ISSN isflD-3iss Pages 1-246 ■ Year 2021, Vol. 68, No. 1 Acta ChimicaSlc Acta Chimica Slc Slovenica ActaC 68/2021 Bra* ôv: si* i |sí L' Ä/^JSF^ http://acta.chem-soc.si 9771580315594 EDITOR-IN-CHIEF KSENIJA KOGEJ University of Ljubjana, Facuty of Chemstry and Chemical Technology, Večna pot 113, SI-1000 Ljubljana, Slovenija E-mail: ACSi@fkkt.uni-lj.si, Telephone: (+386)-1-479-8538 ASSOCIATE EDITORS Alen Albreht, National Institute of Chemistry, Slovenia Aleš Berlec, Jožef Stefan Institute, Slovenia Janez Cerkovnik, University of Ljubljana, Slovenia Mirela Dragomir, Jožef Stefan Institute, Slovenia Ksenija Kogej, University of Ljubljana, Slovenia Krištof Kranjc, University of Ljubljana, Slovenia Matjaž Kristl, University of Maribor, Slovenia Franc Perdih, University of Ljubljana, Slovenia Aleš Podgornik, University of Ljubljana, Slovenia Helena Prosen, University of Ljubljana, Slovenia Irena Vovk, National Institute of Chemistry, 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, University of Ljubljana Janez Košmrlj, University of Ljubljana, Slovenia Blaž Likozar, National Institute of Chemistry, Slovenia Mahesh K. Lakshman, 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 ADVISORY EDITORIAL BOARD Chairman Branko Stanovnik, Slovenia Members Udo A. Th. Brinkman, The Netherlands Attilio Cesaro, Italy Vida Hudnik, Slovenia Venceslav Kaucic, Slovenia Zeljko Knez, Slovenia Radovan Komel, 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: Academic Search Complete, Central & Eastern European Academic Source, Chemical Abstracts Plus, Chemical Engineering Collection (India), Chemistry Citation Index Expanded, Current Contents (Physical, Chemical and Earth Sciences), Digitalna knjižnica Slovenije (dLib.si), DOAJ, ISI Alerting Services, PubMed, Science Citation Index Expanded, SciFinder (CAS), Scopus and Web of Science. Impact factor for 2019 is IF = 1.263. 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Acta ChimicaSlc ActaChimicaSlc GraphicalSlovenica ctaC Contents Year 2021, Vol 68, No. 1 feature article scientific paper 17—24 Inorganic chemistry Zinc(II) Complexes Derived from Schiff Bases: Syntheses, Structures, and Biological Activity Ling-Wei Xue, Xu Fu, Gan-Qing Zhao and Qing-Bin Li 25—36 Chemical, biochemical and environmental engineering Effect of Vacuum Frying Conditions on Quality of French Fries and Frying Oil Esra Devseren, Dilara Okut, Mehmet Koç, Özgül Özdestan Ocak, Haluk Karataç and Figen Kaymak-Ertekin Graphical Contents 37—43 Inorganic chemistry Extraction-Chromogenic System for Cobalt Based on 5-Methyl-4-(2-thiazolylazo)Resorcinol and Benzalkonium Chloride Danail G. Georgiev Hristov, Petya Vassileva Racheva, Galya Konstantinova Toncheva and Kiril Blazhev Gavazov 44—50 Inorganic chemistry Synthesis, Crystal Structures, Characterization and Catalytic Property of Copper(II) Complexes Derived from Hydrazone Ligands Yao Tan and Yan Lei [¡I 51-64 Organic chemistry Multi-component Reactions of Cyclohexan-1,3-diketones to Produce Fused Pyran Derivatives with Antiproliferative Activities and Tyrosine Kinases and Pim-1 Kinase Inhibitions Rafat Milad Mohareb, Rehab Ali Ibrahim and Ensaf Sultan Alwan 65-71 Organic chemistry 6-Bromo-2'-(2-chlorobenzylidene)nicotinohydrazide and 6-Bromo-2'-(3-bromo-5-chloro-2-hydroxybenzylidene)nicotinohydrazide Methanol Solvate: Synthesis, Characterization, Crystal ... Hai-Yun Zhu 72—87 Organic chemistry New Approaches for the Synthesis of Heterocyclic Compounds Derived from Cyclohexan-1,3-dione with Anti-proliferative Activities Rafat Milad Mohareb, Yara Raafat Milad and Ayat Ali Masoud 88—101 Biochemistry and molecular biology Synthesis, Crystallographic Structure, Hirshfeld Surface Analysis, Drug-likeness Properties and Molecular Docking Studies of New Oxime-pyridine Compounds Tufan Topal 102—108 Inorganic chemistry Copper(II) and Zinc(II) Complexes Derived from N,N'-Bis(4-bromosalicylidene)propane-1,3-diamine: Syntheses, Crystal Structures and Antimicrobial Activity Yu-Mei Hao 109—117 Organic chemistry Ultrasound-Assisted Synthesis, Antioxidant Activity and Computational Study of 1,3,4-Oxadiazol-2-amines Hamid Beyzaei, Soheila Sargazi, Ghodsieh Bagherzade, Ashraf Moradi, and Elahe Yarmohammadi NCI analysis 118—127 Biochemistry and molecular biology Antidiabetic Potential of Stem Bark Extract of Enantia chlorantha and Lack of Modulation of Its Therapeutic Efficacy in Diabetic Rats Co-Administered ... Latifat Bolanle Ibrahim, Patience Funmilayo Idowu, Opemipo Adekanye Moses, Mutiu Adewunmi Alabi and Emmanuel Oladipo Ajani 128—136 Applied chemistry Application of Silica Supported Calix[4]arene Derivative as Anti-reversion Agent in Tire Tread Formulation Hediye Mohamadi, Fereshteh Motiee, Saeed Taghvaei-Ganjali and Mandana Saber-Tehrani 137—143 General chemistry Physicochemical Properties of octane Isomers in View of the Structural Numbers Anton Perdih 144—150 Organic chemistry Structure of Biologically Active Benzoxazoles: Crystallography and DFT Studies Una Glamočlija, Selma Špirtovic-Halilovic, Mirsada Salihovic, Iztok Turel, Jakob Kljun, Elma Veljovic, Selma Zukic and Davorka Završnik 151-158 Biomedical applications Apoptotic Effect of Homobrassinin and Thiazino[6,5-b] indol is Associated with Downregulation of Heat Shock Proteins in Human ovarian Adenocarcinoma Cells CxX- CX^XV^O- homobrassinin 2-(4'-f[jorpheny1amino)-4H-1.3-tiazino|6,5-b|irido1e Zuzana Solarova, Martin Kello and Peter Solar 159-169 Physical chemistry Characterization of Hydration Behaviour and Modeling of Film Formulation Arunima Pramanik, Rudra Narayan Sahoo, Souvik Nandi, Ashirbad Nanda and Subrata Mallick 170-177 Materials science 4-(4,5-Diphenyl-1H-imidazole-2-yl)phenol: Synthesis and Estimation of nonlinear optical Properties using Z-Scan Technique and Quantum Mechanical Calculations Fatemeh Mostaghni 178—184 Inorganic chemistry on the Validity of Minimum Magnetizability Principle in Chemical Reactions Hiteshi Tandon, Tanmoy Chakraborty and Vandana Suhag 185—192 Physical chemistry Electrodeposition and Growth of Iron from an Ethylene Glycol Solution Vusala Asim Majidzade, Akif Shikhan Aliyev, Mahmoud Elrouby, Dunya Mahammad Babanly and Dilgam Babir Tagiyev 2e (degrees) 193—204 Inorganic chemistry Manganese(II) ^-Diketonate Complexes with Pyridin-4-one, 3-Hydroxypyridin-2-one and 1-Fluoropyridine Ligands: Molecular Structures and Hydrogen-bonded Networks Anze Cavic and Franc Perdih 205—211 Organic chemistry Co2 Improved Synthesis of Benzimidazole with the Catalysis of a New Calcium 4-Amino-3-hydroxybenzoate Ruo-Xuan Gao, Yuan-Yuan Gao, Ning Zhu and Li-Min Han 212—221 Inorganic chemistry Catecholase-Like Activity and Theoretical Study in Solid State of a new Ru(III)-Schiff Base Complex Niladri Biswas, Sandeepta Saha, Ennio Zangrando, Antonio Frontera and Chirantan Roy Choudhury 222—228 Biochemistry and molecular biology Protective Effects of 3-benzoyl-7-hydroxy Coumarin on Liver of Adult Rat Exposed to Aluminium Chloride Ahmet Özkaya and Kenan Türkan 229-238 General chemistry Physico-Chemical Properties of the Pyrolytic Residue Obtained by Different Treatment Conditions of Meat and Bone Meal Marija Zupančič and Nataša Čelan Korošin 239—246 Inorganic chemistry Hydrogen Bonds in Bis(1ff-benzimidazole-KN3) cadmium(II) Dibenzoate: Hirshfeld Surface Analysis and AIM Perspective Jia-Jun Wang, Li-Nan Dun, Bao-Sheng Zhang, Zhong-Hui Wang, He Wang, Chuan-Bi Li and Wei Liang DOI: 10.17344/acsi.2020.6488 Acta Chim. Slov. 2021, 68, 1-16 /^creative ^commons | Feature article Let the Biocatalyst Flow Polona Znidarsic-Plazl1,2'* 1 University of Ljubljana, Faculty of Chemistry and Chemical Technology, SI-1000 Ljubljana, Slovenia 2 University of Ljubljana, Chair of Microprocess Engineering and Technology -COMPETE, SI-1000 Ljubljana, Slovenia * Corresponding author: E-mail: polona.znidarsic@fkkt.uni-lj.si Received: 11-04-2020 Abstract Industrial biocatalysis has been identified as one of the key enabling technologies that, together with the transition to continuous processing, offers prospects for the development of cost-efficient manufacturing with high-quality products and low waste generation. This feature article highlights the role of miniaturized flow reactors with free enzymes and cells in the success of this endeavor with recent examples of their use in single or multiphase reactions. Microfluidics-based droplets enable ultrahigh-throughput screening and rapid biocatalytic process development. The use of unique micro-reactor configurations ensures highly efficient contacting of multiphase systems, resulting in process intensification and avoiding problems encountered in conventional batch processing. Further integration of downstream units offers the possibility of biocatalyst recycling, contributing to the cost-efficiency of the process. The use of environmentally friendly solvents supports effective reaction engineering, and thus paves the way for these highly selective catalysts to drive sustainable production. Keywords: Microreactor, enzyme, flow biocatalysis, continuous process, process intensification, process integration 1. Introduction The introduction of so-called "flow chemistry" in synthetic organic chemistry laboratories and also in industrial chemical production at the beginning of the 21st century brought a new paradigm in chemical processing. The availability of miniaturized flow reactors enabled the synthesis of complex molecules under controlled reaction conditions that yielded products of better quality and with fewer undesirable side reactions, as well as the ability to perform chemical reactions that are not possible in traditional batch operations.1 Over the past two decades, mi-croflow technology has matured from early devices and concepts to today's wide range of commercial devices and a variety of applications that also enable very efficient process analytics and control.2 Microflow systems are now important tools in chemical processes, from single-step to end-to-end processing, from (photo) catalytic to separation processes, from (nano)materials synthesis to pharmaceutical and fine chemicals production, and in environmental applications. Recent guidelines in the production of fine chemicals and the pharmaceutical industry to replace batch by continuous processes have further spurred interest in the implementation of "flow chemistry".3-5 Moreover, the quest to reduce the environmental factor, i.e. the E-factor (mass of waste per mass of product), which reaches the highest values in the fine chemicals and phar-ma industries, requires profound changes in production systems.6 Biocatalytic processes, along with continuous processing, have been identified as one of the crucial key areas of green engineering research for sustainable production in these sectors. They also play an important role in biomass valorization and circular economy.7,8 Biocatalysts were already known decades ago as environmentally friendly catalysts operating under mild conditions and with very high regio-, stereo-, and reaction selectivity, making them ideal catalysts for green chemistry.9 Nevertheless, it has been a major challenge to use these sensitive biomolecules and cells in harsh industrial environments, as they often need to convert non-natural substrates at concentrations several orders of magnitude higher than under natural conditions, and also require non-aqueous media for their solubilization. In addition, the frequently observed substrate or product inhibition, poor operational stability, and short shelf-life of biocatalysts prevented a wider application of biocatalytic processes in industrial production. However, in the last two decades, the understanding of protein and cell structure and function has improved tremendously. Genetic manipulations, metabolic Znidarsic-Plazl: Let the Biocatalyst Flow 2 Acta Chim. Slov. 2021, 68, 1-16 flux analysis, and the application of new techniques and materials for biocatalyst immobilization have led to unprecedented opportunities to develop more efficient and robust biocatalysts. Moreover, the use of novel solvents such as ionic liquids and deep eutectic solvents has led to efficient medium engineering that allows for more environmentally friendly production and high substrate availability.10-12 However, to achieve successful biocatalyst application, enzyme/cell, substrate, and medium engineering need to be complemented by reaction, reactor, and process engineering based on a thorough understanding of the reaction system and the specifics of the biological catalyst.10-11,13 In this regard, new concepts of reactor and unit operation design that incorporate continuous operation and miniaturization also provide new opportunities for efficient use of novel biocatalysts.14 The traditional use of large stirred tank reactors operated in a batch mode, or in some cases packed bed or fluidized bed reactors with biocatalysts retained in the particles appears to be sine qua non of industrial biotransformations.15 The implementation of microflow reactors in biocatalytic process development and operation has been much slower than for their chemical process counter-parts.14,16 Even the term "flow biocatalysis" was introduced in the scientific literature only a few years ago. The first review paper devoted to biotransformations in micro-structured reactors written by Bolivar, Wiesbauer, and Ni-detzky in 2011, reported a relatively small number of published studies in this field, mostly using dissolved enzymes, and the challenges of biocatalyst reversible immobiliza-tion.17 In the last decade, flow biocatalysis with a special focus on micro- and mesoscale devices has gained increasing attention in the academic and slowly in the industrial community, as evidenced by several comprehensive review articles,14,16,18-25 special issues of scientific journals, book chapters,26-29 special sections at scientific conferences with industry participation (Biotrans, Flow Chemistry Europe, Implementation of Microreactor Technology in Biotechnology -IMTB, etc.), and specialized webinars such as the "Flow Biocatalysis" organized by European Society of Applied Biocatalysis in October 2020. Although biocatalyst immobilization is gaining momentum by the application of novel materials and tech-niques,23,24,26-30 the majority of industrial biotransformations are carried out in aqueous environments with dissolved enzymes or free cells, which is also reflected in a modest market share of immobilized enzymes in the overall enzyme market.30,31 This is usually associated with additional immobilization costs and an often perceived decrease in biocatalyst activity related to either additional mass transfer limitations or biocatalyst deactivation. Due to the typically low solubility of organic substrates in water, the natural and most common environment for biocatalysis, substrates are either engineered by varying the substrate structure, by adding an immiscible liquid phase (typically organic solvent), or in the case of a single-liquid phase, by applying organic co-solvents. To lower the E-factor, the reduced use of organic solvents has been considered in the last two decades, as well as the application of neoteric solvents, such as ionic liquids (IL) and deep eutectic solvents (DES).6,10 Besides, non-conventional media typically used in environmentally friendly separation processes, such as supercritical CO2 (scCO2) and aqueous two-phase systems (ATPSs), are also gaining attention in biocatalytic processes and offer new opportunities for green biochemical production. This feature article addresses the advantages of continuous microflow-based processes for the efficient utilization of non-immobilized biocatalysts, and for rapid biocatalytic process development. Recent achievements in microreaction technology involving dissolved enzymes or suspended cells in the presence of one or more fluids are discussed with an emphasis on the implementation of green solvents for more sustainable production. Process integration enabling the recycling of biocatalysts, as well as opportunities for analytics integration and capacity expansion will also be considered. 2. Microreactors With Biocatalysts in a Single Liquid Phase The use of continuous operation in microfluidic devices offers several advantages over batch processing, especially when tuning of process variables can prevent biocatalyst deactivation. Most commonly used are simple tubes with diameters ranging from submillimeter to a few mm, typically used in analytical devices such as high-performance liquid chromatography (HPLC), or more sophisticated meander chips (Figure 1a), which are microfabricated from glass or various polymer materials. The intense mixing in stirred tank reactors required to transport substrates and products to and from the active site can lead to interfacial effects that can damage the biocatalyst,32 while a high flow rate required for the same purpose in conventional plug flow reactors can lead to insufficient residence time for completion of the reaction.33 This can be circumvented by the use of microflow systems, where ^m-scale diffusion paths allow for very efficient mass and heat transport, the latter also allowing for very precise temperature control, which is very important for processes involving thermo-sensitive biocatalysts. Diffusion efficiency can be visualized by the dye distribution at the Y-shaped outlet of the microchannel of 12.5 mm length, 205 ^m width, and 100 ^m height, where the dyed and pure water were pumped separately into the Y-shaped inlets. As shown in Figure 1b,34 laminar flow of aqueous methylene blue solution and water in the channel for 0.3 s (flow rate of 50 ^L/ min) resulted in moderate diffusion of the dye into the water (and vice versa), while 3 s residence time (flow rate of 5 ^L/min) allowed diffusion throughout the entire channel. Žnidaršič-Plazl: Let the Biocatalyst Flow 3 Acta Chim. Slov. 2021, 68, 1-16 The definition of process parameters such as fluid flow rate, enzyme and substrate inlet concentration, reactor geometry, etc. could be established based on mathematical modeling comprising transport phenomena and reaction kinetics.35 The advantages of moving from batch to continuous production using, among other devices, a microreactor with dissolved enzyme have been demonstrated for the production of the antidiabetic drug sitagliptin.33 This chemo-enzymatic production, developed a decade ago by Merck and Codexis, is one of the flagship industrial uses of engineered enzymes, in which a highly efficient and solvent-tolerant amine transaminase was developed based on substrate walking, modeling, and mutation approach followed by directed evolution.36 Replacing the environmentally problematic rhodium-catalyzed asymmetric enamine hydrogenation with a biocatalytic step resulted in a product with 99.5 % enantiomeric excess, a 10 to 13% increase in overall yield, a 53% increase in productivity, a 19% reduction in overall waste, elimination of all heavy metals, and a reduction in overall manufacturing costs. In addition, the enzymatic reaction could be carried out in multipurpose vessels, eliminating the need for dedicated high-pressure hydrogenation equipment. 