii/ S0014827X02000149 Table 2. Antioxidant assay for some prepared new compounds. ¿(control) — ¿(test) Compound (%)lnhibition = ------xl00 5j0> ¿(control) Absorbance of samples ABTS (% Inhibition) Control of ABTS 0.47 0% Ascorbic acid 0.06 87.0% 3 0.42 10.6% 7 0.31 34.0% 8 0.35 25.5% 9 0.36 23.4% 10 0.37 21.3% 13 0.31 34.0% 15 0.19 59.6% 16 0.35 25.5% 17 0.34 27.6% 18 0.35 25.5% 19 0.34 27.6% Abu-Melha : Synthesis and Biological Evaluation 930 Acta Chim. Slov. 2017, 64, 919-930 9. R. N. Mahajan, F. H. Havaldar, P. S. Fernandes, Indian J. Chem. Soc., 1991, 68, 245-249. 10. P. G. Baraldi, S. Manfredini, R. Romagnoli, L. Stevanato, A. N. Zaid, R. Manservigi, Nucleos. Nucleot. Nucleic Acid, 1998, 17, 2165-2171. DOI: 10.1080/07328319808004307 11. K. M. El-Mahdy, A. M. El-Kazak, M. Abdel-Megid, M. Seada, O. Farouk, Acta Chim. Slov., 2016, 63, 18-25. D0I:10.17344/acsi.2015.1555 12. C. Parkanyi, D. S. Schmidt, J. Heterocycl. Chem., 2000, 37, 725-729. D0I:10.1002/jhet.5570370409 13. K. Zaima, I. Koga, N. Iwasawa, T. Hosoya, Y. Hirasawa, T. Kaneda, I. S. Ismail, N. H. Lajis, H. Morita, J. Nat. Med., 2013, 67, 9-16. D0I:10.1007/s11418-012-0638-y 14. A. B. A. El-Gazzar, A. M. S. Youssef, M. M. Youssef, A. A. Abu-Hashem, F. A. Badria, Eur. J. Med. Chem. 2009, 44, 609-624. D0I:10.1016/j.ejmech.2008.03.022 15. F. Badria, M. Ameen, M. Akl, Z. Naturforschung, 2007, 62(9/10), 656-660. DOI:10.1515/znc-2007-9-1005 16. A. A. Fadda, A. El-Shafei, A. M. Khalil, T. A. E. Ameen, F. A. Badria, Bioorg. Med. Chem. 2009, 17, 5096-105. D0I:10.1016/j.bmc.2009.05.053 17. S. S. El-Morsy, A. A. Fadda, M. S. El-Houssini, Indian J. Chem. Soc. 1988, 65, 699-701. 18. A. A. Fadda, H. A. Etman, F. A. Amer, M. Barghout, K. S. Mohamed, J. Chem. Tech. & Biotech. 1995, 62, 170-177. D0I:10.1002/jctb.280620210 19. A. A. Fadda, A. M. El Defrawy, S. A. El-Hadidy, Amer. J. Org. Chem. 2012, 2, 87-96. D0I:10.5923/j.ajoc.20120204.03 20. K. Karrer, J. R. Rtjbini, Pharmacology, 1965, 13, 124-130. DOI 1:0.1159/000135602 21. E. Lissi, B. Modak, R. Torres, J. Escobar, A. Urzua, Free Radical Res. 1999, 30, 471-477. DOI:10.1080/10715769900300511 Povzetek S pomočjo 1,4-dihidro-4-okso-1,8-naftiridin-3-karbohidrazida (1) kot izhodne spojine smo pripravili serijo substitu-iranih 1,8-naftiridinskih derivatov ter jih uporabili kot citotoksične in antioksidativne spojine. Spojino 1 smo reagirali z različnimi reagenti in tako pripravili ustrezne 3-heteroarilkarbonil-1,8-naftiridinske derivative 3-19, ki smo jih testirali za in vitro citotoksičnost proti Ehrichovim ascitnim karcinomom in za antioksidativno aktivnost. Spojina 15 je pokazala najboljše citotoksične in antioksidativne lastnosti. Abu-Melha : Synthesis and Biological Evaluation DOI: 10.17344/acsi.2017.3619 Acta Chim. Slov. 2017, 64, 931-937 ¿^creative tS1 commons Scientific paper Preparation and Characterization of Chromium Doped Ni-Cu-Zn Nano Ferrites Bajarang Laxman Shinde,1 Laxman Appa Dhale,2 Venkat S. Suryavanshi3 and Kishan Shankarrao Lohar2* 1 Department of Chemistry, Waghire College, Saswad Dist: Pune, 412301 (M.S.) India 2 Department of Chemistry, Shrikrishna Mahavidyalaya, Gunjoti, 413606, Dist: Osmanabad (M.S.) India 3 Department of Chemistry, Shri Chatrapati Shivaji College, Omerga, Dist: Osmanabad 413 613 (M.S.) India * Corresponding author: E-mail: kslohar@rediffmail.com Received: 07-06-2017 Abstract Chromium doped Ni-Cu-Zn nano ferrites with chemical formula Ni„.Cu„.Zn„ Je. Cr O. (x = 0.0, 0.2, 0.4, 0.6, 0.8, and I 0.2 0.2 0.6 2-x x 4 v 1.0) were prepared by using sol-gel auto combustion method. The prepared precursors of Chromium substituted Ni-Cu-Zn ferrites were sintered at 500 °C for 4h. Compositional stoichiometry were confirmed from EDAX patterns. The XRD data revealed that the all samples possess a single phase cubic spinel structure. The Lattice constant, X-ray density, hopping lengths and crystallite size determined from XRD data decreases with increase in Cr3+ concentration. The IR spectra show two major absorption bands, high frequency band v1 ~ 600 cm1 and low frequency band v2 ~ 450 cm1 attributed to the stretching vibration of tetrahedral and octahedral sites respectively. The surface morphology of the prepared samples was studied by Scanning Electron Microscopy and Transmission Electron Morphology. Keywords: Spinel ferrite, Sol-gel auto-combustion; Thermal analysis, Morphology, X-ray diffraction 1. Introduction Nano ferro spinels are of great interest for addressing relationship between physical properties and their crystal structure. Due to their reduced sizes, these nanopar-ticles may possess novel and/or improved properties in comparison to the bulk materials. This has renewed interest to study different properties of pure and mixed spinel ferrite systems in nanocrystalline regime. Transition metal ferrites both doped and undoped are attractive candidates for a wide range of applications including catalysis,1-4 and several devices such as antennas, permanent magnets, memory storage devices, microwave devices, telecommunication, computer etc.5 The polycrystalline ferrites such as Ni-Cu-Zn ferrites have very important structural properties dependent on several factors such as method of preparation, substitution of cations, sintering temperature, sintering time, sintering atmosphere, porosity and micro-structure.6,7 Ni-Cu-Zn ferrites were considered as one of the most versatile magnetic materials to manufacture Multilayer chip inductors (MLCIs) mainly because of their high electrical resistivity, low sintering temperature and high permeability.89 Recently due to better magnetic properties, controlled shape of nanocrystalline Ni-Cu-Zn ferrites have been the dominant materials for MLIC at high frequency and low sintering temperature.10-16 These oxides can be sintered at relatively low temperatures with a wide range of compositions. Recently, several methods were used to synthesize highly crystalline and uniformly sized magnetic nanoparticles of Ni-Cu-Zn ferrite.17-21 Among these methods, the sol-gel method has gained scientific and technological importance during the last three decades.22-25 The sol-gel auto-combustion synthesis method have many advantages as compared to the conventional methods such as low temperature processing and/or better homogeneity for the synthesis of multi-component materials. Considering the importance of Ni-Cu-Zn ferrite, we investigated the preparation of chromium doped Ni-Cu-Zn nano ferrite by sol-gel auto-combustion method using citric acid as fuel at low temperature with a view to study the influence of the substitution of Cr3+ ion on the structural properties of the system. Shinde et al.: Preparation and Characterization 932 Acta Chim. Slov. 2017, 64, 931-937 2. Experimental 2. 1. Materials and Synthetic Procedure Nanocrystalline chromium substituted Ni-Cu-Zn ferrites, with composition Ni02Cu02Zn06Fe2-xCrO4 (x = 0.0, 0.2, 0.4, 0.6, 0.8 and 1.0) were synthesized by the sol-gel auto-combustion method. Analytical reagent grade nickel nitrate (Ni(NO3)2 • 6H2O), copper nitrate (Cu(NO3) • 6H2O), zinc nitrate (Zn(NO3)2 • 6H2O), chromium nitrate (Cr(NO3)3 • 9H2O) and iron nitrate (Fe(NO3)3 • 9H2O) were used for synthesis. Citric acid (C6H8O7 • H2O) was used as fuel. The reaction procedure was carried out in an air atmosphere without the protection of inert gases. Metal nitrates and citric acid were used in the molar ratio 1:3. The metal nitrates were dissolved in desired stoichiometric proportions together in the minimum amount of double-distilled water to obtain a clear solution. An aqueous solution of citric acid was mixed with the metal-nitrate solution, and ammonia solution was slowly added to adjust the pH ~ 7. The mixed solution was placed on a hot plate with continuous stirring at 90 °C. During evaporation, a very viscous brown gel was formed. When all of the water molecules were removed from the mixture, the viscous gel began to froth. After few minutes, the gel was ignited and burnt with glowing flints. The decomposition reaction continued until the entire citrate complex was consumed. The auto-combustion was completed within a minute, yielding brown-colored ash as the precursor. Sintering temperature was determined from TGA / DTA and prepared powders of all the precursor samples were sintered at 500 °C for 4h to obtain the final product. 2. 2. Characterization of Samples Simultaneous thermo gravimetric (TGA) and differential thermal analysis (DTA) of precursors were carried on SDT Q600 V20.9 Build 20 instrument in air atmosphere at heating rate 10 °C / min, within temperature range 25 °C to 800 °C. The compositional stoichiometry was determined by energy dispersive X-ray spectroscopy (EDAX, Inca Oxford, attached to the SEM).The crystallo-graphic structures were identified by X-ray powder diffraction with Cu-Ka radiation (X = 1.5405 A) by Phillips X-ray diffractometer (Model 3710). The infrared spectra of all the samples were recorded at room temperature in the range 300 to 800 cm-1 using Perkin Elmer infrared spectrophotometer. Morphology and structure of the powder samples were studied on JEOL-JSM-5600 N Scanning Electron Microscope (SEM) and on Philips (model CM 200) Transmission Electron Microscope (TEM). Figure 1: TGA-DTA plot for precursor of Ni02Cu02Zn06Fe2 xCrxO4 (x = 0.0) . . . x x two weight loss steps. The first weight loss step in the temperature range of 30-100 °C, corresponding to endothermic peak around 80 °C, which is due to the loss of coordination water in the precursor. The second weight loss step observed in the temperature range of 310-425 °C corresponding to the exothermic peak around 380 °C, is as a result of the decomposition of unreacted starting citric acid remained after combustion. The released heat in the process of exothermic decomposition has been observed to be sufficient for complete conversion of the metal precursors to metal oxides.26 Figure 2. TGA-DTA plot for precursor of Ni0.2Cu0.2Zn0.6Fe2_ Cr O (x = 1.0) x x 4 x ' Almost no weight loss was observed above 425 °C, representing the presence of only Ni0 2Cu0 2Zn0 6Fe2-xCrxO4 ferrites in this temperature range. 3. Results and Discussion 3. 1. Thermal Analysis (TGA / DTA) The typical TGA/DTA curves of Ni0.2Cu0.2Zn0.6 Fe2-Cr O4 (x = 0.0; Figure 1 and x = 1.0; Figure 2), illustrates 3. 2. Structural Analysis 3. 2. 1. Elemental Analysis The compositional stoichiometry of Ni02Cu02Zn06 Fe2-xCrxO4 ferrite nanoparticles was determined by EDAX. Shinde et al.: Preparation and Characterization Acta Chim. Slov. 2017, 64, 931-937 933 Figure 3. Plots of Observed and Theoretical elemental % versus composition (x) Figure 4. The XRD patterns for Ni„ ,Cu„ Zn„ .Fe, Cr O, ° r 0.2 0.2 0.6 2-x x 4 The EDAX confirmed the homogeneous mixing of the Fe, Ni, Cu, Zn, Cr and O atoms in pure and doped ferrite samples. Figure 3 shows the plots of observed and theoretical percentage of Fe, Ni, Cu, Zn, Cr and O values versus composions (x). The observed elemental % (obtained from EDAX) values are in close agreement with theoretical % (the starting composition used for the preparation) values. The EDAX analysis is affected by the surface crystalline defects of the nanoparticles. This can also be taken into account to explain the difference between the values of the atomic ratio as determined by EDAX and the expected value.2728 3. 2. 2. X-ray Diffraction X-ray diffraction (XRD) patterns of the Ni0 2Cu02Zn06Fe2-xCrxO4 (x = 0.0, 0.2, 0.4, 0.6, 0.8 and 1.0) spinel ferrite system are shown in Figure 4. The XRD patterns confirmed the formation of single phase cubic spinel structure without additional peaks corresponding to any other phases. The crystal structures of Ni-Cu-Zn ferrite are identified as cubic spinel (space group: fd3m) with the corresponding (220), (311), (222), (400), (422), (333) and (440) planes. Lattice parameter (a) of all the samples was determined by using the following equation: 29 a=dVh2+k2+]2 (1) Lattice constant (a) values with an accuracy of ± 0.002 A were calculated for each sample using XRD pattern and are listed in Table 1. It is observed that the lattice constant (a) decreases with increase in Cr3+ concentration. In the present ferrite system Fe3+ ions (0.67A) ions are replaced by the relatively small Cr3+ ions (0.64A). The decrease in the lattice constant is related to the difference in ionic radii of Fe3+ and Cr3+. 30-32 The X-ray density of all the samples was obtained by the following relation: (2) Where, '8' is formula unit, 'M' is molecular weight, 'N' is Avogadro's number, 'a' is lattice constant. The values of X-ray density are presented in table 1. It is seen from Table1that, like lattice parameter, X-ray density also decreased with increasing Cr3+ content 'x'. The decrease in X-ray density is attributed to decrease in lattice constant. It is observed that X-ray density increase for x = 1.0. This is related to the molecular weight of the sample overtakes the volume (a3). The average crystallite diameter 'Dxrd' of powder estimated from the most intense (311) peak of XRD and using the Debye-Scherrer method, 29 (3) Where, P1/2 is the full width of half maximum in (28), '8' is the corresponding Bragg angle and C = 0.9.The values of the crystallite size are given in Table 1. The crystallite size is decreases from 30.3 nm to 8.9 nm with increasing Cr3+substitution. The decrease in the crystallite size indicates that the addition of the Cr3+ obstruct the crystal growth.33 Due to the surface temperature and the molecular concentration at the surface of the crystal results into decrease in the crystal growth. The percentage porosity 'P' of all the samples was calculated using the values of X-ray density and bulk density: ci. -X. ¡00', (4) Shinde et al.: Preparation and Characterization 934 Acta Chim. Slov. 2017, 64, 931-937 Table 1. Lattice constant (a), X-ray density (dx) and hopping lengths (LA) and (LB), particle size (Dxrd) and porosity (p) of Ni0.2Cu0.2Zn0.6Fe2-iCriO4 ' " Composition 'a' 'd' lb P Particle size (nm) 'x' (A) (g/cm3) (A) (A) (%) Dxrd TEM 0.0 8.418 5.329 3.645 2.976 25.4 30.3 30.6 0.2 8.415 5.316 3.644 2.975 26.1 24.9 25.2 0.4 8.406 5.317 3.640 2.972 27.7 19.1 19.5 0.6 8.394 5.323 3.634 2.967 28.7 16.4 15.9 0.8 8.389 5.314 3.632 2.966 28.4 12.6 12.8 1.0 8.367 5.339 3.623 2.958 32.0 8.9 9.5 Where, dB and dx are the bulk and X-ray densities respectively. It is observed that porosity increased from 25.4% (x = 0.0) to 32% (x = 1.0) with the Cr3+ substitution. In the present series of Ni0 2Cu0 2Zn0 6Fe2-xCrxO4, both the molecular weight and the volume of the unit cell decrease with increasing Cr3+ substitution, but the rate of the decrease of molecular weight is more than that of volume. Therefore, the density decreases with Cr3+ substitution, this resulted in increase in porosity. Apart from this, the increase in porosity is mainly attributed to decrease in crystallite size, which increases the grain boundaries of the particle and accordingly the porosity.27 The distance between the magnetic ions i.e. hopping lengths (La and LB) in the tetrahedral A-site and octahedral B -site was calculated using following relation: 34 (5) (6) It has been observed from Table 1 that the hopping lengths (La and LB) decreased with Cr3+ substitution. Decrease in both the hopping lengths with Cr3+ substitution is due to the decrease in lattice constant. 3. 2. 3. Infrared Spectroscopy The infrared spectroscopy is an important tool to probe various ordering phenomena that provide information on the position of ions and vibrational modes of crystals. The substitution of metal ion in ferrites may give rise to structural change within the unit cell without affecting the structure as a whole. Such structural changes brought about by metal ions strongly influence the lattice vibrations.35 The IR spectra as shown in Figure 5, were recorded at room temperature in the frequency range 300-800 cm-1. For ferrites, generally it is found two assigned absorption bands appear around 600 cm-1: v1, which is attributed to stretching vibration of tetrahedral groups Fe3+-O2- and around 400 cm-1: v2, which is attributed to the octahedral groups complex Fe3+-O2-. It is observed from Table 2 and Figure 6 that the higher frequency band (v1) is appeared in the range of 568-610 cm-1 whereas lower frequency band (v2) is appeared in the range of 388-491 cm-1. These bands are characteristics features of spinel structure. It explains that the normal mode of vibration of tetrahedral cluster is higher than that of octahedral cluster. It should be attributed to the shorter bond length of tetrahedral cluster and longer bond length of octahedral cluster.35 Figure 5. IR spectra for the series Ni02Cu02Zn06Fe2 xCrxO4 The differences in band position and intensity with Cr3+ substitution may be related to; the decrease in the Fe^ -O22- intermolecular distance increases the metal-oxygen vibrational energies, which arises from the decrease in the number of Fe3+- O^- complexes caused by the increase of the number of Cr3+- O^complexes35 and, formation of Me2+- O22- at A and B sites (Me = Ni2+, Cu2+, Zn2+). The force constants corresponding to the tetrahedral and octahedral complexes are calculated by using the standard formulae given below: 36 Kt = 7.62 x M1 x v\ x 10"2 Kn = 10.62 x^xuï x 10"2 (7) (8) Shinde et al.: Preparation and Characterization ... Acta Chim. Slov. 2017, 64, 931-937 935 Table 2. Band position (vt and v2), Force constant (K0 and K^ and Bond length (RA and RB) of system Nio.2CUo.2Zno.6Fe2-ICri04 Comp. Band positions Force constants Bond lengths 'x' v1 (cm-1) v2 (cm-1) KO x 105 Kt x 105 Ra(A) Rb(A) (dyne/cm) (dyne/cm) 0.0 568.1 388.9 0.9380 1.6075 3.864 3.065 0.2 574.6 418.1 1.0834 1.8388 3.862 3.064 0.4 585.8 424.7 1.1182 1.8978 3.861 3.063 0.6 597.2 447.2 1.2399 2.0884 3.859 3.063 0.8 603.9 474.2 1.3937 2.3203 3.857 3.062 1.0 610.5 491.9 1.4999 2.4789 3.855 3.061 Where, KO is the force constant of octahedral site, Kt is the force constant of tetrahedral site, Mj molecular weight of tetrahedral site, M2 molecular weight of octahedral site, vj the corresponding center frequency on tetrahedral site, and v2 the corresponding center frequency on octahedral site. The molecular weights Mj and M2 for each sample are calculated from the cation distribution. The force constant is the second derivative of the potential energy with respect to the site radius with the other independent parameters kept constant. The bond lengths RA and RB have been calculated using the formula given by Gorter.37 The molecular weights of the tetrahedral Mj and octahedral M2 sites have been calculated using the cation. The values of Ra, Rb and the force constants Kt and KO are listed in Table: 2. 3. 2. 4. Scanning Electron Microscopy (SEM) Typical Scanning electron micrograph (SEM) of the sample x = 0.6 is shown in Figure 6. Each composition is characterized by a typical porous structure and small rounded grains. It is observed from SEM images that the structure is affected by the Cr3+ substitutions. It can be observed from the SEM images that the prepared samples are amorphous and porous in nature. The decrease in the grain size and an increase in porosity are observed with increasing Cr3+ substitutions. The observed changes in grain size suggest that the substitution of Cr3+in Ni-Cu-Zn ferrite solid solution occurs during sol-gel combustion process which enables a better homogeneity in the powders and, hence, a more controlled microstructure is obtained. 3. 3. 5. Transmission Electron Microscopy (TEM) TEM image of the typical sample x = 0.4 is presented in Figure 7. Figure 6. Scanning electron micrograph of Ni02Cu02Zn06Fe2 xCrxO4 (x = 0.6) ...xx Figure 7. TEM image for Ni02Cu02Zn06Fe2- xCrxO4(x = 0.4) The particles were well distributed and slightly agglomerated. The agglomeration is the indication of high reactivity of the prepared sample with the heat treatment and it may also be come from the magnetostatic interaction between particles. Since Cr3+ ions provide stability to the Ni-Cu-Zn lattice; it is believed that they also inhibit the process of grain growth through coagulation at the stage where the sol-gel is formed and hence samples of small particle size are produced.38 Selected area electron diffraction (SAED) patterns of the respective TEM image is also shown in Figure 8. Shinde et al.: Preparation and Characterization ... 936 Acta Chim. Slov. 2017, 64, 931-937 Figure 8. SAED pattern for Ni02Cu02Zn06Fe2- ICriO4 (x = 0.4) The Bragg' rings observed in these SAED patterns corresponding to specific 'd' values, that match perfectly with the 'd' values calculated from XRD. The superimposition of the bright spot with Debye ring pattern indicates polycrystalline nature of the sample which is in accordance with XRD. Like XRD; SAED also confirmed that the sample does not possess any type of impurity or second phase. 0 10 20 30 40 50 Particle Size {rim} Figure 9. Particles size distribution for Ni„,Cu„,Zn„,Fe, Cr O. (x = o 0.2 0.2 0.6 2- x x 4 v 0.4) (based on TEM micrograph, Figure 7) Particles size distributions for NLCu^nJe, 0.2 0.2 0.6 2- xCrxO4 (x = 0.4) shown in figure 9. Nanocrystalline particles observed were in the range 6-55 nm. Particles with size between 10-30 nm were most abundant (more than 70%). The samples particle size obtained from TEM are in close agreement to the size calculated from the De-bye-Scherrer method. 4. Conclusion Nanocrystalline Ni02Cu02Zn06Fe2-xCr04 ferrites with x = 0.0, 0.2, 0.4, 0.6, 0.8, and 1.0 were prepared by sol gel auto combustion method using citric acid as a fuel. The EDAX pattern confirmed the homogeneous mixing in pure and doped ferrite samples with desired composition. Structural analysis with XRD reveals that the system confirms the formation of single phase cubic spinel structure of Chromium doped Ni-Cu-Zn Nano Ferrites. Lattice constant and X-ray density decreased with Cr3+ substitution. The crystallite size is observed in the range of 8.9-30.3 nm, which is in close agreement with crystallite size obtained from TEM. It is concluded from IR spectra that higher frequency band (v1) is appeared in the range of 568-610 cm-1 whereas lower frequency band (v2) is appeared in the range of 388-491 cm-1 confirming characteristics features of spinel structure. It is observed from SEM that the prepared samples are amorphous and porous in nature. TEM images of the samples confirmed the particle size of obtained ferrite samples is in nm dimensions. The prepared Chromium doped Ni-Cu-Zn Nano Ferrites may be used as catalyst for organic transformations and in several devices such as antennas, memory storage devices, microwave devices etc. 5. References 1. Mathew, T., Shylesh, S., Devassy, B. M., Vijayaraj, et al., Applied Catalysis A: General, 2004, 273(1), 35-45. DOI:10.1016/j.apcata.2004.06.011 2. Lu, H. C., Chang, J. E., Vong, W. W., Chen, et al., International Journal of Physical Sciences, 2011, 6(4), 855-865. 3. Lim, C. W., Lee, I. S., Nano Today, 2010, 5(5), 412-434. DOI:10.1016/j.nantod.2010.08.008 4. Kooti, M., Afshari, M., Scientia Iranica, 2012, 19(6), 19911995. DOI:10.1016/j.scient.2012.05.005 5. H. Su, H. Zhang, X. Tang, Z. Zhong, Y. Jing, Mater. Sci. Eng. B, 2009, 162, 22-25. DOI:10.1016/j.mseb.2009.01.030 6. Wang, Sea-Fue, et al., Journal of Magnetism and Magnetic Materials, 2015, 394, 470-476. DOI:10.1016/j.jmmm.2015.07.037 7. Ahmadpour Ghader, Ali Ghasemi, Ebrahim Paimozd, Current Nanoscience, 2012, 8.4, 598-602. DOI: 10.2174/157341312801784393 8. T. Nakamura, J. Magn. Magn. Mater., 1997, 168, 285-291. DOI:10.1016/S0304-8853(96)00709-3 9. R. Lebourgeois, S. Duguey, J. P. Ganne, J. M. Heintz, J. Magn. Magn. Mater., 2007, 312, 328-330. DOI:10.1016/j.jmmm.2006.10.698 10. J. H. Jean, C. H. Lee, W. S. Kou, J. Am. Ceram. Soc, 1999, 82, 343. DOI:10.1111/j.1551-2916.1999.tb20068.x 11. V. V. Awati, S. M. Rathod, Sagar E. Shirsath, Maheshkumar L. Mane, J. Alloys Compds, 2013, 553, 157-162. DOI:10.1016/j.jallcom.2012.11.045 Shinde et al.: Preparation and Characterization Acta Chim. Slov. 201?, 64, 931-937 937 12. T. Krishnaveni, B. Rajini Kanth, V. Seetha Rama Raju, S.R. Murthy, J. Alloys Compds, 2006, 414, 282-286. DOI:10.1016/j.jallcom.2005.07.029 13. T. Krishnaveni, B. Rajini Kanth, et al., Journal of Alloys and Compounds, 2006, 414, 282-286. DOI:10.1016/j.jallcom.2005.07.029 14. Yen-Pei Fu, Ko-Yin Pan, Cheng-Hsiung Lin, Materials Letters, 2002, 57, 291-296. DOI:10.1016/S0167-577X(02)00780-2 15. Li, Bo, et al., Materials Science and Engineering: B, 2003, 99. 1, 252-254. DOI:10.1016/S0921-5107(02)00489-0 16. Zhu, Haikui, et al., Ceramics International, 2014, 40.7, 1098510989. DOI: 10.1016/j.ceramint.2014.03.102 17. M. A. Gabal, Y. M. Al Angari, A. Y. Obaid, Advanced Powder Technology, 2014, 25, 457-461. DOI:10.1016/j.apt.2013.07.009 18. M. Epifani, E. Melissano, G. Pace, M. Schiopa, J. Europ. Cer-am. Soc, 2007, 27, 115-123. DOI: 10.1016/j.jeurceramsoc.2006.04.084 19. N. L. Freitas, J. P. Continho, M. C. Silva, H. L. Lira, et al., Materials Science Forum, 2010, 660, 943-947. DOI:10.4028/www.scientific.net/MSF.660-661.943 20. Tiago Pinheiro Braga, Bárbara Maria Campos Sales, et al., Catal. Sci. Technol., 2011, 1, 1383-1392. DOI:10.1039/c1cy00176k 21. Y. Ahn, E. J. Choi, S. Kim, H. N. Ok, Mater. Lett., 2001, 50 (1), 47-52. DOI:10.1016/S0167-577X(00)00412-2 22. Lohar K. S., Pachpinde A. M., Langade M. M., et al., Journal of Alloys and Compounds, 2014, 604, 204-210. DOI:10.1016/j.jallcom.2014.03.141 23. B. G. Toksha, Sagar E. Shirsath, M. L. Mane, S. M. Patange, S. S. Jadhav, K. M. Jadhav, J. Phys. Chem. C, 2011, 115, 2090520912 DOI:10.1021/jp205572m 24. Sagrario M. Montemayor, L. A. Garcia-Cerda, J. R. Torres-Lu-bian, O. S. Rodriguez-Fernandez, J Sol-Gel Sci Techn, 2007, 42,181-186 25. J. Azadmanjiri, Mater. Chem. Phys., 2008, 109, 109-112 DOI: 10.1016/j.matchemphys.2007.11.001 26. A. Mali, A. Ataie, Ceramics International, 2004, 30, 19791983. DOI:10.1016/j.ceramint.2003.12.178 27. Hashim, Mohd, et al., Powder technology, 2012, 229, 37-44. DOI:10.1016/j.powtec.2012.05.054 28. K. S. Lohar, S. M. Patange, S. E. Shirsath et al., Int. J. of Modern Phy. B, 2011, 25-16, 2157-2166. 29. B. D. Cullity, "Elements of X-ray diffraction", (Addison-Wesley Publ. Comp. Inc., Reading, Massachusetts, U.S.A.), 1956, pp. 99. 30. S. M. Patange, Sagar E Shirsath, et al., Appl. Phys. A: Mater Sci. Proces, 2009, A95, 429-434. DOI: 10.1007/s00339-008-4897-0 31. N. Kumari, V. Kumar, K. Singh, J. Alloys Compd., 2015, 622, 628-634. DOI:10.1016/j.jallcom.2014.10.083 32. Yuksel Koseoglu, Ceramics international, 2015, 41, 64176423. DOI:10.1016/j.ceramint.2015.01.079 33. M. Ashtara, A. Munirb, M. Anis-ur-Rehmanb, A. Maqsooda, Materials Research Bulletin, 2016, 79, 14-21. 34. B. Vishwanathan and V. R. K. Murthy, Ferrite Material Science and Technology, New Delhi: Narsoa Publishing House, 1990, pp. 15-61. 35. S. S. Bellad, R. B. Pujar, B. K. Chougule, Indian J. Pure Appl. Phys., 1998, 36, 598-601. 36. A. A. Yousef, M. E. El-Zain, S. A. Mazen, et al., J. Phys. Condens. Matter, 1994, 6(29), 5717-5724. DOI: 10.1088/0953-8984/6/29/014 37. E. W. Gorter, Philips Res. Rep., 1954, 9, 295-320. 38. Muhammad Javed Iqbal, Barkat-ul-Ain, Mater. Sci. Engg. B, 2009, 164, 6-11. Povzetek Nanoferite Ni-Cu-Zn dopirane s kromom in s kemijsko sestavo Ni02Cu02Zn06Fe2iCriO4 (x = 0.0, 0.2, 0.4, 0.6, 0.8, 1.0) smo pripravili z modificirano sol-gel metodo. Tako pripravljene s kromom substituirane prekurzurje Ni-Cu-Zn feritov smo sintrali štiri ure pri 500 °C. Kemijsko sestavo smo potrdili z energijsko disperzivno rentgensko analizo (EDAX). Podatki rentgenske praškovne analize (XRD) pa razkrivajo, da so imajo vsi vzorci le eno fazo in kubično spinelno strukturo. Konstante osnovne celice, razdalje med magnetnimi ioni in velikost kristalitov, ki smo jih določili z rentgensko praškovno analizo se zmanjšujejo z večanjem koncentracije Cr3+ ionov. V infrardečih spektrih prevladujeta dva trakova: trak pri visokih frekvencah v1 ~ 600 cm1 in nizkih frekvencah v2 ~ 450 cm1, ki ju lahko pripišemo valenčnim nihanjem v tetraedričnih in oktaedričnih okoljih. Morfologijo površine pripravljenih vzorcev smo preučevali z uporabo vrstične elektronske mikroskopije (SEM) in presevne elektronske mikroskopije (TEM). Shinde et al.: Preparation and Characterization 938 DOI: 10.17344/acsi.2017.3637 Acta Chim. Slov. 2017, 64, 938-944 /^creative ^commons Scientific paper The Impact of a Silane Pigment Treatment on the Properties of Thickness-sensitive Spectrally Selective Paints Miha Steinbücher,1 Peter Venturini,1 Jože Hafner,2 Matevž Zupančič,3 Peter Gregorčič3 and Iztok Golobič3* 1Helios Group, Količevo 2, SI-1230 Domžale, Slovenia 2 Slovenian National Building and Civil Engineering Institute, Dimičeva 12, SI-1000 Ljubljana, Slovenia 3 University of Ljubljana, Faculty of Mechanical Engineering, Aškerčeva 6, SI-1000 Ljubljana, Slovenia * Corresponding author: E-mail: iztok.golobic@fs.uni-lj.si phone: +38614771420 Received: 17-06-2017 Abstract Thickness-sensitive, spectrally selective paints based on a silane treatment of pigments were prepared with different pigment-volume concentrations. The critical pigment-volume concentration was determined by means of electrochemical impedance spectroscopy, while the pigment particle size distribution was determined with ultrasound spectroscopy. The selectivity versus thickness relation of a paint with a near-critical pigment-volume concentration was studied spectro-scopically through performance criteria. Its nonlinearity was shown to be related to the surface topography. This relation was further supported by hydrophobicity measurements. Heat-gathering tests in a simulated solar collector supported the spectroscopic determination of an optimal dry-film thickness. Keywords: TSSS paints, pigment treatment, spectral selectivity, topography, hydrophobicity 1. Introduction Spectrally selective paints play a crucial role in solar collector panels needed for efficient solar-thermal conversion systems.1 Such systems require spectrally selective absorbers with high absorption of the sunlight and low infrared emittance resulting in little heat lose to environment via convection and radiation. The established measure for the solar irradiation absorptivity as a material property is the solar absorptivity (a, aS, aS), defined by the following equation: (i) where S(X) is the normalized direct solar irradiation distribution function2 and R(X) is a hemispheric reflectance spectra of the material surface. On the other hand, the established measure of the fraction of black-body emissivity at a given temperature for a material is the thermal emissivity (e, eT eT), defined by the following equation: (2) where r(k, T) is the black-body radiation distribution function at a given absolute temperature (T). Since high solar absorptivity and a low thermal emissivity are desired for solar-energy exploitation, the performance criterion PCC was introduced as a measure of the spectral selectivity, combining both aS and eT: PCr = -a + Ce C s T (3) where C is a constant appropriate for the collector type used. The most commonly used values are C = 0.5 for high-temperature collectors, C = 0.25 for low-temperature collectors and C = 0.34 for unglazed collectors. The C values as well as classification of collectors are based on experimental work.3 Coatings, made from organic spectrally selective materials,4, 5 represent one of efficient options to produce Steinbücher et al.: The impact of a silane pigment treatment ... Acta Chim. Slov. 2017, 64, 938-944 939 spectrally selective absorbers. They can be divided into thickness-insensitive, spectrally selective,6-10 and thickness-sensitive, spectrally selective (TSSS) paints.11 The development of both types is interlinked in the fields of absorptive pigments and their dispersions,12 binders,13 and durability.14, 15 Typically, black TSSS coatings are based on an interaction between a thin layer of highly absorptive paint applied without a primer and a thermal infrared reflective substrate, such as aluminum, copper or other metals. Their selectivity depends to a large extent on their thickness, as both aS and eT increase non-linearly with thickness. However, the thickness of the TSSS coatings is seldom measured, as their usual dry-film thicknesses is of the same magnitude as the surface roughness of the commonly used substrates, but thickness measurements commonly used in the paint industry cannot be reliable.16 The grammage, i.e., the weight of coating per nominal surface, roughness disregarded, is used instead. The other factor influencing selectivity is the substrate. However, its influence on aS is not significant with coatings applied in usual thicknesses (aS > 0.9). On the other hand, eT is very (and almost linearly) dependent on the substrate eT. Since the development of solar selective absorbers with both high conversion efficiency and high-temperature stability remains a challenging issue due to the cost and the materials problems,17 the main aim of this work is examination of the selectivity of in-situ silane-treated-pig-ment TSSS (stpTSSS) paints. Therefore, we are focused in study of performance criteria as a function of pigment concentration as well as on the optical properties of the pigment, the binder and the surface topology. Here, the surface-topography study is supported by hydrophobicity analysis performed through measurements of the static contact angle of a water droplet. 2. Materials and Methods The stpTSSS paints were produced according to a previously disclosed procedure.4 The SUNCOLOR TS S Black Al (Helios Group, SI) paint was used for the particle size determination, topography studies, contact-angle measurements and tests of the heat-gathering properties. Solarect Z (Helios Group, SI) was used as a reference TSSS paint without silane-treated pigments. The paints were applied by spraying, bar or coil coating, diluted with the appropriate Helios SUNCOLOR Thinner (Helios Group, SI) or SUNCOLOR Thinner (Helios Group, SI). The substrates used were acid-pretreated, 0.2-mm-thick, copper (eT = 0.03) sheets and degreased, 0.4-mm-thick aluminum sheets (eT = 0.01-0.02). The coatings were cured in a calibrated Kambic LSP-190 C laboratory oven with ± 5 °C precision at temperatures > 100 °C. The coatings' grammage was determined by weighing the difference of uncoated and coated substrates with known surfaces. The masses were determined with a preci- sion of ± 0.0001 g. The surfaces of the rectangular samples were determined by measuring their length and width with a precision of ± 0.5 mm. The resulting grammage measurement error for a substrate of 10.0 cm x 10.0 cm, typically used in this work, is 0.01 g/m2. The aS values were determined by recording the UV-VIS-NIR spectra with a Perkin Elmer Lambda 950 spectrometer with a 150-mm Spectralon integration sphere and processing the spectral data according to equation (1). The error in the measurement of aS << 0.01.18 The eT values were determined by recording the IR spectra with a Bruker IFS66/S spectrometer using an OPTO-SOL integration sphere and processing the spectral data according to equation (2). The error in the measurement is not documented. The Electrochemical Impedance Spectroscopy (EIS) measurements were performed with a Parstat 2273 poten-tiostat and a Tait cell. The measuring system has a resolution of 1pA. The EIS19 data were plotted as a Nyquist chart showing the imaginary impedance (Z") versus the real impedance (Z'). The higher arc shows a higher coating capacity and thus better barrier properties and a lower permeability. The EIS data were used for a critical pigment volume concentration (cPVC) determination of the coatings according to the previously described method.20,21 The particle sizes were determined with a Dispersion Technology DT-1200 acoustic spectrometer. The method based on acoustic attenuation versus an ultrasound frequency measurement allows for a particle size determination in a realistic, undiluted sample, which is in contrast to the more common dynamic light scattering. The particle size distribution is calculated from the recorded ultrasound spectrum, the dry-matter content, and densities using a unimodal or bimodal model. The uncertainty of the measurement for the sample relevant to this work is below 20 nm. The substrate and coating topographies were determined with an AFM Park XE100 atomic force microscope with a spatial resolution of 500 pm and 150 pm of background noise. The uncertainty of the measurement for the samples relevant to this work is below 1 nm. The static contact angles for water were measured with a Kruss DSA 100 drop shape analysis system with the error of the measurement being < 1°. Three measurements were made for each sample and then averaged. The thermal response to irradiation was determined with stagnation-temperature measurements of a model absorber in a glazed or unglazed thermally insulated housing. The illumination was provided by filtered Atlas metal halide lamps; the power was regulated by changing the distance between the lamps and the absorber. The light flux was determined with a Kipp & Zonen CM11 pyranometer with a spectral range of 285-2800 nm and a zero offset < 7 W/m2. The temperature was measured by means of type-K thermocouples, and a Agilent 34970A and a Windows PC were used for the data acquisition. Low-Fe-content glass with T = 0.92 was used for the glazing. Steinbucher et al.: The impact of a silane pigment treatment 940 Acta Chim. Slov. 2017, 64, 938-944 3. Results and Discussion 3. 1. PVC Optimization Paints with different pigment volume concentrations (PVCs) were produced according to the already known procedure and applied to a copper substrate by air spraying.22 The silane coating of pigment in-situ during the dispersion process allows for a better dispersion and smaller particle size, as shown in Fig. 1. Figure 1. SEM analysis of surfaces of TSSS coatings made of silane treated (top) and untreated (bottom) pigments. Samples were made with the same PVC and the same application process. The aS and eT values of the resulting coatings are shown in Figs. 2 and 3 with the aS and eT curves for the Solarect Z24 paint with PVC 27 as a reference. Both paints are based on the same binder and pigment and are formulated with the same PVC. The previously known relationship between the PVC and the spectral selectivity of a TSSS paint is also relevant for the stpTSSS paints. Paints with a higher PVC exhibit higher aS and lower eT for the same grammage and substrate. The advantage of the new material is even clearer in a comparison of the PC values. Fig. 4 shows the PC0 5 values derived from the above data. 0.94 0.92 0.90 0.88 0.86 0.84 0.82 □ □ ® A° O ^ □ o A O O . □ / A / O PVC 27 A PVC 31 □ PVC 41 /° O PVC 55 o A/ —Solarect Z24 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 Grammage (g/m2) Figure 2. aS as a function of grammage for stpTSSS at different PVC with a non-silane treated reference, increase of PVC leads to higher a values. 0.00 0.50 1.00 1.50 2.00 2.50 Grammage (g/m2) 3.00 3.50 Figure 3. eT as a function of grammage for stpTSSS at different PVC with a non-silane treated reference, increase of PVC leads to lower eT values. It is clear that the new coatings display better selectivity at a given PVC compared to the reference, but the silane-pigment-treating technology offers a further advance. The reference coating is limited to its PVC with the mechanical properties. The stpTSSS, on the other hand, can be produced at significantly higher PVCs without any apparent loss of adhesion or cohesion. Kunic et al (2009) described simple mechanical tests such as cross-cut adhe-sion23 to determine the mechanical stability in organic selective coatings. However, such simple testing method cannot give a full picture to important properties like film permeability, which significantly influence the coating's durability. It is well known that properties of coating film, including ones Steinbucher et al.: The impact of a silane pigment treatment Acta Chim. Slov. 2017, 64, 938-944 941 Figure 4. PC05 values as a function of grammage for stpTSSS at different PVC with a non-silane treated reference. Changes in PVC doesn't significantly influence the optimal grammage, which is about 1.5 g/m2. tested by cross-cut adhesion test, abruptly changes at cPVC.24 cPVC is the PVC where there is just enough binder to provide a complete adsorbed layer on the pigment surfaces and to fill the voids between pigment particles in the film. In a film with PVC > cPVC, pigment particles are not completely wetted and there are voids and/or pores between them. To determine the cPVC of stpTSSS paints we used electrochemical impedance spectroscopy (EIS) technique. A ladder of coatings with PVCs from 25 to 55 was prepared and applied in a 20-^m thickness with a bar-coater on a steel substrate. The EIS measurements of the cured coatings were performed and the results are shown as a Nyquist chart in Fig. 5. The Nyquist chart shows that the film permeability decreases with the PVC until the cPVC is reached, after which it drops significantly. A precise determination of the PVC allowed for the development of the most selective, but still durable, coating. All subsequent experiments were performed with a coating corresponding to the coating 5 in Fig. 5. -4.00E+04 E U S N -2.50E+04 -2,00E+04 -L50E+04 0,00E+00 5 _---------- 3 —--- -— 1 L 6,7,8,9,10 3. 2. Spectral Selectivity The questions of the selectivity of the coatings and selectivity optimization were already partially addressed in the PVC dependence. Fig. 4 shows clearly that there is an optimal thickness of the coating from the point of view of selectivity. Fig. 6 shows the combined UV-VIS-NIR and IR spectra of a stpTSSS paint at different thicknesses, expressed as grammages. The spectral data show strong pigment absorption in the UV and VIS regions, gradually diminishing in the NIR region. In the Thermal Infrared (TIR) region significant absorption bands are visible at 3-3.5 ^m (C-H bond stretching), 5.8 ^m (C=O bond stretching), 7.9 ^m (Si-C bond stretching), 9 ^m (broad, Si-O bond stretching), all corresponding to the paint's binder. The absorption band at 4.2 ^m is caused by atmospheric CO2 and is numerically subtracted in the eT calculations. The increase in the absorptivity with thickness points to well-established light-absorption and scattering phenomena as the reason for the selectivity.25 The PC data in Fig. 4, on the other hand, show a more distinct nonlinearity compared to the reference coating and point to another mechanism behind the relation between the selectivity and the thickness of the coating. We believe the explanation lies in the surface topography of a thinly applied near cPVC coating. 0,00E+00 I.00E+04 2.00E+04 3,00E+04 4,00E+04 Z' (ii/cm2) Figure 5. Nyquist chart depicting EIS measurement data as Z'' versus Z' of below (1-5) and above critical (6-10) PVC stpTSSS paints. Figure 6. Combined UV-VIS-NIR and IR spectra of a stpTSSS paint applied at different grammages. 3. 3. Topography To interpret the topographies correctly, more data are needed, i.e., the distribution of particle sizes (PSD) in the coating. Fig. 7 shows the particle size distribution obtained by acoustic spectrometry in liquid stpTSSS paint (i.e., before application). Clearly, the bimodal distribution can be explained with pigment particles that have a median particle size of 300 nm and a polymer particle dispersion with a median particle size of 10 ^m. The latter are dissolved in the curing process and not present in a cured coating. Steinbucher et al.: The impact of a silane pigment treatment 942 Acta Chim. Slov. 2017, 64, 938-944 0.01 0.1 1 10 100 d (ftm) Figure 7. Particle size distribution in liquid TSSS paint. To study the topography of the TSSS coatings applied at thickness close to the optimal one, optical microscopy is of little help, as the surface roughness of practical substrates approaches or exceeds the thickness of the coating. AFM imaging was used instead. The samples were prepared by air-spraying the paint on an aluminum substrate. The image of the substrate in Fig. 8 shows a na-no-rough surface. From the image of the stpTSSS coating with a grammage of 1.2 g/m2 in Fig. 9, a nanostructure with a pattern corresponding to the particle size determined in a liquid coating superimposed on a non-flat substrate can be seen. In some spots a less structured surface due to a larger concentration of the binder can be seen. The AFM image of the surface of the stpTSSS coating with a grammage of 1.7 g/m2 in Fig. 10 shows a less structured surface. The uneven surface of the substrate is better filled with the coating and the pigment particles are less obviously exposed. The trend is further evident in Fig. 11, showing an AFM image of the stpTSSS coating with a grammage of 2.4 g/m2. The surface is smoother and the pigment particles are enclosed in a binder, clearly visible below the cPVC coating. Figure 8. AFM image of aluminum substrate. Figure 9. AFM image of stpTSSS coating with 1.2 g/m2 grammage on aluminum substrate. Figure 10. AFM image of stpTSSS coating with 1.7 g/m2 grammage on aluminum substrate. Figure 11. AFM image of stpTSSS coating with 2.4 g/m2 grammage on aluminum substrate. From the above images we can conclude that the PC versus thickness relation for the stpTSSS coatings shown in Steinbucher et al.: The impact of a silane pigment treatment Acta Chim. Slov. 2017, 64, 938-944 943 Fig. 4 is influenced by the change of the PVC in the top layer of the coating as a significant amount of binder is used for the substrate wetting, thus exposing more pigment particles. 3. 4. Hydrophobicity The nanostructure of the stpTSSS is also evident in its hydrophobicity. We present the contact angles for the water of coatings applied at different grammages in Fig. 12. As the binder has a contact angle for water of 96°, if applied on a thick enough layer to eliminate the influence of a substrate, it is clear from the data that the large contact angles for the stpTSSS coating are the result of structured surfaces. It could also be reasoned that the wetting of the pigment particles in the top layer of the coating is not perfect at lower grammages, resulting in lower contact angles for the more structured surfaces. Figure 12. Contact angles for water for stpTSSS coatings applied at different grammages. 3. 5. Heat-gathering Properties The spectroscopic measurements (Fig. 6) and nano-topography (Figs. 8-11) must of course correspond with Figure 13. Stagnation temperatures and samples' spectral selectivity in heat gathering tests. practical results to be of industrial significance. To check the practicability of the stpTSSS coatings the heat-gathering properties were tested. The coatings were prepared with air spraying on an aluminum substrate. Fig. 13 shows the spectral selectivity data of the samples and the stagnation temperatures with glazed and unglazed setups at various illuminations. The above results clearly show that the best heat-gathering properties are exhibited by the most selective sample, i.e., the one with the lowest PC value. Relatively low temperatures were achieved in the setups used, which means PC025 is the appropriate criteria for coating selection. 4. Conclusions Silane-treated-pigment TSSS paints (stpTSSS) produced according to a recently disclosed procedure are the first Task-X-certified26, organic, spectrally selective coatings. They have now entered industrial production on a major European producer's solar absorber surfaces coil-coating line. The selectivity of stpTSSS paints was studied. The performance criteria (PC) of the coating were found to decrease with the pigment-volume concentration (PVC). The critical PVC (cPVC) was determined by means of EIS. The nonlinearity of the PC versus grammage relation was shown to be related not only to the optical properties of the pigment and the binder used, but also to the surface topography. Thin, near cPVC coatings exhibit a significantly different surface topography than the thicker coatings. We believe that sub-optimal pigment wetting by the binder in the thinly applied coatings' top layer is caused by a disproportionate amount of binder being used for the substrate wetting, resulting in a PVC gradient in the coating. This phenomenon, which is not readily observable in thicker coatings, leaves the subject open for further studies. Contact-angle measurements for water support the surface-topography findings, i.e., the contact angles increase with the thickness. This can be readily explained by the coatings' compositions and topographies. The spectroscopic measurements of the paint's selectivity correlate well with the results obtained with the stagnation temperature built up in a model collector. 5. Acknowledgements Authors wish to thank the staff of Laboratory for the Spectroscopy of Materials at National Institute of Chemistry (of Slovenia) under leadership of Prof. Dr. Boris Orel for spectral selectivity measurements and the staff of Physical Analytical Laboratory at Helios d.d. under leadership Steinbucher et al.: The impact of a silane pigment treatment 944 Acta Chim. Slov. 2017, 64, 938-944 of Dr. Saša Skale for measurements. The work was partially financed through KC-TIGR (Competence Centre sustainable and innovative construction); operation part financed by the European Union, European Regional Development Fund. MS thanks Public Agency for Technology of the Republic of Slovenia (TIA) for financing through MR-08 program, grant number P-MR-08/21; operation part financed by the European Union, European Social Fund. The authors also acknowledge the financial support from the state budged by the Slovenian Research Agency (Programme Nos. P2-0223 and P2-0392). 6. References 1. F. Cao, K. McEnaney, G. Chen, Z.F. Ren, Energy Environ. Sci., 2014, 7, 1615-1627. DOI:10.1039/c3ee43825b 2. ISO 9845-1, Solar energy - Reference solar spectral irradiance at the ground at different receiving conditions - Part 1: Direct normal and hemispherical solar irradiance for air mass 1,5, ISO, Geneve, 1992. 3. SOLABS, Development of unglazed solar absorbers (resorting to coloured selective coatings on steel material) for building facades, and integration into heating systems, EU project, Contract no. ENK6- CT-2002-00679. 4. M. Koželj, B. Orel, M. Steinbucher, I. Jerman, M. Vodlan, Aminosilane-modified pigments for spectrally selective paints, methodfor their preparation and application in paints, SI Patent Number 23055, date of patent November 30, 2010. 5. M. Koželj, B. Orel, M. Steinbucher, I. Jerman, M. Vodlan, Coil-coated spectrally selective coatings on copper or aluminum with pigments modified by aminosilane, EU Patent Number 2432835 B1, date of patent January 27, 2016. 6. M. Hoeflaak, Optimization of spectrally selective coatings for flat plate solar collectors, in: 8th International conference "Optimising paint formulation: preservation, stabilization and care", Amsterdam, Netherlands, 1988. 7. B. Orel, H. Spreizer, L. Slemenik Perše, M. Fir, A. Šurca Vuk, D. Merlini, M. Vodlan, Sol. Energy Mat. Sol. Cells, 2007, 91, 93-107. DOI:10.1016/j.solmat.2006.07.013 8. B. Orel, H. Spreizer, L. Slemenik Perše, M. Fir, A. Šurca Vuk, D. Merlini, M. Vodlan, M. Kohl, Sol. Energy Mat. Sol. Cells, 2007, 91, 108-119. DOI:10.1016/j.solmat.2006.07.012 9. E. AlShamaileh, Sol. Energy 2010, 84, 1637-1643. DOI:10.1016/j.solener.2010.06.003 10. J. Manara, M. Reidinger, M. Rydzek, M. Arduini-Schuster, Prog. Org. Coat., 2011, 70, 199-204. DOI:10.1016/j.porgcoat.2010.09.024 11. S. Wijewardane, D.Y. Goswami, Renewable Sustainable Energy Rev., 2012, 16, 1863-1873. DOI:10.1016/j.rser.2012.01.046 12. I. Jerman, M. Koželj, B. Orel, Sol. Energy Mat. Sol. Cells, 2010, 94, 232-245. DOI:10.1016/j.solmat.2009.09.008 13. B. Japelj, A. Šurca Vuk, B. Orel, L. Slemenik Perše, I. Jerman, J. Kovač, Sol. Energy Mat. Sol. Cells, 2008, 92, 1149-1161. DOI:10.1016/j.solmat.2008.04.003 14. R. Kunič, M. Koželj, B. Orel, A. Šurca Vuk, A. Vilčnik, L. Slemenik Perše, D. Merlini, S. Brunold, Sol. Energy Mat. Sol. Cells, 2009, 93, 630-640. DOI:10.1016/j.solmat.2008.12.026 15. R. Kunič, M. Mihelčič, B. Orel, L. Slemenik Perše, B. Bizjak, J. Kovač, S. Brunold, Sol. Energy Mat. Sol. Cells, 2011, 95, 29652975. DOI:10.1016/j.solmat.2011.05.014 16. ISO 2808, Paints and varnishes - Determination of film thickness, ISO, Geneve, 2007. 17. H. D. Liu, T. R. Fu, M. H. Duan, Q. Wan, C. Luo, Y. M. Chen, D. J. Fu, F. Ren, Q. Y. Li, X. D. Cheng, B. Yang, X. J. Hu, Sol. Energy Mat. Sol. Cells, 2016, 157, 108-116. DOI:10.1016/j.solmat.2016.05.035 18. Measurement of Total Solar Reflectance of Paint Panels using PerkinElmer UV/Vis/NIR Spectrophotometers and UV WinLab Software, Perkin Elmer, Inc., Waltham, USA, 2010. 19. S. Skale, V. Doleček, M. Slemnik, Corros. Sci., 2007, 49, 10451055. DOI:10.1016/j.corsci.2006.06.027 20. R. E. Lobnig, W. Villalba, K. Goll, J. Vogelsang, I. Winkels, R. Schmidt, P. Zanger, J. Soetemann, Prog. Org. Coat., 2006, 55, 363-374. DOI:10.1016/j.porgcoat.2005.11.015 21. R. E. Lobnig , V. Bonitz, K. Goll, M. Single, W. Villalba, J. Vogelsang, I. Winkels, R. Schmidt, P. Zanger, Prog. Org. Coat., 2007, 60, 1-10. DOI:10.1016/j.porgcoat.2007.03.003 22. N. Selvakumar, H. C. Barshilia, Sol. Energy Mat. Sol. Cells, 2012, 98, 1-23. DOI:10.1016/j.solmat.2011.10.028 23. ISO 2409, Paints and Varnishes - Cross-cut test, ISO, Geneve, 2007. 24. Z. W. Wicks, F. N. Jones, S. P. Pappas, D. A. Wicks, Organic Coatings: Science and Technology, 3rd Edition, Wiley, New Jersey, USA, 2007, pp. 461-471. DOI:10.1002/047007907X 25. M. Klanjšek Gunde, J. Kozar Logar, Z. Cmjak Orel, B. Orel, Thin Solid Films, 1996, 277, 185-191. DOI: 10.1016/0040-6090(95)08023-6 26. B. Carlsson, M. Kohl, Recommended qualification test procedure for solar absorber surface durability, IEA Solar Heating and Cooling Program, Paris, France, 2005. Povzetek Članek obravnava od debeline odvisne spektralno selektivne premaze, ki so bili pripravljeni z različnimi volumskimi koncentracijami pigmenta temelječih na silansko obdelanih pigmentih. Kritična volumska koncentracija pigmenta je bila določena s pomočjo elektrokemijske impedančne spektroskopije, porazdelitev velikosti delcev pa z ultrazvočno spektroskopijo. Odvisnost selektivnosti od debeline premaza s skoraj kritično volumsko koncentracijo pigmenta je bila preučevana spektroskopsko na podlagi zmogljivostnega kriterija. Pokazalo se je, da je nelinearnost slednjega povezana s površinsko topografijo, kar je bilo dodatno potrjeno z meritvami hidrofobnosti. Meritve sposobnosti zbiranja toplote v simuliranem toplotnem zbiralniku so potrdile spektroskopsko določitev optimalne debeline suhega filma. Steinbucher et al.: The impact of a silane pigment treatment DOI: 10.17344/acsi.2017.3642 Acta Chim. Slov. 2017, 64, 945-958 'commons 945 Scientific paper Graphene Oxide/Co3O4 Nanocomposite: Synthesis, Characterization, and Its Adsorption Capacity for the Removal of Organic Dye Pollutants from Water In this work, graphene oxide/Co3O4 nanocomposite was synthesized via hydrothermal decomposition of [Co(en)3] (NO3)3 complex onto graphene oxide nanosheets. The as-prepared nanocomposite (denoted as GO/Co3O4) was structurally characterized by Fourier transform infrared (FT-IR) spectroscopy, X-ray diffraction (XRD), Raman spectroscopy, scanning electron microscopies (TEM and SEM), energy dispersive X-ray (EDX) spectroscopy, magnetic measurements, and N2 adsorption-desorption analysis. The results demonstrated successful immobilization of Co3O4 nanoparticles with an average diameter size of around 12.5 nm on the surface of graphene oxide nanosheets. The adsorption performance of GO/Co3O4 nanocomposite was investigated towards different organic dyes in aqueous solutions. The results displayed that the adsorption rate of the GO/Co3O4 nanocomposite was 98% for methylene blue (MB) in 12 min, and 66% and 45% for Rhodamine B (RhB) and methyl orange (MO) in 40 min, respectively. The effects of various important parameters including adsorbent dosage, contact time, pH, and temperature on the adsorption process were investigated in detail. The equilibrium adsorption data were better fitted by Langmuir isotherm. Adsorption kinetics is well-modeled using pseudo-second-order model. Different thermodynamic parameters indicated that the adsorption process was physisorption and spontaneous. The findings of the present work highlighted facile fabrication of GO/Co3O4 and its application for rapid and efficient removal of MB from wastewater. Keywords: Graphene oxide nanosheets, Co3O4 nanoparticles, Hydrothermal decomposition, Nanocomposite, Adsorption performance, Organic dyes. Kolsoum Pourzare, Saeed Farhadi* and Yaghoub Mansourpanah Department of Chemistry, Lorestan University, Khoramabad68151-44316, Iran * Corresponding author: E-mail: sfarhadi1348@yahoo.com Tel: +98 06633120611, fax: +98 06633120618. Received: 18-06-2017 Abstract 1. Introduction Among these technologies, adsorption is the most widely used method due to its versatility, wide applicability, and economic feasibility.7 Activated carbon, clays, zeolites, polymeric materials, etc. have been applied to adsorb dyes from wastewater. However, these adsorbents suffer from either low adsorption capacities or separation problem. Hence, the adsorbent having both mentioned characteristics is immensely desired in both science and technology societies.8 In material science research, a great deal of attention has been focused on graphene, a carbon allotrope with a two-dimensional sheet-like structure with many unique features such as high electrical conductivity, mechanical flexibility, chemical and thermal stability, high surface functionality, and large surface area.7 Due to strong interplane interactions, graphene and its derivatives tend to aggregate in a layer-by-layer manner which, as a result, a significant part of their surface area is lost. The use of the Many chemical industries such as paper, plastics, cosmetics, leather, printing, food, textile, etc. use dyes for coloring their products and release the various types of dyes into water bodies which prevent the penetration of sunlight, retard the photosynthetic reactions, and affect aquatic life.1-4 Most dye molecules have aromatic rings in their structures, which make them highly toxic, non-biodegradable, carcinogenic, and mutagenic to both human being and aquatic life.5 Hence, it is essential to remove or minimize dyes to permissible levels, without disturbing the quality of water to be able to use it in diverse industrial and agricultural applications.6 A wide array of wastewater treatment techniques including membrane filtration, cen-trifugation, photodegradation, chemical coagulation, and adsorption have been developed for removing dyes from wastewater. Pourzare et al.: Graphene Oxide/Co3O4 Nanocomposite: 946 Acta Chim. Slov. 2017, 64, 945-958 dispersion of single-layered GO as an absorbent allows one to utilize the surface area to the utmost extent, but these GO sheets are difficult to collect from water.9 This problem can be solved via chemical modification of graphene by the process of attaching organic groups or inorganic particles onto graphite oxide (GO) surfaces which can result not only in physical separation of the resultant functionalized graphene sheets but also in the possible formation of a stable dispersed phase of graphene in the synthesis process10-12 and keep the surface area and pore volume at high levels which is required for applications such as adsorption processes and photocatalysis.13 In recent years, various transition metal oxides nanoparticles such as Fe2O3, ZnO, Fe3O4, TiO2, etc., have been deposited on GO nanosheets. For instance, Ami-no-functionalized Fe3O4 (NH2-Fe3O4) particles were deposited on graphene oxide sheets and were used to adsorb Methylene Blue (MB) and Neutral Red (NR) from aqueous solution by Xie et al.14 The adsorption test of dyes demonstrated that it only took 30 min for MB and 90 min for NR to reach equilibrium. Luo et al. fabricated magnetic cyclodextrin/graphene oxide (MCGO) materials and investigated their application as excellent adsorbents for methylene blue.15 MCGO demonstrated extremely fast MB-removal from wastewater with high removal efficiency within 50 min. Li et al. prepared a magnetic CoFe2O4-functionalized graphene sheet (CoFe2O4-FGS) nanocomposite via a facile hydrothermal method and used it to adsorb methyl orange.16 The observed maximum adsorption capacity at 10 mg L-1 initial concentration was 71.54 mg g-1. In another study, Fan et al. prepared a magnetic chitosan-GO (MCFO) nanocomposite through co-valent bonding of chitosan to the surface of Fe3O4 nanopar-ticles followed by covalent functionalization of GO with magnetic chitosan which acted as a good adsorbent to adsorb MB from aqueous solutions.17 Yao et al. also fabricated Fe3O4/SiO2/GO nanocomposite through a covalent bonding technique and used it as an adsorbent for the removal of MB from aqueous solution.18 Maximum MB adsorption capacities were 97, 102.6, and 111.1 mg g-1at 25, 45, and 60 °C, respectively. Spinel-type cobalt oxide (Co3O4) is an important magnetic p-type semiconductor oxide and its synthesis and properties have attracted considerable attention owing to its prominent applications in heterogeneous catalysis, energy storage and conversion, sensors, devices, etc.19-25 For this reason, various nanostructures of CoO, 34 such as nanoparticles, nanoplates, nanorods, nanotubes, nanodiscs, nanoflowers, nanocubes and hollow microspheres structures have been prepared by using different synthesis methods.26-33 To the best of our knowledge, the investigation of adsorption properties of GO/Co3O4 nanocomposites has not been reported yet. In this study, we presented a solvothermal approach for the production of GO/Co3O4 nanocomposite. The resulting products was characterized by FT-IR, XRD, Raman, FE-SEM, EDX, TEM, and VSM techniques and its adsorption properties in removing dye molecules from aqueous solutions were investigated. 2. Experimental 2. 1. Materials Methyl orange (C14H14N3NaO3S, MO), methylene blue (C16H18ClN3S, MB), Rhodamine B (C28H31ClN2O3, RhB), Cobalt (II)-chlorid hexahydrate, and all other materials and solvents were purchased from Merck chemical Co (Germany). All chemical materials were of analytical grade and were used as received without further purification. 2. 2. Synthesis of Co3O4 Nanoparticles First, [Co(en)3](NO3)3 complex was prepared via the simple reaction of an aqueous solution of [Co(en)3]Cl3 with concentrated nitric acid according to the reported method.34 To prepare Co3O4 nanoparticles, [Co(en)3] (NO3)3 complex was decomposed at 250 °C for 1 h in an electric furnace under ambient air. The decomposition product was collected for characterization. 2.3. Synthesis of Graphene Oxide/Co3O4 Nanocomposite (GO/Co3O4) Graphene oxide (GO) was prepared by the oxidation of graphite powder under acidic conditions according to modified Hummers method using a mixture of H2SO4, NaNO3, and KMnO/5-36 For the synthesis of GO/Co3O4 nanocomposite, 50 mg GO was dispersed into 20 mL de-ionized water by sonication for 1 h to achieve a uniform dispersion of GO. 100 mg as-prepared Co3O4 nanoparti-cles were dispersed in deionized water for 15 min and were gradually added into GO suspension. The mixture was sonicated for 30 min and transferred into an autoclave for hydrothermal treatment at 180 °C for 24 h. The resultant product was separated by centrifugation and washed with deionized water, and dried in an oven at 60 °C for 12 h. 2. 4. Methods of Characterization Fourier-transform infrared spectra were obtained on Shimadzu FT-IR 8400S (Japan) with temperature controlled high sensitivity detector (DLATGS detector) in the scan range of 500-4000 cm-1 using KBr pellet. The XRD patterns were obtained on a Rigaku D-max C III, X-ray dif-fractometer using Ni-filtered Cu Ka radiation (X = 1.5406 A) for phase determination samples. Optical absorption spectra of dyes were obtained on a Cary 100 UV-Vis spectrophotometer in the wavelength range of 200-800 nm. A vibrating sample magnetometer (VSM, Magnetic Danesh-pajoh Kashan Co., Iran) was employed to measure magnetic parameter at room temperature. Particle size was ob- Pourzare et al.: Graphene Oxide/Co3O4 Nanocomposite: Acta Chim. Slov. 2017, 64, 945-958 947 served by a transmission electron microscope (Philips CM120) at the accelerating voltage of 100 kV. SEM images were obtained on MIRA3 TESCAN Field Emission Scanning Electron Microscope equipped with Energy Dispersive X-ray (EDX) analyzer for the elemental analysis of the sample. N2 adsorption-desorption measurements were performed at 77 K (Micromeritics Tristar ASAP 3000) using Brunauer-Emmett-Teller (BET) method. 2. 5. Adsorption Tests Adsorption experiments were performed by using 30 ml solution with known MB concentration and varying the amount of GO/Co3O4 as the adsorbent from 10 to 30 mg in 40 min. The initial pH of MB solution was adjusted in the range of 4-12 by dropwise adding 0.1 mol/L NaOH or 0.1 mol/L HCl solutions. After adsorption was completed, the solution was separated from the precipitate by centrifuga-tion at 5000 rpm for 5 min. The concentrations of the dye in the solutions after different time intervals were determined with a UV-visible spectrophotometer at the wavelength of 664 nm (X ). The amount of MB adsorbed onto GO/Co3O4 nanocomposite (qt) and its removal rate (R%) were calculated by the following equations: qt = (C0-Ct)V/m, R% = (C0-Ct)100/C0 = = (A0-At)100/A0 (1) where C0 and Ct (mg/L) are liquid-phase concentrations of dye at initial and at time t, respectively, V (L) is the volume of the solution and m (g) is the mass of the used adsorbent. A0 and At are the absorbance of MB before and after the adsorption, respectively. 3. Results and Discussion 3. 1. Characterization of the GO/CoO, 3 4 Nanocomposite The FT-IR spectra of the samples are shown in Figure 1. For the starting [Co(en)3](NO3)3 complex, the characteristic stretching bands of NH2, CH2, NO3 were appeared at about 3100-3400, 2951 and 1380 cm-1, respec-tively.37 As can be seen in the spectrum of Co3O4 (Figure 1(b)), almost all bands associated with the complex obviously disappeared when the complex was decomposed at 250 °C and only two strong bands at 569 and 663 cm-1 were observed which confirmed the spinel structure of Co3O4. The former band was attributed to the stretching vibration mode of Co(III)-O and the latter band could be assigned to Co(II)-O bond.38 The formation of Co3O4 from the [Co(en)3](NO3)3 complex can be related to the explosive decomposition of the complex via an intramolecular redox process occurring between the ethylenedi-amine (en) ligands and NO3- ions as reducing and oxidiz- ing agents, respectively. This reaction resulted in the formation of solid Co3O4 and gaseous products i.e. CO2, H2O and NOx (NO, N2O and NO2).38 Although the exact reaction is unclear and intermediates and gaseous products had not been identified directly, the formation of Co3O4 can be expressed as follows: [Co(en)3](NO3)3(s) — Co3O4(s) + CO2(g) + ^O(g) + NOx (NO(g) +^O(g) +NO2(g)). In FT-IR spectrum of GO (Figure 2(c)) obvious characteristic peaks of GO could be seen, including C=O stretching vibrations of COOH groups (1726 cm1), graphitic C=C stretching vibrations (1618 cm-1), O-H deformation vibrations of tertiary C-OH (1398 cm-1), C-O stretching vibrations of epoxy/alkoxy groups (1026 cm-1) and O-H stretching vibrations ( 3100-3700 cm-1).39 However, as can be seen in Figure 2(d), after hydrothermal treatment the band of C=O was disappeared and the intensity of O-H and C-O bands were decreased, which indicated the removal of oxygen-containing functional groups and reduction of GO. This finding confirms the formation of reduced graphene oxide (rGO) in the composite and the restoration of a graphitic structure in graphene.40 Moreover, the two strong absorption peaks in the spectrum of GO/Co3O4 at lower Figure 1. FT-IR spectra of (a) [Co(en)3](NO3)3 complex, (b) Co3O4, (c) GO, and (d) GO/Co3O Pourzare et al.: Graphene Oxide/Co3O4 Nanocomposite: 948 Acta Chim. Slov. 2017, 64, 945-958 frequencies (in the 400-600 cm-1 range) could be assigned to the stretching vibrations of Co-O bonds of Co3O4 phase. This result confirmed the formation of GO/Co3O4 nano-composite. In order to further identify the chemical composition and structure of the prepared samples, powder X-ray diffraction (XRD) was conducted. Figure 2 displays the XRD patterns of Co3O4 and GO/Co3O4 nanocomposite. The diffraction patterns in Figures 2(a) and (b) are similar and can be indexed to the Co3O4 phase (JCPDS No. 781970). Moreover, no characteristic diffraction peaks for GO were observed in the pattern indicating that GO nanosheets were not stacked during the synthesis process. The reason can be attributed to the fact that Co O 3 4 nanoparticles anchored on the surfaces of GO prevented the exfoliated GO nanosheets from restacking. However, a broad characteristic peak for graphene nanosheets at about 20 = 23° appeared, suggesting that GO was reduced to graphene during the formation of the nanocomposite. The average domain size of Co3O4 nanoparticles was calculated to be approximately 13 nm by the Scherrer formula: Dxrd = 0.9À/(P cos 0), where DXRD is average crystalline size; À, and 0 are wavelength of Cu Ka radiation, full width at half maximum of the diffraction peak, and Bragg angle, respec- tively.41 £ GO « VI 1 ^mm W S © 3 S 1 JjjU 20 30 40 5» SO 20(deg.) 70 80 Figure 2. XRD patterns of (a) Co3O4and (b) GO/Co3O4 nanocomposite. Raman spectroscopy is a powerful tool to characterize significant structural changes of GO during the composite synthesis. Figure 3 presents the Raman spectra of GO and GO/Co3O4 nanocomposite. According to the Raman spectrum of GO in Figure 3(a), the obvious peaks at 1318 and 1584 cm-1 can be attributed to the disordered structure (D band, sp3 carbon atoms of disorders and defects) and graphite structure (G band, sp2 carbon atoms in graphitic sheets) of GO, respectively.42 In the spectrum of GO/Co3O4, D and G peaks still exist, while other two obvious peaks at 477 and 683 cm-1 can be attributed to Figure 3. Raman spectra of (a) GO and (b) GO/Co3O4 nanocomposite. Co3O4. Compared to GO, it was clear that D and G bands of GO/Co3O4 were down shifted by 10 cm-1. The red shifts of D and G bands for GO/Co3O4 provided evidence for charge transfer between GO and Co3O4, which indicated a strong interaction between them. Raman spectra further confirmed the successful synthesis of GO/Co3O4 composite. SEM images indicating the microstructural features of GO, Co3O4, GO/Co3O4 nanocomposite are shown in Figure 4. SEM image of pure GO in Figure 4(a) shows layered structure of GO having large stacks, possibly consisting of hundreds of graphene oxide nanosheets. It should also be noted that the surfaces of GO sheets were quite flat and smooth. Figure 4(b) shows SEM micrograph of sphere-like Co3O4 nanoparticles. The SEM images of GO/ Co3O4 in Figures 4(c) and (d) clearly show graphene oxide nanosheets were successfully decorated with Co3O4 nanoparticles. It can be clearly seen that the Co3O4 nanoparticles were well deposited on GO which were a flexible interleaved structure. Some wrinkles are found on the surface, which may be important for preventing aggregation of GO and maintaining high surface area, which could be a great benefit to its adsorption ability. On the contrary with pure GO sheets, the surfaces of GO nanosheets in the nanocomposite were rough, and the edges were highly crumpled. The morphologies and microstructures of the as-prepared Co3O4 and GO/Co3O4 samples were further Pourzare et al.: Graphene Oxide/Co3O4 Nanocomposite: Acta Chim. Slov. 2017, 64, 945-958 949 Figure 4. SEM images of (a) GO, (b) Co3O4, and (c,d) GO/Co3O4. Figure 5. TEM images of (a,b) Co3O4, and (c,d) GO/Co3O4 nanocomposite. Pourzare et al.: Graphene Oxide/Co3O4 Nanocomposite: 950 Acta Chim. Slov. 2017, 64, 945-958 analyzed by TEM. Figures 5(a) and (b) show the typical TEM images of the Co3O4, displaying that the product consists of a large quantity of nearly uniform monodis-persed spheres with the diameter size in the range of 1025 nm which loosely aggregated. As can be seen in Figures 5(c) and (d), the almost transparent graphene sheets are fully exfoliated and decorated homogeneously with sphere-like Co3O4 nanoparticles having an average diameter of 12 nm in consistent with the average particle size calculated from Debye-Scherer formula. No obvious aggregation was seen in Figures 5(c) or 4(d). The GO sheets could not only prevent agglomeration of the Co3O4 nanoparticles and enable a good dispersion of these spherical particles, but also substantially enhance the specific surface area of the composite. Further investigation was carried out by energy dispersive X-ray spectroscopy (EDX) to characterize the composition of the as-prepared GO/Co3O4 nanocompos-ite as shown in Figure 6. The presence of C, O and Co elements in the composites could be proven by the EDX elemental spectrum of GO/Co3O4. The inset of Figure 6 shows a representative SEM image of the nanocomposite with corresponding EDX elemental mappings. As presented in the inset of Figure 6, the distribution of corresponding elemental mappings confirmed the existence of C, O, and Co. From the maps, it can be seen that the elements were uniformly distributed over the nanocomposite, confirming the homogeneity of the sample. The results further indicated that Co3O4 nanocrystals had been successfully loaded on the surface of GO. 10 •5? £ ûû 05- 1.0 —ocytco. $ „ .......-■■■"'"I / IS -loogo -8000 -6000 4000 -2000 0 2000 «00 6000 8c00 1000c H(Oe} Figure 7. Magnetic hysteresis loops of (a) Co3O4 nanoparticles and (b) GO/Co3O4 nanocomposite. of Co3O4 sample shows a ferromagnetic behavior. The fine hysteresis loop of GO/Co3O4 nanocomposite exhibited a typical weak ferromagnetic behavior at room temperature. The saturation magnetization (Ms) of Co3O4 nanoparticles sample is 1.3 emu g-1. In contrast, the GO/Co3O4 nanocomposite has smaller Ms value (0.18 emu g-1), due to the presence of non-magnetic GO component, weakening its magnetic property. Nitrogen adsorption experiments were used to evaluate the pore size and structure of samples. Figure 8 shows the nitrogen adsorption-desorption isotherms and the corresponding pore size distributions curves (the insets) for GO, Co O and GO/Co O samples. The isotherms in 3 4 3 4 Figure 8(a)-(c) can be classified to type IV with H4 hysteresis loop for GO and H3-hysteresis loops for Co3O4 and GO/Co3O4 samples (according to the IUPAC classification), which indicate the presence of mesopores. The interconnected porous network could mainly contribute to the formation of mesopores of GO and the aggregation of GO nanosheets could result in the formation of the mesopores. Some textural properties of the samples were listed in Table 1. As shown in Table 1, the materials were mesoporous. The BET surface area and pore volume of GO/Co3O4 were higher than the values of GO. It can be concluded that the addition of Co3O4 had a great effect on the structure of GO, greatly increasing the surface area and pore volume, which were all favorable factors for improving the adsorption performance. Table. 1 Textural properties of GO, Co3O4 and GO/Co3O4 samples. Figure 6. EDX spectrum of GO/Co3O4 nanocomposite. The inset shows the corresponding EDX elemental mappings. Entry Sample SBET (m2/g) Vp (cm3/g) Dp (nm) 1 GO 79.15 0.126 1.26 2 CÛ3O4 103.67 0.338 1.27 The magnetization curves of Co3O4 and GO/Co3O4 3 GO/Co3O4 107.27 0.336 1.27 samples were measured at room temperature, as shown in Figure 7. Obviously, the shape of magnetic hysteresis loop SBET: BET surface area. V : Total pore volume. D : Average pore diameter calculated using BJH method. Pourzare et al.: Graphene Oxide/Co3O4 Nanocomposite: 951 Acta Chim. Slov. 2017, 64, 945-958 Figure 8. N2 adsorption-desorption isotherms of (a) GO, (b) Co3O4 and (c) GO/Co3O4 samples. The insets show the corresponding pore size distribution curves. 3. 2. Adsorption Studies The adsorption activity of GO/Co3O4 was examined by dispersing composite powder (30 mg) into an aqueous solution (30 ml, 25 mg L-1) of organic dyes (MB, RhB, and MO), and the concentrations of the dye solutions were determined at given intervals by UV-vis absorption spectra. From Figure 9, it can be clearly seen that GO/Co3O4 had different adsorption abilities towards MB, RhB, and MO. The removal percentage of MB of up to 98% could be achieved in 12 min, while only 66% and 45% of RhB and MO were removed within 40 min. To better understand adsorption ability of the nanocomposite, MB was chosen as the removal target to study the adsorption performance in more detail, including adsorption kinetic parameters, adsorption isotherms, and thermodynamic parameters. Figure 10 shows the adsorption abilities of pure Co3O4 and GO samples toward MB, RhB, and MO dyes under our reaction conditions. By using Co3O4 alone, it is clear from Figure 10(a)-(c) the decrease in intensities of characteristic UV-Vis absorption bands of these dyes is almost negligible within 40 min, indicating that it has no ability to adsorb dyes even after long contact times. Figure 10(d)-(f) shows that the GO sample has different adsorption ability towards the dyes. It can be seen that the intensity of the absorption bands of MB and RhB decreases with increasing contact time. The adsorption efficiencies of GO nansheets sample toward these two dyes are about 90% and 50%, respectively, albeit after long adsorption times of 40 min (Figure 10(d) and (e)). On the other hand, as can be seen in Figure 10 (f), the decrease in intensity of characteristic absorption band of MO dye is trace within 40 min. In Figure 10 (g) the adsorption abilities of Co3O4, GO and GO/Co3O4 samples toward MB, RhB, and MO dyes were compared. The removal percentages of three dyes in the presence of Co3O4 sample as an adsorbent were almost negligible (less than 5%) in 40 min. The removal rates of GO sample for MB and RhB cationic dyes are 90 and 50% after long adsorption time of 40 min and the removal of Pourzare et al.: Graphene Oxide/Co3O4 Nanocomposite: 952 Acta Chim. Slov. 2017, 64, 945-958 Figure 9. The adsorption capability of GO/Co3O4 nanocomposite toward different dyes: (a) MB; (b) RhB and (c) MO. (d) The removal efficiency of MB, RhB, and MO dyes. Figure 10. The adsorption capabilities of (a)-(c) Co3O4 nanoparticles and (d)-(f) grapheme oxide (GO) nanosheets toward MB, RhB, and MO dyes. (g) Adsorption efficiency (%) of the dyes in the presence of different adsorbent samples. Pourzare et al.: Graphene Oxide/Co3O4 Nanocomposite: Acta Chim. Slov. 2017, 64, 945-958 953 MO dye is almost negligible at the same time. It is clear that with respect to the removal percentages and adsorption times, the GO/Co3O4 nanocomposite is more suitable and superior. It is suggested that the well dispersed Co3O4 nanoparticles on the graphene surface could act as spacers and thus prohibit the grapheme sheets to restack. This directly results in significant increase of the geometry surface area of graphene, which can be of great benefit to adsorption processes. This result is consistent with BET surface area data in Figure 8. 3. 3. Kinetic Studies and Effects of Contact Time Figure 11 shows the effect of GO/Co3O4 adsorbent dosage on the removal of MB. It is obvious that the percentage of dye removed by the adsorbent increased during the initial stage due to the highest amount of available vacant surface sites and was then slow for all samples until a state of equilibrium was reached after 12 min. It is also observed in Figure 11 that the percentage of the adsorbed dye at equilibrium increased sharply from 43% to 98% with increasing adsorbent dosage from 10 to 30 mg that could be attributed to increase of contact area and availability of more adsorption sites. Meanwhile, to further investigate the adsorption behavior of GO/Co3O4, pseudo-second-order kinetics model 1»» »0 SO > TO B = ai « <59 JO 40 JO :o , -—♦— -♦ JO tJtf 10 tag 1« ii 14 Time (min]l 1« It 10 11 was applied. The pseudo-second-order kinetic model is expressed by the following equation: t/q = 1/k2qe2 + t/qe (2) where k2 is rate constant of the pseudo-second-order model (g mg-1 min-1), qe and qt (mg g-1) are the amounts of the dye adsorbed at equilibrium and at various times t (min), respectively. The values of k2 and qe at different amount of adsorbent can be determined from the intercept and slope of plots of t/qt versus t (Figure 12), respectively, and the results are given in Table 2. It is observed that the experimental adsorption capacity (qeexp: 24.63) value was close to the calculated adsorption capacity (qecal: 26.31). Also, large correlation coefficients (R2 = 0.998) suggested that the adsorption kinetic followed the pseudo-second-order model. The values of qe,cal were increased from 26.31 to 38.46 mg g-1, when the initial amount of adsorbent was decreased from 30 mg to 10 mg, due to the affinity for the adsorption surface sites at lower adsorbent concentration. Figure 11. The effect of with different dosages of GO/Co3O4 on the removal of MB Figure 12. Pseudo-second-order kinetics plot of MB adsorption on GO/Co3O4 nanocomposite. The comparative removal efficiency of MB with different adsorbents in the aqueous medium is presented in Table 3. It was observed that in term of removal efficiency (%) and contact time, GO/Co3O4 nanocomposite was the most efficient adsorbent (98% adsorption of methylene blue) compared to other reported adsorbents. This may be Table 2. Adsorption kinetic parameters for the adsorption of MB onto GO/Co3O4. Adsorbent dosage (mg) Oe.exp (mg g-1) k2 (g mg1 min-1)a Oe.cal (mg g-1) R2 30 24.63 0.05159 26.31 0.998 20 25.15 0.0371 28.57 0.994 10 32.52 0.0193 38.46 0.989 aPseudo-second-order kinetics Pourzare et al.: Graphene Oxide/Co3O4 Nanocomposite: ... 954 Acta Chim. Slov. 2017, 64, 945-958 Table 3. The comparative removal efficiency of MB with different adsorbents. Entry Adsorbent material efficiency (%) Removal cmb (mg L-1) Time (min) Ref. 1 G-CNT hybrid 2 h6p2wi8o62/mof-5 3 M-MWCNTs 4 (4-Hap)4 [Mo8O26] hybrid 5 h3pw12o40@mil-ioi 6 GO/Co3O4 nanocomposite 97 97 82 100 97.5 98 10 10 20 10 20 25 180 10 120 120 30 12 [40] [49] [51] [54] [55] This work due to the fact that, in the case of GO/Co3O4 nanocomposite, the main driving force for adsorption was electrostatic forces of attraction between cationic MB molecules and negatively charged oxygen-containing surface groups along with n-n interaction between localized n electrons in the conjugated aromatic rings of the adsorbent and adsorbate which is comparatively stronger than n-n interaction, electrostatic attraction, van der Waals interaction, and hydrogen bonding alone.43,44 3. 4. Adsorption Isotherm An adsorption isotherm declares the relationship between the mass of dye adsorbed at a given temperature under equilibrium conditions per unit mass of adsorbent (qe, mg g-1) and the liquid phase dye concentration (Ce, mg L-1).45 In this study, to investigate the nature of electrostatic interaction of dye molecules with GO/Co3O4 nanocom-posite, Langmuir and Freundlich models were applied, in which the experiments were conducted by varying the amount of adsorbent from 10 to 25 mg at 25 °C while keeping the concentration of methylene blue solution constant (25 mg L-1). Langmuir adsorption model supposes that maximum adsorption occurs on a saturated monolay-er of solute molecules and all adsorption sites on the ad- sorbent surface are homogeneous and is given by the following equation: 46 c/q = 1/kLqm+c/qm (3) where kL is Langmuir adsorption constant, ce, qe, and qm, are MB concentration at equilibrium (mg L-1), the amount of MB adsorbed at equilibrium (mg g-1), and the maximum adsorption capacity (mg g-1), respectively. The values of qm and k^ are computed from the slope and intercept of the linear plot of ce/qe versus ce. The separation factor (RL) is used to evaluate the favorability adsorption on the adsorbent, which is defined by the following equation: 47 Rl = 1/(1+ KLCO) (4) The parameter can show that the isotherm is irreversible (RL = 0), favorable (0 < RL< 1), linear (RL = 1) or unfavorable (RL> 1). In this work, the value of RL calculated for the initial concentrations of MB was 0.11, which illustrated that the adsorption of MB onto GO/Co3O4 was favorable. Freundlich model is based on the assumption that the multilayer of the adsorption process occurs on a heterogeneous surface and is given by the following equa- tion:47 Figure 13. Adsorption isotherm plots for the adsorption of MB onto GO/Co3O4 nanocomposite: (a) Langmuir isotherm, (b) Freundlich isotherm. Pourzare et al.: Graphene Oxide/Co3O4 Nanocomposite: Acta Chim. Slov. 2017, 64, 945-958 955 Table 4. Isotherm parameters for the adsorption of MB onto GO/Co3O4. Langmuir isotherm Freundlich isotherm K (L mg1) qm (mg g-1) Rl R2 Kf (mg g-1 (L mg-1)1/n) n R2 0.32 40 0.11 0.973 16.36 3.98 0.910 logqe = 1/n log ce + log kf (5) where KF is a Freundlich constant and n is the heterogeneity factor. The isotherms based on the experimental data are shown in Figures 13(a) and (b), and the parameters obtained from linear regression are summarized in Table 4. According to the obtained data, correlation coefficient R2 in Langmuir model (0.973) was higher than that of Freundlich model (0.910), which exhibited that Langmuir model was suitable for describing the adsorption equilibrium of MB on GO/Co3O4 nanocomposite. 3. 5. Effect of pH The effect of pH on the adsorption process of dyes is important because industrial dyes are discharged in waste-waters at a pH different from the environmental pH.48 In general, the solution pH can affect the surface charge of the adsorbent, the degree of ionization/ dissociation of dye molecules as well as dissociation of functional groups on the active sites of the adsorbent.49,50 Figure 14 shows the effect of initial solution pH on MB adsorption onto GO/ Co3O4. Both, the adsorption capacity and removal rate of MB became significant with increasing solution pH from 4 to 12. This phenomenon can be explained by the fact that at higher pH values, the surface of GO/Co3O4 may become negatively charged, which can attract positively charged MB cations through electrostatic forces. A similar trend was observed for the adsorption of methylene blue onto magnetic cyclodextrin/graphene oxide,15 or polydopamine microspheres,47 magnetite-loaded multi-walled carbon nanotubes,51 and magnetic graphene oxide.52 3. 6. Thermodynamic Parameters Thermodynamic studies for the adsorption of MB onto GO/Co3O4 were carried out at different temperatures. Thermodynamic parameters, namely, Gibbs free energy (AG°), enthalpy (AH°) and entropy (AS°) were calculated using following equations: lnKL = -AH°/(RT) + AS°/R AG°= -RT lnK (6) where KL (L/g) is Langmuir constant, R is universal gas constant (8.314 J mol-1 K) and T is absolute temperature (in Kelvin). Plotting lnKL versus 1/T gave a straight line with slope and intercept equal to -AH°/R and AS°/R, respectively (Figure 15). The positive value of AH° (Table 5) showed endothermic nature of adsorption process that it was in accordance with increasing adsorption capacity collaborated with the increase of temperature. The negative value of AG° for different temperatures showed the feasibility and spontaneous nature of adsorption. The activation energy, Ea, was calculated by using Arrhenius equation: lnk = ln A - E /RT (7) Figure 14. Effect of initial pH on adsorption capability and removal of MB with GO/Co3O4 nanocomposite. Figure 15.Van't Hoff plots for the adsorption of MB onto GO/ Pourzare et al.: Graphene Oxide/Co3O4 Nanocomposite: ... Co3O4. 956 Acta Chim. Slov. 2017, 64, 945-958 where k is pseudo-second-order rate constant, E , A, R, and T are activation energy, Arrhenius factor, universal gas constant, and temperature in Kelvin, respectively. The linear plot of lnk against 1/T provided slope equal to -Ea/R. The value of Ea ranging from 5 to 40 kJ mol-1 are characteristic for physisorption while ranging from 40 to 800 kJ mol-1 indicates chemisorptions.53 The activation energy for the adsorption of MB onto GO/Co3O4 nanocomposite was found to be 7.37 kJ mol-1, which indicated that the process was governed by physical adsorption. Table 5. Thermodynamic parameters for the adsorption of MB onto T (K) AG° (KJ/mol) AH° (KJ/mol) AS° (J/mol k) 298 -2.96 13.01 53.62 308 -3.49 - - 318 -4.03 - - 3. 7. Recyclability of GO/Co3O4 Nanocomposite Recovery and regeneration ability of the adsorbent, that can reduce the cost of the treatment process, is crucial for its practical application. For this purpose, the used adsorbent was regenerated with ethanol solution, and the absorption-desorption cycle was repeated four times and the obtained results are shown in Figure 16. In the first three cycles, the removal efficiencies were 98%, 95%, and 93%, respectively, and then remained at 90% at the fourth cycle. These results show that the adsorbent had the potential of reusability. Recycle Figure 16. Effect of recycle times of GO/Co3O4 on MB removal rate. 4. Conclusion GO/Co3O4 nanocomposite was synthesized successfully by a hydrothermal route. The results obtained from XRD, FTIR, FESEM, EDS, and TEM techniques showed that Co3O4 nanoparticles were deposited onto GO na-nosheets. Compared with Co3O4 nanoparticles, GO/Co3O4 nanocomposite showed weaker ferromagnetic behavior. The results revealed that GO/Co3O4 nanocomposite demonstrated rapid uptake of cationic methylene blue (MB) and the adsorption process followed pseudo-second-order kinetic model as well as Langmuir isotherms. Rapid adsorption rate was mainly attributed to the electrostatic interaction of oppositely charged adsorbate-adsorbent species along with their n-n interaction. Thermodynamic parameters showed that the adsorption was spontaneous. Recyclability tests indicated that GO/Co3O4 nanocompos-ite could be recycled and utilized several times without losing adsorption capacity. 5. Acknowledgements The authors gratefully acknowledge Lorestan University Research Council and Iran Nanotechnology Initiative Council (INIC) for their financial supports. 6. References 1. C. A. Demarchi, M. Campos and C. A. Rodrigues, J. Environ. Chem. Eng. 2013, 1, 1350-1358. D01:10.1016/j.jece.2013.10.005 2. J. Wang, C. Zheng, S. Ding, H. Ma and Y. Ji, Desalination 2011, 273, 285-291. D0I:10.1016/j.desal.2011.01.042 3. F. Marahel, M. A. Khan, E. Marahel, I. Bayesti and S. Hosseini, Des. Water Treat. 2015, 53, 826-835. 4. N. M. Julkapli, S. Bagheri and S. B. A. Hamid, Scientific World J. 2014, 2014. DOI: 10.1080/19443994.2013.846240 5. H. Hou, R. Zhou, P. Wu and L. Wu, Chem. Eng. J. 2012, 211, 336-342. D0I:10.1016/j.cej.2012.09.100 6. A. K. Sarkar, A. Pal, S. Ghorai, N. Mandre and S. Pal, Carbo-hydr. Polym. 2014, 111, 108-115. D0I:10.1016/j.carbpol.2014.04.042 7. M. Yusuf, F. Elfghi, S. A. Zaidi, E. Abdullah and M. A. Khan, RSC Adv. 2015, 5, 50392-50420. 8. L. Zhou, C. Gao and W. Xu, ACS Appl. Mater. Interfaces. 2010, 2, 1483-1491. D0I:10.1021/am100114f 9. Y. Chen, L. Chen, H. Bai and L. Li, J. Mater. Chem. A. 2013, 1, 1992-2001. D01:10.1039/C2TA00406B 10. S. Park and R. S. Ruoff, Nat. nanotechnol. 2009, 4, 217-224. D0I:10.1038/nnano.2009.58 11. X. Wang, X. Li, L. Zhang, Y. Yoon, P. K. Weber, H. Wang, J. Guo and H. Dai, Sci. 2009, 324, 768-771. D01:10.1126/science.1170335 12. T. Ramanathan, A. Abdala, S. Stankovich, D. Dikin, M. Herrera-Alonso, R. Piner, D. Adamson, H. Schniepp, X. Chen and R. Ruoff, Nat. Nanotechnol. 2008, 3, 327-331. D0I:10.1038/nnano.2008.96 13. B. Li and H. Cao, J. Mater. Chem. 2011, 21, 3346-3349. D01:10.1039/C0JM03253K GO/Co O 3 4 Pourzare et al.: Graphene Oxide/Co3O4 Nanocomposite: Acta Chim. Slov. 2017, 64, 945-958 957 14. G. Xie, P. Xi, H. Liu, F. Chen, L. Huang, Y. Shi, F. Hou, Z. Zeng, C. Shao and J. Wang, J. Mater. Chem. 2012, 22, 10331039. DOI:10.1039/C1JM13433G 15. L. Li, L. Fan, H. Duan, X. Wang and C. Luo, RSC Adv. 2014, 4, 37114-37121. 16. N. Li, M. Zheng, X. Chang, G. Ji, H. Lu, L. Xue, L. Pan and J. Cao, J. Solid State Chem. 2011, 184, 953-958. DOI:10.1016/j.jssc.2011.01.014 17. L. Fan, C. Luo, X. Li, F. Lu, H. Qiu and M. Sun, J. Hazard. Mater. 2012, 215, 272-279. DOI:10.1016/j.jhazmat.2012.02.068 18. Y. Yao, S. Miao, S. Yu, L. P. Ma, H. Sun and S. Wang, J. Colloid Interface Sci. 2012, 379, 20-26. DOI:10.1016/j.jcis.2012.04.030 19. F. Jiao and H. Frei, Angew. Chem. 2009, 121, 1873-1876. DOI:10.1002/ange.200805534 20. H. Sun, H. Tian, Y. Hardjono, C. E. Buckley and S. Wang, Catal. Today. 2012, 186, 63-68. DOI:10.1016/j.cattod.2011.09.001 21. J. A. Koza, Z. He, A. S. Miller and J. A. Switzer, Chem. Mater. 2012, 24, 3567-3573. DOI:10.1021/cm3012205 22. Z. Wang and L. Zhou, Adv. Mater. 2012, 24, 1903-1911. DOI:10.1002/adma.201200469 23. G. Wang, H. Liu, J. Horvat, B. Wang, S. Qiao, J. Park and H. Ahn, Chem. Eur. J. 2010, 16, 11020-11027. DOI:10.1002/chem.201000562 24. T. Zhu, J. S. Chen and X. W. Lou, J. Mater. Chem. 2010, 20, 7015-7020. DOI:10.1039/c0jm00867b 25. L. Jin, X. Li, H. Ming, H. Wang, Z. Jia, Y. Fu, J. Adkins, Q. Zhou and J. Zheng, RSC Adv. 2014, 4, 6083-6089. 26. S. Farhadi and K. Pourzare, Mater. Res. Bull. 2012, 47, 15501556. DOI:10.1016/j.materresbull.2012.02.028 27. S. Farhadi, K. Pourzare and S. Bazgir, J. Alloys Compd. 2014, 587, 632-637. DOI:10.1016/j.jallcom.2013.10.259 28. L. Wang, J. Deng, Z. Lou and T. Zhang, Sens. Actuators B Chem. 2014, 201, 1-6. DOI:10.1016/j.snb.2014.04.074 29. J. Xu, L. Gao, J. Cao, W. Wang and Z. Chen, Electrochim. Acta. 2010, 56, 732-736. DOI:10.1016/j.electacta.2010.09.092 30. D. Vickers, L. Archer and T. Floyd-Smith, Colloids Surf. A Physicochem. Eng. Aspects, 2009, 348, 39-44. DOI:10.1016/j.colsurfa.2009.06.025 31. J. S. Chen, T. Zhu, Q. H. Hu, J. Gao, F. Su, S. Z. Qiao and X. W. Lou, ACS appl. mater. interfaces. 2010, 2, 3628-3635. DOI:10.1021/am100787w 32. S. Farhadi, Z. Heydari-Chegeni and M. Mousavi, J. Alloys Compd. 2017, 692, 923-933. DOI:10.1016/j.jallcom.2016.09.136 33. F. Hong, Y. Ni, Y. Zhong, H. Wu, J. Alloys Compd. 2016, 659, 112-121. DOI:10.1016/j.jallcom.2015.11.049 34. G. S. Girolami, T. B. Rauchfuss, R.J. Angelici, Synthesis and technique in inorganic chemistry: a laboratory manual, University Science Books, Sausalito, CA, 1999. 35. Y. Yao, Z. Yang, H. Sun and S. Wang, Ind. Eng. Chem. Res. 2012, 51, 14958-14965. DOI:10.1021/ie301642g 36. P. Bradder, S. K. Ling, S. Wang and S. Liu, J. Chem. Eng. Data. 2010, 56, 138-141. DOI:10.1021/je101049g 37. K. Nakamoto, Infrared and Raman spectra of inorganic and coordination compounds, Wiley Online Library, 1986. 38. S. Farhadi, M. Javanmard and G. Nadri, Acta Chim. Slov. 2016, 63, 335-343. DOI:10.17344/acsi.2016.2305 39. B. Pejova, A. Isahi, M. Najdoski and I. Grozdanov, Mater. Res. Bull. 2001, 36, 161-170. DOI:10.1016/S0025-5408(00)00479-7 40. L. Ai and J. Jiang, Chem. Eng. J. 2012, 192, 156-163. DOI:10.1016/j.cej.2012.03.056 41. H. P. Klug and L. E. Alexander, X-ray diffraction procedures, Wiley New York. 1954. 42. Y. Zhao, S. Chen, B. Sun, D. Su, X. Huang, H. Liu, Y. Yan, K. Sun and G. Wang, Sci. Rep. 2015, 5, 7629-7635. DOI: 10.1038/srep07629 43. P. Sharma and M. R. Das, J. Chem. Eng. Data. 2012, 58, 151158. DOI:10.1021/je301020n 44. Y. Li, Q. Du, T. Liu, X. Peng, J. Wang, J. Sun, Y. Wang, S. Wu, Z. Wang and Y. Xia, Chem. Eng. Res. Des. 2013, 91, 361-368. DOI:10.1016/j.cherd.2012.07.007 45. L. Sun, S. Hu, H. Sun, H. Guo, H. Zhu, M. Liu and H. Sun, RSC Adv. 2015, 5, 11837-11844. 46. L. Yao, S. K. Lua, L. Zhang, R. Wang and Z. Dong, J. Hazard. Mater. 2014, 280, 428-435. DOI:10.1016/j.jhazmat.2014.08.026 47. J. Fu, Z. Chen, M. Wang, S. Liu, J. Zhang, J. Zhang, R. Han and Q. Xu, Chem. Eng. J. 2015, 259, 53-61. DOI:10.1016/j.cej.2014.07.101 48. X. Rong, F. Qiu, C. Zhang, L. Fu, Y. Wang and D. Yang, Powder Technol. 2015, 275, 322-328. DOI:10.1016/j.powtec.2015.01.079 49. X. Liu, W. Gong, J. Luo, C. Zou, Y. Yang and S. Yang, Appl. Surf. Sci. 2016, 362, 517-524. DOI:10.1016/j.apsusc.2015.11.151 50. Z. Chen, J. Zhang, J. Fu, M. Wang, X. Wang, R. Han and Q. Xu, J. Hazard. Mater. 2014, 273, 263-271. DOI:10.1016/j.jhazmat.2014.03.053 51. L. Ai, C. Zhang, F. Liao, Y. Wang, M. Li, L. Meng and J. Jiang, J. Hazard. Mater. 2011, 198, 282-290. DOI:10.1016/j.jhazmat.2011.10.041 52. J. H. Deng, X.-R. Zhang, G.-M. Zeng, J.-L. Gong, Q.-Y. Niu and J. Liang, Chem. Eng. J. 2013, 226, 189-200. DOI:10.1016/j.cej.2013.04.045 53. A. Farghali, M. Bahgat, W. El Rouby and M. Khedr, J. Alloys Compd. 2013, 555, 193-200. DOI:10.1016/j.jallcom.2012.11.190 54. Y. Q. Zhang, C.-C. Wang, T. Zhu, P. Wang and S.-J. Gao, RSC Adv. 2015, 5, 45688-45692. 55. T. T. Zhu, Z.-M. Zhang, W.-L. Chen, Z.-J. Liu and E.-B. Wang, RSC Adv. 2016, 6, 81622-81630. Pourzare et al.: Graphene Oxide/Co3O4 Nanocomposite: ... 958 Acta Chim. Slov. 2017, 64, 945-958 Povzetek Nanokompozit grafenovega oksida (GO)/Co3O4 smo sintetizirali s hidrotermalnim razpadom spojine [Co(en)3](NO3)3 na nanoplasteh grafenovega oksida. Tako pripravljen nanokompozit (GO/Co3O4) smo karakterizirali z infrardečo spektroskopijo (FT-IR), rentgensko praškovno difrakcijo (XRD), ramansko spektroskopijo, vrstično in presevno elektronsko mikroskopijo (TEM, SEM), energijsko disperzivno rentgensko spektroskopijo (EDX), magnetnimi meritvami in N2 adsorpcijsko - desorpcijsko analizo. Rezultati so pokazali, da so se nanodelci Co3O4, s povprečnim premerom 12,5 nm pripeli na plasti grafenovega oksida. Adsorpcijsko učinkovitost GO/Co3O4 smo preučevali napram različnim organskim barvilom v vodnih raztopinah. Rezultati adsorpcijske učinkovitosti nanokompozita GO/Co3O4 so: metilen modro: 98 % v 12 minutah; rodamin B 66 % v 40 minutah, metiloranž 45 % v 40 minutah. Podrobno smo preučevali vplive različnih parametrov kot so množina adsorbenta, čas, pH vrednosti in temperatura na adsorpcijski proces. Podatki adsorpcijskega ravnotežja najbolje sledijo Langmuirjevi izotermi, adsorpcijsko kinetiko pa lahko opišemo s psevdo modelom drugega reda. Različni termodinamski parametri kažejo na to, da je proces adsorpcije spontan. Poudarimo lahko tudi lažjo pripravo nanokompozita GO/Co3O4 in njegovo uporabo pri odstranjevanju barvila metilen modro iz odpadnih vod. Pourzare et al.: Graphene Oxide/Co3O4 Nanocomposite: DOI: 10.17344/acsi.2017.3657 Acta Chim. Slov. 2017, 64, 959-967 ¿^creative tS1 commons Scientific paper The Integration of Submicroscopic Representations Used in Chemistry Textbook Sets into Curriculum Topics Špela Hrast* and Vesna Ferk Savec Faculty of Education, University of Ljubljana, Kardeljeva ploščad 16, 1000 Ljubljana, Slovenia * Corresponding author: E-mail: spela.hrast@pef.uni-lj.si Received: 21-06-2017 Abstract To support the understanding of chemistry concepts and processes at the particulate level, various representations are included in learning materials. This paper focuses on how submicroscopic representations (SMRs) are integrated into Slovenian chemistry textbook sets with respect to the curriculum topics for 8th and 9th Grade. Textbook set analysis is based on four holistic SMRs descriptors (direct, indirect, combined descriptor, and SMRs without descriptors), which support learners' recognition of SMRs' informational value at different levels by providing different accompanying SMRs add-ons. The textbook sets analysis revealed that the number of SMRs varies significantly with regard to different curriculum topics. The overall proportion of the descriptors that enable the learner a direct recognition of SMRs is low in all curriculum topics. Interestingly, the descriptors that do not enable the learners a direct recognition of SMRs prevail in textbook sets. To obtain more detailed insight into the criteria based on which the textbook authors integrate SMRs with various descriptors into textbook sets, further studies are necessary. Keywords: Chemistry, curriculum, submicroscopic representations, textbook 1. Introduction Textbooks are an important resource in supporting the effective teaching and learning of chemistry, as one school subject in the larger field of science education. They can be used for studying at school as well as at home.1 In order to be used as teaching materials at schools, the textbook sets for chemistry should be synchronised with the National Curriculum for Chemistry at certain educational levels2,3 and confirmed by the National Commission for Textbook Approval at the Ministry of Education of the Republic of Slovenia.4 To support the quality of the textbook sets in chemistry education, significant attention has been paid to textbook analysis. For example, Abraham et al.5 studied eighth graders' degree of understanding of five selected chemistry concepts found in textbooks and attempted to identify related misconceptions; Sanger and Greenbowe6 analysed the college chemistry textbooks as sources of misconceptions and errors in electrochemistry; Abd-El-Khalick, Waters, and Le7 studied representations of nature of science in high school chemistry textbooks over the past four decades; Devetak, Vogrinc, and Glazar8 studied explanations of states of matter in Slovenian science textbooks from 1th to 8th Grade; and Souza and Porto9 analysed iconographic and textual aspects of chemistry textbooks which had significant diffusion in the context of Brazilian universities. Johnstone10 suggested that representing chemistry concepts and processes is based on representations at three levels: macroscopic (observable phenomena), submicroscopic or particulate (different representations of atomic, molecular, and particle structures), and symbolic (mathematical and chemical symbols). The understanding of chemistry is based on creating mental images for corresponding phenomena on the particulate level. Such mental images are considered to be internal representations that can be visualised through the use of special symbolic systems, so-called external representations of the particulate nature of matter,11,12 which are referred to as submicroscopic representations (SMRs) in this paper. Few macroscopic observations can be understood without recourse to sub-microscopic representation or models.13 Various visualisations are used to support students when connecting the three levels of concept representations,14-16 as the interpretation of the macroscopic phenomenon at the particu-late level is perceived to be an important part in contemporary chemistry teaching.17 Hrast and Ferk Savec: The Integration of Submicroscopic Representations 960 Acta Chim. Slov. 201?, 64, 959-967 2. The Context and the Purpose of the Study This paper focuses on SMRs in Slovenian chemistry textbook sets with respect to the topics of the National Chemistry Curriculum for 8th and 9th Grade.3 Previous research has been dealing with misconceptions related with SMRs.18 The novelty of the present research is in focusing on the descriptors, which accompanying SMRs. Namely, based on their own acceptance of the simultaneous use of SMRs as a part of a triple representation of chemistry concepts (submicroscopic, macroscopic and symbolic levels), chemistry educators, such as authors of textbooks, can assume that students can also easily comprehend and efficiently learn with the use of SMRs.10 However, the understanding of the kinds of information and inferences that the visualisations in various learning materials provide requires explicit instruction and practice.19 The research indicates that students' successful learning with SMRs is significantly impacted by their representational competence in chemistry.20-22 The representational competence includes a distinct set of skills for constructing, selecting, interpreting, and using disciplinary representations for communicating, learning, or problem solving.23 Moreover, in the textbook sets the authors can unintentionally devote more emphasis to the implementation of SMRs in particular topics, whereas in others the partic-ulate representations can be neglected. The following research questions (RQ) were stated: 1st RQ: How does the number of SMRs in Slovenian chemistry textbook sets for 8th and 9th Grade change with respect to curriculum topics? 2nd RQ: What holistic descriptors of SMR add-ons are used to support learners in the recognition of SMRs' informational value in specific curriculum topics of Slovenian chemistry textbook sets for 8th and 9th Grade? 3rd RQ: How does the number of specific holistic descriptors of SMR add-ons in Slovenian chemistry textbook sets for 8th and 9th Grade change with respect to curriculum topics? Table 1. The list of the analysed textbook sets Textbook set* title Author(s) Publisher Year of publication (Edition) Textbook/ workbook Number of Pages Textbook/ workbook age Grade/ Learner's Kemija danes 1 Gabrič, A., Glažar, S. A., Graunar, M., Slatinek-Žigon, M. DZS 2014 (1st Ed.)/ 2013 (1st Ed.) 125/106 8/13 Kemija 8, Sajovic, I., Wissiak Grm, K., Godec, A., Zavod RS 2014 264 8/13 i-učbenik Kralj, B., Smrdu, A., Vrtačnik, M., Glažar, S. za šolstvo Moja prva kemija Vrtačnik, M., Wissiak Grm, K. S., Glažar, S. A., Godec, A. Modrijan 2015 (1st Ed.)/ 2014 (1st Ed.) 240/92, 61 8, 9/13, 14 Peti element 8 Devetak, I., Cvirn Pavlin, T., Jamšek, S. ROKUS KLET T 2010 (1st Ed.)/ 2010 (1st Ed.) 103/71 8/13 Pogled v kemijo 8 Kornhauser, A., Frazer, M. M K 2003 (1st Ed.)/ 2004 (1st Ed.) 140/126 8/13 Od atoma do molekule Smrdu, A. JUTRO 2012 (2nd Ed.)/ 2012 (2nd Ed.) 128/160 8/13 Kemija Graunar, M., Podlipnik, M., Mirnik, J. (textbook) DZS 2016 (1st Ed.)/ danes 2 Dolenc, D., Graunar, M., Modec, B. (notebook) 2016 (1st Ed.) 152/96 9/14 Kemija 9, Jamšek, S., Sajovic, I., Wissiak Grm, K., Zavod RS 2014 271 9/14 i-učbenik Godec, A., Boh, B., Vrtačnik, M., Glažar, S. za šolstvo Peti element 9 Devetak, I., Cvirn Pavlin, T., Jamšek, S. ROKUS Klett 2011 (1st Ed.)/ 2011 (1st Ed.) 77/ 79 9/14 Pogled v kemijo 9 Kornhauser, A., Frazer, M. M K 2005 (1st Ed.)/ 2006 (1st Ed.) 140/115 9/14 Od molekule do makromolekule Smrdu, A. Jutro 2013 (2nd Ed.)/ 2013 (2nd Ed.) 128/152 9/14 The term "textbook set (*)" refers to all materials for students in the written or electronic form. Hrast and Ferk Savec: The Integration of Submicroscopic Representations ... Acta Chim. Slov. 201?, 64, 959-967 961 3. Methods 3. 1. Sample In this study, we focused on the chemistry textbook sets in primary school (8th and 9th Grade), which are in Slovenia obligatory written based on the objectives of National Chemistry Curriculum and consequently confirmed by the National Commission for Textbook Approval at the Ministry of Education, Science and Sport in the 2016/17 school year. National Chemistry Curriculum for 8th and 9th Grade3 for each of the ten topics (Chemistry is a World of Matter, Atom and the Periodic System of Elements, Compounds and Bonding, Chemical Reactions, The Elements in the Periodic Table, Acids, Bases and Salts, Hydrocarbons and Polymers, Organic Compounds Containing Oxygen, Organic Compounds Containing Nitrogen, The Mole) specifies specific objectives and points out suggested contents how to implement them in chemistry teaching. Teachers are free to distributed the above listed curriculum topics in 70 hours in Grade 8 and 64 hours in Grade 9 with regard to their opinion. A list of analysed textbook sets is shown in Table 1. As can be derived from the Table 1, in the present study 2826 pages were analysed. Table 2. Examples of SMR add-ons Descriptor Examples of SMR add-ons Direct (D) Explanatory key I Model of carton atom ^ Model of hydrogen atom A Hydrogen atoms are represented with white cycles, carbon atoms are represented with black cycles. Indirect (I) Molecular model of propane Molecular model ropanc Explanatory key Mtxlïl ■olYailxjn nom Combined (C) nun Molecular model of propane kyy W Explanatory key M odd of carton atom Model of hydrogen a Molecular model of propane (qiy xV Molccular model of propane Hydrogen atoms arc represented with white cycles, carbon atoms are represented with black cycles. Without (W) Hrast and Ferk Savec: The Integration of Submicroscopic Representations 962 Acta Chim. Slov. 2017, 64, 959-967 3. 2. Instruments For the purpose of this research, a rubric24 for the evaluation of SMRs in the textbook sets was used. This rubric was based on the assumption that in practice the learner perceives each SMR as one whole. The four main holistic descriptors accompanying SMRs were used: i.e. direct descriptor (D), indirect descriptor (I), combined descriptor (C), and SMRs without descriptors (W), which support learners' recognition of SMRs' informational value on different levels by providing different accompanying add-ons of SMRs. To ensure the validity of the rubric, 283 pages (10% of all analysed textbook set pages) were analysed by both authors, and the four main types of holistic descriptors were defined. To reduce bias issues related to the use of the rubric for categorisation of SMR descriptors, through discussion and agreement, a 95% inter-rater reliability of the rubric was established. The direct SMR add-ons enable the learner a direct and unambiguous recognition of particles. Thereby, various types of explanatory keys can be used: for example, pictorial, textual, integrated structural, or other symbolic notations used in the explanatory key. Indirect SMR add-ons do not enable the learner a direct recognition of particles. The nature of the particles can be derived based on the compound's name, structure-properties relation or symbolic SMR add-ons, or by other means, but only in cases that the learners have the necessary chemical knowledge, that enables the recognition of its informational value. Combined SMR add-ons enable the learner a direct recognition and provide other information. It is a combination of the direct and the indirect descriptor. Examples of SMR add-ons are presented in Table 2. In order to visualize descriptors explicitly, the type of descriptors represents the only variable and SMR add-ons are accompanying SMR of the same compound (propane molecule). 3. 3. Data analysis The rubric described in the instrument section was used in the analysis of the chemical representations of the entire sample of chemistry textbook sets, which are presented in Table 1. The textbook sets were analysed individually. The SMRs were categorised with regard to curriculum topics of the National Chemistry Curriculum for 8th and 9th Grade.3 The core topics in which SMRs were categorised are the following: (1) Chemistry is a World of Matter (orig. Kemija je svet snovi); (2) Atom and the Periodic System of Elements (orig. Atom in periodni sistem elementov); (3) Compounds and Bonding (orig. Povezovanje delcev/gradnikov); (4) Chemical Reactions (orig. Kemijske reakcije); (5) The Elements in the Periodic Table (orig. Elementi v periodnem sistemu); (6) Acids, Bases and Salts (orig. Kisline, baze in soli); (7) Hydrocarbons and Polymers (orig. Družina ogljikovodikov s polimeri); (8) Organic Compounds Containing Oxygen (orig. Kisikova družina organskih snovi); (9) Organic Compounds Containing Nitrogen (orig. Dušikova družina organskih spojin) and (10) The Mole (orig. Množina snovi). Eventually, the number of SMRs in each of the topics were counted and the frequencies calculated. Analysed SMRs involved SMRs of molecules, atoms and ions. 4. Results and Discussion The results of the analysis are presented with regard to the research questions. 4. 1. The Number of SMRs in Slovenian Chemistry Textbook Sets for 8th and 9th Grade Change With Respect to Curriculum Topics (Related to RQ1) The number of images about the SMRs in chemistry textbook sets is given in Table 3. Most frequently, the SMRs were used in the topics "Hydrocarbons and Polymers" (28.68%; 407 SMRs) and "Organic Compounds Containing Oxygen" (20.23%; 287 SMRs). The lowest frequencies of the use of SMRs were found in the following Table 3. The proportion of SMRs and curriculum objectives in the particular topics of the textbook sets The topics of the National Chemistry Curriculum for 8th and 9th Grade N SMRs f (%) Curriculum objectives N f (%) Chemistry is a World of Matter (1) 179 12.61 5 8.62 Atom and the Periodic System of Elements (2) 29 2.04 4 6.90 Compounds and Bonding (3) 150 10.57 5 8.62 Chemical Reactions (4) 69 4.86 6 10.34 The Elements in the Periodic Table (5) 16 1.13 7 12.07 Acids, Bases and Salts (6) 160 11.28 7 12.07 Hydrocarbons and Polymers (7) 407 28.68 9 15.52 Organic Compounds Containing Oxygen (8) 287 20.23 7 12.07 Organic Compounds Containing Nitrogen (9) 116 8.17 5 8.62 The Mole (10) 6 0.42 3 5.17 SUM 1419 100.00 58 100.00 Hrast and Ferk Savec: The Integration of Submicroscopic Representations ... Acta Chim. Slov. 2017, 64, 959-967 963 topics: The Mole (0.42%; 6 SMRs), The Elements in the Periodic Table (1.13%; 16 SMRs) and Atom and the Periodic System of Elements (2.04%; 29 SMRs). It was expected that the change in the number of the SMRs would be proportionally related to the number of objectives in the specific chemistry topics, as the objectives in the Chemistry Curriculum for 8th and 9th Grade3 are written operationally and can be interpreted by the use of representations on all three representational levels (macroscopic, submicroscopic, and symbolic). This has been proven true for the topics in which the highest proportion of the SMRs has been used. The highest proportion of SMRs in the chemistry textbook sets for the curriculum topic "Hydrocarbons and Polymers" (28.68%; 407 SMRs) is proportional to the highest proportion of objectives in this topic (15.52%; 9 objectives). Similarly, the proportion of SMRs in the chemistry textbook sets for the curriculum topic "Organic Compounds Containing Oxygen" (20.23%; 287 SMRs) are proportional to the proportion of objectives in this topic (12.07%; 7 objectives). Chemistry concepts and processes of these topics are traditionally explained by combining all three representational levels, which is also encouraged with the notations of the curriculum objectives that directly indicate the relationship between structure, properties, and application of substances. For example, one of the objectives in the curriculum topic "Hydrocarbons and Polymers" states: "Students should know that carbon and hydrogen are the fundamental elements of organic compounds - hydrocarbons, and they can identify the causes for the abundance and the variety of organic compounds".24 Despite the high proportion of associated objectives (10.34%; 6 objectives) in the curriculum, the textbook set analysis revealed surprisingly a low number of SMRs used in the curriculum topic "Chemical Reactions" (4.86%; 69 SMRs). Furthermore, the manner in which notations of the objectives are written indicates the need for their explanation by the combined use of the three levels of their representation. For example, it would be expected that for the achievement of the objectives such as "Students should be able to define reactants and products of chemical reactions"26 and "Students should get acquainted with chemical equations as notations of chemical reactions"26 would be to a greater proportion presented in the textbooks, not only with photos of the phenomena and/or examples of experiments with their symbolic notations but also with the underlying SMRs. More frequent use of the triple-nature representations of chemistry concepts would also be expected in the curriculum topic "The Elements in the Periodic Table". However, as mentioned before, the number of SMRs in this topic are one of the lowest among the curriculum topics (Table 3). One of the reasons for the lower proportions of SMRs could also be in the nature of some of the objectives. In particular, some objectives explicitly refer to the macroscopic representational level, e.g. "Students should get to know natural resources of elements and com-pounds"27 or to the development of stoichiometric skills, e.g. "Students should know how to calculate a mass percentage of the elements in the compounds".27 Consequently, in these cases the use of SMRs could easily be neglected. 