33 The study on the multistep synthesis of sitagliptin monophosphate from chloropyrazine encompassed the design of a continuous end-to-end manufacturing process comprising microreac-tors and microseparators, and optimization of the biocatalytic step with dissolved transaminase based on a steady-state plug-flow model, taking into account enzyme recycling. Based on the evaluated optimized productivity and a comprehensive techno-economic analysis of this process, a net present value of $150 million over 20 years was calculated. Besides, an assessment of the environmental impact of the process demonstrated its sustainability with an E-factor of 53, which outperforms conventional pharma batch processes with a typical E-factor of 200.33 Another obstacle that is very often perceived in bio-catalysis is the alteration of enzyme microenvironment by the reaction, which can lead to its deactivation. The product may inhibit enzymes or be toxic to cells, while the pH change affects not only the activity and stability of enzymes, but also ionization and stability of substrates, products, and other components in the reaction mixture. Besides, high substrate concentrations can inhibit the biocatalyst, which can be prevented by using a continuous stirred tank reactor with low steady-state substrate concentration. To address this problem in tubular reactors, Szita's group developed a "side-entry reactor" (Figure 1c) in which the principle of a fed-batch substrate feed strategy was efficiently introduced into a microflow reactor. When tested for the transketolase-catalyzed reaction of lithium hydroxypyruvate and glycolaldehyde to L-eryth-rulose, a 4.5-fold increase in outlet product concentration and a 5-fold increase in throughput were achieved compared to a single-input reactor.37 Un upgraded version with the integrated optical pH sensors enabled not only monitoring of pH but also adjustment of this parameter via the side entries. As a result, the pH drop in the penicillin G acylase-catalyzed synthesis of 6-aminopenicillanic acid was significantly attenuated and the product yield was increased by up to 29% compared to the process without pH adjustment. This contribution represents a further step towards fully instrumented and controlled microfluidic reactors for biocatalytic process development.38 Figure 1: a) A Y-Y-shaped meander chip with 2 inlets and outlets; b) the outlet of the Y-Y channel presented in a) into which stained and pure water were pumped separately; the residence time in the channel was 0.3 and 3 s at the indicated flow rates of 50 ^L/min and 5 |iL/ min, respectively; reproduced with permission from Milozic et al., Chem. Biochem. Eng. Q., 2014 28, 215-223;34 c) a microfluidic side-entry reactor scheme with the indicated inlets (A, B), and side entries (1-10); all channels in the reactor had a cross section of 1 mm x 0.5 mm, and the length of individual sections of the main channel was 60 mm; reproduced with permission from Lawrence et al., Bio-technol. J. 2017, 12, 1600475.37 3. Microreactors With Biocatalysts in the Multi-Liquid Phase System The introduction of another liquid phase into the (typically aqueous) phase containing the biocatalyst opens, among others, the possibility of preventing its inactivation by compartmentalizing the inhibitor (substrate, product) from the biocatalyst, as well as shifting the reaction equilibrium toward product synthesis by in situ product removal.19 Another important result of controlled flow in the micro-flow systems is the prevention of stable emulsion formation, which often hinders product isolation after multi-liquid phase processing in conventional stirred tank reactors.39 Liquid-liquid two-phase flow in microflow devices can be efficiently controlled, resulting in a variety of fluid flow regimes (Figure 2) and efficient transport between compartments. Since diffusion times are proportional to the square of the characteristic length, typical mixing times in microfluidic devices based on diffusion are in the range of milliseconds, which is several orders of magnitude better than in conventional reactors. The flow pattern in microscale channels is a function of operating conditions, such as flow rates, phase ratio and fluid properties. In addition, the flow is influenced by the roughness and Žnidaršič-Plazl: Let the Biocatalyst Flow 4 Acta Chim. Slov. 2021, 68, 1-16 wettability of the channel wall, as well as by the geometry of the inlet channels (Y-, X-, or T-shaped) and the main channel diameter or aspect ratio for cylindrical or rectangular channels, respectively.19 Biocatalytic reactions within microflow systems typically involve various alkanes in addition to the aqueous phase. Among non-aqueous media, ionic liquids and recently also deep eutectic solvents (DES), both liquids consisting of ions with melting temperatures below 100 'C, are gaining increased attention in biocatalysis because they offer very high solubility of organic substrates. DESs are considered to be the fourth generation of ILs, although they do not consist entirely of ionic species.40 While the synthesis of ILs requires chemical synthesis, often performed efficiently in microflow reactors,41 DESs are prepared by simply mixing at least two inexpensive, nontoxic, and readily available components that are capable of self-associating in a specific molar ratio to form a new eutectic phase. The most typical compounds that constitute DESs are choline and urea, although amines, sugars, alcohols, polyols and organic acids are also used.42 Both solvent classes are nonvolatile, nonflammable, highly viscous, and can be prepared in a plethora of variations, resulting in properties that can be tailored as needed, which also makes them attractive for application in biocatalytic processes.40,42 Aqueous two-phase systems (ATPSs) are another green solvents that are gaining importance in biotransformations. They are mostly used in bioseparations to integrate solid phase removal and extraction of the biomole-cule of interest based on selective partitioning between phases. Typically, they are formed from two polymers such as polyethylene glycol (PEG) and dextran (Dex) or a polymer and an inorganic salt, e.g. phosphate and sulfate, dissolved in water, although some hydrophilic ILs are also capable of forming IL-ATPSs when mixed with aqueous solutions of inorganic salts.43 They provide a benign environment for the biocatalyst along with the possibility of reducing substrate and/or product inhibition by compart-mentalization in the other of the two phases. The industrial use of ATPSs is still hampered by their drawbacks such as slow diffusive mass transfer, long settling time for phase separation, and batch processing,44 so processing in mi-crofluidic systems present a promising tool for wider use of this green technology.45 Recently, we reported the use of the microfluidics for the generation of a temperature-dependent aqueous micellar two-phase system (AMTPS) containing a surfactant in the time frame of a few seconds (Figure 2 d). The ability to change temperature almost instantaneously, and further integration with a microsettler and micro-ultrafiltration unit enabled sustainable and efficient purification of a high value-added value protein from algal biomass extract.46 The introduction of an additional liquid phase offers a wealth of flow patterns and attractive features that greatly expand the applications of liquid-liquid two-phase mi-crofluidics. Three-liquid phase systems are widely used for Figure 2: Typical liquid-liquid two-phase flow in microchannels: a) parallel flow of aqueous and organic phase, b) formation of water-in-oil droplets, c) slug or Taylor flow of the hydrophobic ionic liquid in aqueous phase, d) mixed flow of the aqueous micellar two-phase system described by Serucnik et al.46 with core-annular flow and annular flow in the centre of the channel surrounded by droplets. various purposes, such as kinetic studies, microparticle synthesis, sample purification, and pharmaceutical crystallization. In addition to the parallel flow of all three phases (Figure 3 a2) and the generation of double emulsions (Figure 3 b2), which are of interest for the sorting of bio-catalyst and other applications, a novel hybrid slug flow-laminar flow system (Figure 3 c2) was reported, where one layer is the laminar aqueous flow and the other layer is the slug flow. This flow was successfully stabilized by installing a partition wall between the two channels.47 In the following chapters, the application of multi-liquid systems in biocatalytic processes will be highlighted. 3. 1. Microreactors With a Parallel Flow of Immiscible Liquids With Biocatalysts Due to their small dimensions and low applied flow rates, laminar flows are typical of microflow systems in which immiscible liquid phases flow in parallel to form a stable and continuous interface through which mass transfer occurs.47 The use of parallel flows has been achieved in microchannels with 2 Y-Y-shaped (Fig. 1a) or three ^-shaped inlets and outlets. The main advantage of such processing is the possibility to separate the phases at the output of the microchips with multiple inlets and outlets presented also in Figs. 1a, 1b, so that no further phase separator is required. To achieve this, precise tuning of the flow rates of both phases is required so that the interface can be positioned in the middle of the channel, while exiting channels can be chemically modified to become more or less hydrophobic. In a comprehensive review on enzymatic reactions utilizing non-aqueous media, several examples of enzymatic reactions with liquid-liquid (Fig. 2a and 3 a1) and liquid-liquid-liquid (Fig. 3 a2) parallel flow were given.19 A pioneering work by Maruyama et al. on the environ- Žnidaršič-Plazl: Let the Biocatalyst Flow 5 Acta Chim. Slov. 2021, 68, 1-16 Figure 3: Schematic diagram and characteristics of multi-liquid microfluidics comprising liquid-liquid (L-L) or liquid-liquid-liquid (L-L-L) flow: (a1) L-L: laminar flow shown also in Fig. 1a (a2) L-L-L: three-layer laminar flow, (b1) L-L: droplet flow shown also in Fig.1b, (b2) L-L-L: double emulsions, (cl) L-L: slug flow shown also in Fig. lc, and (c2) L-L-L: hybrid slug flow-laminar flow. Reproduced with permission from Wang et al., Lab Chip 2020, 20, 1891-1897, published by The Royal Society of Chemistry.47 mentally relevant laccase-catalyzed dechlorination of p-chlorophenol revealed 50-fold better specific productivity than in a laboratory-scale vessel with gentle shaking, achieving nearly 70% dehalogenation of the toxic substrate in 2 s.48 In later studies, the most frequently used enzyme was Candida antarctica lipase B (CaLB), which acts at the interface of the two phases, so that in a parallel flow the reaction surface is well defined. This enabled very accurate modelling and reactor performance prediction for the es-terification of isoamyl alcohol and acetic anhydride with substrate and product convection in the flow direction, diffusion in all directions, and reaction at the interface of the Y-Y- shaped microchannel.49 Along with aqueous buffers, alkanes are most commonly used as the second liquid phase in parallel flow.19 The use of an ionic liquid as the second phase was demonstrated in the enantioselective separation of (SJ-ibuprofen from a racemic mixture based on an enzymatic reaction. A thin film of ionic liquid between two aqueous phases with different lipases in each flow within the ^-^-shaped microchannel provided a high interfacial area and processing time of only 30-60 s to achieve efficient enantioselective transport of this drug, which exhibits different pharmaceutical and/or toxicological effects depending on its optical purity.50 Urease-catalyzed hydrolysis in an aqueous two-phase system of PEG and Dex using parallel laminar flow in a Y-Y-shaped microfluidic device, schematically shown in Figure 4, showed a 500-fold increase in the apparent reaction rate compared to conventional ATPS in a beaker under gentle stirring. The very short residence time in the channel was increased by 4 consecutive reaction cycles, resulting in a 4-fold increase in conversion.44 A theoretical study of the enzymatic production of cephalexin, an important ^-lactam antibiotic, using an ATPS based on PEG and phosphate in a microscale device comprising a thin dialysis membrane that provides flow stabilization and prevents transport of the enzyme and enzymesubstrate complex from the salt phase to the PEG phase. In the synthesis catalyzed by penicillin acylase, the effect of counter-current and co-current arrangements on cephalexin yield in microreactors with parallel flow of ATPS was discussed, as well as the possibility of transport enhancement by a direct-current (DC) electric field applied perpendicular to the interface. Based on the mathematical model comprising also mass transport across the membrane induced by an imposed electric field, the counter-current arrangement within the microreactor-separator was found to be suitable for cephalexin synthesis under most of the conditions studied.51' 3. 2. Microfluidics-Based Droplets With Biocatalysts Droplet-based microfluidic systems, which use passive microfluidic structures to rapidly generate and manipulate subnanoliter-volume droplets in microchannel environments, have changed the paradigm of biochemical ex-perimentation.52 Compartmentalization of liquids into droplets within an immiscible carrier liquid, usually stabilized with a surfactant molecule, has been successfully ap- Žnidaršič-Plazl: Let the Biocatalyst Flow Acta Chim. Slov. 2021, 68, 1-16 Figure 4. Schematic illustration of the ATPS enzymatic reaction and product separation in microchannel a) and at interface b) with a simple double Y-branched microfluidic device. Reproduced with permission from Meng et al., Chem. Eng. J. 2018, 335, 392-400.44 6 plied in numerous fields including single-cell and biomol-ecule analysis, diagnostics, drug delivery, protein crystallization, and chemical reactions.22,52,53 Discussed herein are their applications in biocatalytic process development phases, as well as for process intensification. 3. 2. 1. Droplets in Biocatalyst Screening and Characterization Selecting the most promising among the plethora of mutants generated by genetic manipulation or random mutagenesis is often the rate-limiting step in modern approaches to industrially relevant biocatalysts. Microfluid-ics-based ultrahigh-throughput screening of native or engineered enzymes and cells using droplets currently represents the most powerful tool for very rapid biocatalyst discovery and evolution at remarkably low cost.22,52-55 Furthermore, microfluidic platforms developed for directed evolution of enzymes in droplets, allowing screening of 107 mutants per round of evolution, have revolutionized the area of enzyme engineering.56 Briefly, aqueous droplets in oil generated, as shown in Figs. 2b and 3 b1, at frequencies up to 2 kHz are capable of encapsulating a single enzyme or cell together with the substrate, which is often barcoded. After the reaction, which is performed during on- or off-chip incubation, the droplets are typically re-emulsified into water-in-oil-in-water droplets, as shown in Fig. 3 b2, and re-injected into the sorter and dispersed in an oil stream leading to the Y-shaped junction (Fig. 1b). Here, droplets are flowed into one of the channels, while those containing an active biocatalyst are selected by a detector and directed into the other channel. Most commonly, fluorescence-activated droplet sorting (FADS) based on laser activation is used.52-57 As an example, a reliable and convenient ultrahigh-throughput screen- ing platform based on flow cytometric droplet sorting (FCDS), shown in Figure 5, was demonstrated to efficiently isolate novel esterases from metagenomic libraries by processing 108 single cells per day.58 Further encapsulation of single cells producing an enzyme of interest in microfluidic-based droplets along with a fluorogenic substrate and optionally lysing agents ensures that product formation occurs in the same compartment as the catalyst-encoding gene. The fluorescent product-containing droplets can then be sorted using FADS enabling ultrahigh-throughput directed evolution.59-61 As an example, a droplet-microfluidic screening platform was used to improve a previously optimized artificial aldolase by an additional factor of 30, resulting in a rate increase of over 109-fold .59 Evolutionary units in the form of monodisperse double emulsions or gel-shell beads (GSBs) containing a protein mutant and its coding DNA represent further step towards extremely fast biocatalyst engineering.62 Another ultra-high throughput protein screening platform called Split-and-Mix Library on Beads (SpliMLiB) was presented by Hollfelder's group. Directed evolution workflows were accelerated by DNA libraries constructed on the surface of microbeads suitable for direct functional screening in water-in-oil emulsion droplets with cell-free expression.63 To expand the application of this technique beyond non-fluorogenic substrates/products, assays based on ab-sorbance are being investigated.64 Future detection modes will include fluorescence-based approaches (anisotropy, Förster resonance energy transfer, lifetime) and label-free approaches based on light scattering (including Raman scattering) or droplet morphology.55 Reports on the application of positive dielectrophoresis-based Raman-activated droplet sorting for culture-free and label-free screening of enzyme function in vivo,65 and droplet sorting by inter- Žnidaršič-Plazl: Let the Biocatalyst Flow 7 Acta Chim. Slov. 2021, 68, 1-16 Figure 5 Workflow of the ultrahigh-throughput screening platform based on flow cytometric droplet sorting to mine novel enzymes from metagen-omic libraries. A. Collection of environmental microbes. B. Extraction of metagenomic DNA. C. Digestion and cloning of metagenomic DNA into an expression vector. D. Transformation of recombinant plasmids into a host strain for encoded protein expression. E. Encapsulation of single cells into water-in-oil-in-water double emulsion droplets, along with the screening substrate. F. Flow cytometric analysis and sorting of positive droplets. G. Secondary screening based on 96-well plate assays. H. Identification of novel enzymes. Reproduced with permission from Ma et al., Environ. Microbiol. 2020, DOI: 10.1111/1462-2920.15257.58 facial tension66 confirm these expectations. A sophisticated Raman-activated droplet sorting device uses periodically applied positive dielectrophoresis force to capture fast-moving cells, followed by simultaneous microdroplet encapsulation and sorting. The label-free method of sorting droplets by pH requires no active components and provides a robust platform for enzyme sorting in high-throughput applications.65 Another promising approach in this regard is the coupling of droplet microfluidics with electrospray ionization - mass spectroscopy (ESI-MS), which provides a label-free high-throughput screening platform. The system also enabled effective in vitro transcription-translation within the droplets analyzed directly by MS, demonstrating opportunities to greatly accelerate the screening of enzyme evolution libraries.67 Few nL or even pL surfactant-stabilized monodisperse droplets can be regarded as moving reactors that allow an extraordinarily large number of experiments to be performed simultaneously. As they move along channels, the reaction in the droplets can be monitored e.g. via laser-induced fluorescence measurements of product concentration that provide a time-dependence of the reac-tion.54 Because they consume minute amounts of reagents to provide the necessary information on reaction kinetics and biocatalyst inhibition, they significantly outperform conventional microtiter plates in terms of cost and time.52,53 In addition, droplet microfluidics offers the potential to generate and analyze enormous amounts of kinetic data through a high degree of integration with detection modalities. Some excellent reviews of droplet applications comprising examples of controlled microflu-idic systems used for e.g. automated analysis of enzyme kinetics, screening of protein crystallization conditions and protein solubility can be consulted for further information on this topic.52,53,57 However, the requirements for advanced droplet dispensing control and accurate sequential addition of samples or reagents to droplets at a high volumetric flow rate remain a challenging task. To address this, droplet array technologies have begun to offer a pathway to high-throughput screening.52 After many years of intensive research, and despite the enormous potential for industrial use, few commercial applications have been developed, and significant development in the field is still needed to make them reliable and widely applicable.68 There is a strong belief that high-throughput, high-sensitivity droplet-based microfluidics will become the gold standard for optimizing computationally engineered en-zymes.61 Exploration of 3-D printing technologies, robotics, and artificial intelligence is paving the way for smart platforms that could change the paradigm and drive the development of industrial biocatalytic processes.22,52 Žnidaršič-Plazl: Let the Biocatalyst Flow 8 Acta Chim. Slov. 2021, 68, 1-16 3. 2. 2. Droplets in Biocatalytic Processing Microfluidics-based droplets are characterized by a very high surface-to-volume ratio that allows high mass transfer between the phase containing the biocatalyst and the phase containing substrate and/or product, so their use can lead to process intensification when the reaction is limited by mass transfer. The benefits of using microfluid-ics-based droplets were demonstrated in the bio-hydration of acrylonitrile to acrylamide using Rhodococcus ruber whole cells containing nitrile hydratase, which is one of the important large-scale biotransformations. Conventional processing in stirred tank reactors is hindered by the low aqueous solubility of acrylonitrile, the low concentration of free cells, limitations on external mass transfer resulting in reduced apparent reaction rates, and by the limited ability to increase impeller speed and thus mass transfer due to potential interfacial effects leading to cell damage.32 To circumvent these problems, acrylonitrile was dispersed into small droplets of 25 to 35 ^m using a specially designed membrane dispersion microreactor. This enabled approximately 30% higher product yield in 5-times less time and also proved to extend the life of the free cells.69,70 The very high surface-to-volume-ratio of the 190 ^m droplets generated in an X-junction microchannel (Fig. 1b) was also advantageous for the Candida antarctica lipase B (CaLB)-catalyzed synthesis of isoamyl acetate, allowing the "natural" production of this important aroma. The amphiphilic enzyme positioned together with the substrate in the hydrophilic ionic liquid tends to attach to the surface of the organic phase forming droplets. The high interfacial area as well as the in-situ product removal into the organic liquid droplets allowed much higher volumetric productivities than reported in the literature for this esterification. Furthermore, the incorporation of a hydro-phobic membrane-based separator allowed separation of the enzyme from the product in the organic phase and several successful recyclations of the biocatalyst.71 The same reaction has been studied in flow reactors developed by Corning®, which allow efficient mixing of two-phase systems without the need for high energy or high pressure drop devices. The key component of the system is a fluidic module made of special glass, which consists of a chain of identical cells with variable cross-sections and internal elements. The fluid is forced to split and then recombine in each cell, leading to the renewal of the interface in two-phase systems such as liquid-liquid (Figure 6 a). The ease of their scalability from laboratory to production scale and customization to meet specific requirements provides a cost-effective solution for a broad portfolio of reactions in organic synthesis as well as for extraction.72,73 Application of the low-flow module to li-pase-catalyzed esterification in an aqueous- n-heptane two-phase system enabled efficient interfacial mass transfer and in situ product removal, resulting in unprecedented volumetric productivities.74 Further scale-up of the Figure 6: Reactors with two-phase flow: a) a close-up of a liquid-liquid flow regime obtained in a Low Flow Advanced Flow™ Reactor developed by Corning®, and b) a scheme of the microfluidic system with an enzyme recycle: a - a T-shaped element, b - reaction micro-capillary, c - settler, d- reservoir of the top phase with dissolved reactants, e - reservoir of the recycle stream, f - product reservoir, g - peristaltic pump, h - dialysis micromodule, i - waste, j - dialysate solution, k - microdialyzer ports. Reproduced with permission from Vobecka et al., Chem. Eng. J. 2020, 396, 125236.75 process in a 70-mL modular reactor demonstrated excellent process scalability.22 The droplets generated by microfluidics were also used for the preparation of semipermeable silica micropar-ticles that allowed compartmentalization of enzymes. The porous shell allowed selective diffusion of substrate and product while protecting the enzymes from degradation by proteinases and maintaining their functionality over multiple reaction cycles. The system was tested for ^-glu-cosidase encapsulation and for the combined compart-mentalization of glucose oxidase and horseradish peroxi-dase, which form a controlled reaction cascade for the glucose detector. The microparticles were trapped in a mi-crofluidic array device in which the enzyme activity could be tested in a single microparticle, which also provided information on reaction kinetic parameters and stability.76 3. 