4. 2. Holistic descriptors of SMR add-ons used to support learners in the recognition of SMRs' informational value in specific curriculum topics of Slovenian chemistry textbook sets for 8th and 9th Grade (related to RQ2) The analysis revealed that various topics of the Chemistry Curriculum for 8th and 9th Grade3 include not only different numbers of SMRs but that those SMRs also include different add-ons. The descriptors of the SMR addons have already been presented in Table 2. As expected, the analysis of the textbooks revealed that the authors of the textbook sets used various proportions of different types of descriptors of SMR add-ons in specific topics, which is presented in Table 4 and Table 5. In the top three topics in which SMRs are most frequently used, i.e. "Hydrocarbons and Polymers", "Organic Compounds Containing Oxygen" and "Chemistry is a World of Matter", the use of indirect SMRs add-ons prevail (56.27%, 229 SMRs; 69.69%, 200 SMRs; 36.87%, 66 SMRs, respectively). However, it is interesting that more than one third of SMRs (35.87%, 146 SMRs; 26.13, 75 SMRs; 35.20%, 63 SMRs, respectively) do not include any descriptors. In these cases, the recognition of the informational value of particular SMRs depends entirely on learners' previous experience, knowledge, and their representational competence. This is surprising for the first curriculum topic "Chemistry is a World of Matter", as it should introduce some of the examples of simple compounds and the states of matter on the particle level, where students' development of their representational competence needs to be systematically supported by explicit instruction and prac-tice.18 In "Hydrocarbons and Polymers" and "Organic Compounds Containing Oxygen" other types of SMR descriptors occur rarely, but in the first topic of the curriculum "Chemistry is a World of Matter", in addition to the indirect descriptor of SMR add-ons also the significant proportion of the combined descriptors (26.26%; 47 SMRs) were found. In these cases, the recognition of the informational value of a particular SMR is supported by SMR add-ons and the learners' previous experience, knowledge, and their representational competence. As mentioned earlier, in the topics of the curriculum "Atom and the Periodic System of Elements", "The Elements in the Periodic Table", and "The Mole", the lowest frequencies of the use of SMRs were found. However, the analysis of different types of SMRs add-ons in the second curriculum topic "Atom and the Periodic System of Ele- Hrast and Ferk Savec: The Integration of Submicroscopic Representations 964 Acta Chim. Slov. 2017, 64, 959-967 ments" reveals that more than half of the SMRs cases are accompanied by indirect descriptors (55.17%; 16 SMRs) of SMR add-ons, more than one third of SMRs enable the learner a direct recognition of particles (10.34%; 3 SMRs) and provide other information - combined descriptor (31.03%; 9 SMRs). In this case the recognition of the informational value of a particular SMR is substantially supported by SMR add-ons, which greatly enables students easier understanding of the atomic structure, ions formation from the atoms and, consequently, the understanding of the relation between atomic structure and their position in the Periodic Table of Elements with regard to the objectives of the curriculum.3 In the fifth topic of the curriculum, "The Elements in the Periodic Table", the SMR cases with indirect descriptors of SMR add-ons (62.50%; 10 SMRs) prevail. The second most frequently used SMRs are SMRs without SMR add-ons (25.00%; 4 SMRs). In the last curriculum topic "The Mole" two thirds of SMRs do not have SMRs add-ons (66.67%; 4 SMRs) and the rest have an indirect descriptor (33.33%; 2 SMRs). For the recognition of SMRs' informational value learners need to rely on their previous experience, knowledge, and representational competence developed in earlier topics of the curriculum. In all of the remaining topics "Acids, Bases and Salts", "Compounds and Bonding", "Organic Compounds Containing Nitrogen" and "Chemical reactions" the indirect SMR add-ons prevail (45.63%, 73 SMRs; 58.67%, 88 SMRs; 56.03%, 65 SMRs; 59.42%, 41 SMRs, respectively). Moreover, in these topics, which are placed in different parts of the curriculum, a high proportion of SMRs without any SMR add-ons was found, which do not support recognition of SMRs' informational value. 4. 3. Holistic descriptors of SMR add-ons used to support learners in the recognition of SMRs' informational value in particular curriculum topics of Slovenian chemistry textbook sets for 8th and 9th Grade (related to RQ3) In the textbook set analysis, particular attention has been devoted to studying whether the authors systematically planned the integration of SMS in the textbooks in order to support students' development of their representational competence. Specifically, in order to do so, Hinze, Rapp, Williamson, et al.,19 pointed out that explicit instruction for that purpose and practice with the use of particular representations, e.g. SMRs, is necessary. In the context of the textbook sets, it would mean, that particular kinds of SMRs with add-ons would be carefully selected and their integration into the textbook sets continuously upgraded throughout the curriculum. Table 4. The proportion of SMRs within the particular topics of the textbook sets - part 1 Descriptor of SMR add-ons The first five topics of the National Chemistry Curriculum for 8th and 9th Grade3 General Chemistry is a World of Matter Atom and the Periodic System of Elements Compounds and Bonding Chemical Reactions The Elements in the Periodic in Table N f (%) N fc (%) N fc (%) N fc (%) N fc (%) N fc (%) Direct (D) 63 4.44 3 1.68 3 10.34 5 3.33 16 23.19 2 12.50 Indirect (I) 790 55.67 66 36.87 16 55.17 88 58.67 41 59.42 10 62.50 Combined (C) 147 10.36 47 26.26 9 31.03 26 17.33 6 8.70 0 0.00 Without (W) 419 29.53 63 35.20 1 3.45 31 20.67 6 8.70 4 25.00 SUM 1419 100.00 179 100.00 29 100.00 150 100.00 69 100.00 16 100.00 Table 5. The proportion of SMRs within the particular topics of the textbook sets - part 2 The last five topics of the National Chemistry Curriculum for 8th and 9th Grade3 Organic Organic Acids, Bases Hydrocarbons Compounds Compounds and Salts and Polymers Containing Contenting Oxygen Nitrogen N fc (%) N fc (%) N fc (%) N fc (%) N fc (%) Direct (D) 11 6.88 17 4.18 6 2.09 0 0.00 0 0.00 Indirect (I) 73 45.63 229 56.27 200 69.69 65 56.03 2 33.33 Combined (C) 38 23.75 15 3.69 6 2.09 0 0.00 0 0.00 Without (W) 38 23.75 146 35.87 75 26.13 51 43.97 4 66.67 SUM 160 100.00 407 100.00 287 100.00 116 100.00 6 100.00 Descriptor of SMR add-ons Hrast and Ferk Savec: The Integration of Submicroscopic Representations ... Acta Chim. Slov. 2017, 64, 959-967 965 Figure 1. The proportion of SMRs with particular descriptors within all of the topics in the textbook sets [Curriculum topics: Chemistry is a World of Matter (1); Atom and the Periodic System of Elements (2); Compounds and Bonding (3); Chemical Reactions (4); The Elements in the Periodic Table (5); Acids, Bases and Salts (6); Hydrocarbons and Polymers (7); Organic Compounds Containing Oxygen (8); Organic Compounds Containing Nitrogen (9); The Mole (10)] From Figure 1, it can be seen how the overall proportion of different descriptors of SMR add-ons are distributed through the topics of the curriculum. The textbook set analysis revealed that the overall proportion of direct and combined descriptors of SMR add-ons is significantly low through all the curriculum topics (range from 0.00% to 3.31%). That can be connected with the Johnstone's claim10 that the experienced chemists do the transition between levels of representations very easily and they assumed that learners can do this as easily as they do. These simplified assumptions about learners' easy comprehension and efficient learning with SMRs are reflecting in the low frequency of SMR descriptors, where the informational value of a particular SMR enables the learner a direct recognition of particles, in textbook sets. It can also be observed from Figure 1, that the overall percentages of SMRs with indirect descriptors or without any add-ons throughout the curriculum are higher than the percentages of SMRs with direct and combined descriptors of SMR add-ons, whereby the figures in the topics "Hydrocarbons and Polymers" and "Organic Compounds Containing Oxygen" stand out (range from 5.29% to 16.14%). From the perspective of the integration of SMRs into the textbook sets based on the assumption about their continuous upgrading throughout the curriculum topics (from the first to last topic), this is a very interesting finding. It can be related to the fact that in these chapters the highest proportion of SMSs is used from all curriculum topics, as proposed earlier, probably as a consequence of the number of curriculum objectives for these topics (Table 3). However, the ratio among the different descriptors of SMR add-ons used in these chapters speaks in favour of indirect SMR add-ons, which do not enable the learner a direct recognition of particles, as well as SMRs without add-ons, which do not provide any additional information to support the learner's recognition process. The reason for the use of such representations at that point of the textbook sets could either be that the authors assume that the learners are already able to recognise the informational value of SMRs indirectly, because their representational competence has been adequately developed in previous topics, or that the authors integrate SMRs with add-ons into the textbook sets without considering how the correct recognition of the informational value by learners might affect the learning process based on it. As only a low proportion of direct and combined SMR add-ons was found in the textbook set analysis, it seems worthy to encourage textbook authors to devote special attention to optimally equip the SMRs with addons to support students' development of the representational competence across the curriculum. When the learners' perception of SMRs is not a possible initial obstacle for learning, it will be easier to overcome the range of students' misconceptions, related to particle nature of chemical concepts, i.e. in making the distinctions between elements, compounds, and mixtures; appreciations of the reversibility of state changes; seeing atoms and ions as the building blocks of matter; appropriate use of basic chemistry terminology, understanding that Broensted acids and bases are not substances but molecules and ions etc.28-31 On the other hand it is important to pay attention to scientific accuracy of SMRs to enable appropriate understanding of chemical concepts and processes. Hrast and Ferk Savec: The Integration of Submicroscopic Representations 966 Acta Chim. Slov. 2017, 64, 959-967 5. Conclusions When learning chemistry, learners are often challenged by various representations at the submicroscopic level that are included in learning materials, such as textbook sets, to develop understanding of chemistry concepts and processes at the particulate level. Previous research19,23 has demonstrated that the understanding of visualisations in various learning materials based on recognising the informational value of various representations requires explicit instruction and practice. This paper presents an analysis of the chemistry textbook sets for 8th and 9th Grade in Slovenia from the perspective of the integration of SMRs into various National Chemistry Curriculum3 topics. In particular, it was found that the number of SMRs in Slovenian chemistry textbook sets varies significantly with regard to different curriculum topics. In the topics where the highest proportion of the SMRs has been used ("Hydrocarbons and Polymers", "Organic Compounds Containing Oxygen"), the number of the SMRs is proportionally related to the number of objectives in the specific chemistry topics. Despite the high proportion of objectives in the curriculum, the textbook sets analysis indicates a surprisingly low number of the SMRs used in the curriculum topics "Chemical Reactions" and "The Elements in the Periodic Table". Regarding the holistic descriptors of SMR add-ons, that are used to support learners in the recognition of SMRs' informational value in various curriculum topics, it was found that the descriptors that do not enable the learners a direct recognition of SMRs prevail, especially in the topics "Hydrocarbons and Polymers" and "Organic Compounds Containing Oxygen", which are in the final part of the curriculum. In contrast, the textbook set analysis revealed that the overall proportion of the descriptors that enable the learner a direct recognition of the informational value of SMRs is very low through all the curriculum topics. The results can serve as a foundation for a discussion with textbook authors about the role of SMRs with add-ons in supporting students' development of representational competence across curriculum as well as in the learning process, as it seems valuable that the particular kinds of SMRs with add-ons would be carefully selected and their integration into the textbook sets continuously upgraded throughout the curriculum. Further studies are necessary to obtain more detailed insight into the criteria based on which the authors integrate SMRs with various descriptors into textbook sets. From the learner's point of view, in future studies, it would be valuable to focus on research possibilities provided by contemporary technology, such as Eyetracker, for following the learner's information processing and their use of SMRs in learning with traditional as well as with e-learning materials. Thus far, for example, the importance of various features and notations of visualisations for the learners has been examined with eye trackers by William- son et al.,32 who studied students' use of ball-and-stick images versus electrostatic potential maps when considering electron density, positive charge, proton attack, and hydroxide attack; O'Keefe et al.,33 who examined how the integration of multiple representations was associated with learning in a multimedia simulation; Ferk Savec et al.34 examined some of the features of the explanatory key, such as coloured versus black-and-white explanatory keys, and pictorial versus textual explanatory keys, etc. The results of such studies contribute to the quality of the textbook sets and consequently also to the quality of chemistry teaching and learning, as they can be used for studying in various learning environments. 6. References 1. V. Gkitzia, K. Salta, C. Tzougraki, Chem. Educ. Res. Pract. 2011, 12, 5-14. DOI:10.1039/C1RP90003J 2. A. Bačnik, N. Bukovec, A. Poberžnik, T. Požek Novak, Z. Keuc, H. Popič, M. Vrtačnik, in: N. Purkat (Ed.): Učni načrt. Program gimnazija. Kemija, Ministrstvo za šolstvo in šport, Zavod RS za šolstvo, Ljubljana, 2008, 59 pp. 3. A. Bačnik, N. Bukovec, M. Vrtačnik, A. Poberžnik, M. Križaj, V Stefanovik, K. Sotlar, S. Dražumerič, S. Preskar, in: A. Štrukelj (Ed.): Učni načrt. Program osnovna šola. Kemija, Ministrstvo za šolstvo in šport, Zavod RS za šolstvo, Ljubljana, 2011, 31 pp. 4. Ministrstvo za izobraževanje, znanost in šport, Trubar učbeniški sklad, https://soca1.mss.edus.si/Trubar/default.aspx, (assessed: June 9, 2017) 5. M. R. Abraham, E. B. Grzybowski, J. W. Renner, E. A. Marek, J. Res. Sci. Teach. 1992, 29, 105-120. DOI:10.1002/tea.3660290203 6. M. J. Sanger, T. J. Greenbowe, J. Chem. Educ. 1999, 76, 853860. DOI:10.1021/ed076p853 7. F. Abd-El-Khalick, M. Waters, A. P. Le, J. Res. Sci. Teach. 2008, 45, 835-855. DOI:10.1002/tea.20226 8. I. Devetak, J. Vogrinc, S. A. Glažar, Int. J. Env. Sci. Ed. 2010, 5, 217-235. 9. K. A. F. Souza, P. A. Porto, Sci. Educ. 2012, 21, 705-727. DOI:10.1007/s11191-012-9442-z 10. A. H. Johnstone, J. Comput. Assist. Learn. 1991, 7, 75-83. DOI:10.1111/j.1365-2729.1991.tb00230.x 11. J. K. Gilbert, in: J. K. Gilbert (Ed.): Visualization in Science Education, Springer, Dordrecht, 2005, pp. 9-27. DOI:10.1007/1-4020-3613-2_2 12. J. K. Gilbert, M. Reiner, M. Nakhleh. Visualization: Theory and practice in science education. Springer, New York, 2008, 325 pp. DOI: 10.1007/978-1-4020-5267-5 13. J. Oversby, in: J. K. Gilbert, C. J. Boulter (Eds.): Developing models in science education, Kluwer Academic Publishers, Dordrecht, 2000, pp. 227-251. DOI:10.1007/978-94-010-0876-1_12 14. H. D. Barke, H. Wirbs, Chem. Educ. Res. Pract. 2002, 3, 185200. DOI:10.1039/B2RP90015G Hrast and Ferk Savec: The Integration of Submicroscopic Representations ... Acta Chim. Slov. 2017, 64, 959- 967 967 15. V. Ferk Savec, I. Sajovic, K. S. Wissiak Grm, in: J. K. Gilbert (Ed.): Multiple Representations in Chemical Education, Springer, Berlin, 2009, pp. 309-331. DOI:10.1007/978-1-4020-8872-8_14 16. S. M. Al-Balushi, S. H. Al-Hajri, Chem. Educ. Res. Pract. 2014, 15, 47-58. D0I:10.1039/C3RP00074E 17. I . Eilks, in: V. G. Tsaparlis, H. Sevian (Eds.): Concepts of Matter in Science Education, Springer, New York, 2013, pp. 213-230. D0I:10.1007/978-94-007-5914-5_10 18. I. Devetak, J. Vogrinc, S. A. Glažar, Res. Sci. Educ. 2009, 4, 82-94. 19. S. R. Hinze, D. N. Rapp, V. M. Williamson, M. J. Shultz, G. Deslongchamps, K. C. Williamson, Learn. Instr. 2013, 26, 12-21. D0I:10.1016/j.learninstruc.2012.12.002 20. V. Ferk, M. Vrtacnik, A. Blejec, A. Gril, Int. J. Sci. Educ. 2003, 25, 1227-1245. D0I:10.1080/0950069022000038231 21. R. Kozma, J. Russell, in: J. Gilbert (Ed.): Visualization in Science Education, Kluwer, London, 2005, pp. 121-146. D0I:10.1007/1-4020-3613-2_8 22. A. T. Stull, M. Hegarty, B. L. Dixon, M. Stieff, Cogn. Instr. 2012, 30, 404-434. D0I:10.1080/07370008.2012.719956 23. M. Stieff, S. Scopelitis, M. E. Lira, D. Desutter, Sci. Educ. 2016, 100, 344-363. D0I:10.1002/sce.21203 24. Š. Hrast, V. Ferk Savec, J. Balt. Sci. Educ. 2017, 16, in press. 25. A. Bačnik, N. Bukovec, M. Vrtačnik, A. Poberžnik, M. Križaj, V. Stefanovik, K. Sotlar, S. Dražumerič, S. Preskar, in: A. Štrukelj (Ed.): Učni načrt. Program osnovna šola. Kemija, Ministrstvo za šolstvo in šport, Zavod RS za šolstvo, Ljubljana, 2011, pp. 11. 26. A. Bačnik, N. Bukovec, M. Vrtačnik, A. Poberžnik, M. Križaj, V. Stefanovik, K. Sotlar, S. Dražumerič, S. Preskar, in: A. Štrukelj (Ed.): Učni načrt. Program osnovna šola. Kemija, Ministrstvo za šolstvo in šport, Zavod RS za šolstvo, Ljubljana, 2011, pp. 9. 27. A. Bačnik, N. Bukovec, M. Vrtačnik, A. Poberžnik, M. Križaj, V. Stefanovik, K. Sotlar, S. Dražumerič, S. Preskar, in: A. Štrukelj (Ed.): Učni načrt. Program osnovna šola. Kemija, Ministrstvo za šolstvo in šport, Zavod RS za šolstvo, Ljubljana, 2011, pp. 10. 28. K. Taber, Chemical misconceptions: Prevention, diagnosis and cure (Vol. 1), Royal Society of Chemistry, London, 2002, 246 pp. 29. H. D. Barke, A. Hazari, S. Yitbarek. Students' misconceptions and how to overcome them. Springer, Berlin, Heidelberg, 2009, pp. 21-36. D0I:10.1007/978-3-540-70989-3_3 30. V. Kind, Beyond appearances: Students' misconceptions about basic chemical ideas, School of Education, Durham University, UK, 2009, 84 pp. 31. H. Barke, N. Harsch, Afr. J. Chem. Educ. 2014, 4, 82-94. 32. V. M. Williamson, M. Hegarty, G. Deslongchamps, K. C. Williamson III, M. J. Shultz, J. Chem. Educ. 2015, 90, 159-164. D0I:10.1021/ed200259j 33. P. A. O'Keefe, S. M. Letourneau, B. D. Homer, R. N. Schwartz, J. L. Plass, Comput. Human. Behav. 2014, 35, 234-242. D0I:10.1016/j.chb.2014.02.040 34. V. Ferk Savec, Š. Hrast, I. Devetak, G. Torkar, Acta Chim. Slov. 2016, 63, 864-873. D0I:10.17344/acsi.2016.2835 Povzetek V podporo razumevanju naravoslovnih pojmov in procesov na ravni delcev so v učna gradiva vključene različne predstavitve (reprezentacije). Članek se osredotoča na preučevanje, kako so submikroskopske reprezentacije (SMR-ji) integrirane v slovenske kemijske učbeniške komplete v povezavi z vsebinami učnega načrta za osnovno šolo. Izhodišče za analizo učbeniškega gradiva so predstavljali štirje holistični deskriptorji SMR-jev (direktni, indirektni, kombinirani deskriptor in SMR-ji brez deskriptorjev), ki glede na specifične opisnike SMR-jev do različne mere podpirajo učenca pri prepoznavanju informacijske vrednosti SMR-jev. Analiza učbeniških kompletov je pokazala, da se število SMR-jev močno spreminja glede na različne vsebine učnega načrta. Delež deskriptorjev, ki učencem omogočajo direktno prepoznavanje SMR-jev, je nizek v vseh vsebinah učnega načrta. Zanimivo je, da deskriptorji, ki ne omogočajo direktne prepoznave SMR-jev, prevladujejo v učbeniških kompletih. Z namenom pridobitve poglobljenega vpogleda v kriterije, na podlagi katerih avtorji učbenikov integrirajo SMR-je z različnimi deskriptorji v učbeniške komplete, so potrebne nadaljnje raziskave. Hrast and Ferk Savec: The Integration of Submicroscopic Representations 968 DOI: 10.17344/acsi.2017.3662 Acta Chim. Slov. 2017, 64, 968-979 ^creative ty com mor Scientific paper Optimization of UPLC Method for Simultaneous Determination of Rosuvastatin and Rosuvastatin Degradation Products Jure Zakrajšek,1* Katarina Bevc-Černilec,1 Simona Bohanec1 and Uroš Urleb2 1 Lek Pharmaceuticals d.d., Development Center Slovenia, Verovškova 57, 1526 Ljubljana, Slovenia 2 Biologics Technical development and Manufacturing, Novartis, Verovškova 57, 1526 Ljubljana, Slovenia * Corresponding author: E-mail: jure.zakrajsek@sandoz.com; Tel.: +386 1 580 26 30, Fax.: +386 1 568 13 93 Received: 23-06-2017 Abstract An ultra-performance liquid chromatographic method for simultaneous determination of rosuvastatin and rosuvastatin degradation products was developed and optimized by using fractional factorial experimental design. Optimized method is capable to accurately determine all potential degradation products of rosuvastatin. During the optimization the effect of four chosen chromatographic factors was evaluated. The analytical method operational design region was modeled using Umetrics MODDE software and optimal chromatographic conditions were predicted. The results of the model show that the most important factors to reach good separation between the peaks of rosuvastatin impurities are the pH of buffer solution and the amount of ACN and THF in the mobile phase. The final optimized method using QbD approach was validated for linearity, accuracy and precision for determination of rosuvastatin and rosuvastatin degradation products in rosuvastatin pharmaceutical dosage forms. Limit of detection and quantification were determined for two known specified impurities. The use of experimental designs enabled us to obtain the maximum amount of information about the analytical method design region. Optimization of the method was done without additional experiments, only weighing the responses and rebuilding the statistical model. This approach is very cost-effective when evaluating a variety of different factors and their interactions. Keywords: Fractional factorial design; experimental design; UPLC method optimization; rosuvastatin 1. Introduction Rosuvastatin belongs to the statin class of pharmaceutical substances that are used for lowering of low-density lipoprotein cholesterol (LDL-C). Rosuvastatin is mainly administered as a calcium salt of the active hydroxy acid in tablets with 2.5, 5, 10, 20 or 40 mg of rosuvastatin (RSV). Rosuvastatin substance is chemically not stable and degrades into many known and unknown degradation products under different stress conditions (thermal, oxidative, and/or photolytic)1. The structural formulas of rosuvastatin and its known impurities are shown in Figure 1. It is a white amorphous powder only sparingly soluble in water and slightly soluble in ethanol. It contains a polar methane sulphonamide group that interacts with the HMG-CoA reductase.2-3 Efforts for applying concepts of quality by design (QbD) principles to analytical method development have increased in recent years in order to achieve more accurate, robust and rigged analytical methods which are used for better control strategy of production processes.4-6 While quality by design principles are well known and adopted for the development of pharmaceutical products,7,8 QbD concept has not yet been fully adopted for analytical method development and optimization. Different experimental designs are used for the evaluation of the effects of different factors and their interactions for process optimization and design space modeling. Compatibility studies between the drug substance and other excipients performed by experimental designs are also described in literature.9 Maximum amount of obtained information with the smallest possible number of experiments was achieved by this approach.9 Effect of dif- Zakrajšek et al.: Optimization of UPLC Method for Simultaneous Acta Chim. Slov. 2017, 64, 968-979 969 Figure 1. Structural formula of rosuvastatin (RSV) and its known impurities (RSV diastereoisomer, RSV oxo, RSV lactone, RSV dehydro). ferent process and formulation parameters on chemical stability of the final drug product was also studied by using fractional factorial designs.10 The same principles of QbD can be applied to analytical method development/optimization in order to develop more robust analytical methods and determine the analytical method operation design region.11,12 The proposed concept is designed around the analytical target profile (ATP), which predefines the requirements for the analytical method. During the development phase of the analytical method one must show that the analytical method confirms to the criteria set in the ATP.13 Use of experimental designs (DoE) in order to assess the multidimensional combination and interactions of factors that could affect the measurements is suggested.14 Different experimental designs can be used and are described in the literature to evaluate the effect of different chromatographic parameters such as a buffer pH value, column temperature, percentage of organic modifier in the mobile phase and others.15-17 The same concepts can be applied not only to chromatographic analytical methods, but also to other analytical techniques such as Karl Fisher titration for water content determination.18 To assure the quality of pharmaceutical dosage forms containing rosuvastatin calcium as active ingredient, the assay of rosuvastatin and its degradation products needs to be controlled by a validated analytical method. Various analytical methods have been reported in the literature for determination of rosuvastatin in different pharmaceutical Zakrajšek et al.: Optimization of UPLC Method for Simultaneous ... 970 Acta Chim. Slov. 2017, 64, 968-979 dosage forms including combinational products. These include HPTLC,19 HPLC with ultraviolet detection20-24 and UPLC with ultraviolet detection.25 Different HPLC methods with ultraviolet detection have been published for the determination of rosuvasta-tin degradation products.25-27 Several methods have also been published for the determination of rosuvastatin in biological fluids using RP-HPLC/UV detection28 or LC/MS.29 The objective of this work was to develop a new analytical method that could simultaneously be used for the determination of rosuvastatin and rosuvastatin degradation products in rosuvastatin pharmaceutical dosage forms. The method has to be stability indicating30 and capable to detect all changes in product quality during stability testing at various stability conditions according to regulatory requirements (accelerated, long-term or stress stability).31 Simultaneous determination of rosuvastatin and its degradation products with one analytical method is a great benefit regarding time and resources, taking into account the number of samples that need to be analyzed during the stability studies for regulatory purposes. In addition the method should be accurate, precise and linear with acceptable limit of detection and qualification for all specified impurities (RSV oxo and RSV lactone).32 The development of the method was done by using QbD principles. The optimization step of the development was performed using experimental designs and analytical method operational design region modeling. A degraded sample from two different stress conditions (photolytic and acidic conditions) was used for the final optimization step. 2. Experimental 2. 1. Chemicals and Reagents Anhydrous acetic acid (CH3COOH), ammonium acetate (CH3COONH4), tetrahydrofuran (THF) purchased from Merck KGaA (Darmstadt, Germany), acetonitrile (CH3CN - ACN) purchased from J.T. Baker (Avantor Performance Materials, Center Valley, PA) were used for preparation of mobile phases and solvents. Acetonitrile and tetrahydrofuran were of HPLC grade, all other chemicals were of analytical grade. Milli-Q water purification system (Millipore Corp., Bedford, MA) was used to obtain highly purified water used for all aqueous solutions. In-house rosuvastatin tert-octylammonium working standard, rosuvastatin oxo tert-octylammonium and rosu-vastatin lactone identification standards were used for preparation of standard solutions used for validation and analysis of drug product. Stock buffer solutions with different pH were prepared by weighing 1.54 g of ammonium acetate and dissolving in 1000 mL of highly purified water. pH of buffer solution was adjusted to defined pH value using anhydrous acetic acid. Mixture of stock buffer solution : acetonitrile = 600 : 400 (v/v) was used as solvent for standard and sample preparation. 2. 2. Equipment Experiments were performed on a Waters Acquity UPLC separation module, equipped with a quaternary gradient pump, temperature controlled column heater, sampler manager and dual wavelength UV detector (Waters Corporation, Milford, MA). Instrument control was performed using Empower 3 Software for chromatography (Waters Corporation, Milford, MA). Same software was used for data acquisition and processing of results. Waters Acquity UPLC HSS C18 analytical chromatographic column (100 x 2.1mm, 1.8 pm) provided by Waters Corporation (Milford, MA) was used for all optimization experiments. pH measurements and adjustments were performed with Mettler-Toledo SevenMulti pH meter using a Mettler-Toledo InLab Expert Pro pH electrode (Mettler-Toledo LLC, Columbus, OH). Mobile phases were vacuum filtered prior the use through Omnipore™ 0.1 pm JV filter, purchased from Millipore (Billerica, MA). Suntest chamber Atlas SUNTEST XLS+ (Atlas, Mount Prospect, IL) was used to expose sample solutions to artificial sunlight according to ICH guideline for Photo-stability.33 Samples and standard solutions were filtered through Millipore Millex-GV Hydrophilic PVDF 0.22 pm disk filters, purchased from Millipore (Billerica, MA), before the analysis was performed using UPLC method. 2. 3. Analytical Method Fast gradient UPLC method was developed for simultaneous determination of rosuvastatin and its degradation products in rosuvastatin formulations with a single injection of the sample and UV detection at two different wavelengths. The initial chromatographic conditions are summarized in Table 1. The initial method was optimized due to the insufficient resolution between rosuvastatin peak (RSV) and RSV diastereoisomer as presented in the chromatogram of peak identification solutions shown in Figure 2. We have optimized the initial chromatographic parameters using a fractional factorial experimental design. The goal of the optimization was to achieve better resolution between rosuvastatin peak and rosuvastatin diastereoisomer and not worsen the resolution between other peaks of degradation products within the chro-matogram. Zakrajšek et al.: Optimization of UPLC Method for Simultaneous ... Acta Chim. Slov. 2017, 64, 968-979 Table 1. Chromatographic conditions of the initial UPLC method for assay determination of rosuvastatin. Mobile phase A ammonium acetate buffer pH 3.6 : acetonitrile : tetrahydrofuran = 750 : : 200 : 40 (v/v/v) Mobile phase B ammonium acetate buffer pH 3.6 : acetonitrile : tetrahydrofuran = 250 : : 700 : 50 (v/v/v) Column Waters Acquity UPLC HSS C18, 100 x 2.1 mm, 1.8 |m Column temperature 30 °C Flow rate 0.5 mL/min Detection wavelength 242 nm (for degradation products) 280 nm (for assay determination) Injection volume 12 |L 971 Time %A %B 0 88 12 Gradient parameters 8.5 88 12 19.5 30 70 20 88 12 M(E " t.bo 2ÎK) jB *J? si» ÏK MO abo a.bo' is'« ii!oo " ij'oo ialoo um " isloo is'oô it!« is:on iq:mi »00 Figure 2. Chromatogram of peak identification solution analyzed with initial chromatographic conditions. 2. 4. Standard Solutions Stock standard solution of rosuvastatin working standard (0.5 mg/mL) was prepared by dissolving appropriate amount of rosuvastatin working standard in solvent. Working concentrations of rosuvastatin standard solutions were achieved by diluting the stock standard solution with solvent to concentration 2.5 ^g/mL (used for the quantitation of degradation products) and concentration 0.25 ^g/mL (used for signal-to-noise determination). Stock standard solution was used for assay determination of rosuvastatin. Different concentrations of standard solution used in the validation were achieved by diluting the stock standard solution with solvent. Stock solutions of rosuvastatin oxo (100 ^g/mL) and rosuvastatin lactone (100 ^g/mL) standards were prepared by dissolving appropriate amount of rosuvastatin oxo or rosuvastatin lactone standard in solvent. Stock solutions were used to prepare spiked samples at appropriate concentration levels used in the validation study. All standard solutions were filtered through Millipore Millex-GV Hydrophilic PVDF 0.22 |m filter into vials. 2. 5. Analysis of Samples Samples were prepared by dissolving 10 rosuvastatin tablets in appropriate volume of solvent to acquire a concentration 0.5 mg/mL of rosuvastatin. In addition, ultrasonic bath was used to achieve complete disintegration of the tablets. All sample solutions were filtered through Millipore Millex-GV Hydrophilic PVDF 0.22 |m filter into vials and analyzed with the analytical method. To evaluate a resolution between unknown impurity X and RSV lactone impurity a degraded sample of rosuvastatin tablets was prepared. Two stock sample solutions were exposed to different degradation conditions. One sample solution was exposed to artificial sunlight under which the unknown impurity X was formed. Hydrochloric acid was added to the second sample solution, as rosuvastatin lactone impurity is known to form under acidic conditions. Both samples were mixed in 1 : 1 (v/v) Zakrajšek et al.: Optimization of UPLC Method for Simultaneous 972 Acta Chim. Slov. 2017, 64, 968-979 ratio to obtain a sample that was used for optimization purposes. 3. Results and Discussion 3. 1. Chromatographic Condition Optimization Screening of the influence of four chromatographic factors on different chromatographic responses was performed as the initial step of analytical method optimization. A randomized fractional factorial experimental design (24-1) of resolution IV with central point was used. Buffer pH, amount of acetonitrile in mobile phase A, the amount of organic modifier tetrahydrofuran in mobile phase A and column temperature were selected as factors Table 2. Factors and corresponding levels for 24 1 fractional factorial design used for screening analysis. Factor name Abbr. Settings Initial (f1) Buffer pH pH 3.3 to 3.9 3.6 (f2) Amount of THF THF 30 to 50 mL 40 mL (f3) Amount of ACN ACN 175 to 225 mL* 200 mL (f4) Column temperature Temp 25 to 35 °C 30 °C *Change in ACN volume was compensated with defined buffer solution volume so that the total volume of buffer solution and ACN was not changed. of interest, and were used to generate the fractional factorial experimental design. All factors and their corresponding levels are shown in Table 2. Fractional factorial experimental design was generated using Umetrics MODDE 11.0 software. Eleven experiments presented in Table 4 were carried out according to the generated experimental design. Three central point experiments (experiment N9, N10 and N11) were also included for the determination of experimental error. All experiments were carried out in a randomized order (run order) in order to eliminate any systematic errors. Six chromatographic responses presented in Table 3 were selected and measured for all performed experiments: resolution between rosuvastatin (RSV) peak and RSV diastereoisomer (Res 1), resolution between impurity X peak and RSV lactone peak (Res 2), resolution between RSV lactone peak and RSV dehydro impurity peak (Res 3), number of theoretical plates of rosuvastatin peak (N), symmetry factor for rosuvastatin peak (T) and retention time or rosuvastatin peak (Rt). The results of experiments are presented in Table 4. All obtained and collected response measurements were processed with Umetrics MODDE software. Partial least squares (PLS) multivariate method of simultaneously estimating the models for all the responses was used for fitting and optimizing the statistical model. PLS method Table 3. Responses and used suitability criteria for method optimization and sweet spot analysis. Response name Resolution RSV, RSV diastereoisomer Resolution impurity X, RSV lactone Resolution RSV lactone, RSV dehydro Number of theoretical plates of RSV peak Symmetry factor for RSV peak Retention time or RSV peak Suitability criteria Min Target Max Res 1 2.5 3.0 - Res 2 1.5 2.0 - Res 3 6.0 6.5 - N 7000 7500 - T 0.8 1.0 1.6 Rt - 6.0 6.5 Table 4. Randomized 241 fractional factorial design and results of observed responses. Exp Run Factors Responses No Order f1 f2 f3 f4 Res 1 Res 2 Res 3 N T Rt N1 1 - - - - 3.39 1.39 5.48 7976 1.59 9.432 N2 11 + - - + 2.98 1.26 3.30 8379 1.66 8.907 N3 3 - + - + 2.55 n.a.* 7.74 6930 1.51 5.493 N4 4 + + - - 2.66 1.69 5.09 7401 1.53 6.951 N5 8 - - + + 2.30 1.68 6.19 6361 1.45 4.368 N6 2 + - + + 2.07 1.57 3.25 6537 1.45 4.383 N7 7 - + + - 2.22 1.64 6.63 5643 1.37 3.87 N8 10 + + + - 2.07 1.83 4.86 6138 1.37 4.037 N9 9 0 0 0 0 2.63 1.54 5.78 7526 1.51 5.846 N10 5 0 0 0 0 2.64 1.57 5.77 7186 1.54 5.838 N11 6 0 0 0 0 2.63 1.60 5.78 7217 1.54 5.844 * Resolution could not be measured due to the coelution of the two peaks. Zakrajšek et al.: Optimization of UPLC Method for Simultaneous ... Acta Chim. Slov. 2017, 64, 968-979 973 simultaneously deals with numerous responses, taking their covariances into account. This provides an overview of the relationship between the responses and factors to determine the proper effect on all responses obtained within the statistical model.34 This provides an overview of the relationship between the responses and factors to determine the proper effect on all responses obtained within the statistical model. The main effects of factors were identified for each response using the statistical model and are presented in Figure 3. Higher amount of ACN and THF in the mobile phase A have negative effect on theoretical plates of RSV peak (N), as presented in Figure 3. In addition, higher level of these two factors also has a negative effect on symmetry factor of RSV peak (T) and retention time of RSV peak (Rt). Higher amount of ACN and THF in the mobile phase A with the combination of higher column temperature also have negative effect on the resolution between RSV peak and RSV diastereoisomer (Res 1). On the contrary, higher amount of ACN and THF in the mobile phase A have positive effect on resolution between impurity X peak and RSV lactone peak (Res 2), by improving the resolution between these two peaks. The most significant factors affecting the resolution between RSV lactone peak and RSV dehydro peak (Res 3) are buffer solution pH with negative effect and the amount of THF in the mobile phase A with positive effect. Reducing the amount of ACN in the mobile phase A and reducing the buffer solution pH would lead to better responses of all measured resolutions. All non-significant factors were excluded from the statistical model and the model was refitted. Using MODDE integrated sweet spot analysis tool, the analytical operational design region was modeled. Appropriate suitability criteria were assigned to all the responses measured (see Table 3). Using the analysis tool the optimal chromatographic conditions were predicted (Figure 4). The analytical method operational design region is presented for all four evaluated factors from the sweet spot diagram in Figure 4. The green area represents the part of the operational design region where all from statistical model calculated responses fulfil the criteria set for individual responses. This area is called the sweet spot. The black cross represents the optimal conditions as predicted by MODDE software tool. The outcome of the sweet spot analysis is in line with our preliminary conclusions obtained from the main effects plot (Figure 3). The sweet spot calculated by MODDE software tool is achieved by lowering buffer solution pH, reducing the amount of ACN in the mobile phase A and raising the column temperature. No additional change was made to the amount of the THF in the mobile Figure 3. Main effects for all observed responses for UPLC method optimization for the determination of rosuvastatin and rosuvastatin degradation products. Zakrajšek et al.: Optimization of UPLC Method for Simultaneous ... 974 Acta Chim. Slov. 2017, 64, 968-979 Buffer pH = 3.3 Buffer pH = 3.6 Buffer pH = 3.9 S Sweet spol 175 180 135 190 195 200 305 210 215 220 175 ISO 165 190 195 205 305 210 215 220 175 130 135 ISO 195 200 205 210 215 220 225 Amount of ACN Amount of ACN Amount of ACN Figure 4. Sweet spot diagram for a PLS model of the analytical operational design region. Optimal chromatographic conditions proposed by MODDE are marked with a cross. The initial chromatographic conditions are marked with a star at the center of the figure. 173 100 1s5 190 195 200 205 210 215 220 225 Amount of ftCN Figure 5. Sweet spot diagram for a PLS model of the analytical operational design region with weighted responses. Zakrajšek et al.: Optimization of UPLC Method for Simultaneous ... Acta Chim. Slov. 2017, 64, 968-979 975 phase. The chromatographic parameters of the initial sweet spot analysis are presented in Table 5 (initial sweet spot conditions) and in Figure 4 (marked with a star). Using the suggested sweet spot chromatographic conditions, the degraded sample mixture was analyzed. Chromatograms of degraded sample mixture (shown as a blue lines) and peak identification solution obtained with chromatographic conditions of the initial sweet spot and final sweet spot conditions (shown as black lines) are presented in Figure 6. The resolution between impurity X peak and RSV lactone peak was not satisfactory when proposed initial sweet spot conditions were used while considerably better resolution between RSV and RSV diastereoisomer peak was achieved. Since we had a statistical model for the analytical method operational design region no additional experiments were performed. MODDE sweet spot analysis was performed once again by weighing the importance of different responses measured. Resolution between RSV peak and RSV diastereoisomer peak (Res 1) and resolution between impurity X peak and RSV lactone peak (Res 2) were assigned with the highest weights, while the weights for all other responses were reduced. This way the sweet spot tool Table 5. Initial chromatographic conditions compared to initial sweet spot conditions and final sweet spot conditions. Factor name Abbr. (f1) Buffer pH pH (f2) Amount of THF THF (f3) Amount of ACN ACN (f4) Column temperature Temp Initial Initial Final chromatographic sweet spot sweet spot conditions conditions conditions 3.6 3.3 3.3 40 mL 40 mL 45 mL 200 mL 185 mL 185 mL 30 °C 35°C 35°C Figure 6. Chromatogram of the peak identification solution (black line) and degraded sample mixture (blue line) analyzed with initial sweet spot conditions (top) and final sweet spot conditions (bottom). Zakrajšek et al.: Optimization of UPLC Method for Simultaneous ... 976 Acta Chim. Slov. 2017, 64, 968-979 was forced to find a spot with a satisfactory resolution between the critical pairs of impurities and not achieving the acceptance criteria for other responses. Additionally, the criteria for resolution between impurity X peak and RSV lactone peak (Res 2) was also tightened. After running the sweet spot analysis again with the weighted responses, the new suggested sweet spot was calculated. The analytical method operational design region was modeled again and is presented in Figure 5. pH buffer solution value was fixed to 3.3 and column temperature to 35 °C. New optimal chromatographic conditions proposed by MODDE optimizer are marked with a cross. The resolution between the unknown impurity X and RSV lactone could be improved with higher amounts of THF in the mobile phase. Suggested chromatographic parameters are presented in Table 5 (final sweet spot conditions). The chromatogram obtained with the final sweet spot chromatographic conditions (Figure 6 bottom) shows significantly improved resolution between the impurity X peak and RSV lactone peak. The resolution between RSV peak and RSV diastereoisomer is significantly better than with the initial method. Despite all the method changes overall runtime of the analysis method was not prolonged. 3. 2. Analytical Method Validation The optimized analytical method was validated for determination of rosuvastatin and rosuvastatin degradation products in rosuvastatin tablets. Linearity, precision, accuracy, limit of detection and quantification were determined for main compound rosuvastatin and both specified degradation products RSV oxo and RSV lactone. The acceptance criteria for different validation parameters were set in line with ICH requirements.32 3. 2. 1. Linearity and Working Range The linearity of the method for determination of ro-suvastatin was determined by using nine different standard solutions of rosuvastatin working standard. All solu- tions were prepared in three replicates. The covered concentration range was from 0.25 mg/mL to 0.75 mg/mL (50-150% of target concentration. The linearity of rosuvastatin was determined using nine different solutions of rosuvastatin working standard prepared in three replicates for determination of related substances. The concentration range covered was from 0.15 ^g/mL to 6.0 ^g/mL (0.03- 1.2%). For rosuvastatin lactone and rosuvastatin oxo impurities, the linearity was determined using six different standard solutions prepared in three replicates. The concentration range covered for rosuvastatin lactone was 0.15 ^g/mL to 6.0 ^g/mL (0.03-1.2 %) and for rosuvastatin oxo 0.25 ^g/mL to 3.0 ^g/mL (0.05-0.6 %). The obtained linear regression results are presented in Table 6. The response of all components was found to be linear in the tested concentration range. Good correlation coefficient (> 0.99) was obtained for all components. 3. 2. 2. Precision Repeatability of the analytical system for determination of rosuvastatin was check at the target concentration of rosuvastatin (0.5 mg/mL) by six replicate injections of the sample solution. Repeatability was also validated for known rosuvastatin impurities RSV lactone and RSV oxo. The results of individual impurities are expressed as % (percentage) of RSV lactone and % of RSV oxo. The obtained results and RSD of the measurements are presented in Table 7. Intermediate precision was performed by injecting six sample solution replicates. The study was done by different analyst that performed the analysis on different days, different chromatographic systems and different UPLC columns. All together six different variations were performed. Assay of rosuvastatin, % of RSV lactone and % of RSV oxo were measured. The results are presented in Table 7. The results in Table 7 demonstrate good precision of the method for assay determination, RSD value < 1.0%. The precision for individual impurities was found to be acceptable as the criteria for RSD (n = 6 or 36) of the impurities measured at this level was set to < 20 %. Table 6. Average linear regression data for RSV, RSV lactone and RSV oxo obtained for optimized UPLC method. Component Target and concentration range [pg/mL] Slope Intercept bias in % Intercept R2 RSV for assay determination 500 250-750 2929137 6892266 0.47 0.9998 RSV for degradation products 1.3 0.15-6.0 7662070 999981 5.05 0.9981 RSV lactone 2.9 0.15-5.9 8030538 -14466 -0.06 0.9996 RSV oxo 2.5 0.25-3.0 5354275 -51492 -0.71 0.9994 Zakrajšek et al.: Optimization of UPLC Method for Simultaneous ... Acta Chim. Slov. 2017, 64, 968-979 977 Table 7. Repeatability and intermediate precision results for RSV, RSVlactone and RSV oxo. Component Precision % content1 % RSD2 Intermediate precision % content3 % RSD4 RSV for assay determination 99.61 0.96 98.89 0.71 RSV lactone 0.111 2.23 0.105 10.72 RSV oxo 0.091 4.59 0.080 12.30 1 average of 6 determinations; 2 determined on 6 measurements; 3 average of 36 determinations; 4 determined on 36 measurements values were within < 100 ± 3 % and the RSD, ,, of the ob- (n=3) tained recovery values was below 2 %, demonstrating good accuracy of the method for determination of rosuvastatin in rosuvastatin drug products. The accuracy for degradation products determination was also satisfactory for all components. The calculated recovery was within 80-120% for all components and the RS-D(n3) of the obtained recovery values was below 20%. 3. 2. 4. Limit of Detection The detection limits were determined on the basis of signal-to-noise (S/N) ratio > 3 : 1 according to ICH guidelines. The signal-to-noise values and calculated LOD concentrations are presented in Table 9. The calculated obtained LOD concentration are 0.009 ^g/mL for rosuvastatin, 0.009 ^g/mL for RSV lac- Table 8. Results of recovery experiments at five concentration levels of RSV, RSV lactone and RSV oxo, obtained with optimized UPLC method. Component Level 1 Level 2 Level 3 RSV for assay determination Concentration range Mean recovery1 % RSD2 70 % (350 ||g/mL) 101.31 0.16 100 % (500 |ig/mL) 101.18 0.29 130 % (650 |g/mL) 101.17 0.25 RSV for degradation products determination Concentration range Mean recovery1 % RSD2 0.03 % (0.15 |ig/mL) 102.56 9.82 0.20 % (1.0 ||g/mL) 101.47 4.07 0.24 % (1.2 |g/mL) 103.88 3.25 RSV lactone Concentration range Mean recovery1 % RSD2 0.03 % (0.15 |ig/mL) 110.14 4.63 1.0 % (5.0 |g/mL) 105.49 0.54 1.2 % (6.0 |g/mL) 104.84 0.75 RSV oxo Concentration range Mean recovery1 % RSD2 0.05 % (0.25 |ig/mL) 108.97 6.79 0.5 % (2.5 |g/mL) 99.76 0.38 0.6 % (3.0 |g/mL) 100.01 0.51 1 average of 3 determinations; 2 determined on 3 measurements Table 9. Results of signal-to-noise values and LOD for RSV, RSV lactone and RSV oxo impurities obtained with optimized UPLC method. Component Concentration % of RSV Concentration ^g/mL Average S/N ratio (n = 6) LOD ^g/mL RSV 0.01 0.05 16.5 0.009 RSV lactone 0.01 0.05 15.9 0.009 RSV oxo 0.02 0.1 10.6 0.028 3. 2. 3. Accuracy The accuracy of the proposed analytical method was checked at three concentration levels. For rosuvastatin determination the range was from 70%-130% of the target assay determination concentration. For the determination of degradation products, the accuracy for rosuvastatin was checked in range 0.03 % (0.15 ^g/mL)-0.24 % (1.2 ^g/mL), for RSV lactone in range 0.03 % (0.15 ^g/mLH.2 % (6.0 ^g/mL) and for RSV oxo in range 0.05 % (0.25 ^g/mL)-0.6 % (3.0 ^g/mL). All samples were prepared by spiking the appropriate amount of a component into a placebo solution. Spiked samples were prepared in three replicates and analyzed by the proposed optimized analytical method. The recovery factor was calculated and obtained results are presented in Table 8. As presented in Table 8 the recovery for the determination of rosuvastatin is acceptable. Individual recovery Zakrajšek et al.: Optimization of UPLC Method for Simultaneous ... 978 Acta Chim. Slov. 2017, 64, 968-979 Table 10. Results of signal-to-noise values and LOQ for RSV, RSV lactone and RSV oxo impurities obtained with optimized UPLC method. Concentration Concentration Average S/N ratio LOQ Component % of RsV ^l (n = 6) ^L RSV 0.03 0.15 51.8 0.029 RSV lactone 0.03 0.15 48.8 0.031 RSV oxo 0.05 0.25 25.9 0.097 tone and 0.028 ^g/mL for RSV oxo impurity. The determined S/N ratios are higher than required 3, however lower quantification limits are not needed since the reporting limit for impurities is 0.05% with respect to the concentration of rosuvastatin in the sample. 3. 2. 5. Limit of Quantification The quantitation limits were determined on the basis of signal-to-noise (S/N) ratio > 10 : 1 according to ICH guidelines. The signal-to-noise values and calculated LOQ concentrations are presented in Table 10. The calculated obtained LOQ concentration are 0.029 ^g/mL for rosuvastatin, 0.031 ^g/mL for RSV lactone and 0.097 ^g/mL for RSV oxo impurity. The determined S/N ratios are higher than required 10, but lower quantification limits are not needed as the reporting limit for impurities is 0.05% with respect to the concentration of rosuvastatin in the sample. 4. Conclusions The optimization of a new analytical method capable of simultaneous determination of rosuvastatin assay and its degradation products in rosuvastatin drug products was performed with a single fractional factorial experimental design. Only 11 experiments were needed for the optimization, while at least 16 experiments would be needed to cover the same analytical method operational region of the first optimization step with a traditional one factor at time (OFAT) approach. During the optimization, it was demonstrated that it is necessary to do the optimization of analytical methods with a sample that contains all possible degradation products. The degradation of the sample in this article was performed by artificial sun-light and acid hydrolysis as this was proven to be the most stability indicating condition for rosuvastatin. It was shown that with the appropriate statistical model of the analytical method operational region one can also do the optimization with no additional experiments, if the outcome of the first trial is not satisfactory. The responses can be weighted and the sweet-spot analysis run again on the same set of experiments. The final analytical method optimized with QbD approach was validated according to ICHQ2R1 guideline.32 The method was found to be linear, accurate and precise for both rosuvastatin assay determination and determination of rosuvastatin degradation products. The validated method was successfully applied for rosuvastatin drug products. The final optimized method is stability indicating and is capable to detect all changes in the rosuvastatin pro-duct(s) that are stored at different storage and stress stability conditions. It enables to determine the content of rosu-vastatin and its degradation products in a single injection run. This optimization reflects in saving of time and resources since one stability study includes hundreds of samples tested during the product's shelf life. 5. References 1. R. P. Shah, A. Sahu, S. Singh, Anal Bioanal Chem 2013, 405, 3215-3231. D0I:10.1007/s00216-013-6760-1 2. A. G. Olsson, F. McTaggart, A. Raza, Cardiovasc Drug Rev 2002, 20, 303-328. D0I:10.1111/j.1527-3466.2002.tb00099.x 3. K. C. Ferdinand, Expert opinion on pharmacotherapy 2005, 6, 1897-1910. DOI:10.1517/14656566.6.11.1897 4. F. G. Vogt, A. S. Kord, J. Pharm. Sci. 2011, 100, 797-812. D0I:10.1002/jps.22325 5. J. Ermer, P. J. Borman, J. Carolan, P. Faulkner, C. Finkler, O. Grosche, M. Hanna-Brown, J. Hoffmann, I. Gill, A. Lenhart, P. W. Nethercote, A. Rignall, T. Sokoliess, G. Wegener, M. Pohl, Pharmind 2010, 72, 256-264. 6. P. Borman, P. Nethercote, M. Chatfield, D. Thompson, K. Truman, Pharm. Technol. 2007, 31, 142-152. 7. ICH Guideline: Pharmaceutical Development Q8(R2), CHMP/ ICH/167068/04. 8. Pharmaceutical cGMPs for the 21st Century - A Risk-Based Approach, U.S. Food and Drug Administration (FDA). 9. S. Bohanec, T. Rozman Peterka, P. Blazic, R. Jurecic, J. Grmas, A. Krivec, J. Zakrajsek, Acta Chim. Slov. 2010, 57, 895-903. 10. P. Petelin, M. Homar, A. Bajc, J. Kerc, S. Bohanec, Acta Chim. Slov. 2012, 59, 156-162. 11. M. Schweitzer, M. Pohl, M. Hanna-Brown, P. Nethercote, P. Borman, G. Hansen, K. Smith, J. Larew, Pharm. Technol. 2010, 34, 52-59. 12. P. Borman, J. Roberts, C. Jones, M. Hanna-Brown, R. Szucs, S. Bale, Sep. Sci. 2010, 2, 2-8. 13. T. W. Graul, K. L. Barnett, S. J. Bale, I. Gill, M. Hanna-Brown, in: D. J. am Ende (Ed.): Chemical Engineering in the Pharmaceutical Industry: R&D to Manufacturing, Wiley, Hoboken, NJ, US, 2011, pp. 545-562. Zakrajšek et al.: Optimization of UPLC Method for Simultaneous ... Acta Chim. Slov. 2017, 64, 968- 979 979 14. M. Moder, S. Bohanec, J. Zupan, Acta Chim. Slov. 1997, 44, 181-196. 15. S. Karmarkar, R. Garber, Y. Genchanok, S. George, X. Yang, R. Hammond, J. Chromatogr. Sci. 2011, 49, 439-446. DOI:10.1093/chrsci/49.6.439 16. D. Awotwe-Otoo, C. Agarabi, P. J. Faustino, M. J. Habib, S. Lee, M. A. Khan, R. B. Shah, J. Pharm. Biomed. Anal. 2012, 62, 61-67. DOI:10.1016/j.jpba.2012.01.002 17. M. Hanna-Brown, P. Borman, S. Bale, R. Szucs, J. Roberts, C. Jones, Sep. Sci. 2010, 2, 12-20. 18. L. Zhou, J. M. Socha, F. G. Vogt, S. Chen, A. S. Kord, Am. Pharm. Rev. 2010, 74-84. 19. S. J. Varghese, T. K. Ravi, J AOAC Int 2010, 93, 1222-1227. 20. S. Ashour, S. Omar, Int J Biomed Sci 2011, 7, 283-288. 21. S. K. Banerjee, N. M. Vasava, Bull. Pharm. Res 2013, 3, 29-33. 22. A. K. Gajjar, V. D. Shah, Eurasian journal of analytical chemistry 2010, 5, 265-283. 