3. Segmented-Flow Microreactors With Biocatalysts Segmented flow with alternating fluid segments (e.g., slug flow in Fig. 1c and Fig 2c) is much easier to achieve than stable parallel flow in a long channel that allows complete conversion, so this type of flow prevails in biocatalytic processing in liquid-liquid systems.19 It also allows very efficient mass transfer between phases, based on convec-tive motion in each segment that renews the interface, which increases the concentration gradient of the product and facilitates diffusive penetration through the inter-face.77 A typical setup consists of the mixing unit (T- or Y-shaped mixer), tubes with lengths that provide the appropriate residence times, and phase separators based on gravity, membranes, etc. Compared to batch processes, where the intensive mixing of several phases required for efficient mass transfer regularly leads to emulsification and Žnidaršič-Plazl: Let the Biocatalyst Flow 9 Acta Chim. Slov. 2021, 68, 1-16 thus phase separation problems, this obstacle is reduced in microflow reactors.39,78 Applications of slug flow include various reactions and enzymes, from reactions catalyzed by alcohol-dehy-drogenase (ADH),79-82 to hydroxynitrile lyase-catalyzed C-C bond formation,83 a reduction with pentaerythritol tetranitrate reductase,84 terpene production catalyzed by aristolochene synthase,77 penicillin acylase-catalyzed antibiotic synthesis,75,85,86 and lipase-assisted biodiesel pro-duction,87,88 among others. An interesting reactor design was reported by Karande et al. who combined different sized capillaries from 2.5 mm i.d. to 0.5 mm i.d. to comply with lower substrate concentration along the tubular reactor, where the ADH-catalyzed reaction takes place. This allowed optimization of the conversion of selected aldehyde to corresponding alcohol dissolved in an organic phase and contacted in a slug flow regime with an aqueous phase containing enzyme and cofactor, as well as a cofactor de-hydrogenase-based regeneration system.79 To circumvent the interfacial deactivation of ADH in segmented flow, the addition of surfactant and immobilization of the enzyme in porous beads carried along the tubular reactor within the aqueous segments were tested. Both approaches resulted in very efficient stabilization of the enzyme, with surfactant addition being preferred due to better enzyme activity, less complexity, and ease of implementation in slug flow microreactors.80 Significant mechanical energy savings have been reported for lipase-catalyzed soybean oil hydrolysis using a slug flow microreactor. The hydrodynamically well-controlled slug flow generated in a T-shaped microfluidic channel and continued in submillimeter reaction capillaries, ensured uniform residence time of all slugs and enabled the recovery of well-defined products. Further integration with two microfluidic separators resulted in phase separation and the possibility to reuse the dissolved en- 89 zyme.89 Recently, lipase-catalyzed biodiesel production in a slug-flow microreactor has received considerable attention. Very pure biodiesel with glycerol content below the detection limit was produced in an integrated system with two microchips connected in series. The first Y-shaped microchannel was used for biodiesel production with methanol in one feed and an emulsion of oil, lipase and surfactant in the second. In the Y-shaped microchannels connected in series, simultaneous purification, i.e., glycer-ol removal, was achieved with DES based on choline chloride and ethylene glycol.87 In another study by the same group, DES based on choline chloride and glycerol was used for biodiesel production based on lipase-catalyzed transesterification of edible and waste sunflower oil with methanol. The reaction, which was carried out in a Y-Y-shaped microchannel as well as in a mm-scale tube, resulted in a 3-4-fold higher productivity than in the stirred tank reactor operated in batch mode.88 Environmentally friendly ATPS prepared from PEG and phosphate buffer was used for an enzyme-catalyzed synthesis of the ^-lactam antibiotic cephalexin, which is produced industrially on a multi-tones annual scale. The microfluidic setup shown in Figure 6 b included a slug-flow microreactor that supported efficient mass transfer between penicillin acylase dissolved in the bottom ATPS phase and the substrates in the ATPS's top phase, as well as in situ product separation in the latter. Integration of the settler resulted in phase separation and enabled further recycling of the reaction phase containing the dissolved enzyme. The recycling circuit also included a microdyalizer operated in counter-current regime to remove phenylgly-cine, which tended to clog the system. The system could be operated continuously for 5 h, and the operating time appeared to be limited only by the washout of the enzyme.75 The same group has also applied various ATPSs in the production of 6-aminopenicillanic acid (6-APA) from penicillin G using dissolved penicillin acylase. Criteria for the selection of ATPS were optimal separation of 6-APA from the enzyme, high buffering capacity to reduce undesirable pH decrease due to dissociation of phenylacetic acid - a byproduct of the reaction, relatively low cost of ATPS components, and the possibility of electrophoretic transport of fine droplets and reaction products to both accelerate phase separation and increase the 6-APA concentration in the product stream. The possibility of electropho-retic transport of the salt-rich droplets in the system was verified in a simple microfluidic device.85 A continuation of this study led to the development of electric-field-enhanced selective separation of the reaction byproduct in a membrane micro contactor. Application of DC electric field resulted in enhanced mass transfer through a semipermeable membrane for rapid, continuous, and selective separation of electrically charged low-molecular-weight phenylacetic acid from the original reaction mixture containing free penicillin acylase. Furthermore, the electroos-motic flow through the membrane, which counter-directs the transport of phenylacetic acid, was advantageously used to concentrate the separated product in the acceptor phase.86 The importance of minimizing stable emulsion formation, typical of the stirred tank batch processing, was highlighted for the enzymatic reduction of hydrophobic ketone in a biphasic methyl tert-butyl ether (MTBE) -buffer carried out in a segmented flow formed in a Y-shaped mixer and guided in a poly(fluorenylene ethy-nylene) (PFE) coil of 0.8 mm diameter, and compared with the batch process. While the conversions in both process operations were similar under comparable conditions, emulsification and precipitation were strongly suppressed when the biocatalytic reactions were carried out in flow mode, significantly simplifying and minimizing the effort required for biphasic biocatalytic reaction systems.90 The pioneering work of Karande et al.80 inspired the study of segmented flow, in which segments containing a Žnidaršič-Plazl: Let the Biocatalyst Flow 10 Acta Chim. Slov. 2021, 68, 1-16 heterogeneous biocatalyst surrounded by another liquid phase flowed in the microreactor. A segmented hydro-gel-organic solvent system was developed based on superabsorbent polymer consisting of partially neutralized cross-linked polyacrylic acid, in whose matrix enzymes and whole cells could be embedded. Such a "fluid heterogeneous phase" was investigated with the ADH-catalyzed reduction of acetophenone and the aldoxime dehy-dratase-catalyzed dehydration of octanal oxime. Especially for solvent-labile catalytic systems, this approach offers an alternative for the application of immobilized biocatalysts in a continuously running process beyond conventional packed bed and wall-coated reactors.90 4. Microreactors With Biocatalysts Containing Gaseous and Liquid Phase Enzymes can be used as highly selective catalysts for the oxyfunctionalization of unactivated carbons in organic synthesis. Insufficient oxygen supply is often a bottleneck in O2-dependent reactions, which is why a high influx of the gas phase and intensive mixing are required in conventional stirred tank reactors. In biocatalytic processes, this can lead to enzyme deactivation and gas stripping of substrate and product.32,91 To circumvent this, a tube-in-tube reactor (TiTR), in which the gaseous substrate enters the reaction chamber along the entire length of the tube, is a promising alternative. A flow-through chemistry apparatus developed a decade ago allows contact between gasses and liquids via a semipermeable Teflon AF-2400 membrane of a submillimeter i.d.92 The application of such a high-pressure reactor setup providing oxygen supply across the membrane surface from the outside of the reactor system was demonstrated for the synthesis of 3-phenylcatechol using a continuous segmented flow of the aqueous phase with the enzyme and decanol with the substrate as shown in Figure 7. 2-Hy-droxybiphenyl- 3-monooxygenase was applied as a biocatalyst for the hydroxylation reaction and also required co-factor regeneration, which was provided by formate dehydrogenase dissolved in an aqueous phase. Very high Figure 7. Scheme of a tube-in-tube reactor used for enzymatic hydroxylation using gaseous oxygen as a substrate. Reproduced with permission from Tomaszewski et al., Org. Process Res. Dev. 2014, 18, 1516-1526.91 volumetric productivities were obtained when the reactor was of sufficient length providing the required residence times, emphasizing the potential of the TiTR as a promising technology for the realization of gas-dependent enzymatic reactions. The same reactor configuration was also used to study the kinetics of oxygen-dependent reactions catalyzed by glucose oxidase, where the challenges of conventional systems can be avoided by creating a bubble-free aeration system. The TiTR setup was fully automated and computer controlled, allowing characterization of an oxygen-dependent enzyme within 24 hours with minimal manual labor, outperforming the conventional batch setup approach. By pressurizing the system, the dissolved oxygen concentration can reach 25-times the values achievable by air supply under atmospheric conditions. Operation in the low dispersed flow regime allowed the generation of time-series data with an enzymatic catalyst, despite its low diffusivity, and the resulting data were in good agreement with experiments conducted in a batch system.93 Direct introduction of the gas phase into enzymatic microreactors, allowing efficient supply of gaseous substrate, has been reported for many biocatalytic processes involving immobilized enzymes that were reviewed else-where.14,16,20-28 A report on the generation of a three-phase slug flow in a microchannel used for dissolved enzyme-catalyzed reaction revealed the benefit of introducing an inert gas phase (nitrogen) into a liquid-liquid slug flow to stabilize the liquid-liquid interface, and improve uniformity and reproducibility of the flow. In this way, uniform reaction-transport properties were created in a heterogeneous reaction system with an unstable interface in a long microchannel, as demonstrated in the lipase-cata-lyzed hydrolysis of soybean oil.94 Among the commercial meso-scale flow reactors enabling efficient direct gas-liquid contact, Corning® Advanced Flow Reactors, such as presented in Figure 6a, are commonly used in chemical industry, but to the author's knowledge, no report of enzymatic reaction with the gas phase in these reactors has been reported. On the other hand, the Coflore™ agitated cell reactor (ACR) and the agitated flow reactor (ATR) have been used for the chiral resolution of DL-alanine using non-immobilized whole Pichia pastoris cells with D-amino acid oxidase, where reaction is oxygen limited due to the gas-liquid mass transfer constraints of the conventional vessels. Comparison of a batch process performed in a 250 mL stirred tank reactor at various stirring speeds with a 100 mL Coflore ACR, a dynamically mixed plug flow reactor that uses to promote mixing, revealed a slightly increased reaction rate in the flow reactor. Further comparison of 1 L stirred tank batch reactor with 1 L Coflore ATR tubular reactor using lateral movement showed much greater improvement in volumetric productivity for the flow reactor due to improved gas-liquid mass transfer. In addition, virtually the same results were obtained with AFR when scaling up from 1 to 10 L Žnidaršič-Plazl: Let the Biocatalyst Flow 11 Acta Chim. Slov. 2021, 68, 1-16 without seeing the losses already evident when moving from 1 to 4 L in conventional batch reactor.95 5. Process Analytics in Microreactors With Biocatalysts As described in the chapter on droplet microfluidic platforms used for fast biocatalyst screening, evolution and characterization, highly automated and controlled devices using numerous analytical techniques are under development. Miniaturization and integration of several analytical techniques such as chromatography, electrophore-sis, or flow injection analysis in devices referred to as micro Total Analysis Systems (^TAS) were the first applications of microfluidics starting in the 1990s.96 In contrast, most studies presented in this review use off-line analytical techniques such as HPLC or spectrophotometry. Optical sensors for non-invasive and non-destructive monitoring of e.g. oxygen, pH, carbon dioxide, glucose, and temperature reviewed by Gruber et al. (2017) have great potential for on-line and at-line monitoring, both of which have some advantages and disadvantages as listed in Table 1.97 An example of in-line analysis of dissolved oxygen and substrate or product concentration in the microchannel outlet stream is shown in Figure 8. Dissolved oxygen concentration was measured using a fiber microsensor within the needle inserted into the stream, while substrate or product concentration was evaluated using the flow-through miniaturized optical detector that measures ab-sorbance at the specific wavelength of interest. Oxygen can also be monitored on-line by introducing sensory nanoparticles into the fluid and monitoring them using a fluorescence microscope, or by creating measurement points in the channel.98 As mentioned in Chapter 2, on-line pH measurement has been established Figure 8. Monitoring of the microchannel outlet stream regarding the dissolved oxygen concentration using fiber microsensor within the needle inserted in the flow, and the substrate or product concentration based on flow-through miniaturized spectrophotometer. based on a similar approach.97 The use of novel manufacturing capabilities offered by 3D printing technology and the integration of novel materials will pave the way for better process monitoring that will also enable process control, which is crucial for the efficient application of biocatalysts. 6. Conclusions and Future Perspectives If four technological advances evolved in the last decades of the previous century have been recognized as crucial for the acceptance of enzymes as "alternative catalysts" in industry, viz the development of i) techniques for large-scale isolation and purification of enzymes, ii) techniques for large-scale immobilization of biocatalysts, iii) biocatalytic processes in organic solvents, and iv) recombinant DNA technology enabling biocatalyst engineering,9 the fifth technological advance that can now be added is the development of continuous processing in miniaturized devices designed to efficiently harness these unique cata- Table 1: Summary of the advantages of on-line and at-line monitoring in microfluidic systems as proposed by Gruber et al., Lab Chip, 2017, 17, 2693, published by The Royal Society of Chemistry.97 Advantages Disadvantages on-line Real time analysis possible Possible interaction of sensors with the flow or reactants Rapid feedback allows real time process control Sensors need to be recalibrated and replaced over time No manual sampling required Increase of system complexity (fabrication, design, operation, maintenance) Measurement at real temperature Cross sensitivity with other analytes or interferences can be difficult to quantify No sampling required Limitation to a specific analytical problem and a certain concentration range Less risk of contamination Production flow undisrupted by sampling or redirecting at-line Significant number of assays/analytical methods available Changes in sample before analysis possible Can be cost-efficient Analysis limited to on-site equipment Flow cells available Certain sample volume necessary Feedback available quickly Risk of contamination through sampling Žnidaršič-Plazl: Let the Biocatalyst Flow 12 Acta Chim. Slov. 2021, 68, 1-16 lysts. The application of microflow systems for biotransformations with free biocatalysts offers several advantages, from reduced shear stress on fragile molecules and cells, to reduced mechanical energy requirements for efficient mixing, to very efficient contacting of multiple phases that allows compartmentalization of biocatalyst and often inhibitory substrates and/or products. Microfluidics-based droplets manipulated in highly automated microfluidic devices provide a revolutionary tool for ultrahigh-through-put biocatalyst evolution and efficient biocatalytic process development. Furthermore, continuous process operation in microflow reactors also allows for easy downstream process integration, enabling enzyme or cell recycling and thus very high total catalyst turnover number, defined as the total moles of product produced per mole of enzyme over the lifetime of the enzyme. The use of environmentally friendly solvents in such production systems can ensure that the goals of green chemistry as well as the bioecono-my are achieved. The requirements for fully controlled mi-croflow systems are driving the intensive development of integrated analytics, with new manufacturing technologies such as 3D printing together with novel materials offering endless possibilities. The use of model-based approaches that allow quantification of mass transfer in various reaction systems and microreactor configurations, as well as apparent reaction rates at different process conditions, will help to exploit the potential of microreactor technology. The use of engineering tools, such as characteristic times analysis and dimensionless numbers evaluation, could be of great value in this endeavor.22,79,80 Industrial implementation of flow biocatalysis requires the knowledge transfer between the various disciplines involved in process development. Understanding the fundamental phenomena underlying the structure and function of biocatalysts, biocatalytic reaction mechanisms and kinetics, and the performance of microreactors is therefore a basic requirement and should be implemented in the curricula of chemical, biochemical, and engineering study programs. Acknowledgements Financial support from the Slovenian Research Agency (Grants P2-0191, N2-0067 and J4-1775) and from the EC H2020 project COMPETE (Grant 811040) is gratefully acknowledged. The author would like to thank M. Serucnik, L. Ostanek Jurina, B. Peric, T. Pilpah and M. Kle-mencic from University of Ljubljana for providing graphical material. Abbreviations ACR Agitated cell reactor ADH Alcohol-dehydrogenase ATR Agitated flow reactor 6-APA 6-Aminopenicillanic acid AMTPS Aqueous micellar two-phase system ATPS Aqueous two-phase system CaLB Candida antarctica lipase B DC Direct current DES Deep eutectic solvent Dex Dextran E-factor Environmental factor ESI-MS Electrospray ionization - mass spectroscopy FADS Fluorescence-activated droplet sorting FCDS Flow cytometric droplet sorting GSB Gel-shell beads HPLC High-performance liquid chromatography IL Ionic liquid MTBE Methyl tert-butyl ether PEG Polyethylene glycol PFA Perfluoroalkoxy PFE Poly(fluorenylene ethynylene) scCO2 Supercritical CO2 SpliMLiB Split-and-Mix Library on Beads TiTR Tube-in-tube reactor 7. References 1. D. T. McQuade, P. H. Seeberger. Applying flow chemistry: methods, materials, and multistep synthesis. J. Org. Chem. 2013, 78, 6384-6389. DOI: 10.1021/jo400583m. DOI:10.1021/jo400583m 2. K. F. Jensen. Flow chemistry—Microreaction technology comes of age. 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Izpostavljeni članek osvetljuje vlogo miniaturiziranih pretočnih reaktorjev s prostimi encimi in celicami v teh prizadevanjih na osnovi nedavnih primerov njihove uporabe v eno ali večfaznih reakcijah. Kapljična mikroflu-idika omogoča izvedbo ultra visokozmogljivostnih presejalnih testov in hiter razvoj biokatalitskih procesov. Uporaba mikroreaktorjev edinstvenih konfiguracij zagotavlja zelo učinkovito kontaktiranje večfaznih sistemov, kar se odraža v intenzifikaciji procesov in izognitvi problemom, prisotnih pri konvencionalnem šaržnem procesiranju. Nadaljnja integracija z zaključnimi procesi nudi možnosti recikliranja biokatalizatorjev, kar prispeva k ekonomski učinkovitosti procesov. Uporaba okolju prijaznih topil podpira učinkovito reakcijsko inženirstvo in tlakuje pot tem visoko selektivnim katalizatorjem k postavitvi trajnostne proizvodnje. © 0 Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Žnidaršič-Plazl: Let the Biocatalyst Flow DOI: 10.17344/acsi.2020.5817 Acta Chim. Slov. 2021, 68, 17-24 /^creative ©'commons Scientific paper Zinc(II) Complexes Derived from Schiff Bases: Syntheses, Structures, and Biological Activity Ling-Wei Xue,* Xu Fu, Gan-Qing Zhao and Qing-Bin Li College of Chemistry and Chemical Engineering, Pingdingshan University, Pingdingshan Henan, 467000 P.R. China * Corresponding author: E-mail: pdsuchemistry@163.com Received: 01-06-2020 Abstract Three new zinc(II) complexes, [Zn2I2(L!)2] (1), [Zn(HL2)2(NCS)2] (2), and [ZnIL3] (3), where L1 is the anionic form of 2-[(6-methylpyridin-2-ylimino)methyl]phenol (HL1), HL2 is the zwitterionic form of 2-(cyclopropylim-inomethyl)-5-fluorophenol (HL2), and L3 is the anionic form of 5-bromo-2-[(3-morpholin-4-ylpropylimino)methyl] phenol (HL3), have been prepared and characterized by elemental analyses, IR, UV and NMR spectra, and single crystal X-ray crystallographic determination. Complex 1 is a dinuclear zinc complex, and complexes 2 and 3 are mononuclear zinc complexes. The Zn atoms in the complexes are in tetrahedral coordination. The effect of the complexes on the antimicrobial activity against Staphylococcus aureus, Escherichia coli, and Candida albicans were evaluated. Keywords: Zinc complex, Schiff base, Crystal structure, Antimicrobial activity 1. Introduction The rapid increasing interest in the synthesis and structural studies of Schiff bases is due to their bioactivity and coordination properties.1 Schiff bases are active against fungal, cancer, convulsant, oxidant and diuretic activities.2 Metal complexes of Schiff bases have attracted considerable attention due to their versatile biological activity, such as antifungal, antibacterial and antitumor.3 And, in general, the metal complexes have higher biological activities than the free Schiff bases. It has been shown that the Schiff base complexes derived from salicylaldehyde and its derivatives with primary amines, bearing the N2O, N2S, NO2 or NSO donor sets, have potential antimicrobial activities.4 Zinc is an important biological element, its complexes derived from Schiff bases have received particular attention due to their interesting antimicrobial potential.5 Recent research indicated that the halide and pseudohalide groups can severely increase the antimicrobial activities.6 Our research group has reported some metal complexes with effective antimi- HL1 Scheme 1. The Schiff bases crobial activities.7 In pursuit of new and efficient antimicrobial agents, in the present work, three new zinc(II) complexes, [Zn2I2(L1)2] (1), [Zn(HL2)2(NCS)2] (2), and [ZnIL3] (3), where L1 is the anionic form of 2-[(6-methylpyridin-2-ylim-ino)methyl]phenol (HL1), HL2 is the zwitterionic form of 2-(cyclopropyliminomethyl)-5-fluorophenol (HL2), and L3 is the anionic form of 5-bromo-2-[(3-morpholin-4-ylpro-pylimino)methyl]phenol (HL3) (Scheme 1), are reported. To our knowledge, only two complexes with HL1,8 and no complexes with HL2 and 2. Experimental 2. 1. Material and Methods Salicylaldehyde, 4-fluorosalicylaldehyde, 4-bromo-salicylaldehyde, 2-amino-6-methylpyridine, cyclopro-pylamine, and 3-morpholin-4-ylpropylamine were purchased from Fluka. Other reagents and solvents were analytical grade and used without further purification. Ele- OH HL2 HL3 HL3 have been reported so far. Xue et al.: Zinc(II) Complexes Derived from Schiff Bases: 18 Acta Chim. Slov. 2021, 68, 17-24 mental (C, H, and N) analyses were made on a PerkinElm-er Model 240B automatic analyser. Zinc analysis was carried out by EDTA titration. Infrared (IR) spectra were recorded on an IR-408 Shimadzu 568 spectrophotometer. UV-Vis spectra were recorded on a Lambda 900 spectrometer. X-ray diffraction was carried out on a Bruker SMART 1000 CCD area diffractometer. JH and 13C NMR spectra were recorded on a Bruker 300 MHz spectrometer. 2. 2. Synthesis of the Ligands 2. 2. 1. 2-[(6-Methylpyridin-2-ylimino)methyl] phenol (HL1) Salicylaldehyde (1.22 g, 0.01 mol) and 2-ami-no-6-methylpyridine (1.06 g, 0.01 mol) were reacted in methanol (50 mL) for 30 min at 20 °C. The solvent was removed by distillation to give yellow product of HL1. Analysis Calcd. (%) for C13H12N2O: C 73.56, H 5.70, N 13.20. Found (%): C 73.41, H 5.82, N 13.05. IR data (KBr, cm-1): 1623 (CH=N). UV in acetonitrile (À, e): 273 nm, 1.03 x 104 L mol-1 cm-1; 350 nm, 2.77 x 103 L mol-1 cm-1. 1H NMR (300 MHz, d6-DMSO): 5 12.11 (s, 1H, OH), 8.63 (s, 1H, CH=N), 7.68-7.64 (m, 3H, ArH and PyH), 7.46 (t, 1H, ArH), 7.10 (t, 1H, ArH), 6.99 (d, 1H, ArH), 6.81 (d, 1H, PyH), 2.50 (s, 3H, CH3). 13C NMR (126 MHz, DMSO) 6 162.32, 161.87, 160.11, 159.23, 138.12, 133.03, 132.72, 121.85, 121.36, 119.77, 115.61, 112.22, 23.70. 2. 2. 2. 2-(Cyclopropyliminomethyl)-5-fluorophenol (HL2) 4-Fluorosalicylaldehyde (1.40 g, 0.01 mol) and cy-clopropylamine (0.57 g, 0.01 mol) were reacted in methanol (50 mL) for 30 min at 20 °C. The solvent was removed by distillation to give yellow product of HL2. Analysis Calcd. (%) for C10H10FNO: C 67.03, H 5.62, N 7.82. Found (%): C 67.16, H 5.54, N 7.96. IR data (KBr, cm-1): 1627 (CH=N). UV in acetonitrile (À, e): 233 nm, 1.51 x 104 L mol-1 cm-1; 265 nm, 8.38 x 103 L mol-1 cm-1; 346 nm, 5.10 x 103 L mol-1 cm-1. 1H NMR (300 MHz, d6-DMSO): 5 12.15 (s, 1H, OH), 8.67 (s, 1H, CH=N), 7.61 (d, 1H, ArH), 6.87 (d, 1H, ArH), 6.43 (s, 1H, ArH), 1.82 (m, 1H, CH), 0.71 (m, 2H, CH2), 0.43 (m, 2H, CH2). 13C NMR (126 MHz, DMSO) 6 167.13, 162.77, 161.10, 132.05, 120.54, 109.23, 103.82, 35.11, 6.77. 2. 2. 3. 5-Bromo-2-[(3-morpholin-4- ylpropylimino)methyl]phenol (HL3) 4-Bromosalicylaldehyde (2.01 g, 0.01 mol) and 3-morpholin-4-ylpropylamine (1.44 g, 0.01 mol) were reacted in methanol (50 mL) for 30 min at 20 °C. The solvent was removed by distillation to give yellow product of HL3. Analysis Calcd. (%) for C14H19BrN2O2: C 51.39, H 5.85, N 8.56. Found (%): C 51.31, H 5.77, N 8.71. IR data (KBr, cm-1): 1633 (CH=N). UV in acetonitrile (À, e): 228 nm, 1.36 x 104 L mol-1 cm-1; 274 nm, 6.73 x 103 L mol-1 cm-1; 335 nm, 3.35 x 103 L mol-1 cm-1. 1H NMR (300 MHz, d6-DMSO): 5 11.78 (s, 1H, OH), 8.58 (s, 1H, CH=N), 7.51 (d, 1H, ArH), 7.47 (s, 1H, ArH), 7.11 (d, 1H, ArH), 3.68 (t, 2H, CH2), 3.60 (q, 4H, CH2), 2.43 (t, 2H, CH2), 2.27 (q, 4H, CH2), 1.72 (m, 2H, CH2). 13C NMR (126 MHz, DMSO) 5 162.11, 156.35, 133.20, 125.07, 124.31, 120.89, 114.34, 67.18, 62.83, 59.22, 54.71, 29.67. 2. 3. Synthesis of the Complexes 2. 3. 1. [Zn2l2(L1)2] (1) Then, a methanol solution (20 mL) of ZnI2 (0.319 g, 1.0 mmol) was added to the methanol solution of HL1 (0.212 g, 1.0 mmol). The mixture was stirred for 1 h at 20 °C to give a colorless solution. Colorless block-shaped single crystals suitable for X-ray diffraction were formed by slow evaporation of the solution in air for several days. The yield was 45% (based on HL1). Analysis Calcd. (%) for C26H22I2N4O2Zn2: C 38.69, H 2.75, N 6.94, Zn 16.20. Found (%): C 38.82, H 2.63, N 6.85, Zn 16.37. IR data (KBr, cm-1): 1615 (CH=N). UV in acetonitrile (X, e): 310 nm, 3.13 x 103 L mol-1 cm-1; 410 nm, 2.32 x 103 L mol-1 cm-1. 1H NMR (300 MHz, d6-DMSO): 5 8.71 (s, 1H, CH=N), 7.81-7.45 (m, 4H, ArH and PyH), 7.10 (t, 1H, ArH), 6.92 (d, 1H, ArH), 6.82 (d, 1H, PyH), 2.50 (s, 3H, CH3). 13C NMR (126 MHz, DMSO) 5 163.41, 162.02, 161.05, 159.33, 138.17, 132.92, 132.65, 121.82, 121.71, 120.14, 115.66, 112.31, 23.70. 2. 3. 2. [Zn(HL2)2(NCS)2] (2) A methanol solution (20 mL) of Zn(ClO4)2-6H2O (0.372 g, 1.0 mmol) and ammonium thiocyanate (0.076 g, 1.0 mmol) was added to the methanol solution of HL2 (0.179 g, 1.0 mmol). The mixture was stirred for 1 h at 20 °C to give a colorless solution. Colorless block-shaped single crystals suitable for X-ray diffraction were formed by slow evaporation of the solution in air for several days. The yield was 37% (based on HL2). Analysis Calcd. (%) for C22H20F2N4O2S2Zn: C 48.94, H 3.73, N 10.38, Zn 12.11. Found (%): C 49.13, H 3.82, N 10.28, Zn 12.35. IR data (KBr, cm-1): 2073 (NCS), 1653 (CH=NH). UV in acetonitrile (X, e): 270 nm, 3.56 x 103 L mol-1 cm-1; 305 nm, 1.91 x 103 L mol-1 cm-1; 350 nm, 8.33 x 102 L mol-1 cm-1; 397 nm, 2.06 x 102 L mol-1 cm-1. 1H NMR (300 MHz, d6-DM-SO): 5 10.82 (s, 1H, NH), 8.75 (s, 1H, CH=N), 7.61 (d, 1H, ArH), 6.87 (d, 1H, ArH), 6.46 (s, 1H, ArH), 1.83 (m, 1H, CH), 0.71 (m, 2H, CH2), 0.43 (m, 2H, CH2). 13C NMR (126 MHz, DMSO) 5 167.35, 162.45, 163.27, 135.86, 132.13, 119.71, 109.43, 104.51, 34.78, 6.77. 2. 3. 3. [ZnIL3] (3) A methanol solution (20 mL) of ZnI2 (0.319 g, 1.0 mmol) was added to the methanol solution of HL3 (0.326 g, Xue et al.: Zinc(II) Complexes Derived from Schiff Bases: ... 19 Acta Chim. Slov. 2021, 68, 17-24 1.0 mmol). The mixture was stirred for 1 h at 20 °C to give a colorless solution. Colorless block-shaped single crystals suitable for X-ray diffraction were formed by slow evaporation of the solution in air for several days. The yield was 54% (based on HL3). Analysis Calcd. (%) for C14H18BrIN2O2Zn: C 32.43, H 3.50, N 5.40, Zn 12.61. Found (%): C 32.27, H 3.63, N 5.45, Zn 12.82. IR data (KBr, cm-1): 1637 (CH=N). UV in acetoni-trile (À, s): 225 nm, 3.78 x 103 L mol-1 cm-1; 242 nm, 3.11 x 103 L mol-1 cm-1; 278 nm, 1.85 x 103 L mol-1 cm-1; 357 nm, 9.28 x 102 L mol-1 cm-1. 1H NMR (300 MHz, d6-DMSO): 5 8.67 (s, 1H, CH=N), 7.50 (d, 1H, ArH), 7.45 (s, 1H, ArH), 7.11 (d, 1H, ArH), 3.67 (t, 2H, CH2), 3.62 (q, 4H, CH2), 2.37 (t, 2H, CH2), 2.27 (q, 4H, CH2), 1.73 (m, 2H, CH2). 13C NMR (126 MHz, DMSO) 6 164.32, 158.11, 132.87, 125.12, 124.26, 121.81, 114.51, 67.25, 63.02, 59.38, 54.66, 29.71. 2. 4. X-Ray Diffraction Data were collected from selected crystals mounted on glass fibres. The data were collected with Mo_K"a radiation (0.71073 Â) at 298(2) K with a Bruker SMART 1000 CCD area diffractometer. The data for the complexes were processed with SAINT9 and corrected for absorption using SADABS.10 Multi-scan absorption corrections were applied with ^-scans.11 The structures were solved by direct method using SHELXS-97 and refined by full-matrix least-squares techniques on F2 using anisotropic displacement parameters.12 The imino H atom in complex 2 was located form a difference Fourier map and refined with N-H distance of 0.90(1) A. All other hydrogen atoms were placed at the calculated positions. Idealized H atoms were refined with isotropic displacement parameters set to 1.2 (1.5 for methyl groups) times the equivalent isotropic U values of the parent carbon atoms. The crystallographic data for the complexes are listed Table 1. Supplementary material has been deposited with the Cambridge Crystallographic Data Centre (nos. 1448092 (1), 1975485 (2), 1975486 (3)); deposit@ccdc.cam.ac.uk or http://www.ccdc.cam.ac.uk). 2. 5. Antimicrobial Assay Qualitative determination of antimicrobial activity was done using the disk diffusion method.13 The antibac- Table 1. Crystallographic data and experimental details for the complexes 1 2 3 Molecular formula Qg^^N^Z^ C22H2„F2N4O2S2Zn CnH^Brl^Zn Formula weight 807.02 539.91 518.48 Crystal size, mm 0.27x0.26x0.26 0.23x0.22x0.21 0.17x0.15x0.15 Radiation (À, Â) MoKa (0.71073) MoKa (0.71073) MoKa (0.71073) Crystal system Triclinic Monoclinic Monoclinic Space group P-1 P2/c P2i/c Unit cell dimensions: a, Â 8.056(2) 10.920(1) 15.065(2) b, Â 8.659(2) 7.021(1) 9.023(1) c, Â 11.034(2) 16.122(1) 12.860(1) a, ° 78.264(2) 90 90 P, ° 74.640(2) 97.347(1) 105.203(1) Y, ° 69.431(2) 90 90 V, Â3 689.7(3) 1225.9(3) 1687.0(3) Z 1 2 4 P calcd, g cm-3 1.943 1.463 2.041 F(000) 388 552 1000 T ■ T L mm> L max 0.4109, 0.4222 0.7678, 0.7847 0.4463, 0.4840 Absorption coefficient, mm-1 4.007 1.213 5.659 9 Range for data collection, ° 1.93-25.49 1.88-25.50 1.40-25.50 Reflections collected 4159 6294 33117 Independent reflections (Rint) 2571 (0.0315) 2287 (0.0495) 3135 (0.0809) Reflections with I > 2a(T) 1832 1473 2431 Data/parameters 2571/164 2287/154 3135/190 Restraints 0 1 0 Goodness-of-fit on F2 1.040 1.024 1.058 Final R indices (I > 2a(I)) R = 0.0457 Ri = 0.0688 Rj = 0.0404 wR2 = 0.0878 wR2 = 0.1835 wR2 = 0.0936 R indices (all data) R = 0.0736 Rj = 0.1057 Rj = 0.0605 wR2 = 0.1026 wR2 = 0.2073 wR2 = 0.1081 Apmax Apmim e Â-3 1.38, -0.55 1.47, -0.39 0.84, -0.64 Xue et al.: Zinc(II) Complexes Derived from Schiff Bases: ... 20 Acta Chim. Slov. 2021, 68, 17-24 terial activity was tested against B. subtilis, E. coli, P. fluorescence and S. aureus using MH medium (Mueller-Hin-ton medium). The MICs (minimum inhibitory concentrations) of the test compounds were determined by a colori-metric method using the dye MTT [3-(4,5-dimethylthi-azol-2-yl)-2,5-diphenyltetrazolium bromide]. A stock solution of the synthesized compound (50 ^g mL-1) in DMSO was prepared and quantities of the test compounds were incorporated in specified quantity of sterilized liquid MH medium. A specified quantity of the medium containing the compound was poured into micro-titration plates. A suspension of the microorganism was prepared to contain approximately 105 cfu mL-1 and applied to micro-titration plates with serially diluted compounds in DMSO to be tested and incubated at 37 °C for 24 h. After the MICs were visually determined on each of the micro-titration plates, 50 ^L of PBS containing 2 mg of MTT per millilitre was added to each well. Incubation was continued at room temperature for 4-5 h. The content of each well was removed and 100 ^L of isopropanol containing hydrochloric acid was added to extract the dye. After 12 h of incubation at room temperature, the optical density (OD) was measured with a micro-plate reader at 550 nm. 3. Results and Discussion 3. 1. Chemistry The Schiff bases were readily prepared by the reaction of equimolar quantities of aldehyde and amines in methanol, which were used directly for the preparation of the zinc complexes at ambient temperature. The zinc complexes are stable at room temperature in the solid state and soluble in common organic solvents, such as methanol, ethanol, chloroform, and acetonitrile. The results of the elemental analyses are in accord with the composition suggested for the complexes. 3. 2. IR and Electronic Spectra The IR spectra of the complexes were analyzed and compared with those of their free Schiff bases. The intense absorption bands at 1623, 1627 and 1633 cm-1 in the spectra of the Schiff bases HL1, HL2 and HL3, respectively, can be assigned to the C=N stretching. In the complexes, these bands are shifted to 1615, 1653 and 1637 cm-1 upon com-plexation with the zinc atoms, which can be attributed to the coordination of the imine nitrogen to the metal cen-tre.14 The absorption of this band for complex 2 is located at higher wavelength than complexes 2 and 3, which is due to the protonation of the imino group. The typical absorption at 2073 cm-1 in the spectrum of complex 2 is assigned to the vibration of the NCS ligand.15 UV-Vis spectra of the free Schiff bases and the complexes were recorded in HPLC grade acetonitrile solution. The spectra of the Schiff bases exhibit bands at 220-280 nm and 330-360 nm attributed to n^n* and n^n* transitions. In the spectra of the complexes the charge transfer bands at 220-280 nm remain intact, in agreement with the n^n* transitions of the Schiff base ligands. The remaining bands at 350-410 nm in the spectra of the complexes are assigned to the metal to ligand charge transfer (MLCT) transition.16 3. 3. NMR Spectra The 1H NMR spectra of the Schiff bases exhibit OH (phenolic) proton resonances at 11.78-12.15 ppm, imine proton resonances at 8.58-8.67 ppm and aromatic proton resonances in the range 6.43-7.68 ppm, respectively. On coordination, the signal due to OH proton disappears, indicating deprotonation of the phenolic OH and subsequent coordination of the phenoxide oxygen to the metal atoms. Involvement of the imine nitrogen in coordination has shifted the resonance signal of the imine proton by 0.7-0.9 ppm. The peaks observed in 13C NMR spectra of the Schiff bases and the complexes are in expected range and values are given in experimental section. The carbonyl C and imine C atoms in the complexes have shifted compared to their ligands, confirming the coordination through carbonyl O and imine N atoms.17 3. 4. Crystal Structure Description of the Complexes Complex 1 is a phenolato-bridged dinuclear zinc(II) compound (Figure 1), with Zn—Zn separation of 3.096(2) A. The crystal of the complex possesses a crystallographic inversion symmetry, with the inversion center located at the middle of the two zinc atoms. The Zn atom in the complex is coordinated by one pyridine N and two phenolate O atoms from two Schiff base ligands, and one I atom, generating tetrahedral geometry. The Schiff base acts as a bi-dentate ligand, and forms a six-membered chelate ring with the metal center through the phenolate O and imine N. The bond distances subtended at the metal atoms are Figure 2. Molecular packing structure of complex 1. Xue et al.: Zinc(II) Complexes Derived from Schiff Bases: ... 21 Acta Chim. Slov. 2021, 68, 17-24 comparable to those observed in similar zinc(II) complexes with Schiff bases.18 In the crystal structure of the complex, molecules are stacked via n—n interactions (Table 3) along the b axis (Figure 2). Complex 2 is a thiocyanate-coordinated mononuclear zinc(II) compound (Figure 3). The Zn atom in the complex is coordinated by two phenolate O atoms from two zwitterionic Schiff base ligands, and two thiocyanate N atoms, generating tetrahedral geometry. The Schiff base Figure 3. Molecular structure of complex 2. Atoms labeled with the suffix A are at the symmetry position - x, y, 1/2 - z. Figure 4. Molecular packing structure of complex 2. acts as a mono dentate ligand, with the phenol H atom transferred to the imino N group. The bond distances subtended at the metal atoms are comparable to those observed in similar zinc(II) complexes with Schiff bases and thiocyanate ligands.19 In the crystal structure of the complex, molecules are stacked via n—n interactions (Table 3) including the pyridine ring N(2)-C(8)-C(12)-C(11)-C(10)-C(9) and the chelate ring Zn(1)-N(1)-C(8)-N(2), along the b axis (Figure 4). Complex 3 is an iodide-coordinated mononuclear zinc(II) compound (Figure 5). The Zn atom in the complex is coordinated by one phenolate O, one imino N, and one morpholine N atoms of the Schiff base ligand, and one I atom, generating tetrahedral geometry. The Schiff base acts as a tridentate ligand, with the morpholine ring adopts chair configuration. The bond distances subtended at the metal atoms are comparable to those observed in similar zinc(II) complexes with Schiff bases and thiocyanate ligands.20 Figure 5. Molecular structure of complex 3. In the crystal structure of the complex, molecules are stacked via n—n interactions (Table 3) including the phenyl ring C(1)-C(2)-C(3)-C(4)-C(5)-C(6) and the chelate ring Zn(1)-O(1)-C(2)-C(1)-C(7)-N(1), along the b axis (Figure 6). 3. 5. Antimicrobial Activity The results of the antimicrobial activity are summarized in Table 4. A comparative study of minimum inhibitory concentration (MIC) values of the Schiff base and the zinc complex indicated that the complex has more effective activity against Staphylococcus aureus, Escherichia coli, and Candida albicans than the free Schiff base. Generally, Xue et al.: Zinc(II) Complexes Derived from Schiff Bases: ... 