23. M. A. Mukthinuthalapati, V. Bukkapatnam, S. P. Bandaru, Adv Pharm Bull 2014, 4, 405-411. 24. Z. M. Turabi, O. h. A. Khatatbeh, International Journal of Pharmaceutical Sciences and Drug Research 2014, 6, 154-159. 25. G. V. R. Reddy, B. V. Reddy, S. W. Haque, H. D. Gautam, P. Kumar, A. P. Kumar, J. H. Park, Química Nova 2011, 34, 250255. DOI:10.1590/S0100-40422011000200015 26. T. N. Mehta, A. K. Patel, G. M. Kulkarni, G. Suubbaiah, J AOAC Int 2005, 88, 1142-1147. 27. S. Palvai, S. C. Seelam, K. Dhanalakshmi, N. Reddy, International Journal of Pharmacy & Therapeutics 2013, 4, 182-187. 28. Y. Shah, Z. Iqbal, L. Ahmad, A. Khan, M. I. Khan, S. Nazir, F. Nasir, J Chromatogr B Analyt Technol Biomed Life Sci 2011, 879, 557-563. DOI:10.1016/j.jchromb.2011.01.004 29. S. J. Varghese, T. K. Ravi, J AOAC Int 2013, 96, 307-312. DOI:10.5740/jaoacint.11-117 30. R. Maheswaran, Pharm. Technol. 2012, 36, 73-80. 31. ICH Guideline: Stability Testing of New Drug Substances and Products Q1A(R2), CPMP/ICH/2736/99. 32. ICH Guideline: Validation of Analytical Procedures: Text and Methodology Q2(R1), CPMP/ICH/381/95. 33. ICH Guideline: Stability Testing: Photostability Testing of New Drug Substances and Products Q1B, CPMP/ICH/279/95. 34. L. Eriksson, E. Johansson, N. Kettaneh-Wold, C. Wikstrom, S. Wold: Design of Experiments - Principles and Applications, Umetrics AB, Umea, Sweden, 3rd ed., 459, 2008. Povzetek Z uporabo delnega faktorskega načrta smo ovrednotili vpliv štirih kromatografskih parametrov in izvedli optimizacijo visoko ločljivostne tekočinske kromatografske metode za hkratno določitev rosuvastatina in njegovih razkrojnih produktov. Nova optimizirana metoda omogoča natančno določitev vsebnosti rosuvastatina in vseh razkrojnih produktov rosuvastatina v farmacevtskih pripravkih, ki vsebujejo rosuvastatin. S pomočjo programa Umetrics MODDE smo izdelali model, ki opisuje delovno območje analitske metode, in napovedali optimalne pogoje kromatografske ločbe. Rezultati statističnega modela so pokazali, da so najpomembnejši parametri, ki zagotavljajo najboljšo ločbo med rosuvastatinom in njegovimi nečistotami, pH puferne raztopine in količina ACN ter THF v mobilni fazi. Analitsko metodo za določitev rosuvastatina in njegovih razkrojnih produktov, optimizirano s pomočjo QbD pristopa, smo validirali, pri čemer smo ovrednotili njeno linearnost, točnost in natančnost. Za dve znani specificirani nečistoti smo določili mejo določitve in mejo zaznave. Uporaba programa za statistično načrtovanje poskusov nam je omogočila izvedbo optimizacije le z utežitvijo spremljanih odzivov in ponovnim vrednotenjem delovnega območja analitske metode, brez izvedbe dodatnih poskusov. S statističnim načrtovanjem poskusov smo dobili kar največ informacij o delovnem območju analitske metode z najmanjšim možnim številom izvedenih poskusov. S takšnim pristopom se zmanjšajo tudi stroški razvoja metode, saj nam statistično načrtovanje poskusov omogoča hkratno obravnavo različnih parametrov in njihovih medsebojnih interakcij. Zakrajšek et al.: Optimization of UPLC Method for Simultaneous ... 980 DOI: 10.17344/acsi.2017.3696 Acta Chim. Slov. 2017, 64, 980-987 /^creative tS1 com mor Scientific paper Ordering Effects and Percolation in the Structure Formation Process of the Oriented Polyolefin Porous Films Galina Kazimirovna Elyashevich,3* Dmitrii Vladimirovich Novikov,3 Ivan Sergeevich Kuryndin,3 Andreja Jelen2 and Vili Bukosek1* 1 Faculty for Natural Sciences and Engineering, University of Ljubljana,Snezniska c. 5, Ljubljana, 1000 Slovenia 2 Jozef Stefan Institute, Jamova cesta 39, Ljubljana, 1000 Slovenia 3 Institute of Macromolecular Compounds, Russian Academy of Sciences,Bolshoi pr. 31, St. Petersburg, 199004 Russia * Corresponding author: E-mail: elya@hq.macro.ru; vili.bukosek@ntf.uni-lj.si Received: 12-07-2017 Abstract Structure transitions and mechanism of the formation of superlattices lamellae in microporous polyolefin (polyethylene and polypropylene) films obtained in the process based on polymer melt extrusion followed by annealing, uniaxial extension and thermal fixation have been studied by statistical analysis of electron microscopy images of the film surface. The structure of the porous films prepared in the multistage process has been studied by SEM, gravimetry and permeability measurements. It has been shown that the pore formation at the stage of uniaxial extension is accompanied by the ordering of lamellae and their self-organization controlled by spin draw ratio and annealing temperature. It was established that an increase of these parameters lead to the transition of disorder-order type. The effect of preparation conditions on the ordering process of regular spatial lattices of lamellae has been discussed. Keywords: Percolation, self-organization, through permeability, porous films, polyethylene, polypropylene 1. Introduction At present time a large number of porous systems containing microscopic pores have been prepared and studied; and among them, polyolefin films are the most promising materials. Owing to high chemical resistance to various media, they are widely used as gas and liquid permeable membrane materials in medical, chemical, and food industries. Microporous polymer films have a number of advantages over porous systems based on inorganic substances, namely: easy manufacturing, small thickness and hence lower resistance to mass transfer, and high elasticity. Flexible-chain polyolefins such as high density polyethylene1,2 and polypropylene3-6 are capable to form the oriented lamellar structures during crystallization of polymer melts under extension. The thermal treatment of these samples at temperatures close to the melting temperature of a polymer leads to an increase in the degree of crystal-linity and a decrease in the number of tie chains in amorphous regions between lamellae, and, finally, almost all tie chains become stressed. In result of such structure formation the materials (films and fibers) acquire so-called "hard elastic" mechanical properties, that is, high elastic modulus and ability to large reversible deformations.4,7 Due to a very low number of tie chains in amorphous regions the pores appear between lamellae at uniaxial extension of hard elastic samples in air at room temperature. Porous films with well developed networks of through flow channels may be used as microfiltration membranes, separators in galvanic cells, and elastic substrates in composite systems.8-13 Up to now, there have been few published papers5,14 concerning the analysis of the porousfilm preparation process, at which transition from individual pores to a connected network of through flow channels is observed. In14 it was shown that the formation of through pores in the samples prepared in the process based on melt extrusion occurs via the percolation mechanism.15 The task of the work was the investigation of the effect of pore formation process conditions on the percolation transitions and or- Elyashevich et al.: Ordering Effects and Percolation Acta Chim. Slov. 2017, 64, 980-987 981 dering effects at the self-organization of lamellar structure in the polyolefin porous films. 2. Experimental The process used to prepare the microporous polyolefin films consists of four stages: (1) melt extrusion, (2) annealing of extruded films, (3) uniaxial extension of the annealed films (pore formation stage), and (4) thermal fixation of the porous structure.2,5,14 The films are characterized by a multilevel (multifractal) structure.5 Such structure is formed by stacks of crystalline lamellae arranged normally to the orientation (extension) direction of samples, so that molecular chains in crystallites are oriented in the extension direction. Lamellae stacks are connected by thin "bridges" (stressed ties) and form the framework of the solid-phase percolation cluster.16 In the process of uniaxial extension, discontinuities (pores) appear in such filmsas a result of moving apart and bending of lamellae between the bridges of tie chains. It was shown that the number and size of pores grow with an increase in the degree of extension that leads to the coalescence of pores and the formation of through flow channels, i.e., the films become filtration membranes permeable to liquids. It was also found that the overall porosity (P) and the permeability (G) of the membranes can be controlled by varying the spin draw ratio (X) at the polymer melt extrusion, the annealing temperature (T ) of extruded films and degree of uniaxial extension (e) at the pore formation stage.17 Two structure self-ordering types are implemented in the prepared microporous films. The first type includes the cooperative percolation over pores (the formation of through channels) or the geometrical phase transition occurring upon reaching a critical degree of the overall porosity. The second type is associated with the periodic spatial superlattice of lamellae due to the disorder-order transition and ordering of particle aggregates (stacks of lamel-lae).The percolation transition leads to the formation of a liquid-permeable porous structure and the self-organization of lamellae at increasing the control parameters of the process: X, T and e. 1 ann Commercial grades of linear PE (M = 170000, M / M = 4-5) and isotactic PP (M = 380000, M /M = 4-5) n ' v w w n ' was used for porous films preparation. The melt was crystallized in air. The degree of melt orientation was varied by the spin draw ratio X. Extruded films were annealed under isometric conditions, i.e. at fixed ends of the film, to prevent their shrinkage at heating. The porous structure of membranes was formed at uniaxial extension of annealed films at room temperature. At the final stage, thermal fixation of the porous samples was performed for relaxation of inner stresses resulting from extension. The permeability of porous films G was determined by measuring the liquid (ethanol) flow rate through the film.8 An important characteristic of porous systems is overall porosity, i.e., the fraction of the sample volume occupied by pores. In the microporous films under investigation all type of pores, namely, the open-cell, closed and through pores, contribute to the overall porosity P which was determined by the gravimetric method. P = [(P-Pp) / p] x 100 ' (1) Where p is the density of a dense (nonporous) film, being equal to 950 and 900 kg/m3 for PE and PP films, respectively; and pp is the density of a porous film determined by weighing. The images of the surface of membrane samples were obtained on a FE-SEM SUPRA 35 scanning electron microscope (Ziess, Germany). Statistic analysis of SEM images was carried out using the cluster two-phase model on a square lattice with the ratio r/£ ~ 0.1, where r is the distance between sites and ^ is the correlation length of phase clusters. Within this model, we calculated the average lattice density Op of porous phase clusters (porous phase fraction in two-dimensional space) and the radial distribution functions g(R) of the cluster lattice density. The effective size d and fractal dimension D of porous phase clusters were determined by the initial region (R (2) where L^ and L „ are the periods of alternation of the lattice densities of the solid phase and the porous phase clusters, respectively, along the s axis. The regular spatial lattice of particles corresponds to the parameter 9 = 0; with an increase in the coordination disorder, the parameter 9 increases. The periods Ls|| and Lp|| were determined using the functions g(R)||calculated in the direction of this axis. The thickness L of solid phase particles was calculated as L = 0.8R . , where R . is the 1 || mm min position of the first minimum of the corresponding function g(R)||. Elyashevich et al.: Ordering Effects and Percolation 982 Acta Chim. Slov. 2017, 64, 980-987 3. Results and Discussion The microporous films prepared by the method used in this study are characterized by a relief-like strongly developed surface. This pattern arises at uniaxial extension stage in result of stress release due to the through pores appearance. As can be seen from SEM images, the surface relief of the PE and PP films (Figure 1)is designed by two types of structural elements; namely, extended large comblike structures arranged perpendicular to the direction of orientation of the film and thin "bridges" aligned parallel to the film orientation direction and connecting the comblike structures. Figure 1. SEM images of the PE and PP porous films surface Deeper regions (pores) are seen between these structural formations on the sample surface. The PP samples are characterized by smaller sizes of the structural elements of the surface relief and by their considerably larger number as compared to the PE films. These characteristics of the surface structure indicate that the PP films have a more developed surface relief than the PE ones. This is confirmed by the measurements of specific surface, which is equal to 41 and 83 m2/g for PE and PP microporous films, respectively. 3. 1. Effect of Spin Draw Ratio on the Ordering Transitions Due to the orientation efforts at the melt extrusion the periodic superlattice of oriented lamellae is formed in the films via the universal mechanism of particle self-ordering, controlled by the spin draw ratio X. At the stages of extrusion and annealing which is performed at isometric conditions, lamellae are organized in an ensemble of oriented supramolecular structures. Under uniaxial extension of the films prepared at these stages this ensemble transforms to a spatial network of smaller particles, i.e., lamellae stacks (superlattices of lamellae), due to irreversible splitting of structons. The superlattice formation can be considered as the disorder-order transition, which leads to ordering of the multilevel supramolecular polymer structure. At increasing of X the size and degree of the aggregates ordering increase. The transformation of the microporous films' surface texture with variations in spin draw ratio is caused by the change in the relative contribution from the stacks of lamellae and stressed ties connecting them to the spatial distribution of the density of the solid phase cluster. A typical property of the microporous polyolefin films under study is the percolation effects which are associated with the formation of a fractal structure of the surface. The fractal cluster of the solid phase is arisen near the threshold of percolation in result of the coalescence of pores and appearance of through flow channels. Percolation mechanism of the through pores formation is demonstrated by the dependence of the permeability G (flow rate through the membrane) on X which exhibits a percolation threshold of X for permeability at threshold value of the overall porosity P (the sample volume fraction occupied with pores) (Figure 2). For PE films the threshold is reached at P* = (30 ± 2) %, for PP ones P* = (23 ± 2) %. The difference between the values of P* for PE and PP is connected with the structure details of PE films. This result is in accordance with percolation theory15 which gives for percolation threshold 2530%. The transition through the percolation threshold at P >P* is accompanied bythe formation of a percolation cluster of the porous phase in the material. On the film surface, this transition corresponds to an increase in the correla- Elyashevich et al.: Ordering Effects and Percolation Acta Chim. Slov. 2017, 64, 980-987 983 50 30 30 PE 1 y y^l 30 60 X 90 " PP 1 f / / / / / X . J* y tlij^ a / I i E 300 t= nj sz CM 200 £ o too 500 E • - ro j: CM 303 ,§. O" 100 Figure 2. Plots of overall porosity P (1) and permeability G (2) of porous PE and PP films vs. spin draw ratio X. tion length a decrease in the total anisotropy of the density distribution ps of the solid phase clusters, and a disordering of the average density distribution of the porous phase clusters. The above phenomena have a common character and are observed in the computer implementation of the lattice clusters models. The orientational order of lamellae and the degree of orientation of polymer chains increase with increasing X. This growth is nonlinear, which is indicated by both the nonmonotonic dependence of the correlation length on X for investigated samples and a significant scatter of the data on birefringence in PE porous films, obtained by changing the parameter X.18 It can be assumed that the nonlinear in X orientation behavior reflects different conditions of their structure formation depending on parameters of melt extrusion process. In this case, the transition from one mode to another is accompanied by a change in the orientation of molecules and supramolecular structures themselves and also porous phase clusters. Of particular interest is the disorder-order orientation transition on the X scale, associated with the formation of oriented through channels. Microporous PE films were prepared at variation of X in the range 24-69. The structural parameters and porous structure transformation have been investigated by statistical treatment of the SEM images. It was obtained that the lattice density fraction Os of the solid phase on the surface of microporous PE films corresponds in two-dimensional mapping to the infinite cluster of the particles at Os > O*, where O* is the critical value at the percolation threshold (0.45 ± 0.03).19 In its turn, the porous structure of the films is transformed upon varying parameter X, and the transition from discrete pores (Op < O*) to through channels (Op > O*) occurs at X > 29. The existence of the critical value of X, which provides percolation in PE membranes, was previously found in.14 Radial functions g(R) of the clusters density distribution of the phases in the general case is typical for the structurally nonuniform systems and nonmonotonically approach the straight line g(R) = 1 (Figure 3a) as R increases. This straight line is reached at R = where ^ is the correlation radius of density fluctuations.19 It should be expected that the values of ^ for two mutually complementary percolation clusters coincide (Figures 3b, 3c). Figure 3. Distribution functions g(R) of the clusters density along the directions: solid phase (1) and porous phase (2) for X = 24 (a), 36 (b) and 69 (c). At the same time as lattice density Op of the cluster of the particles decreases, the value of ^ increases (Table 1). For the PE film obtained at X = 69 the periodic oscillation of the functions g(R) near the value g(R) = 1, and Table 1. The structural parameters of the porous PE films. Solid phase .m d, .m Pores n d, .m 24 0.71 0.6 0.36 0.35 29 0.25 0.13 0.30 0.18 36 0.54 0.28 0.46 0.38 42 0.58 0.80 0.42 0.62 69 - 0.09 0.57 0.09 \ Elyashevich et al.: Ordering Effects and Percolation 984 Acta Chim. Slov. 2017, 64, 980-987 functions g(R) almost coincide both for the solid phase and for pores which indicates the formation of the uniform periodic structure of microporous PE films. The infinite cluster is transformed into the two-dimensional qua-silattice of oriented particles, and the superlattice is constructed of ordered stacks of lamellae, so that each stack involves ~2.5 lamellae. The mean sizes of the particles (stacks of lamellae) and pores in the direction perpendicular to the orientation axis coincide. The formation of through channels at increasing of X is associated with ordering the particles in the orientation direction of the samples. Functions g(R) of clusters of the phases along the orientation direction indicate that the periodicity of alternation of the particles with period L|| appears at the scale of the correlation radius It was calculated that the value of ^substantially exceeds both the thickness of a single lamella (30 nm) and the long period (35 nm).20 Thus, the anisometric particles that are revealed by SEM on the film surface are the fragments of the solidified polymer material involving the stacks of lamellae. The pattern of the surface is similar to the internal lamellar structure of porous samples; however, they are characterized by a larger scale of structural ele-ments.20,21 Periods L^ of alternation of the particles and pores in the orientation direction for the porous sample prepared at X = 69 coincide and correspond to the average period L = 170 nm. Structure transformations in PP porous films were investigated for the samples prepared at X = 44 and 78. In the samples, prepared at X = 78, the 3D regular lattice of lamellae stacks and also the system of oriented through flow channels providing the highest permeability to the sample are formed. In 2-dimensional images the regular scale lattice of lamellae is characterized by a coincidence of alternation periods of lattice density for solid (s) and porous (p) phases clusters along (Ls|| = Lp|| = 110 nm) and perpendicular (L± = L =110 nm) to extension direction (axe s), both.In this case overall porosity of the films equal 50%, so solid and porous phases in the lattice may be considered as co-dimensional. In the film prepared at X = 44 regular 3D lattice of lamellar is absent. Along axes, the condition £ = L .. = L .. = sIL pll 110 nm is valid. However, in transverse direction there is no any coordination between radial functions g(R)s± and g(R)p±of a lattice density clusters of phases distribution. In this case the solid and porous phases are non-dimensional ones; the overall porosity is 41%. Non-dimensional character of solid and porous phases is the reason of permeability decreasing of this membrane in 1.5 times in comparison withthe film (X = 78) which is characterized by regular 3D lattice of lamellae. The transition "co-dimensional - non-dimensional" solid and porous phases is related to the change of topo-logical structure of the films, namely, to transition from the model of statistical network of pores to model of oriented network of through flow channels. 3. 2. The influence of the Annealing Temperature on the Structure Transformation at Pore Formation The process of the pore formation at uniaxial extension is regulated by self-organization of lamellae and, consequently, strongly depends on the lamellar structure formed at extrusion and annealing stages. Figure 4 shows the relationship of the basic parameters (T and X), ensuring the achievement of percolation values of the overall porosity (23 and 30% for PE and PP films, respectively). This figure shows, under what combination of values X and T ° ann appears through permeability in porous polyolefin films. Figure 4. Relation between X and T at the threshold values of overall porosity for PE and PP films. Uniaxial extension is 200%. The figure evidences: when extrusion was performed at lower X, the percolation value P* is achieved at higher Tann, and vice versa; if on the extrusion stage greater X was used, then annealing can be performed at lower temperatures Tann. However, this regularity is disrupted for PE. The fact is that structural rearrangement, consisting in increasing the size of lamellae occurs when annealing was carried out at temperatures at which the mobility of the chains in Elyashevich et al.: Ordering Effects and Percolation Acta Chim. Slov. 2017, 64, 980-987 985 the PE crystals appears. According to dynamic mechanical analysis (DMA) for obtained PE films, this temperature is in the range 90-100 °C (Figure 5). Annealing of the films at lower temperatures does not permit to realize the perfecting of the lamellar structure and transition to a hard-elastic state, which is a prerequisite for the formation the through channels, upon subsequent uniaxial extension. The weakening of the connection between X and T ° ann for PE films at T close to 100 °C shows, that further in- ann crease X does not allows to decrease T below this tem- ann perature. For PP films character of relation between thresholds X and T does not change, because in this case ann used values X correspond T which are higher than appearance of chains mobility in crystals 130-140 °C according to DMA (Figure 5). Figure 5. The temperature dependences of tg 5: PE and PP films. To establish a correlation between the density of the porous phase Op (the surface area occupied by all types of pores) and overall porosity P, the PP films obtained at different T for which there is a linear relationship between ann these parameters were selected. In the case of the preva- 0.40 0.35 a 0.30 0.25 / - / / / ' .1 / / m - € . « / / •...— / 1 1 1 30 40 P, % 50 Figure 6. The dependences of the average lattice density Op of orous phase clusters on the surface of PE (1) and PP (2) films on overall porosity P. The dashed line corresponds to Op = P. lence of the through oriented channels (after the percolation threshold) occurs a linear correlation between two and three dimensional porosity (Figure 6). The dependence of permeability of porous films G on the parameter t = (P - P*)/P* characterizing the degree of deviation of the porosity parameter P from the percolation threshold P* was studied. The parameter P in the region P >P* was varied by changing of Tmn; P linearly increases with Tann. It was shown that the dependence G(t) is a power-law function G ~ t' and is characterized by the critical indices ' = 1.5 and ' = 1.9 for X = 44 and X = 78, respectively. The change of the critical index ' with increasing X can be associated with the transition from percolation in a random inhomogeneous medium (' = 1.5) to anisotropic percolation over oriented through channels (' = 1.9). Such a transition should be a consequence of an increase in the cooperativity of the lamellae ordering process at the stage of pore formation and the development of a regular spatial 3D lattice of particles. An analysis of the two-dimensional SEM images of the membrane surface makes it possible to clear up the character of pore formation process with increasing porosity P or the degree of deviation t from the percolation threshold P*. The areas occupied by pores increase that corresponds to the pore growth under steady-state conditions at which the lamellar structure of membranes is ordered. The investigation of correlations between the two-dimensional network density Op of porous phase clusters and the volume porosity P of PP membranes shows that Op is independent of P in the case of X = 44; at X = 78 the linear correlation between these parameters is observed. The equality Op = P is valid for homogeneous systems. Porous phase clusters in the membranes under study are topolog-ically inhomogeneous due to the presence of three pore types: through, closed, and open cell. The establishment of the linear dependence Op(P) in going from X = 44 to X = 78 suggests that, among three pore types, through channels begin to dominate. With an increase in Tann, lamellae undergo ordering: the larger is the value of X, the higher is the rate of decrease in the degree of ordering the lamellae 9 which approaches zero at T — T . The transition from a less ordered cluster ann m of the solid phase to a more ordered cluster is associated with the convergence of the values of the periods L^ and Ls|| and with the formation of a superlattice of lamellae. Similar self-organization of particles in membranes occurs with an increase in the parameter X at a fixed temperature T. ann The effect of the annealing temperature Tann on the degree of ordering the lamellae 9 in the membranes is illustrated in Figure7 (curve 2). In the range Tann< 439-440 K with an increase in Tann, lamellae undergo ordering. In this case, the larger is the value of X, the higher is the rate of decrease in the parameter 9. In the temperature range Elyashevich et al.: Ordering Effects and Percolation 986 Acta Chim. Slov. 2017, 64, 980-987 T > 440 K the parameter 9 is calculated for aggregates of lamellae. The permeability G of the membranes prepared at the parameter X = 78 increases to infinity with an increase in the annealing temperature Tann (Figure 7, curve 1). This behavior of the dependence G(Tmn) is explained by the fact that, in the range Tann< 440 K, the permeability increases as a result of the increase in the degree of ordering of the particle lattice (with a decrease in 9) at a constant pore size d, whereas at T > 440 K, it increases due to 1 ann the increase in the pore size d when 9- const. Figure 7. The dependences of the ratio G/G for the permeability of porous films (1) and the parameter 9 (2) on the temperature Tann. The statistical analysis of SEM images of PP porous films prepared at X = 44 and 78 shows that the films surfaces exhibit polymorphism represented by two regions with different topologies: regularly arranged particles similar to those ones in the films annealed at T < 442 K, and the ann regions characterized by larger supermolecular structure formed at T > 442 K whose size increases with T . The ann ann transformation of the film surface microrelief from the first type to the second one of regions manifests itself as a sharp increase in the period L^ of alternation of the density of the solid phase cluster (Figure 8). The increase of L in the films annealed at T = s|| ann 442-444 K is associated with the formation of stacks of lamellae constituting the framework of the solid phase percolation cluster.16 The aggregation of lamellae leads to the separation of the curves Ls||(Tann) at the point of bifurcation at a temperatures T > 440 K into two branches: for ann the individual lamellae (curve 3) and for the aggregates of lamellae. It should be noted that the alternation of the solid phase density Ls|| for the individual lamellae, within the error of calculation, does not depend on T and X. ann Thus, two self-assembly mechanisms with increasing T were detected: the gradual and bifurcation ones; the ann latter is characterized by a higher cooperativity of the Figure 8. The dependences of period Ls|| in PP porous films on the annealing temperature T for the aggregates of lamellae at X = 44 (1), and X = 78 (2), and for the individual lamellae (3). lamella ordering process. The choice of this or that mechanism by the system depends on the melt spin draw ratio X during extrusion. 3. 3. The Uniaxial Extension Stage The self-organization of elements forming the the film surface relief, which is similar to the lamellar structure of the polymer, occurs at the stage of uniaxial extension of the films (pore formation). It should be noted that uniaxial extension of the annealed PE and PP films leads to the formation of pores even at small degrees of deformation £, immediately from the beginning of the extension process that may be observed visually in the change of transparent annealed films to opaque milk-white microp-orous material because of the light scattering on the walls of pores.22 Uniaxial extension of the polyolefin samples along the direction of extrusion initiates an ordering process of particles of the solid phase - crystalline lamellae - and the ties connecting them. The self-organization of solid phase particles is accompanied by the increase in anisotropy of mechanical properties of the samples, change in their texture from biaxial to axial, an increasing in the degree of homogeneity of the spatial structure and a significant growth in the permeability, which reaches a value of 250 l/ (m2 h atm) that is characteristic for the membranes with a regular 3D lattice lamellae2,5,17. 4. Conclusion The transformations of supermolecular organization in the process of porous structure formation under study Elyashevich et al.: Ordering Effects and Percolation Acta Chim. Slov. 2017, 64, 980- 987 987 have been analyzed using statistical treatment of the SEM images of the PE and PP films surfaces at variations of their orientation degree. The porous structure of these films is originated at uniaxial extension stage following the extrusion and annealling. Basic characteristics of the porous structure - permeability and overall porosity - are significantly affected by both the degree of orientation X of the polymer melt at extrusion stage and also the annealing temperature T of the extruded films. The dependences of 1 ann 1 permeability on Tann and X are characterized by the percolation threshold for the appearance of through flow channels. At uniaxial extension the permeability increases with degree of extension due to increasing of number and sizes of through pores. This growth is accompanied by a rising of the ordering degree of the structure elements - self-organization and the formation of the superlattice of crystalline lamellae. The sample structure is transformed from biaxial topology of the random network of pores to uniaxial one of the oriented through channels. The self-organization of porous phase is the result of formation the regular spatial lamellar 3D lattice at the pore formation stage. This transformation may be considered as the disorder-order transition which leads to the ordering of the multilevel supermolecular structure of the film. 5. Acknowledgments This study was supported by Joint Research Projects of the Russian Academy of Sciences and the Faculty for Natural Sciences and Engineering, University of Ljubljana (Slovenia), BI-RU/14-15-037 and BI-RU/16-18-017. 6. References 1. J. Kim, S. S. Kim, M. Park, M. Jang, J. Membr. Sci. 2008, 318, 201-209. DOI:10.1016/j.memsci.2008.02.050 2. G. K. Elyashevich, E. Yu. Rozova, E. A. Karpov, RF Patent Number 2,140,936, date of patent April 15, 1997. 3. J. Loboda-Cackovic, H. Cackovic, R. Hosemann, Macromol. Sci. Part B. Phys. 1979, 16, 127-144. DOI: 10.1080/00222347908212286 4. I. K. Park, H. D. Noether, Colloid Polym. Sci. 1975, 253, 824839. DOI:10.1007/BF01452402 5. G. K. Elyashevich, I. S. Kuryndin, V. K. Lavrentyev, A. Yu. Bo-brovskii, V. Bukosek, Phys. Solid State 2012, 54, 1907-1916. DOI: 10.1134/S1063783412090090 6. A. V. Yefimov, V. P. Lapshin, V. I. Fartunin, , P. V. Kozlov, N. F. Bakeyev, Polymer Science 1983, 25, 692-700. 7. E. A. Karpov, V. K. Lavrentyev, E. Yu. Rozova, G. K. Elyashevich, Polymer Science 1995, A.37, 1247-1253. 8. R. E. Kesting. Synthetic polymer membranes. A structural perspective. 2nd Edn., John Wiley & Sons, New York- Chich-ester, 1985. 9. M. Mulder. Basic Principles of Membrane Technology, Kluw-er Acad. Publ., Dordrecht, 1991. 10. S.Y. Lee, S.Y. Park, H.S. Song, Polymer 2006, 47, 3540-3547. DOI: 10.1016/j.polymer.2006.03.070 11. S. S. Zhang, J. Power Sources 2007, 295, 351-364. DOI: 10.1016/j.jpowsour.2006.10.065 12. I. Novak, G. K. Elyashevich, I. Chodak, A. S. Olifirenko, M. Steviar, M. Spirkova, N. N. Saprykina, E. N. Vlasova, A. Kleinova, Eur. Polym. J. 2008, 44, 2702-2707. DOI:10.1016/j.eurpolymj.2008.05.015 13. A. Bobrovskii, V. Shibaev, G. Elyashevich, E. Rosova, A. Shimkin, V. Shirinyan, A. Bubnov, M. Kaspar, V. Hamplova, M. Glogarova, Liq. Cryst. 2008, 35, 533-539. DOI: 10.1080/02678290802015697 14. G. K. Elyashevich, A. G. Kozlov, E. Yu. Rozova, Polymer Science, Ser. A. 1998, 40, 567-573. 15. D. Stauffer, A. Aharony, Introduction to percolation theory, Taylor and Francis, London, 1994. 16. D. V. Novikov, V. K. Lavrentyev, G. K. Elyashevich, V. Bukosek, Phys. Solid State 2012, 54, 1903-1906. DOI: 10.1134/S1063783412090223 17. I. S. Kuryndin, V. K. Lavrentyev, V. Bukosek, G. K. Elyashevich, Polymer Sci. 2015, A57, 717-722. 18. A. Bobrovskii, V. Shibaev, G. Elyashevich, E. Rosova, A. Shimkin, V. Shirinyan, K.-L. Cheng, Polym. Adv. Technol. 2010, 21, 100-112. 19. J. M. Ziman, Models of Disorder, Cambridge University Press, London, 1979. 20. M. Raab, J. Scudla, A. G. Kozlov, V. K. Lavrentyev, G .K. Elyashevich, J. Appl. Polym. Sci. 2001, 80, 214-222. 21. G. K. Elyashevich, I. S. Kuryndin, E. Yu. Rosova, Polym. Adv. Technol. 2002, 13, 725-736. DOI:10.1002/pat.251 22. A. A. Zinchik, I. S. Kuryndin, K. V. Ezhova, G. K. Elyashevich, Proc. SPIE 2016, 9890, 989016 (1-7). DOI: 10.1117/12.2227776. Povzetek Na mikroporoznih poliolefinskih (polietilenskih in polipropilenskih) filmih, pripravljenih v postopku ekstruzije iz taline polimera, čemur je sledilo žarjenje, enoosno raztezanje ter toplotno stabiliziranje, so bili s statistično analizo slik elektronske mikroskopije površja filmov, raziskani strukturni prehodi in mehanizem nastajanja super-rešetk lamel. Strukturo poroznih filmov, pripravljenih v večstopenjskem postopku, smo preučevali z meritvami SEM, gravimetrije in prepustnosti. Pokazano je bilo, da nastajanje por v fazi enoosnega raztezanja spremlja urejanje lamel, njihovo samoorganizacijo nadzira predilno raztezno razmerje ter temperatura žarjenja. Ugotovljeno je bilo, da povečanje teh pokazateljev vodi do prehoda vrste nered - red. Podana je razprava o vplivu pogojev priprave na postopek urejanja pravilne prostorske rešetke lamel. Elyashevich et al.: Ordering Effects and Percolation 988 DOI: 10.17344/acsi.2017.3709 Acta Chim. Slov. 2017, 64, 988-1004 ©creative com mar Scientific paper Microwave-assisted One-pot Efficient Synthesis of Functionalized 2-Oxo-2-phenylethylidenes-linked 2-Oxobenzo[1,4]oxazines and 2-Oxoquino[1,4]oxalines: Synthetic Applications, Antioxidant Activity, SAR and Cytotoxic Studies Vashundhra Sharma,11 Pradeep K. Jaiswal,11 Dharmendra K. Yadav,2,3 Mukesh Saran,4 Jaroslav Prikhodko,5 Manas Mathur,4 Ajit K. Swami,4 Irina V. Mashevskaya5 and Sandeep Chaudhary1,6* 1 Department of Chemistry, Malaviya National Institute of Technology, Jawaharlal Nehru Marg Jaipur-302017, India. 2 Department of Biochemistry, All India Institute of Medical Sciences (AIIMS), Jodhpur, Rajasthan-342005, India. 3 College of Pharmacy, Gachon University of Medicine and Science, Room # 502, Hambakmoeiro 191, Yeonsu-gu, Incheon city, 406-799, Korea. 4 Department of Advance Molecular Microbiology, Seminal Applied Sciences Pvt. Ltd. Jaipur-302015, India. 5 Department of Organic Chemistry, Faculty of Chemistry, Perm State University, 15 Bukireva, Perm 614990,Russian Federation. 6 Materials Research Centre, Malaviya National Institute of Technology, Jawaharlal Nehru Marg, Jaipur-302017, India. * Corresponding author: E-mail: E-mail: schaudhary.chy@mnit.ac.in Fax: 911412529029; Tel: 911412713319; $Both authors have equal contribution. Received: 21-07-2017 Abstract A microwave-assisted, environmentally benign green protocol for the synthesis of functionalized (Z)-3-(2-oxo-2-phenylethylidene)-3, 4-dihydro-2ff-benzo[fo][1,4]oxazin-2-ones (11a-n) in excellent yields (upto 97%) and (Z)-3-(2-oxo-2-phenylethylidene)-3,4-dihydroquinoxalin-2(1H)-ones (14a-h) (upto 96% yield) are reported. The practical applicability of developed methodology were also confirmed by the gram scale synthesis of 11a, 14c and 14e; synthesis of anticancer alkaloid Cephalandole A 16 (89% yield). All the synthesized compounds 11a-n, 14a-h and 16 were assessed for their in vitro antioxidant activities in DPPH radical scavenging and FRAP assay. In DPPH assay, compounds 11a, 14c and 14e, the most active compounds of the series, were found to show IC50 value of 10.20 ± 0.08 |ig/mL, 9.89 ± 0.15 |ig/mL and 8.97 ± 0.13 |ig/mL, respectively in comparison with standard reference (ascorbic acid, IC50 = 4.57 |g/mL). Whereas, in FRAP antioxidant assay seven compounds (11c, 11e, 11i, 11k, 11l, 14d and 14h) displayed higher antioxidant activity in comparison to the reference standard BHT (C05FRAP = 546.2 |M). Moreover, the cytotoxic studies of the compounds 11a, 14c, 14e and 14h were found to be non-toxic in nature in 3T3 fibroblast cell lines using MTT assay. Keywords: Benzo[1,4]oxazines; 2-oxobenzo[1,4]oxazines; 2-oxoquino[1,4]oxalines; Antioxidant; Microwave-Assisted Organic Synthesis (MAOS); DPPH; FRAP; Structure-Activity Relationship. Sharma et al.: Microwave-assisted one-pot efficient synthesis Acta Chim. Slov. 2017, 64, 988-1004 989 1. Introduction Benzo[1,4]oxazines 1-8, a sub-class of benzo fused heterocycles, are endowed with a wide range of biological activities such as anti-inflammatory,1 analgesic,2 antibacterial,3 neuroprotective,4 D2 receptor antagonists,5 antimycobacterial,6,7 antihypertensive,8 antifungal,9 herbicidal,10 antiarrhythmic,11 thrombin inhibitor and fibrinogen receptor antagonists,12 5-HT receptor antagonists,13 potent inhibitor of tumor-driven angiogenesis14a and selective non-steroidal mineralocorticoid receptor antagonists1"® etc. Some marine secondary metabolites such as, Arcticoside 5a (potent antifungal agent) and C-1027 chromophore- III & V 5b (potent antitumor antibiotic),15 which were isolated from a culture of an arctic marine actinomycete Streptomyces strain; possess as benzo[1,4]oxazines substructures in their active scaffolds (figure 1). Owing to the several biological activities having benzo[1,4]oxazines moieties in their scaffold or in whole molecule, several syntheses of benzo[1,4]oxazines, 2-oxo- Figure 1. Structures of biologically active benzo[1,4]oxazines 1-8. Sharma et al.: Microwave-assisted one-pot efficient synthesis ... 990 Acta Chim. Slov. 2017, 64, 988-1004 benzo[1,4] oxazines and its related structural motifs have been reported in the literature using metal as well as metal-free catalyst.16-18 Earlier, Kikelj et al. reported the first synthesis of 3-unsubstituted 3,4-dihydro-1,4-benzoxaz-in-2-ones via catalytic hydrogenation of 4-benzyl-3,4-di-hydro-1,4-benzoxazin-2-one.16a Since then, several metal-catalysed synthesis of substituted benzo[1,4]oxazines have been reported in the literature16b-d [figure 2; entry 1-iii]. 2-aminophenols or substituted 2-nitrophenols16f or 2-halophenols,16g-j were most commonly used as starting materials towards the synthesis of benzo[1,4]oxazine derivatives. With 2-aminophenols as the starting substrate, various protection and deprotection steps are re-quired.16k4 Xia et al. (2008) reported sulphamic acid as an efficient catalyst for the synthesis of benzo[1,4]oxazines derivatives in one pot reaction condition providing good yield.16e In spite of this efficient methodology, sulphamic acid is associated with several drawbacks with respect to its hazardous nature towards animals as well as environment, such as the high toxicity of sulphamic acid in animals (LD50 = 1312 mg/kg in mouse via oral route; LD50 = 3160 mg/kg in rat via oral route; toxicity value of LC50 = 70.3 mg/l in fish Pimephales promelas species); acute oral and inhalation toxicity to human etc. Its disposal also induces toxicity to the environment.1® Thus, none of the reported methodologies were environmentally benign as these were associated with several drawbacks such as the use of toxic catalysts, toxic starting materials, hazardous organic solvents, multistep and complicated reaction assembly, limited number of appropriate substrates for diverse synthesis, tedious workup and low yields etc.19-21 Therefore, an efficient, environmentally benign and more green approach for the synthesis of benzo[1,4]oxazines is still a challenging area of research. Moreover, it has also been observed that several chalcones and its analogues,22a-c quinolines,22d-f and cou-marin-derived scaffolds,228-11 which have 2-oxobenzo[1,4] oxazine-like substructure in their scaffold, were found to be potent antioxidants under several in vitro antioxidant assays. So, in our endeavour to search for new class of potent antioxidants we have developed inclinations towards 2-oxo-benzo[1,4]oxazine based analogues (prototype A: figure 1), because it has almost similar substructure as present in coumarins, chalcones and quinolone or its analogues. In this context, we were interested to explore the green synthesis and antioxidant activity of non-naturally occurring 2-oxobenzo[1,4]oxazines derived analogues, because to the best of our knowledge the antioxidant activity of 2-oxobenzo[1,4]oxazine class of molecules have never been studied earlier. During the past few decades, Microwave-Assisted Organic Synthesis (MAOS) has been identified as an efficient green protocol for accelerating drug discovery process.23a-d Moreover, it is well documented that Mi- Figure 2. Previous and present reports for the synthesis of substituted benzo[1,4]oxazine derivatives. Sharma et al.: Microwave-assisted one-pot efficient synthesis ... Acta Chim. Slov. 2017, 64, 988-1004 991 crowave irradiation (MW) is a form of electromagnetic energy having lower frequency (300-300000 MHz) and it has several advantages over conventional heating conditions such as: reduction of the reaction times as well as decrease side reactions, increase yields of desired products and improve reproducibility. Therefore, industrial as well as academic research groups are frequently using MAOS for rapid reaction optimization, accelerating the efficient synthesis of new chemical entities and also for the novel methodology development.23e-g Hence, utilizing this concept; herein, we report a very simple, mild and highly efficient green protocol for the synthesis of highly functionalized 2-oxobenzo[1,4]oxazines 11a-n (upto 97% yield) and 2-oxoquino[1,4]oxalines 14a-h (upto 96% yield) under microwave irradiations using readily available starting materials. The main advantage of this protocol is the avoidance of any toxic reagent, solvent or catalyst. Although, compounds 11a-i and 14a-h are already reported in the literature, but they were prepared by other routes,17 and their antioxidant activities have never been evaluated till now. Therefore, for the first time, we have evaluated the in vitro antioxidant activities of all the synthesized compounds 11a-n, 14a-h and 16 in DPPH radical scavenging assay using ascorbic acid (IC = 4.57 ^g/mL) as standard reference and ferric reducing antioxidant power (FRAP) assay taking BHT (546.0 ± 13.6 ^M) as standard reference. To the best of our knowledge, this is the first report of microwave-assisted synthesis and in vitro antioxidant activities of functionalized 2-oxobenzo[1,4]oxazines 11a-n, 2-oxoquino[1,4]oxalines 14a-h and 16 in excellent yields having high level of functional group compatibility. 2. Results and Discussion We started our initial investigation towards the development of an environmentally benign, sustainable protocol for the synthesis of 2-oxobenzo[1,4]oxazine with a typical model reaction between 2,4-dioxo-4-phenylbutanoic acid 9a and 2-aminophenol 10a in isopropanol (1.0 mL) under nitrogen atmosphere at room temperature for 3 h which furnished the condensation product 11a in only 18% yield (entry 1, Table 1). Carrying out the above reaction at 90 °C for 3 h afforded 11a in better yield [(45%), entry 2, Table 1, Method 1]. The product obtained was fully characterized by its spectroscopic data (1H and 13C NMR, HRMS and IR). Since we observed an increase in the yield of 11a as we change solvent from isopropanol to DMF and conventional to MW irradiation condition; we switched over to more polar DMSO solvent. Thus, the above reaction was carried out in DMSO solvent, at 150 °C for 10 min under microwave irradiation, which furnished 11a in 77% yield (entry 7, Table 1, Method B). Decreasing or increasing the Table 1. Optimization study: Synthesis of 2-oxobenzo[1,4]oxazines 11a by the reaction of 2,4-dioxo-4-phenylbutanoic acid 9a and 2-aminophenol 10a.a Entry Solvent Temp (°C) Method Ab Time (min) Yield (%)d Method Bc Time (min) Yield (%)d 1 Isopropanol rt 180 18 - - 2 Isopropanol 90 180 45 10 52 3 Isopropanol 90 300 55 30 58 4 DMF 90 180 51 30 64 5 DMF 120 180 58 20 62 6 DMF 150 120 50 15 69 7 DMSO 150 180 59 10 77 8 DMSO 150 240 61 5 51 9 DMSO 150 300 67 15 72 10 DMSO 180 120 65 2 56 11 Diethylene glycol 150 180 61 5 94 12 Diethylene glycol 150 120 54 3 80 13 Diethylene glycol 150 300 67 7 93 14 Diethylene glycol 170 180 64 2 85 15 Diethylene glycol 160 180 65 2 82 16 Diethylene glycol 170 300 63 5 81 a Reaction conditions: 9a (0.1 mmol), 10a (0.1 mmol) in solvent (1.0 mL), 5-300 min, N2 atmosphere. b Method A: Conventional heating; c Method B: Microwave Irradiation; d Isolated yield after recrystalization/column chromatography. Sharma et al.: Microwave-assisted one-pot efficient synthesis ... 992 Acta Chim. Slov. 2017, 64, 988-1004 a unless otherwise mentioned, all the reactions were carried out with substrates 9a-f (0.2 mmol), substituted 2-aminophenols 10a-c (0.2 mmol) in diethylene glycol (2.0 mL) at 150 °C under microwave irradiation. b Isolated yield. Scheme 1. Microwave-assisted one-pot green synthesis of 2-oxobenzo[1,4]oxazine analogues (11a-n).a,b Sharma et al.: Microwave-assisted one-pot efficient synthesis ... Acta Chim. Slov. 2017, 64, 988-1004 993 time for the above reaction in DMSO solvent was not found to be fruitful. (Entries 8-10, Table 1). Then, we planned to perform our model reaction in more polar diethylene glycol as solvent. To our surprise, after 5 min at 150 °C, we obtained 11a in 94% yield (entry 11, Table 1). Furthermore, in spite of increasing the reaction temperature from 150 °C to 170 °C or increasing/ decreasing the reaction time; we were successful in obtaining 11a in the yield range of 81-93% (entries 12-16, Table 1). Finally, based on above screening studies, diethylene glycol as solvent, 150 °C temperature for 5 min was found to be the best optimized reaction condition under microwave irradiations (entry 11, Table 1). After optimization study, we further investigated the scope and generality of this reaction. Several alkyl/alkoxy/ halide/nitro-substituted 2,4-dioxo-4-phenylbutanoic acids 9a-f were reacted with alkyl/halide/nitro-substitut- a Unless otherwise mentioned, all reactions were carried out with substrates 9a-g or 12 (0.2 mmol) and 1,2-diamino benzene 13 (0.2 mmol) in diethylene glycol (2.0 mL) at 150 °C under microwave irradiation. b Isolated yield. Scheme 2. Microwave-assisted one-pot synthesis of functionalized 2-oxoquino[1,4]oxalines 14a-h.a Sharma et al.: Microwave-assisted one-pot efficient synthesis ... 994 Acta Chim. Slov. 2017, 64, 988-1004 ed 2-aminophenol 10a-c in diethylene glycol under our optimized conditions (Scheme 1). The desired 2-oxoben-zo[1,4]oxazines 11a-n were purified either by flash column chromatography method or by recrystallization (see experimental section). As evident from scheme 1; substituted 2, 4-di-oxo-4-phenylbutanoic acid 9a-f reacted smoothly with substituted 2-aminophenol 10a-c, and furnished substituted 2-oxobenzo[1,4]oxazines 11a-n in 78-97% yield range. It has been observed that nitro-based 2-oxobenzo[1,4]ox-azines 11k-n were obtained in comparatively lesser yields (78-83%) with rest of the compounds 11a-j. This is due to poor solubility of nitro-based 2-oxobenzo[1,4]oxazines 11k-n in ethyl acetate which makes the purification of these compounds via column chromatography very tedious and cumbersome. In this study, the most characteristic feature observed was that a broad range of functional groups, like Cl, Br, OMe and NO2 are well compatible under our optimized reaction conditions. Thus, these groups can further be manipulated to obtain new therapeutic molecules. After successful implementation of our methodology on 11a-n series; we extended its synthetic application towards the synthesis of its congener class of bioactive het-erocycles i.e. 2-oxoquino[1,4]oxalines 14a-h; which were synthesized from 9a-g with phenyl-1,2-diamine 13 using our optimised methodology in the excellent yield (9096%), as depicted in Scheme 2. Furthermore, the practicality of this methodology was demonstrated via gram scale synthesis of compounds 11a, 14c and 14e. Thus, the reaction of 9a (2.00 g, 10.40 mmol), 9c (2.26 g, 10.00 mmol) or 9e (2.71 g, 10.00 mmol) with either 10a (1.13 g, 10.40 mmol) or 13 (1.08 g, 10.00 mmol) in diethylene glycol under MW irradiation at 150 °C for 7-9 min furnished the target compounds, 11a (2.49 g, 90.41%); 14c (2.60 g, 87.32%) and 14e (3.09 g, 90.14%), respectively. (Scheme 3) We have further demonstrated practicality of our developed methodology for the synthesis of anticancer indole alkaloid, Cephalandole A, which was isolated from Taiwanese orchid Cephalanceropsis gracilis (Orchidaceae). Its crude extract showed good activity against CNS (SF-268; IC50 = 12.2 ^M), breast (MCF-7; IC50 = 7.57 ^M) and lung (NCI-H460; IC50 = 7.8 ^M) carcinoma cell lines.24 3-indoleglyoxylic acid (15) on reaction with aminophenol (10a) in diethylene glycol under MW at 150 °C for 10 min furnished indole alkaloid Cephalandole A (16) in 89% yield (Scheme 4). The spectral data was found to be the same as the literature data. 2. 1. Material and Methods for Antioxidant Activity 2. 1. 1. In vitro antioxidant DPPH radical scavenging activity24a-e In DPPH radical scavenging method the synthesized compounds at different concentrations ranging from 10 to 100 ^g mL-1 was mixed with 1.5 mL of a DPPH methanolic solution (20 mg L-1). Pure methanol was taken as control and ascorbic acid (vitamin C) was used as a reference compound. The percent of DPPH decoloration of the sample was calculated according to the formula. Decoloration % = [1 - (Abs sample / Abs control)] x 100 Scheme 3. Gram scale synthesis of 11a, 14c and 14h. Scheme 4. Synthesis of Cephalandole A (16) Sharma et al.: Microwave-assisted one-pot efficient synthesis ... Acta Chim. Slov. 2017, 64, 988-1004 995 The decoloration was plotted against the sample concentration and a logarithmic regression curve was established in order to calculate the IC . The results are expressed as antiradical efficiency (AE), which is 1000-fold inverse of the IC50 value AE = 1000/ IC50. 2. 1. 2. In vitro Ferric Reducing Antioxidant Power (FRAP) Assay24f The FRAP reagent was prepared by mixing freshly prepared 10.0 mM of ferric-tripyridyltriazine (TPTZ) solution, 20.0 mM FeCl3 x 6H2O solution and 300 mM sodium acetate buffer (pH 3.6) in a ratio of 1:1:10 (v/v/v). Sample was added to 3 mL of FRAP reagent and this reaction mixture was incubated for 30 min at 37 °C temperature. The absorbance of prepared reaction mixture was measured at 593 nm. A freshly prepared solution of FeSO was used for calibration of standard curve. The 4 FRAP antioxidant capacities were expressed in terms of C0.5FRAP (the concentration of samples with respect to the antioxidant ability equivalent to that of FeSO4 at 0.5 mmol/L). 2. 2. Antioxidant Activity: In vitro antioxidant DPPH Radical Scavenging Assay, FRAP Assay and Structure-activity Relationship25 The DPPH radical scavenging assay is generally utilised for the screening of antioxidant activity of diverse heterocycles.25a DPPH is a stable free radical, which can easily accept an electron or a hydrogen radical to become a stable molecule. Literature reports illustrate that DPPH assay works in two ways; a single electron transfer (SET) or a hydrogen atom transfer (HAT) mechanism.25b DPPH in the methanolic medium has odd electron configuration which shows a strong absorption band at 515 nm and the absorbance decreases in the presence of free radical scavengers which results in the colour change from deep purple to yellow.25c-d The radical quenching ability strongly depends on the structural accessibility of the radical trapping site. The electron density as well as steric hindrance plays a vital role in the antioxidant activity because they may prevent the test molecule from reaching to the DPPH radical site and thus results in lower activity.25e Table 2. Antioxidant activity of synthesized compounds 11a-n, 14a-h and 16 by DPPH radical scavenging and FRAP assay.25 S. No. Compound No. Antioxidant activity" DPPH assay (IC50) (^g/mL) FRAP assay (C0.5FRAP ^M) 1 11a 10.20 ± 0.08 611.5 ± 23.2 2 11b 19.70 ± 0.31 763.2 ± 38.1 3 11c 29.80 ± 0.17 306.8 ± 25.8 4 11d 65.32 ± 0.97 >1000 5 11e 25.02 ± 0.21 421.7 ± 37.9 6 11f 23.45 ± 0.14 845.9 ± 35.1 7 11g 67.40 ± 0.28 >1000 8 11h 78.50 ± 1.41 921.6 ± 29.6 9 11i 34.42 ± 0.62 291.7 ± 23.1 10 11j 42.98 ± 0.76 >1000 11 11k 21.27 ± 0.11 489.2 ± 18.5 12 11l 56.12 ± 1.03 348.8 ± 31.4 13 11m 15.70 ± 0.14 598.5 ± 23.4 14 11n 78.76 ± 1.43 >1000 15 14a 27.36 ± 0.44 638.4 ± 37.6 16 14b 91.36 ± 2.04 >1000 17 14c 9.89 ± 0.15 612.8 ± 17.8 18 14d 28.24 ± 0.46 498.4 ± 22.4 19 14e 8.97 ± 0.13 689.3 ± 30.0 20 14f 43.54 ± 0.88 592.7 ± 41.6 21 14g 38.97 ± 0.97 >1000 22 14h 14.27 ± 0.23 358.3 ± 17.7 23 16 11.87± 0.14 NDb 24 Ascorbic acid 4.57 - 25 BHT - 546.0 ± 13.6 a Results are expressed as a mean ± standard deviation (n = 3). DPPH radical scavenging activities are expressed as IC50 concentrations of the compounds (^g/mL) required to inhibit 50 % of the radicals and the maximum inhibition values; b ND means not done. Sharma et al.: Microwave-assisted one-pot efficient synthesis ... 996 Acta Chim. Slov. 2017, 64, 988-1004 The FRAP assay was measured using the method as described by Benzie and Strain.25f This methodology demonstrates that the antioxidant molecule reacts to a complex of ferric tripyridyltriazine [Fe3+-TPTZ] and produces a colored ferrous tripyridyltriazine [Fe2+-TPTZ] complex. Generally, the reducing nature of antioxidant molecule is associated with their action by breaking the free radical chain via donating a hydrogen atom. All the synthesized 2-oxobenzo[1,4]oxazines 11a-n, 2-oxoquino[1,4]oxalines 14a-h and Cephalandole A 16 were screened for their in vitro antioxidant activities using DPPH radical scavenging activity assay using ascorbic acid as standard reference as well as in FRAP assayusing BHT as standard reference (Table 2)..25,26 The in vitro antioxidant screening of compounds 11a-f, having no substitution at benzoxazine aromatic ring; it had been observed that the compound 11a was found to be the most active compound having IC50 value of 10.20 ± 0.08 ^g/mL (entry 1, Table 2) in comparison with standard reference ascorbic acid (IC50 = 4.57 ^g/mL). When mono-halo substituents were present at side chain of aromatic ring as in case of 11c, 11e and 11f having -Cl, -F and -Br substituent respectively; these molecules exhibited slightly lesser antioxidant activity in comparison with 11a (Table 2; entry 3, 5 and 6). Further, 2-oxobenzo[1,4]oxazine having di-halo substituents on side chain of aromatic ring (11d) exhibited decrease of antioxidant activity drastically (Table 2; entry 4) in comparison to mono-halo substituted analogues 11c, 11e and 11f. In addition, electron-donating substituents at side chain of aromatic ring in compound 11b showed better antioxidant activity in comparison to halo-substituted 2-oxobenzo[1,4]oxazine analogues 11c-11f. Moreover, in 2-oxobenzo[1,4]oxazines having -CH3 or -NO2 substituent at para-position of benzoxazine aromatic nucleus (11g-11n); the antioxidant activity was found to be lesser (Table 2; entry 7-14) in comparison with unsubstituted analogues 11a-11f, except compound 11k and 11m; which exhibited better activity profile (Table 2; entry 11 and 13). Whereas, 2-oxobenzo[1,4]oxazine having di-chloro substituents at side chain of aromatic ring (Table 2; entry 14); 11n was found to be the least active compound among 2-oxobenzo[1,4]oxazine series. Furthermore, the -CH3 substituent at benzoxazine nucleus along with electron-donating methyl substituent or halo-substituent at side chain of aromatic ring as in compounds 11g-j; these were found to show moderate to poor antioxidant activities (Table 2; entry 7-10) in comparison to other analogues of the series. In the case of 2-oxoquino[1,4]oxalines 14a-h derivatives, compounds 14c and 14e were found to be the best compounds of this series and have shown the antioxidant activities having the IC50 value of 9.89 ± 0.15 ^g/mL and 8.97 ± 0.13 ^g/mL, respectively, in comparison to ascorbic acid (Table 2; entry 17 and 19). The 2-oxoquino[1,4] oxalines 14f having fluoro substituent showed lesser antioxidant activity profile (IC50 value of 43.54 ± 0.88 ^g/mL). It can be speculated that due to larger electronegativity of fluorine atom, which accumulates the electron density, restricts the delocalization of bonds due to which, the free electrons of 14f are not easily available for quenching of DPPH radical. Furthermore, extending the side chain of phenyl ring to more electron rich naphthyl ring in 14h (IC50 = 14.27 ± 0.23 ^g/mL) showed promising activity. In addition, the un-substituted 2-oxoquino[1,4]oxaline 14a and dichloro-substituted side chain of aromatic ring having 2-oxo-quino[1,4]oxaline 14d showed lesser activity (Table 2; entry 15 and 18) in comparison with 14c and 14e. The electron-donating substituents at side chain of aromatic ring (compound 14b and 14g) showed poor activity profile (Table 2: entry 16 and 21) in comparison with their corresponding halo-substituted analogues 14c and 14e. All the synthesized compounds were also assessed in the ferric to ferrous reduction assay (FRAP assay) taking BHT as standard reference. (Table 2) In the present study, the trend for ferric ion reducing activities of all the compounds 11a-n and 14a-h, with respect to standard reference BHT indicates that the seven compounds (11c, 11e, 11i, 11k, 11l, 14d and 14h) were found more potent antioxidant than BHT. The compound which have the mono-halo (such as: F, Cl, Br) substituent at the side chain of aromatic ring in 2-oxobenzo[1,4]oxazin (compound 11c, 11e, 11i, 11k and 11l) exhibited higher antioxidants activity than BHT, whereas 2-oxoquino[1,4]oxaline 14d and 14h, which have 2,4-dichloro substituent at side chain of aromatic ring or naphthyl substituent displayed better antioxidant activity than standard reference BHT. Compounds 11a, 11m, 14c and 14f showed comparable FRAP antioxidant activity than standard reference BHT. Rest of the compounds showed moderate to low FRAP antioxidant activity. Moreover, for the first time the antioxidant activity of Cephalandole A 16 was also evaluated and found to possess moderate antioxidant activity having IC50 value of 11.87 ± 0.14 ^g/mL in comparison to ascorbic acid (Table 2; entry 23) in DPPH radical scavenging assay. These results showed that the mono-halo substitution at side chain of aromatic ring in nitrogen congener of 2-oxo-benzo[1,4]oxazines i.e. 2-oxoquino[ 1,4] oxalines 14c and 14e along with un-substituted 2-oxobenzo[1,4]oxazine 11a were found to be the most active compounds of the series showing promising antioxidant activities in DPPH radical scavenging. Furthermore, in the FRAP antioxidant assay, seven compounds (11c, 11e, 11i, 11k, 11l, 14d and 14h), which have mono-halo substitution at side chain of aromatic ring in 2-oxobenzo[1,4]oxazine (11c, 11e, 11i and 11k, 11l) and dihalo substituent as well as naphthyl substituent at 2-oxoquino[1,4]oxalines (14d and 14h), showed higher antioxidant activity in comparison with BHT, respectively. 2. 3. Cytotoxicity Compounds 11a, 14c, 14e and 14h (which displayed good antioxidant activity in DPPH radical scavenging as- Sharma et al.: Microwave-assisted one-pot efficient synthesis ... Acta Chim. Slov. 2017, 64, 988-1004 997 say) were also assessed for their cytotoxic studies using MTT assay taking 25-250 ^g/mL concentration in 3T3 fibroblast cell lines.27 The screening results showed that these compounds were found non-toxic even at 250 ^g/mL and displays allowable values of cell viability. (Figure 3) 3. Experimental Section 3. 1. General All glass apparatus were oven dried prior to use. Melting points were taken in open capillaries on complab melting point apparatus and are presented uncorrected. Microwave reactor (CEM Discover) was used for operation of reactions. Infrared spectra were recorded on a Perkin-El-mer FT-IR Spectrum 2 spectrophotometer 1H NMR and 13C NMR spectra were recorded on ECS 400 MHz (JEOL) NMR spectrometer using CDCl3, CD3OD and CD3SOCD3 as solvent and tetramethylsilane as internal reference. Electrospray ionization mass spectrometry (ESI-MS) and HRMS were recorded on Xevo G2-S QTof (Waters, USA) Spectrometer. Column chromatography was performed over Merck silica gel (particle size: 60-120 Mesh) procured from Qualigensa (India), flash silica gel (particle size: 230400 Mesh). All chemicals and reagents were obtained from Sigma Aldrich (USA), Merck (India) or Spectrochem (India) and were used without further purification. 3. 2. General Procedure for the Synthesis of (Z)-3-(2-oxo-2-phenylethylidene)-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-one (11a) in optimization study as given in table 1: (1) Method A (conventional heating condition): A solution of 9a (19.2 mg, 0.10 mmol) and 10a (10.9 mg, 0.10 mmol) in given solvent (1.0 mL) was heated at given time and temperature (as shown in Table 1). The progress of the reaction was monitored by TLC using 9:1 hexane/ethyl acetate as an eluent. After completion of reaction, the reaction mixture was extracted with ethyl acetate (3 x 50 mL) and distilled water. The organic layer was combined and dried over anhydrous Na2SO4 and the organic solvent was removed under reduced pressure to give the crude product. The crude products were purified either by recrystallization using EtOAc/hexane (v/v = 20:80) or by flash column chromatography method over silica gel using 9:1 hexane/ethyl acetate as an eluent which afforded the pure desired 2-oxobenzo[1,4]oxazine 11a having good yields (18-67%). (2) Method B (microwave irradiation condition): To a solution of 9a (19.2 mg, 0.10 mmol) in given solvent (1.0 mL) was added 10a (10.9 mg, 0.10 mmol), and the reaction mixture was irradiated under microwave at given temperature and time (as shown in Table 1). The progress of the reaction was monitored by TLC using 9:1 hexane/ethyl acetate as an eluent. After completion of the reaction, the reaction mixture was extracted with ethyl acetate (3 x 50 mL) and distilled water. The organic layer was combined and dried over anhydrous Na2SO4 and the organic solvent was removed under reduced pressure to give the crude product. The crude product were purified either by recrystallization using EtOAc/hexane (v/v = 20:80) or by flash column chromatography method over silica gel using 9:1 hexane/ethyl acetate as an eluent which afforded the pure desired 2-oxobenzo[1,4]oxazine 11a product having good yields (51-94%). 3. 3. General Procedure for the synthesis of functionalized (Z)-3-(2-oxo-2-phenylethylidene)-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-ones (11a-n) and (Z)-3-(2-oxophenylethylidene)-3,4- 100 ^ sa IOC 100 100 100 ills «14c H14i ■ 14h 94.41 Concentration (/ig ml; Figure 3. Percentage cell viability test. Sharma et al.: Microwave-assisted one-pot efficient synthesis ... 998 Acta Chim. Slov. 2017, 64, 988-1004 dihydroquinoxalin-2(1H)-ones (14a-h) as given in scheme 1 and scheme 2: To a solution of compound 9a-f (0.20 mmol; 1 eq., as given in Scheme 1) or 9a-g (0.20 mmol; 1 eq. as given in Scheme 2) in diethylene glycol (2.0 mL) was added compound 10a-c (0.20 mmol; 1 eq., as given in Scheme 1) or 12 and 13 (0.20 mmol; 1 eq., as given in Scheme 2) and the reaction mixture was irradiated under microwave at 150 °C temperature for about 5-10 min depending upon the substrate utilized. The progress of the reaction was monitored by TLC using 9:1 hexane/ethyl acetate as an eluent. After completion of reaction, the reaction mixture was extracted with ethyl acetate (3 x 50 mL) and distilled water. The organic layer was combined and dried over anhydrous Na2SO4 and the organic solvent was removed under reduced pressure to give the crude product. The crude products were purified either by recrystallization using EtOAc/hexane (v/v = 20:80) or by flash column chromatography method over silica gel using 9:1 hexane/ethyl acetate as an eluent which afforded the pure desired (Z)-3-(2-oxo-2-phenylethylidene)-3,4-dihydro-2H-benzo[b] [1,4]oxazin-2-ones 11a-n and (Z)-3-(2-oxo-2-phenyle-thylidene)-3,4-dihydroquinoxalin-2(1H)-ones 14a-h having good yields (78-97%). 3. 4. Characterization data of (Z)-3-(2- oxo-2-phenylethylidene)-3,4-dihydro-2H-benzo[b] [1,4]oxazin-2-ones, (Z)-3-(2-oxo-2-phenylethylidene)-3,4-dihydroquinoxalin-2(1H)-ones (11a-n and 14 a-h) and Cephalandole A (Z)-3-(2-oxo-2-phenylethylidene)-3,4-dihydro-2H-ben-zo[b][1,4]oxazin-2-one (11a)17a-c Yellow solid; yield: 94%, Rf (EtOAc/hexane; 20:80) = 0.85; m.p. 185-186 °C; FT-IR (KBr, v /cm-1) 3434, 1 v max ' 1754, 1614, 1594, 1270; 1H NMR (400 MHz, CDCl3) 5 8.00 (d, J = 7.4 Hz, 2H, C4'H, C8'H), 7.55 - 7.46 (m, 3H, CTH, C5'H, C7'H), 7.21-7.05 (m, 5H, C5H, C6H, C7H, C8H); 13C NMR (100 MHz, CDCl3) 5 191.6 (>C=O), 156.3 (O=C-O-), 141.3 (C9), 139.1 (C3'), 138.3 (C6'), 132.8 (C3), 128.8 (C10), 127.7 (C4\ C8'), 126.0 (C5\ C7'), 124.0 (C6), 123.8 (C7), 117.2 (C5), 116.0 (c8), 94.7 (-C=C); HRMS (ESI) calcd. for C16H11NO3 [M+H]+: 266.0739; found 266.0734. 16 11 3 (Z)-3-(2-oxo-2-(p-tolyl)ethylidene)-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-one (11b)17b d Yellow solid; yield: 95%, Rf (EtOAc/hexane; 20:80) = 0.80; m.p. 160-162 °C; FT-IR (KBr, v /cm-1) 3437, max 2925, 1759, 1622, 1110; 1H NMR (400 MHz, CDCl3) 5 7.91 (d, J = 7.6 Hz, 2H, C4'H, C8'H), 7.27 (m, 2H, C5'H, C7'H), 7.20-7.16 (m, 2H, C5H, C8H), 7.10-7.06 (m, 2H, C6H, C7H), 7.03 (d, J = 1.2 Hz, 1H, CHH), 2.41 (s, 3H, -CH3); 13C NMR (100 MHz, CDCl3) 5 191.4 (>C=O), 156.5 (O=C-O-), 143.6 (C6'), 141.2 (C9), 138.8 (C3'), 135.7 (c3), 129.5 (C10), 127.9 (C4\ 8'), 125.9 (C5\ C7'), 123.9 (C6), 123.8 (C7), 117.2 (C5), 115.9 (C8), 94.8 (-C=C-), 21.8 (-CH3); HRMS (ESI) calcd. for C17H13NO3 [M+H]+: 280.0895; f3ound 280.0899. 17 13 3 (Z)-3-(2-(4-chlorophenyl)-2-oxoethylidene)-3,4-dihy-dro-2H-benzo[b][1,4]oxazin-2-one (11c)17ad Yellow solid; yield: 90%, Rf (EtOAc/hexane; 20:80) = 0.80; m.p. 155-157 °C; FT-IR (KBr, v /cm-1) 3437, 1759, max 1633, 1585, 752; 1H NMR (400 MHz, CDCl3) 5 7.92 (d, J = 8.4 Hz, 2H, C4'H, C8'H), 7.44 (d, J = 8.4 Hz, 2H, C5'H, C7'H), 7.22-7.09 (m, 4H, C5H, C6H, C7H, C8H), 6.98 (s, 1H, CTH); 13C NMR (100 MHz, CDCl3) 5 190.1 (>C=O), 156.1 (O=C-O-), 141.4 (C6'), 139.4 (C9), 139.1 (C3'), 136.6 (C3), 129.1 (C10), 129.0 (C4\ C8'), 126.0 (C5\ C7'), 124.3 (C6), 123.6 (c7), 117.3 (C5), 116.1 (C8), 94.2 (-C=C-); HRMS (ESI) calcd. for C16H10ClNO3 [M+2H]+: 301.0349; found 301.0345. (Z)-3-(2-(2,4-dichlorophenyl)-2-oxoethylidene)-3,4-di-hydro-2H-benzo[b][1,4]oxazin-2-one (11d) Yellow solid; yield: 97%, Rf (EtOAc/hexane; 20:80) = 0.85; m.p. 160-162 °C; FT-IR (KBr, v /cm-1) 3436, 1758, max 1620, 1577, 1101; 1H NMR (400 MHz, CDCl3) 5 12.85 (s, 1H, -NH- ), 7.53 (d, J = 7.6 Hz, 1H, C4'H), 7.46 (s, 1H, C8'H), 7.32 (d, J = 8.0 Hz, 1H, C5'H), 7.20 - 7.12 (m, 5H, C6H, C7H, C8H, C7'H), 6.76 (s, 1H, CHH); 13C NMR (100 MHz, CDCl3) 191.1 (>C=O), 155.1 (O=C-O-), 140.8 (c9), 138.4 (C6'), 136.8 (C3'), 136.6 (C4'), 131.8 (C10), 130.0 (C3), 129.9 (C8'), 126.7 (C5'), 125.4 (C7'), 123.9 (C6), 122.7 (C7), 116.6 (c5), 115.6 (C8), 97.5 (Ch: -C=C-); HRMS (ESI) calcd. for C16H9Cl2NO3 [M+2H]+: 334.9959; found 334.9953. 16 9 2 3 (Z)-3-(2-(4-fluorophenyl)-2-oxoethylidene)-3,4-dihy-dro-2H-benzo[b][1,4]oxazin-2-one (11e)17eJ Yellow solid; yield: 94%, Rf (EtOAc/hexane; 20:80) = 0.80; m.p. 152-154 °C; FT-IR (KBr, v /cm-1) 3434, 2925, max 1757, 1622, 1596, 1156; 1H NMR (400 MHz, CDCl3) 5 8.03 (dd, J = 5.6, 8.8 Hz, 2H, C4'H, C8'H), 7.22-7.11 (m, 6H, C5H, C6H, C7H, C8H, C5'H, C7'H), 7.00 (s, 1H, CHH); 13C NMR (100 MHz, CDCl3) 5 190.1(>C=O), 166.9 (C6'), 156.2 (O=C-O-), 141.3 (C9), 139.2 (C3'), 134.6 (C3), 130.3 (C10), 125.9 (C4\ C8'), 124.6 (C6), 123.7 (C7), 117.3 (C5), 116.0 (C5', C7'), 115.8 (C8), 94.3 (C1^:-C=C-) ; HRMS (ESI) calcd. for C,,H FNO, [M+H]+: 284.0645; found 284.0649. 16 10 3 Sharma et al.: Microwave-assisted one-pot efficient synthesis ... Acta Chim. Slov. 2017, 64, 988-1004 999 (Z)-3-(2-(4-bromophenyl)-2-oxoethylidene)-3,4-dihy-dro-2H-benzo[fo][1,4]oxazin-2-one (11f)17i Yellow solid; yield: 93%, Rf (EtOAc/hexane; 20:80) = 0.85; m.p. 200-202 °C; FT-IR (KBr, v /cm-1) 3437, 1754, 1 v max ' 1624, 1585, 1277, 1111; 1H NMR (400 MHz, CDCl3) 8 7.85 (d, J = 8.0 Hz, 2H, C4'H, C8'H), 7.60 (d, J = 8.0 Hz, 2H, C5'H, C7'H), 7.24-7.09 (m, 4H, C5H, C6H, C7H, C8H), 6.97 (s, 1H, CTH); 13C NMR (100 MHz, CDCl3) 8 190.3 (>C=O), 156.1 (O=C-O-), 141.4 (C9), 139.4 (C3'), 137.1 (C3), 132.1 (C5\ C7'), 129.2 (C10), 127.8 (C4\ C8'), 126.0 (C6'), 124.3 (C6), 123.6 (C7), 117.3 (C5), 116.1 (C8), 94.2 (Cr: -C=C-); HRMS (ESI) calcd. for C16H10BrNO3 [M+2H]+: 344.9844; found 344.9849. (Z)-6-methyl-3-(2-oxo-2-phenylethylidene)-3,4-dihy-dro-2H-benzo[fo][1,4]oxazin-2-one (11g)17c Yellow solid; yield: 88%, Rf (EtOAc/hexane; 20:80) = 0.90; m.p. 157-158 °C; FT-IR (KBr, v /cm-1) 3436, max 1750, 1618, 1572, 1123, 740; 1H NMR (400 MHz, CDCl3) 8 8.00-7.98 (m, 2H, C4'H, C8'H), 7.56-7.45 (m, 3H, C13H, C5'H, C7'H), 7.07-7.02 (m, 2H, C8H, C6'H), 6.88 (d, J = 8.5 Hz, 2H, C5H, C7H), 2.34 (s, 3H, -CH3); 13C NMR (100 MHz, CDCl3) 8 191.5 (>C=O), 156.5 (O=C-O-), 139.4 (C9), 139.2 (C3'), 138.4 (C6), 136.1 (C3), 132.7 (C6'), 128.8 (C4\ C8'), 127.7 (C5\ C7'), 124.8 (C7), 123.4 (C10), 116.8 (C5), 116.2 (C8), 94.5 (CL: -C=C-), 21.08 (-CH3); HRMS (ESI) calcd. for C17H13NO3 [M+H]+: 280.0895; found 280.0899. (Z)-6-methyl-3-(2-oxo-2-(p-tolyl) ethylidene)-3, 4-di-hydro-2H-benzo[fo][1,4]oxazin-2-one (11h)17e Yellow solid; yield: 89%, Rf (EtOAc/hexane; 20:80) = 0.80; m.p. 162-164 °C; FT-IR (KBr, v /cm-1) 3434, max 1762, 1602, 1313, 1047; 1H NMR (400 MHz, CDCl3) 8 7.90 (d, J = 8.1 Hz, 2H, C4'H, C8'H), 7.27 (d, J = 8.0 Hz, 2H, C5'H, C7'H), 7.06 (d, J = 8.1 Hz, 1H, C8H), 7.02 (s, 1H, CTH), 6.88 (d, J = 9.4 Hz, 2H, C5H, C7H), 2.42 (s, 3H, C6': -CH3), 2.35 (s, 3H, C6: -CH3); 13C NMR (100 MHz, CDCl3) 8 191.3 (>C=O), 156.6 (O=C-O-), 143.5 (C6'), 139.3 3C9), 139.0 (C3'), 136.0 (C6), 135.8 (C3), 129.5 (C5\ C7'), 127.8 (C4\ C8'), 124.6 (C7), 123.5 (C10), 116.8 (C5), 116.1 (C8), 94.6 (C1': -C=C-), 21.7 (C6: -CH3), 21.0 (C6': -CH3); HRMS (ESI) calcd. for C18H15NO3 [M+H]+: 294.1052; found 294.1055. (Z)-3-(2-(4-chlorophenyl)-2-oxoethylidene)-6-methyl-3,4-dihydro-2H-benzo[fo][1,4]oxazm-2-one (11i)17h Yellow solid; yield: 91%, Rf (EtOAc/hexane; 20:80) = 0.85; m.p. 145-147 °C; FT-IR (KBr, v /cm-1) 3437, 1767, max 1629, 1582; 1H NMR (400 MHz, CDCl3) 8 7.94-7.92 (m, 2H, C4'H, C8'H), 7.46-7.43 (m, 2H, C5'H, C7'H), 7.08 (d, J = 9.2 Hz, 1H, C8H), 6.97 (s, 1H, C1'H), 6.92-6.90 (m, 2H, C5H, C7H), 2.35 (s, 3H, -CH3); 13C NMR (100 MHz, CDCl3) 8 190.1 (>C=O), 156.3 (O=C-O-), 139.5 (C9), 139.4 (C6'), 139.0 (C3'), 136.7 (C6), 136.1 (C3), 129.1 (C4\ C8'), 129.0 (C5\ C7'), 125.0 (C7), 123.2 (C10), 116.9 (C5), 116.3 (C8), 94.1 (CT: -C=C-), 21.1 (-CH3); HRMS (ESI) calcd. for C17H-12ClNO3 [M+2H]+: 315.0506; found 315.0509. (Z)-3-(2-(4-bromophenyl)-2-oxoethylidene)-6-methyl-3,4-dihydro-2H-benzo[fo][1,4]oxazin-2-one (11j) Yellow solid; yield: 96%, Rf (EtOAc/hexane; 20:80) = 0.85; m.p. 179-181 °C; FT-IR (KBr, v /cm-1) 3435, max 2923, 1763, 1624, 1543, 1052; 1H NMR (400 MHz, CDCl3) 8 7.87-7.84 (m, 2H, C4'H, C8'H), 7.63-7.59 (m, 2H, C5'H, C7'H), 7.08 (d, J = 9.2 Hz, 1H, C8H), 6.96 (s, 1H, CTH), 6.92-6.90 (m, 2H, C5H, C7H), 2.35 (s, 3H, -CH3); 13C NMR (100 MHz, CDCl3) 8 190.2 (>C=O), 156.3 (O=C-O-), 139.5 (C9), 139.4 (C6'), 137.1 (C3'), 136.2 (C6), 132.0 (C3), 129.2 (C5\ C7'), 127.7 (C4\ C8'), 125.1 (c7) 123.2 (C10), 117.0 (C5), 116.3 (C8), 94.0 (CL: -C=C-), 21.1 (-CH3); HRMS (ESI) calcd. for C17H12BrNO3 [M+h]+: 358.0001; found 358.0007. (Z)-3-(2-(4-bromophenyl)-2-oxoethylidene)-6-nitro-3,4-dihydro-2H-benzo[fo][1,4]oxazin-2-one (11k) Yellow solid; yield: 83%, Rf (EtOAc/hexane; 20:80) = 0.70; m.p. 195-197 °C; FT-IR (KBr, v /cm-1) 3435, 2925, max 1759, 1525, 1023; 1H NMR (400 MHz, CDCl3) 8 8.03-7.97 (m, 2H, C4'H, C8'H), 7.89-7.86 (m, 2H, C5'H, C7'H), 7.66-7.63 (m, 2H, C5H, C7H), 7.32 (d, J = 9.2 Hz, 1H, C8H), 7.07 (s, 1H, CTH); 13C NMR (100 MHz, CDCl3) 8 190.8 (>C=O), 154.9 (O=C-O-), 145.3 (C6), 145.0 (C9), 138.0 (C3'), 136.5 (C3), 132.3 (C5\ C7'), 129.4 (C4\ C8'), 128.6 (C6'), 124.5 (C10), 119.2 (C7), 118.0 (C5), 111.6 (C8), 96.4 (Cr: -C=C-); HRMS (ESI) calcd. for C16H9BrN2O5 [M+2H]+: 389.9695; found 389.9699. (Z)-6-nitro-3-(2-oxo-2-(p-tolyl) ethylidene)-3, 4-dihy-dro-2H-benzo[fo][1,4]oxazin-2-one (11l) Yellow solid; yield: 81%, Rf (EtOAc/hexane; 20:80) = 0.75; m.p. 220-223 °C; FT-IR (KBr, v /cm-1) 3433, 1760, max 1624, 1524, 1109; 1H NMR (400 MHz, DMSO-d6) 8 8.73 (d, J = 2.4 Hz, 1H, C4'H), 7.96-7.92 (m, 3H, C5'H, C7'H, C8'H), 7.46-7.37 (m, 3H, C5H, C7H, C8H), 6.95 (s, 1H, CTH), 2.40 (s, 3H, -CH3); 13C NMR (100 MHz, DMSO-d6) 189.9 (>C=O), 155.7 (0=C-O-), 145.9 (C6), 143.8 (C9), 139.2 (C6'), 138.5 (C3'), 135.2 (C3), 130.1 (C4\ C8'), 128.1 (C5\ C7'), 119.5 (C10), 118.9 (C7), 117.7 (C5), 112.6 (C8), 94.8 (C1': -C=C-), 21.7 (-CH3); HRMS (ESI) calcd. for C17H12N2O5 [M+H]+: 325.0746; found 325.0741. (Z)-3-(2-(4-chlorophenyl)-2-oxoethylidene)-6-nitro-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-one (11m) Yellow solid; yield: 78%, Rf (EtOAc/hexane; 20:80) = 0.70; m.p. 239-240 °C; FT-IR (KBr, v /cm-1) 3435, max 2924,1622, 1525, 1272; 1H NMR (400 MHz, DMSO-d6) 8 8.73 (s, 1H, C5H), 8.03 (d, J = 7.2 Hz, 2H, C4'H, C8'H), 7.91 (d, J = 9.1 Hz, 1H, C7H), 7.59 (d, J = 7.2 Hz, 2H, C5'H, C7'H), 7.42 (d, J = 9.0 Hz, 1H, C8H), 6.90 (s, 1H, CTH); 13C NMR (100 MHz, DMSO-d6) 8 188.9 (>C=O), 156.0 (O=C-O-), 146.0 (C6), 144.7 (C9), 139.8 (C6'), 138.2 Sharma et al.: Microwave-assisted one-pot efficient synthesis ... 1000 Acta Chim. Slov. 2017, 64, 988-1004 (C3'), 137.1 (C3), 129.9 (C4\ 08'), 129.6 (C5\ C7'), 125.8 (C10), 119.0 (C7), 117.8 (CS), 113.1 (C8), 94.4 (CE: -C=C-); HRMS (ESI) calcd. for C16H9ClN2O5 [M+2H]+: 346.0200; found 346.0204. (Z)-3-(2-(2,4-dichlorophenyl)-2-oxoethylidene)-6-ni-tro-3,4-dihydro-2H-benzo[fo][1,4]oxazin-2-one (11n) Yellow solid; yield: 80%, Rf (EtOAc/hexane; 20:80) = 0.75; m.p. 185-187 °C; FT-IR (KBr, v /cm-1) 3588, 2930, 1 v max /tt 1769, 1585, 1685, 1108; 1H NMR (400 MHz, CDCl3) 8 8.05 -7.99 (m, 2H, C7H, C8'H), 7.55(d, J = 8.4 Hz, 1H, C5'H, C7'H), 7.48 (d, J = 2.0 Hz, 1H, C7'H), 7.37-7.33 (m, 2H, C5H, C8H), 6.89 (s, 1H, CEH); 13C NMR (100 MHz, CDCl3) 8 192.4 (>C=O), 154.5 (O=C-O-), 145.2 (C6), 145.1 (C9), 138.0 (C6'), 137.6 (C3'), 136.9 (C4'), 132.7 (C3), 131.0 (C8'), 130.8 (C5'), 127.6 (C7'), 124.2 (C10), 119.4 (C7), 118.1 (C5), 111.7 (C8), 100.4 (CE: -C=C-); HRMS (ESI) calcd. for C16H8Cl2N2O5 [M+2H]+: 379.9810; found 379.9815. (Z)-3-(2-oxo-2-phenylethylidene)-3,4-dihydroquinox-alin-2(1H)-one (14a)17a-b Yellow solid; yield: 94%, Rf (EtOAc/hexane; 20:80) = 0.85; m.p. 268-269 °C; FT-IR (KBr, v /cm-1) 3060, 1688, max 1619; 1H NMR (400 MHz, DMSO-d6) 8 10.0 (s, 1H, -NH-), 8.07-8.05 (m, 2H, C4'H, C8'H), 7.55-7.48 (m, 3H, C5'H, C6'H, C7'H), 7.21 - 7.12 (m, 4H, C5H, C6H, C7H, C8H); 7.03 (s, 1H, CTH); 13C NMR (100 MHz, DMSO-d6) 8 187.9 (>C=O), 155.2 (-NH-C=O), 145.4 (C3), 138.4 (C3'), 131.2 (C6'), 128.2 (C10), 126.5 (C5\ C7'), 123.9 (C4\ C8'), 123.6 (C9), 123.1 (C6), 116.1 (C7), 115.1 (C5), 114.9 (C8), 89.0 (CE: -C=C-); HRMS (ESI) calcd. for C16H-12N2O2 [M+H]+: 265.0899; found 265.0893. (Z)-3-(2-oxo-2-(p-tolyl)ethylidene)-3,4-dihydroquinox-alin-2(1H)-one (14b)17k Yellow solid; yield: 96%, Rf (EtOAc/hexane; 20:80) = 0.80; m.p. 221-222 °C, FT-IR. (KBr, v /cm-1) 3045, max 1677, 1615; 1H NMR (400 MHz, CDCl3) 8 10.26 (s, 1H, -NH-), 7.96 (d, J = 8.0 Hz, 2H, C4'H, C8'H), 7.31-7.29 (m, 2H, C5'H, C7'H), 7.21-7.12 (m, 4H, C5H, C6H, C7H. C8H); 7.01 (s, 1H, CEH), 2.43 (s, 3H, -CH3); 13C NMR (100 MHz, CDCl3) 8 190.5 (>C=O), 158.0 (-NH-C=O), 144.6 (C6'), 142.8 (C3), 136.3 (C3'), 130.3 (c10), 129.4 (C5\ C7'), 127.7 (C4\ C8'), 125.6 (C9), 124.9 (C6), 123.9 (C7), 116.2 (C5), 115.9 (C8), 90.9 (CE: -C=C-), 21.7 (C6': -CH3); HRMS (ESI) calcd. for C17H14N2O2 [M+H]+: 279.1055; found 279.1059. (s, 1H, CrH); 13C NMR (100 MHz, DMSO-d6) 5 187.5 (>C=O), 156.1 (-NH-C=O), 146.7 (C3), 138.1 (C6'), 137.3 (C3'), 129.3 (C10), 127.5 (C4\ C8'), 124.8 (C5\C7'), 124.2 (C9), 124.1 (C6), 117.3 (C7), 117.2 (C5), 115.9 (C8), 89.9 (CE: -C=C-); HRMS (ESI) calcd. for C16H11ClN2O2 [M+2H]+: 300.0509; found 300.0503. (Z)-3-(2-(2,4-dichlorophenyl)-2-oxoethylidene)-3,4-di-hydroquinoxalin-2(1H)-one (14d)171 Yellow solid; yield: 95%, Rf (EtOAc/hexane; 20:80) = 0.80; m.p. 260-262 °C, FT-IR (KBr, v /cm-1) 3054, 1 v max ' 1682, 1618; 1H NMR (400 MHz, DMSO-d6) 5 11.84 (s, 1H, -NH-), 7.63-7.49 (m, 4H, C4'H, C5'H, C7'H, C8H), 7.19-7.15 (m, 3H, C5H, C6H, C7H), 6.44 (s, 1H, CEH); 13C NMR (100 MHz, DMSO-d6) 5 187.8 (>C=O), 154.8 (-NH-C=O), 145.2 (C6'), 138.2 (C3), 134.7 (C3'), 130.7 (C8'), 130.3 (C4'), 129.2 (C10), 127.1 (C5'), 126.6 (C9), 123.8 (C7'), 123.5 (C6), 123.1 (C7), 116.3 (C5), 114.9 (C8), 92.8 (CE: -C=C-); HRMS (ESI) calcd. for C16H10Cl-2N2O2 [M+2H]+: 334.0119; found 334.0113. (Z)-3-(2-(4-bromophenyl)-2-oxoethylidene)-3,4-dihy-droquinoxalin-2(1H)-one (14e)17g Yellow solid; yield: 93%, Rf (EtOAc/hexane; 20:80) = 0.75; m.p. 281-282 °C, FT-IR (KBr, v /cm-1) 3044, 1678, max 1606; 1H NMR (400 MHz, DMSO-d6) 5 11.81 (s, 1H, -NH-), 7.92-7.89 (m, 2H, C4'H, C8'H), 7.71-7.69 (m, 2H, C5'H, C7'H), 7.44 (s, 1H, C8H), 7.18-7.14 (m, 3H, C5H, C6H. C7H); 6.79 (s, 1H, CEH); 13C NMR (100 MHz, DMSO-d6) 5 186.5 (>C=O), 155.1 (-NH-C=O), 145.7 (C3), 137.5 (C3'), 131.2 (C5\ C7'), 128.5 (C10), 126.5 (C4\ C8'), 125.1 (C6'), 123.8 (C9), 123.1 (C6), 116.2 (C7), 115.1 (C5), 114.9 (C8), 88.8 (CE: -C=C-); HRMS (ESI) calcd. for C.,H..BrN,O, [M+2H]+: 344.0004; found 344.0009. 16 11 2 2 (Z)-3-(2-(4-fluorophenyl)-2-oxoethylidene)-3,4-dihy-droquinoxalin-2(1H)-one (14f)17g Yellow solid; yield: 91%, Rf (EtOAc/hexane; 20:80) = 0.80; m.p. 252-253 °C, FT-IR (KBr, v /cm-1) 3053, max 1680, 1614; 1H NMR (400 MHz, DMSO-d6) 5 12.0 (s, 1H, -NH-), 8.02-8.00 (m, 2H, C4'H, C8'H), 7.48 (s, 1H, C8H), 7.33-7.28 (m, 2H, C5'H. C7'H), 7.12-7.11 (m, 3H, C5H, C6H, C7H); 6.76 (s, 1H, CEH);13C NMR (100 MHz, DM-SO-d6) 5 187.1 (>C=O), 164.4 (C6'), 155.7 (-NH-C=O), 145.7 (C3), 135.3 (C3'), 129.9 (C4\ 8'), 129.8 (C10), 126.7 (C9), 124.1 (C6), 123.7 (C7), 116.6 (C5), 113.8 (C8), 115.4 (C5\C7'), 88.9 (CE: -C=C-); HRMS (ESI) calcd. for C, H,,FNO [M+H]+: 283.0805; found 283.0809. 16 11 2 2 (Z)-3-(2-(4-chlorophenyl)-2-oxoethylidene)-3,4-dihy-dro-quinoxalin-2(1H)-one (14c)17aJk Yellow solid; yield: 91%, Rf (EtOAc/hexane; 20:80) = 0.85; m.p. 267-268 °C, FT-IR (KBr, v /cm-1) 3052, 1686, max 1614; 1H NMR (400 MHz, DMSO-d6) 8 11.8 (s, 1H, -NH-), 7.98 (d, J = 8.8 Hz, 2H, C4\ C8'), 7.57-7.44 (m, 3H, C5'H, C7'H, C8H), 7.19-7.14 (m, 3H, C5H, C6H, C7H); 6.80 (Z)-3-(2-(4-methoxyphenyl)-2-oxoethylidene)-3,4-di-hydroquinoxalin-2(1H)-one (14g)16e Yellow solid; yield: 90%, Rf (EtOAc/hexane; 20:80) = 0.75; m.p. 241-242 °C, FT-IR (KBr, v /cm-1) 3058, 1689, max 1618; 1H NMR (400 MHz, DMSO-d6) 8 11.95 (s, 1H, -NH-), 7.95 (d, J = 9.2 Hz, 2H, C4'H, C86H), 7.45-7.44 (m, 1H, C8H), 7.14-7.03 (m, 5H, C5H, C6H, C7H, C5'H, C7'H); Sharma et al.: Microwave-assisted one-pot efficient synthesis ... Acta Chim. Slov. 2017, 64, 988-1004 1001 6.77 (s, 1H, CTH), 3.83 (s, 3H, -OCH3); 13C NMR (100 MHz, DMSO-d6) 5 187.7 (>C=O), 162.3 (C6'), 155.9 (-NH-C=O), 144.9 (C3), 131.3 (C4\C8\ C10), 129.2 (C3'), 126.5 (C9), 124.3 (C6), 123.7 (C7), 116.3 (C5), 115.3 (C8), 113.9 (C5\ C7'), 88.9 (CE: -C=C-), 55.4 (-OCH3); HRMS (ESI) calcd. for C17H14N2O3 [M+H]+: 295.1004; found 295.1009. (Z)-3-(2-(naphthalen-2-yl)-2-oxoethylidene)-3,4-dihy-droquinoxalin-2(1H)-one (14h)17m Yellow solid; yield: 94%, Rf (EtOAc/hexane; 20:80) = 0.85; m.p. 263-264 °C, FT-IR (KBr, v /cm-1) 3093, 1 v max ' 1694, 1614; 'H NMR (400 MHz, DMSO-d6) 5 11.67 (s, 1H, -NH-), 8.17-8.05 (m, 2H, C4'H, C9'H), 7.81-7.72 (m, 3H, C5'H, C6'H, C8'H), 7.52-7.39 (m, 4H, C6H, C8H, C7'H, C10'H); 7.25-7.14 (m, 2H, C5H, C7H), 6.89 (s, 1H, CTH); 13C NMR (100 MHz, DMSO-d6) 5 188.3 (>C=O), 156.3 (-NH-C=O), 146.5 (C3), 144.1 (C3'), 139.9 (C12'), 138.3 (C1E), 129.6 (C9'), 129.4 (C10), 128.5 (C4'), 128.4 (C5'), 127.5 (C9), 127.3 (C8'), 125.3 (C7'), 125.1 (C6'), 124.7 (C6), 124.4 (C7), 117.1 (C10'), 116.3 (C5), 115.9 (C8), 90.1 (Cr: -C=C-); HRMS (ESI) calcd. for C20H-14N2O2 [M+H]+: 315.1055; found 315.1059. Synthesis of Cephalandole A (16): To a solution of 3-Indoleglyoxylic acid 15 (226.9 mg, 1.20 mmol) in dieth-ylene glycol was added 10a (130.8 mg, 1.20 mmol) and the reaction mixture was irradiated under MW at 150 °C temperature for 10 min and the progress of reaction was monitored by TLC. After that, the reaction mixture was extracted with ethyl acetate (3 x 50 mL) and distilled water. The organic layer was combined and dried over anhydrous Na-2SO4 and the organic solvent was removed under reduced pressure to give the crude product. The crude product was further purified by flash column chromatography method over silica gel using hexane/ethyl acetate (8:2; v/v) as an eluent which afforded the pure desired Cephalandole A 16 having good yield (280.5 mg, 89%). Yellowish solid; m.p. 232-233 °C; 1H NMR24c (400 MHz, DMSO-d6) 5 11.98 (s, 1H), 8.76-8.74 (m, 1H), 8.69 (s, 1H), 7.85 (d, J = 6.4 Hz, 1H), 7.54-7.39 (m, 4H), 7.27-7.25 (m, 2H); HRMS (ESI) calcd. for QHNO [M+H]+: 263.0742; ; found 263.0749. 16 10 2 2 tionalized 2-oxobenzo[1,4]oxazines 11a-n and 2-oxoqui-no[1,4]oxalines 14a-h in excellent yields. This reaction tolerates a broad range of substrates, and provides a straightforward access to functionalized 2-oxobenzo[1,4]oxazines and 2-oxoquino[1,4]oxalines. The practical applicability of developed methodology was confirmed by the gram scale synthesis of 11a, 14c and 14e, along with the synthesis of Cephalandole A (16) (89% yield). All the synthesized compounds were screened for their in vitro antioxidant activities using DPPH radical scavenging and FRAP assays. Compounds 11a, 14c and 14e, the most active compounds of the series, were found to show IC50 value of 10.20 ± 0.08 |g/mL, 9.89 ± 0.15 |g/mL and 8.97 ± 0.13 |g/mL, respectively as compared to standard reference ascorbic acid (IC50 = 4.57 |g/mL) in DPPH assay, whereas in FRAP assay, seven compounds (11c, 11e, 11i, 11k, 11l, 14d and 14h) exhibited higher antioxidant activity in comparison with BHT. Cytotoxic studies revealed that the non-toxic nature of compounds 11a, 14c, 14e and 14h even at 250 |g/mL concentration. To the best of our knowledge, this is the first report of microwave-assisted synthesis and in vitro antioxidant activities of functionalized 2-oxobenzo[1,4] oxazines 11a-n; and 2-oxoquino[1,4]oxalines 14a-h and Cephalandole A 16 in excellent yields. The potential in vitro antioxidant activity combined with ease of preparation qualifies these compounds as candidates for further lead optimization studies. 6. Acknowledgement S. C. acknowledges SERB, New Delhi for Fast Track young scientist scheme (Grant No. CS-037/2013); DST, New Delhi for DST-RFBR Indo-Russian Joint Research Project (INT/RUS/RFBR/P-169) and CSIR, New Delhi for CSIR-EMR Grant [02 (0189)/14/EMR-II]. V. S. and P. K. J. thanks MNIT, Jaipur and CSIR, New Delhi respectively for providing financial assistance in the form of institute fellowship and RA fellowship, respectively. Materials Research Centre, MNIT, Jaipur is gratefully acknowledged for providing analytical facilities. 4. Supplementary Data The characterization spectra of synthesized 2-oxo-benzo[1,4]oxazines 11a-n, 2-oxoquino[1,4]oxalines 14a-h and Cephalandole A (16) are provided in supplementary material via the "Supplementary Content'' section of this article's webpage. 5. Conclusions In summary, we have developed a simple and highly efficient MW-assisted protocol for the synthesis of func- 7. References 1. (a) A. Khalaj, M. Abdollahi, A. Kebriaeezadeh, N. Adibpour, Z. Pandi, S. Rasoulamini, Ind. J. Pharmacol. 2002, 34, 184-188, (b) I. V. Mashevakaya, L. V. Anikina, Yu. B. Vikharev, V. A. Safin, S. V. KoEtsova, A. N. Maslivets, Pharm. Chem. J. 2001, 35, 414-417 and references cited therein. D01:10.1023/A:1013724004277 2. (a) R. D. Kamble, S. V. Hese, R. J. Meshram, J. R. Kote, R. N. Gacche, B. S. Dawane, Med. Chem. Res. 2015, 24, 1077-1088; DOI: 10.1007/s00044-014-1165-z (b) N. Gokhan, H. Erdogan, N. T. Durlu, R. Demirdamar, Farmaco, 1999, 54, 112-115. Sharma et al.: Microwave-assisted one-pot efficient synthesis ... 1002 Acta Chim. Slov. 2017, 64, 988-1004 DOI: 10.1016/S0014-827X(98)00111-6 3. M. Beach, R. Frechette, WO Patent Appl. 9728167, 1997. 4. (a) P. Lestage, B. Lockhart, M. B. Fleury, M. Largeron, WO Patent Appl., 9962889, 1999. (b) E. Blattes, B. Lockhart, P. Lestage, L. Schwendimann, P. Gressens, M. B. Fleury, M. Largeron, J. Med. Chem. 2005, 48, 1282-1286. D01:10.1021/jm040874m 5. L. D. Wise, D. J. Wustrow, T. Belliotti, WO Patent Appl., 9745419, 1997. 6. K. Waisser, M. Perina, J. Kunes, V. Klimesova, J. Kaustova, Farmaco, 2003, 58, 1137-1149. DOI: 10.1016/j.farmac.2003.07.004 7. S. Konda, S. Raparthi, K. Bhaskar, R. K. Munaganti, V. Gu-guloth, L. Nagarapu, D. M. Akkewar, Bioorg. Med. Chem. Lett. 2015, 25, 1643-1646 and references cited therein. D0I:10.1016/j.bmcl.2015.01.026 8. F. Touzeau, A. Arrault, G. Guillaumet, E. Scalbert, B. P. Feiffer, M. C. Rettori, P. Renard, J. Y. Mérour, J. Med. Chem. 2003, 46, 1962-1979. D0I:10.1021/jm021050c 9. K. Waisser, L. Kubicova, V. Buchta, P. Kubanova, K. Bajerova, L. Jiraskova, O. Bednarik, O. Bures, P. Holy, Folia Microbiol. 2002, 47, 488-492. D0I:10.1007/BF02818786 10. F. A. Macias, D. Marin, A. Oliveros-Bastidas, J. M. G. Molinillo, J. Agric. Food. Chem. 2006, 54, 9357-9365. D0I:10.1021/jf062168v 11. E. N. Koini, P. Papazafiri, A. Vassilopoulos, M. Koufaki, Z. Horváth, I. Koncz, L. Virág, G. J. Papp, A. Varró, T. Calo-geropoulou, J. Med. Chem. 2009, 52, 2328-2340. D0I:10.1021/jm801228h 12. J. Ilas, Z. Jakopin, T. Borstnar, M. Stegnar, D. Kikelj, J. Med. Chem. 2008, 51, 5617-5629. D0I:10.1021/jm8003448 13. S. M. Bromidge, R. Arban, B. Bertani, S. Bison, M. Borriello, P. Cavanni, G. D. Forno, R. Di-Fabio, D. Donati, S. Fontana, M. Gianotti, L. J. Gordon, E. Granci, C. P. Leslie, L. Moccia, A. Pasquarello, I. Sartori, A. Sava, J. M. Watson, A. Worby, L. Zonzini, V. Zucchelli, J. Med. Chem. 2010, 53, 5827-5843. D0I:10.1021/jm100482n 14. (a) D. S. La, J. Belzile, J. V. Bready, A. Coxon, T. De Melfi, N. Doerr, J. Estrada, J. C. Flynn, S. R. Flynn, R. F. Graceffa, S. P. Harriman, J. F. Larrow, A. M. Long, M. W. Martin, M. J. Morrison, V. F. Patel, P. M. Roveto, L. Wang, M. M. Weiss, D. A. Whittington, Y. Teffera, Z. Zhao, A. J. Polverino, J. C. Harmange, J. Med. Chem, 2008, 51, 1695-1705; D0I:10.1021/jm701129j (b) T. Hasui, N. Matsunaga, T. Ora, N. Ohyabu, N. Nishig-aki, Y. Imura, Y. Igata, H. Matsui, T. Motoyaji, T. Tanaka, N. Habuka, S. Sogabe, M. Ono, C. S. Siedem, T.P. Tang, C. Gauthier, L. A. De Meese, S. A. Boyd, S. Fukumoto, J. Med. Chem. 2011, 54, 8616-8631. D0I:10.1021/jm2011645 15. K. Moon, C. H. Ahn, Y. Shin, T. H. Won, K. Ko, S. K. Lee, K. B. Oh, J. Shin, S. Nam, D. C. Oh, Mar. Drugs 2014, 12, 2526-2538. D0I:10.3390/md12052526 16. (a) N. Zidar, D. Kikelj, Tetrahedron, 2008, 64, 5756-5761; D0I:10.1016/j.tet.2008.04.010 (b) C. Ramesh, B. R. Raju, V. Kavala, C.-W. Kuo, C.-F. Yao, Tetrahedron, 2011, 67, 1187-1192; D0I:10.1016/j.tet.2010.11.095 (c) P. Jangili, J. Kashanna, B. Das, Tetrahedron Lett., 2013, 54, 3453-3456; D0I:10.1016/j.tetlet.2013.04.090 (d) C. Huo, J. Dong, Y. Su, J. Tang, F. Chen, Chem. Commun. 2016, 52, 13341-13344; D0I:10.1039/C6CC05885J (e) M. Xia, B. Wu, G.-F. Xiang, Synthetic Commun. 2008, 38, 1268-1278; D0I:10.1080/00397910701873250 (f) E. Feng, H. Huang, Y. Zhou, D. Ye, H. Jiang, H. Liu, J. Org. Chem. 2009, 74, 2846-2849; D0I:10.1021/jo802818s (g) G. Feng, J. Wu, W.-M. Dai, Tetrahedron Lett. 2007, 48, 401-404; D0I:10.1016/j.tetlet.2006.11.084 (h) Y. Yuan, G. Liu, L. Li, Z. Wang, L. Wang, J. Comb. Chem. 2007, 9, 158-170; D0I:10.1021/cc060094u (i) X. Xing, J. Wu, G. Feng, W.-M. Dai, Tetrahedron 2006, 62, 6774-6781; D0I:10.1016/j.tet.2006.05.001 (j) N. G. Kundu, Chaudhuri, G. A. Upadhyay, J. Org. Chem. 2001, 66, 20-29; D0I:10.1021/jo000826j (k) N. Henry, G. Guillaumet, M. D. Pujol, Tetrahedron Lett. 2004, 45, 1465-1468; D0I:10.1016/j.tetlet.2003.12.030 (l) E. E. Stepanova, A. V. Babenysheva, A. N. Maslivets, Russ. J. Org. Chem. 2011, 47, 937-940; D0I:10.1134/S1070428011060182 (m) E. E. Stepanova, Z. G.Aliev, A. N. Maslivets, Russ. J. Org. Chem. 2013, 49, 1762-1767 and references cited therein; D0I:10.1134/S1070428013120105 (n) Z. G. Aliev, O. P. Krasnykh, A. N. Maslivets, L. O. Atov-myan, Russ. Chem. Bull. 2000, 49, 2045-2047; D0I:10.1023/A:1009532227461 (0) E. N. Koini, P. Papazafiri, A.Vassilopoulos, M. Koufaki, Z. Horvath, I. Koncz, L.Virag, G. J. Papp, A. Varro, Calo-geropoulou, T. J. Med. Chem. 2009, 52, 2328-2340. D0I:10.1021/jm801228h 17. For known compounds, see: (a) M. Xia, Faming Zhuanli Shen-qing Gongkai Shuomingshu, 2008, CN 101108860 A; (b) I. V. Mashevskaya, I. A. Tolmacheva, E. V.Voronova, T. F. Odego-va, G. A. Aleksandrova, A. F. Goleneva, S. V. Kol'tsova, A. N. Maslivets, Pharm. Chem. J. (Translation of Khimiko-Farmat-sevticheskii Zhurnal), 2002, 36, 32-34; (c) Y. Iwanami, T. Seki, T. Inagaki, Bull. Chem. Soc. Jpn. 1971, 44, 1316-1321; D0I:10.1246/bcsj.44.1316 (d) E. N. Kozminykh, N. M. Igidov, G. A. Shavkunova, V. O. Kozminykh, Russ. Chem. Bull. (Translation of Izvestiya Akademii Nauk, Seriya Khimicheskaya), 1997, 46, 1285-1290; (e) V. L. Gein, N. A. Rassudikhina, N. V. Shepelina, M. I. Vakhrin, E. B. Babushkina, E. V. Voronina, Pharm. Chem. J. 2008, 42, 529-532; D0I:10.1007/s11094-009-0175-5 (f) M. Xia, B. Wu, G. Xiang, J. Fluor. Chem. 2008, 129, 402408; D0I:10.1016/j.jfluchem.2008.01.019 (g) I. V. Mashevskaya, I. G. Mokrushin; K. S. Bozdyreva, A. N. Maslivets, Russ. J. Org. Chem. 2011, 47, 253-257; D0I:10.1134/S1070428011020151 (h) J. Reynisson, W. Court, C. O. Neill, J. Day, L. Patterson, E. McDonald, P. Workman, M. Katan, S. A. Eccles, Bioorg. Med. Chem. 2009, 17, 3169-3176; D0I:10.1016/j.bmc.2009.02.049 (1) V. A. Maslivets, A. N. Maslivets, Russ. J. Org. Chem. 2012, 48, 1234-1238 and references cited therein; Sharma et al.: Microwave-assisted one-pot efficient synthesis ... Acta Chim. Slov. 2017, 64, 988-1004 1003 D01:10.1134/S1070428012090151 (j) X. Lia, N. Liua, H. Zhanga, S. E. Knudsonb, R. A. Slaydenb, P. J. Tongea, Bioorg. Med. Chem. Lett. 2010, 20, 6306-6309; D0I:10.1016/j.bmcl.2010.08.076 (k) V. V. Khalturina, Yu. V. Shklyaev, Z. G. Aliev, A. N. Masliv-ets, Russ. J. Org. Chem. 2009, 45, 1519-1522; D0I:10.1134/S1070428009100169 (l) R. C. Rastogi, R. H. Khan, K. R. Baruah, C. S. Sarmah, Ind. J. Hetero. Chem. 1992, 1, 247-248; (m) K. Noriaki, R. Naomi, U. Kimihisa, N. Yuji, T. Kotaro, S. Miho, K. Hideaki, Y. Hiro-shi, N. Satoshi, M. Yuzuru, Jpn. Kokai Tokkyo Koho 2000, JP 2000154139 A 20000606. 18. (a) G. Choudhary, R. T. Naganaboina, R. K. Peddinti, RSC Adv., 2014, 4, 17969-17979, and references cited therein; (b) F. A. Macias, D. Marin, A. Oliveros-Bastidas, J. M. G. Molinillo, Nat. Prod. Rep. 2009, 26, 478-489; D0I:10.1039/b700682a (c) J. Ilas, P. A. Stefanic, M. S. Dolenc, D. Kikelj, Tetrahedron 2005, 61, 7325-7348, and references therein. D0I:10.1016/j.tet.2005.05.037 19. For selected examples see: (a) K. C. Nicolaou, K. Sugita, P. S. Baran, Y. L. Zhong, Angew. Chem., Int. Ed. 2001, 40,207-210; D0I:10.1002/1521-3773(20010105)40:1<207::AID-ANIE 207>3.0.C0;2-K (b) K. C. Nicolaou, P. S. Baran, Y. L. Zhong, K. Sugita, J. Am. Chem. Soc. 2002, 124, 2212-2220; D0I:10.1021/ja012124x (c) M. Largeron, A. Neudorffer, M. Vuilhorgne, E. Blattes, M. -B. Fleury, Angew. Chem. Int. Ed. 2002, 41, 824-827. D0I:10.1002/1521-3773(20020301)41:5<824::AID-ANIE 824>3.0.CO;2-Z 20. (a) C. Trebaul, J. Roncali, F. Garnier R. Guglielmetti, Bull. Chem. Soc. Jpn. 1987, 60, 2657; D0I:10.1246/bcsj.60.2657 (b) R. B. Moffett, J. Med. Chem. 1966, 9, 475-478; D0I:10.1021/jm00322a006 (c) A. Chilin, A. Confente, G. Pastorini, A. Guiotto, Eur. J. Org. Chem. 2002, 12, 1937-1940; D0I:10.1002/1099-0690(200206)2002:12<1937::AID-EJ0C 1937>3.0.C0;2-W (d) D. N. Nicolaides, D. R. Gautam, K. E. Litinas, D. J. Hadji-pavlou-Litina C. A. Kontogiorgis, J. Heterocycl. Chem. 2004, 41, 605-611; D0I:10.1002/jhet.5570410421 (e) D. N. Nicolaides, R. W. Awad, E. A. Varella, J. Heterocycl. Chem. 1996, 33, 633-637; D0I:10.1002/jhet.5570330318 (f) I. Yavari, S. Souri, M. Sirouspour, H. Djahaniani, Synthesis. 2006, 3243-3249; (h) R. Ballini, A. Palmieri, M. A. K Talaq, S. Gabrielli, Adv. Synth. Catal. 2009, 351, 2611-2614; (i) D. Albanese, A. Donghi, D. Landini, V. Lupia, M. Penso, Green Chem. 2003, 5, 367-369; (j) D. Albanese, D. Landini, V. Lupi, M. Penso, Adv. Synth. Catal. 2002, 344, 299-302. 21. (a) Q. -Y. Zhang, B. -K. Liu, W. -Q. Chen, Q. Wu, Lin, X. -F. A Green Chem. 2008, 10, 972-977; D0I:10.1039/b806960c ( b) K. M. H. Nguyen, L. Schwendimann, P. Gressens, M. Largeron, Org. Biomol. Chem. 2015, 13, 3749-3756. 22. (a) M. R. El Sayed Aly, H. H. Abd, El Razek Fodah, S. Y. Saleh, Eur. J. Med. Chem. 2014, 76, 517-530; D01:10.1039/C50B00049A (b) Y. P. Qian, Y. J. Shang, Q. F. Teng, J. Chang, G. J. Fan, X. Wei, R. R. Li, H. P. Li, X. J. Yao, F. Dai, B. Zhou, Food Chemistry 2011, 126, 241-248; (c) N. A. Shakil, M. K. Singh, M. Sathiyendiran, J. Kumar, J. C. Padaria, Eur. J. Med. Chem. 2013, 59, 120-131; (d) V. Oliveri, G. I. Grasso, F. Bellia, F. Attanasio, M. Viale, G. Vecchio, Inorg. Chem. 2015, 54, 25912602; (e) L. Savegnago, A. I. Vieira, N. Seus, B. S. Goldani, M. R. Castro, E. J. Lenardäo, D. Alves, Teterahedron Lett., 2013, 54, 40-44; (f) A. Detsi, D. Bouloumbasi, K. C. Prou-sis, M. Koufaki, G. Athanasellis, G. Melagraki, A. Afantitis, O. Igglssi-Markopoulou, C. Kontogiorgis, D. J. Hadjipavlou Litina, J. Med. Chem. 2007, 50, 2450-2458; (g) F. Pérez-Cruz, S. Vazquez-Rodriguez, M. J. Matos, A. Herrera-Morales, F. A. Villamena, A. Das, B. Gopalakrishnan, C. Olea-Azar, L. San-tana, E. Uriarte, J. Med. Chem. 2013, 56, 6136-6145; (h) G. Mazzone, A. Galano, J. R. Alvarez-Idaboy, N. Russo, J. Chem. Inf. Model., D0I:10.1021/acs.jcim.6b00006 23. (a) C. O. Kappe, Angew. Chem. Int. Ed. 2013, 52, 7924-7928; D01:10.1002/anie.201304368 (b) A. Sharma, P. Appukkuttan, E. V. Eycken, Chem. Comm. 2012, 48, 1623-37; D0I:10.1039/C1CC15238F (c) C. E. Bell, A.Y. Shaw, F. De Moliner, C. Hulme, Tetrahedron 2014, 70 (1), 54-59; D0I:10.1016/j.tet.2013.11.035 (d) A. Srinivas, M. Sunitha, K. Raju, B. Ravinder, S. Anusha, T. Rajasri, P. Swapna, D. Sushmitha, D. Swaroopa, G. Nikitha, C. G. Rao, Acta Chim. Slov. 2017, 64, 319-331; D0I:10.17344/acsi.2016.3153 (e) L. Moradi, M. A. Sadegh, Acta Chim. Slov. 2017, 64, 506512; D0I:10.17344/acsi.2017.3417 (f) A. Srinivas, M. Santhosh, M. Sunitha, P. Karthik, K. Srinivas, K.V. Reddy, Acta Chim. Slov. 2016, 63, 827-836. D0I:10.17344/acsi.2015.2124 24. (a) P.-L. Wu, Y.-L. Hsu, C.-W. Jao, J. Nat. Prod. 2006, 69, 14671470; D0I:10.1021/np060395l (b) J. Mason, J. Bergman, T. Janosik, J. Nat. Prod., 2008, 71, 1447-1450; D0I:10.1021/np800334j (c) L. Gross, F. Mohn, N. Moll, G. Meyer, R. Ebel, W. M. Ab-del-Mageed, M. Jaspars, Nat. Chem. 2010, 2, 821-825; D0I:10.1038/nchem.765 (d) L. Gross, Nat. Chem. 2011, 3, 273-278. D01:10.1038/nchem.1008 25. (a) N. G. Baydar, G. Ozkan, S. Yasar, Food Control. 2007, 18, 113-1136; D0I:10.1016/j.foodcont.2005.09.001 (b) M. S. Blois, Nature 1958, 181, 1199-1200; D0I:10.1038/1811199a0 (c) O. P. Sharma, T. K. Bhat, Food Chem. 2009, 113, 12021205; D0I:10.1016/j.foodchem.2008.08.008 (d) P. C. Eklund, O. K. Langvik, J. P. Warna, T. O. Salmi, S. M. Willfor, R. E. Sjoholm, Org. Biomol. Chem. 2005, 3, 33363347; D0I:10.1039/b506739a (e) A. Faria, C. Calhau, V. de Freitas, N. Mateus, J. Agric. Food. Chem. 2006, 54, 2392-2397; D0I:10.1021/jf0526487 (f) I. F. Benzie, J. J. Strain, Anal. Biochem. 1996, 239, 70-76. D01:10.1006/abio.1996.0292 26. (a) W. Brand-Williams, M. E. Cuvelier, C. Berset, Food Sci. Sharma et al.: Microwave-assisted one-pot efficient synthesis ... 1004 Acta Chim. Slov. 2017, 64, 988-1004 Tech. 1995, 28, 25-30; 6882. DOI:10.1021/jf020540a (b) I. Parejo, F. Viladomat, J. Bastida, A. Rosas-Romero, N. 27. M. Danihelova, M. Veverka, E. Sturdik, S. Jantova, Interdiscip. Flerlage, J. Burillo, C. Codina, J. Agri. Food. Chem. 2002, 50, Toxicol. 2013, 6, 209-216. DOI:10.2478/intox-2013-0031ww Povzetek V prispevku je opisana okolju prijazna in s pomočjo mikrovalov spodbujena sinteza funkcionaliziranih (Z)-3-(2-okso-2-feniletiliden)-3, 4-dihidro-2H-benzo[fo][1,4]oksazin-2-onov (11a-n) z odličnimi izkoristki (do 97%) in (Z)-3-(2-ok-so-2-feniletiliden)-3,4-dihidrokinoksalin-2(1H)-onov (14a) (do 96% izkoristek). Uporabna vrednost razvite metodologije je prikazana na sintezi spojin 11a, 14c in 14e na gramski skali ter na sintezi protitumorskega alkaloida cefalandola A (16, 89% izkoristek). Vse sintetizirane spojine, 11a-n, 14a-h in 16, so bile preizkušene na in vitro antioksidativno aktivnost, DPPH radikalsko lovljenje in FRAP test. Pri DPPH testiranju so se kot najbolj aktivne pokazale spojine 11a, 14c in 14e z IC50 vrednostmi 10.20 ± 0.08 |ig/mL, 9.89 ± 0.15 |ig/mL in 8.97 ± 0.13 |ig/mL glede na standardno referenco (askorbinska kislina, IC50 = 4.57 |g/mL), medtem, ko je pri FRAP antioksidativnem testu sedem spojin (11c, 11e, 11i, 11k, 11l, 14d in 14h) izkazalo višjo antioksidativno aktivnost kot referenčni standard BHT (C05FRAP = 546.2 |M). Poleg tega so študije citotoksičnosti spojin 11a, 14c, 14e in 14h hh pri MTT testiranju pokazale, da so te spojine netoksične do celičnih linij naravnih 3T3 fibroblastov. Sharma et al.: Microwave-assisted one-pot efficient synthesis ... DOI: 10.17344/acsi.2017.3731 Acta Chim. Slov. 2017, 64, 1005-1019 ¿^creative tS1 commons Scientific paper Magnetically Recyclable Fe3O4/GO-NH2/H3PMo12O40 Nanocomposite: Synthesis, Characterization, and Application in Selective Adsorption of Cationic Dyes from Water Saeed Farhadi,*1 Mohammad Hakimi2 and Mansoureh Maleki2 1 Chemistry Department, Lorestan University, Khoramabad 68151-44316, Iran. 2 Chemistry Department, Payame Noor University, Tehran 19395-4697, Iran. * Corresponding author: E-mail: sfarhadi1348@yahoo.com Tel.: +986633120611 fax: +986633120618 Received: 03-08-2017 Abstract In this study, the PMo12O403- polyanion was immobilized chemically on amino functionalized magnetic graphene oxide nanosheets. The as-prepared ternary magnetic nanocomposite (Fe3O4/GO-NH2/H3PMo12O40) was characterized by powder X-ray powder diffraction (XRD), fourier transformation infrared spectroscopy (FTIR), Raman spectroscopy, energy dispersive spectroscopy (EDX), field emission scanning electron microscopy (FESEM), BET surface area measurements, magnetic measurements (VSM) and atomic force microscopy (AFM). The results demonstrated the successful loading of H3PMo12O40 (~36.5 wt.%) on the surface of magnetic graphene oxide. The nanocomposite showed a higher specific surface area (77.07 m2/g) than pure H3PMo12O40 (<10 m2/g). The adsorption efficiency of this nanocomposite for removing methylene blue (MB), rhodamine B (RhB) and methyl orange (MO) from aqueous solutions was evaluated. The nanocomposite showed rapid and selective adsorption for cationic dyes from mixed dye solutions. The adsorption rate and capacity of Fe3O4/GO-NH2/H3PMo12O40 were enhanced as compared with GO, GO-NH2, Fe3O4/GO-NH2, and H3PMo12O40 samples due to enhanced electrostatic attraction and hydrogen-bonding interactions. The nanocomposite is magnetically separated and reused without any change in structure. Thus, it could be a promising green adsorbent for removing organic pollutants in water. Keywords: Graphene oxide nanosheets; Magnetic nanocomposite; Polyoxometalates; Organic dyes; Adsorption; Fe3O4 nanoparticles. 1. Introduction Industrial activities release an increasing amount of contaminants, such as metal ions, organic dyes, and cleaning agents, which has raised public concern.1,2 So, waste-water treatment has attracted much attention in the past decades because of grievous effluent discharge of some organic dyes from plating, textile, and printing paper, plastic, cosmetic, pharmaceutical, and food industries that are resistant to biological degradation, making them quite difficult to remove from the wastewater.3,4 Organic dyes are not only highly visible and, even in a small amount, decrease gas solubility in water, but also toxic, carcinogenic, and mutagenic for human beings.5-9 Owing to their complex aromatic molecular structures, dyes are generally sta- ble to light, heat and oxidizing agents.10 Therefore, effective removal of dyes from dye-wastewater is essential. Among the various technologies such as photo catalytic degradation,11 electrochemical degradation,12 and adsorption,13 adsorption is considered one of the most efficient and economical methods for water purification.14 Many polymeric and inorganic adsorbents such as carbonaceous nanoma-terials,15 porous metal oxides,16 clays,17 chitosan,18 zeolites,19 and so on20,21 were developed for removing pollutants from aqueous solutions. However, such adsorbents are associated with certain problems that limit their practical applications, such as low adsorption capacity, slow adsorption rate, and difficult separation of the adsor-bents.22 Furthermore, some of them are only effective for wastewater including low concentrations of dyes and they Farhadi et al.: Magnetically Recyclable Fe3O4/GO-NH2/H3PMo12O4i 1006 Acta Chim. Slov. 2017, 64, 1005-1019 are generally poor at selectively removing the targeted organic dye wastes. Hence, in this regard, it is extremely imperative to find a new desirable adsorption material, which not only is capable of reducing the organic dyes in dye-wastewater with high efficiency and fast adsorption rate but also can achieve selective separation and recovery of raw materials. Polyoxometalates (POMs), as an outstanding class of anionic metal oxide clusters, have attracted great attention due to their earth-abundant source, rich topology and versatility, controllable shape and size, oxo-enriched surfaces, high electronegativity etc.,23 which have various applications in many fields, such as catalysis,24 optics,25 magnetism,26 biological medicine,27 and dye adsorption.28 The strong attraction of POMs to cationic dyes suggests that they are potential and suitable adsorbents for selectively capturing cationic dyes. However, there are still obvious disadvantages for POMs as adsorbents: (i) their relatively small surface area seriously obstructs the accessibility to the active sites and (ii) their excellent solubility in aqueous solution determines that they cannot be reused and recycled in the process of wastewater treatment. Therefore, plenty of remarkable work has been done to encapsulate POMs into porous solid matrices, such as activated car-bon29 and silica30 for creating composite materials. Unfortunately, these methods sometimes lead to low POM loading; it is thus of vital significance to search for an applicable solid matrix to immobilize POMs, which might greatly improve their adsorption ability for target dyes. Among various materials, graphene oxide (GO) has been proven as an effective sorbent for the removal of inorganic and organic pollutants owing to its large theoretical specific surface area (~2630 m2/g) and the presence of several active sites on its surface.31-39 In addition, in comparison with other carbonaceous nanomaterials, GO may be more environmental friendly and have better biocom-patibility.40 However, it is difficult to separate it from aqueous solution because of its small particle size, causing serious health and environmental problems once it is discharged into the environment.41 The centrifugation method needs a very high rate and the traditional filtration method may cause blockages of filters. Compared with traditional centrifugation and filtration methods, the magnetic separation method is considered as a rapid and effective technique for separating nanomaterials from aqueous solution.42,43 Hence, magnetite/graphene composites with large specific surface area (enhancing the removal of water pollutants) and magnetic separation (facilitated by the recycling of the composites) have begun to be used in the field of environmental treatment.44-48 On the basis of the above discussion, in this work, amino functionalized magnetic graphene oxide (Fe3O4/ GO-NH2) was synthesized by a facile method and used as a novel support for immobilizing Keggin-type PMo12O 403-anions. This magnetically recoverable ternary nanocom-posite material (Fe3O4/GO-NH2/H3PMo12O40) was pre- pared by a simple acid-base electrostatic interaction between H3PMo12O40 and amino groups of Fe3O4/GO-NH2. For one thing, PMo12O 403- anion with highly electronegative and hydrophilic properties and structural stability could be utilized as a potential adsorbent for removal of the cationic dyes in dye-wastewater. For another, magnetic GO possesses outstanding porosity and extremely large surface area, and it is insoluble in water, which is an appropriate solid matrix to anchor Keggin-type PMo12O 403-anions. The combination of polyoxoanions and Fe3O4/ GO-NH2 could improve the surface area and avoid the dissolution of POM. The hybrid nanomaterial exhibited superior adsorption rate and selective adsorption ability for the cationic dyes. Remarkably, this material exhibited a large-scale adsorption capacity of 426.7 mg/g for MB. Hence, it is a promising and environmental friendly adsorbent for removing and separating organic pollutants in dye-waste-water. 2. Experimental 2. 1. Materials and Characterization Techniques Graphite powder (C, 99.95%), 3-aminopropyltriet-hoxysilane (APTES, 99%), phosphomolybdic acid (H3P-Mo12O40, 98%), toluene, sulfuric acid (H2SO4, 98%), and potassium permanganate (KMnO4, 98%) were purchased from Merck Chemical Co. All other chemicals were commercially purchased and used without further purification. The infrared spectra were recorded at room temperature using a Shimadzu FT-IR 160 spectrophotometer in the 4000-400 cm-1 region with KBr pellets. Powder XRD patterns were recorded on a Rigaku D-max C III X-ray diffrac-tometer using Ni-filtered Cu Ka radiation (X = 1.54184 A). The morphology of samples was studied using a MIRA3 TESCAN scanning electron microscope equipped with an energy dispersive X-ray analyzer (EDX) for the elemental analysis. AFM images were recorded by multi-mode atomic force microscopy (ARA-AFM, model Full Plus, ARA Research Co., Iran). Magnetic measurements were carried out at room temperature using a vibrating sample magnetometer (VSM, Magnetic Daneshpajoh Kashan Co., Iran) with a maximum magnetic field of 10 kOe. Optical adsorption spectra were obtained using a Cary 100 Varian UV-Vis spectrophotometer in a wavelength range of 200800 nm. The Brunauer-Emmett-Teller (BET) surface area was measured by N2 adsorption measurements at 77 K using a Nova 2000 instrument. The concentration of Mo in the composite was determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES, model OEC-730). A controllable Serial-Ultrasonics apparatus (James 6MD, England) operating at an ultrasonic frequency of 100 kHz with a nominal output power of 50 W was used to disperse samples. Farhadi et al.: Magnetically Recyclable Fe3O4/GO-NH2/H3PMo12O40 ... Acta Chim. Slov. 2017, 64, 1005-1019 1007 2. 2. Preparation of Graphene Oxide Graphene oxide (GO) was prepared by the modified Hummers method through the oxidation of graphite powder.49,50 Graphite powder (2.0 g) and NaNO3 (1.0 g) were mixed with 40 mL of concentrated H2SO4 in a 500 mL flask and stirred for 1 h in an ice bath. Then KMnO4 (6.0 g) was added into the vigorously stirred suspension slowly below 15 °C. The ice bath was then removed, and the mixture was stirred at room temperature until it slowly became brownish slurry. It was diluted with 100 mL of water. The reaction temperature was rapidly increased to 98 °C with effervescence, and the color changed to brown. After that, 200 mL of water and 20 mL of H2O2 (30 wt.%) were added. For purification, the mixture was centrifuged and washed with 10% HCl and then deionized water several times to remove the residual metal ions and acid. After centrifuging and drying at room temperature, GO was obtained as a powder. 2. 3. Preparation of Magnetic Graphene Oxide (Fe3O4/GO) 0.25 g of GO was dispersed in 90 mL of water by son-ication for 1 h. Then, 0.84 g of (NH4)2Fe(SO4)2 and 2.08 g of (NH4)Fe(SO4)2 were added to the GO dispersion and its pH was adjusted at 12 by adding 1 mol/L NaOH. The mixture was stirred at 50 °C for 2 h, filtered and washed with water and ethanol three times. The resulting solid was Fe3O4 /GO. 2. 4. Preparation of Aminopropyl Functionalized Magnetic Graphene Oxide (Fe3O4/GO-NH2) To a round bottom flask, 0.40 g of the as-synthesized Fe3O4/GO dispersed in 50 mL of water, 5 mL of APTES, and 150 mL of ethanol were added. The mixture was stirred for 30 min at room temperature and refluxed at 80 °C for 24 h. After the reaction, the solid was separated by a magnet and washed with ethanol to remove the unreacted APTES. The final product was dried at 80 °C in vacuum for 12 h to obtain Fe3O4/GO-NH2. 2. 5. Preparation of the Fe3O4/GO-NH2/ H3PMo12O40 Hybrid Nanomaterial 1 g of the as-prepared Fe3O4/GO-NH2 was dispersed in 80 mL of water and sonicated for 1 h. Then, 1 g of H3P-Mo12O40 in 200 mL of ethanol was added and sonicated for another 1 h. The resulting mixture was stirred at room temperature for 24 h, filtered, and washed with deionized water and ethanol three times to remove the unreacted H3PMo12O40. The final product was dried at 60 °C in open air to obtain magnetic Fe3O4/GO-NH2/H3PMo12O40 hybrid nanomaterial. Elemental analysis (ICP-AES) showed that the Mo content in Fe3O4/GO-NH2/H3PMo12O40 was 32.55%. According to the elemental analysis results and molecular weight of H3PMo12O40, the loading amount (wt.%) of H3PMo12O40 in Fe3O4/GO-NH2/H3PMo12O40 was estimated to be 36.5%. 2. 6. Dye Adsorption Tests The aqueous stock solutions of dyes (500 mg/L) were prepared by dissolving solid dyes (MB, RhB, and MO) in deionized water. Working solutions of dyes, when required, were prepared by successive dilution of the stock solution with deionized water. The adsorption experiments of dyes were performed in a 100 mL glass beaker and the adsorption reaction temperature was maintained at 25 °C. In a typical experiment, 25 mg of the as-prepared Fe3O4/GO-NH2/H3PMo12O40 hybrid nanomaterial was added into 50 mL of dye aqueous solution (C0 = 25 mg/L) and stirred in the dark and at predetermined time intervals, a small portion (3 mL) of the dye solution was pipetted out and the solid adsorbent was separated by an external magnet. The concentration (Ct) of the dye in the clear solution was determined by measuring the absorbance of the solution at fixed wavelengths, 664 nm for MB, 554 nm for RhB, and 463 nm for MO, using a UV-Vis spectropho-tometer. A similar experiment was also performed with 50 mL of different concentrations of MB solutions (5, 15, 25, 35, and 50 mg/L). Also, the hybrid nanomaterial was transferred into the mixtures of MB/MO (v:v 1/1, 50 mL, 25 mg/L), MO/RhB, and MB/MO/RhB. UV-Vis spectros-copy was performed to determine the selective adsorption ability of hybrid nanomaterial at given time intervals. Furthermore, the adsorption capability of the Fe3O4, Fe3O4/ GO, Fe3O4/GO-NH2, and H3PMo12O40 samples toward MB dye solution was evaluated under similar conditions as described above. 25 mg of the as-prepared adsorbent was added into 50 mL of 25 mg/L MB solution and stirred in the dark. Dye removal ability (%) was calculated by measuring the dye absorbance at 664 nm before and after adsorption process at room temperature. Finally, Fe3O4/GO-NH2/H3PMo12O40 was investigated for its recycling property for the removal of MB after Fe3O4/GO-NH2/H3PMo12O40 was separated, washed with ethanol, and dried in a vacuum oven. In all experiments, the adsorption efficiency (r|%) and adsorption capacity (qt; mg/g) were calculated according to the following equations: n% = [(C0 - Ct)/C0] x 100 = [(A0 - At)/A0] x 100 (1) qt = [(C0 - Ct) x V]/m () Where C0 (mg/L) and A0 are the initial dye concentration and absorbance before removal at initial time t = 0, Ct (mg/L) and At are the concentration and absorbance of dye remaining in the solution after treatment with adsorbent at time t, qt is the amount of adsorbed MB molecules on the adsorbent (in mg/g). V (in L) is the initial volume of the MB solution and m (in g) is the mass of the adsorbent. Farhadi et al.: Magnetically Recyclable Fe3O4/GO-NH2/H3PMo12O4i 1008 Acta Chim. Slov. 2017, 64, 1005-1019 3. Results and Discussion 3. 