22 Acta Chim. Slov. 2021, 68, 17-24 Figure 6. Molecular packing structure of complex 3. this is caused by the greater lipophilic nature of the complex than the ligand. Such increased activity of the metal chelates can be explained on the basis of chelating theo-ry.21 On chelating, the polarity of the metal atoms will be reduced to a greater extent due to the overlap of the ligand orbital and partial sharing of positive charge of the metal atoms with donor atoms. Further, it increases the delocali-zation of ^-electrons over the whole chelate ring and enhances the lipophilicity of the complex. This increased li-pophilicity enhances the penetration of the complex into lipid membrane and blocks the metal binding sites on enzymes of micro-organisms. The complexes have stronger activities against Staphylococcus aureus, Escherichia coli, and Candida albicans than the free Schiff bases. For Staphylococcus aureus and Escherichia coli, the activities of the complexes are less than the control drug Tetracycline. But for Candida albi- Table 2. Selected bond distances (Â) and angles (°) for the complexes 1 Zn(1)-O(1)#1 1.990(5) Zn(1)-N(1) 2.014(5) O(1)-Zn(1)-N(1)#1 122.3(2) O(1)-Zn(1)-N(1) 88.1(2) N(1)-Zn(1)-I(1) 122.9(2) Zn(1)-O(1) 2.080(4) Zn(1)-I(1) 2.541(1) O(1)-Zn(1)-O(1)#1 81.0(2) O(1)-Zn(1)-I(1)#1 113.3(1) O(1)-Zn(1)-I(1) 113.1(1) 2 Zn(1)-O(1) 1.929(4) O(1)-Zn(1)-O(1)#2 117.1(2) O(1)-Zn(1)-N(2)#2 103.8(2) Zn(1)-N(2) 1.958(6) O(1)-Zn(1)-N(2) 109.8(2) N(2)-Zn(1)-N(2)#2 112.7(4) 3 Zn(1)-I(1) 2.5319(8) Zn(1)-O(1) 1.905(4) Zn(1)-N(1) 1.989(5) Zn(1)-N(2) 2.111(4) O(1)-Zn(1)-N(1) 96.47(18) O(1)-Zn(1)-N(2) 120.94(19) N(1)-Zn(1)-N(2) 94.06(19) O(1)-Zn(1)-I(1) 116.24(13) N(1)-Zn(1)-I(1) 119.21(14) N(2)-Zn(1)-I(1) 107.99(12) Symmetry codes: #1 - x, 1 - y, 2 - z; #2 - x, 1 - y, 2 - z. Table 3. Parameters between the planes for the complexes Cg Distance between ring centroids (Â) Dihedral angle (°) Perpendicular distance of Cg(I) on Cg(J) (Â) Beta angle (°) Gamma angle (°) Perpendicular distance of Cg(J) on Cg(I) (Â) 1 Cg(1)-Cg(2)« Cg(2)-Cg(2)« Cg(2)-Cg(3)#2 3.879 3.657 3.664 4.877 0 2.391 3.4456 3.3852 -3.4737 32.19 22.24 18.96 27.34 22.24 18.56 3.2826 3.3852 -3.4655 2 Cg(4)-Cg(4)#3 4.152 0 -3.428 34.36 34.36 -3.428 3 Cg(5)-Cg(6)#4 Cg(6)-Cg(6)*s 4.143 4.260 2.088 0 -3.295 3.654 37.02 30.91 37.33 30.91 -3.308 3.654 1: Cg(1), Cg(2) and Cg(3) are the centroids of Zn(1)-N(1)-C(8)-N(2), N(2)-C(8)-C(12)-C(11)-C(10)-C(9) and C(1)-C(2)-C(3)-C(4)-C(5)-C(6), respectively. 2: Cg(4) is the centroid of C(1)-C(2)-C(3)-C(4)-C(5)-C(6). 3: Cg(5) and Cg(6) are the centroids of Zn(1)-O(1)-C(2)-C(1)-C(7)-N(1) and C(1)-C(2)-C(3)-C(4)-C(5)-C(6), respectively. Symmetry codes: #1: 1-x, 1-y, -z; #2: 2-x, 1-y, -z; #3: -x, 1-y, -z; #4: 1-x, 1-y, 1-z; #5: 1-x, 2-y, 1-z. Xue et al.: Zinc(II) Complexes Derived from Schiff Bases: ... 23 Acta Chim. Slov. 2021, 68, 17-24 cans, the complexes have stronger activities than Tetracycline. Complex 2 has the most activity against Staphylococcus aureus with MIC value of 2 ^g/mL. The three zinc complexes have higher activities against Staphylococcus aureus and lower activities against Escherichia coli and Candida albicans than the zinc(II) and manganese(II) complexes with the ligand N'-(1-(pyridin-2-yl)ethyhdene)isonico-tinohydrazide.22 Further work needs to be carried out to investigate the structure-activity relationship. Table 4. MIC values (^g/mL) for the antimicrobial activities of the tested compounds Compound Staphylococcus Escherichia Candida aureus coli albicans HL1 128 256 > 1024 HL2 128 128 > 1024 HL3 64 256 > 1024 1 4 16 128 2 2 32 256 3 4 32 256 Tetracycline 0.25 2.0 > 1024 4. Conclusions In summary, three new zinc(II) complexes with hal-ide and pseudohalide ligands derived from Schiff bases have been prepared and characterized. The structures of the complexes are confirmed by single crystal X-ray crys-tallographic determination. The Zn atoms in the complexes are in tetrahedral coordination. The complexes have better activities on the bacteria Staphylococcus aureus and Escherichia coli than the control drug Tetracycline. Moreover, the complexes have stronger activities against Candida albicans than Tetracycline. Interestingly, complex 2 has the most activity against Staphylococcus aureus with MIC value of 2 ^g/mL. 5. Acknowledgments This research was supported by the National Sciences Foundation of China (nos. 20676057 and 20877036) and Top-class foundation of Pingdingshan University (no. 2008010). 6. References 1. L. N. Obasi, J. C. Ezeorah, V. Ossai, A. Jude, U. S. Oruma, A. Ibezim, M. Lutter, L. Rhyman, K. Jurkschat, N. Dege, P. Ramasami, J. Mol. 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Povzetek Trije novi cinkovi (II) kompleksi, [Zn2I2(L1)2] (1), [Zn(HL2)2(NCS)2] (2) in [ZnIL3] (3), kjer je L1 anionska oblika 2-[(6-metilpiridin-2-ilimino)metil]fenola (HL1), HL2 je zwitterionska oblika 2-(ciklopropiliminometil)-5-fluorofenola (HL2) in L3 je anionska oblika 5-bromo-2-[(3-morfolin-4-ilpropilimino)metil]fenol (HL3), so bili pripravljeni in kar-akterizirani z elementno analizo, IR, UV in NMR spektroskopijo ter rentgensko difrakcijo na monokristalih. Kompleks 1 je dvojedrni cinkov kompleks, kompleksa 2 in 3 pa sta enojedrna cinkova kompleksa. Atomi Zn v kompleksih so v tetraedrski koordinaciji. Preučevana je bila tudi protimikrobna aktivnost kompleksov proti Staphylococcus aureus, Escherichia coli in Candida albicans. © (D Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Xue et al.: Zinc(II) Complexes Derived from Schiff Bases: ... DOI: 10.17344/acsi.2020.5886 Acta Chim. Slov. 2021, 68, 25-36 /^.creative o'commons Scientific paper Effect of Vacuum Frying Conditions on Quality of French Fries and Frying Oil Esra Devseren,1 Dilara Okut,1 Mehmet Koç,2 Ozgul Ozdestan Ocak,1 Haluk Karataç3 and Figen Kaymak-Ertekin1* 1 Ege University, Faculty of Engineering, Department of Food Engineering, 35100, Bornova, Izmir, Turkey 2 Adnan Menderes University, Faculty of Engineering, Department of Food Engineering, 09010, Aydin, Turkey 3 Arçelik A. §., R&D Center, 34940, Istanbul, Turkey * Corresponding author: E-mail: figen.ertekin@ege.edu.tr Phone: +90.232.3113006 Fax: +90.232.3427592 Received: 02-06-2020 Abstract Vacuum frying conditions were investigated with respect to physical, chemical and sensorial properties of French fries and frying oil, besides determining the effect of frying conditions in terms of frying temperature and time. In order to determine the optimum frying conditions of the French fries optimization study was carried out according to Central Composite Rotatable Design. The results were evaluated to determine optimum vacuum frying conditions targeting minimum oil content, 30-45 N in range of hardness, minimum acrylamide content and maximum overall preference. The optimum vacuum frying condition was selected as 124.39 °C of frying temperature and 8.36 min of frying time for French fries. The French fries obtained at optimum conditions for vacuum frying preserved the desired color, textural properties and flavor and it has low oil content and reduced acrylamide formation. In addition, the frying oil quality was preserved with vacuum frying. Keywords: French fries; vacuum frying; browning index; hardness; acrylamide; microstructure 1. Introduction Deep fat frying is one of the earliest and most popular way of cooking process where the food is immersed into the hot oil (150-200 °C) at atmospheric pressure. Due to attractive color, texture and flavor of fried foods, deep fat frying is commonly applied both at home and in the food industry.1 Nowadays, deep fat frying has been remarkably interested by researchers due to rising consumption of fast food and pre-cooked food. Unfortunately, many studies related with deep fat frying reported that excessive oil uptake and high frying temperature had negative effects on health such as coronary heart diseases, cancer, diabetes and hypertension.2 As a result of simultaneous heat and mass transfer during deep-fat frying chemical and physical changes in foods have occurred for instance; moisture loss, oil uptake, crust formation, starch gelatinization and color change with the Maillard reaction, oxidation, hydrolysis and polymerization of oil. In addition to these changes, acrylamide was also formed during deep-fat frying process.3 Although consumer awareness of the relationship between nutrition and health increases, deep fat frying still continues to be one of the main cooking methods.4 That is why, the researchers have tried to develop alternative frying methods to eliminate the negative effects of deep fat frying and get healthier fried products. Vacuum frying is one of the new technologies as an alternative to atmospheric pressure frying and it is performed by immersing the food into the oil in a closed system at pressures lower than atmospheric pressure.5,6 Even though vacuum frying process carried out under the pressure of 6.65 kPa,7-10 it is increased up to 27.5 kPa in some studies.6,11-12 The vacuum frying process is performed at low temperature in low-oxygen frying medium.9,13 Thus, the vacuum frying is superior to atmospheric frying in terms of the protection of nutritional composition, color and flavor of the fried product, reusability of frying oil and acrylamide formation.13 Acrylamide, classified as a potential carcinogen by the International Agency for Research on Cancer, is formed in foods rich in both carbohydrates and proteins Devseren et al.: Effect of Vacuum Frying Conditions 26 Acta Chim. Slov. 2021, 68, 25-36 during cooking process at high temperatures.14 Many studies in literature showed that vacuum frying of potato chips led to form less acrylamide compared to those of atmospheric pressure.3,15-16 Although reduction of acrylamide in vacuum fried products is clear, the effect of vacuum on oil uptake is highly complex. According to Garayo and Morei-ra,13 potato chips had 30% less fat content with vacuum frying than atmospheric frying, while the potato chips with similar color and textural properties obtained with both frying methods. In contrast to this study, Troncoso et al.,8 found that vacuum frying resulted in an increase in oil content of potato chips compared to atmospheric frying. The quality of frying oil is also an important parameter affecting the quality of the fried food. Limited number of studies have been in literature investigating the effect of vacuum application on quality of frying oil.9,17 Crosa et al.,9 investigated the effect of vacuum frying and traditional frying on sunflower oil degradation, fatty acid composition and alpha-tocopherol content. It was determined a significant increase in the usage time of the oil with vacuum frying process compared with atmospheric frying process. Similarly, Aladedunye and Przybylski,17 reported that vacuum frying reduced the total polarity by 76% compared to atmospheric frying and lower oxidative degradation was observed. They also found that the rate of tocoph-erol degradation was three and twelve slower with vacuum frying than with other methods. During frying, the texture of the fruits and vegetables becomes initial soft and then hard due to progressive development of a dehydration crust. In addition, there is less structural change with vacuum frying than with atmospheric frying.14 Visual observations in apple slices showed that the surface of vacuum fried products was less expanded than atmospheric frying.6 Dueik et al.,18 determined that the maximum power of fried carrot chips is not affected by frying technology and temperature, so that vacuum fried chips have the same crispness as atmospheric fried ones. Da Silva and Moreira,19 did not found significant differences in the textural parameters of sweet potato, mango and green beans between atmospheric and vacuum frying. In this study, a vacuum cooking equipment having frying function was developed to investigate the effect of frying conditions in terms of temperature and time on physical, chemical and sensorial properties of French fries and frying oil. In order to determine the optimum frying conditions (temperature and time), optimization study was carried out according to Central Composite Rotatable Design (CCRD) and the effects of the temperature and time of vacuum frying were investigated. ed as frying medium due to preferring mostly at home. Prior to frying process, frozen French fries were sorted with respect to their size (1 x 1 x 8 cm) and stored at -24 °C. The moisture and oil content of the frozen French fries were determined 71.7 ± 1.7% and 3.9 ± 0.5%, respectively. 2. 2. Developed Vacuum Frying Equipment The vacuum frying equipment was designed to allow frying at wide range of temperature and vacuum pressure. Tomruk et al.20 already gave the constructional and working details of the equipment used in this study. The developed equipment consisted of three parts that were vacuum vessel, condenser and vacuum pump as shown in Fig. 1. The vacuum vessel was capacity of 6 liter and a basket, which can be moved up and down, was added to the vacuum vessel and it was difference from Tomruk et al.20. Also, a thermocouple (PT 100) was placed in the vacuum vessel to determine temperature of frying oil. Electrical heater was 1.5 kW and the vacuum pump (0.55 Hp) was oily type. The condenser includes refrigerant (R-404a). In this developed equipment, while the vacuum level and cooking time were controlled by PLC system, the heater was programmed by PID control system. Fig. 1. Developed vacuum frying equipment 2. Experimental 2. 1. Material Frozen French fries and sunflower oil were obtained from a local supermarket in Izmir. Sunflower oil was select- 2. 3. Frying Process In this study, French fries were fried under vacuum. French fries to sunflower oil ratio was 1:6 (w/v). Vacuum frying process was consisted of eight steps: (1) heating oil to selected temperature, (2) loading of French fries in a bas- Devseren et al.: Effect of Vacuum Frying Conditions ... 27 Acta Chim. Slov. 2021, 68, 25-36 ket and closing the lid, (3) applying vacuum until 13.3 kPa, (4) immersing the French fries into the hot oil, (5) frying French fries for selected time, (6) lifting the basket from the oil, (7) waiting for 30 seconds to allow the surface oil flow down, (8) pressurizing the vessel. The level of vacuum in this study was 13.3 kPa for all vacuum frying experiments. This level of vacuum was maximum vacuum pressure can be applied in the developed equipment. For vacuum frying experiments, French fries were fried at 120-150 °C for 5-15 min according to Central Composite Rotatable Design (CCRD). Experimental CCRD design was given in Table 1. All the frying experiments were done in duplicate. (Micro-CT) equipment (Scanco Medical ^CT 50, Switzerland). 3D models were created with the images received cross-section of the sample by using X-rays. The average pore diameter (^m) of the raw and fried samples were also measured.24 Sensory evaluation Sensory properties of fried French fries were evaluated in terms of appearance, color, texture, flavor (taste and smell) and overall acceptance. Sensory analysis was performed according to Altug and Elmaci,25 with 10 panelists. All panelists were non-smokers. The intensity of the properties was determined using a 5-point scale (1 being the lowest and 5 being the highest). 2. 4. French Fries Analysis Moisture Content For determination of moisture content in raw and fried French fries, samples were crushed with sand and dried in vacuum oven (Vacucell, U.S.A) at 65 °C for 24-48h until constant weight.21 Color Color of the raw and fried French fries were determined with a Minolta Chroma Meter (Konica Minolta, CR-400, Osaka, Japan). Hunter L, a, and b values were recorded to calculate the browning index (BI) of French fries (Eq. 1).22,23 Bl = ([100 * ((a + 1.75L)/(5.645L + (1) Texture The firmness of raw and fried French fries was measured by using the Texture Analyzer equipment (TA-XT2, Stable Micro Systems, UK) equipped with Multiple Chip Rig probe. Puncture test was simultaneously applied to 10 potato samples and the maximum power (N) was recorded. Puncture test was performed under these conditions: pre-test speed: 2 mm/s, test speed: 1 mm/s, post-test speed: 10 mm/s, distance: 5 mm and trigger force: 20 g. Acrylamide content Acrylamide content of French fries was analyzed with LC/MS-MS (ThermoFisher Scientific, USA). This method consisted of three sections, which were acrylamide standards preparation, sample preparation and purification.23 011 content Oil content of samples was gravimetrically determined by using hexane as the solvent with Soxhelet system.21 Microstructure Microstructure of the raw and fried French fries were determined by using Micro Computer Tomography 2. 5. Frying Oil Analysis Free Fatty Acids Free fatty acids (FFA) content of frying oil was determined using a titrimetric method and expressed as free oleic acid percentage.26 Peroxide Value (PV) Peroxide value (PV) expressed in milliequivalents of active oxygen per kilogram oil (mEq O2/kg oil), was determined. 26 Total Polar Compounds (TPC) Total polar compounds estimation was based on dielectric constant changes directly measured on hot oil with Deep Frying Oil Tester testoT270, when frying oil temperature was approximately 80 ( ± 5) °C. Experimental Design and Statistical Analysis Response surface methodology (RSM) was used to investigate the main effects of frying temperature and time on physical, chemical and sensorial properties of French fries fried under vacuum and optimize the responses. All experimental data were fitted to a second-order polynomial model and regression coefficients were obtained for each response. Significant terms in the models were found by analysis of variance (ANOVA). Design Expert Ver. 7.0.0 (Stat-Ease, 2005) and were used to fit response surfaces, where significant differences (p < 0.05) were detected. All of the analysis were done in triplicates. Results are shown as mean ± standard deviation. Optimization Numerical methods (desirability function) were used for optimization. At least five attempts were carried out at the optimum point as determined by the model (the optimum process conditions) and the optimum point was confirmed experimentally. In this study, desirability functions were evaluated for the criteria of minimum oil content, 30-45 N in range of hardness, minimum acrylamide content and maximum overall preference. These criterions Devseren et al.: Effect of Vacuum Frying Conditions ... 28 Acta Chim. Slov. 2021, 68, 25-36 were chosen according the literature data. Furthermore, response surface graphs and contour lines that helps to determine the optimum point is plotted using models obtained by regression analysis. 3. Results and Discussion 3. 1. Effect of Vacuum Frying on French Fries The physical, chemical and sensorial properties of the French fries at different experimental conditions are given in Table 1. ANOVA and regression analysis were evaluated to fit the model and determined the statistical significance of the model terms, as shown in Table 2. The quadratic polynomial model represented significantly the experimental values of responses at p < 0.05 level, besides the lack of fit of models were not significant. The counter plots of the predicted model of BI, hardness, oil content, acrylamide content, moisture content, overall acceptance, average pore diameter and peroxide value are given in Fig. 2. Moisture content was an important quality criteria for fried foods due to its effect on final product texture, microstructure and sensorial acceptability.27 The moisture content of fried foods varied with initial moisture content and dimensions of the product, and frying temperature and time.24 The moisture content of French fries decreased with an increase in frying temperature and time as shown in Table 1. Garayo and Moreira,13 explained this circumstance as follows; the boiling point of water reduced with a decrease in pressure, so the water in the potato begun to vaporize faster under vacuum. Lisinska and Golubows-ka,28 have reported that French fries had a moisture content of 44.7% because of atmospheric frying at 180 °C for 7 min. Besides, Romani et al.,29 observed that moisture content of French fries changed from 43.38% to 29.37% at 180 °C for 10 min depending on type of fryer and potato to oil ratio. These results were in consistent with our findings for vacuum frying. The ANOVA results also showed that, the moisture contents of French fries fried under vacuum significantly changed with frying temperature and time as shown in Table 2. In addition, it can be clearly seen in Fig.2. The oil content of fried products was also one of the most important quality features in many ways such as number of calories supplied by the food and growing trend on consuming foods produced with healthier methods. Beside the health issues about oil consumption, it had specific functional effects on flavor, appearance and smooth mouthfeel, which contributes the sensorial quality of the product.30 For this reason, many studies focused on producing fried products with low oil content that still protecting the desirable texture and flavor. Oil absorption was affected by the quality of frying oil, frying temperature, time and pressure, shape and chemical composition of food and applied pre-treatments.4 In literature, critical S T3 -3 G ft T3 -a 3 a t 3 S3 £ "o H o. H «j > T3 « S •a a '3 o S & (/i p« 3 o « 150 1° ^ £ 0 ^ tr « r2 (A m Ln ON Ln m NO NO M ^ ^O N to fO M ON c^ c^ no m ^H NO NO m NO c^ c^ C> C> C> C> O O o o o o ^H m Ln ON ^ oo m m H ^ m m NO 0.05). In addition, the frying oil has a polar substance content of 8.08 ± 0.20% before frying process, this was indicated that the vacuum frying process does not produce significant differences in the total polar material content. In our study, the results of the total polar material content were found to be in accordance with the current regulation of frying oil in all countries. The free fatty acid content of the frying oil is widely used as an indicator of oil degradation. Free fatty acidity can be followed quickly and reliably during frying.65 It is usually formed by the decomposition of triglycerides as a result of hydrolysis in the oil with the effect of air and humidity at high temperature.66 According to the frying oil regulation in Turkey, the acids number can be maximum 2.5 mg KOH /g oil.64 The acids number is equal to two times free fatty acids content. This limit value may vary between countries. The maximum permissible free fatty acid values of some countries are as follows; 1.25% in Austria , 2.5% in Belgium , 1% in Germany, 1.25% in Japan and 2.25% in the Netherlands.67 In addition, frying oil is changed according to the free fatty acid content in the industry and the point determined for changing the oil varies depending on the product. This value is 0.5% for potato chips and 1% for French fries. The effects of vacuum frying process variables on the free fatty acid content of frying oil were investigated and the free fatty acid content of frying oil were found between 0.0323% and 0.0486% oleic acids. These values were very close to each other. The free fatty acid content of the oil was determined as 0.0310 ± 0.0019% before frying process. It was observed that the free fatty acid content of the oil increased with the frying process, but the frying oil was suitable for reuse according to the regulations. According to ANOVA analysis, the effect of vacuum frying process conditions on the free fatty acidity of frying oil could not be explained by the selected quadratic model (p > 0.05). This is because the values of free fatty acid of frying oils are very close to each other. Frying temperature and time did not have a significant effect on the free fatty acid content of vacuum frying oil (Table 2). The peroxide value increases with the progression of primary oxidation in oil. As oxidation progresses, primary oxidation products (hydroperoxide) are broken down to Devseren et al.: Effect of Vacuum Frying Conditions ... 