1. Characterization of the Fe3O4/GO-NH2/ H3PMo12O40 Hybrid Nanomaterial The GO is an inexpensive, stable, environmentally benign, easily available solid material, and contains rich oxidation functional groups, such as hydroxyl, epoxide, carboxyl, and carbonyl groups. Due to these functional groups, GO can well disperse in polar solvents and forms a homogenous colloidal suspension, which facilitates the contact between reactant and catalytic active site.51 On the other hand, these rich functional groups can be easily reacted with organic molecules to generate stable covalent functional GO.19-21 These suggest that attaching organic bases on GO might afford an efficient, reusable and environmentally benign base support for heteropolyacids such as PMo12. In this work, Keggin-type H3PMo12O40 was immobilized onto 3-aminopropyl functionalized GO nanos-heets decorated with magnetic Fe3O4 nanoparticles. The preparation of the Fe3O4/GO-NH2/H3PMo12O40 hybrid is illustrated in Figure 1. The surface of GO-NH2 is positively charged by the protonation of -NH2, which helps in anchoring the PMo12O403- anion due to electrostatic attraction. To confirm the successful construction of the Fe3O4/ GO-NH2/H3PMo12O40 hybrid, elemental analysis was employed. I2t re3vealed12 th40at Fe3O4/GO-NH2/H3PMo12O40 contains 36.5% Mo, indicating that H3PMo12O40 molecules were anchored on GO nanosheets. Due to the electrostatic attraction of the absorbed PMo12O403 and -NH3+ group, the hydrogen bonds between -NH3+ and H3PMo12O40 molecules are reinforced.52-54 These are the possible reasons for the significant enhancement of Fe3O4/GO-NH2/H3P-Mo12O40 adsorptivity. Moreover, the structure and composition of the hybrid nanomaterial was characterized by XRD, FT-IR, Raman spectra, EDX, SEM, AFM, VSM, and BET surface area analyses. Figure 2 displays the XRD patterns for Fe3O4, Fe3O4/ GO-NH2, and Fe3O4/GO-NH2/H3PMo12O40 samples. All the diffraction patterns in Figure 2(a)-(c) are similar and can be indexed to the Fe3O4 phase (JCPDS No. 41-1488). No characteristic diffraction peaks of the H3PMo12O40 appeared which presumably was due to the low content incorporation of H3PMo12O40. Also this result implies that the Keggin unit homogeneously disperses into the GO a to tn c ID c) Graphene nanosheet Graphene nanosheet 5 10 30 40 50 60 2theta(deg,) Figure 2. XRD patterns of (a) pure Fe3O4, (b) Fe3O4/GO-NH2 and (c) Fe,O4/GO-NH,/H,PMo O . Figure 1. The preparation procedure to attach H3PMo12O40 on the surface of aminopropyl functionalized magnetic graphene oxide. Farhadi et al.: Magnetically Recyclable Fe3O4/GO-NH2/H3PMo12O40 ... Acta Chim. Slov. 2017, 64, 1005-1019 1009 nanosheets, which will help to enhance the adsorption activity of the hybrid nanomaterial. Moreover, no characteristic diffraction peaks for GO are observed in the pattern indicating that the GO nanosheets do not stack during the synthesis. The reason can be attributed to the Fe3O4 and H3PMo12O40 nanoparticles anchored on the surfaces of GO to prevent the exfoliated GO nanosheets from restacking.55 However, a broad characteristic peak for graphene nanosheets at about 20 = 23 appeared, suggesting that the GO was reduced to graphene during the functionalization process. The FT-IR spectra of synthesized materials are shown in Figure 3. The FT-IR spectrum of the GO in Figure 3(a) shows the C=O and graphitic C=C stretching bands at about 1735 and 1626 cm-1, respectively.56 Also, the strong band at 1030 cm-1 is related to the stretching vibrations of the C-OH bonds. In the spectrum of Fe3O4/ GO-Si(CH2)3NH2 sample (abbreviated as Fe3O4/GO-NH,,) in Figure 3(b), the strong band at about 588 cm-1 is assigned to the Fe-O stretching vibration of the spinel-type Fe3O4 structure.57 The presence of Si-O bond was confirmed by a strong band at about 1116 cm-1 assigned to the Si-O-C stretching vibration.58,59 Also, two broad bands at 3430 and 1630 cm-1 can be ascribed to the stretching and bending modes of -NH2 groups, respectively. The presence of the anchored propyl chain of APTES was confirmed by C-H stretching vibrations at 2926 and 2850 cm-1.59 The FT-IR spectrum of the Fe3O4/GO-NH2 exhibits peaks at 1223 and 1028 cm-1 corresponding to C-N and C-OH, respectively.60 These show the successful grafting of APTES onto the magnetic GO sheet through covalent bonds. It is well known that Keggin-type PMo12O403- poly-oxometalate contains a cluster of Mo(VI) ions linked by oxygen atoms with a tetrahedral phosphate group. Oxygen atoms form four physically distinct bonds (P-Oa, Mo-Ot, Mo-Ob-Mo, and Mo-Oc-Mo bonds), which have distinct infrared signatures as shown in Figure 3(c): 1066 cm-1 for asymmetric stretch vibration of P-Oa (Oa corresponds to oxygen atom of tetrahedral phosphate group), 966 cm-1 for asymmetric stretch vibration of Mo = Ot (Ot corresponds to the terminal oxygen atoms), 870 cm-1 for bending vibration of Mo-Ob-Mo (Ob corresponds to oxygen atom bridging the two tungsten atoms), and 786 cm-1 for bending vibration of Mo-Oc-Mo (Oc represents oxygen atom at the corners of the Keggin structure).61 The FT-IR spectrum of the hybrid nanomaterial sample is shown in Figure 3(d), the absorption peaks of PMo12O 403- cluster at 1053, 945, 875, and 798 cm-1 corresponding to the P-Oa, Mo=Ot, Mo-O -Mo and Mo-O -Mo band vibrations, and the vi- bc brational bands of Fe3O4/GO-NH2 located around 1605, 1383, 1042, 746, and 543 cm-1 were all observed in the IR spectrum of the hybrid nanomaterial which demonstrates the coexistence of PMo12O403-, Fe3O4, and GO-NH2 in the hybrid nanomaterial. The red/blue shift of peaks of the Fe3O4/GO-NH2/H3PMo12O40 compared with the parent H3PMo12O40 can be attributed to strong attraction between negatively charged PMo12O 403- and positively charged Fe3O4/GO-NH3+ surface.62,63 Raman spectroscopy is a powerful tool to characterize the significant structural changes in GO during the Fe3O4/GO-NH2/H3PMo12O40 hybrid synthesis. r I l 4-000 3600 3000 2S00 2000 1S00 Wavelength (cm-1} 1000 GOO Figure 3. IR spectra of (a) pure GO, (b) Fe3O4/GO-NH2 and (c) H,PMo O and (d) Fe,O./GO-NH,/H,PMoO. Figure 4 presents the Raman spectra of GO, Fe3O4/ GO-NH2 and Fe3O4/GO-NH2/H3PMo12O40. All the s3am4-ples display the characteristic D (sp3 carbon atoms of disorders and defects) and G bands (sp2 carbon atoms in graphitic sheets).64,65 As compared with the D and G bands of pure GO (D, 1318 cm-1; G, 1590 cm-1), the two bands of Fe3O4/GO-NH2 shift to D, 1315 cm-1 and G, 1588 cm-1, suggesting the successful silylanization of GO with APTES. After anchoring the H3PMo12O40 on the Fe3O4/GO-NH2, further shifts in D and G bands are found (D, 1311 Farhadi et al.: Magnetically Recyclable Fe3O4/GO-NH2/H3PMo12O4i 1010 Acta Chim. Slov. 2017, 64, 1005-1019 cm-1 and G, 1592 cm-1). These shifts in Raman peaks can be attributed to strong interaction between PMo12O403- anion and positively charged Fe3O4/GO-NH3+ in Fe3O4/GO-NH2/H3PMo12O40 hybrid,66 which is consistent with the FT-IR analysis. In addition, it is shown that the D/G intensity ratios of Fe3O4/GO-NH2 and Fe3O4/GO-NH2/H3P-Mo12O40 are higher than that of GO, suggesting a higher level of disorder of the graphene layers during the func-tionalization process.67 Also, this is proposed to be caused by the reduction of GO during the syntheses of GO-NH2 and Fe3O4/GO-NH2/H3PMo12O40 hybrid as confirmed by the XRD data.68 The peaks of Keggin structure did not appear, which indicated that H3PMo12O40 species on the Fe3O4/GO-NH2 sample were in a high-dispersed state. This result was identical to the XRD results. SEM images indicating the microstructural features of GO, Fe3O4, Fe3O4/GO-NH2, and Fe3O4/GO-NH2/H3P-Mo12O40 hybrid nanomaterial are shown in Figure 5. The SEM micrograph of pure GO in Figure 5(a) shows the highly porous and layered structure of GO having large stacks, possibly consisting of hundreds of GO nanosheets. It should also be noted that the surfaces of the GO sheets are quite flat and smooth. Figure 5(b) shows the SEM micrograph of sphere-like Fe3O4 nanoparticles. It is worthy to observe that the lateral size of graphitic nanosheets was not disrupted with the oxidation, The SEM image of Fe3O4/GO-NH2 in Figure 5(c) clearly shows GO nanos- 800 1000 1200 1400 1600 Raman shift (cm~''| 1800 Figure 4. Raman spectra of (a) GO, (b) Fe3O4/GO-NH2 and (c) Figure 5. SEM images of (a) GO, (b) Fe3O4, (c) Fe3O4-GO, (d) Fe3O4/GO-NH2, and (e)-(f) Fe3O4/GO-NH2/H3PMo12O40 hybrid nanomaterial. Farhadi et al.: Magnetically Recyclable Fe3O4/GO-NH2/H3PMo12O40 ... Fe3O4/GO-NH2/H3PMo12O40. Acta Chim. Slov. 2017, 64, 1005-1019 1011 heets were successfully decorated with the Fe3O4 nanopar-ticles consisting of small and elongated grains. It was found that the average size of Fe3O4 nanoparticles was in the range of 20-30 nm. Figure 5(d)-(f) shows the morphology of the Fe3O4/GO-NH2/H3PMo12O40 hybrid nano-material. As can be seen, agglomerated Fe3O4 and PMo12 nanoparticles completely covered the surfaces of large graphene sheets. It should also be noted that the micropores between the graphene sheets were evenly filled up with the Fe3O4 nanoparticles as clearly seen in the images of stack edges given in Figure 5(d)-(f). All the micrographs of the hybrid nanomaterial clearly indicated that the surface properties of the modified GO product were strongly affected. Opposite to pure GO, the surfaces of GO nanosheets in the hybrid nanomaterial are rough, and the edges are highly crumpled. It can be concluded that microstructural properties of Fe3O4/GO-NH2/H3PMo12O40 hybrid nanomaterial encourage preparing the highly porous, magnetically active and structurally layered nano-materials. AFM is a beneficial tool for studying various morphological features and parameters, since it has the advantage of probing in deep insights of surface topography qualitatively due to its both lateral and vertical nanometer scale spatial resolution. The AFM images in Figure 6 display the surface morphology of the Fe3O4/GO-NH2/H3P-Mo12O40. As observed in Figure 6(a), the AFM image reveals the appearance of sphere-like nanoparticles and their respective particle size and morphology clearly were close to those determined by the SEM images. As can be seen from Figure 6(b), the surface of the composite showed a porous and uniform packed structure with size (height) of particles on the grapheme sheet to be approximately 20 nm. Thus, the Fe3O4/GO-NH2/H3PMo12O40 could provide a rough and coarse surface with porosity for adsorption uses. The results are in good agreement with BET results and SEM images. Further investigation was carried out by energy dispersive X-ray spectroscopy (EDX) to characterize the composition of the as-prepared Fe3O4/GO-NH2/H3PMo12O40 hybrid nanomaterial. Figure 7(a)-(i) show the EDX spectrum and a representative SEM image of the hybrid nano-material with corresponding EDX elemental mappings. The presence of C, N, Si, O, Fe, P, and Mo elements in the composite can be proved by the EDX elemental spectrum (Figure 7(a)). As presented in Figure 7(b)-(f), the corresponding elemental mapping distribution shows the existence of C, N, Si, O, Fe, P, and Mo. From the maps, it can be seen that the elements are uniformly distributed over the hybrid nanomaterial, confirming the homogeneity of the sample. The P and Mo elements were from H3PMo12O40 and the results further indicate that the H3PMo12O40 particles were successfully supported on the surface of the Fe3O4/GO-NH2. The magnetic properties of the pure Fe3O4 and Fe3O4/ GO-NH2/H3PMo12O40 samples were investigated by VSM at room temperature, and the magnetic hysteresis loops are depicted in Figure 8. It is clear that both Fe3O4 and the Fe3O4/GO-NH2/H3PMo12O40 composite are soft magnetic materials due to their coercivity (Hc) of zero.69 Moreover, they are also superparamagnetic materials, as their magnetic hysteresis loops passed through the origin of the coordinates. The saturation magnetization values of Fe3O4 and Fe3O4/GO-NH2/H3PMo12O40 are 27.50 and 9.16 emu/g, respectively. The saturation magnetization of the magnetic composite decreases by approximately 70% compared with that of pure Fe3O4, which can be attributed to the less magnetic source component (Fe3O4) per gram in the composite sample. However, the saturation magnetization of the composite could satisfy the requirements of easy separation in the suspension solution using an extra magnet after reaction as shown in the inset of Figure 8. Thus, the Fe3O4/GO-NH2/H3PMo12O40 composite can be easily separated using a magnetic separation process after being used for the removal of dye pollutants from aqueous solutions. Figure 6. (a) AFM image and (b) height profile of Fe3O4/GO-NH2/H3PMo12O40 in a three dimensional image. Farhadi et al.: Magnetically Recyclable Fe3O4/GO-NH2/H3PMo12O4i 1012 Acta Chim. Slov. 2017, 64, 1005-1019 Figure 7. (a) EDX spectrum, and (b)-(i) a representative SEM image of the Fe3O4/GO-NH2/H3PMo12O40 hybrid nanomaterial with corresponding EDX elemental mappings. Figure 8. Magnetic hysteresis loop of (a) Fe3O4 and (b) Fe3O4/GrO-NH2/H3PMo12O40 at room temperature. The inset shows the behaviour of the nanocomposite under an external magnetic field. N2 adsorption/desorption measurements were performed to investigate specific surface area and the pore size distribution of the Fe3O4/GO-NH2/H3PMo12O40. As can be seen in Figure 9(a), the nitrogen adsorption isotherm is a typical type IV curve with a fine H1-type hysteresis loop in the range of ca. 0.8-1.0 p/p0, indicating the existence of mesoporous structure.70,71 The BET surface area is measured to be 76.36 m2/g that is much higher than the value of pure POM (<10 m2/g). In addition, the total pore volume is 0.01 cm3/g and according to the corresponding Barrett-Joyner-Halenda (BJH) pore size distribution curve in Figure 9(b), the pore size distribution of the Fe3O4/GO-NH2/H3PMo12O40 shows a peak centered at around 2.28 nm. Such porosity of Fe3O4/GO-NH2/H3P-Mo12O40 composite can improve the adsorption performance. It can be concluded that introduction of magnetic Fe3O4/GO-NH2 has a good effect on the structure of H3P- Farhadi et al.: Magnetically Recyclable Fe3O4/GO-NH2/H3PMo12O40 ... Acta Chim. Slov. 2017, 64, 1005-1019 1013 160 S a) 140 f 7 f rf" 120 6 S j ^loo- 5 's 1 so- a. S 4 Ifl •O m ■o 2 60 3 a o 40 2 > 20- 1 0 1- 0 0.5 Relative pressure (P/Pq) 50 100 pore size (A0} Figure 9. (a) N2 adsorption-desorption isotherm and (b) pore size distribution of Fe3O4/GO-NH2/H3PMo12O40. Mo12O40 and increases the surface area and porosity of POM which are all useful factors for improving the adsorption performance. 3. 2. Dye Adsorption Studies To evaluate the adsorption capability of Fe3O4/GO-NH2/H3PMo12O40 hybrid nanomaterial for removing or- a) b) 0 c) • SCO 600 700 Wavelength(nin) 800 300 400 500 600 700 800 Wavelength (nm) d) 800 0 5 10 15 20 25 30 35 40 Time (min) Figure 10. UV-vis spectral changes of dyes aqueous solutions over Fe3O4/GrO-NH2/H3PMo12O40 at different time intervals: (a) MB dye, (b) RhB dye, (c) MO dye and (d) Comparison of adsorption efficiency (%) of dyes as function of time. Conditions: [dye]= 25 mg/L, 50mL; [Adsorbent]= 25 mg/50 mL at 25 °C. Farhadi et al.: Magnetically Recyclable Fe3O4/GO-NH2/H3PMo12O4i 1014 Acta Chim. Slov. 2017, 64, 1005-1019 ganic dyes from contaminated water, three organic pollutants (MB, RhB, and MO) with different sizes and charges were selected for experiments. The adsorption was monitored using the characteristic absorption peak, which is 664, 553 and 463 nm for MB, RhB, and MO, respectively. The decrease in the intensity of these characteristic peaks with adsorption time indicates the decrease in the dye amount in the solution. The time dependent UV-Vis absorption spectra of dyes in the presence of the Fe3O4/GO-NH2/H3PMo12O40 are shown in Figure 10(a)-(c). The digital images and UV-Vis spectroscopic results show that the characteristic absorption peaks of cationic MB and RhB dyes at 664 and 553 nm almost completely disappeared within 3 and 30 min, respectively. As it can be seen in Figure 10(c), the characteristic absorbance band of MO at 463 nm decreased slightly even after 40 min with negligible fading of the orange color. The results show that the hybrid nanomaterial is a poor absorbent for anionic MO dye from aqueous solution. As compared in Figure 10(d), it is clear that the Fe3O4/GO-NH2/H3PMo12O40 has different adsorption abilities toward different organic dyes. The adsorption efficiency for MB is 100% in 3 min and for RhB is 96% in 30 min. The removal of MO dye is less than 25% after 40 min. Particularly, the adsorption rate of 50 mL of 25 mg/L MB and RhB solutions quickly reached 100% and 85%, respectively, in the first three minutes. The different effects on removal of dyes can be related to the structure of the dye molecules and the adsorbent material. Firstly, POMs are a kind of hydrophilic metal-oxo cluster compounds. The hydrophilic/hydrophobic property of the hybrid framework is modulated by loading the H3PMo12O40 molecules, which allows the ingress and egress of the dye molecules. Secondly, POMs with a large number of negative charges in the hybrid may have a stronger force with the positive charges of dyes. So, the hybrid has demonstrated a good adsorption property toward the cationic dye molecules MB and RhB. However, the removal percent of RhB is relatively low due to the large volume of the RhB molecules, which caused a steric hindrance with the active adsorption sites on the hybrid. Although MO molecules are small enough for ingress and egress, a little uptake capacity of MO was observed as the negative charge of this dye molecule. There are like-charges that repel each other between the POM caged in hybrid and MO. To further demonstrate the role of anionic H3PMo12O40 cluster in the hybrid nanomaterial, a series of control experiments were carried out using Fe3O4, Fe3O4/GO, Fe3O4/ GO-NH2 and pure H3PMo12O40 samples as adsorbents for removing MB dye. As shown in Figure 11, the adsorption efficiencies of these samples toward the MB dye are 40-78% albeit after long adsorption times of 30-40 min. Apparently, the removal efficiencies and rates of these materials are smaller than those of the Fe3O4/GrO-NH2/H3PMo12O40 hybrid nanomaterial for cationic MB dye (100% in 3 min). Therefore, there is still merit in exploring the hybrid nano-material as efficient adsorbent toward cationic MB dye. Figure 11. Adsorption efficiency (%) of the MB dye in the presence of different adsorbent samples. 3. 3. Selective Adsorption Ability of the Hybrid Nanomaterial for the Mixed Organic Dyes Selective adsorption and separation of the specific dye are more attractive and challenging in the process of dye-wastewater treatment. In this study, in view of the large uptake capacity of MB and RhB in Fe3O4/GO-NH2/ H3PMo12O40, it can be anticipated that the composite material may also have an outstanding adsorption and separation behavior in the treatment of mixture of dyes. The selective uptake of dyes was tested using the MB/MO mixture (50 mL, C0(MB) = C0(MO) = 25 mg/L) and MB/RhB mixture (50 C0(mb) = C0(mo) = 25 mg/L) with 25 mg of Fe3O4/GO-NH2/H3PMo12O40 as adsorbent. The process was monitored by UV-Vis spectroscopy. As MB and MO are similar in molecule size, the preferable uptake of MB from the MB/MO mixture may be assigned to the anionic nature of the composite Fe3O4/GO-NH2/H3PMo12O40, as shown in Figure 12(a). For comparison, cationic RhB was selected to mix with anionic MO (50 mL, C0(MO) = C0(RhB) = 25 mg/L). Results revealed that RhB was also preferably adsorbed on the composite material from the MO/RhB mixture as illustrated in Figure 12(b), which may imply that the uptake of dyes is heavily influenced by molecule size along with charges.72,73 To further validate this point, a ternary mixture of MB, RhB, and MO (50 mL, C0(MB) = C0(M0) = C0(RhB) = 25 mg/L) with 25 mg of the adsorbent was investigated. As exhibited in Figure 12(c), the representative peaks of MB and RhB all disappeared quickly in mixed dyes and only the characteristic absorption peaks of MO were left, suggesting that Fe3O4/GO-NH2/H3PMo12O40 could selectively capture cationic dyes when utilized in the corresponding ternary mixture. The same conclusion is displayed in the insets of Figure 12(a)-(c) and only the color of MO can be seen in the final solutions of mixed dyes. It can be attributed to the negative charge of this dye mol- Farhadi et al.: Magnetically Recyclable Fe3O4/GO-NH2/H3PMo12O40 ... Acta Chim. Slov. 2017, 64, 1005-1019 ioi5 Wave! ci i ntli (mil) Figure 12. Selective adsorption ability of Fe3O4/GO-NH2/H3PMo12O40 toward mixed dyes solution of (a) MB+MO, (b) RhB+MO, (c) MB+RhB+MO. Conditions : C0 (MB) = C0 (RhB) = C0 (MO) = 25 mg/L and adsorbent dose = 25 mg/50mL ecule, which repels each other between MO and the H3P-Mo12O40 cluster in the hybrid nanomaterial. The slightly decreasing absorbance of MO is more likely to be adsorbed on the surface of adsorbent. The results further confirmed that the electrostatic attraction is the key factor for the occurrence of adsorption. Thus, Fe3O4/GO-NH2/H3PMo12O40 composite nanomaterial is an environmental friendly, active adsorbent for removing different cationic organic pollutants after the immobilization of POM anion. 3. 4. The Reusability and Stability of the Hybrid Nanomaterial The stability and reusability of the adsorbents are an important standard for practical application. To verify whether the composite material is stable and recycled during the adsorption experiments, the cycle tests of Fe3O4/GO-NH2/H3PMo12O40 on removing MB were explored. After each cycle, the adsorbent was completely separated by a magnet because of the magnetic property in water. Subsequently, the fast release process of the adsorbed MB was achieved by thoroughly washing the adsorbent with a dilute solution of NaCl and ethanol three times. Then, desorbed adsorbent was added to 50 mL of 25 mg/L MB solution under stirring. As described in Figure 13, the composite nanomaterial showed almost identically rapid adsorption of MB. After four cycles, the regenerated adsorbent was still able to remove 94% MB from the aqueous solution. Thus, we may conclude that the composite nanomaterial can be reusable during the adsorption experiment. The stability of this material is further discussed. As depicted in Fig. 14(a) and (b), the XRD pattern and FT-IR spectrum of the recovered adsorbent after the fourth run are consistent with those of the as-synthesized composite (see Figures 2(c) and 3(d)). These observations confirmed that the structure of the Fe3O4/GO-NH2/H3PMo 12O40 hybrid nanomaterial is stable under the reaction conditions and is not affected by the reactants. The morphology of the recycled adsorbent particles was also analyzed. Fig. 14(c) Farhadi et al.: Magnetically Recyclable Fe3O/GO-NH2/H3PMo12O4i 1016 Acta Chim. Slov. 2017, 64, 1005-1019 Figure 13. Recyclability of the Fe3O4/GO-NH2/H3PMo12O40 hybrid nanomaterial in the removal of MB dye. shows a representative SEM image of the adsorbent after four cycles. It could be observed that the recovered adsorbent almost kept its initial size and morphology (see Figure 5(e) and (f)) and the surface of GO nanosheets was still decorated with Fe3O4 and H3PMo12O40 particles, revealing the strong binding between the Fe3O4 nanoparticles and H3PMo12O40 with GO nanosheets. We can also observe representative peaks of C, N, O, Si, Fe, P, and Mo from the EDX of the recovered Fe3O4/GO-NH2/H3PMo 12O40 (Figure 14(d)). Considering the above mentioned experiment results, we can conclude that the structure of the compound remained intact, which further confirms its excellent stability and recyclability. The Fe3O4/GO-NH2/H3PMo12O40 hybrid exhibited the maximum adsorption capacity of 426.7 mg/g when 100 mg of Fe3O4/GO-NH2/H3PMo12O40 was soaked in 100 mL of 500 mg/L aqueous MB solution for 2 h. As can be seen in Table 1, Fe3O4/GO-NH2/H3PMo12O40 exhibits Figure 14. (a) XRD pattern, (b) FT-IR spectrum, (c) SEM image, and (d) EDX spectrum of the recovered Fe3O4/GO-NH2/H3PMo12O40 hybrid nanomaterial after the fourth run. Farhadi et al.: Magnetically Recyclable Fe3O4/GO-NH2/H3PMo12O40 ... Acta Chim. Slov. 2017, 64, 1005-1019 ioi7 much higher uptake capacity of MB, compared with the adsorbents that were reported.74-87 On the basis of the above observations and the characteristics of Fe3O4/GO-NH2/H3PMo12O40 nanocomposite, we infer that the following factors could contribute to the increased adsorption capacity of this nanocomposite. On the one hand, the anchored aminopropyl groups and the presence of the well dispersed Fe3O4 and H3PMo12O40 nanoparticles on the GO nanosheets surface could act as spacers and thus prohibit the graphene sheets to restack. This directly results in significant increase of the specific surface area of graphene structures, which can be of great benefit to adsorption processes. This result is consistent with BET surface area data in Figure 9. On the other hand, the anchored PMo12O403-polyanions with a large number of negative charges in the ternary hybrid have a stronger attraction force with the positive charges of cationic dyes (e.g. MB). In fact, higher adsorption capacity of the Fe3O4/GO-NH2/H3PMo12O40 is due to synergistic effect between GO nanosheets and PMo12O403- polyanions. Thus, this material is a promising adsorbent for the treatment of toxic organic pollutants in the dye-wastewater. Table 1. Comparison of the adsorption capacities of MB onto some typical adsorbents. Entry Adsorbent material Adsorption Ref. capacity (mg/g) 1 Activated carbon 135 74 2 Nano- ZIF - 8 13.3 74 3 Zeolite 10.86 75 4 Fe3O4/graphene 33.66 76 5 ^pw^/zim 810 74 6 H3PW12O40/MnIIIporphyrin Graphene 10.5 77 7 153.83 78 8 CoFe2O4/MWCNT 14.3 79 9 H6P2WA2/MOF-5 51.81 80 10 H3PW12O40/MIL-101(Fe) 473.7 81 11 H34PW1112V/40MIL-101(Cr) 371 82 i2 (4-Hap)4[Mo8O2j 916.04 83 13 MOF/graphite oxide 18 84 14 Carbon nanotubes 46.2 85 15 MOF-235 187 86 16 Grapheme oxide (GO) 144.92 87 17 Calcium alginate/GO 188.81 87 18 Fe3°4/G°-NH2/H3PMo12°40 426.7 This work 4. Conclusions In summary, the novel ternary hybrid nanomaterial Fe3O4/GO-NH2/H3PMo12O40 was synthesized by a simple acid-base interaction between aminopropyl functional-ized magnetic graphene oxide nanosheets and H3PMo12O40 and tested as a new adsorbent in the removal of MB, RhB, and MO dyes from aqueous solutions. The results confirmed that this adsorbent displayed much higher adsorp- tion capacity for cationic dyes and lower adsorption capacity for anionic dyes. This adsorption selectivity is due to the favorable electrostatic interactions between the adsorbents and cationic dyes. High adsorption capacity, compared with other adsorbents, accompanied by the ease of separation by an external magnetic field make the prepared hybrid a powerful separation tool to be utilized in wastewater treatment. 5. Acknowledgements The authors gratefully acknowledge the Lorestan University Research Council and Iran Nanotechnology Initiative Council (INIC) for their financial support. 6. References 1. S. Kaur, R. Gopal, W. J. Ng, S. Ramakrishna and T. Matsuura, MRS Bull., 2008, 33, 21-26. DOI:10.1557/mrs2008.10 2. H. W. Liang, X. Cao, W. J. Zhang, H. T. Lin, F. Zhou, L. F. Chen and S. H. Yu, Adv. Funct. Mater., 2011, 21, 3851-3858. DOI: 10.1002/adfm.201100983 3. L. S. Zhong, J. S. Hu, H. P. Liang, A. M. Cao, W. G. Song and L. J. Wan, Adv. Mater., 2006, 18, 2426-2431. DOI: 10.1002/adma.200600504 4. J. B. Zimmerman, J. R. Mihelcic, S. James, Environ. Sci. Tech-nol, 2008, 42, 4247-4254. DOI:10.1021/es0871457 5. G. Crini, Bioresour. Technol., 2006, 97, 1061-1085. DOI:10.1016/j.biortech.2005.05.001 6. R. D. Ambashta and M. Sillanpaa, J. Hazard. Mater., 2010, 180, 38-49. DOI:10.1016/j.jhazmat.2010.04.105 7. S. H. Chen, J. Zhang, C. L. Zhang, Q. Y. Yue, Y. Li and C. Li, Desalination, 2010, 252, 149-156. DOI: 10.1016/j.desal.2009.10.010 8. A. Mittal, A. Malviya, D. Kaur, J. Mittal and L. Kurup, J. Hazard. Mater. 2007, 148, 229-240. DOI: 10.1016/j.jhazmat.2007.02.028 9. S. B. Wang, Y. Boyjoo, A. Choueib and Z. H. Zhu, Water Res. 2005, 39, 129-138. DOI:10.1016/j.watres.2004.09.011 10. J. B. Zimmerman, J. R. Mihelcic and S. James, Environ. Sci. Technol. 2008, 42, 4247-4254. DOI:10.1021/es0871457 11. Z. Xiong, L. L. Zhang, J. Ma and X. S. Zhao, Chem. Commun., 2010, 46, 6099-6101. DOI:10.1039/c0cc01259a 12. S. Prakash, A. M. Rajesh and V. K. Shahi, Chem. Eng. J., 2011, 168, 108-114. DOI:10.1016/j.cej.2010.12.047 13. L. Ai, H. Yue and J. Jiang, Nanoscale, 2012, 4, 5401-5408. DOI:10.1039/C2NR31333B 14. B. Wang, H. Wu, L. Yu, R. Xu, T. T. Lim, and X. W. Lou, Adv. Mater., 2012, 24, 1111-1116. DOI: 10.1002/adma.201104599 15. J. C. Crittenden, R. R. Trussell, D. W. Hand, K. J. Howe and, G. Tchobanoglous, Water Treatment: Principles and Design, 2nd ed.; Wiley: Chichester, U.K., 2005. 16. Y. J. Xu, G. Weinberg, X. Liu, O. Timpe, R. Schlogl and D. S. Farhadi et al.: Magnetically Recyclable Fe3O/GO-NH2/H3PMo12O4i 1018 Acta Chim. Slov. 2017, 64, 1005-1019 Su, Adv. Funct. Mater., 2008, 18, 3613-3619. DOI: 10.1002/adfm.200800726 17. T. Zhu, J. S. Chen and X. W. Lou, J. Phys. Chem. C, 2012, 116, 6873-6878. D0I:10.1021/jp300224s 18. M. Valix, W. H. Cheung and G. Mckay, Langmuir, 2006, 22, 4574-4582. D0I:10.1021/la051711j 19. N. K. Lazaridis, G. Z. Kyzas, A. A. Vassiliou and D. N. Bikiaris, Langmuir, 2007, 23, 7634-7643. D0I:10.1021/la700423j 20. E. Alvarez-Ayuso, A. Garcia-Sanchez and X. Querol, Water Res., 2003, 37, 4855-4862. D0I:10.1016/j.watres.2003.08.009 21. M. S. Mauter, M. Elimelech, Environ. Sci. Technol. 2008, 42, 5843-585. D0I:10.1021/es8006904 22. M. Liu, C. Chen, J. Hu, X. Wu and X. Wang, J. Phys. Chem. C, 2011, 115, 25234-25240. D0I:10.1021/jp208575m 23. J. W. Zhang, J. H. Luo, P. M. Wang, B. Ding, Y. C. Huang, Z. L. Zhao, J. Zhang and Y. G. Wei, Inorg. Chem., 2015, 54, 2551-2559. D0I:10.1021/ic502622k 24. S. S. Wang and G. Y. Yang, Chem. Rev., 2015, 115, 4893-4962. D0I:10.1021/cr500390v 25. J. S. Li, X. J. Sang, W. L. Chen, L. C. Zhang, Z. M. Zhu, Y. G. Li, Z. M. Su and E. B. Wang, J. Mater. Chem. A, 2015, 3, 14573-14577. D0I:10.1039/C5TA03259H 26. U. Kortz, A. Muller, J. V. Slageren, J. Schnack, N. S. Dalal and M. Dressel, Coord. Chem. Rev., 2009, 253, 2315-2327. D0I:10.1016/j.ccr.2009.01.014 27. J. T. Rhule, C. L. Hill, D. A. Judd and R. F. Schinazi, Chem. Rev., 1998, 98, 327-358. D0I:10.1021/cr960396q 28. A. X. Yan, S. Y, Y. G. Li, Z. M. Zhang, Y. Lu, W. L. Chen and E. B. Wang, Chem. Eur. J., 2014, 20, 6927-6933. D0I:10.1002/chem.201400175 29. V. K. Gupta, T. A. Saleh, Environ. Sci. Pollut. Res., 2013, 20, 2828-2843. D0I:10.1007/s11356-013-1524-1 30. Y. Chen, S. Zhao and Y. F. Song, Appl. Catal. A: Gen., 2013, 466, 307-314. D0I:10.1016/j.apcata.2013.06.030 31. A. Tayyebi and M. Outokesh, RSC Adv., 2016, 6, 13898-13913. 32. M. Yusuf, F. Elfghi, S. A. Zaidi, E. Abdullah and M. A. Khan, RSC Adv., 2015, 5, 50392-50420. 33. Z. H. Cheng, J. Liao, B. Z. He, F. Zhang, F. A. Zhang, X. H. Huang and L. Zhou, ACS Sustain. Chem. Eng. 2015, 3, 16771685. D0I:10.1021/acssuschemeng.5b00383 34. J. Li, C. L. Chen, R. Zhang and X. K. Wang, Sci. China Chem., 2016, 59, 150-158. D0I:10.1007/s11426-015-5452-4 35. G. X. Zhao, L. Jiang, Y. D. He, J. X. Li, H. L. Dong and X. K. Wang, W. P. Hu, Adv. Mater., 2011, 23, 3959-3963. D0I:10.1002/adma.201101007 36. J. Wang, Z. M. Chen and B. L. Chen, Environ. Sci. Technol., 2014, 48, 4817-4825. D0I:10.1021/es405227u 37. H. Wang, X. Z. Yuan, G. M. Zeng, Y. Wu, Y. Liu, Q. Jiagn and S. S. Gu, Adv. Colloid Interface Sci., 2015, 221, 41-59. D0I:10.1016/j.cis.2015.04.005 38. H. Wang, X. Z. Yuan, Y. Wu, H. J. Huang, X. Peng, G. M. Zeng, H. Zhong, J. Liang and M. M. Ren, Adv. Colloid Interface Sci., 2013, 195-196, 19-40. D0I:10.1016/j.cis.2013.03.009 39. H. Wang, X. Z. Yuan, Y. Wu, H. J. Huang, G. M. Zeng, Y. Liu, X. L. Wang, N. B. Lin and Y. Qi, Appl. Surf. Sci., 2013, 279, 432-440. D0I:10.1016/j.apsusc.2013.04.133 40. H. Chen, B. Gao and H. Li, J. Hazard. Mater., 2015, 282, 201207. D0I:10.1016/j.jhazmat.2014.03.063 41. P. Wang, Q. Shi, Y. Shi, K. K. Clark, G. D. Stucky and A. A. Keller, J. Am. Chem. Soc., 2008, 131,182-188. D0I:10.1021/ja806556a 42. S. Zhang, H. Li, Z. Wang, J. Liu, H. Zhang, B. Wang and Z. Yang, Nanoscale, 2015, 7, 8495-8502. D0I:10.1039/C5NR00527B 43. X. J. Deng, L. L. Lu, H. W. Li and F. Luo, J. Harzard. Mater., 2010, 183, 923-930. 44. M. C. Liu, T. Wen, X. L. Wu, C. L. Chen, J. Hu, J. X. Li and X. K. Wang, Dalton Trans., 2013, 43, 14710-14717. D0I:10.1039/c3dt50955a 45. J. Zhu, S. Wei, H. Gu, S. B. Rapole, Q. Wang, Z. Luo, N. Haldo-laarachchige, D. P. Young and Z. Guo, Environ. Sci. Technol., 2012, 46, 977-98. D0I:10.1021/es2014133 46. Y. Liu, C. Luo, G. Cui and S. Yan, RSC Adv., 2015, 5, 5415654164. 47. X. Bai, R. Feng, Z. Hua, L. Zhou and H. Shi, Environ. Eng. Sci., 2015, 32, 370-378. D0I:10.1089/ees.2014.0015 48. F. Gu, M. Liang, D. Han and Z. Wang, RSC Adv., 2015, 5, 39964-39972. 49. Y. J. Yao, S. D. Miao, S. Z. Liu, L. P. Ma, H. Q. Sun and S. B. Wang, Chem. Eng. J., 2012, 184, 326-332. D0I:10.1016/j.cej.2011.12.017 50. N. I. Kovtyukhova, P. J. Ollivier, B. R. Martin, T. E. Mallouk, S. A. Chizhik, E. V. Buzaneva and A. D. Gorchinskiy, Chem. Mater., 1999, 11, 771-778. D0I:10.1021/cm981085u 51. X. Gong, G. Liu, Y. Li, D. Y. W. Yu and W. Y. Teoh, Chem. Mater., 2016, 28, 8082-8118. D01:10.1021/acs.chemmater.6b01447 52. L. Zhao, Y. Chi, Q. Yuan, N. Li, W. Yan and X. Li, J. Colloid Interface Sci., 2013, 390, 70-77. D0I:10.1016/j.jcis.2012.08.059 53. Z. Zhang, F. Zhang, Q. Zhu, W. Zhao, B. Ma and Y. Ding, J. Colloid Interface Sci., 2011, 360, 189-194. D0I:10.1016/j.jcis.2011.04.045 54. M. Masteri-Farahania, J. Movassagh, F. Taghavi, P. Eghbali and F. Salimi, Chem. Eng. J., 2012, 184, 342-346. D0I:10.1016/j.cej.2011.12.094 55. Y. Fu and X. Wang, Ind. Eng. Chem. Res., 2011, 50, 7210-7218. D0I:10.1021/ie200162a 56. G. H. Moon, Y. Park, W. Kim and W. Choi, Carbon, 2011, 49, 3454-3462. D01:10.1016/j.carbon.2011.04.042 57. L. Q. Guo, P. R. Ye, J. Wang, F. F. Fu and Z. J. Wu, J. Hazard. Mater., 2015, 298, 28-35. D0I:10.1016/j.jhazmat.2015.05.011 58. Q. S. Zhao, D. F. Chen, Y. Li, G. L. Zhang, F. B. Zhang and X. B. Fan, Nanoscale, 2013, 5, 882-885. D0I:10.1039/C2NR33290F 59. M. A. Ghasemzadeh, B. Molaei, M. H. Abdollahi-Basir and F. Zamani, Acta Chim. Slov., 2017, 64, 73-82. D0I:10.17344/acsi.2016.2823 60. S. K. Singh, M. K. Singh, P. P. Kulkarni, V. K. Sonkar, J. J. A. Gracio and D. Dash, ACS Nano, 2012, 6, 2731-2740. D0I:10.1021/nn300172t Farhadi et al.: Magnetically Recyclable Fe3O4/GO-NH2/H3PMo12O40 ... Acta Chim. Slov. 2017, 64, 1005-1019 ioi9 61. C. Rocchiccioli-Deltcheff, M. Fournier, R. Franck and R. Thouvenot, Inorg. Chem., 1983, 22, 207-216. D01:10.1021/ic00144a006 62. H. Zhang, A. J. Xie, Y. H. Shen, L. G. Qiu and X. Y. Tian, Phys. Chem. Chem. Phys., 2012, 14, 12757-12763. D0I:10.1039/c2cp41561e 63. D. Zhou and B. H. Han, Adv. Funct. Mater., 2010, 20, 27172722. DOI: 10.1002/adfm.200902323 64. Y. B. Sun, D. D. Shao, C. L. Chen, S. B. Yang and X. K. Wang, Environ. Sci. Technol. 2013, 47, 9904-9910. DOI: 10.1021/es401174n 65. Y. Sun, D. Shao, C. Chen, S. Yang and X. Wang, Environ. Sci. Technol., 2013, 47, 9904-9910. D0I:10.1021/es401174n 66. S. Alwarappan, A. Erdem, C. Liu and C.-Z. Li, J. Phys. Chem. C, 2009, 113, 8853-8857. D0I:10.1021/jp9010313 67. Y.-X. Ma, Y.-F. Li, G.-H. Zhao, L.-Q. Yang, J.-Z. Wang, X. Shan and X. Yan, Carbon, 2012, 50, 2976-2986. D0I:10.1016/j.carbon.2012.02.080 68. L. M. Cui, Y. G. Wang, L. Gao, L. H. Hu, L. G. Yan, Q. Wei and B. Du, Chem. Eng. J., 2015, 281, 1-10. D0I:10.1016/j.cej.2015.06.043 69. W. Zhang, M. Wang, W. Zhao and B. Wang, Dalton Trans., 2013, 42, 15464-15474. D0I:10.1039/c3dt52068d 70. X. Q. Tian, C. M. Cheng, L. Qian, B. Z. Zheng, H. Y. Yuan, S. P. Xie, D. Xiao and M. M. F. Choi, J. Mater. Chem., 2012, 22, 8029-8035. D0I:10.1039/c2jm16057a 71. H. Pang, Y. H. Ma, G. C. Li, J. Chen, J. S. Zhang, H. H. Zheng and W. M. Du, Dalton Trans., 2012, 41, 13284-13291. D0I:10.1039/c2dt31916k 72. L. Li, X. L. Liu, H. Y. Geng, B. Hu, G. W. Song and Z. S. Xu, J. Mater. Chem. A, 2013, 1, 10292-10299. D0I:10.1039/c3ta11478c 73. X. Zhao, X. Bu, T. Wu, S.-T. Zheng, L. Wang and P. Feng, Nat. Commun., 2013, 4, 1-9. 74. R. Li, X. Q. Ren, J. S. Zhao, X. Feng, X. Jiang, X. X. Fan, Z. G. Lin, X. G. Li, C. G. Hu and B. Wang, J. Mater. Chem. A, 2014, 2, 2168-2173. D01:10.1039/C3TA14267A 75. C. D. Woolard, J. Strong and C. R. Erasmus, Appl. Geochem., 2002, 17, 1159-1164. D0I:10.1016/S0883-2927(02)00057-4 76 Y. Yao, S. Miao, S. Liu, L. P. Ma, H. Sun and S. Wang, Chem. Eng. J., 2012, 184, 326-332. D0I:10.1016/j.cej.2011.12.017 77. C. Zou, Z. J. Zhang, X. Xu, Q. H. Gong, J. Li and C. D. Wu, J. Am. Chem. Soc., 2012, 134, 87-90. D0I:10.1021/ja209196t 78 T. Liu, Y. Li, Q. Du, J. Sun, Y. Jiao, G. Yang, Z. Wang, Y. Xia, W. Zhang and K. Wang, Colloids Surf., B, 2012, 90, 197-203. DOI: 10.1016/j.colsurfb.2011.10.019 79. A. A. Farghali, M. Bahgat, W. M. A. El Rouby and M. H. Khe-dr, J. Solution Chem. 2012, 41, 2209-2225. D0I:10.1007/s10953-012-9934-0 80. X. X. Liu, W. P. Gong, J. Luo, C. T. Zou, Y. Yang and S. J. Yang, Appl. Surf. Sci., 2016, 362, 517-524. D0I:10.1016/j.apsusc.2015.11.151 81. T. T. Zhu, Z.-M. Zhang, W.-L. Chen, Z.-J. Liu and E.-B. Wang, RSC Adv., 2016, 6, 81622-81630 82. A. X. Yan, S. Y, Y. G. Li, Z. M. Zhang, Y. Lu, W. L. Chen and E. B. Wang, Chem. Eur. J., 2014, 20, 6927-6933. D01:10.1002/chem.201400175 83. Y.-Q. Zhang, C.-C. Wang, T. Zhu, P. Wang and S.-J. Gao, RSC Adv. 2015, 5, 45688-45692. 84. L. Li, X. L. Liu, H. Y. Geng, B. Hu, G. W. Song and Z. S. Xu; J. Mater. Chem., A, 2013, 1, 10292-10299. D0I:10.1039/c3ta11478c 85. Y. Yao, F. Xu, M. Chen, Z. Xu and Z. Zhu, Bioresour. Technol., 2010, 101, 3040-3046. D0I:10.1016/j.biortech.2009.12.042 86. E. Haque, J. W. Jun and S. H. Jhung, J. Hazard. Mater., 2011, 185, 507-511. D0I:10.1016/j.jhazmat.2010.09.035 87. Y. Li, Q. Du, T. Liu, J. Sun, Y. Wang, S. Wu, Z. Wang, Y. Xia and L. Xia, Carbohydr. Polym., 2013, 95, 501-507. D0I:10.1016/j.carbpol.2013.01.094 Povzetek V raziskavi poročamo o vezavi polianionov PMo12O403- na amino funkcionalizirane nanoplasti grafenovega oksida (GO). Tako pripravljen ternarni magnetni nanokompozit (Fe3O4/GO-NH2/H3PMo12O40) smo karakterizirali z naslednjimi metodami: rentgensko praškovno difrakcijo (XRD) infrardečo spektroskopijo (FTIR), ramansko spektroskopijo, energijsko disperzivno spektroskopijo (EDS), vrstično elektronsko mikroskopijo z emisijo polja (FE-SEM), meritvami površine (BET), magnetnimi meritvami (VSM) in mikroskopijo na atomsko silo (AFM). Rezultati so pokazali uspešno vezavo H3PMo12O40 (utežni delež ~36.5 %) na površino magnetnega grefenovega oksida. Specifična površina nanokompozita je bila višja (77.07 m2/g) od čistega H3PwMo12O40 (<10 m2/g). Preučevali smo adsorpcijsko učinkovitost nanokompozita v primerih odstranjevanja nekaterih barvil (metilen modro, rodamin B in metiloranž) iz vodnih raztopin. Z uporabo nanokompozita smo prikazali hitro in selektivno adorpcijo za kationska barvila iz mešanih raztopin barvil. Stopnja in kapaciteta adsorpcije Fe3O4/GO-NH2/H3PMo12O40 sta bili povečani v primerjavi z GO, GO-NH2, Fe3O4/GO-NH2 in H3P-Mo12O40 zaradi povečane elektrostatske privlačnosti in interakcij preko vodikovih vezi. Nanokompozit lahko ločimo z magneti in ponovno uporabimo, pri čemer v kompozitu ne pride do strukturnih sprememb. Menimo, da nanokompozit Fe3O4/GO-NH2/H3PMo12O40 predstavlja obetaven "zeleni" adsorbent za odstranjevanje organskih onesnaževal iz voda. Farhadi et al.: Magnetically Recyclable Fe3O/GO-NH2/H3PMo12O4i 1020 DOI: 10.17344/acsi.2017.3773 Acta Chim. Slov. 2017, 64, 1020-1029 /^creative tycommor Scientific paper Eco-Friendly Multi-Component Synthesis of y-Spiroiminolactones in Water Hamid Reza Safaei1* and Mohsen Shekouhy2 1 Department of Applied Chemistry, Shiraz Branch, Islamic Azad University, P.O. Box 71993-5, Shiraz, Iran 2 Department of Chemistry, College of Sciences, Shiraz University, 71454 Shiraz, Iran * Corresponding author: E-mail: safaei@iaushiraz.ac.ir Tel.: +98 713 6402715; fax: +98 713 6412488 Received: 20-08-2017 Abstract Y-Spiroiminolactone derivatives can be synthesized through an one-pot three-component reaction of cyclic carbonyl compounds (isatin, acenaphthoquinone, ninhydrin), activated a-methylene carbonyl groups and isocyanides in water using sodium dodecyl sulfate (SDS) as a commercially available and cheap surfactant compound. All products were obtained in good to excellent yields without formation of any by-products in water as a green and benign reaction medium. Moreover, presented method was successfully applied for the synthesis of some novel bis(spiroiminolactone) derivatives. Keywords: Y-spiroiminolactone, water, multi-component reaction, isocyanide, surfactant 1. Introduction It is well-known that heterocyclic compounds have played an important role in drug discovery and pharmaceuticals synthesis. Among various kinds of heterocycles, iminolactones have been intensively studied and widely applied as useful intermediates for the synthesis of buteno-lides.1 They have been shown wide range of biological ac- tivities such as antimicrobial,2 antifungal,3 anti-inflamma-tory,4 anticancer,5 and antiviral.6 Moreover, iminolactones have been used for the synthesis of sphingofungin F7a that exhibits inhibitory effects toward serine palmitoyl transferase (SPT), which induces apoptosis in both yeast and mammalian cells by blocking the sphingosine biosynthesis pathway.7b Scheme 1. The one-pot multi-component synthesis of y-spiroiminolactones in water. Safaei and Shekouhy: Eco-Friendly Multi-Component Synthesis of ... Acta Chim. Slov. 2017, 64, 1020-1029 1021 For a number of reasons, the development of simple and more benign chemical processes for the synthesis of biologically active compounds in water is one of the major challenges for chemists. Some of these reasons are the fact that water is a safe, inexpensive, readily available, and environmentally benign reaction medium.8 However, the insolubility of most organic compounds in water is the major drawback of the application of water as a safe reaction medium in organic synthesis. Furthermore, some active sites in organic compounds are either decomposed or deactivated in water. One useful way to overcome this problem is the application of surfactant-combined catalysts that was first introduced by Kobayashi.9a-e As people's concerns about their living environment increases continuously, the design of new multi-component reactions (MCRs) with ecofriendliness, green procedures had drawn significant attention, especially in organic synthesis and drug discovery with environmentally benign solvents (specially water) and reagents.9b In continuation to our recent studies about the synthesis of heterocyclic compounds in water,10 we wish to report an eco-friendly synthesis of y-spiroiminolactones (4a-n) via a one-pot three component reaction of cyclic 1,2-ketones (1a-d), activated a-methylene carbonyl compounds (2a-c), and isocyanide derivatives (3a-c) in water. (Scheme 1). 2. Results and Discussion Initially, in order to find the best reaction condition, the one-pot condensation reaction between isatin (1a) (1 mmol, 0.147 g), 5,5-dimethylcyclohexane-1,3-dione (2a) (1 mmol, 0.140 g) and cyclohexyl isocyanide (3a) (1 mmol, 0.109 g) was selected as a model reaction. The reaction yield and duration time were monitored in the presence of sodium dodecyl sulfate (SDS) as a very cheap and readily available surfactant compound under various reaction conditions. The obtained results are summarized in Table 1. Table 1. The one-pot condensation reaction between isatin (1a) (1 mmol, 0.147 g), 5,5-dimethylcyclohexane-1,3-dione (2a) (1 mmol, 0.140 g) and cyclohexyl isocyanide (3a) (1 mmol, 0.109 g) in the presence of SDS as a surfactant in water (5 mL) under various reaction conditions. Entry SDS (mol%, g) Temp. (°C) Time (h) Yield (%)a 1 5, 0.014 Reflux 48 51 2 10, 0.028 Reflux 36 85 3 15, 0.042 Reflux 24 91 4 20, 0.056 Reflux 24 91 5 25, 0.070 Reflux 24 90 6 15, 0.042 r.t. 48 - 7 15, 0.042 40 48 15 8 15, 0.042 70 48 73 9 - Reflux 48 - a Isolated yield. As it is shown in Table 1, the best result was obtained in the presence of 15 mol% of SDS at reflux temperature (Table 1, entry 3). Moreover, the model reaction was studied in the absence of SDS in water at reflux temperature. At this condition the reaction was not proceeded even after a long time (48 h) and a gummy solid was formed (Table 1, entry 9). This observation establishes the crucial role of SDS as a surfactant to produce an appropriate reaction medium. Moreover, the model reaction was investigated in other solvents such as EtOH, MeOH, CH2Cl2, THF, CH3CN, EtOAc and M-hexane, and only trace amounts of products were detected (Table 2). Table 2. The one-pot condensation reaction between isatin (1a), 5,5-dimethylcyclohexane-1,3-dione (2a), and cyclohexyl isocyanide (3a) with the presence of SDS in various solvents at reflux conditions.a Entry Solvent (5 mL) Time (h) Yield (%)b 1 «-hexane 24 Trace 2 EtOAc 24 Trace 3 ch3cn 24 Trace 4 EtOH 24 28 5 MeOH 24 21 6 CH2CI2 24 Trace 7 THF 24 Trace a Molar ratio: isatin (1 mmol, 0.147 g), 5,5-dimethylcyclohexane-1,3-dione (1 mmol, 0.140 g), cyclohexyl isocyanide (1 mmol, 0.109 g), and SDS (15 mol%, 0.042 g). b Isolated yield. In the next step, to establish the generality and efficiency of the presented method a variety of starting materials were examined. In this regard a variety of reactive cyclic carbonyl compounds such as isatins, acenaphtho-quinone and ninhydrin (1a-d), 1,3-dicarbonyl compounds (2a-c) and isocyanides (3a-c) were examined under optimized conditions (Scheme 1). The obtained results are summarized in Table 3. All reactions were completed after 24 h and desired products were obtained in good to excellent yields (7292%). As it is shown in Table 3, lower yields of products were obtained in the case of ninhydrin (Table 3, entries 10 and 11) that may be due to the higher water solubility of ninhydrin and its lower tendency to diffuse to micelles. We think that two factors play crucial role in this reaction and directly affect the yields and the rates. One is the use of SDS as a surfactant and the other one is water as a solvent. Recent investigations indicate that water molecules repel small covalent organic molecules and force them to form aggregates in order to decrease the organic surface area.11a Forced aggregates of organic reactants are raised in energy more than their unaggregated ground states and are closer in energy to the activated complexes or transition states and this phenomenon significantly leads to the Safaei and Shekouhy: Eco-Friendly Multi-Component Synthesis of 1022 Acta Chim. Slov. 2017, 64, 1020-1029 Table 3. The one-pot multi-component synthesis of y-spiroiminolactone derivatives in the presence of SDS in water after 24 h. Entry Reactant 1 Reactant 2 Reactant 3 Product 4 Yield (%)a 1a 2a 3a 91 1a 2a 3b 92 1a 2a 3c 89 1b 2a 3a 83 1b 2a 3b 89 1b 2a 3c 82 1a 2b 3a 90 1b 2b 3a 89 1a 2b 3b 91 10 1d 2a 3a 75 1 2 3 4 5 6 7 8 9 Safaei and Shekouhy: Eco-Friendly Multi-Component Synthesis of Acta Chim. Slov. 2017, 64, 1020-1029 1023 Entry Reactant 1 Reactant 2 Reactant 3 Product 4 Yield (%)a a Isolated yields. enhancement of the reaction rate. Furthermore, hydrogen bonding between polar transition states and water molecules leads to the stabilization of transition state and decreases the activation gap of the reaction.lla Therefore, the use of SDS as a surfactant has major influence on the rate of the reactions. This compound forms many micelles in water with a lipophilic inner site that will act as micro reactors and prepare appropriate places for organic materials to meet each other. So briefly, SDS makes micelles as nano-reactors for organic reactions in water, therefore organic materials with higher energy states penetrate to these nano-reactors and collide with each other to re-act.11b In recent years, the synthesis of bis-heterocyclic compounds has been attracted chemists because of unique biological activities such as antibacterial, fungicidal, tuber-culostatic, antiamoebic, and plant growth regulation.12 Therefore, there are numerous reports on the synthesis of various types of these compounds.13 Considering these facts, we applied our method for the synthesis of some novel bis(spiroiminolactone) derivatives. For this purpose, compound (6) was synthesized as a bis(isatin) compound via the reaction of isatin (1a, 2 eq) and 1,4-bis(chlorome-thyl)benzene (5, 1 eq) in DMF. Then it was condensed with carbonyl compounds processing the a-methylene group (2a-c) and cyclohexyl isocyanide (3a, 2 eq) in water, and in the presence of SDS (Scheme 2). As it is shown in Scheme 3, our proposed mechanism for the synthesis of y-spiroiminolactones (4a-n) via a one-pot three-component reaction between cyclic carbonyl compounds (1a-d), activated a-methylene carbonyl groups (2a-c) and isocyanides (3a-c) consists of two steps. In the first step, intermediate (5) forms from the Knoeve-nagel condensation of cyclic carbonyl compounds (1a-d) and activated a-methylene carbonyl derivatives (2a-c). In the next step, desired products (4a-n) are obtained via the Michael-type addition of isocyanides (3a-c) to the intermediate (5). It is followed by an intermolecular cyclization reaction. Based on the Jafari report14 the catalytic effect of mi-cellar SDS in our presented method can be explained as the following. Cyclic carbonyl compounds (1a-d), activat- ■ Time(h) ■ Isolated Yield (%) 91 91 90 go 91 1 2 Î 4 5 Run Figure 1. The one-pot condensation reaction between isatin (1a) (1 mmol, 0.147 g), 5,5-dimethylcyclohexane-1,3-dione (2a) (1 mmol, 0.140 g) and cyclohexyl isocyanide (3a) (1 mmol, 0.109 g) in the presence of recovered SDS in water (5 mL) under reflux condition. Safaei and Shekouhy: Eco-Friendly Multi-Component Synthesis of 1024 Acta Chim. Slov. 2017, 64, 1020-1029 Scheme 2. The one-pot three component synthesis of some novel bis(spiroiminolactone) derivatives in water in the presence of SDS. ed a-methylene groups (2a-c) and isocyanides (3a-c), which are expected to produce y-spiroiminolactones (4a-n), are all hydrophobic molecules in aqueous media. In the micellar solution of SDS, the hydrophobic moieties escape from water molecules, which encircle the micelle core of SDS. Therefore, they are activated by hydrogen bonding and are pushed by water molecules into the hydrophobic core of the micellar droplets, where the reactions take place more easily. Water is also a sufficiently polar medium to shift the keto-enol equilibrium to the enol form, which are highly hydrophilic species. This explanation is also schematically presented by Scheme 3. The possibility of catalyst recycling was examined by using the model reaction under the optimized conditions. After completion of the reaction, the insoluble products were filtered, water was evaporated under reduced pressure, and the obtained solid was washed with diethyl ether and dried under reduced pressure for 24 h. The recovered SDS was reused five times and no loss of efficiency was observed (Figure 1). Safaei and Shekouhy: Eco-Friendly Multi-Component Synthesis of Acta Chim. Slov. 2017, 64, 1020-1029 1025 Scheme 3. Proposed mechanism for the synthesis of y-spiroiminolactones (4a-n) via a one-pot three-component reaction between cyclic carbonyl compounds (1a-d) (isatin, acenaphthoquinone, ninhydrin), activated a-methylene groups (2a-c) and isocyanides (3a-c) in the presence of SDS in water. 3. Conclusion In summary, an efficient one-pot procedure for the synthesis of novel Y-spiroiminolactone derivatives is described. This method avoids the use of hazardous catalysts or solvents. The advatages the presented method are efficiency, generality, high yields of desired products, short reaction times, clean reaction profile, ease of product isolation, simplicity, and agreement with some green chemistry protocols, which all make it a useful and attractive process for the synthesis of y-spiroimi-nolactone derivatives. Moreover, the presented method was successfully applied for the synthesis of more complex structures such as novel bis(spiroiminolactone) derivatives. 4. Experimental The chemicals used in this work were obtained from Merck and Sigma-Aldrich companies and were used without purification. Melting points were measured on an Elecrtothermal 9100 apparatus and are uncorrected. 'H and 13C NMR spectra were recorded on a BRUKER DRX-250 AVANCE spectrometer at 250 and 62.5 MHz respectively. Chemical shifts are given as S values in CDCl3 against TMS as an internal standard and J values are given in Hz. IR spectra were recorded using a Shimadzu FT-IR apparatus. Microanalyses were performed on a Per-kin-Elmer 240-B microanalyzer. Safaei and Shekouhy: Eco-Friendly Multi-Component Synthesis of ... 1026 Acta Chim. Slov. 2017, 64, 1020-1029 Preparation of 1-(4-((2,3-dioxoindolin-1-yl)methyl) benzyl)indoline-2,3-dione (6) In a double neck 250 mL round-bottom flask connected to an argon gas inlet, NaH (110 mmol, 2.63 g) was added to a solution of isatin (100 mmol, 14.71 g) in dried DMF 50 mL at 0 oC and resulting mixture was stirred at that temperature under argon atmosphere for 0.5 h, and then warmed to room temperature. Then, 1,4-bis(chlo-romethyl)benzene (50 mmol, 8.75 g) was added and resulting solution stirred at 80 oC for 8h under argon atmosphere. After this time, the reaction mixture was cooled to room temperature and immediately added to the cold water (500 mL). Resulting precipitates were filtered and dried. Crude products were recrystallized from hot acetic acid and 1-(4-((2,3-dioxoindolin-1-yl)methyl)benzyl)indo-line-2,3-dione (16.00 g, 81%) was obtained as an orange solid. mp 243-245 °C. IR (KBr) v 3050, 2960, 1720, 1670 cm-1. 1H NMR (250 MHz, CDCl3) 5 (ppm) 5.83 (s, 4H), 7.20 (s, 4H), 7.28 (t, J = 7.5 Hz, 2H), 7.45 (d, J = 7.5 Hz, 2H), 7.64 (t, J = 7.5 Hz, 2H), 7.74 (d, J = 7.5 Hz, 2H). 13C NMR (62.5 MHz, CDCl3) 5 (ppm) 46.2, 111.1, 121.3, 123.5, 128.2, 129.3, 133.8, 135.0, 151.3, 166.9, 186.3. Anal. Calcd. for C, 72.72; H, 4.07; N, 7.07 (%). 24 16 2 4 x ' Found: C, 72.80; H, 4.01; N, 7.11 (%). MS (m/z): 396 (M+). General procedure for the preparation of y-spiroiminol-actones in the presence of SDS in water Isocyanide derivative (1 mmol) was added to a mixture of 1,3-dicarbonyl compound (1 mmol), reactive carbonyl compound (isatins, acenaphthoquinone or ninhydrin) (1 mmol) and SDS (0.04 g, 15 mol%) in water (5 mL), and the resulting mixture was refluxed and stirred for 24 h. After this time, the reaction mixture was cooled to room temperature and precipitates were filtered and washed with water (10 mL), and recrystallized from hot ethanol to afford the pure product. For the synthesis of bis(spiroiminolactone) derivatives, 2 mmol of isocyanide and 1,3-dicarbonyl compounds should be applied for each 1 mmol of bis(isatin) compound. 2-(cyclohexylimino)-6,6-dimethyl-6,7-dihydro-2H- spiro [benzofuran-3,3'-indoline]-2',4(5H)-dione (4a) White solid, mp 231-234 °C (dec.) (lit. 230 oC (dec.)).10a IR (KBr) v 3395, 2858, 1745, 1731, 1654 cm-1. 1H NMR (250 MHz, CDCl3) 5 (ppm) 0.96 (s, 3H), 0.99 (s, 3H), 1.13-1.28 (m, 10H), 1.92-2.07 (distorted AB system, 2H), 2.32 (d, J = 17.5 Hz, 1H), 2.42 (d, J = 17.5 Hz, 1H), 3.92-4.03 (m, 1H), 6.82-6.97 (m, 3H), 7.24 (t, J = 7.5 Hz, 1H), 8.01 (s, 1H). 13C NMR (62.5 MHz, CDCl3) 5 (ppm) 23.1, 23.8, 26.3, 29.6, 31.1, 37.0, 49.7, 55.4, 66.4, 107.6, 121.2, 121.3, 126.1, 126.6, 136.5, 137.9, 140.9, 148.3, 174.2, 191.2. Anal. Calcd. for C HNO; C, 72.99; H, 6.92; N, 23 26 2 3 7.40 (%). Found: C, 73.09; H, 6.98; N, 7.51 (%). MS (m/z): 378 (M+). 2-(tert-butylimino)-6,6-dimethyl-6,7-dihydro-2H-spiro [benzofuran-3,3'-indoline]-2',4(5H)-dione (4b) White solid, mp 224-226 °C (dec.) (lit. 227 oC (dec.)).10a IR (KBr) v 3390, 2950, 1750, 1731, 1615 cm-1. 'H NMR (250 MHz, CDCl3) 5 (ppm) 0.96 (s, 3H), 0.99 (s, 3H), 1.05 (s, 9H), 2.01 (d, J = 17.5 Hz, 1H), 2.13 (d, J = 17.5 Hz, 1H), 2.47 (d, J = 17.5 Hz, 1H), 2.68 (d, J = 17.5 Hz, 1H), 6.44 (d, J = 7.5 Hz, 1H), 6.75 (t, J = 7.5 Hz, 1H), 7.31-7.43 (m, 2H), 8.04 (s, 1H). 13C NMR (62.5 MHz, CDCl3) 5 (ppm) 26.7, 29.1, 30.0, 37.4, 50.1, 52.8, 67.4, 108.0, 121.6, 121.7, 126.5, 127.0, 136.9, 138.3, 144.4, 148.7, 174.6, 191.6. Anal. Calcd. for C21H24N2O3: C, 71.57; H, 6.86; N, 7.95 (%). Found: C, 71.50; H, 6.82; N, 8.02 (%). MS (m/z): 352 (M+). 6,6-dimethyl-2-(2,3,3-trimethylbutan-2-ylimino)-6,7-di-hydro-2H-spiro[benzofuran-3,3'-indoline]-2',4(5H)-di-one (4c) White solid, mp 217-220 oC (dec.) (lit. 220 °C (dec.)).10a IR (KBr) v 3350, 2985, 1724, 1700, 1603 cm-1. 1H NMR (250 MHz, CDCl3) 5 (ppm) 1.06 (s, 3H), 1.10 (s, 3H), 1.12 (s, 9H), 1.56 (s, 6H), 2.08 (d, J = 17.5 Hz, 1H), 2.18 (d, J = 17.5 Hz, 1H), 2.55 (d, J = 18.0 Hz, 1H), 2.68 (d, J = 18.0 Hz, 1H), 6.75 (d, J = 7.5 Hz, 1H), 6.90 (t, J = 7.5 Hz, 1H), 7.16-7.32 (m, 2H), 8.73 (s, 1H). 13C NMR (62.5 MHz, CDCl3) 5 (ppm) 22.9, 24.2, 26.3, 29.6, 33.5, 37.0, 49.7, 67.0, 72.3, 107.6, 121.2, 121.3, 126.1, 126.6, 136.0, 136.5, 137.9, 148.3, 174.2, 191.2. Anal. Calcd.for C25H32N2O3: C, 73.50; H, 7.90; N, 6.86 (%). Found: C, 73.51; H, 7.93; N, 6.84 (%). MS (m/z): 394 (M+). 5'-chloro-2-(cyclohexylimino)-6,6-dimethyl-6,7-dihydro -2H-spiro[benzofuran-3,3 '-indoline]-2',4(5H)-dione (4d) White solid, mp 256-258 °C (dec.) (lit. 254 °C (dec.)).10a IR (KBr) v 3400, 2975, 1733, 1710, 1680 cm-1. 1H NMR (250 MHz, CDCl3) 5 (ppm) 0.97(s, 3H), 0.99 (s, 3H), I.12-1.30 (m, 10H), 1.96- 2.04 (distorted AB system, 2h), 2.27 (d, J = 17.5 Hz, 1H), 2.40 (d, J = 17.5 Hz, 1H), 3.413.50 (m, 1H), 6.78 (d, J = 7.5 Hz, 1H), 7.18 (d, J = 7.5 Hz, 1H), 7.40 (s, 1H), 8.75 (s, 1H).13C NMR (62.5 MHz, CDCl3) 5 (ppm) 23.4, 25.0, 26.5, 29.8, 32.8, 37.2, 49.9, 55.6, 68.9, 109.1, 120.2, 125.5, 129.1, 129.6, 136.8, 137.8, 141.1, 148.5, 174.4, 195.4. Anal. Calcd. for C23H25ClN2O3: C, 66.90; H, 6.10; N, 6.78 (%). Found: C, 66.93; H, 6.15; N, 6.86 (%). MS (m/z): 412 (M+). 2-(tert-butylimino)-5'-chloro-6,6-dimethyl-6,7-dihydro-2H-spiro[benzofuran-3,3'-indoline]-2',4(5H)-dione (4e) White solid, m.p. = 234-236 °C (dec.) (lit. 238 °C (dec.)).10a IR (KBr) v 3250, 2985, 1744, 1632, 1600 cm-1. 1H NMR (250 MHz, CDCl3) 5 (ppm) 0.97 (s, 3H), 0.99 (s, 3H), 1.11 (s, 9H), 2.05-2.21 (distorted AB system, 2H), 2.53 (d, J = 17.5 Hz, 1H), 2.65 (d, J = 17.5 Hz, 1H), 6.77 (d, J = 7.5 Hz, 1H), 7.13 (d, J = 7.5 Hz, 1H), 7.41 (s, 1H), 8.51 (s, 1H). 13C NMR (62.5 MHz, CDCl3) 5 (ppm) 26.3, 28.7, 29.6, 37.0, 49.7, 52.4, 68.9, 105.9, 120.0, 125.3, 128.9, 129.4, 136.5, 137.6, 144.0, 148.3, 176.1, 195.1. Anal. Calcd. for Safaei and Shekouhy: Eco-Friendly Multi-Component Synthesis of Acta Chim. Slov. 2017, 64, 1020-1029 1027 C21H23ClN2O3: C, 65.20; H, 5.99; N, 7.24 (%). Found: C, 652122; H, 5.91; N, 7.28 (%). MS (m/z): 386 (M+). 5'-chloro-6,6-dimethyl-2-(2,3,3-trimethylbutan-2-ylimi-no)-6,7-dihydro-2H-spiro[benzofuran-3,3'-indoline]-2',4(5H)-dione (4f) White solid, mp 220-223 °C (dec.) (lit. 224oC (dec.)).10a IR (KBr) v 3200, 2970, 1725, 1685, 1600 cm-1. 1H NMR (250 MHz, CDCl3) 5 (ppm) 0.98 (s, 3H), 1.05 (s, 3H), 1.12 (s, 9H), 1.55 (s, 6H), 2.10-2.27 (distorted AB system, 2H), 2.58 (d, J = 17.5 Hz, 1H), 2.68 (d, J = 17.5 Hz, 1H), 6.74 (d, J = 7.5 Hz, 1H), 7.13 (d, J = 7.5 Hz, 1H), 7.41 (s, 1H), 8.68 (s, 1H). 13C NMR (62.5 MHz, CDCl3) 5 (ppm) 23.1, 24.4, 26.5, 29.8, 33.7, 37.2, 49.9, 65.9, 71.6, 109.1, 120.2, 125.5, 129.1, 131.3, 134.4, 136.8, 137.8, 148.5, 178.9, 195.5. Anal. Calcd. for C25H31ClN2O3: C, 67.20; H, 6.81; N, 6.53 (%). Found: C, 67.33; H 6.98; N, 6.36 (%). MS (m/z): 428 (M+). 2-(cyclohexylimino)-6,7-dihydro-2H-spiro[benzofuran-3,3'-indoline]-2',4(5H)-dione (4g) White solid, mp 231-234 °C (dec.) (lit.235 oC (dec.)).10a IR (KBr) v 3390, 2855, 1740, 1730, 1655 cm-1. 1H NMR (250 MHz, CDCl3) 5 (ppm) 1.02-1.31 (m, 10H), 1.93-2.12 (m, 4H), 2.38 (t, J = 7.5 Hz, 2H), 3.98 (m, 1h), 6.74 (d, J = 7.5 Hz, 1H), 6.90 (t, J = 7.5 Hz, 1H), 7.15-7.26 (m, 2H), 8.04 (s, 1H). 13C NMR (62.5 MHz, CDCl3) 5 (ppm) 19.7, 21.9, 23.4, 26.1, 31.3, 34.5, 55.6, 69.7, 107.8, 117.8, 124.3, 126.8, 128.2, 138.1, 141.1, 146.4, 179.7, 196.3. Anal. Calcd. for C21H22N2O3: C, 71.98; H, 6.33; N, 7.99 (%). Found: C, 71.95; H, 6.38; N, 7.95 (%). MS (m/z): 350 (M+). 5'-chloro-2-(cyclohexylimino)-6,7-dihydro-2H-spiro [benzofuran-3,3'-indoline]-2',4(5H)-dione (4h) White solid, mp 243-245 °C (dec.) (lit. 241 oC (dec.)).10a IR (KBr) v 3395, 2850, 1746, 1732, 1650 cm-1. 1H NMR (250 MHz, CDCl3) 5 (ppm) 0.98-1.30 (m, 10H), 1.95-2.34 (m, 4H), 2.31 3t, J = 7.5 Hz, 2H), 3.94 (m, 1h), 6.73 (d, J = 7.5 Hz, 1H), 7.14 (d, J = 7.5 Hz, 1H), 7.40 (s, 1H), 8.04 (s, 1H). 13C NMR (62.5 MHz, CDCl3) 5 (ppm) 19.7, 21.9, 23.4, 26.6, 31.3, 34.5, 58.3, 69.2, 109.1, 120.2, 125.5, 128.2, 129.1, 133.0, 137.8, 141.1, 144.1, 178.9, 195.9. Anal. Calcd. for C21H21ClN2O3: C, 65.54; H, 5.50; N, 7.28 (%). Found: C, 65.58; H, 5.51; N, 7.34 (%). MS (m/z): 384 (M+). 2-(tert-butylimino)-6,7-dihydro-2H-spiro[benzofuran-3,3'-indoline]-2',4(5H)-dione (4i) White solid, mp 225-228 °C (dec.), (lit. 225 oC (dec.)).10a IR (KBr) v 3392, 2955, 1752, 1731, 1615 cm-1. 1H NMR (250 MHz, CDCl3) 5 (ppm) 1.07 (s, 9H), 2.08-2.20 (m, 4H), 2.28 (t, J = 7.5 Hz, 2H), 6.79 (d, J = 7.5 Hz, 1H), 6.91 (t, J = 7.5 Hz, 1H), 7.17 (d, J = 7.5 Hz, 1H), 7.29 (t, J = 7.5 Hz, 1H), 9.01 (s, 1H). 13C NMR (62.5 MHz, CDCl3) 5 (ppm) 19.3, 21.5, 28.5, 34.1, 52.2, 69.0, 107.4, 121.1, 124.5, 126.4, 127.8, 137.7, 143.8, 145.1, 177.9, 195.5. Anal. Calcd. for C19H20N2O3: C, 70.35; H, 6.21; N, 8.64 (%). Found: C, 70.39; H, 6.27; N, 8.66 (%). MS (m/z): 324 (M+). 2-(cyclohexylimino)-6,6-dimethyl-6,7-dihydro-2H-spiro [benzofuran-3,2'-indene]-1',3',4(5H)-trione (4j) White solid, mp 238-240 °C (dec.) (lit. 242 oC (dec.)).10a IR (KBr) v 3390, 2855, 1743, 1735, 1730 cm-1. 1H NMR (250 MHz, CDCl3) 5 (ppm) 0.97 (s, 3H), 0.99 (s, 3H), 1.04-1.38 (m, 10H), 1.98-2.13 (distorted AB system, 2H), 2.47 (d, J = 18.0 Hz, 1H), 2.59 (d, J = 17.5 Hz, 1H), 4.01 (m, 1H), 6.64 (d, J = 7.5 Hz, 2H), 6.98 (d, J = 7.5 Hz, 2H). 13C NMR (62.5 MHz, CDCl3) 5 (ppm) 22.3, 23.0, 25.5, 28.8, 33.5, 36.2, 48.9, 54.6, 68.5, 115.4, 133.2, 133.5, 136.7, 136.9, 141.7, 157.2, 196.8, 205.1. Anal. Calcd. for C24H25NO4: C, 73.64; H, 6.44; N, 3.58 (%). Found: C, 73.66; H, 6.440; N, 3.60 (%). MS (m/z): 391 (M+). 2-(tert-butylimino)-6,7-dihydro-2H-spiro[benzofuran-3,2'-indene]-1',3',4(5H)-trione (4k) White solid, mp 244-246 °C (dec.) (lit. 248 oC (dec.)).10a IR (KBr) v 3390, 2850, 1745, 1738, 1730 cm-1. 1H NMR (250 MHz, CDCl3) 5 (ppm) 1.04 (s, 9H), 1.87-2.32 (m, 4H), 2.40 (t, J = 7.5 Hz, 2H), 6.79 (d, J = 7.5 Hz, 2H), 7.01 (d, J = 7.5 Hz, 2H). 13C NMR (62.5 MHz, CDCl3) 5 (ppm) 20.0, 23.2, 29.2, 34.8, 52.9, 67.8, 115.3, 123.4, 134.5, 137.9, 145.1, 158.3, 196.1, 206.7. Anal. Calcd. for C20H19NO4: C, 71.20; H, 5.68; N, 4.15 (%). Found: C, 71.25; H, 5.62; N, 4.20 (%). MS (m/z): 337 (M+). 2-(cyclohexylimino)spiro[indeno[1,2-b]furan-3,3'-indo-line]-2',4(2H)-dione (4l) White solid, mp 284-287 °C (dec.) (lit. 284 oC (dec.)).10a IR (KBr) v 3390, 2855, 1738, 1695 cm-1. 1H NMR (250 MHz, DMSO-d6) 5 (ppm) 1.01-1.33 (m, 10H), 3.96 (m, 1H), 6.75 (d, J = 7.5 Hz, 1H), 6.90 (t, J = 7.5 Hz, 1H), 7.15-7.26 (m, 4H), 7.64 (t, J = 7.5 Hz, 1H), 7.82 (d, J = 7.5 Hz, 1H), 9.93 (s, 1H). 13C NMR (62.5 MHz, DMSO-dfi) 5 (ppm) 23.1, 23.8, 31.1, 55.4, 64.0, 107.9, 117.2, 121.3, 122.1, 124.4, 125.1, 126.6, 127.0, 128.3, 131.1, 132.5, 139.9, 145.0, 147.8, 152.3, 170.2, 195.7. Anal. Calcd. for C24H20N2O3: C, 74.98; H, 5.24; N, 7.29 (%). Found: C, 74.93; H, 5.26; N, 7.33 (%). MS (m/z): 384 (M+). 2'-(tert-butylimino)-2H-spiro[acenaphthylene-1,3'-in-deno[1,2-b]furan]-2,4'(2'H)-dione (4m) White solid, m.p. >300 oC (lit. > 300 oC).10a IR (KBr) v 3395, 2857, 1730, 1695 cm-1. 1H NMR (250 MHz, CDCl3) 5 (ppm) 1.05 (m, 9H), 7.12 -7.44 (m, 5H), 7.527.69 (m, 1H), 7.78-7.91 (m, 1H), 7.98-8.20 (m, 3H). 13C NMR (62.5 MHz, CDCl3) 5 (ppm) 31.2, 54.5, 76.6, 121.5, 124.0, 125.1, 125.8, 126.5, 127.0, 127.8, 128.8, 129.5, 130.1, 132.0, 132.9, 134.0, 134.1, 134.8, 140.5, 145.3, 147.9, 186.5, 195.8. Anal. Calcd. for CHNO,: C, 79.37; H, 4.87; N, 26 19 3 3.56 (%). Found: C, 79.39; H, 4.80; N, 3.51 (%). MS (m/z): 393 (M+). Safaei and Shekouhy: Eco-Friendly Multi-Component Synthesis of 1028 Acta Chim. Slov. 2017, 64, 1020-1029 2'-(cyclohexylimino)-6',6'-dimethyl-6',7'-dihydro-2H, 2'H-spiro[acenaphthylene-1,3'-benzofuran]-2,4'(5'H)-dione (4n) White solid, mp 231-233 °C (dec.) (lit. 229 oC (dec.)).10a IR (KBr) v 2890, 1740, 1620, 1612. 1H NMR (250 MHz, CDCl3) 5 (ppm) 0.87 (s, 3H), 0.90 (s, 3H), 0.97-1.05 (m, 10H), 1.98-2.14 (distorted AB system, 2h), 2.35-2.44 (distorted AB system, 2H), 4.05 (m, 1H), 7.36-7.42 (m, 1H), 7.65-7.75 (m, 1H), 7.81-7.95 (m, 1H), 7.98-8.23 (m, 3H). 13C NMR (62.5 MHz, CDCl3) 5 (ppm) 21.4, 26.9, 28.5, 30.2, 35.5, 48.5, 50.1, 54.0, 93.2, 108.6, 112.8, 117.2, 122.0, 124.9, 127.7, 128.4, 130.9, 132.6, 149.0, 153.2, 163.0, 185.7, 191.5 195.7. Anal. Calcd. for C27H27NO3: C, 78.42; H, 6.58; N, 3.39 (%). Found: C, 78.38; H, 6.61; N, 3.42 (%). MS (m/z): 413 (m+). 2'-(cyclohexylimino)-6',6'-dimethyl-6',7'-dihydro-2H, 2'H-spiro[acenaphthylene-1,3'-benzofuran]-2,4'(5'H) -dione (7a) White solid, mp >300 °C. IR (KBr) v 3050, 2970, 1740, 1730, 1660 cm-1. 1H NMR (250 MHz, DMSO-dg) 5 (ppm) 0.96 (s, 6H), 0.99 (s, 6H), 1.07-1.28 (m, 20H), 1.9 62.14 (distorted AB system, 4H), 2.56 (d, J = 18.0 Hz, 2H), 2.67 (d, J = 18.0 Hz, 2H), 3.92-4.03 (m, 2H), 4.64 (d, J = 12.0 Hz, 2H), 4.89 (d, J = 12.0 Hz, 2H), 6.76 (d, J = 7.5 Hz, 2H), 6.92 (t, J = 7.5 Hz, 2H), 7.05 (s, 4H), 7.15-7.26 (m, 4H). 13C NMR (62.5 MHz, DMSO-dfi) 5 (ppm) 23.4, 24.0, 26.5, 29.8, 31.3, 37.2, 39.9, 49.9, 55.6, 68.2, 107.9, 116.9, 121.9, 124.0, 127.1, 129.3, 131.6, 136.1, 139.2, 141.0, 147.7, 174.8, 196.1. Anal. Calcd. for C,HNO: C, 75.50; H, ' 54 58 4 6 ' ' ' 6.81; N, 6.52 (%). Found: C, 75.41; H, 6.88; N, 6.43 (%). MS (m/z): 859 (M+). 2'-(cyclohexylimino)-6',6'-dimethyl-6',7'-dihydro-2H, 2'H-spiro[acenaphthylene-1,3'-benzofuran]-2,4'(5'H) -dione (7b) White solid, mp >300 °C. IR (KBr) v 3060, 2950, 1748, 1735, 1665 cm-1. 1H NMR (250 MHz, DMSO-dg) 5 (ppm) 0.99-1.29 (m, 20H), 1.96-2.12 (m, 8H), 2.37 (t, J = 7.5 Hz, 4H), 3.99 (m, 2H), 4.590 (d, J = 12.0 Hz, 2H), 4.73 (d, J = 12.0 Hz, 2H), 6.75 (d, J = 7.5 Hz, 2H), 6.90 (t, J = 7.5 Hz, 2H), 7.06 (s, 4H), 7.15-7.26 (m, 4H). 13C NMR (62.5 MHz, DMSO-d6) 5 (ppm) 19.9, 22.0, 23.5, 26.8, 27.8, 34.7, 46.2, 55.8, 65.6, 108.1, 121.4, 123.3, 126.6, 126.9, 127.3, 129.4, 135.4, 139.3, 141.1, 145.7, 174.1, 196.7. Anal. Calcd. for Q„H„NO: C, 74.79; H, 6.28; N, 6.98 (%). Found: C, 50 50 4 6 74.74; H, 6.20; N, 7.05 (%). MS (m/z): 802 (M+). 2'-(cyclohexylimino)-6',6'-dimethyl-6',7'-dihydro-2H, 2'H-spiro[acenaphthylene-1,3'-benzofuran]-2,4'(5'H) -dione (7c) White solid, mp >300 °C. IR (KBr) v 3060, 2950, 1748, 1735, 1665 cm-1. 1H NMR (250 MHz, DMSO-dg) 5 (ppm) 1.00-1.35 (m, 20H), 3.90 (m, 2H), 4.59 (d, J = 12.0 Hz, 2H), 4.74 (d, J = 12.0 Hz, 2H), 6.79 (d, J = 7.5 Hz, 2H), 6.90 (t, J = 7.5 Hz, 2H), 7.11 (s, 4H), 7.14-7.26 (m, 8h), 7.64 (t, J = 7.5 Hz, 2H), 7.82 (d, J = 7.5 Hz, 2H). 13C NMR (62.5 MHz, DMSO-d6) 5 (ppm) 23.5, 24.2, 31.5, 43.3, 55.8, 66.0, 107.9, 119.2, 121.7, 124.8, 127.2, 127.3, 127.4, 128.7, 129.9, 131.5, 131.8, 132.9, 140.1, 143.6, 146.3, 153.4, 176.0, 193.5. Anal. Calcd. for C HNO; C, 77.22; H, 5.32; N, 56 46 4 6 ' ' ' ' ' 6.43 (%). Found; C, 77.13; H, 5.40; N, 6.31 (%). MS (m/z); 870 (M+). 5. Acknowledgements The research for this paper was financially supported by the Islamic Azad University, Shiraz Branch, Iran, through project entitled Isocyanide-based one-pot three component synthesis of novel spiro-iminolactone derivatives in the presence of SDS in aqueous medium. The authors appreciate the IAU (Shiraz Branch) for its support. 6. References 1. (a) D. Villemin, L. Liao, Synth. Commun. 2003, 33, 15751585. DOI:10.1081/SCC-120018778 (b) Y. Tang, C. Li, Tetrahedron Lett. 2006, 47, 3823-3825. DOI:10.1016/j.tetlet.2006.03.166 2. G. Grossmann, M. Poncioni, M. Bornand, B. Jolivet, M. Neuburger, U. Sequin, Tetrahedron 2003, 59, 3237-4251. DOI:10.1016/S0040-4020(03)00483-6 3. (a) S. M. Hein, J. B. Gloer, B. Koster, D. Malloch, J. Nat. Prod. 2001, 64, 809-812. DOI:10.1021/np000617u (b) M. Pour, M. Spulak, V. Balsanek, J. Kunes, P. Kubanova, V. Butcha, Bioorg. Med. Chem. 2003, 11, 2843-2866. DOI:10.1016/S0968-0896(03)00220-7 4. S. Padakanti, M. Pal, K. R. Yeleswarapu, Tetrahedron 2003, 59, 7915-7920. DOI:10.1016/j.tet.2003.08.021 5. (a) S. Takahashi, A. Kubota, T. Nakata, Tetrahedron Lett. 2002, 43, 8661-8664. DOI:10.1016/S0040-4039(02)02182-2 (b) F. Bellina, E. Falchi, R. Rossi, Tetrahedron 2003, 59, 90919100. DOI:10.1016/j.tet.2003.09.061 6. (a) S. Hanessian, R. Y. Park, R. Y. Yang, Synlett 1997, 351-352. DOI:10.1055/s-1997-803 (b) A. Choudhury, F. Jin, D. Wang, Z. Wang, G. Xu, D. Nguyen, J. Castoro, M. E. Pierce, P. N. Confalone, Tetrahedron Lett. 2003, 44, 247-250. DOI:10.1016/S0040-4039(02)02532-7 7. (a) F.-F. Gan, Sh.-B. Yang, Y.-Ch. Luo, W.-B. Yang, P.-F. Xu, J. Org. Chem. 2010, 75, 2737-2740. DOI:10.1021/jo100183d (b) M. M. Zweerink, A. M. Edison, G. B. Well, W. Pinto, R. L. Lester, J. Biol. Chem. 1992, 267, 25032-25038. 8. (a) P. A. Grieco, Organic Synthesis in Water, Blackie Academic and Professional, London, 1998. DOI: 10.1007/978-94-011-4950-1 (b) P. T. Anastas, T. C. Williamson, Green Chemistry, ed., ACS Symposium Series 626, American Chemical Society, Wash- Safaei and Shekouhy: Eco-Friendly Multi-Component Synthesis of Acta Chim. Slov. 2017, 64, 1020-1029 1029 ington, DC, 1996. (c) V. J. Li, T. H. Chan, Organic Reactions in Aqueous Media, Wiley, New York, 1997. (d) P. Anastas J. C. Warner, Green Chemistry: Theory and Practice, Oxford University Press, Oxford, 1998. (e) C. J. Li, Chem. Rev. 2005, 105, 3095-3166. DOI:10.1021/cr030009u (f) H. Mehrabi, H. Abusaidi, J. Iran Chem. Soc. 2010, 7, 890894. DOI:10.1007/BF03246084 (g) N. Azizi, A. Khajeh Amiri, M. Bolourtchian, M. R. Saidi, J. Iran Chem. Soc. 2009, 6, 749-753. DOI:10.1007/BF03246165 (h) Sh. Rostamizadeh, H. Estiri, M. Azad, J. Iran Chem. Soc. 2016, 13, 1367-1374. DOI:10.1007/s13738-016-0851-9 (i) M. Riahi, E. Farsani, F. Assady, B. Jalilian Yadollahi, H. Amiri Rudbari, J. Iran Chem. Soc. 2015, 12, 1207-1212. DOI:10.1007/s13738-014-0583-7 (j) M. Shiri, J. Iran Chem. Soc. 2013, 10, 1019-1023. DOI:10.1007/s13738-013-0239-z (k) B. Zeynizadeh, M. Zabihzadeh, Z. Shokri, J. Iran Chem. Soc. 2016, 13, 1487-1492. DOI:10.1007/s13738-016-0864-4 9. (a) S. Kobayashi, K. Manabe S. Nagayama, in Modern Car-bonyl Chemistry, ed. J. Otera, Wiley-VCH, Weinheim, 2000. (b) K. Manabe, Y. Mori, T. Wakabayashi, S. Nagayama, S. Kobayashi, J. Am. Chem. Soc. 2000, 122, 7202-7207. DOI:10.1021/ja001420r (c) S. Kobayashi, S. Nagayama, T. Busujima, J. Am. Chem. Soc. 1998, 120, 8287-8288. DOI:10.1021/ja980715q (d) M. Shekouhy, A. Khalafi-Nezhad, Green Chem. 2015, 17, 4815-4829. DOI:10.1039/C5GC01448D (e) M. Shekouhy, Catal. Sci. Tech. 2012, 2, 1010-1020. DOI:10.1039/c2cy00493c (f) L. J. Yan, Y. Ch. Wang, ChemistrySelect 2016, 1, 69486960. DOI: 10.1002/slct.201601534 (g) A. R. Moosavi-Zare, M. A. Zolfigol, R. Salehi-Moratab, E. Noroozizadeh, Can. J. Chem. 2017, 95, 194-198. DOI:10.1139/cjc-2016-0374 (h) A. R. Moosavi-Zare, M. A. Zolfigol, E. Noroozizadeh, R. Salehi-Moratab, M. Zarei, J. Mol. Cat. A: Chem. 2016, 420, 246-253. DOI:10.1016/j.molcata.2016.04.021 (i) A. R. Moosavi-Zare, M. A. Zolfigol, E. Noroozizadeh, M. Zarei, R. Karamian, M. Asadbegy, J. Mol. Cat. A: Chem. 2016, 425, 217-228. DOI:10.1016/j.molcata.2016.10.011 (j) M. A. Ghasemzadeh, M. H. Abdollahi-Basir, Acta. Chim. Slov. 2016, 63, 627-637. DOI: 10.17344/acsi.2016.2386 (k) F. Moradgholi, J. Lari, M. Vahidi Parsa, M. Mirkarrazi, Acta Chim. Slov. 2016, 63, 781-789. DOI: 10.17344/acsi.2016.2634 (l) E. Noroozizadeh, A. R. Moosavi-Zare, M. A. Zolfigol, A. Zare, M. Zarei, Can. J. Chem. 2017, 95, 16-21. DOI: 10.1139/cjc-2016-0258 10. (a) H. R. Safaei, N. Shioukhi, M. Shekouhy, Monatsh. Chem. 2013, 144, 1855-1863. DOI:10.1007/s00706-013-1060-1 (b) H. R. Safaei, M. Shekouhy, Sh. Khademi, V. Rahmanian, M. Safaei, J. Ind. Eng. Chem. 2014, 20, 3019-3024. DOI: 10.1016/j.jiec.2013.11.037 11. (a) R. N. Butler, A. G. Coyne, Chem. Rev. 2010, 110, 63026337. DOI:10.1021/cr100162c (b) B. Samiey, C.-H. Cheng, J. Wu, J. Chem. 2014, 1-14. DOI: 10.1155/2014/908476 12. (a) H. Singh, L. D. S. Yadav, B. K. Bhattacharya, J. Indian. Chem. Soc.1979, 56, 1013-1017. (b) N. C. Desai, Indian J. Chem. Sect. B 1993, 32, 343-346. (c) X. M. Feng, R. Chen, X. C. Liu, Z. Y. Zhang, Chin. J. Appl. Chem. 1991, 8, 28-33. (d) P. S. Upadhyay, R. N. Vansdadia, A. J. Baxi, Indian J. Chem. Sect. B 1990, 29, 793-796. (e) Z. Y. Zhang, X. Chen, L. L. Wei, Z. L. Ma, Chem. Res. Chin. Univ. 1991, 7, 129-135. (f) A. R. Bhat, F. Athar, A. Azam, Eur. J. Med. Chem. 2009, 44, 426-431. DOI:10.1016/j.ejmech.2007.11.005 (g) D. B. Reddy, B. Seenaiah, S. Eswaraiah, T. Seshamma, M. V. R. Reddy, J. Indian. Chem. Soc. 1989, 66, 893-899. (h) P. F. Iqbal, H. Parveen, A. R. Bhat, F. Hayat, A. Azam, Eur. J. Med. Chem. 2009, 44, 4747-4751. DOI:10.1016/j.ejmech.2009.06.016 13. R. M. Shaker, Arkivoc 2012, 1, 1-44. 14. A. A. Jafari, F. Moradgholi, F. Tamaddon, Eur. J. Org. Chem. 2009, 2009, 1249-1255. Povzetek V prispevku je opisana priprava y-spiroiminolaktonskih derivatov z enostopenjsko trokomponentno reakcijo cikličnih karbonilnih spojin (kot so: isatin, acetonaftokinon, ninhidrin), spojin z aktivirano a-metilensko karbonilno skupino in izocianidov v vodi, ob uporabi natrijevega dodecil sulfata (SDS) kot komercialno dostopne in cenene površinsko aktivne snovi. Vsi produkti so bili pridobljeni z dobrimi in nekateri celo z odličnimi izkoristki, brez tvorbe stranskih produktov. Kot reakcijski medij je bila uporabljena voda kot zeleno in nenevarno topilo. Predstavljena metoda je bila tudi uspešno uporabljena za pripravo nekaterih novih bis(spiroiminolaktonskih) derivatov. Safaei and Shekouhy: Eco-Friendly Multi-Component Synthesis of 1030 DOI: 10.17344/acsi.2017.3805 Acta Chim. Slov. 2017, 64, 1030-1041 /^creative tycommor Scientific paper Synthesis, Nematicidal and Antifungal Properties of Hybrid Heterocyclics Avula Srinivas,1* Malladi Sunitha,1 Pulluri Karthik1 and K. Vasumathi Reddy2 1 Department of Chemistry, Vaagdevi Degree & PG College 2 Department of Zoology, Vaagdevi Degree & PG College Kishanpura, Warangal, Telangana, India 506001 * Corresponding author: E-mail: avula.sathwikreddy@gmail.com Received: 28-08-2017 Abstract A new series of 5-((3aB,5S,6S,6aB)-6-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2,2-dimethyltetrahydro-furo[2,3-d][1,3]dioxol-5-yl)-3-(4-fluorophenyl)-2,6-diphenyl-3,3a,5,6-tetrahydro-2H-pyrazolo[3,4-d]thiazoles 10a-r was synthesized by the reaction of chalcone derivatives of 2-((3a_R,5S,6S,6a_R)-6-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)-3-phenylthiazolidin-4-one 9 with aryl/alkyl hydrazines. The chemical structures of newly synthesized compounds were elucidated by IR, NMR, MS and elemental analysis. The compounds 10a-r were evaluated for their nematicidal activity against Dietylenchus myceliophagus and Caenorhab-ditis elegans by aqueous in vitro screening technique. Among them, compounds containing N-benzylpyrazole moiety (10d, 10j, 10p), and N- methylpyrazole moiety (10f, 10i, 10r) showed significant nematicidal activity against both tested nematodes with LD50 160-210 ppm, almost equal to oxamyl standard. Further, these compounds 10a-r were screened for their antifungal (MZI, MIC, and MFC) activity against four fungal organisms viz, Candida albicans (ATCC 102331), Aspergillus fumigates (HIC 6094), Trichophyton rubrum (IFO 9185) and Trichophyton mentagrophytes (IFO 40996). Most of the new compounds showed appreciable activity against the tested fungi, and emerged as potential molecules for further development. Keywords: Hybrid heterocyclics, click reaction, Knovenagel condensation, cyclisation, nematicidal activity, antifungal activity 1. Introduction 1,2,3-Triazoles are one of the most important classes of heterocyclic organic compounds, which are reported to be present in a plethora of biological activities in diverse therapeutic areas.1 The 1,2,3-triazole motif is associated with diverse pharmacological activities, such as antibacterial, antifungal, hypoglycemic, antihypertensive and analgesic properties. Polysubstituted five-membered aza heterocycles rank as the most potent glycosidase inhibitors.2 Further, this nucleus in combination (or linked) with various other classes of compounds, such as amino acids, steroids, aromatic compounds, carbohydrates etc., became prominent in having various pharmacological properties.3 1,2,3-Triazole modified carbohydrates have became easily available after the discovery of the Cu(I) catalyzed azide-alkynes 1,3-dipolar cycloaddition reaction4 and quickly became a pronounced class of non-nat- ural sugars. The chemistry and biology of triazole modified sugars is dominated by triazole glycosides.5 Therefore, the synthesis and investigation of biological activity of 1,2,3-triazole glycosides is an important objective, which also received a considerable attention by the medicinal chemists. Thiazoles are familiar group of heterocyclic compounds possessing a wide variety of biological activities and their utility as medicine is very much estabilished.6 Thiazole nucleus is also an integral part of all the available pencillins which have revolutionized the therapy of bacterial diseases.7 Furthurmore, there has been considerable interest in the chemistry of thiazolidine-4-one ring system which is the core structure in various synthetic pharmaceuticals displaying a broad spectrum of biological activi-ties.8-10 The thiazolidine-4- one ring also occurs in nature; thus actithiazic acid isolated from Streptomycis strains exhibits highly specific in vitro activity against mycobacteri- Srinivaset al.: Synthesis, Nematicidal and Antifungal Properties Acta Chim. Slov. 2017, 64, 1030-1041 1031 um Tuberculosis.11 Thiazolidine-4-ones are known to exhibit diverse bioactivities, such as antiplatelet activating factor,12 antihistaminic,13 COX inhibitory,14 Ca+2 channel blocking,15 PAF antagonist,16 cardioprotective,17 anti-isch-emic18 and nematicidal activities.19 Moreover, pyrazoles and their derivatives could be considered as possible antimicrobial agents.20 The other derivatives display antide-pressant,21 antiarthritic22 and cerebroprotecting23 properties. Some aryl pyrazoles were reported to be non-nucleo-side human immunodeficiency virus (HIV-1) reverse transcriptase inhibitor,24 COX-2 inhibitor,25-27 activator of the nitric oxide receptor and to have soluble guanylate cy-clase activity.28 Nematodes are tiny worms, some of them are plant parasites, and can play an important role in the predisposition of the host plant to the invansion by secondary pathogens.29 Plants attacked by nematodes show retarded growth and development, as well as loss in the quality and quantity of the harvest. The namaticides currently still in use are slated for reduction due to the environmental problems, and human and animals health concerns. For example, effective namaticides, such as dibromochloro-propane (DBCD) and ethylene dibromide (EDB) have been withdrawn from the market due to their deleterious effects on humans and the environment. Methyl bromide, the most effective and widely used fumigant for soil-borne pests including nematodes, has already been banned. The use of nonfumigant nematicides, based on or-ganophosphates and carbamates, is expected to increase concomitantly with the withdrawal of methyl bromide, which will bring about new environmental concerns. In fact, the highly toxic Aldicarb used to control insects and nematodes has been detected in ground water.30 Therefore, alternative nematode control methods or less toxic nemat-icides need to be developed.31 One way of searching for such nematicidal compounds is to screen naturally occurring compounds in plants. Several such compounds, e.g. alkaloids, sesquiterpenes, diterpenes, polyacetylenes have nematicidal activity.32-33 For example, a-terthienyl is a highly effective nematicidal compound. Other compounds with nematicidal activity have been isolated from plants, mainly from the family Asteraceae.32-33 However, compounds of plant origin and their analogs have not been developed into commercial nematicides yet; hence there is a need to develop commercial syntheses. Following the successful introduction of antimicrobial and nematicidal agents, inspired by the biological profile of triazoles, thiazolidinones, pyrazoles and their increasing importance in pharmaceutical and biological fields and in continuation of our work on biologically active molecules34-48 and in order to enhance the biological activity of triazoles, thiazolidinones and pyrazole moieties, it was thought to be of interest to accomodate tri-azole, thiazolidinones and pyrazole moieties in single molecular framework. In this article we report the synthesis of a new class of hybrid heterocycles 10a-g in good yields and their evaluation for in vitro antifungal and nemati-cidal activity. 2. Results and Discussion The key intermediate 8 required for the synthesis of title compound was prepared according to the procedure outlined in the Scheme 1. Diacetone D-glucose (1) prepared from D-(+)-glucose by treating with acetone in the presence of a catalytic amount of sulphuric acid according to the literature procedure,49 reduction of 2 (prepared by Swern oxidation of 1) with NaBH4 in aq. ethanol at 0 °C for 1 h gave 3 (77%), which on subsequent propargylation in DMF in the presence of NaH for 1 h afforded propargyl ether 4 (80%). Now the propargyl ether converted into tri-azole 5 (82%) by using 1,3-dipolar cycloaddition with p-chlorophenyl azide was carried out at ambient temperature in the presence of CuSO4 and sodium ascorbate in a mixture of 1:1 t-BuOH-H2O as reported by Sharpless. Acid hydrolysis of 5,6-acetonide 5 in 60% AcOH furnished the diol 6 (85%), which on oxidative cleavage with NaIO4 gave the aldehyde 7. Subsequently one-pot synthesis of tri-azole linked to thiazolidenone glycosides was carried out by the condensation reaction between 7, primary aromatic amine and a thioglycolic acid in the presence of ZnCl2 under microwave irradiation/conventional heating (Scheme 2). In the classical method, the reactions were performed in dry toluene at reflux for a long time (2-4 h), often leading to degradation processes and consequent low yields of isolated products, whereas the application of microwave assisted technology, the reaction is completed in only 5-10 minutes and the compounds, isolated by conventional work-up, are obtained in satisfactory yields, often higher Table 1. Synthesis of compounds 10a-r Compound R R1 Yield (%) 10a p-Me-C6H4- c6h5- 69 10b p-Me-C6H4- p-MeOC6H4- 70 10c p-Me-C6H4- P-Cl-C6H4- 67 10d p-Me-C6H4- CA-CH2- 72 10e p-Me-C6H4- (CH3VCH- 76 10f p-Me-C6H4- CH3- 66 10g P-q-c6h4- CA- 68 10h P-Cl-C6H4- p-MeOC6H4- 70 10i p-Cl-C6H4- P-Cl-C6H4- 76 10j P-Cl-C6H4- C6H5-CH2- 69 10k P-Cl-C6H4- (CH3VCH- 67 10l p-Cl-C6H4- CH3- 71 10m p-NO2-C6H4- CA- 74 10n p-NO2-C6H4- p-MeOC6H4- 76 10o P-NO2-C6H4- P-Cl-C6H4- 72 10p p-NO2-C6H4- C6H5-CH2- 74 10q P-N02-C6H4- (CH3VCH- 71 10r P-NO2-C6H4- CH3- 67 Srinivaset al.: Synthesis, Nematicidal and Antifungal Properties 1032 Acta Chim. Slov. 2017, 64, 1030-1041 124 î 3 '"«-V w'V W>v d'' "'oA cf "-(A Sheme 1 „-Po \ - 'Ha c;i Sheme 2 Cl N-n Kii-il R = a) 4-CH-CH b) 4-Cl-CH; c) 4-NO-C Hc ' 3 6 5 y 6 5 ' 2 6 5 Reagents and conditions: a) COCl2, CH2Cl2, Et3N, -78 °C rt, 1.5 h, 83%; b) NaBH4, EtOH, H2O, (192:1), 0 °C rt, 78%; c) Propargyl bromide, NaH, DMF, 0 oC rt; d) p-Chlorophenylazide, sodium ascorbate, CuSO4 ■ 5H2O, f-BuOH/H2O, 0 °C rt, 75%; e) 60%, AcOH, rt, 69%; f) NaIO4, CH2Cl2, 0 °C rt, 75%; g) Ar-NH2, SHCH2COOH, ZnCL toluene2, 80 °C, 85%; h) 4-F-C H-CHO, AcOH / 2, ' 6 4 NaOAc, reflux, 82-88%; i) R'-NHNH2 ■ HCl, AcOH/ NaOAc reflux, 76-67%. MA" than those achieved by traditional methods.35 Compound 8 was then reacted with p-fluorobenzaldehyde in the presence of anhydrous NaOAc in glacial AcOH at reflux temperature to give chalcone derivatives of triazole linked thi-azolidenone glycosides 9. Furthermore, the compounds upon cyclocondensation with alkyl/aryl hydrazines in the presence of anhydrous NaOAc in glacial AcOH at reflux temperature gave 10a-r in good yields. The versatility of the reaction is demonstrated by the fact that both aromatic hydrazines, such as phenyl hydrazines, and aliphatic hy-drazines, such as benzylhydrazine, isopropylhydrazine, methylhydrazine, afforded their corresponding 10a-r in good yields. The structures of synthesized compounds were confirmed by IR, NMR, MS and elemental analysis. Furthermore, the compounds were subjected to nemati-cidal and anti bacterial testing. 3. Antifungal Activity The newly prepared compounds 10a-r were screened for their antifungal activity against four fungal organisms, viz Candida albicans (ATCC 10231), Aspergillus fumigates (HIC 6094), Trichophyton rubrum (IFO 9185) and Trichophyton mentagrophytes (IFO 40996) in dimethyl sulfoxide (DMSO) by agar diffusion method.50 Amphotericin B was Srinivaset al.: Synthesis, Nematicidal and Antifungal Properties ... Acta Chim. Slov. 2017, 64, 1030-1041 1033 used as the standard drug and the zones of fungal inhibition values are reported in Table 3. In addition, the MIC and MFC values determined by the broth dilution method51 are recorded in Table 4. 4. Nematicidal Activity The compounds synthesized 10a-g in this study were also screened for their nematicidal activity against Dietylenchus myceliophagus and Caenorhabditis elegans by aqueous in vitro screening technique52 at various concentrations. The nematicidal activity of each tested compound was compared with the standard drug Levamisole. The results have been expressed in terms of LD i.e. median le- thal dose at which 50% of nematodes became immobile (dead). The screened data reveal that compounds 10e and 10f are the most effective against Dietylenchus myceliophagus and Caenorhabditis elegans with LD50 190 and 220 x 10-6 respectively, whereas the other tested compounds showed moderate activity. The LD50 values of the compounds screened are presented in Table 2. 5. Experimental Commercial grade reagents were used as supplied. Solvents except analytical reagent grade were dried and purified according to literature when necessary. Reaction progress and purity of the compounds were checked by Table 2. Median lethal dose LD50 (ppm) of compounds 10a-r Compd. D. myceliophagus C. elegans Compd. phagus D. mycelio- C. elegans 10a 850 670 10j 180 200 10b 950 870 10k 520 670 10c 240 360 10l 190 210 10d 190 210 10m 800 620 10e 550 600 10n 920 810 10f 160 200 10o 230 350 10g 810 650 10p 190 190 10h 900 820 10q 510 600 10i 290 380 10r 180 200 Oxamyl 150 180 Oxamyl 150 180 LD, median lethal dose (the concentration at which 50% nematodes became immobile) Table 3. Inhibitory zone diameters (mm) of 10a-r against tested fungal strains Compound Mean zone of inhibition (MZI) in mma C. albicans A. fumigatus T. rubrum T. mentagrophytes 10a 11 10 - 09 10b 09 15 13 12 10c 13 11 11 10 10d 22 16 15 16 10e - 13 - - 10f 20 20 20 18 10g - - 17 - 10h 14 - - - 10i - - 15 - 10j 21 17 14 16 10k 10 - 11 12 10l 18 15 13 16 10m 08 11 10 09 10n 13 - - - 10o 15 - - - 10p 19 16 11 13 10q - - 09 - 10r 21 14 15 14 Amphotericin B 25 20 20 18 Amphotericin B (100 ^g/disc) was used as the positive reference; compounds 10a-r (300 ^g/disc). - indicates no sensitivity or MZI lower that 7 mm. a Values are mean (n = 3). Srinivaset al.: Synthesis, Nematicidal and Antifungal Properties 1034 Acta Chim. Slov. 2017, 64, 1030-1041 Table 4. Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) in ^g/mL of compounds 10a-r 1 nmnn C. albicans A. fumigatus T. rubrum T. mentagrophytes VyUlllUU« MIC MFC MIC MFC MIC MFC MIC MFC 10a - - 25.0 25.0 25.0 50.0 25.0 25.0 10b 25.0 25.0 25.0 50.0 12.5 12.5 12.5 25.0 10c 12.5 25.0 6.25 12.5 6.25 12.5 6.25 12.5 10d 3.12 6.25 25.0 25.0 12.5 25.0 25.0 25.0 10e 12.5 25.0 - - - - - - 10f 3.12 3.12 3.12 6.25 3.12 6.25 6.25 12.5 10g 6.25 6.25 12.5 25.0 12.5 12.5 6.25 12.5 10h 25.0 50.0 - - 25.0 50.0 25.0 50.0 10i 12.5 12.5 25.0 25.0 12.5 12.5 25.0 50.0 10j 3.12 6.25 6.25 12.5 6.25 12.5 12.5 25.0 10k 12.5 25.0 25.0 25.0 - - 25.0 50.0 10l 12.5 25.0 6.25 50.0 6.25 25.0 - - 10m 12.5 25.0 12.5 50.0 6.25 25.0 12.5 12.5 10n 6.25 12.5 12.5 25.0 12.5 25.0 - - 10o 25.0 25.0 25.0 50.0 25.0 50.0 6.25 12.5 10p 12.5 25.0 - - - - - - 10q 25.0 25.0 25.0 50.0 12.5 25.0 12.5 25.0 10r 12.5 25.0 12.5 25.0 12.5 25.0 12.5 25.0 Amphotericin B 6.25 12.5 3.12 6.25 3.12 12.5 3.12 12.5 - Indicates fungi are resistant to the compound >100 ^g/mL concentration. thin-layer chromatography (TLC) on pre-coated silica gel F254 plates from Merck and compounds were visualized either by exposure to UV light or dipping in 1% aqueous potassium permanganate solution. Silica gel chromatographic columns (60-120 mesh) were used for separations. Microwave reactions were carried out in mini lab microwave catalytic reactor (ZZKD, WBFY-201). Optical rotations were measured on Perkin-Elmer 141 polarimeter by using a 2 mL cell with a path length of 1 dm with CHCl3 or CDCl3 as solvent. All melting points are uncorrected and measured using Fisher-Johns apparatus. IR spectra were recorded as KBr disks on a Perkin-Elmer FT IR spectrometer. The 1H NMR and 13C NMR spectra were recorded on a Varian Gemini spectrometer (300 MHz for 1H and 75 MHz for 13C). Chemical shifts are reported as 5 on ppm scale against TMS as the internal reference and coupling constants (J) are reported in Hz units. Mass spectra were recorded on a VG micro mass 7070H spectrometer. Elemental analyses (C, H, N) were determined by a Perkin-Elmer 240 CHN elemental analyzer and are within ±0.4% of theoretical values. 2-((3afl,5S,6S,6afl)-6-((1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2,2-dimethyltetrahydrofu-ro[2,3-d][1,3]dioxol-5-yl)-3-phenylthiazolidin-4-ones 8a-c. To a solution of diol 6 (0.200 g, 0.48 mmol) in CH2Cl2 (5 mL), NaIO4 (0.130 g, 0.61 mmol) was added at 0 °C and stirred at room temperature for 6 h. The reaction mixture was filtered and washed with CH2Cl2 (2 x 10 mL). It was dried (Na2SO4) and evaporated to give aldehyde 7 (0.150 g) in quantitative yield as a yellow liquid, which was used as such for the next reaction. To a stirred mixture of 7 (0.150 g, 0.395 mmol), aromatic amine (0.395 mmol) and anhydrous thioglycolic acid (0.160 g, 0.211 mmol) in dry toluene (5 mL), ZnCl2 (0.100g, 0.751 mmol) was added after 2 min and irradiated in microwave bath reactor at 280 W for 4-7 minutes at 110 °C. After cooling, the filtrate was concentrated to dryness under reduced pressure and the residue was tak-en-up in ethyl acetate. The ethyl acetate layer was washed with 5% sodium bicarbonate solution and finally with brine. The organic layer was dried over Na2SO4 and evaporated to dryness at reduced pressure. The crude product thus obtained was purified by column chromatography on silica gel (60-120 mesh) with hexane/ethyl acetate as the eluent. 2-((3afl,5S,6S,6afl)-6-((1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2,2-dimethyltetrahydrofu-ro[2,3-d][1,3]dioxol-5-yl)-3-p-tolylthiazolidin-4-one (8a) m.p. 191-193 °C; 1H NMR (300 MHz, CDCl3) 5 8.26 (d, J = 8.7 Hz, 2H, Ar-H), 8.04 (s,1H,Ar-H), 7.54 (d, J = 9.2 Hz, 2H, Ar-H), 7.39 (d, J = 8.33 Hz, 2H, Ar-H), 7.15 (d, J = 8.3 Hz, 2H, Ar-H), 5.76 (d, J = 3.6 Hz,1H, C1H), 4.96 (d, J = 5.2 Hz,1H, CH-S), 4.66 (t, J = 3.9 Hz,1H, C2H), 4.54 (s, 2H, OCH2), 3.96-3.91 (m, 1H,C4H), 3.76 (s, 2H, CH2), 3.26 (dd, J = 9.1 Hz, J2 = 4.2 Hz, Hi, C3H), 2.3 (s, 3H,CH3), 1.53 (s, 3H ,CH3), 1.36 (m, 3H,CH3); 13C NMR (75 MHz, CDCl3) 8 172.6, 143.2, 137.4, 133.6, 132.3, 131.2, 128.4, 127.9, 124.8,122.9, 119.2, 111.2, 103.8, 81.2, 78.1, 74.1, 65.9, 51.4, 26.1, 16.1; MS: m/z (M++Na) 565. Anal. Calcd for CH ClN O S: C, 57.51; H, 5.51; N, 10.32. Found: C, 26 2/ 4 5 57.32; H, 5.35; N, 10.09. Srinivaset al.: Synthesis, Nematicidal and Antifungal Properties ... Acta Chim. Slov. 2017, 64, 1030-1041 1035 3-(4-Chlorophenyl)-2-((3afl,5S,6S,6afl)-6-((1-(4-chlo-rophenyl)-1H-imidazol-4-yl)methoxy)-2,2-dimeth-yltetrahydrofuro[2,3-d ][1,3]dioxol-5-yl)thiazoli-din-4-one (8b) m.p. 216-218 °C; 1H NMR (300 MHz, CDCl3) 5 8.02 (s,1H,Ar-H), 7.50 (d, J = 9.2 Hz, 4H, Ar-H), 7.41 (d, J = 8.9 Hz, 4H,Ar-H), 5.72 (d, J = 3.6 Hz,1H, C1H), 4.94 (d, J = 5.2 Hz, CH-S), 4.60 (t, J = 3.9 Hz,1H, C2H), 4.51 (s, 2H, OCH2), 3.96-3.91 (m, 1H, C4H), 3.76 (s, 2H, CH2), 3.31 (dd, J = 9.1 Hz, J2 = 4.2 Hz, 1H, C3H), 1.55 (s, 3H, CH3), 1.32 (m, 3H, CH3); 13C NMR (75 MHz, CDCl3) 5 170.6, 139.4, 134.8, 133.23, 129.4, 128.6, 125.6, 122.2, 119.4, 111.2, 104.9, 81.5, 74.5, 66.3, 52.6, 34.6, 26.5; MS: m/z (M++H) 563. Anal. Calcd for C25H24Cl2N4O5S: C, 53.29; H, 4.29; N, 9.94. Found: C, 53.21; H, 4.16; N, 9.83. 2-((3afl,5S,6S,6afl)-6-((1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2,2-dimethyltetrahydrofu-ro[2,3-d][1,3]dioxol-5-yl)-3-(4-nitrophenyl)thiazoli-din-4-one (8c) m.p. 201-205 °C; 1H NMR (300 MHz, CDCl3) 5 8.26 (d, J = 8.7 Hz, 2H), 8.04 (s, 1H, Ar-H), 7.51 (d, J = 9.2 Hz, 2H, Ar-H), 7.42 (d, J = 8.5 Hz, 2H, Ar-H), 6.82 (d, J = 9.8 Hz, 2H, Ar-H), 5.71 (d, J = 3.6 Hz, 1H, C1H), 4.96 (d, J = 5.2 Hz, CH-S), 4.62 (t, J = 3.9 Hz,1H, C2h), 4.53 (s, 2H, OCH2), 3.96-3.91 (m, 1H,C4H), 3.76 (s, 2H, CH2), 3.28 (dd, J = 9.1 Hz, J2 = 4.2 Hz, 1H, C3H), 1.52 (s, 3H,CH3), 1.34 (m, 3H,CH3); 13C NMR (75 MHz, CDCl3) 5 170.6, 147.5, 144.4, 143.2, 134.8, 131.2, 128.6, 124.6, 122.4, 119.8, 111.8, 104.9, 81.5, 78.2, 74.8, 66.9, 52.4, 34.6, 26.8; MS: m/z (M++H) 574. Anal. Calcd for C25H24ClN5O7S: C, 52.31; H, 4.21; N, 12.20. Found: C, 52.26; H, 4.19; N, 12.11. (Z)-5-Benzylidene-2-((3afl,5S,6S,6afl)-6-((1-(4-chloro-phenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2,2-dimeth-yltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)-3-phenylthi-azolidin-4-ones 9a-c. A mixture of compound 8 (0.01 mol), p-fluorobenzaldehyde (0.02 mol) and sodium acetate (0.01 mol) in anhydrous glacial acetic acid (20 mL), was refluxed for 3 h. The reaction mixture was concentrated and then poured into ice cold water, the solid thus separated was filtered, washed with water and crystallized from glacial acetic acid to afford pure compounds. (Z)-2-((3afl,5S,6S,6afl)-6-((1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2,2-dimethyltetrahydrofu-ro[2,3-d][1,3]dioxol-5-yl)-5-(4-fluorobenzylidene)-3-p-tolylthiazolidin-4-one (9a). This compound was obtained as brown solid, m.p. 231-235 °C; 1H NMR (300 MHz, CDCl3) 5 8.22 (d, J = 8.7 Hz, 2H, Ar-H), 8.06 (s, 1H, Ar-H), 7.84 3s, 1H, CH =C), 7.54 (d, J = 9.2 Hz, 2H, Ar-H), 7.39 (d, J = 8.33 Hz, 2H, Ar-H), 7.15 (d, J = 8.3 Hz, 2H, Ar-H), 6.91-6.87 (m, 5H, Ar-H and CH-S), 5.76 (d, J = 3.6 Hz, 1H, C1H), 4.66 (t, J = 3.9 Hz,1H, C2H), 4.54 (s, 2H, OCH2), 3.96-33.91 (m, 1H,C4H), 3.26 (dd, J = 9.1 Hz, J2 = 4.2 Hz, 1H, C3H), 2.3 (s, 3H,CH3), 1.53 (s, 3H,CH3), 1.36 (m, 3H,CH3); 13C NMR (75 MHz, CDCl3) 5 172.6, 143.2, 137.4, 133.6, 132.3, 131.2, 128.4, 127.9, 124.8, 122.9, 119.2, 111.2, 103.8, 81.2, 78.1, 74.1, 65.9, 51.4, 26.1, 16.1; MS: m/z (M++H) 649. Anal. Calcd for C H nClFN„O S: C, 61.06; H, 33 30 4 5 4.66; N, 8.63. Found: C, 60.82; H, 4.45; N, 8.43. (Z)-3-(4-Chlorophenyl)-2-((3afl,5S,6S,6afl)-6-((1-(4-chlorophenyl)-1H- 1,2,3-triazol-4-yl)methoxy)-2,2-di-methyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)-5-(4-flu-orobenzylidene)thiazolidin-4-one (9b). The compound was obtained as dark yellow solid, m.p. 232-235 °C; 1H NMR (300 MHz, DMSO-d6) 5 8.06 (s, 1H, Ar-H), 7.82 (s, 1H, CH =C), 7.52 (d, J = 9.2 Hz, 4H, Ar-H), 7.43 (d, J = 8.9 Hz, 4H,Ar-H), 6.91-6.87 (m, 5H, Ar-H and CH-S), 5.72 (d, J = 3.6 Hz, 1H, C1H), 4.60 (t, J = 3.9 Hz,1H, C2H), 4.51 (s, 2H, OCH2), 3.96-3.91 (m, 1H, C4H), 3.31 (dd, J = 9.1 Hz, J2 = 4.2 Hz, 1H, C3H), 1.55 (s, 3H, CH3), 1.32 (m, 3H, CH3); 13C NMR (75 MHz, DMSO-dfi) 5 170.6, 138.4, 134.8, 13332, 130.8, 129.4, 128.6, 124.6, 122.2, 119.4, 111.2, 104.9, 81.5, 74.5, 66.3, 52.6, 34.6, 26.5; MS: m/z (M++H) 669. Anal. Calcd for C32H27Cl2FN4O5S: C, 57.40; H, 4.06; N, 8.37. Found: C, 57.331; Hi, 4.01; N, 8.03. (Z)-2-((3afl,5S,6S,6afl)-6-((1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2,2-dimethyltetrahydrofu-ro[2,3-d][1,3]dioxol-5-yl)-5-(4-fluorobenzylidene)-3-(4-nitrophenyl)thiazolidin-4-one (9c). The compound was obtained as brown solid, m.p. 216-218 °C; 1H NMR (300 MHz, DMSO-d6) 5 8.26 (d, J = 8.7 Hz, 2H), 8.04 (s,1H, Ar-H), 7.84 (s, 1H, CH =C), 7.51 (d, J = 9.2 Hz, 2H, Ar-H), 7.42 (d, J = 8.5 Hz, 2H,Ar-H), 6.91-6.87 (m, 5H, Ar-H and CH-S), 6.82 (d, J = 9.8 Hz, 2H, Ar-H), 5.71 (d, J = 3.6 Hz, 1H, C1H), 4.62 (t, J = 3.9 Hz, 1H, C2H), 4.53 (s, 2H, OCH2), 3.96-3.91 (m, 1H,C4H), 3.76 (s, 2H, CH2), 3.28 (dd, J = 9.1 Hz, J2 = 4.2 Hz, 1H, C3H), 1.52 (s, 3H,CH3), 1.34 (m, 3H,CH3); 13C NMR (75 MHz, CDCl3) 5 170.6, 147.5, 144.4, 143.2, 138.4, 134.8, 130.6, 131.2, 128.6, 124.6, 122.4, 119.8, 111.8, 104.9, 81.5, 78.2, 74.8, 66.9, 52.4, 26.8; MS: m/z (M++Na) 692. Anal. Calcd for C31H29ClFN5O7S: C, 55.56; H, 4.36; N, 10.30. Found: C, 55.26; H, 4.29; N, 10.10. 5-((3afl,5S,6S,6afl)-6-((1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2,2-dimethyltetrahydrofu-ro[2,3-d][1,3]dioxol-5-yl)-3-(4-fluorophenyl)-2,6-di-phenyl/ 2-alkyl-6-phenyl-3,3a,5,6-tetrahydro-2H-pyra-zolo[3,4-d]thiazoles 10a-r. A mixture of compound 9 (5 mmol), alkyl/aryl hydrazine (5 mmol) and anhydrous sodium acetate (5 mmol) in glacial acetic acid (20 mL), was refluxed for 7 h. The reaction mixture was concentrated and cooled to room temperature, the solid thus separated was filtered, washed thoroughly with water. The crude product thus obtained was purified by column chromatography on silica gel with hexane/ethyl acetate as eluent to afford pure compounds. 5-((3afl,5S,6S,6afl)-6-((1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2,2-dimethyltetrahydrofu- Srinivaset al.: Synthesis, Nematicidal and Antifungal Properties ... 1036 Acta Chim. Slov. 2017, 64, 1030-1041 ro[2,3-d][1,3]dioxol-5-yl)-3-(4-fluorophenyl)-2-phenyl-6-p-tolyl-3,3a,5,6-tetrahydro-2H-pyrazolo[3,4-d]thi-azole (10a). This was obtained by reacting compound 9a (1 g) and phenylhydrazine (0.25 g) as described in the typical procedure and isolated as a brown solid, yield 72%, m.p. 248-250 °C; 1H NMR (300 MHz, DMSO-d.) 5 8.20 (d, J = 8.7 Hz, 2H, Ar-H), 8.04 (s, 1H, Ar-H), 7.51 (d, J = 9.2 Hz, 2H, Ar-H), 7.40 (s, 1H, CH-S), 7.29 (d, J = 8.33 Hz, 2H, Ar-H), 7.18-7.15 (m, 5H, Ar-H), 7.05 (d, J = 8.3 Hz, 2H, Ar-H), 6.91-6.87 (m, 4H, Ar-H), 5.76 (d, J = 3.6 Hz,1H, C1H), 5.62 (d, J = 2.2 Hz, 1H, S-CH), 5.25 (d, J = 2.2 Hz, 1H, CH-N), 4.66 (t, J = 3.9 Hz,1H, C2H), 4.54 (s, 2H, OCH2), 3.96-3.91 (m, 1H, C4H), 3.26 (dd, J( = 9.1 Hz, J2 = 4.2 Hz, 1H, C3H), 2.3 (s, 3H,CH3), 1.53 (s, 3H,CH3), 1.36 (m, 3H, CH3); 13C NMR (75 MHz, DMSO-d.) 5 172.6, 143.2, 141.4, 137.4, 133.6, 132.3, 131.2, 129.3, 1225.3, 128.4, 127.9, 124.8, 122.9, 119.2, 111.2, 103.8, 81.2, 78.1, 74.1, 65.9, 51.4, 26.1, 21.3, 16.1; MS: m/z (M++Na) 761. Anal. Calcd for C,.H,,ClFN,O.S: C, 63.36; H, 4.90; N, 11.37. 39 36 6 4 ' ' ' ' ' Found: C, 63.12; H, 4.75; N, 11.29. 5-((3a.,5S,6S,6a.)-6-((1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2,2-dimethyltetrahydrofu-ro[2,3-d][1,3]dioxol-5-yl)-3-(4-fluorophenyl)-2-(4-me-thoxyphenyl)-6-p-tolyl-3,3a,5,6-tetrahydro-2H-pyra-zolo[3,4-d]thiazole (10 b). This was obtained by reacting compound 9a (1 g) and p-methoxyphenylhydrazine (0.35 g) as described in the typical procedure and isolated as a yellow solid, yield 62%, m.p. 268-272 °C; 1H NMR (300 MHz, DMSO-d6) 5 8.18 (d, J = 8.7 Hz, 2H, Ar-H), 8.04 (s, 1H, Ar-H), 7.51 (d, J = 9.2 Hz, 2H, Ar-H), 7.40 (s, 1H, CH-S), 7.29 (d, J = 8.33 Hz, 2H, Ar-H), 7.12-7.10 (m, 4H, Ar-H), 7.05 (d, J = 8.3 Hz, 2H, Ar-H), 6.91-6.87 (m, 4H, Ar-H), 5.76 (d, J = 3.6 Hz,1H, C1H), 5.62 (d, J = 2.2 Hz, 1H, S-CH), 5.25 (d, J = 2.2 Hz, 1H, CH-N), 4.66 (t, J = 3.9 Hz,1H, C2H), 4.54 (s, 2H, OCH2), 3.96-3.94 (m, 1H,C4H), 3.91 (s, 3H,OMe), 3.26 (dd, J( = 9.1 Hz, J2 = 4.2 Hz, 1H, C3H), 2.3 (s, 3H,CH3), 1.53 (s, 3H, CH3), 1.36 (m, 3H, CH3); 13C NMR (75 MHz, DMSO-d.) 5 172.6, 151.7, 143.2, 141.4, 137.4, 133.6, 132.3, 131.2, 129.3, 125.3, 128.4, 127.9, 124.8, 122.9, 119.2, 111.2, 103.8, 81.2, 78.1, 74.1, 65.9, 55.8, 51.4, 26.1, 21.3, 16.1; MS: m/z (M++H) 789. Anal. Calcd for C„„H ClFNOS: C, 62.46; H, 4.98; N, 10.92. Found: C, 40 38 6 5 ' ' 62.12; H, 4.88; N, 10.87. 2-(4-Chlorophenyl)-5-((3a.,5S,6S,6a.R)-6-((1-(4-chlo-rophenyl)-1 H-1,2,3-triazol-4-yl)methoxy)-2,2-dimeth-yltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)-3-(4-fluoro-phenyl)-6-p-tolyl-3,3a,5,6-tetrahydro-2H -pyra-zolo[3,4-d]thiazole (10c). This was obtained by reacting compound 9a (1 g) and p-chlorophenylhydrazine (0.45 g) as described in the typical procedure and isolated as a brown solid, yield 76%, m.p. 248-251 °C; 1H NMR (300 MHz, DMSO-d) 5 8.21 (d, J = 8.7 Hz, 2H, Ar-H), 8.09 (s,1H,Ar-H), 7.515 (d, J = 9.2 Hz, 2H, Ar-H), 7.42 (s, 1H, CH-S), 7.29 (d, J = 8.33 Hz, 2H, Ar-H), 7.10-7.08 (m, 4H, Ar-H), 7.05 (d, J = 8.3 Hz, 2H, Ar-H), 6.91-6.87 (m, 4H, Ar-H), 5.76 (d, J = 3.6 Hz, 1H, C1H), 5.62 (d, J = 2.2Hz, 1H, S-CH), 5.25 (d, J = 2.2 Hz, 1H, (CH-N), 4.66 (t, J = 3.9 Hz, 1H, C2H), 4.54 (s, 2H, OCH2), 3.96-3.94 (m, 1H, C4H), 3.26 (dd, Jt = 9.1 Hz, J2 = 4.2 Hz, 1H, C3H), 2.3 (s, 3H,CH3), 1.53 (s, 3H, CH3), 1.36 (m, 3H, CH3); 13C NMR (75 MHz, DMSO-d.) 5 1722.6, 151.7, 143.2, 137.4, 133.6, 132.3, 131.2, 129.3, 126.3, 125.3, 128.4, 127.9, 124.8, 122.9, 119.2, 111.2, 103.8, 81.2, 78.1, 74.1, 65.9, 51.4, 26.1, 21.3, 16.1; MS: m/z (M++ H) 773. Anal. Calcd for C H Cl FN O S: C, 60.54; 39 35 2 6 4 H, 4.58; N, 10.86. Found: C, 60.32; H, 4.28; N, 10.47. 2-Benzyl-5-((3a.,5S,6S,6a.)-6-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2,2-dimethyltetrahy-drofuro[2,3-d][1,3]dioxol-5-yl)-3-(4-fluorophenyl)-6-p-tolyl-3,3a,5,6-tetrahydro-2H-pyrazolo[3,4-d]thiazole (10d). This was obtained by reacting compound 9a (1 g) and benzylhydrazine (0.65 g) as described in the typical procedure and isolated as a brown solid, yield 66%. m.p. 288-291°C; 1H NMR (300 MHz, DMSO-d.) 5 8.21 (d, J = 8.7 Hz, 2H, Ar-H), 8.09 (s, 1H, Ar-H), 7.55 (d, J = 9.2 Hz, 2H, Ar-H), 7.42 (s, 1H, CH-S), 7.29 (d, J = 8.33 Hz, 2H, Ar-H), 7.10-7.08 (m, 5H, Ar-H), 7.05 (d, J = 8.3 Hz, 2H, Ar-H), 6.91-6.87 (m, 4H, Ar-H), 5.92 (s, 2H, CH2Ph), 5.76 (d, J = 3.6 Hz, 1H, C1H), 5.62 (d, J = 2.2 Hz, 1H, S-CH), 5.25 (d, J = 2.2 Hz, 1H, CH-N), 4.66 (t, J = 3.9 Hz, 1H, C2H), 4.54 (s, 2H, OCH2), 3.96-3.94 (m, 1H, C4H), 3.26 (dd, J( = 9.1 Hz, J2 = 4.2 Hz, 1H, C3H), 2.3 (s, 3H, CH3), I.53 (s, 3H, CH3), 1.36 (m, 3H, CH3); 13C NMR (75 MHz, DMSO-d.) 5 171.6, 151.6, 143.2, 137.4, 133.6, 132.3, 131.2, 129.3, 125.3, 128.4, 127.9, 124.8, 122.9, 119.2, 111.2, 103.8, 81.2, 78.1, 74.1, 65.9, 58.1, 51.4, 26.1, 21.3, 16.1; MS: m/z (M++ H) 753. Anal. Calcd for C H ClFN O S: C, 63.78; 40 38 6 4 H, 5.08; N, 11.16. Found: C, 63.52; H, 4.96; N, 10.97. 5-((3a.,5S,6S,6a.)-6-((1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2,2-dimethyltetrahydrofu-ro[2,3-d][1,3]dioxol-5-yl)-3-(4-fluorophenyl)-2-isopro-pyl-6-p-tolyl-3,3a,5,6-tetrahydro-2H-pyrazolo[3,4-d] thiazole (10e). This was obtained by reacting compound 9a (1 g) and isopropylhydrazine (0.35 g) as described in the typical procedure and isolated as a yellow solid, yield 76%, m.p. 248-251 °C; 1H NMR (300 MHz, DMSO-d.) 5 8.19 (d, J = 8.7 Hz, 2H, Ar-H), 8.06 (s, 1H, Ar-H), 7.55 (d, J = 9.2 Hz, 2H, Ar-H), 7.42 (s, 1H, CH-S) 7.29 (d, J = 8.33 Hz, 2H, Ar-H), 7.05 (d, J = 8.3 Hz, 2H, Ar-H), 6.91-6.87 (m, 4H, Ar-H), 5.76 (d, J = 3.6 Hz, 1H, C1H), 5.62 (d, J = 2.2 Hz, 1H, S-CH), 5.25 (d, J = 2.2 Hz, 1H, CH-N), 4.66 (t, J = 3.9 Hz, 1H, C2H), 4.54 (s, 2H, OCH2), 3.96-3.94 (m, 1H, C4H), 3.26 (dd, Jt = 9.1 Hz, J2 = 4.2 Hz, 1H, C3H), 2.802.78 (m, 1H, CH), 2.3 (s, 3H,CH3), 1.53 (s, 3H, CH3), 1.36 (m, 3H, CH3), 0.96 (d, J = 6.3 Hz, 6H, 2 x CH3); 13C NMR (75 MHz, DMSO-d.) 5 171.6, 151.6, 143.2, 137.4, 133.6, 132.3, 131.2, 129.3, 128.4, 127.9, 124.8, 122.9, 119.2, 111.2, 103.8, 81.2, 78.1, 74.1, 65.9, 58.1, 51.4, 50.6, 26.1, 21.3, 16.1; MS: m/z (M++ H) 705. Anal. Calcd for Srinivaset al.: Synthesis, Nematicidal and Antifungal Properties ... Acta Chim. Slov. 2017, 64, 1030-1041 1037 C H saFNAS: C, 61.31; H, 5.43; N, 11.92. Found: C, 36 38 6 4 ' ' 60.98; H, 5.26; N, 11.77. 5-((3a.R,5S,6S,6a.R)-6-((1-(4-Chlorophenyl)-1.H-1,2,3-triazol-4-yl)methoxy)-2,2-dimethyltetrahydrofu-ro[2,3-d][1,3]dioxol-5-yl)-3-(4-fluorophenyl)-2-meth-yl-6-p-tolyl-3,3a,5,6-tetrahydro-2H-pyrazolo[3,4-d]thi-azole (10 f). This was obtained by reacting compound 9a (1 g) and methylhydrazine (0.25 g) as described in the typical procedure and isolated as a yellow solid, yield 66%, m.p. 228-231 °C; 1H NMR (300 MHz, DMSO-d.) 5 8.19 (d, J = 8.7 Hz, 2H, Ar-H), 8.06 (s, 1H, Ar-H), 7.55 (d, J = 9.2 Hz, 2H, Ar-H), 7.42 (s, 1H, CH-S), 7.29 (d, J = 8.33 Hz, 2H, Ar-H), 7.05 (d, J = 8.3 Hz, 2H, Ar-H), 6.91-6.87 (m, 4H, Ar-H), 5.76 (d, J = 3.6 Hz, 1H, C1H), 5.62 (d, J = 2.2 Hz, 1H, S-CH), 5.25 (d, J = 2.2 Hz, 1H, CH-N), 4.66 (t, J = 3.9 Hz, 1H, C2H), 4.54 (s, 2H, OCH2), 3.96-3.94 (m, 1H, C4H), 3.71 (s, 3H, N-CH3), 3.26 (dd, J = 9.1 Hz, J2 = 4.2 Hz, 1H, C3H), 2.3 (s, 3H, CH3), 1.53 (s, 3H, CH3), 1.36 (m, 3H, CH3); 13C NMR (75 MHz, DMSO-d.) 5 171.6, 151.6, 143.2, 137.4, 133.6, 132.3, 131.2, 129.3, 128.4, 127.9, 124.8, 122.9, 119.2, 111.2, 103.8, 81.2, 78.1, 74.1, 65.9, 58.1, 51.4, 39.3, 26.1, 16.1; MS: m/z (M++ Na+) 689. Anal. Calcd for C H ClFN o„S: C, 60.30; H, 5.06; N, 12.42. Found: C, 34 34 6 4 60.22; H, 4.96: N, 12.22. 6-(4-Chlorophenyl)-5-((3a.R,5S,6S,6a.R)-6-((1-(4-chlo-rophenyl)-1 H-1,2,3-triazol-4-yl)methoxy)-2,2-dimeth-yltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)-3-(4-fluoro-phenyl)-2-phenyl-3,3a,5,6-tetrahydro-2H-pyra-zolo[3,4-d]thiazole (10g). This was obtained by reacting compound 9b (1 g) and phenylhydrazine (0.25 g) as described in the typical procedure and isolated as a brown solid, yield 65%, m.p. 249-251°C; 1H NMR (300 MHz, DMSO-d.) 5 8.16 (d, J = 8.7 Hz, 2H, Ar-H), 8.02 (s, 1H, Ar-H), 7.49 (d, J = 9.2 Hz, 2H, Ar-H), 7.36 (s, 1H, CH-S), 7.22 (d, J = 8.33 Hz, 2H, Ar-H), 7.13-7.11 (m, 5H, Ar-H), 7.02 (d, J = 8.3 Hz, 2H, Ar-H), 6.81-6.77 (m, 4H, Ar-H), 5.66 (d, J = 3.6 Hz, 1H, C1H), 5.58 (d, J = 2.2 Hz, 1H, 5-CH), 5.21 (d, J = 2.2 Hz, 1H, CH-N), 4.62 (t, J = 3.9 Hz, IH, C2H), 4.51 (s, 2H, OCH2), 3.94-3.91 (m, 1H, C4H), 3.26 (dd, J = 9.1 Hz, J2 = 4.2 Hz, 1H, C3H), 1.53 (s, 3H, CH3), 1.36 (m, 3H, CH3); 13C NMR (75 MHz, DMSO-d.) 5 17436, 143.0, 141.4, 137.4, 133.6, 132.3, 131.2, 129.3, 125.3, 128.4, 127.9, 126.1, 124.8, 122.9, 119.2, 111.2, 103.8, 81.2, 78.1, 74.1, 65.9, 51.4, 26.1, 16.1; MS: m/z (M++H) 759. Anal. Calcd for C H ClFN O S: C, 60.08; H, 4.38; N, 39 36 6 4 II.07. Found: C, 60.02; H, 4.11; N, 10.95. 6-(4-Chlorophenyl)-5-((3afl,5S,6S,6afl)-6-((1-(4-chlo-rophenyl)-1 H-1,2,3-triazol-4-yl)methoxy)-2,2-dimeth-yltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)-3-(4-fluoro-phenyl)-2-(4-methoxyphenyl)-3,3a,5,6-tetrahy-dro-2H-pyrazolo[3,4-d]thiazole (10h). This was obtained by reacting compound 9b (1 g) and p-methoxy-phenylhydrazine (0.35 g) as described in the typical proce- dure and isolated as a yellow solid, yield 68%, m.p. 274-276 °C; 1H NMR (300 MHz, DMSO-d.) 5 8.18 (d, J = 8.7 Hz, 2H, Ar-H), 8.04 (s, 1H, Ar-H), 7.51 (d, J = 9.2 Hz, 2H, Ar-H), 7.40 (s, 1H, CH-S), 7.29 (d, J = 8.33 Hz, 2H, Ar-H), 7.12-7.10 (m, 4H, Ar-H), 7.05 (d, J = 8.3 Hz, 2H, Ar-H), 6.91-6.87 (m, 4H, Ar-H), 5.76 (d, J = 3.6 Hz, 1H, c1h), 5.62 (d, J = 2.2 Hz, 1H, S-CH), 5.25 (d, J = 2.2 Hz, 1H, CH-N), 4.66 (t, J = 3.9 Hz, 1H, C2H), 4.54 (s, 2H, OCH2), 3.96-3.94 (m, 1H, C4H), 3.91 (s, 3H, OMe), 3.26 (dd, J = 9.1 Hz, J2 = 4.2 Hz, 1H, C3H), 1.53 (s, 3H,CH3), 1.36 (m, 3H, CH3); 13C NMR (75 MHz, DMSO-d.) 5 172.6, 151.7, 143.2, 141.4, 137.4, 133.6, 132.3, 131.2, 129.3, 126.1, 125.3, 128.4, 127.9, 124.8, 122.9, 119.2, 111.2, 103.8, 81.2, 78.1, 74.1, 65.9, 55.8, 51.4, 26.1, 16.1; MS: m/z (M++H) 789. Anal. Calcd for C H Cl FN O S: C, 59.32; H, 4.47; N, 39 35 2 6 5 10.64. Found: C, 58.92; H, 4.18; N, 10.47. 2,6-Bis(4-chlorophenyl)-5-((3a.R,5S,6S,6a.R)-6-((1-(4-chlorophenyl)-1H- 1,2,3-triazol-4-yl)methoxy)-2,2-di-methyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)-3-(4-flu-orophenyl)-3,3a,5,6-tetrahydro-2H-pyrazolo[3,4-d]thi-azole (10i). This was obtained by reacting compound 9b (1 g) and p-chlorophenylhydrazine (0.45 g) as described in the typical procedure and isolated as a brown solid, yield 76%, m.p. 268-271 °C; 1H NMR (300 MHz, DMSO-d.) 5 8.23 (d, J = 8.7 Hz, 2H, Ar-H), 8.11 (s, 1H, Ar-H), 7.58(d, J = 9.2 Hz, 2H, Ar-H), 7.46 (s, 1H, CH-S), 7.29 (d, J = 8.33 Hz, 2H, Ar-H), 7.11-7.09 (m, 4H, Ar-H), 7.07 (d, J = 8.3 Hz, 2H, Ar-H), 6.91-6.87 (m, 4H, Ar-H), 5.76 (d, J = 3.6 Hz, 1H, C1H), 5.62 (d, J = 2.2 Hz, 1H, S-CH), 5.25 (d, J = 2.2 Hz, 1H, CH-N), 4.66 (t, J = 3.9 Hz, 1H, C2H), 4.54 (s, 2H, OCH2), 3.96-3.94 (m, 1H, C4H), 3.26 (dd, J = 9.1 Hz, J2 = 4.2 Hz, 1H, C3H), 1.53 (s, 3H, CH3), 1.36 (m, 3H, CH3); 13C NMR (75 MHz, DMSO-d.) 5 172.6, 151.7, 143.2, 137.4, 133.6, 132.3, 131.2, 129.3, 12(5.3, 125.3, 128.4, 127.9, 124.8, 122.9, 119.2, 111.2, 103.8, 81.2, 78.1, 74.1, 65.9, 51.4, 26.1, 16.1; MS: m/z (M++ Na) 815. Anal. Calcd for C,aH„Cl FNO.S: C, 57.47; H, 4.08; N, 10.56. Found: C, 38 32 3 6 4 57.22; H, 3.95; N, 10.32. 2-Benzyl-6-(4-chlorophenyl)-5-((3afl,5S,6S,6afl)-6-((1-(4-chlorophenyl) - 1H-1,2,3-triazol-4-yl)methoxy)-2,2-dimethyltetrahydrofuro[2,3-d ][1,3]dioxol-5-yl) -3-(4-fluorophenyl)-3,3a,5,6-tetrahydro-2H-pyra-zolo[3,4-d]thiazole (10j). This was obtained by reacting compound 9b (1 g) and benzylhydrazine (0.65 g) as described in the typical procedure and isolated as a brown solid, yield 71%, m.p. 258-261 °C; 1H NMR (300 MHz, DMSO-d.) 5 8.25 (d, J = 8.7 Hz, 2H, Ar-H), 8.11 (s, 1H, Ar-H), 7.59 (d, J = 9.2 Hz, 2H, Ar-H), 7.46 (s, 1H, CH-S), 7.32 (d, J = 8.33 Hz, 2H, Ar-H), 7.12-7.10 (m, 5H, Ar-H), 7.08 (d, J = 8.3 Hz, 2H, Ar-H), 6.96-6.90 (m, 4H, Ar-H), 5.96 (s, 2H, CH2Ph), 5.77 (d, J = 3.6 Hz, 1H, C1H), 5.65 (d, J = 2.2 Hz, 1H, S-CH), 5.28 (d, J = 2.2 Hz, 1H, (CH-N), 4.66 (t, J = 3.9 Hz, 1H, C2H), 4.54 (s, 2H, OCH2), 3.96-3.94 (m, 1H, C4H), 3.26 (dd, J = 9.1 Hz, J2 = 4.2 Hz, 1H, C3H), 1.53 Srinivaset al.: Synthesis, Nematicidal and Antifungal Properties 1038 Acta Chim. Slov. 2017, 64, 1030-1041 (s, 3H, CH3), 1.36 (m, 3H, CH3); 13C NMR (75 MHz, DMSO-d6) S 171.6, 151.6, 143.2, 137.4, 133.6, 132.3, 131.2, 129.3, 125.3, 128.4, 127.9, 124.8, 122.9, 119.2, 111.2, 103.8, 81.2, 78.1, 74.1, 65.9, 58.1, 51.4, 26.1, 16.1; MS: m/z (M++ H) 773. Anal. Calcd for C H CLFNAS: C, 60.54; H, ' 39 35 2 6 4 4.56; N, 10.86. Found: C, 60.34; H, 4.36; N, 10.37. 6-(4-Chlorophenyl)-5-((3a.R,5S,6S,6a.R)-6-((1-(4-chlo-rophenyl)-1 H-1,2,3-triazol-4-yl)methoxy)-2,2-dimeth-yltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)-3-(4-fluoro-phenyl)-2-isopropyl-3,3a,5,6-tetrahydro-2H-pyra-zolo[3,4-d]thiazole (10k). This was obtained by reacting compound 9b (1 g) and isopropylhydrazine (0.35 g) as described in the typical procedure and isolated as a yellow solid, yield 69%, m.p. 238-239 °C; 1H NMR (300 MHz, DMSO-d6) S 8.32 (d, J = 8.7 Hz, 2H, Ar-H), 8.16 (s,1H,Ar-H), 7.65 (d, J = 9.2 Hz, 2H, Ar-H), 7.52 (s, 1H, CH-S), 7.39 (d, J= 8.33 Hz, 2H, Ar-H), 7.09 (d, J = 8.3 Hz, 2H, Ar-H), 6.95-6.92 (m, 4H, Ar-H), 5.76 (d, J = 3.6 Hz, 1H, C1H), 5.62 (d, J = 2.2 Hz, 1H, S-CH), 5.25 (d, J = 2.2 Hz, Hi, CH-N), 4.66 (t, J = 3.9 Hz, 1H, C2H), 4.54 (s, 2H, OCH2), 3.96-3.94 (m, 1H, C4H), 3.26 (dd, J) = 9.1 Hz, J2 = 4.2 Hz, 1H, C3H), 2.80-2.78 (m, 1H, CH), 1.53 (s, 3H, CH3), 1.36 (m, 3H, CH3), 0.96 (d, J = 6.3 Hz, 6H, 2xCH3); 13C NMR (75 MHz, DMSO-d.) S 171.6, 151.6, 143.2, 137.4, 133.6, 132.3, 131.2, 129.3, 128.4, 127.9, 124.8, 122.9, 119.2, 111.2, 103.8, 81.2, 78.1, 74.1, 65.9, 58.1, 51.4, 50.6, 26.1, 16.1; MS: m/z (M++ H) 725. Anal. Calcd for C H„Cl FN o„S: C, 57.93; H, 4.86; N, 11.58. Found: C, 35 35 2 6 4 57.72; H, 4.66; N, 11.46. 6-(4-Chlorophenyl)-5-((3afl,5S,6S,6afl)-6-((1-(4-chlo-rophenyl)-1 H-1,2,3-triazol-4-yl)methoxy)-2,2-dimeth-yltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)-3-(4-fluoro-phenyl)-2-methyl-3,3a,5,6-tetrahydro-2H-pyra-zolo[3,4-d]thiazole (10l). This was obtained by reacting compound 9b (1 g) and methylhydrazine (0.25 g) as described in the typical procedure and isolated as a yellow solid, yield 76%, m.p. 241-243 °C; 1H NMR (300 MHz, DMSO-d6) S 8.29 (d, J = 8.7 Hz, 2H, Ar-H), 8.16 (s,1H, Ar-H), 7.59 (d, J = 9.2 Hz, 2H, Ar-H), 7.48 (s, 1H, CH-S), 7.39 (d, J = 8.33 Hz, 2H, Ar-H), 7.09 (d, J = 8.3 Hz, 2H, Ar-H), 6.91-6.88 (m, 4H, Ar-H), 5.76 (d, J = 3.6 Hz, 1H, C1H), 5.62 (d, J = 2.2 Hz, 1H, S-CH), 5.25 (d, J = 2.2 Hz, 1H, CH-N), 4.66 (t, J = 3.9 Hz, 1H, C2H), 4.54 (s, 2H, OCH2), 3.963.94 (m, 1H, C4H), 3.71 (s, 3H, N-CH3), 3.26 (dd, Jt = 9.1 Hz, J2 = 4.2 Hz, 1H, C3H), 1.53 (s, 3H, CH3), 1.36 (m, 3H, CH3); 13C NMR (75 MHz, DMSO-d.) S 171.6, 151.6, 143.2, 137.4, 133.6, 132.3, 131.2, 129.3, 128.4, 127.9, 124.8, 122.9, 119.2, 111.2, 103.8, 81.2, 78.1, 74.1, 65.9, 58.1, 51.4, 39.3, 16.1; MS: m/z (M++ H) 684. Anal. Calcd for C„H CLFNAS: C, 56.14; H, 4.42; N, 12.28. Found: C, 32 30 2 6 4 55.92; H, 4.26: N, 12.02. 5-((3afl,5S,6S,6afl)-6-((1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2,2-dimethyltetrahydrofu- ro[2,3-d][1,3]dioxol-5-yl)-3-(4-fluorophenyl)-6-(4-ni-trophenyl)-2-phenyl-3,3a,5,6-tetrahydro-2H-pyra-zolo[3,4-d]thiazole (10m). This was obtained by reacting compound 9c (1 g) and phenylhydrazine (0.25 g) as described in the typical procedure and isolated as a brown solid, yield 75%, m.p. 241-243 °C; 1H NMR (300 MHz, DMSO-d6) S 8.26 (d, J = 8.7 Hz, 2H, Ar-H), 8.02 (s, 1H, Ar-H), 7.4 9 (d, J = 9.2 Hz, 2H, Ar-H), 7.22 (d, J = 8.33 Hz, 2H, Ar-H), 7.08-7.05 (m, 6H, Ar-H and CH-S), 7.02 (d, J = 8.3 Hz, 2H, Ar-H), 6.81-6.77 (m, 4H, Ar-H), 5.66 (d, J = 3.6 Hz, 1H, C1H), 5.58 (d, J = 2.2 Hz, 1H, S-CH), 5.21 (d, J = 2.2 Hz, 1H, CH-N), 4.62 (t, J = 3.9 Hz,1H, C2H), 4.51 (s, 2H, OCH2), 3.94-3.91 (m, 1H, C4H), 3.26 (dd, J = 9.1 Hz, J2 = 4.2 Hz, 1H, C3H), 1.53 (s, 3H, CH3), 1.36 (m, 3H, CH3); 13C NMR (75 MHz, DMSO-d.) S 174.6, 143.0, 141.4, 137.4, 136.2, 133.6, 132.3, 131.2, 129.3, 128.4, 127.9, 126.1, 124.8, 122.9, 119.2, 111.2, 103.8, 81.2, 78.1, 74.1, 65.9, 51.4, 26.1, 16.1; MS: m/z (M++H) 770. Anal. Calcd for C38H33ClFN7O6S: C, 59.26; H, 4.32; N, 12.73. Found: C, 593.16; H, 4.10; N, 12.53. 5-((3afl,5S,6S,6afl)-6-((1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2,2-dimethyltetrahydrofu-ro[2,3-d][1,3]dioxol-5-yl)-3-(4-fluorophenyl)-2-(4-me-thoxyphenyl)-6-(4-nitrophenyl)-3,3a,5,6-tetrahy-dro-2H-pyrazolo[3,4-d]thiazole (10n). This was obtained by reacting compound 9c (1 g) and p-methoxy-phenylhydrazine (0.35 g) as described in the typical procedure and isolated as a yellow solid, yield 76%, m.p. 264-266 °C; 1H NMR (300 MHz, DMSO-d.) S 8.28 (d, J = 8.7 Hz, 2H, Ar-H), 8.04 (s, 1H, Ar-H), 7.51 (d, J = 9.2 Hz, 2H, Ar-H), 7.40-7.35 (m, 5H, Ar-H and CH-S), 7.29 (d, J = 8.33 Hz, 2H, Ar-H), 7.05 (d, J = 8.3 Hz, 2H, Ar-H), 6.91-6.87 (m, 4H, Ar-H), 5.76 (d, J = 3.6 Hz, 1H, C1H), 5.62 (d, J = 2.2 Hz, 1H, S-CH), 5.25 (d, J = 2.2 Hz, 1H, CH-N), 4.66 (t, J = 3.9 Hz, 1H, C2H), 4.54 (s, 2H, OCH2), 3.96-3.94 (m, 1H, C4H), 3.91 (s, 3H, OMe), 3.26 (dd, J = 9.1 Hz, J2 = 4.2 Hz, 1H, C3H), 1.53 (s, 3H, CH3), 1.36 (m, 3H, CH3); 13C NMR (75 MHz, DMSO-d.) S 172.6, 151.7, 143.2, 141.4, 137.4, 133 .6, 132.3, 131.2, 129.3, 126.1, 125.3, 128.4, 127.9, 124.8, 122.9, 119.2, 111.2, 103.8, 81.2, 78.1, 74.1, 65.9, 55.8, 51.4, 26.1, 16.1; MS: m/z (M++H) 800. Anal. Calcd for C39H35ClFN7O7S: C, 58.53; H, 4.41; N, 12.25. Found: C, 58.22; H, 4.16; N, 12.01. 