34 Acta Chim. Slov. 2021, 68, 25-36 produce secondary oxidation products (aldehydes and ke-tones).9 Therefore, while peroxide value give information about the quality of the oil, but it is insufficient in determining the usage period of the oil. The peroxide value of the frying oil at different temperatures and times under vacuum, are given in Table 1. When the peroxide values were examined, it was determined that increasing in temperature and time increased the peroxide values, but the effect of time was very low. It was observed that the values of peroxide varied between 0.113 and 0.481 meq O2 /kg and the highest value was reached in the condition at the highest temperature (150 °C). According to the results of variance analysis, the effect of temperature and time of the frying oil on the peroxide number was statistically significant (p < 0.05). 3. 3. Optimization The results were evaluated by using Design Expert version 7.0-packaged software and optimum vacuum frying conditions targeting minimum oil content, 30-45 N in range of hardness, minimum acrylamide content and maximum overall preference. Second-order polynomial model was used for each response for determining the optimum point. The desirability function approach was applied to obtain the optimum point solution given in Table 3. The optimum vacuum frying condition was selected as 124.39 °C of frying temperature and 8.36 min of frying time for French fries frying. The oil content, hardness, acrylamide content and overall preference at optimum conditions were predicted as 16.35%, 32.03 N, 87.4 ppb and 4.09, respectively. The results of the five validation experiments at optimum vacuum frying conditions are also given in Table 3, comparatively as average results and the estimated values. The oil content, hardness, acrylamide content and overall preference of the obtained samples were found to be significant (p < 0.05) different from the predicted values determined by Design Expert. It was determined that the hardness value was within the defined limits (30-45 N), but it was higher than the predicted value. Overall preference score was found to be higher than the predicted value, oil content and acrylamide content were obtained to be lower than the predicted values. These indicated a considerable increase in positive way. 4. Conclusion Vacuum frying methods were applied to investigate the effect of frying conditions on physical, chemical and sensorial quality of French fries. That is why a vacuum frying equipment prototype was designed to work under both vacuum and atmospheric pressure. Effect of frying independent variables (temperature and time) on French fries and frying oil was investigated according to CCRD experimental design. In addition, vacuum frying process conditions in terms of frying temperature and time were optimized to produce French fries with targeting minimum oil content, 30-45 N in range of hardness, minimum acryl-amide content and maximum overall preference. The optimum vacuum frying condition was selected as 124.39 °C of frying temperature and 8.36 min of frying time for French fries frying. The results showed that vacuum frying method protected the characteristic color, texture and sensorial quality of French fries, while the acrylamide content of French fries was low. The encountered problems, when targeted to achieve the desired color and texture in French fries frying at atmospheric frying, such as high acrylamide content, darkening of color and high oil content could be eliminated with vacuum frying. As a conclusion, vacuum frying can produce French fries containing low acrylamide content but having the same quality characteristics with those obtained under the atmospheric frying. Acknowledgements Funding provided by Ministry of Science, Industry and Technology, Republic of Turkey SAN- TEZ project (Project no: 0724.STZ.2014) and Ar^elik A.§ is appreciated. 4. References 1. R. G. Moreira, J. Palau, X. Sin, J Food Process Eng. 1995, 18, 307-320. DOI: 10.1111/j.1745-4530.1995.tb00369.x 2. D. Dana, I. S. Saguy, Adv Colloid Interfac, 2006, 128-130, 267-272. 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D0I:10.1016/j.foodres.2006.10.004 67. J. B. Rossell, Frying: Improving Quality. Woodhead Publishing Limited, Cambridge, 2001, pp. 1-355. D01:10.1201/9781439822951 Povzetek Testirali smo različne pogoje vakuumskega cvrtja pomfrita in preverjali njegove fizikalne, kemijske in organoleptične lastnosti glede na uporabljeno olje ter trajanje in temperaturo cvrtja. Da bi določil optimalne pogoje cvrtja smo pri študiji uporabili načrt s centralno zasnovo z rotacijo. Optimalni pogoji so vključevali minimalno vsebnost olja, trdoto v območju 30-45 N, minimalno vsebnost akrilamida in celovito ustreznost. Optimalne rezultate cvrtja pomfrita smo dosegli s temperaturo 124.39 °C in trajanjem 8.36 min. Tako pripravljen pomfrit je ohranil željeno bravo, teksturne lastnosti in okus, vseboval pa je tudi malo olja in znižano tvorbo akrilamidov. Poleg tega se je pri vakuumskem cvrtju tudi ohranila kvaliteta olja. © 0 Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Devseren et al.: Effect of Vacuum Frying Conditions ... DOI: 10.17344/acsi.2020.6035 Acta Chim. Slov. 2021, 68, 37-43 /^creative ^commons Scientific paper Extraction-Chromogenic System for Cobalt Based on 5-Methyl-4-(2-thiazolylazo) Resorcinol and Benzalkonium Chloride Danail G. Georgiev Hristov,1 Petya Vassileva Racheva,1,2 Galya Konstantinova Toncheva3 and Kiril Blazhev Gavazov1A* 1 Department of Chemical Sciences, Medical University of Plovdiv, 120 Buxton Brothers St., Plovdiv 4004, Bulgaria 2 Research Institute at the Medical University of Plovdiv, 15A Vasil Aprilov Bld., Plovdiv, Bulgaria 3 Department of General and Inorganic Chemistry with Methodology of Chemical Education, University of Plovdiv Paisii Hilendarski, 24 Tsar Assen St., Plovdiv 4000, Bulgaria * Corresponding author: E-mail: kgavazov@abv.bg Received: 04-14-2020 Abstract The interaction between Co11 and 5-methyl-4-(2-thiazolylazo)-resorcinol (MTAR) was studied in a water-chloroform system, in the presence or absence of benzalkonium chloride (BZC) as a cationic ion-association reagent. The optimum pH, concentration of the reagents and extraction time for the extraction of Co were found. In the presence of BZC, the extracted ion-associate could be represented by the formula (BZ+)[CoIII(MTAR2-)2], where MTAR is in its deprotonat-ed form. The following extraction-spectrophotometric characteristics were determined: absorption maximum, molar absorptivity, Sandell's sensitivity, limit of detection, limit of quantification, constant of extraction, distribution ratio and fraction extracted. In the absence of BZC, the extraction is incomplete and occurs in a narrow pH range. The extracted chelate contains one deprotonated and one monoprotonated ligand: [Com(MTAR2-)(HMTAR-)]. Keywords: Cobalt; 4-(2-thiazolylazo)resorcinol; benzalkonium chloride; ternary complex; solvent extraction; spectro-photometry 1. Introduction Cobalt is a group 9 transition metal that occupies position 27 in the periodic table. Because of its unique properties it is important for industry, agriculture, medicine and high technology development. As a highly wear- and corrosion-resistant metal which retains these properties even at high temperature, cobalt is an essential constituent of alloys for special uses.1,2 It is also applied in the manufacture of sintered cutting tools, catalysts, permanent magnets, pigments, siccative's and rechargeable (e.g., lithium-ion) batteries. Since the need of these batteries increases, some business experts predict a 47x increase in global demand for cobalt in 2030 compared to 2017.3 Cobalt is a relatively rare element. It is present in rocks, soils and sea water at very low concentrations and its average content in the continental crust is approximately 17.3 mg/kg.4 There are several commercially important cobalt-containing minerals, such as heterogenite, lin-naeite, cobaltite, smaltite, erythrite, carrolite, skutterudite and asbolite. However, in most deposits, this element is not in sufficient quantity to be economically minable alone and is obtained as a by-product of the metallurgy of copper, nickel, silver, gold, lead and zinc.1,4,5 Cobalt is an essential trace element for all animals, including humans, and an active nutrient for bacteria, fungi and algae. It is utilized by animals only in the form of vitamin B-12, synthesized by certain bacteria and archaea in the presence of enough cobalt. Cobalt deficiency (or vitamin B-12 deficiency) in humans can lead to pernicious anemia and nerve damage.6 On the other hand, excess cobalt can provoke numerous negative effects on central metabolism.7,8 That is why its content in various samples should be monitored.8-11 There are many analytical methods for cobalt. Azo dyes such as 4-(2-pyridylazo)resorcinol (PAR)12-23 and Hristov et al.: Extraction-Chromogenic System 38 Acta Chim. Slov. 2021, 68, 37-43 4-(2-thiazolylazo)resorcinol (TAR)24-27 have long been used for its preconcentration and subsequent determination. Due to the ability of these reagents to form anionic chelates with cobalt cations, analytical procedures are often associated with the addition of auxiliary reagents providing the necessary hydrophobicity and extraction characteristics. The following cationic reagents have been used as components of Co-PAR and Co-TAR ternary complexes: diphenylguanidine,15 dicyclohexyl-18-crown-6,17 tetrade-cyl(trihexyl)phosphonium chloride,21 xylometazoline hydrochloride,19 tributylammonium bromide,22 tetrapheny-larsonium chloride,28,29 tetraphenylphosphonium chlo-ride,28,29 nitron,30 tetrazolium salts,27,31-33 and quaternary ammonium salts.16,18,20,34-36 5-Methyl-4-(2-thiazolylazo)resorcinol (MTAR)37 is a TAR derivative that has been used in our laboratory for the liquid-liquid extraction of Viv,v 38-42 and Ni11.43 Its interaction with Co11 in water-ethanol medium has been studied by Kiryukhina.44 This reagent has also been used for the reversed-phase capillary high-performance liquid chromatographic determination of Co11.45 There are no reports on the liquid-liquid extraction of Co complexes with MTAR, nor on the ternary Co-MTAR complexes with auxiliary reagents. The objective of this work was to investigate the complex formation and liquid-liquid extraction of Co with MTAR in the presence and absence of benzalkonium chloride (BZC). BZC (Figure 1) is a mixture of quaternary ammonium chlorides used in pharmaceuticals, cosmetics and cleaning products due to its valuable bacteriostatic, bactericidal, fungicidal, algicide, spermicide and surfactant properties.46,47 Its ability to form ion-pairs with bulky anions has been used in liquid-liquid extraction methods for its determination.48 L^J h3c ch3 Fig. 1. Structural formula of BZC. The index n can be 8, 10, 12, 14, 16 or 18 (constituents with n = 12, 14, and 16 predominate). 2. Experimental 2. 1. Reagents and Apparatus Cobalt standard solution (1000 mg dm-3, Co(NO3)2) was obtained from Merck, Germany. Working solutions (cCo = 4.0 x 10 4 mol dm 3) were prepared by appropriate dilution. MTAR (95%) and BZC (> 95%) were also Merck products. Neutral or slightly basic aqueous solutions of MTAR (2.0 x 10-3 mol dm-3) were prepared by the addition of KOH.40,41 BZC was dissolved in water (cBzc = 2 x 10-2 mol dm-3 and 4 x 10-4 mol dm-3). Chloroform was redistilled and used repeatedly. The acidity of the aqueous phase was maintained constant by the addition of buffer solution, prepared by mixing 2 mol dm-3 aqueous solutions of acetic acid and ammonia. The resulting pH was checked by a WTW InoLab 720 pH-meter (Germany) with a precision of ±0.01 pH units. UV/vis spectrophotometers Ultrospec3300 pro and Camspec M508 UV-Vis (UK), equipped with 10-mm path-length cells, were employed for absorbance measurements. Distilled water was used in all experiments. 2. 2. General Procedure Solutions of Co11, buffer (pH 3.7-9.2), MTAR and BZC were sequentially transferred into a separatory funnel. Water was added to a total volume of 10 cm3. Then 10 cm3 of chloroform were buretted and the funnel was shaken for a fixed time interval. After a short wait for phase separation, a portion of the organic extract was transferred through a filter paper into the spectrophotometer cell. Ab-sorbance was measured against chloroform or a simultaneously prepared blank. 2. 3. Determination of the Distribution Ratio and Fraction Extracted The distribution ratio D was found from the equation D = A1/(A3 - A1), where A1 is the absorbance measured after a single extraction under the optimum conditions in the presence of BZC (Table 1) and A3 is the absorbance measured after a triple extraction under the same conditions. The total volume in both cases (single extraction and triple extraction) was 25 cm3.39-41 The fraction extracted was calculated from the equation E(%) = 100 x D/(D+1). 3. Results and Discussion 3. 1. Absorption Spectra and Effect of pH Kiryukhina44 reported that Co11 reacts with MTAR in a water-ethanol medium to form a 1:2-complex with an Table 1. Optimum extraction-spectrophotometric conditions. Extraction system ^max' nm PH cMTAR, mol dm-3 cBZC, mol dm-3 Shaking time, min Co - MTAR - water -chloroform Co - MTAR - BZC- water -chloroform 509 550 5.5 7.5 4.0 x 10-4 1.6 x 10-4 1.4 x 10-4 3a 3 a quantitative extraction cannot be achieved Hristov et al.: Extraction-Chromogenic System 39 Acta Chim. Slov. 2021, 68, 37-43 absorption maximum Xmax = 520 nm. She found that the optimum pH for complex formation was 6 - 8 but did not pay attention to the change in spectral characteristics as the pH changed. Our extraction studies have shown that the extracted into organic phase complex in the Co-MTAR-water-chlo-roform system has an absorption maximum at 509 nm (Figure 2, spectrum 1). In the presence of BZC, the absorption maximum shifts to the higher wavelengths (about 549 -550 nm; spectrum 2) and the optimum pH range widens significantly and shifts to higher pH values (Figure 3). Figure 2. Absorption spectra in chloroform of the Co-MTAR binary complex (1) against blank (1') and Co-MTAR-BZC ternary complex (2) against corresponding blank (2'). Cco = 4 x 10-5 mol dm-3; tex = 3 min; cMTAR = 4.0 x 10-4 mol dm-3 (1, 1') or 1.6 x 10-4 mol dm-3 (2, 2'); pH = 5.5 (1, 1') or 7.5 (2, 2'); cBZC = 1.4 x 10-4 mol dm-3 (2, 2'). Figure 3. Absorbance of the Co-MTAR (1) and Co-MTAR-BZC (2) complexes and corresponding blanks (1' and 2') vs pH of the aqueous phase. co = 4 x 10-5 mol dm-3 (1, 2); Cmtar = 4 x 10-4 mol dm-3 (1, 1', 2, 2'); cBZC = 1 x 10-3 mol dm-3 (2, 2'); X = 509 nm (1,1') or 550 nm (2,2'); tex = 3 min. 3. 2. Effect of MTAR Concentration and the MTAR-to-Co Molar Ratio The effect of MTAR concentration on the absorbance at 509 and 550 nm is shown in Figure 4, curves 1a-c. Processing of the data received in the presence or absence of BZC by the straight-line method of Asmus49 (Figure 5) showed that in both cases the MTAR-to-Co molar ratio is 2:1. When the concentration of MTAR increases in the Figure 4. Effect of MTAR (1a,b,c) and BZC (2) concentration. (1a) cCo = 4 x 10-5 mol dm-3, pH 5.2, tex = 3 min, X = 509 nm; (1b) cCo = 8.5 x 10-5 mol dm-3, cBZC = 4 x 10-4 mol dm-3, pH 6.0, tex = 3 min, X = 509 nm; (1c) cCo = 4.0 x 10-5 mol dm-3, cBZC = 1.4 x 10-4 mol dm-3, pH 7.5, tex = 3 min, X = 550 nm; (2) cCo = 4 x 10-5 mol dm-3, Cmtar = 2 x 10-4 mol dm-3, pH 7.5, tex = 3 min, X = 550 nm. a) b) c) Figure 5. Determination of the MTAR-to-Co molar ratio by the straight-line method of Asmus. The experimental conditions are given in Figure 4, curves 1a-c, respectively. Hristov et al.: Extraction-Chromogenic System 40 Acta Chim. Slov. 2021, 68, 37-43 presence of BZC, the absorption maximum shifts to lower wavelengths. This can be attributed to the simultaneous extraction of the binary Co-MTAR complex (Xmax = 509 nm). 3. 3. Effect of BZC Concentration and the BZC-to-Co Molar Ratio The effect of BZC concentration is shown in Figure 4, curve 2. This curve allowed the BZC-to-Co molar ratio (1:1) to be determined by many methods: straight-line method of Asmus,49 Bent and French limited logarithm method50,51 (Figure 6), You and Jones method52 (Figure 7) and mobile equilibrium method53 (Figure 8). Successful determination of the composition by all these methods is rarely possible since they have certain limitations related to the stability of the species and the presence of side pro-cesses.51,54 In addition to the aforementioned methods, the composition was determined by the Job's method of continuous variations (Figure 9).55 The obtained curve shows that the complex is rather stable. 1.6 0 -1-"-1-1- 4 4.4 4 8 5.2 5.6 6 - Log cBZC Figure 6. Determination of the BZC-to-Co molar ratio by the Bent-French limited logarithm method. 12 0 123456789 10 cazc'cCo Figure 7. Determination of the BZC-to-Co molar ratio by the You and Jones method. 1 .2 -.-i--- -3 -7 -6 -5 4 Log eWc Figure 8. Determination of the BZC-to-Co molar ratio by the mobile equilibrium method. 0 0 1 0.2 0 3 0.4 0.5 0.6 0.7 0.8 0.9 1 CB7.C HcCa+cB7c\ Figure 9. Determination of the BZC-to-Co molar ratio by the Job's method of continuous variations and Kex by the Likussar-Boltz method. k = Cco + Cbzc = 8 x 10-5 mol dm-3, Cmtar = 2 x 10-4 mol dm-3, pH 7.5, X = 547 nm. 3. 4. Suggested Chemical Formulae and Equations In order to propose correct formulae for the extracted species, the data for their composition must be synchronized with the information accumulated in the literature for the possible oxidation of CoII to CoIII by the atmospheric oxygen. Numerous studies in this area allow us to summarize that in the presence of azo dyes17,27-31,56-61 such as PAR and TAR, (i) the oxidation proceeds rapidly (in seconds);58,61 (ii) the oxidation is quantitative;60 (iii) Co11 is oxidized even in the presence of reducing agents such as ascorbic acid, sulfite or hydrazine;57,61 (iv) the protonation state of the azo dye in the complex can be estimated by the position and intensity of the spectral bands in the visible range. The MTAR complex extracted in the absence of BZC has a composition of Co:MTAR = 1:2. Because of the requirement of electroneutrality, it is reasonable to assume that its formula is [Com(HL-)(L2-)]° (L2- and HL- are the deprotonated and monoprotonated forms of the ligand H2L = MTAR). A similar Co-PAR complex has been isolat- Hristov et al.: Extraction-Chromogenic System 41 Acta Chim. Slov. 2021, 68, 37-43 ed and studied by Mochizuki et al.58 The chloroform-extracted (at pH close to 4) Co-TAR binary complex29 can probably be represented by the same formula. It can be seen from Figure 3 (curve 1) that the Co-MTAR binary complex is formed in a narrow pH range (about pH 5.5), which corresponds well with the onset of the decrease in absorbance (curve 1 ') associated with the conversion of the neutral MTAR into a monoanion: HL- + H+ (1) The course of curve 1' is in good agreement with the pKa value determined by Menek et al.62 (Table 2; pKp-OH = 5.7), characterizing Eq. 1. As the pH increases the neutral complex is transformed into an anionic complex with a bathochromically shifted absorption maximum: [CoIII(HL-)(L2-)]0 — [Com(L2-)2]- + H+ (2) The anionic complex can also be formed by the direct interaction (Eq. 3) of the metal with the dominant form of the reagent under optimal conditions (i.e., HL-). CoII(aq) + 2 HL- [CoIII(L2-)2] + 2H+ + e- (3) The formation of the ternary ion-association complex can be described by Eq. 4, in which BZ+ is the cation of BZC. [CoIII(L2-)2]- + BZ+ — (BZ+)[CoIII(L2-)2] (4) The overall process of complex formation and extraction at the optimum pH-range (see Figure 3, curve 2) is shown in Eq. 5. CoII(aq) + 2 HL (aq) + BZ+(aq) - (BZ+)[CoIII(L2-)2] (org) + 2H+(aq) + e- (5) It involves metal assisted deprotonation of the ligand HL-, oxidation of the initially formed labile CoII-complex to inert CoIII-complex in the presence of air,16,58,63 and ion-association between BZ+ and the anionic complex.54,64 The spectral characteristics and composition of the ternary complex (Co:MTAR:BZC = 1:2:1), and the optimal pH-range of its existence suggests that it contains two depro-tonated ligands, as in the literature concerning similar ternary PAR- and TAR-complexes.27-31 These azo dyes have pKa values (Table 2) that are close to those reported for MTAR. 3. 5. Extraction Characteristics The conditional equilibrium constant characterizing Eq. 5 was calculated by the mobile equilibrium method53 (Figure 8), Likussar-Boltz method67 (Figure 9) and Holme-Langhmyir method.68 The obtained values are given in Table 3, along with the values for fraction extracted (E) and distribution ratio (D). Table 3. Extraction characteristics. Extraction characteristic Value 5.6 ± 0.2a (N = 3) Extraction constant (Log Kex) 5.7 ± 0.3a (N = 5) 5.8 ± 0.1c (N = : 5) Distribution ratio (Log D) 1.4 ± 0.2 (N = 4) Fraction extracted (E), % 96 ± 2 (N = 4) a Likussar-Boltz method. b Molar equilibrium method. c Holme-Langmyhr method. 3. 6. Beer's Law and Analytical Characteristics The relationship between the concentration of CoII in the aqueous phase and the absorbance of the extracted ternary complex was studied under optimum conditions (Table 1). A good linearity was observed in the concentration range of 0.2 - 2.8 ^g cm-3 (R2 = 0.9994, N = 7). The linear regression equation was A = 0.395y + 0.004, where A is the absorbance and y is the concentration (^g cm-3). The standard deviations of the slope and intercept were H2L0 Table 2. Dissociation constants of MTAR, TAR and PAR. Azo dye PKNH pKa PKp-OH PKo-OH Conditions Ref. MTAR - 5.7 11.8 62 TAR 1.25 6.0 9.3 0.1 mol dm-3 NaC1O4 65, 66 0.96 6.23 9.44 0.1 mol dm-3 NaC1O4 - 6.16 9.59 I = 0.2 - 6.15 9.68 PAR 2.7 5.83 12.5 0.1 mol dm-3 KNO3 65, 66 2.66 5.48 12.31 0.1 mol dm-3 NaC1O4 3.1 5.6 11.9 I = 0.1 2.57 6.2 11.5 Hristov et al.: Extraction-Chromogenic System 42 Acta Chim. Slov. 2021, 68, 37-43 0.004 and 0.008, respectively. The limits of detection (LOD) and quantitation (LOQ), calculated as 3- and 10 times standard deviation of the intercept divided by the slope, were LOD = 0.058 ^g cm-3 and LOQ = 0.193 ^g cm-3. The molar absorptivity (e) and Sandell's sensitivity (SS) were e = 2.33 x 104 dm3 mol-1 cm-1 and SS = 2.53 x 10-3 ^g cm-2, respectively. 4. 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V prisotnosti BZC lahko ekstrahirani ionski kelat predstavimo s formulo (BZ+)[Com(MTAR2-)2], kjer je MTAR v deprotonirani obliki. Določili smo tudi naslednje ekstrakcijske in spektrofotometrične parametre: absorpcijski maksimum, molarno absorptivnost, Sandellovo občutljivost, mejo detekcije in kvantifikacije, konstanto ekstrakcije, porazdelitveno razmerje in delež ekstrakcije. V odsotnosti BZC je ekstrakcija nepopolna in poteka v ozkem območju pH. Ekstrahirani kelat vsebuje en deprotoniran in en mono-protoniran ligand, [Conl(MTAR2-)(HMTAR-)]. © ® Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Hristov et al.: Extraction-Chromogenic System 44 DOI: 10.17344/acsi.2020.6044 Acto Chim SlQV 2021, 68, 44-50 ©Commohs Scientific paper Synthesis, Crystal Structures, Characterization and Catalytic Property of Copper(II) Complexes Derived from Hydrazone Ligands Yao Tan* and Yan Lei* School of Environmental and Chemical Engineering, Chongqing Three Gorges University, Chongqing404000, P. R. China * Corresponding author: E-mail: 18696838310@163.com (Yao Tan), leiyan222@126.com (Yan Lei) Received: 07-03-2020 Abstract A new bromido-coordinated mononuclear copper(II) complex [Cu(HL1)Br2] (1), and a new mononuclear copper(II) complex [CuL2(HL2)]ClO4 • 0.5H2O (2), with the hydrazone ligands 4-ferf-butyl-N'-(1-(pyridin-2-yl)ethylidene)benzo-hydrazide (HL1) and 4-bromo-N'-(pyridin-2-ylmethylene)benzohydrazide (HL2), have been synthesized and structurally characterized by physico-chemical methods and single crystal X-ray determination. X-ray analysis indicates that the Cu atom in complex 1 is in distorted square pyramidal coordination, and that in complex 2 is in octahedral coordination. The catalytic property for epoxidation of styrene by the complexes was evaluated. Keywords: Copper complex; hydrazone ligand; crystal structure; catalytic property 1. Introduction Hydrazone compounds, containing the typical -CH=N-NH-C(O)- groups, represent one of the most attractive series of ligands in coordination chemistry. The hydrazone ligands are capable of binding various transition and rare earth metal atoms to form complexes with versatile structures and properties.1 To date, most hydrazone complexes have been reported to have interesting catalytic properties, such as asymmetric epoxidation, oxidation of sulfides, and various type of polymerization.2 Among the complexes, those with Cu centers are of particular interest for their catalytic properties.3 To date, a large number of metal complexes with the hydrazones derived from salicylaldehyde and its analogues are reported. This type of hydrazone ligands usually act as dianionic form during coordination.4 However, the complexes with the hydrazones derived from 2-acetylpyridine and 2-pyridinecarboxaldehyde, which act as mono-anionic form in the complexes are rare.5 Shaabani and coworkers have reported copper(II) complexes with the ligand pyridine-2-carboxaldehyde 4-hydroxy benzol hydrazone, and their interesting antibacterial activities.6 In pursuit of new copper(II) complexes with mono-anionic hydrazone ligands, we report herein the syntheses, X-ray crystal structures, and catalytic properties of a new bromido-co-ordinated mononuclear copper(II) complex [Cu(HL1)Br2] (1), and a new mononuclear copper(II) complex [CuL2(HL2)]ClO4 • 0.5H2O (2), with the hydrazone ligands 4- tert-butyl-N'-(1-(pyridin-2-yl)ethylidene)benzohydra-zide (HL1) and 4-bromo-N'- (pyridin-2-ylmethylene)ben-zohydrazide (HL2) (Scheme 1). Scheme 1. The preparation of the hydrazone ligands HL1 and HL2. Tan and Lei: Synthesis, Crystal Structures, Characterization Acta Chim. Slov. 2021, 68, 44-50 45 2. Experimental 2. 1. Materials Copper bromide, copper perchlorate, 2-acetylpyri-dine, 2-pyridinecarboxaldehyde, 4-tert-butylbenzohydra-zide, and 4-bromobenzohydrazide were purchased from Aldrich. All other reagents with AR grade were used as received without further purification. Caution: Copper perchlorate is potentially explosive. Only small quantities should be used and handled with great care. 2. 2. Physical Measurements Infrared spectra (4000-400 cm-1) were recorded as KBr discs with a FTS-40 BioRad FT-IR spectrophotometer. The electronic spectra were recorded on a Lambda 35 spectrometer. Microanalyses (C,H,N) of the complex were carried out on a Carlo-Erba 1106 elemental analyzer. Solution electrical conductivity was measured at 298K using a DDS-11 conductivity meter. GC analyses were performed on a Shimadzu GC-2010 gas chromatograph. 2. 3. X-ray Crystallography Crystallographic data of the complexes were collected on a Bruker SMART CCD area diffractometer with graphite monochromated Mo-Ka radiation (À = 0.71073 Â) at 298(2) K. Absorption corrections were applied by using the multi-scan program.7 The structures of the complexes were solved by direct methods and successive Fourier difference syntheses (SHELXS-97), and anisotropic thermal parameters for all nonhydrogen atoms were refined by full-matrix least-squares procedure against F2 (SHELXL-97).8 All non-hydrogen atoms were refined anisotropically. The hydrogen atoms were located at calculated positions, and refined isotropically with (Uiso(H) values constrained to 1.2 Uiso(C) and 1.5 Uiso(O and methyl C). The perchlorate anion of complex 2 is disordered over two sites, with occupancies of 0.565(3) and 0.435(3), respectively. The Cl-O and O---O distances are restrained to 1.40(1) and 2.30(2) A, respectively. The atoms of the disordered perchlorate anion are refined as isotropic behavior. The resolution and bond precision of complex 2 are low, which is due to the poor quality of the crystal. There is a large solvent accessible void in the structure of complex 2, which might indicate the presence of solvent molecules. The crystallographic data and experimental details for the structural analysis are summarized in Table 1, and the selected bond lengths and angles are listed in Table 2. 2. 4. Synthesis of [Cu(HL1)Br2] (1) 2-Acetylpyridine (1.0 mmol, 0.12 g) and 4-fert-but-ylbenzohydrazide (1.0 mmol, 0.19 g) were mixed and stirred in methanol (20 mL) for 30 min. Then, copper bromide (1.0 mmol, 0.22 g) was added to the mixture, and the final mixture was further stirred at room temperature for 30 min. The deep blue reaction solution was Table 1. Crystallographic data for the single crystal of the complexes 1 2 Empirical formula C72H84Br8Cu4N12O4 C52H40Br4Cl2CU2N12O13 Formula weight 2074.95 1558.58 Temperature (K) 298(2) 298(2) Crystal system Monoclinic Triclinic Space group P2i/c P1 a (A) 8.0883(11) 10.0284(10) b (A) 13.2359(13) 12.0977(11) c (A) 18.8467(12) 13.9005(15) « (°) 90 83.955(2) P (°) 101.400(2) 77.755(2) Y (°) 90 89.579(2) V (A3) 1977.8(4) 1638.7(3) Z 1 1 F(000) 1028 772 Collected data 11413 9964 Unique data 3651 4390 Observed data [I > 2ff(I)] 2553 1551 Restraints 0 80 Parameters 230 431 Goodness-of-fit on F2 1.022 0.903 Ru wR2 [I > 2c(I)] 0.0456, 0.1013 0.0859, 0.1502 Ru wR2 (all data) 0.0773, 0.1142 0.1806, 0.2141 Tan and Lei: Synthesis, Crystal Structures, Characterization ... 46 Acta Chim. Slov. 2021, 68, 44-50 46 Table 2. Selected bond distances (Â) and bond angles (°) for the complexes Bond d, À Bond d, À 1 Cu(1)-N(1) 2.010(4) Cu(1)-N(2) 1.978(4) Cu(1)-O(1) 2.037(3) Cu(1)-Br(1) 2.5519(9) Cu(1)-Br(2) 2.3801(9) N(2)-Cu(1)-N(1) 78.95(17) N(2)-Cu(1)-O(1) 78.10(15) N(1)-Cu(1)-O(1) 156.59(17) N(2)-Cu(1)-Br(2) 145.42(13) N(1)-Cu(1)-Br(2) 99.22(13) O(1)-Cu(1)-Br(2) 97.01(11) N(2)-Cu(1)-Br(1) 107.43(13) N(1)-Cu(1)-Br(1) 97.18(13) O(1)-Cu(1)-Br(1) 94.04(12) Br(2)-Cu(1)-Br(1) 107.05(3) L Cu(1)-N(1) 2.292(10) Cu(1)-N(2) 2.005(10) Cu(1)-N(4) 2.094(12) Cu(1)-N(5) 1.849(13) Cu(1)-O(1) 2.379(8) Cu(1)-O(2) 2.048(9) N(5)-Cu(1)-N(2) 177.6(5) N(5)-Cu(1)-O(2) 78.4(5) N(2)-Cu(1)-O(2) 99.3(4) N(5)-Cu(1)-N(4) 79.2(5) N(2)-Cu(1)-N(4) 103.1(5) O(2)-Cu(1)-N(4) 157.3(4) N(5)-Cu(1)-N(1) 105.4(5) N(2)-Cu(1)-N(1) 74.2(4) O(2)-Cu(1)-N(1) 100.2(3) N(4)-Cu(1)-N(1) 89.5(4) N(5)-Cu(1)-O(1) 108.7(4) N(2)-Cu(1)-O(1) 71.7(4) O(2)-Cu(1)-O(1) 86.1(3) N(4)-Cu(1)-O(1) 97.4(4) N(1)-Cu(1)-O(1) 145.9(3) evaporated to remove three quarters of the solvents under reduced pressure, yielding blue solid of the complex. Yield: 45%. Well-shaped single crystals suitable for X-ray diffraction were obtained by recrystallization of the solid from methanol. Elemental analysis found: C, 41.15; H, 4.23; N, 7.92%. C72H84Br8Cu4N12O4 calcd: C, 41.68; H, 4.08; N, 8.10%. IR data (KBr, cm-1): 3453 (w, OH), 3183 (w, NH), 1635 (s, C=o), 1603 (s, CH=n), 1502, 1461, 1346, 1165, 1073, 963, 860, 637, 540, 515, 446. UV-Vis data (Xmax, nm): 276, 320, 375, 515, 681. 2. 5. Synthesis of [CuL2(HL2)] ClO4 • 0.5H2O (2) 2-Pyridinecarboxaldehyde (1.0 mmol, 0.11 g) and 4-bromobenzohydrazide (1.0 mmol, 0.21 g) were mixed and stirred in methanol (20 mL) for 30 min. Then, copper perchlorate hexahydrate (1.0 mmol, 0.37 g) was added to the mixture, and the final mixture was further stirred at room temperature for 30 min. The deep blue reaction solution was evaporated to remove three quarters of the solvents under reduced pressure, yielding blue solid of the complex. Yield: 37%. Well-shaped single crystals suitable for X-ray diffraction were obtained by recrystallization of the solid from methanol. Elemental analysis found: C, 40.23; H, 2.68; N, 10.71%. C52H4oBr4Cl2Cu2N12O13 calcd: C, 40.07; H, 2.59; N, 10.78%. IR data (KBr, cm-1): 3482 (w, OH), 3197 (w, NH), 1638 (s, C=O), 1592 (s, CH=N), 1565, 1487, 1445, 1367, 1306, 1293, 1222, 1150, 1107, 1066, 1010, 919, 845, 777, 751, 671, 623, 580, 523, 476. UV-Vis data (Xmax, nm): 293, 378, 656. 2. 6. Styrene Epoxidation The epoxidation reaction was carried out at room temperature in acetonitrile under N2 atmosphere with constant stirring. The composition of the reaction mixture was 2.00 mmol of styrene, 2.00 mmol of chlorobenzene (internal standard), 0.10 mmol of the complex (catalyst) and 2.00 mmol iodosylbenzene or sodium hypochlorite (oxidant) in 5.00 mL freshly distilled acetonitrile. When the oxidant was sodium hypochlorite, the solution was buffered to pH 11.2 with NaH2PO4 and NaOH. The composition of reaction medium was determined by GC with styrene and styrene epoxide quantified by the internal standard method (chlorobenzene). All other products detected by GC were mentioned as others. For each complex the reaction time for maximum epoxide yield was determined by withdrawing periodically 0.1 mL aliquots from the reaction mixture and this time was used to monitor the efficiency of the catalyst on performing at least two independent experiments. Blank experiments with each oxi-dant and using the same experimental conditions except catalyst were also performed. 3. Results and Discussion 3. 1. Chemistry The hydrazones were readily prepared by condensation reaction of 2-acetylpyridine with 4-tert-butylben-zohydrazide, and 2-pyridinecarboxaldehyde with 4-bro-mobenzohydrazide, respectively, in methanol. The com- Tan and Lei: Synthesis, Crystal Structures, Characterization ... Acta Chim. Slov. 2021, 68, 44-50 47 Scheme 2. The preparation of the complexes. plexes 1 and 2 were prepared by the reaction of the hy-drazones with copper bromide (for 1) and copper per-chlorate hexahydrate (for 2) in methanol (Scheme 2). The reaction progresses are accompanied by an immediate color change of the solution from colorless to deep blue. The molar conductivities (AM = 28 O-1 cm2 mol-1 for 1 and 153 O-1 cm2 mol-1 for 2) are consistent with the values expected for non-electrolyte and 1:1 electrolyte.9 3. 2. Crystal Structure Description of Complex 1 Single-crystal X-ray analysis reveals that compound 1 is a bromido-coordinated mononuclear copper(II) complex. The ORTEP plot of the complex is shown in Fig. 1. The copper atom is in a distorted square pyramidal geometry, which is coordinated by the N2O donor atoms of the hydrazone ligand and one Br atom in the basal plane, and one Br atom at the apical position. The distortion of the square pyramidal coordination of the structure can be observed from the bond angles (Table 2) related to the Cu atom. The cis- and trans- angles related to the Cu atom at the basal plane are in the range of 78.10(15)-99.22(13)° and 145.42(13)-156.59(17)°, respectively. The bond angles among the apical and basal donor atoms are in the range of 94.04(12)-107.43(13)°. The bond lengths of Cu-O and Cu-N (Table 2) are close to those in other Cu complexes with Schiff base ligands.9 The question arises as to whether the coordination polyhedron around the five-coordinated metal ion can be described as a distorted square pyramid or a distorted trigonal bipyramid. Further information can be obtained by determining the structural index t which represents the relative amount of trigonality (square pyramid, t = 0; trigonal bipyramid, t = 1); t = (ft - a)/60°, a and ft being the two largest angles Fig. 1. ORTEP diagram of complex 1 with 30% thermal ellipsoid. Fig. 2. Molecular packing structure of complex 1 linked by hydrogen bonds. Tan and Lei: Synthesis, Crystal Structures, Characterization ... 48 Acta Chim. Slov. 2021, 68, 44-50 48 around the central atom.10 The value of t is 0.366. Thus, the coordination geometry of the copper atom is approximately described as a severely distorted square pyramid. The hydrazone ligand coordinates to the Cu atom through neutral state. The molecules are linked through N-H—Br hydrogen bonds (Table 3), to generate chains along the y axis (Fig. 2). 3. 3. Crystal Structure Description of Complex 2 Single-crystal X-ray analysis reveals that compound 2 is a mononuclear copper(II) complex. The ORTEP plot of the complex is shown in Fig. 3. The compound contains a [CuL2(HL2)] cation, a perchlorate anion and half water molecule of crystallization. The ORTEP plot of the complex is shown in Fig. 1b. The copper atom is in a distorted octahedral geometry, which is coordinated by the N2O donor atoms of one neutral hydrazone ligand and one mono-anionic hydrazone ligand. The distortion of the octahedral coordination of the structure can be observed from the bond angles (Table 2) related to the Cu atom. The cis- and trans- angles related to the Cu atom are in the range of 74.2(4)-108.7(4)° and 145.9(3)-177.6(5)°, respectively. The bond lengths of Cu-O and Cu-N (Table 2) are close to those in other Cu complexes with Schiff base ligands.9 The perchlorate anions are Fig. 3. ORTEP diagram of complex 2 with 30% thermal ellipsoid. Fig. 4. Molecular packing structure of complex 2 linked by hydrogen bonds. linked to the complex cations through N-H—O hydrogen bonds (Table 3; Fig. 4). 3. 4. IR and UV-vis Spectra of the Complexes The weak and broad absorptions in the region 33503500 cm-1 are attributed to the O-H bonds of the water molecules. The weak absorptions at 3180-3200 cm-1 are assigned to the stretching vibration of the N-H groups. The intense bands at 1635 cm-1 for 1 and 1638 cm-1 for 2 are assigned to the vibration of the C=O groups. The typical bands for the azomethine groups, v(C=N), are observed at 1595-1603 cm-1 for both complexes.11 The in- Table 3. Hydrogen bond distances (Â) and bond angles (deg) for the complexes D-H-A d(D-H) d(H-A) d(D-A) Angle (D-H-A) 1 N3-H3—Br2#1 0.86 3.05 3.910(4) 178(5) 2 N3-H3A—O4#2 0.86 2.45 3.036(19) 126(5) Symmetry codes: #1: 1 - x, -y, 1 - z; #2: x, -1 + y, z. Tan and Lei: Synthesis, Crystal Structures, Characterization ... Acta Chim. Slov. 2021, 68, 44-50 49 tense bands in the range of 1060-1110 cm-1 for the spectrum of complex 2 are due to the vibration of the Perchlorate anion.12 The weak bands in the range of 400-650 cm-1 are assigned to the vibrations of the Cu-O and Cu-N bonds. In the UV-Vis spectra of the complexes, the bands at 375 nm for 1 and 378 nm for 2 are attributed to the azome-thine chromophore n-n* transition. The bands at higher energy (276 and 320 nm for 1 and 293 nm for 2) are associated with the benzene n-n* transition.13 The weak and less well-defined bands at 681 nm for 1 and 656 nm for 2 are assigned to the d-d transitions. 3. 5. Catalytic Properties of the Complexes The percentage of conversion of styrene, selectivity for styrene oxide, yield of styrene oxide and reaction time to obtain maximum yield using both the oxidants are given in Table 4. The data reveals that the complexes as catalysts convert styrene most efficiently in the presence of both oxidants. Nevertheless, the catalysts are selective towards the formation of styrene epoxides despite of the formation of by-products which have been identified by GC-MS as benzaldehyde, phenylacetaldehyde, styrene epoxides derivative, alcohols etc. From the data it is also clear that the complexes exhibit excellent efficiency for styrene epoxide yield. When the reactions are carried out with PhIO and NaOCl, styrene conversions of complexes 1 and 2 were about 87% and 75%, and 79% and 73%, respectively. It is evident that between PhIO and NaOCl, the former acts as a better oxidant with respect to both styrene conversion and styrene epoxide selectivity. The epoxide yields for the complexes 1 and 2 using PhIO and NaOCl as oxidants are 79% and 72%, and 70% and 65%, respectively. 4. Conclusion Two new mononuclear copper(II) complexes derived from hydrazone ligands were prepared and characterized. Single crystal X-ray analysis indicates that the Cu atom in complex 1 is in distorted square pyramidal coordination, and that in complex 2 is in octahedral coordination. The complexes have effective catalytic property for the epoxidation of styrene, with conversions over 70% and selectivities over 90%. Supplementary Material CCDC 1858019 for 1 and 1858021 for 2 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via http://www.ccdc. cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e-mail: depos-it@ccdc.cam.ac.uk. Acknowledgments This project was supported by the Scientific and Technological Research Program of Chongqing Municipal Education Commission (Grant No. KJQN201801222) and the Chunhui Project from Education Ministry of China (Grant No. Z2015140). 5. References 1. (a) Z. You, H. Yu, Z. Li, W. Zhai, Y. Jiang, A. Li, S. Guo, K. 