2-(4-Chlorophenyl)-5-((3afl,5S,6S,6afl)-6-((1-(4-chloro-phenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2,2-dimethyltet-rahydrofuro[2,3-d][1,3]dioxol-5-yl)-3-(4-fluoro-phenyl)-6-(4-nitrophenyl)-3,3a,5,6-tetrahydro-2H-pyra-zolo[3,4-d]thiazole (10 o). This was obtained by reacting compound 9c (1 g) and p-chlorophenylhydrazine (0.45 g) as described in the typical procedure and isolated as a brown solid, yield 72%, m.p. 285-287 °C; 1H NMR (300 MHz, DM-SO-d1) S 8.23 (d, J = 8.7 Hz, 2H, Ar-H), 8.11 (s, 1H, Ar-H), 7.58 (d, J = 9.2 Hz, 2H, Ar-H), 7.40-7.35 (m, 5H, Ar-H and CH-S), 7.29 (d, J = 8.33 Hz, 2H, Ar-H), 7.07 (d, J = 8.3 Hz, Srinivaset al.: Synthesis, Nematicidal and Antifungal Properties ... Acta Chim. Slov. 2017, 64, 1030-1041 1039 2H, Ar-H), 6.91-6.87 (m, 4H, Ar-H), 5.76 (d, J = 3.6 Hz, 1H, CjH), 5.62 (d, J = 2.2 Hz, 1H, S-CH), 5.25 (d, J = 2.2 Hz, 1H, CH-N), 4.66 (t, J = 3.9 Hz, 1H, C2H), 4.54 (s, 2H, OCH2), 3.96-3.94 (m, 1H, C4H), 3.26 (dd, J = 9.1 Hz, J2 = 4.2 Hz, 1H, C3H), 1.53 (s, 3H, CH3), 1.36 (m, 3H, CH3); 13C NMR (75 MHz, DMSO-d6) 8 1732.6, 151.7, 143.2, 137.4, 133.6, 132.3, 131.2, 129.3, 126.3, 125.3, 128.4, 127.9, 124.8, 122.9, 119.2, 111.2, 103.8, 81.2, 78.1, 74.1, 65.9, 51.4, 26.1, 16.1; MS: m/z (M++ Na) 815. Anal. Calcd for C38H32Cl2FN7O6S: C, 56.72; H, 4.01; N, 12.81. Found: C, 56.42; H, 3.95; N, 12.72. 2-Benzyl-5-((3afl,5S,6S,6afl)-6-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2,2-dimethyltetrahy-drofuro [2,3-d][1,3]dioxol-5-yl)-3-(4-fluoro-phenyl)-6-(4-nitrophenyl)-3,3a,5,6-tetrahydro-2H-pyr-azolo[3,4-d]thiazole (10p). This was obtained by reacting compound 9c (1 g) and benzylhydrazine (0.65 g) as described in the typical procedure and isolated as a yellow solid, yield 71%, m.p. 275-277 °C; 1H NMR (300 MHz, DMSO-d6) 8 8.35 (d, J = 8.7 Hz, 2H, Ar-H), 8.11 (s, 1H, Ar-H), 7.59 (d, J = 9.2 Hz, 2H, Ar-H), 7.46-7.40 (m, 6H, Ar-H and CH-S), 7.32 (d, J = 8.33 Hz, 2H, Ar-H), 7.08 (d, J = 8.3 Hz, 2H, Ar-H), 6.96-6.90 (m, 4H, Ar-H), 5.96 (s, 2H, CH2Ph), 5.77 (d, J = 3.6 Hz, 1H, C1H), 5.65 (d, J = 2.2 Hz, 1H, S-CH), 5.28 (d, J = 2.2 Hz, 1H, CH-N), 4.66 (t, J = 3.9 Hz, 1H, C2H), 4.54 (s, 2H, OCH2), 3.96-3.94 (m, 1H, C4H), 3.26 (dd, J = 9.1 Hz, J2 = 4.2 Hz, 1H, C3H), 1.53 (s, 3H, CH3), 1.36 (m, 3H, CH3); 13C NMR (75 MHz, DMSO-d6) 8 171.6, 151.6, 143.2, 137.4, 133.6, 132.3, 131.2, 129.3, 125.3, 128.4, 127.9, 124.8, 122.9, 119.2, 111.2, 103.8, 81.2, 78.1, 74.1, 65.9, 58.1, 51.4, 26.1, 16.1; MS: m/z (M++ H) 773. Anal. Calcd for CaoH ClFN,O,S: C, 59.73; H, 4.50; ' 39 35 / 6 N, 12.56. Found: C, 59.66; H, 4.41; N, 12.36. 5-((3afl,5S,6S,6afl)-6-((1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2,2-dimethyltetrahydrofu-ro[2,3-d][1,3]dioxol-5-yl)-3-(4-fluorophenyl)-2-isopro-pyl-6-(4-nitrophenyl)-3,3a,5,6-tetrahydro-2H-pyra-zolo[3,4-d]thiazole (10q). This was obtained by reacting compound 9c (1 g) and isopropylhydrazine (0.35 g) as described in the typical procedure and isolated as a brown solid, yield 69%, m.p. 221-223 °C; 1H NMR (300 MHz, DMSO-d6) 8 8.32 (d, J = 8.7 Hz, 2H, Ar-H), 8.16 (s, 1H, Ar-H), 7.65 (d, J = 9.2 Hz, 2H, Ar-H), 7.52-7.48 (m, 5H, Ar-H and CH-S), 7.39 (d, J = 8.33 Hz, 2H, Ar-H), 7.09 (d, J = 8.3 Hz, 2H, Ar-H), 5.76 (d, J = 3.6 Hz, 1H, C1H), 5.62 (d, J = 2.2 Hz, 1H, S-CH), 5.25 (d, J = 2.2 Hz, 1H1 CH-N), 4.66 (t, J = 3.9 Hz, 1H, C2H), 4.54 (s, 2H, OCH2), 3.96-3.94 (m, 1H, C4H), 3.26 (dd, J = 9.1 Hz, J2 = 4.2 Hz, 1H, C3H), 2.80-2.78 (m, 1H, CH), 1.53 (s, 3H, CH3), 1.36 (m, 3H, CH3), 0.96 (d, J = 6.3 Hz, 6H, 2xCH3); 13C NMR (75 MHz, DMSO-d6) 8 171.6, 151.6, 143.2, 137.4, 133.6, 132.3, 131.2, 129.3, 128.4, 127.9, 124.8, 122.9, 119.2, 111.2, 103.8, 81.2, 78.1, 74.1, 65.9, 58.1, 51.4, 50.6, 26.1, 16.1; MS: m/z (M++ H) 736. Anal. Calcd for C35H35ClFN7O6S: C, 57.10; H, 4.79; N, 13.32. Found: C, 56.98; H 4.56; N, 12.99. 5-((3afl,5S,6S,6afl)-6-((1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methoxy)-2,2-dimethyltetrahydrofu-ro[2,3-d][1,3]dioxol-5-yl)-3-(4-fluorophenyl)-2-meth-yl-6-(4-nitrophenyl)-3,3a,5,6-tetrahydro-2H-pyra-zolo[3,4-d]thiazole (10r). This was obtained by reacting compound 9c (1 g) and methylhydrazine (0.25 g) as described in the typical procedure and isolated as a brown solid, yield 67%, m.p. 251-253 °C; 1H NMR (300 MHz, DMSO-d6) 8 8.29 (d, J = 8.7 Hz, 2H, Ar-H), 8.16 (s, 1H, Ar-H), 7.59 (d, J = 9.2 Hz, 2H, Ar-H), 7.48-7.44 (m, 5H, Ar -H and CH-S), 7.39 (d, J = 8.33 Hz, 2H, Ar-H), 7.09 (d, J = 8.3 Hz, 2H, Ar-H), 5.76 (d, J = 3.6 Hz, 1H, C1H), 5.62 (d, J = 2.2 Hz, 1H, S-CH), 5.25 (d, J = 2.2 Hz, 1H1 CH-N), 4.66 (t, J = 3.9 Hz,1H, C2H), 4.54 (s, 2H, OCH2), 3.96-3.94 (m, 1H, C4H), 3.71(s, 3H, N-CH3), 3.26 (dd, J = 9.1 Hz, J2 = 4.2 Hz, 1H, C3H), 1.53 (s, 3H, CH3), 1.36 (m, 3H, CH3); 13C NMR (75 MHz, DMSO-d6) 8 17L6, 151.6, 143.2, 137.4, 133.6, 132.3, 131.2, 129.3, 128.4, 127.9, 124.8, 122.9, 119.2, 111.2, 103.8, 81.2, 78.1, 74.1, 65.9, 58.1, 51.4, 39.3, 16.1; MS: m/z (M++ H) 684. Anal. Calcd for C33H31ClFN7O6S: C, 55.97; H, 4.41; N, 13.85. Found: C, 55.82; H, 4.26: N, 13.72. 6. Conclusion In conclusion, a series of a new class of hybrid het-erocycles 10a-r have been synthesized and evaluated for their nematicidal activity, most of the compounds showed appreciable nematicidal activity. The antifungal activities of these compounds were evaluated against various fungi. Many of the synthesized compounds showed good activity against the tested fungi and therefore have emerged as potential molecules for further development. 7. Acknowledgements The authors are thankful to CSIR-New Delhi for the financial support (Project funding No: 02 (247)15/EMR-II), Director, CSIR- IICT, Hyderabad, India, for NMR and MS spectral analysis and Principal, Vaagdevi Degree & PG College Hanamkonda for his constant encouragement. 8. References 1. (a) K. D. Hani, D. A. Leigh, Chem. Soc.Rev. 2010, 39, 12401251. DOI:10.1039/B901974J (b) C. O. Kappe, E. Van der Eycken, Chem.Soc. Rev. 2010, 39, 1280-1290 D0I:10.1039/B901973C (c) A. H. El-Sagheer, T. Brown, Chem. Soc. Rev. 2010, 39, 1388-1405 D0I:10.1039/b901971p (d) A. Qin, J. W. Y. Lam, B. Z. Tang, Chem. Soc. Rev. 2010, 2522-2544. D0I:10.1039/b909064a (e) M. Meldal, C. W. Tornoe, Chem. Rev. 2008, 108, 29523015. D0I:10.1021/cr0783479 Srinivaset al.: Synthesis, Nematicidal and Antifungal Properties 1040 Acta Chim. Slov. 2017, 64, 1030-1041 (f) H. Nandivada, X. Jiang, J. Lahann, Adv. Mater. 2007, 19, 2197-2208. D01:10.1002/adma.200602739 (g) Y. L. Angell, K. Burgess, Chem. Soc. Rev. 2007, 36, 16741689. D0I:10.1039/b701444a (h) D. Fournier, R. Hoogenboom, U. S. Schubert, Chem. Soc. Rev. 2007, 36, 1369-1380. D0I:10.1039/b700809k (i) J. E. Moses, A. D. Moorhouse, Chem. Soc. Rev. 2007, 36, 1249-1262. D0I:10.1039/B613014N (j) J. F. Lutz, Angew. Chem. Int. Ed. 2007, 46, 1018-1125. DOI: 10.1002/anie.200604050 (k) A. Dondoni, Chem. - Asian J. 2007, 2, 700-708. D0I:10.1002/asia.200700015 (l) H. C. Kolb, K. B. Sharpless, Drug Discovery Today, 2003, 8, 1128-1137. D0I:10.1016/S1359-6446(03)02933-7 2. (a) A. Brick, J. Muldoon, Y.-C. Lin, J. H. Elder, D. S. Goodsell, A. J. Olson, V. V. Fokin, K. B. Sharpless, C.-H. Wong, Chem-BioChem. 2003, 4, 1246-1248. D0I:10.1002/cbic.200300724 (b) M. J. Soltis, H. J. Yeh, K. A. Cole, N. Whittaker, R. P. Wer-sto, E. C. Kohn, Drug Metab. Dispos. 1996,24, 799-806. 3. (a) W.-Q. Fan, A. R. Katritzky, 1,2,3-Triazoles, In Comprehensive Heterocyclic Chemistry II. Edited by A. R. Katritzky, C. W. Rees, V. Scriven, Elsevier, Oxford. 1996, 4, 1-126, pp. 905-1006. (b) M. Whiting, J. Muldoon, Y.-C. Lin, S. M. Silverman, W. Lindstrom, A. J. Olson, H. C. Kolb, M. G. Finn, K. B. Sharp-less, J. H. Elder, V. V. Fokin, Angew. Chem. Int. Ed., 2006, 45, 1435-1439. D0I:10.1002/anie.200502161 (c) Y. Bourne, H. C. Kolb, Z. Radie, K. B. Sharpless, P.Taylor, P. Marchot, Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 1449-1454. D0I:10.1073/pnas.0308206100 (d) W. G. Lewis, G. Green, F. Z. Grynszpan, Z. Radie, P. R. Carlier, P. Taylor, M. G. Finn, K. B. Sharpless, Angew. Chem., Int. Ed. 2002, 41, 1053-1057. D0I:10.1002/1521-3773(20020315)41:6<1053::AID-ANIE 1053>3.0.C0;2-4 4. R. Huisgen, A. Padwa, 1,3-Dipolar Cycloaddition Chemistry, Ed. Wiley, New York, 1984, 1, 1-176. 5. (a) N. A. Al-Maoudi, A. Y. Al-Soud, Tetrahedron Lett. 2002, 43, 4021-4022. D0I:10.1016/S0040-4039(02)00733-5 (b) B. H. M. Kuijpers, S. Groothuys, A. B. R. Keereweer, P. J. L. M. Quaedflieg, R. H. Blaauw, F. L.van Delft, F. P. J. T. Rutjes, Org. Lett. 2004, 6, 3123-3126. D0I:10.1021/ol048841o (c) C. Srinivas, X. Fang, Q. Wang, Tetrahedron Lett. 2005, 46, 2331-2334. D0I:10.1016/j.tetlet.2005.01.175 (d) S. Hotha, R. I. Anegundi, A. A. Natu, Tetrahedron Lett. 2005, 46, 4585-4588. D0I:10.1016/j.tetlet.2005.05.012 (e) S. Hotha, S. Kashyap, J. Org. Chem. 2006, 71, 364-367. D0I:10.1021/jo051731q 6. H. K. Urman, O. Bulay, B. Clayson, P. Shudik, Cancer Lett. 1975, 1, 69-74. D0I:10.1016/S0304-3835(75)95362-8 7. A. A. Field, A. A. Tyrell, G. P. Lampson, M. R. Hilleman, Proc. Natl. Acad. Sci. U.S.A, 1967, 58, 1004-1010. D01:10.1073/pnas.58.3.1004 8. M. G. Vigorita, R. Ottana, F. Monoforte, R. Maccari, A. Trova-to, M. T. Monoforte, M. F. Taviano, Bioorg. Med. Chem. Lett. 2001, 11, 2791-2794. DOI: 10.1016/S0960-894X(01)00476-0 9. M. S. Chande, V. J. Suryanarayana, J. Chem. Res, 2005, 345347. DOI: 10.3184/0308234054506749 10. C. V. Kavitha, S. Basappa, N. Swamy, K. Manteling, S. Doreswamy, M. A. Sridhar, S. Prasad, K. S. Rangappa, Bioorg. Med. Chem. Lett. 2006, 14, 2290-2299. D01:10.1016/j.bmc.2005.11.017 11. B. A. Sobin, J. Am. Chem. Soc, 1952, 74, 2947-2948. D0I:10.1021/ja01131a526 12. Y. Tanabe, Y. Kumuro, N. Imanishi, S. Morooka, M. Enomo-to, A. Kojima, Y. Sanemistu, M. Mizutani, Tetrahedron Lett. 1991, 32, 379-382. D0I:10.1016/S0040-4039(00)92633-9 13. T. Previtera, M. G. Vigorita, M. Bisila, F. Orshini, F. Benetolla, G. Bombieri, Eur. J. Med. Chem. 1994, 29, 317-324. DOI: 10.1016/0223-5234(94)90102-3 14. R. Ottana, E. Mazzon, L. Dugo, F. Moforte, R. Maccari, L. Sau-tebin, G. De Luca, M. G. Vigorita, S. Alcaro, F. Ortuso, Eur. J. Pharmacol. 2002, 448, 71-80. D0I:10.1016/S0014-2999(02)01888-5 15. T. Kato, T. Ozaki, K. Tamura, J. Med. Chem. 1999, 42, 31343146. D0I:10.1021/jm9900927 16. Y. Tanabe, G. Suzukamo, Y. Komuro, N. Imanishi, S. Morro-ka, M. Enomoto, A. Kojima, Y. Sanimistu, M. Mizutani, Tetrahedron Lett. 1991, 32, 379-382. D0I:10.1016/S0040-4039(00)92633-9 17. T. Kato, T. Ozaki, K.Tamura, Tetrahedron Assymm. 1999, 10, 3963-3968. D0I:10.1016/S0957-4166(99)00441-3 18. Y. Adachi, Y. Suzuki, N. Homma, M. Fuzukawa, K. Tamura, I. Nishie, O. Kuromaru, Eur. J. Pharmacol. 1999, 367, 267-273. D0I:10.1016/S0014-2999(98)00938-8 19. C. S. Reddy, A. Srinivas, A. Nagaraj, J. Heterocycl. Chem. 2008, 45, 999-1003. D0I:10.1002/jhet.5570450409 20. K. Y. Lee, J. M. Kim, J. N. Kim, Tetrahedron Lett. 2003, 44, 6737-6740. D0I:10.1016/S0040-4039(03)01648-4 21. P. Erhan, A. Mutlu, U. Tayfun, E. Dilek, Eur. J. Med. Chem. 2001, 36, 539-543. D0I:10.1016/S0223-5234(01)01243-0 22. R. A. Nugen, M. Meghan, J. Med. Chem. 1993, 36, 134-139. D0I:10.1021/jm00053a017 23. H. Kawazura, Y. Takahashi, Y. Shiga, F. Shimada, N. Ohto, A. Tamura, Jpn. J. Pharmacol. 1997, 73, 317-324. D0I:10.1254/jjp.73.317 24. M. J. Genin, C. Bilers, B. J. Kieser, S. M. Poppe, S. M. Swaney, W. G. Tarpley, Y. Yagi, D. L. Romero, J. Med. Chem. 2000, 43, 1034-1040. D0I:10.1021/jm990383f 25. A. G. Habeb, P. N. P. Rao, E. E. Knaus, J. Med. Chem. 2001, 44, 3039-3042. D0I:10.1021/jm010153c 26. H. Hashimoto, K. Imamura, J. I. Haruta, K. Wakitani, J. Med. Chem. 2002, 45, 1511-1517. D0I:10.1021/jm010484p 27. M. Sakya, B. Rast, Tetrahedron Lett. 2003, 44, 7629-7632. D0I:10.1016/j.tetlet.2003.08.054 28. L. David, D. G. B. Selwood, J. Med. Chem. 2001, 44, 78-93. D0I:10.1021/jm001034k 29. U. L. B. Jayasinghe, B. M. M. Kumarihamy, A. G. D. Bandara, E. A. Vasquez, W. Kraus, Nat. Prod. Res. 2003, 17, 259-262. D01:10.1080/1057563021000060121 Srinivaset al.: Synthesis, Nematicidal and Antifungal Properties ... Acta Chim. Slov. 2017, 64, 1030-1041 1041 30. S. Min, L. Xiaoming, J. Shuishui, L. Lingjie, W. Jiali, W. Shi-qiang, Applied Soil Ecology. 2016, 102, 53-60. DOI:10.1016/j.apsoil.2016.02.011 31. J. W. Noling, J. O. Becker, J. Nematol. 1994, 26, 573-586. 32. C. In Ho, K. Jun Heon, S. Sang-Chul, P. Il-Kown, Rus. J. Nematology 2007, 15, 35-40. 33. K. Shigefumi, W. Kojiro, M. Katsura, Agr. Bio. Chem. 1976, 40, 2085-2089. DOI:10.1080/00021369.1976.10862338 34. J. H. Uhlenbroek, J. D. Bijloo, Recl. Trav. Chim. 1958, 77, 1004-1008. DOI: 10.1002/recl.19580771103 35. (a) A. Srinivas, M. Sunitha, B. Ravinder, S. Anusha, T. Rajasri, P. Swapna, D. Sushmitha, D. Swaroopa, G. Nikitha, C. Govind Rao, Acta Chim. Slov. 2017, 64, 319-331. DOI:10.17344/acsi.2016.3153 (b) A. Srinivas, M. Santhosh, M. Sunitha, P. Karthik, K. Srinivas, K. Vasumathi Reddy, Acta Chim. Slov. 2016, 63, 827-836. DOI:10.17344/acsi.2015.2124 36. A. Srinivas, M. Sunitha, C. Govind Rao, Acta Chim. Slov. 2016, 63, 344-350. DOI:10.17344/acsi.2015.2124 37. A. Srinivas, Acta Chim. Soc. 2016, 63, 173-179. DOI:10.17344/acsi.2015.2124 38. A. Srinivas, M. Sunitha, Indian J. Chem., Sect. B 2016, 55B, 102-109. 39. A. Srinivas, M. Sunitha, Indian J. Chem., Sect. B 2016, 55B, 231-239. 40. A. Srinivas, M. Sunitha, C. Govind Rao, Indian J. Chem., Sect. B 2016, 55B, 1239-1242. 41. A. Srinivas, A. Nagaraj, C. S. Reddy, Eur. J. Med. Chem. 2010, 45, 2353-2358. DOI:10.1016/j.ejmech.2010.02.014 42. C. S. Reddy, A. Srinivas, M. Sunitha, A. Nagaraj, J. Heterocycl. Chem, 2010, 47, 1303-1309. DOI:10.1002/jhet.474 43. C. S. Reddy, A. Nagaraj, A. Srinivas, G. P. Reddy, Indian J. Chem., Scet. B, 2010, 49B, 617-622. 44. C. S. Reddy, A. Srinivas, A. Nagaraj, J. Heterocycl. Chem. 2009, 46, 497-502. DOI:10.1002/jhet.100 45. C. S. Reddy, A. Nagaraj, A. Srinivas, G. P. Reddy, Indian J. Chem., Sect. B 2009, 48B, 248-254. 46. C. S. Reddy, A. Srinivas, A. Nagaraj, J. Heterocyclic. Chem. 2008, 45, 1121-1125. 47. C. S. Reddy, G. P. Reddy, A. Nagaraj, A. Srinivas, Org. Commun. 2008, 1, 84-94. 48. A. Srinivas, A. Nagaraj, C. S. Reddy, Indian J. Chem., Sect. B 2008, 47B, 787-791. 49. P. A. Levene, G. M. Meyer, J. Biol. Chem, 1931, 92, 257. 50. National Committee for clinical laboratory standards (NC-CLS). Nat. Comm. Lab. Stands. Villanova, 1982, p. 242. 51. Z. K. Khan, Proc. Int. Workshop UNIDO-CDRI, 1997, 210 (1997). 52. C. W. McBeth, G. B. Bergerson, Phytopathology 1953, 43, 264. Povzetek Z reakcijo med halkonskimi derivati 2-((3aÄ,5S,6S,6aÄ)-6-((1-(4-Worofenü)-1H-1,2,3-triazol-4-il)metoksi)-2,2-di-metiltetrahidrofuro[2,3-d][1,3]dioksol-5-il)-3-feniltiazolidin-4-onov 9 in aril/alkil hidrazini smo pripravili novo serijo 5-((3aÄ,5S,6S,6aÄ)-6-((1-(4-klorofenil)-1H-1,2,3-triazol-4-il)metoksi)-2,2-dimetiltetrahidrofuro[2,3-d][1,3]dioksol-5-ü)-3-(4-fluorofenil)-2,6-difenil-3,3a,5,6-tetrahidro-2H-pirazolo[3,4-d]tiazolov 10a-r. Strukture novih spojin smo določili na osnovi IR, NMR, MS in elementne analize. Za spojine 10a-r smo tudi določili učinkovanje proti ploskim črvom (nematodam) Dietylenchus myceliophagus in Caenorhabditis elegans z in vitro metodo v vodnih raztopinah. Ugotovili smo, da spojine, ki vsebujejo N-benzilpirazolni (10d, 10j, 10p) ali N- metilpirazolni fragment (10f, 10i, 10r), izkazujejo občutno nematocidno aktivnost proti obema testiranima živalskima vrstama z LD50 160-210 ppm, kar je skoraj enako aktivnosti standarda oksamila. Za spojine 10a-r smo tudi raziskali delovanje proti glivam (izmerili smo MZI, MIC in MFC vrednosti) in sicer: Candida albicans (ATCC 102331), Aspergillus fumigates (HIC 6094), Trichophyton rubrum (IFO 9185) in Trichophyton mentagrophytes (IFO 40996). Večina novih spojin je izkazala opazno delovanje proti testiranim glivam, kar daje možnosti nadaljnjega razvoja predstavljenih spojin Srinivaset al.: Synthesis, Nematicidal and Antifungal Properties 1042 DOI: 10.17344/acsi.2017.3838 Acta Chim. Slov. 2017, 64, 1042-1047 ^creative ty com mor Scientific paper Preparation, Structure, Photoluminescent and Semiconductive Properties, and Theoretical Calculation of a Novel Cadmium Complex with Mixed Ligands Xiu-Guang Yi,12 Wen-Tong Chen,1,3,4* Jian-Gen Huang,1 Ding-Wa Zhang1 and Yin-Feng Wang1 1 Institute of Applied Chemistry, Jiangxi Province Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Jinggangshan University, Jian, Jiangxi, 343009, China 2 Research Center for Rare Earths & Nano/micro Functional Materials, Nanchang University Nanchang, Jiangxi 330031, China 3 Key Laboratory of Jiangxi Province for Persistant Pollutants Control and Resources Recycle (Nanchang Hangkong University), Nanchang, Jiangxi 330000, China 4 State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, China * Corresponding author: E-mail: wtchen_2000@aliyun.com Tel.: +86(796)8100490; fax +86(796)8100490 Received: 18-09-2017 Abstract A novel cadmium complex with mixed ligands {[Cd(2,2'-biim)(4,4'-bipy)(H2O)(ClO4)] (ClO4)}n (1) (2,2'-biim = 2,2'-biimidazole; 4,4'-bipy = 4,4'-bipyridine) has been synthesized through hydrothermal reaction and its crystal structure was determined by single-crystal X-ray diffraction technique. Single-crystal X-ray diffraction analyses revealed that complex 1 crystallizes in the space group Pna21 of the orthorhombic system and exhibits a one-dimensional zigzag chain structure consisting of [Cd(2,2'-biim)(4,4'-bipy)(H2O)(ClO4)]n"+ cationic chains and isolated ClO4- anions. Powder photoluminescent characterization reveals that complex 1 has an emission in the green region of the spectrum. Time-dependent density functional theory (TDDFT) calculation showed that the nature of the photoluminescence of complex 1 is originated from the ligand-to-ligand charge transfer (LLCT; from the HOMO of the perchlorate anions to the LUMO of the 4,4'-bipy ligand). A wide optical band gap of 3.25 eV was found by the solid-state UV/vis diffuse reflectance spectrum. Keywords: Cadmium, photoluminescence, semiconductor, TDDFT, LLCT 1. Introduction In recent years, preparation and characterization of coordination complexes have attracted increasing interest not only due to their amazing structural topologies but also their potential applications in the fields of catalyst, sensors, medicine, biology, solar energy conversion, magnetism, photoluminescence materials, and so forth.1-'5 From the perspective of the crystal engineering, the most useful and facile way to construct coordination complexes is to adopt a suitable ligand to connect metal centers. To this end, the ligand is better to possess as much as donor atoms that enable it to bridge metal centers together to yield extended architectures. N-containing heterocyclic ligands (e.g., 2,2'-biim and 4,4'-bipy namely, 2,2'-biimid-azole and 4,4'-bipyridine, respectively), which have several coordination sites and various coordination modes, have been widely applied as such ligands to design novel coordination complexes.6-12 2,2'-biim and 4,4'-bipy have been confirmed to be a good chelating or bridging ligand to build coordination complexes. Being very strong N-ligat-ing ligands, 2,2'-biim and 4,4'-bipy are also important to Yi et al.: Preparation, Structure, Photoluminescent Acta Chim. Slov. 2017, 64, 1042-1047 1043 afford useful supramolecular recognition positions to form interesting supramolecular topologies. Therefore, 2,2'-biim and 4,4'-bipy are important and useful ligands to achieve extended structures or supramolecular geometries. Furthermore, the imidazole or pyridyl rings of the 2,2'-biim or 4,4'-bipy possess delocalized n-electron systems which allow them to become an ideal candidate to prepare photoluminescent materials. In addition, coordination complexes possessing group 12 (IIB) elements (Zn, Cd, Hg) are attractive due to their photoluminescent and semiconductive properties, various coordination numbers and topologies provided by their d10 configuration of the IIB ions, as well as the important role of zinc played in biological systems.13-15 For the sake of exploring the metal ions on the structures and properties of the coordination complexes, we often choose the IIB ions as the central ion source. In order to explore novel IIB coordination complexes with attractive structural topologies and interesting properties, we recently focus on the design and preparation of novel IIB coordination complexes with various organic ligands. We report in this work the preparation, crystal structure, photoluminescent and semiconductive properties, and theoretical calculation of a novel cadmium complex with mixed ligands, i.e. {[Cd(2,2'-biim)(4,4'-bipy)(H2O)(ClO4)](ClO4)}n (1) with a 1-D zigzag chain structure. It should be pointed out that this is the first IIB complex with both 4,4'-bipy and 2,2'-biim ligands. 2. Experimental 2. 1. Materials and Instrumentation. All chemicals and reagents were of reagent grade, commercially available and directly applied for the reaction. Photoluminescent characterization was measured using solid-state powders of the title complex at room temperature on a JY FluoroMax-3 spectrometer. Time-dependent density functional theory (TDDFT) calculation was performed by virtue of the Gaussian03 suite of program packages. Solid-state UV/vis diffuse reflectance measurement was conducted on a computer-controlled TU1901 UV/vis spectrometer equipped with an integrating sphere attachment. Finely-ground powder sample was coated on barium sulfate which acts as a reference for 100% reflectance. 2. 2. Synthesis of {[Cd(2,2'-biim)(4,4'-bipy) (H2O)(ClO4)](ClO4)}n (1) Cd(ClO4)2 ■ 6H2O (1.00 mmol, 0.420 g), 2,2'-biim (1.00 mmol, 0.134 g), 4,4'-bipy (1.00 mmol, 0.156 g) and 10 mL distilled water were put into a 25 mL vial of a Teflon-lined stainless steel autoclave. The autoclave was heated around 433 K under autogenous pressure over a period of ten days and powered off, then cooled to room temperature. Finally, light-yellow block crystals were collected, washed with dis- tilled water, dried in air and used for single-crystal X-ray diffraction as well as property measurements. The yield was 24% (based on cadmium). Caution: perchlorate salts are highly explosive and must be handled with careful! 2. 3. X-ray Structure Determination The single-crystal data of the title complex were collected on a SuperNova X-ray diffractometer equipped with a graphite monochromated Mo-Ka radiation source (X = 0.71073 A) at 293(2) K. The diffraction was performed by means of a « scan mode. Using the CrystalClear software, we reduced the data set and corrected the empirical absorption.16 The crystal structure was successfully solved by using the direct methods and Siemens SHELXTLTM Version 5 software package.17 The non-hydrogen atoms were generated based on the subsequent Fourier difference maps and refined anisotropically. The hydrogen atoms were located theoretically and ride on their parent atoms; Table 1. Crystallographic data and structural analysis for complex 1 Formula C16H16CdCl2N6O9 Mr 619.65 Crystal system orthorhombic Space group Pna21 a (A) 17.7584(5) b (A) 9.8785(3) c (A) 12.3847(4) V (A3) 2172.60(11) Z 4 Reflections collected 6691 Independent, observed reflections (Rint) 3304, 3118 (0.0219) dcalcd. (g/cm3) 1.894 p/mm 1 1.315 F(000) 1232 R1, wR2 0.0271, 0.0634 S 1.027 4p(max, min) (e/A3) 0.388, -0.350 Table 2. Selected bond lengths (A) and bond angles (°) for complex 1 Distance (A) Distance (A) Cd1-N5' 2.290(3) Cd1-N6 2.355(3) Cd1-O1W 2.296(3) Cd1-N4 2.369(4) Cd1-N2 2.303(3) Cd1-O7 2.499(4) Angle (°) Angle (°) N5'-Cd1-O1W 95.24(14) N2-Cd1-N4 72.89(11) N5i-Cd1-N2 170.58(14) N6-Cd1-N4 91.18(13) N2-Cd1-O1W 86.63(13) N5i-Cd1-O7 87.33(12) N6-Cd1-O1W 99.57(13) O1W-Cd1-O7 77.90(14) N2-Cd1-N6 99.89(12) N2-Cd1-O7 84.04(12) N5i-Cd1-N4 103.84(12) N6-Cd1-O7 175.26(12) N4-Cd1-O1W 158.28(12) N4-Cd1-O7 92.53(14) Symmetry code: (i) -x - y + z - Yi et al.: Preparation, Structure, Photoluminescent 1044 Acta Chim. Slov. 2017, 64, 1042-1047 however, the hydrogen atoms of the coordinated water molecule were not located and are not included into the model. The single-crystal structure was finally refined by using the full-matrix least-squares procedure on F2. Crys-tallographic data and structural refinements for the title complex are summarized in Table 1. Selected bond lengths and bond angles for the crystal structure are displayed in Table 2. The hydrogen bonding interactions are presented in Table 3. 3. Results and Discussion Single-crystal X-ray diffraction measurement revealed that complex 1 crystallizes in the space group Pna2l of the orthorhombic system with four formula units in one unit cell and the crystallographically asymmetric unit is comprised of one cadmium ion, one 4,4'-bipy ligand, one 2,2'-biim ligand, one isolated perchlorate anion, one coordinating perchlorate anion and one coordinating water molecule, as presented in Fig. 1. Complex 1 is characterized by a 1-D zigzag chain structure, consisting of [Cd(2,2'-biim)(4,4'-bipy)(H2O)(ClO4)]nn+ cationic chains and isolated ClO4- anions. The cadmium ion displays a slightly distorted octahedral geometry with the equatorial positions inhabited by three nitrogen atoms from one 2,2'-biim and one 4,4'-bipy ligand as well as one oxygen atom from one coordinating water molecule, and the apical sites are occupied by one oxygen atom from one coordinating per-chlorate anion as well as one nitrogen atom from one 4,4'-bipy ligand (Fig. 1). Figure 1. An ORTEP diagram of complex 1 with 30% thermal ellipsoids. Hydrogen atoms are omitted for clarity. Symmetry code: -x - y + z - The neighboring cadmium ions are interconnected by the 4,4'-bipy ligands through the nitrogen atoms to construct a 1D zigzag chain structure running along the b-axis, as depicted in Fig. 2. Different from the bridging 4,4'-bipy ligand, the 2,2'-biim molecule acts as a terminal ligand and chelates to one cadmium ion with the chelating angle N2-Cd1-N4 being of 72.89(11)°. In complex 1, there are two kinds of perchlorate anions, namely, termi- nally coordinating and isolated. The Cd—Cd distance is 11.5886(2) Á because of the distraction of the long rodlike bridging 4,4'-bipy ligand. The bond lengths of Cd-N are in the range of 2.290(3)-2.369(4)Á with a mean value of 2.329(4) Á. This is in agreement with those found in the literature.18-20 The bond lengths of Cd-O for water and for perchlorate ligand are 2.296(3) Á and 2.499(4) Á, respectively. This is also comparable with those reported in the literature.21-24 The bond angle of N5'-Cd1-N2 is 170.58(14)°, close to 180°, while other N-Cd-N angles locate in a range of 72.89(11)°-103.84(12)°, close to 90°. The bond angles of N4-Cd1-O1W and N6-Cd1-O7 are 158.28(12)° and 175.26(12)°, respectively, while other N-Cd-O angles are in a range of 84.04(12)°-99.57(13)°. The bond angle of O-Cd-O is only 77.90(14)°. The dihedral angle of the pyridyl rings of the 4,4'-bipy ligand is 14.90(4)°. The imidazole rings of the 2,2'-biim ligand is nearly copla-nar with a small dihedral angle of 4.64(2)°, which is close to that in another cadmium 2,2'-biim complex (3.23°).25 In complex 1, there are many hydrogen bonding interactions such as N-H—O and C-H—O interactions as listed in Table 3. The 1-D [Cd(2,2'-biim)(4,4'-bipy)(H2O)(ClO4)]B"+ cationic chains and isolated ClO - anions are interlinked 4 together through these hydrogen bonding interactions, electrostatic interactions and van der Waals interactions to Figure 2. The 1-D zigzag chain structure of complex 1. Figure 3. Packing diagram of complex 1 with the dashed lines representing the hydrogen bonding interactions. Yi et al.: Preparation, Structure, Photoluminescent Acta Chim. Slov. 2017, 64, 1042-1047 1045 construct a three-dimensional (3-D) supramolecular structure as shown in Fig. 3. To the best of our knowledge, this is the first IIB complex containing both 4,4'-bipy and 2,2'-biim ligands, although a lot of IIB complexes with either 4,4'-bipy or 2,2'-biim as a ligand have been reported thus far. 26-29 Table 3. Hydrogen bonding interactions D-H-A D-H, A H-A, A D-A, A D-H-A, ° N1-H1B—O2" 0.86 2.38 3.169(6) 153 N1-H1B-O3" 0.86 2.31 3.058(5) 146 N3-H3A—O2" 0.86 2.07 2.905(5) 162 C8-H8A-O7" 0.93 2.54 3.328(6) 142 C10-H10A-O4iii 0.93 2.57 3.298(6) 136 Symmetric codes: (ii) -x, -y, H + z; (iii) x, y, 1 + z. In general, cadmium compounds can exhibit attractive photoluminescent properties and, therefore, they have potential application in the areas of light-emitting diodes, electrochemical displays, photoluminescent materials, sen-sors.30,31 Moreover, complexes containing 4,4'-bipy or 2,2'-biim ligands can generally also show good photoluminescence due to the existence of their delocalized n electrons. As a result, the title complex is expected to possess photoluminescent behavior. Based on these considerations, in the present work, we measured the photoluminescent properties of complex 1 using powder-like samples under room temperature. Fig. 4 gives the photoluminescent excitation and emission spectra of complex 1. As displayed in this figure, the photoluminescent emission spectra of complex 1 show a wide and intensive emission peak, while the photoluminescent excitation spectra show that the effective energy absorption mainly resides in the wavelength span of 250-430 nm. The photoluminescent excitation spectra display a main peak at 424 nm and a shoulder at 355 nm. When it was ex- Figure 4. Photoluminescence spectra of 1 with the red and green lines representing excitation and emission spectra, respectively. cited by the wavelength of 424 nm, the photoluminescent emission spectra yield a strong emission peak at 493 nm in the green region of the spectrum. As a result, complex 1 could be a candidate material for green photoluminescence. Trying to unveil the nature of the photoluminescence spectra of complex 1, we truncated ground state geometry from its single-crystal X-ray diffraction data set and carried out its theoretical calculation in light of the time-dependent density functional theory (TDDFT) based on this ground state geometry. The TDDFT investigation was performed with the B3LYP function and by virtue of the Gaussian03 software package. After successfully calculating, the theoretical electron-distribution diagrams were obtained using the ChemOffice Ultra 7.0 graphics program and the results are given in Fig. 5. It is easy to find out that the electron-density distribution of the singlet state of HOMO is totally resided at the coordinating perchlorate anion with an energy being of -0.213039 Hartrees; however, the electron-density population of the singlet state of the LUMO locates at the 4,4'-bipy ligand and the energy of Figure 5. The electron-density population of complex 1. The isosurfaces correspond to the electronic density differences of -10 e nm 3 (blue) and +10 e nm 3 (red). Yi et al.: Preparation, Structure, Photoluminescent 1046 Acta Chim. Slov. 2017, 64, 1042-1047 the LUMO is calculated to be -0.122838 Hartrees. The energy difference between LUMO and HOMO is 0.090201 Hartrees that is small enough to allow the charge transfer from HOMO to LUMO. Based on these observation, it is proposed that the essence of the photoluminescence of complex 1 could be assigned to the ligand-to-ligand charge transfer (LLCT; from the HOMO of the perchlorate anion to the LUMO of the 4,4'-bipy ligand). Cadmium compounds are well-known not only for their photoluminescent behaviors but also for their semi-conductive properties and the latter enable them to be widely applied in military or civil areas. For example, Hg-CdTe, known as MCT, is one of the most famous military infrared detectors based on the semiconductive properties. Therefore, it could be worthy to measure the semicon-ductive properties of the title complex. Powder-like barium sulfate acts as a reference for 100% reflectance and finely-ground powder sample was coated on the surface of the barium sulfate. After measuring the solid-state UV/vis diffuse reflectance spectra, the data was treated carefully with the Kubelka-Munk function which is known as a/S = (1 - R)2/2R. With regard to this function, a means the absorption coefficient, S refers to the scattering coefficient which is actually wavelength independent when the size of the particle is larger than 5 ^m, and the R is related to the reflectance. From the a/S vs. energy gap diagram, the value of the optical band gap could be determined via extrapolating the linear portion of the absorption edges. In this way, the solid-state UV/vis diffuse reflectance spectra showed that complex 1 has a wide energy band gap of 3.25 eV, as depicted in Fig. 6. As a result, complex 1 could be a possible candidate for the wide optical band gap semiconductors. The slow slope of the optical absorption edge of complex 1 suggests that it must be an indirect transition.32 The energy band gap of 3.25 eV of complex 1 is obviously larger than those of CuInS2 (1.55 eV), CdTe (1.5 eV) and GaAs (1.4 eV), all of them are broadly applied as efficient photovoltaic materials.33,34 i ■ i ■ i 2 3 4 Energy gap/eV Figure 6. Solid-state UV/vis diffuse reflectance spectrum for complex 1. 4. Conclusions In conclusion, a novel cadmium complex with mixed ligands has been synthesized and characterized by single-crystal X-ray diffraction. It exhibits a 1-D zigzag chain structure. It is the first cadmium complex with both 4,4'-bipy and 2,2'-biim ligands. Powder photoluminescent characterization reveals that it displays an emission in the green region. TDDFT calculation revealed that the nature of the photoluminescence is originated from the li-gand-to-ligand charge transfer (LLCT; from the HOMO of the perchlorate anions to the LUMO of the 4,4'-bipy li-gand). A wide optical band gap of 3.25 eV was determined by the solid-state UV/vis diffuse reflectance spectrum. 5. Acknowledgements We gratefully acknowledge the financial support of the NSF of China (21361013, 21362015, 21461013, 51363009), Jiangxi Provincial Science and Technology Support Key Project (20152ACG70021), Jiangxi Provincial Natural Science Foundation (20142BAB205062, 20133ACB20010, 20132BAB203010), Jiangxi Provincial Department of Education's Item of Science and Technology (GJJ150761, GJJ14557, GJJ160745), Jinggangshan University Natural Science Item (JZ0813), the open foundation (ST201522007) of the Key Laboratory of Jiangxi Province for Persistant Pollutants Control and Resources Recycle (Nanchang Hangkong University), and the open foundation (20150019) of the State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences. 6. Supplementary Material Crystallographic data for the structure reported in this paper have been deposited with the Cambridge Crys-tallographic Data Centre as supplementary publication no. CCDC 1569870. Copies of the data can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: (44) 1223 336-033; e-mail: de-posit@ccdc.cam.ac.uk). 7. References 1. Y.-M. So, W.-H. Leung, Coord. Chem. Rev. 2017, 340, 172197. DOI:10.1016/j.ccr.2016.12.009 2. C. C. Mokhtarzadeh, C. E. Moore, A. L. Rheingold, J. S. Fi-gueroa, Angew. Chem. Int. Ed. 2017, 56, 10894-10899. DOI:10.1002/anie.201705877 3. M. Hoarau, C. Hureau, E. Gras, P. Faller, Coord. Chem. Rev. 2016, 308, 445-459. DOI:10.1016/j.ccr.2015.05.011 4. P. Zhang, J. M. Suarez, T. Driant, E. Derat, Y. Zhang, M. Yi et al.: Preparation, Structure, Photoluminescent Acta Chim. Slov. 201?, 64, 1042-1047 1047 Ménand, S. Roland, M. Sollogoub, Angew. Chem. Int. Ed. 2017, 56, 10821-10825. DOI:10.1002/anie.201705303 5. A. J. Howarth, M. B. Majewski, M. O. Wolf, Coord. Chem. Rev. 2015, 282-283, 139-149. DOI:10.1016/j.ccr.2014.03.024 6. X.-X. Lu, Y.-H. Luo, C. Lu, X. Chen, H. Zhang, J. Solid State Chem. 2015, 232, 123-130. DOI:10.1016/j.jssc.2015.09.017 7. X.-F. Yang, M. Liu, H.-B. Zhu, Inorg. Chem. Commun. 2017, 83, 40-43. DOI:10.1016/j.inoche.2017.06.007 8. D. P. Malenov, G. V. Janjic, V. B. Medakovic, M. B. Hall, S. D. Zaric, Coord. Chem. Rev. 2017, 345, 318-341. DOI:10.1016/j.ccr.2016.12.020 9. P. Maslewski, K. Kazimierczuk, Z. Hnatejko, A. Dolçga, Inorg. Chim. Acta 2017, 459, 22-28. DOI:10.1016/j.ica.2017.01.014 10. M.-L. Wei, J.-H. Wang, Y.-X. Wang, J. Solid State Chem. 2013, 198, 323-329. DOI:10.1016/j.jssc.2012.10.025 11. M. Zhu, M.-T. Li, L. Zhao, K.-Z. Shao, Z.-M. Su, Inorg. Chem. Commun. 2017, 79, 69-73. DOI:10.1016/j.inoche.2017.03.020 12. W.-T. Chen, J.-G. Huang, X.-G. Yi, Acta Chim. Slov. 2016, 63, 899-904. DOI:10.17344/acsi.2016.2897 13. L. N. Wang, L. Fu, J. W. Zhu,Y. Xu, M. Zhang, Q. You, P. Wang, J. Qin, Acta Chim. Slov. 2017, 64, 202-207. DOI:10.17344/acsi.2016.3109 14. H. S. Mansur, A. A. P. Mansur, A. Soriano-Araujo, Z. I. P. Lobato, Green Chem. 2015, 17, 1820-1830. DOI: 10.1039/C4GC02072C 15. A. R. D. Voet, H. Noguchi, C. Addy, K. Y. J. Zhang, J. R. H. Tame, Angew. Chem. Int. Ed. 2015, 54, 9857-9860. DOI:10.1002/anie.201503575 16. Rigaku, CrystalClear Version 1.3.5, Rigaku Corporation, 2002. 17. Siemens, SHELXTL™ Version 5 Reference Manual, Siemens Energy & Automation Inc., Madison, Wisconsin, USA, 1994. 18. P. Peng, F.-F. Li, V. S. P. K. Neti, A. J. Metta-Magana, L. Eche-goyen, Angew. Chem. Int. Ed. 2014, 53, 160-163. DOI:10.1002/anie.201306761 19. S. Konar, A. Jana, K. Das, S. Ray, J. A. Golen, A. L. Rheingold, S. K. Kar, Inorg. Chim. Acta 2013, 397, 144-151. DOI:10.1016/j.ica.2012.12.003 20. J. Jin, X. Han, Q. Meng, D. Li, Y.-X. Chi, S.-Y. Niu, J. Solid State Chem. 2013, 197, 92-102. DOI:10.1016/j.jssc.2012.09.016 21. M. Hakimi, Z. Mardani, K. Moeini, F. Mohr, M. A. Fernandes, Polyhedron 2014, 67, 27-35. DOI:10.1016/j.poly.2013.08.065 22. J. A. Wilson, P. E. Kraft, R. L. LaDuca, Inorg. Chim. Acta 2013, 404, 97-104. DOI:10.1016/j.ica.2013.04.027 23. P.-W. Liu, C.-P. Li, Y. Bi, J. Chen, J. Coord. Chem. 2013, 66, 2012-2022. DOI:10.10S0/0095S972.2013.79707S 24. Y.-Y. Liu, J. Liu, J. Yang, B. Liu, J.-F. Ma, Inorg. Chim. Acta 2013, 40S, S5-96. DOI:10.1016/j.ica.2013.03.009 25. X.-F. Wang, X.-Y. Yu, J.-K. Hu, H. Zhang, J. Coord. Chem. 2013, 66, 211S-212S. DOI:10.1080/00958972.2013.798654 26. X. Zhao, F. Liu, L. Zhang, D. Sun, R. Wang, Z. Ju, D. Yuan, D. Sun, Chem. Eur. J. 2014, 20, 649-652. DOI: 10.1002/chem.201304146 27. S.-N. Zhao, S.-Q. Su, X.-Z. Song, M. Zhu,Z.-M. Hao, X. Meng, S.-Y. Song, H.-J. Zhang, Cryst. Growth Des. 2013, U 27562765. DOI:10.1021/cg301S3S7 28. W. Xu, J. Jiang, M. Pan, C. Su, Inorg. Chem. Commun. 2013, S1, 83-86. DOI:10.1016/j.inoche.2013.02.018 29. X. Zhang, G. Ma, F. Kong, Z. Yu, R. Wang, Inorg. Chem. Commun. 2012, 22, 44-47. DOI:10.1016/j.inoche.2012.05.020 30. S. S. M. Rodrigues, D. S. M. Ribeiro, J. X. Soares, M. L. C. Passos, M. L. M. F. S. Saraiva, J. L. M. Santos, Coord. Chem. Rev. 2017, SS0, 127-143. DOI:10.1016/j.ccr.2016.10.001 31. V. Kumar, K.-H. Kim, P. Kumar, B.-H. Jeon, J.-C. Kim, Coord. Chem. Rev. 2017, S42, 80-105. DOI:10.1016/j.ccr.2017.04.006 32. F. Q. Huang, K. Mitchell, J. A. Ibers, Inorg. Chem. 2001, 40, 5123-5126. DOI:10.1021/ic0104353 33. P. Dürichen, W. Bensch, Eur. J. Solid State Inorg. Chem. 1997, ¡4, 1187-1198. 34. R. Tillinski, C. Rumpf, C. Näther, P. Duerichen, I. Jess, S. A. Schunk, W. Bensch, Z. Anorg. Allg. Chem. 1998,624,1285-1290. DOI:10.1002/(SICI)1521-3749(199808)624:8<1285::AID-ZAAC1285>3.0.C0;2-5 Povzetek S hidrotermalno reakcijo smo sintetizirali nov kadmijev kompleks z različnimi ligandi, {[Cd(2,2'-biim)(4,4'-bipy)(H2O) (ClO4)] (ClO4)}n (1) (2,2'-biim = 2,2'-biimidazol; 4,4'-bipy = 4,4'-bipiridin), ter določili kristalno strukturo z monokris-talno rentgensko difrakcijo. Rentgenska strukturna analiza razkriva, da kompleks 1 kristalizira v prostorski skupini Pna2l ortorombskega kristalnega sistema z enodimenzionalnimi cikcak [Cd(2,2'-biim)(4,4'-bipy)(H2O)(ClO4)]nn+ ka-tionskimi verigami in izoliranimi ClO4- anioni. Analiza fotoluminiscenčnih lastnosti prahu kaže, da kompleks 1 emitira zeleno svetlobo. Izračuni na podlagi teorije časovno odvisnega gostotnostnega funkcionala (TDDFT) kažejo, da je vzrok za pojav fotoluminiscence v kompleksu 1 v prenosu naboja z liganda na ligand (LLCT; od HOMO perkloratnega aniona k LUMO 4,4'-bipy liganda). S pomočjo UV/Vis difuzne reflektance v trdnem stanju je bil ugotovljen širok prepovedani pas velikosti 3.25 eV. Yi et al.: Preparation, Structure, Photoluminescent 1048 DOI: 10.17344/acsi.2017.3476 Acta Chim. Slov. 2017, 64, 1048-1055 /^creative ty com mor Short communication Geographical Origin Characterization of Slovenian Organic Garlic Using Stable Isotope and Elemental Composition Analyses Anja Mahne Opatic,12 Marijan Nečemer,4 David Kocman1 and Sonja Lojen1,3 1 Department of Environmental Sciences, "Jožef Stefan" Institute, Jamova cesta 39, Ljubljana, Slovenia_ 2 Jožef Stefan International Postgraduate School, Jamova cesta 39, Ljubljana, Slovenia 3 Faculty of Environmental Science, University of Nova Gorica, Vipavska 13, Nova Gorica, Slovenia 4 Department of Low and Medium Energy Physics, "Jožef Stefan" Institute, Jamova cesta 39, Ljubljana, Slovenia * Corresponding author: E-mail: anja.mahne00@gmail.com Tel: +038631873994 Received: 25-04-2017 Abstract In the present research, the applicability of stable isotope ($13C, $15N, $34S, $18O) and multi-element (P, S, Cl, K, Ca, Zn, Br, Rb, Sr) data for determining the geographical origin of garlic (Allium sativum L.) at the scale of Slovenia was examined. Slovenia is a rather small country (20273 km2) with significant geological and biological diversity. Garlic, valued for its medicinal properties, was collected from Slovenian farms with certified organic production and analyzed by elemental analyzer isotope ratio mass spectrometry combined with energy dispersive X- ray fluorescence spectrometry. Multivariate discriminant analysis (DA) revealed a distinction between four Slovenian macro-regions: the Alpine, Dinaric, Mediterranean and Pannonian. The model was validated through a leave-10%, 20% and 25% out cross validation. The overall success rate of correctly reclassified samples was 77% (on average), indicating that the model and the proposed methodology could be a promising tool for rapid, inexpensive and robust screening to control the provenance of garlic samples. Keywords: Food traceability, Garlic, Geographical origin, Stable isotopes, Elemental composition, Slovenia 1. Introduction Garlic (Allium sativum L.), a bulb vegetable widely used in global cuisine, is one of the most investigated food products. It contains approximately 65% water, 28% carbohydrates, 2% proteins, 1.2% free amino acids and 1.5% fiber. A further 2.3% is comprised of organosulfur compounds, which give garlic its characteristic pungent taste and flavor. These compounds are strongly related to garlic's beneficial effects on health,1 exhibiting antioxidant, antimicrobial, anticarcinogenic and antimutagenic activities as well as containing protective medicinal properties against cardiovascular and respiratory diseases.1,2 In recent years, proof of provenance has played an increasingly significant role in food safety and quality surveillance programmes. It also affects consumers' rights in accordance with national legislation, international standards and guidelines. Thus, geographical origin determi- nation has become another fundamental factor used for evaluating the quality of a product.3 In Slovenia, the quality of garlic is currently defined by the Rules on the Quality of Vegetables.4 At larger scale, in Europe, Commission Regulation (EC) No. 2288/97 lays down the marketing standards for garlic.5 These rules concern the visual appearance of vegetables (size, shape, firmness, cleanliness etc...), packaging and labeling, including the declaration of origin of produce. Nevertheless, rapid, reliable, robust and inexpensive screening methods are still necessary in order to ensure the geographical authenticity and trace-ability of food products and in addition, the main goal of research in this field has been lately focused on defining the parameters and providing appropriate analytical tools. One of the important techniques, where the scope of geographical assignment of food products can be remarkably extended, relies on a combination of isotopic and elemen- Opatic et al.: Geographical Origin Characterization Acta Chim. Slov. 2017, 64, 1048-1055 1049 tal fingerprinting. An important prerequisite is the availability of suitable databases, based on a large number of authentic samples. Moreover, basic knowledge on stable isotope fractionation effects in nature also needs to be increased.6 For garlic, no databank for tracing its origin at national, continental or global scale has been set up yet, in spite of global utilization of garlic as a condiment in cuisine or for medicinal purposes.7 Many authors have attempted to determine the geographical origin of different cereal grains,8-11 Chinese teas12 and honey13 using only stable isotopes of light elements. Samples classification into groups according their geographical origin was relatively successful, indicating that better classification could be achieved by applying not only stable isotope ratios of one or two light elements, but by using multielement stable isotope ratio data. Moreover, the use of stable isotope data in combination with multi-elemental analysis can provide even better discrimination in the case of apple juices,14 teas,15 tomatoes,16 wines17 and honey.18 In spite of this, a combination of multielement stable isotopes and multi-element fingerprinting has been rarely used, especially for tracing the geographical origin of vegetables. There has been scarce research on determining the geographical origin of garlic. Smith (2005)19 proposed trace metal profiling using high-resolution inductively coupled plasma mass spectrometry in order to determine the country of origin of garlic, whilst to authors' very best knowledge there appears to be very little literature data published applying stable isotope ratios or their combination with elemental composition data, e.g. Feher et al., (2017).20 The popularity of garlic in Slovenia is increasing extremely. According to the Statistical office of Republic of Slovenia total production of garlic in Slovenia in 2015 was 980 t, hereof 478 t for market production.21 This was 3 times higher for total production and 35-40 times higher for market production in comparison to 10 years ago. In ten years (i.e., 2005-2015) the garlic import from other countries has decreased for around 20%, and amounted to 1296 t in 2015.22 Slovenia is a small country (20273 km2) characterized by rich geological, climatological and biological diversity. It is situated between the Alps, the Dinaric Mountains, the Pannonian Basin and the Mediterranean Sea, making it ideal for this study. Our primary aim was to examine the possibility of using stable isotope ratio, multielement analyses, and chemometry (specifically, multivariate discriminant analysis (DA)) for characterizing and classifying organically grown Slovenian garlic according to the geographical macro-region in which it was cultivated (Alpine, Dinaric, Pannonian, or Mediterranean). The data gathering at the scale of Slovenia has commenced within the framework of the project "ISO-FOOD- ERA chair for isotope techniques in food quality, safety and traceability" funded by the EU. Furthermore, it is also expected that our findings will initiate the creation of a national database as the proposed methodology could be a promising tool for fast and cheap screening purposes which would allow for seamless implementation into existing food regulations and trade agreements. 2. Materials and Methods 2. 1. Sampling Samples were collected in autumn 2014 and 2015 from several Slovenian certified organic farms in order to ensure authenticity, traceability and equivalent production regimes. In total, 38 samples of garlic from four different Slovenian macro-regions (Fig. 1) were obtained. Fig. 1. Regionalization of Slovenia into four macro-regions23 with sampling sites and years of garlic samples Opatic et al.: Geographical Origin Characterization 1050 Acta Chim. Slov. 2017, 64, 1048-1055 2. 2. Stable Isotope Analysis Samples for simultaneous carbon, nitrogen and sulfur isotope ratio analysis were dried to constant weight in an oven at 60 °C. The dried samples were then homogenized and ground to a fine powder using a mortar. A known weight (10 mg) of garlic powder was folded and compressed in a tin capsule and introduced into the au-tosampler. Analysis was carried out using an Elementar vario PYRO cube elemental analyser (OH/CNS Pyrolyser/ Elemental Analyser) linked to an IsoPrime100 continuous flow isotope ratio mass spectrometer (IsoPrime, Cheadle, Hulme, UK). Each sample was analyzed in three repetitions. The difference between the replicates for any of the elements did not exceed 0.2%o. For oxygen stable isotope analysis, liquid samples were obtained by squeezing fresh garlic through a gauze. Isotopic ratios were then measured after equilibration (40 °C, 6h) with 5% CO2+ He mixture. Analysis was performed using an IsoPrime100 isotope ratio mass spectrometer and MultiFlow preparation system (IsoPrime, Cheadle, UK) according to the ENV 12141:1996.24 The stable isotope compositions are reported according to the IUPAC guidelines25,26 as relative differences in the isotope ratios (isotope-delta values), as seen in Equation (1)25: ill p _ p ¿f" E=- p m (1) The superscripts i and j denote the higher and lower atomic masses of the analysed element E, and Rp and RRef denote the heavy-to-light isotope ratios of the element E in the analysed sample and the reported reference material, respectively. A short-hand notation for elements with only two relevant stable isotopes is used in the text, i.e., S13C, S15N, and the S34S; the S values are expressed in per mil (%o) relative to standards. The S13C values are expressed relative to VPDB (Vienna Peedee belemnite) on a scale normalized by assigning consensus values of -46.6% to L-SVEC lithium carbonate and +1.95% to NBS 19 calcium carbonate. The S18O values of water are expressed relative to the VSMOW2 (Vienna Standard Mean Ocean Water 2), S34S relative to the VCDT (Vienna Canyon Diablo Troilite), and S15N relative to Air (atmospheric N2). By definition, the S value of the reporting standards is 0%. For S13C and S15N measurements, working standards were used, calibrated versus USGS40 (L-glutamic acid; certified S13CVPDB value -26.39 ±0.04%; recommend-edS15N value -4.5 ±0.1%o), USGS41 (L-glutamic acid; certified S13CVPDB value +37.63 ±0.05%; recommended S15NAir value +47.6 ±0.2%), IAEA-CH-6 (sucrose with certified S13C value -10.45 ±0.03%), IAEA-N-1 (ammonium sulfate with certified S15NAir value +0.4 ±0.2%) international reference materials, and IAEA-SO-5 (barium sulfate with recommended S34S +0.5 ±0.2%) reference material for sulfur. The accuracies of the S13C, S15N, and S34S analyses were monitored with commercially available isotope standards (Sercon) with recommended 513C and 515N values, and informative 534S values: Wheat Flour Standard Organic Analytical Standard (OAS; -27.21 ±0.13%; +2.85 ±0.17%; -1.42 ±0.80% for S13C, S15N and S34S, respectively), Sorghum Flour Standard OAS (-13.68 ±0.19%; +1.58 ±0.15%; +10.11 ±1.00%) and Protein (Casein) Standard OAS (-26.98 ±0.13%; +5.94 ±0.08%; +6.32 ±0.80%). All these values were certified by the Elemental Microanalysis Ltd. The measured 518O values were recalculated using the in-house reference materials (distilled seawater with SisO value +0.34 ±0.07%, MilliQ water with S18O value -9.12 ±0.07% and snow with 8mO value -19.73 ±0.09%). All these working standards were calibrated vs. the IAEA international reference material VSMOW2 with recommended 518O„„„„ value 0 ±0.02% and Greenland VSMOw Ice-Sheet Precipitation (GISP; certified S18Ovsmow -24.76 ±0.09%). Reproducibility of the measurements was ±0.1% for 518O, ±0.2% for S13C and ±0.3% for S15N and 534S. 2. 3. Elemental Analysis Multielement determination of macro (P, S, Cl, K, Ca) and micro elemental (Zn, Br, Rb, Sr) content was performed in single measurement using non-destructive energy dispersive X-ray fluorescence spectrometry. Pellets were prepared from 0.5 to 1.0 g of powdered sample material using a pellet die and hydraulic press. For excitation, the disc radioisotope excitation source of Fe-55 (25 mCi) and Cd-109 (20 mCi) from Eckert and Ziegler were used. The emitted fluorescence radiation was measured using an energy dispersive X-ray fluorescence spectrometer consisting of a Si(Li) detector (Canberra), a spectroscopy amplifier (Canberra M2024), analogue-to-digital converter (Canberra M8075) and PC-based multichannel analyser (S-100 Canberra). The spectrometer was equipped with a vacuum chamber (Fe-55) for measuring light elements P-Cl. The energy resolution of the spectrometer was 175 eV at 5.9 keV. The complex X-ray spectra were analyzed using AXIL spectral analysis software. Quantification was then performed utilizing the Quantitative Analysis of Environmental Samples (QAES) software, developed in our laboratory.27,28 The estimated analysis uncertainty was 5% to 10%. The accuracy of the data was checked using the National Institute of Standards and Technology 1573a reference material (tomato leaves). 2. 4. Statistical Analysis Statistical calculations were carried out using the XL-STAT software package (Addinsoft, New York, USA). Simple statistics included analysis of variance by ANOVA with Duncan's tests for comparison of means for normally distributed data, and Kruskal-Wallis one-way analysis of Opatic et al.: Geographical Origin Characterization Acta Chim. Slov. 2017, 64, 1048-1055 io51 variance by ranks (Kruskal-Wallis test) for not normally distributed data in order to reveal statistically significant differences, whilst multivariate DA was used for determination of key factors responsible for discrimination of four Slovenian geographical macro-regions. 3. Results and Discussion 3. 1. Garlic In Table 1 means and standard deviations of the stable isotope and elemental composition of the 38 garlic samples according to their given macro-region origin are reported, whilst Table 2 gives means, standard deviations and ranges (minimum- maximum) of the stable isotope and elemental composition of the 38 garlic samples, irrespective of geographical origin. The data of S15N, S18O, S34S, P, K and Zn were normally distributed and their variances were homoscedastic, while the data of $13C, S, Cl, Ca, Br, Rb, Sr and Si were not normally distributed. According to ANOVA test three parameters (518O, P, K) were statistically significant (p<0.05) for discriminating between the four Slovenian macro-regions. Additionally, the Kruskal- Wallis test revealed that only $13C is statistically significant for determining the origin of the garlic samples. 3. 2. Stable Isotopes We emphasize that only statistically sig- nificant parameters are described below. In this regard, the post-hoc Duncan test revealed that it is possible to differentiate the Mediterranean region from all the other regions on the basis of 518O values. The lowest 518O values were measured in garlic samples from the Dinaric region (average S18O = -2.6%o), whilst the highest S18O values were found in samples from the Mediterranean region (average 518O = -0.8%). The Alpine and Pannonian regions fell in between with average values of 518O = -2.1% and 518O = -1.9%, respectively. The xylem sap of terrestri- al plants reflects the integrated isotopic com-positons of the water sources, since the water is absorbed through the root system with no isotopic fractionation during uptake. After that, transpiration through the leaf stomata occurs, leading to enrichment of 518O in the leaf water.29 The distribution trends of 518O values in garlic samples is similar to that observed in Slovenian groundwater,30 when considering the 518O content of groundwater in the Mediterranean re- ir T3 T3 I J? i—H +1 +1 +1 +1 Li^ Li^ ro 5¡ MD O !S • 5 O Q 53 C^ £ £ $ II .2 ^ i < ¡y í 8 T3 T3 Ib 2 +1 a V C0 2 a 13 V B - Jg o w Oh rí „ to u ^ o •J3 £ V i o 3 1 Q 6 vilam na B; Mg 'k* KRKf\ @ Okus po pomaranči in limeti. ® Brez konzervansov. 0 Brez umetnih barvil, arom in sladil. ® Ena vrečka na dan. Prehransko dopolnilo ni nadomestilo za uravnoteženo in raznovrstno prehrano. Skrbite tudi za zdrav življenjski slog. (^ KRKk Naša inovativnost in znanje za učinkovite in varne izdelke vrhunske kakovosti. ActaChimica Slovenica Acta ChimicaSlovenica Professor Miha Tisler research was mainly devoted to the syntheses of new heterocyclic compounds and their transformations, development of new reagents, structural studies, tautomerism, etc. Figure shows crystal structure of N-[(Z)-2-benzoylamino-3-(4,6-dimethyl-2-pyrimidinylami-no)propenoyl]-L-proline, one of the numerous compounds prepared in his laboratory in Ljubljana which has become internationally known as the school for heterocyclic chemistry (See Editorial). Acta ChimicaSk AcraChimicaSlc Slovenica DdoüvMw«! MaÉ* Kfrflfe ■ km«k ^Ü* i Lii.'ini'.r' 'A V'-.-iii -iJ L ñ;bA- L4.-p j¿ l-rir-j!L\fcko Year 2017, Vol. 64, No. 4 I»»" l-Ul InrMiVol r i. V-. t Acta ChimicaSk /to Chi mica Slovenica L-,n Wrap« Fonlnl»-L\ik i.WirJttJpüdnr fft btnd An fan I UlA i <4*h¿ liv.,Va: »1 Ihqim .Ciiri LfcfcpJl ta Í44IWI ltd to* Fr* M * Ktmnsr ImnK s>=«ki In'. -■« I fcura.Íirarann nSH Ftafc• ri-i.Vj-.iit ft air. I' !i it:, a: n! teimtf ¡kfci dC j#tr.n«» jkul fcbi.t 4k \a£ Uefctkaraw-* ft jfete Ifcnciáre IWpm» Acta ChimicaSk Acta Chimica Slovenica 2 i«),nlH(( \ 52.Í0LJ M'jpdPlf1*!J>l MnMf a. Hi H <«■ tUpK*i Ilk Uu 1'iankkM >1> Wr (m *tn WrrtWrsd taWj JrtttrfVfc^d IÜSmí ™ hqtfmri: bcuwninw.M Bdhí«fúad IfanthVKMint im jfatLw ActaChimicaSk /left/Chimica,S Slovenica ActaChimicaSk Actc Chimica Slovenica rwir ActaChimicaSk Acu Chimica S Slovenica ■ !'. ' '.'I ---.. . - •■'.. zfed In! jj ..-■ IV'-..-,, ftikd ¡Wii- ÍJIIBIÍ MC RJC UWM hfi&i l toll uj ■ *■!. tjritifhi 1 ftn tit fafcf ActaChimicaSk Acta ChimicaSk Slovenica 2 Acta ChimicaSk ActcChimica Slovenica Acta ChimicaSk Actc Chimica Six Slovenica &5X VcvúTtVi DMMmCemlMm \*:r7l IWI*ncfc*