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D0I:10.1023/A:1006989707001 Povzetek Sintetizirali smo nov z bromidom koordiniran enojedrni bakrov(II) kompleks [Cu(HL1)Br2] (1) in nov enojedrni bak-rov(II) kompleks [CuL2(HL2)]ClO4 • 0.5H2O (2) s hidrazonskim ligandom 4-i-butil-N'-(1-(piridin-2-il)etiliden)ben-zohidrazidom (HL1) in 4-bromo-N'-(piridin-2-ilmetilen)benzohidrazidom (HL2). Kompleksa smo okarakterizirali s fiziko-kemijskimi metodami in monokristalno rentgensko difrakcijo. Strukturna analiza je razkrila, da ima Cu atom v kompleksu 1 popačeno kvadratno piramidalno geometrijo, v kompleksu 2 pa oktaedrično geometrijo. Določili smo tudi katalitične aktivnosti za epoksidacijo stirena. © 0 Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Tan and Lei: Synthesis, Crystal Structures, Characterization ... DOI: 10.17344/acsi.2020.6090 Acta Chim. Slov. 2021, 68, 51-64 /^creative ^commons Scientific paper Multi-component Reactions of Cyclohexan-1,3-diketones to Produce Fused Pyran Derivatives with Antiproliferative Activities and Tyrosine Kinases and Pim-1 Kinase Inhibitions Rafat Milad Mohareb,1^ Rehab Ali Ibrahim2 and Ensaf Sultan Alwan1,2 1 Department of chemistry, Faculty of science, Cairo University, Giza, A. R. Egypt 2 Higher Institute of Engineering and Technology, El-Tagammoe El-Khames, New Cairo, Egypt 3 Department of Quality Assurance, Yemen Drug Company for Industry and Commerce (YEDCO), Sanaa, Yemen * Corresponding author: E-mail: raafat_mohareb@yahoo.com Received: 05-06-2020 Abstract In this work the multi-component reactions of either of the arylhydrazocyclohexan-1,3-dione derivatives 3a-c with either of benzaldehyde (4a), 4-chlorobenzaldehyde (4b) or 4-methoxybenzaldehyde (4c) and either malononitrile (5a) or ethyl cyanoacetate (5b) giving the 5,6,7,8-tetrahydro-4H-chromene derivatives 6a-r, respectively, are presented. The reaction of two equivalents of cyclohexan-1,3-dione with benzaldehyde gave the hexahydro-1H-xanthene-1,8(2H)-dione derivative 7. On the other hand, the multi-component reactions of compound 1 with dimedone and benzaldehyde gave 13. Both of 7 and 13 underwent heterocyclization reactions to produce fused thiophene, pyran and thiazole derivatives. Selected compounds among the synthesized compounds were tested against six cancer cell lines where most of them gave high inhibitions; especially compounds 3b, 3c, 6b, 6c, 6d, 6f, 6i, 6m, 6n, 8b, 14a, 15 and 16 being the most cytotoxic compounds. Further tests against the five tyrosine kinases c-Kit, Flt-3, VEGFR-2, EGFR, and PDGFR and Pim-1 kinase showed that compounds 3c, 6c, 6d, 6f, 6n, 14a and 15 were the most potent of the tested compounds toward the five tyrosine kinases and compounds 3c, 6c, 6d, 6n and 15 displayed the highest inhibitions toward Pim-1 kinase. Keywords: Cyclohexan-1,3-dione; dimedone; thiophene; pyran; thiazole; antitumor activity; tyrosine kinases 1. Introduction Pyran derivatives are known as important class of compounds that exist in nature and have many applica-tions1 especially fused pyrans are important core units comprising many natural products. Due to their various kinds of biological activities pyrans and their fused derivatives attracted the attention within the last few years. It was reported that benzo[b]pyran derivatives were excellent anticancer compounds that give good results at very low concentrations.2 Many 2-amino-4H-pyran derivatives have various applications within industry like their uses as photoactive materials,3 pigments,4 and potentially biodegradable agrochemicals.5 In addition, naphthopyrans have many application with optical studies due to their ability to generate a yellow color on being irradiated with UV light (van). In addition, pyranochalcones have many applications like antimutagenic, antimicrobial, antiulcer, and antitumor activities.6-8 Pyrans and their fused derivatives showed different kinds of biological activities. The attachments of heterocyclic ring to the pyran ring improve many of the biological effects of the resulting molecules. Especially the 4H-pyran derivatives exhibited wide range of biological activities with great interests such as antimicrobi-al,9 antiviral,10,11 mutagenicity,12 antiproliferative,13 sex pheromone,14 antitumor,15 cancer therapy,16 and central nervous system activity.17 Some of these compounds were applied in industrial chemistry as they can be used in many cosmetic manufacturing and through the field of agrochemicals.18 Such high importance of pyrans and their derivatives together with the ease of their synthesis with high yields direct many works through their synthesis. Mohareb et al.: Multi-component Reactions 52 Acta Chim. Slov. 2021, 68, 51-64 This encouraged our research group to be attracted toward the synthesis of pyran derivatives through the uses of ß-diketones. The produced compounds showed high antiproliferative activities against cancer cell lines together with high inhibitions toward tyrosine kinases.19-25 Through our present work we adopted multi-component reactions of either arylhydrazonocyclohexan-1,3-dione, aromatic aldehydes and cyanometylene derivatives together with using the produced molecule as a suitable starting material for subsequent heterocyclization to obtain a variety of fused derivatives. The anti-proliferative activities of the synthesized compounds and their inhibitions toward tyrosine kinases were determined. 2. Experimental For newly synthesized compounds melting points were determined and are given as uncorrected values. For all compounds the IR spectra (KBr discs) were measured using a FTIR plus 460 or PyeUnicam SP-1000 spectrophotometer. The 1H NMR spectra were measured using Varian Gemini-300 (300 MHz) and Jeol AS 500 MHz instruments. Measurements were performed in DMSO-d6 as the solvent using TMS as the internal standard and chemical shifts are expressed as 5 ppm. The MS spectra (EI) were measured using Hewlett Packard 5988 A GC/MS system and GCMS-QP 1000 Ex Shimadzu instruments. The microanalytical CHN data were obtained from the Micro-analytical Data Unit at Cairo University and were performed on Vario EL III Elemental analyzer. Screening of compounds against the cancer cell lines and tyrosine kinases were performed through The National Cancer Institute at Cairo University. 2. 1. Synthesis of the Arylhydrazone Derivatives 3a-c A solution of either the diazonium salts (0.01 mol) [prepared by the addition of a solution of sodium nitrite (0.70 g, 0.01 mol) in water (10 mL) to a cold solution of either aniline (0.93 g, 0.01 mol), 4-methylaniline (1.07 g, 0.01 mol) or 4-chloroaniline (1.27 g, 0.01 mol) dissolved in concentrated hydrochloric acid (10 mL, 18 mol) with continuous stirring] was added to a cold solution of any of the compounds 1 (1.12 g, 0.01 mol), in ethanol (50 mL) containing sodium acetate (3.0 g) with stirring. The whole reaction mixture was left at room temperature for 2 h and the formed solid product was collected by filtration. 2-(2-Phenylhydrazono)cyclohexane-1,3-dione (3a) Yellow crystals from ethanol, yield 1.51 g (70%). Mp 145-147 °C. IR (KBr) vmax (cm-1): 3472-3328 (NH), 3055 (CH, aromatic), 1705, 1688 (2C=O), 1640 (C=N), 1634 (C=C); 1H NMR (DMSO-d6, 300 MHz): 5 1.67-1.70 (m, 4H, 2CH2), 2.38-2.45 (m, 2H, CH2), 7.26-7.59 (m, 5H, C6H5), 8.36 (s, 1H, D2O exchangeable, NH); 13C NMR (DM- SO-d6, 75 MHz): 5 24.8, 34.6, 38.2, (3CH2), 120.2, 122.4, 125.8, 127.6 (C6H5), 164.3 (C=N), 166.2, 168.6 (2C=O). Anal. Calcd. for C12H12N2O2: C, 66.56; H, 5.59; N, 12.96. Found: C, 66.80; H, 5.73; N, 13.06. MS: m/z 216 (M+, 36%). 2-(2-(p-Tolyl)hydrazono)cyclohexane-1,3-dione (3b) Brown crystals from ethanol, yield 1.51 g (66%). Mp 170-172 °C. IR (KBr) vmax (cm-1): 3493-3342 (NH), 3055 (CH, aromatic), 1703, 1689 (2C=O), 1638 (C=N), 1632 (C=C); 1H NMR (DMSO-d6, 300 MHz): 5 1.64-1.72 (m, 4H, 2CH2), 2.36-2.43 (m, 2H, CH2), 2.74 (s, 3H, CH3), 7.26-7.59 (m, 4H, C6H4), 8.38 (s, 1H, D2O exchangeable, NH); 13C NMR (DMSO-d6, 75 MHz): 5 24.5, 34.8, 38.6 (3CH2), 39.4 (CH3), 120.6, 123.8, 126.5, 128.3 (C6H4), 164.3 (C=N), 166.7, 168.4 (2C=O). Anal. Calcd. for C13H14N2O2: C, 67.81; H, 6.13; N, 12.17. Found: C, 68.21; H, 6.08; N, 12.36. MS: m/z 230 (M+, 48%). 2-(2-(4-Chlorophenyl)hydrazono)cyclohexane-1,3-di-one (3c) Orange crystals from ethanol, yield 1.85 g (74%). Mp 180-183 °C. IR (KBr) vmax (cm-1): 3485-3326 (NH), 3055 (CH, aromatic), 1701, 1687 (2C=O), 1636 (C=N), 1634 (C=C); 1H NMR (DMSO-d6, 300 MHz): 5 1.62-1.70 (m, 4H, 2CH2), 2.34-2.46 (m, 2H, CH2), 7.24-7.40 (m, 4H, C6H4), 8.38 (s, 1H, D2O exchangeable, NH); 13C NMR (DMSO-d6, 75 MHz): 5 24.5, 34.8, 38.6 (3CH2), 39.4 (CH3), 120.6, 123.8, 126.5, 128.3 (C6H4), 164.3 (C=N), 166.7, 168.4 (2C=O). Anal. Calcd. for C12H11ClN2O2: C, 57.49; H, 4.42; N, 11.17. Found: C, 57.62; H, 4.73; N, 11.29. MS: m/z 250 (M+, 24%). 2. 2. General Procedure for the Synthesis of the 5,6,7,8-Tetrahydro-4H-chromene Derivatives 6a-r Each of either benzaldehyde (1.06 g, 0.01 mol), 4-chlorobenzaldehyde (1.40 g, 0.01 mol) or 4-methoxy-benzaldehyde (1.36 g, 0.01 mol) and either malononitrile (0.66 g, 0.01 mol) or ethyl cyanoacetate (1.13 g, 0.01 mol) were added to a solution of either 3a (2.16 g, 0.01 mol), 3b (2.30 g, 0.01 mol) or 3c (2.50 g, 0.01 mol) in 1,4-dioxane (50 mL) containing triethylamine (1.00 mL). The whole reaction mixture was heated under reflux for 3 h then poured onto ice/water mixture containing a few drops of hydrochloric acid and the formed solid product was collected by filtration. 2-Amino-7-oxo-4-phenyl-8-(2-phenylhydrazono)-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (6a) Yellow crystals from 1,4-dioxane, yield 2.51 g (68%). Mp 95-98 °C. IR (KBr) vmax (cm-1): 3485-3341 (NH2, NH), 3054 (CH, aromatic), 2220 (CN), 1689 (C=O), 1642 (C=N), 1636 (C=C); 1H NMR (DMSO-d6, 300 MHz): 5 2.83-2.94 (2t, 4H, 2CH2), 4.82 (s, 2H, D2O exchangeable NH2), 5.08 (s, 1H, pyran H-4), 7.23-7.48 (m, 10H, 2C6H5), Mohareb et al.: Multi-component Reactions 53 Acta Chim. Slov. 2021, 68, 51-64 8.28 (s, 1H, D2O exchangeable, NH); 13C NMR (DMSO-d6, 75 MHz): 5 37.9, 42.1 (2CH2), 51.2 (pyran C-4), 117.3 (CN), 120.4, 121.3, 121.8, 122.0, 123.6, 124.3,125.8, 126.8 (2C6H5), 130.2, 131.6, 134.8, 136.1 (pyran C), 166.8 (C=N), 167.2 (C=O). Anal. Calcd. for C22H18N4O2: C, 71.37; H, 4.90; N, 15.13.Found: C, 71.52; H, 5.13; N, 15.29. MS: m/z 370 (M+, 28%). 2-Hydroxy-7-oxo-4-phenyl-8-(2-phenylhydrazono)-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (6b) Yellow crystals from 1,4-dioxane, yield 2.59 g (70%). Mp 117-120 °C. IR (KBr) vmax (cm-1): 3528-3330 (OH, NH), 3055 (CH, aromatic), 2220 (CN), 1688 (C=O), 1640 (C=N), 1632 (C=C); 1H NMR (DMSO-d6, 300 MHz): 5 2.81-2.98 (2t, 4H, 2CH2), 5.05 (s, 1H, pyran H-4), 7.257.46 (m, 10H, 2C6H5), 8.26 (s, 1H, D2O exchangeable, NH), 10.27 (s, 1H, D2O exchangeable, OH); 13C NMR (DMSO-d6, 75 MHz): 5 37.6, 42.5 (2CH2), 51.1 (pyran C-4), 116.3 (CN), 120.2, 120.6, 121.9, 122.3, 123.9, 125.2,125.5, 126.3 (2C6H5), 130.4, 131.1, 133.8, 136.5 (pyran C), 166.3 (C=N), 168.4 (C=O). Anal. Calcd. for C22H17N3O3: C, 71.15; H, 4.61; N, 11.31. Found: C, 70.93; H, 4.82; N, 11.42. MS: m/z 371 (M+, 36%). 2-Amino-4-(4-chlorophenyl)-7-oxo-8-(2-phenylhydrazo-no)-5,6,7,8-tetrahydro-4H-chromene-3-carbonit:rile (6c) Yellow crystals from 1,4-dioxane, yield 3.21 g (79%). Mp 93-95 °C. IR (KBr) vmax (cm-1): 3468-3359(NH2, NH), 3055 (CH, aromatic), 2220 (CN), 1688 (C=O), 1646 (C=N), 1630 (C=C); 1H NMR (DMSO-d6, 300 MHz): 5 2.81-2.97 (2t, 4H, 2CH2), 4.84 (s, 2H, D2O exchangeable NH2), 5.13 (s, 1H, pyran H-4), 7.24-7.58 (m, 9H, C6H5, C6H4), 8.32 (s, 1H, D2O exchangeable, NH); 13C NMR (DMSO-d6, 75 MHz): 5 37.5, 42.3 (2CH2), 51.6 (pyran C-4), 117.2 (CN), 120.1, 120.5, 121.5, 122.3, 123.8, 125.1,125.9, 126.3 (C6H5, C6H4), 130.4, 131.6, 133.8, 1358 (pyran C), 166.6 (C=N), 167.8 (C=O). Anal. Calcd. for C22H17ClN4O2: C, 65.27; H, 4.23; N, 13.84. Found: C, 65.42; H, 4.33; N, 14.09. MS: m/z 404 (M+, 72%). 4-(4-Chlorophenyl)-2-hydroxy-7-oxo-8-(2-phenylhy-drazono)-5,6,7,8-tetrahydro-4H-chromene-3-carboni-trile (6d) Yellow crystals from1,4-dioxane, yield 2.63 g (65%). Mp 122-125 °C. IR (KBr) vmax (cm-1): 3542-3348 (OH, NH), 3055 (CH, aromatic), 2220 (CN), 1701 (C=O), 1645 (C=N), 1632 (C=C); 1H NMR (DMSO-d6, 300 MHz): 5 2.83-2.97 (2t, 4H, 2CH2), 5.07 (s, 1H, pyran H-4), 7.227.55 (m, 9H, C6H5, C6H4), 8.23 (s, 1H, D2O exchangeable, NH), 10.29 (s, 1H, D2O exchangeable, OH); 13C NMR (DMSO-d6, 75 MHz): 5 37.2, 42.8 (2CH2), 51.6 (pyran C-4), 117.8 (CN), 120.4, 121.8, 122.2, 122.6, 124.3, 125.6, 125.8, 126.0 (C6H5, C6H4), 130.4, 132.8, 134.8, 135.2 (pyran C), 166.7 (C=N), 168.8 (C=O). Anal. Calcd. for C22H16ClN3O3: C, 65.11; H, 3.97; N, 10.35. Found: C, 65.29; H, 4.16; N, 10.53. MS: m/z 405 (M+, 26%). 2-Amino-4-(4-methoxyphenyl)-7-oxo-8-(2-phenylhy-drazono)-5,6,7,8-tetrahydro-4H-chromene-3-carboni-trile (6e) Brown crystals from 1,4-dioxane, yield 2.40 g (60%). Mp 86-88 °C. IR (KBr) vmax (cm-1): 3478-3338 (NH2, NH), 3055 (CH, aromatic), 2222 (CN), 1687 (C=O), 1643 (C=N), 1630 (C=C); 1H NMR (DMSO-d6, 300 MHz): 5 2.83-2.95 (2t, 4H, 2CH2), 3.70 (s, 3H, OCH3), 4.86 (s, 2H, D2O exchangeable NH2), 5.09 (s, 1H, pyran H-4), 7.227.52 (m, 9H, C6H5, C6H4), 8.33 (s, 1H, D2O exchangeable, NH); 13C NMR (DMSO-d6, 75 MHz): 5 37.3, 42.6 (2CH2), 50.2 (OCH3), 51.2 (pyran C-4), 117.4 (CN), 120.2, 121.0, 121.8, 122.7, 123.2, 125.3,125.6, 126.1 (C6H5, C6H4), 130.1, 132.8, 134.5, 136.2 (pyran C), 166.8 (C=N), 167.9 (C=O). Anal. Calcd. for C23H20N4O3: C, 68.99; H, 5.03; N, 13.99. Found: C, 68.79; H, 4.93; N, 14.27. MS: m/z 400 (M+, 68%). 2-Hydroxy-4-(4-methoxyphenyl)-7-oxo-8-(2-phenylhy-drazono)-5,6,7,8-tetrahydro-4H-chromene-3-carboni-trile (6f) Yellow crystals from 1,4-dioxane, yield 3.00 g (75%). Mp 121-123 °C. IR (KBr) vmax (cm-1): 3552-3329 (OH, NH), 3054 (CH, aromatic), 2220 (CN), 1696 (C=O), 1642 (C=N), 1630 (C=C); 1H NMR (DMSO-d6, 300 MHz): 5 2.81-2.99 (2t, 4H, 2CH2), 3.69 (s, 3H OCH3), 5.13 (s, 1H, pyran H-4), 7.24-7.59 (m, 9H, C6H5, C6H4), 8.24 (s, 1H, D2O exchangeable, NH), 10.32 (s, 1H, D2O exchangeable, OH); 13C NMR (DMSO-d6, 75 MHz): 5 37.5, 42.9 (2CH2), 50.2 (OCH3), 51.6 (pyran C-4), 116.9 (CN), 120.3, 121.6, 122.8, 123.4, 124.7, 125.4, 125.2, 126.4 (C6H5, C6H4), 130.7, 133.2, 134.5, 135.8 (pyran C), 166.8 (C=N), 168.9 (C=O). Anal. Calcd. for C23H19N3O4: C, 68.82; H, 4.77; N, 10.47. Found: C, 68.93; H, 4.80; N, 10.54. MS: m/z 401 (M+, 34%). 2-Amino-7-oxo-4-phenyl-8-(2-(p-tolyl)hydrazono)-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (6g) Brown crystals from 1,4-dioxane, yield 2.84 g (74%). Mp 110-113 °C. IR (KBr) vmax (cm-1): 3492-3326 (NH2, NH), 3055 (CH, aromatic), 2221 (CN), 1687 (C=O), 1641 (C=N), 1630 (C=C); 1H NMR (DMSO-d6, 300 MHz): 5 2.68-2.93 (2t, 4H, 2CH2), 2.80 (s, 3H, CH3), 4.86 (s, 2H, D2O exchangeable NH2), 5.13 (s, 1H, pyran H-4), 7.247.58 (m, 9H, C6H5, C6H4), 8.33 (s, 1H, D2O exchangeable, NH); 13C NMR (DMSO-d6, 75 MHz): 5 37.6, 42.8 (2CH2), 36.8 (CH3), 51.3 (pyran C-4), 117.3 (CN), 120.4, 121.5, 122.4, 122.9, 123.6, 125.8, 125.1, 126.4 (C6H5, C6H4), 130.1, 133.7, 134.8, 135.6 (pyran C), 166.5 (C=N), 167.8 (C=O). Anal. Calcd. for C23H20N4O2: C, 71.86; H, 5.24; N, 14.57. Found: C, 71.72; H, 5.43; N, 14.39. MS: m/z 384 (M+, 42%). 2-Hydroxy-7-oxo-4-phenyl-8-(2-(p-tolyl)hydrazono)-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (6h) Dark brown crystals from1,4-dioxane, yield 2.31 g (60%). Mp 177-179 °C. IR (KBr) vmax (cm-1): 3539-3342 Mohareb et al.: Multi-component Reactions 54 Acta Chim. Slov. 2021, 68, 51-64 (OH, NH), 3055 (CH, aromatic), 2220 (CN), 1692 (C=O), 1645 (C=N), 1631 (C=C); 1H NMR (DMSO-d6, 300 MHz): 5 2.83-2.96 (2t, 4H, 2CH2), 2.72 (s, 3H CH3), 5.11 (s, 1H, pyran H-4), 7.21-7.47 (m, 9H, C6H5, C6H4), 8.26 (s, 1H, D2O exchangeable, NH), 10.31 (s, 1H, D2O exchangeable, OH); 13C NMR (DMSO-d6, 75 MHz): 5 38.1, 42.3 (2CH2), 36.2 (CH3), 51.4 (pyran C-4), 117.8 (CN), 120.1, 120.9, 121.3, 122.8, 124.3, 125.6, 126.1, 126.8 (C6H5, C6H4), 130.9, 132.6, 134.8, 136.4 (pyran C), 166.7 (C=N), 168.5 (C=O). Anal. Calcd. for C23H19N3O3: C, 71.67; H, 4.97; N, 10.90. Found: C, 71.82; H, 4.74; N, 11.25. MS: m/z 385 (M+, 40%). 2-Amino-4-(4-chlorophenyl)-7-oxo-8-(2-(p-tolyl)hy-drazono)-5,6,7,8-tetrahydro-4H-chromene-3-carboni-trile (6i) Pale brown crystals from 1,4-dioxane, yield 2.92 g (70%). Mp 114-116 °C. IR (KBr) vmax (cm-1): 3463-3351 (NH2, NH), 3055 (CH, aromatic), 2220 (CN), 1689 (C=O), 1640 (C=N), 1630 (C=C); 1H NMR (DMSO-d6, 300 MHz): 5 2.65-2.91 (2t, 4H, 2CH2), 2.76 (s, 3H, CH3), 4.88 (s, 2H, D2O exchangeable NH2), 5.08 (s, 1H, pyran H-4), 7.217.50 (m, 8H, 2C6H4), 8.34 (s, 1H, D2O exchangeable, NH); 13C NMR (DMSO-d6, 75 MHz): 5 37.8, 42.9 (2CH2), 36.8 (CH3), 51.6 (pyran C-4), 117.0 (CN), 120.6, 121.8, 122.1, 122.5, 123.4, 125.2, 125.5, 126.1 (2C6H4), 130.3, 132.6, 134.6, 136.1 (pyran C), 166.8 (C=N), 168.1 (C=O). Anal. Calcd. for C23H19ClN4O2: C, 65.95; H, 4.57; N, 13.38. Found: C, 65.73; H, 4.73; N, 13.42. MS: m/z 418 (M+, 28%). 4-(4-Chlorophenyl)-2-hydroxy-7-oxo-8-(2-(p-tolyl)hy-drazono)-5,6,7,8-tetrahydro-4H-chromene-3-carboni-trile (6j) Yellow crystals from1,4-dioxane, yield 2.31 g (55%). Mp 153-155 °C. IR (KBr) vmax (cm-1): 3539-3342 (OH, NH), 3055 (CH, aromatic), 2221 (CN), 1692 (C=O), 1645 (C=N), 1631 (C=C); 1H NMR (DMSO-d6, 300 MHz): 5 2.83-2.96 (2t, 4H, 2CH2), 2.72 (s, 3H CH3), 5.11 (s, 1H, pyran H-4), 7.21-7.47 (m, 8H, 2C6H4), 8.26 (s, 1H, D2O exchangeable, NH), 10.31 (s, 1H, D2O exchangeable, OH); 13C NMR (DMSO-d6, 75 MHz): 5 38.4, 42.8 (2CH2), 36.2 (CH3), 51.2 (pyran C-4), 117.6 (CN), 120.0, 120.6, 122.8, 123.2, 125.0, 125.2, 126.0, 126.5 (2C6H4), 130.2, 132.8, 134.8, 136.5 (pyran C), 166.8 (C=N), 168.5 (C=O). Anal. Calcd. for C23H18ClN3O3: C, 65.79; H, 4.32; N, 10.01. Found: C, 65.81; H, 4.29; N, 9.82. MS: m/z 419 (M+, 58%). 2-Amino-4-(4-methoxyphenyl)-7-oxo-8-(2-(p-tolyl)hy-drazono)-5,6,7,8-tetrahydro-4H-chromene-3-carboni-trile (6k) Brown crystals from 1,4-dioxane, yield 2.92 g (71%). Mp 93-95 °C. IR (KBr) vmax (cm-1): 3463-3351 (NH2, NH), 3055 (CH, aromatic), 2220 (CN), 1689 (C=O), 1640 (C=N), 1630 (C=C); 1H NMR (DMSO-d6, 300 MHz): 5 2.65-2.91 (2t, 4H, 2CH2), 2.76 (s, 3H, CH3), 3.72 (s, 3H, OCH3), 4.88 (s, 2H, D2O exchangeable NH2), 5.08 (s, 1H, pyran H-4), 7.21-7.50 (m, 8H, 2C6H4), 8.34 (s, 1H, D2O exchangeable, NH); 13C NMR (DMSO-d6, 75 MHz): 5 37.8, 42.9 (2CH2), 36.8 (CH3), 50.8 (OCH3), 51.6 (pyran C-4), 116.8 (CN), 120.6, 121.8, 122.1, 122.5, 123.4, 125.2, 125.5, 126.1 (2C6H4),130.3, 132.4, 134.8, 136.1 (pyran C), 166.8 (C=N), 168.1 (C=O). Anal. Calcd. for C24H22N4O3: C, 69.55; H, 5.35; N, 13.52. Found: C, 69.70; H, 5.72; N, 13.68. MS: m/z 414 (M+, 44%). 2-Hydroxy-4-(4-methoxyphenyl)-7-oxo-8-(2-(p-tolyl) hydrazono)-5,6,7,8-tetrahydro-4H-chromene-3-car-bonitrile (6l) Orange crystals from1,4-dioxane, yield 2.82 g (68%). Mp 82-84 °C. IR (KBr) vmax (cm-1): 3548-3328 (OH, Nh), 3055 (CH, aromatic), 2221 (CN), 1692 (C=O), 1641 (C=N), 1631 (C=C); 1H NMR (DMSO-d6, 300 MHz): 5 2.86-2.99 (2t, 4H, 2CH2), 2.75 (s, 3H CH3), 3.67 (s, 3H, OCH3), 5.08 (s, 1H, pyran H-4), 7.26-7.54 (m, 8H, 2 C6H4), 8.26 (s, 1H, D2O exchangeable, NH), 10.30 (s, 1H, D2O exchangeable, OH); 13C NMR (DMSO-d6, 75 MHz): 5 38.8, 42.7 (2CH2), 36.8 (CH3), 50.6 (OCH3), 51.8 (pyran C-4),117.3 (CN), 120.3, 120.9, 122.6, 123.4, 124.8, 125.6, 126.4, 126.9 (2C6H4), 130.1, 133.2, 134.2, 136.4 (pyran C), 166.5 (C=N), 168.8 (C=O). Anal. Calcd. for C24H21N3O4: C, 69.39; H, 5.10; N, 10.11. Found: C, 69.52; H, 4.85; N, 9.96. MS: m/z 415 (M+, 65%). 2-Amino-8-(2-(4-chlorophenyl)hydrazono)-7-oxo-4-phenyl-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (6m) Brown crystals from 1,4-dioxane, yield 2.86 g (71%). Mp 101-103 °C. IR (KBr) vmax (cm-1): 3480-3338 (NH2, NH), 3055 (CH, aromatic), 2223 (CN), 1688 (C=O), 1643 (C=N), 1632 (C=C); 1H NMR (DMSO-d6, 300 MHz): 5 2.68-2.96 (2t, 4H, 2CH2), 4.82 (s, 2H, D2O exchangeable NH2), 5.13 (s, 1H, pyran H-4), 7.24-7.47 (m, 9H, C6H5, C6H4), 8.36 (s, 1H, D2O exchangeable, NH); 13C NMR (DMSO-d6, 75 MHz): 5 37.4, 42.3 (2CH2), 51.2 (pyran C-4), 116.8 (CN), 120.2, 120.9, 121.6, 122.8, 123.0, 124.6, 125.2, 126.8 (C6H5, C6H4), 130.4, 133.0, 134.6, 136.8 (pyran C), 167.2 (C=N), 168.8 (C=O). Anal. Calcd. for C22H17ClN4O2: C, 65.27; H, 4.23; N, 13.84. Found: C, 65.40; H, 4.32; N, 13.79. MS: m/z 404 (M+, 60%). 8-(2-(4-Chlorophenyl)hydrazono)-2-hydroxy-7-oxo-4-phenyl-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (6n) Yellow crystals from 1,4-dioxane, yield 3.03 g (75%). Mp 107-110 °C. IR (KBr) vmax (cm-1): 3528-3358 (OH, NH), 3054 (CH, aromatic), 2223 (CN), 1696 (C=O), 1640 (C=N), 1633 (C=C); 1H NMR (DMSO-d6, 300 MHz): 5 2.83-2.98 (2t, 4H, 2CH2), 5.15 (s, 1H, pyran H-4), 7.237.50 (m, 9H, C6H5, C6H4), 8.28 (s, 1H, D2O exchangeable, NH), 10.35 (s, 1H, D2O exchangeable, OH); 13C NMR (DMSO-d6, 75 MHz): 5 38.5, 42.0 (2CH2), 51.8 (pyran C-4), 117.6 (CN), 120.6, 120.8, 121.5, 122.7, 123.7, 124.9, Mohareb et al.: Multi-component Reactions 55 Acta Chim. Slov. 2021, 68, 51-64 125.8, 126.2 (C6H5, C6H4), 130.4, 133.7, 134.0, 136.0 (pyran C), 166.9 (C=n), 168.6 (C=O). Anal. Calcd. for C22H16ClN3O3: C, 65.11; H, 3.97; N, 10.35. Found: C, 65.08; H, 4.16; N, 10.22. MS: m/z 405 (M+, 48%). 2-Amino-4-(4-chlorophenyl)-8-(2-(4-chlorophenyl)hy-drazono)-7-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (6o) Orange crystals from 1,4-dioxane, yield 2.76 g (63%). Mp 128-131 °C. IR (KBr) vmax (cm-1): 3489-3325 (NH2, NH), 3053 (CH, aromatic), 2222 (CN), 1688 (C=O), 1641 (C=N), 1633 (C=C); 1H NMR (DMSO-d6, 300 MHz): 5 2.61-2.97 (2t, 4H, 2CH2), 4.87 (s, 2H, D2O exchangeable NH2), 5.15 (s, 1H, pyran H-4), 7.23-7.56 (m, 8H, 2C6H4), 8.36 (s, 1H, D2O exchangeable, NH); 13C NMR (DMSO-d6, 75 MHz): 5 37.3, 42.8 (2CH2), 51.2 (pyran C-4), 117.7 (CN), 120.3, 121.5, 122.0, 122.9, 123.6, 124.1, 125.8, 126.6 (2C6H4), 130.6, 133.8, 134.2, 136.0 (pyran C), 166.9 (C=N), 168.4 (C=O). Anal. Calcd. for C22H16Cl2N4O2: C, 60.15; H, 3.67; N, 12.75. Found: C, 59.79; H, 3.59; N, 12.90. MS: m/z 439 (M+, 42%). 4-(4-Chlorophenyl)-8-(2-(4-chlorophenyl)hydrazono)-2-hydroxy-7-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (6p) Pale yellow crystals from 1,4-dioxane, yield 3.43 g (78%). Mp 181-184 °C. IR (KBr) vmax (cm-1): 3550-3329 (OH, NH), 3054 (CH, aromatic), 2222 (CN), 1689 (C=O), 1643 (C=N), 1633 (C=C); 1H NMR (DMSO-d6, 300 MHz): 5 2.80-2.96 (2t, 4H, 2CH2), 5.11 (s, 1H, pyran H-4), 7.257.56 (m, 8H, 2 C6H4), 8.29 (s, 1H, D2O exchangeable, NH), 10.31 (s, 1H, D2O exchangeable, OH); 13C NMR (DM-SO-d6, 75 MHz): 5 38.5, 42.0 (2CH2), 51.6 (pyran C-4), 117.9 (CN), 120.1, 120.6, 121.8, 122.7, 123.2, 124.3, 125.5, 126.8 (2C6H4), 130.5, 133.8, 134.8, 136.1 (pyran C), 166.6 (C=N), 168.9 (C=O). Anal. Calcd. for C22H15Cl2N3O3: C, 60.02; H, 3.43; N, 9.54. Found: C, 60.19; H, 3.80; N, 9.69. MS: m/z 440 (M+, 60%). 2-Amino-8-(2-(4-chlorophenyl)hydrazono)-4-(4-meth-oxyphenyl)-7-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (6q) Yellow crystals from 1,4-dioxane, yield 2.95 g (68%). Mp 86-88 °C. IR (KBr) vmax (cm-1): 3472-3351 (NH2, NH), 3055 (CH, aromatic), 2224 (CN), 1689 (C=O), 1643 (C=N), 1636 (C=C); 1H NMR (DMSO-d6, 300 MHz): 5 2.60-2.99 (2t, 4H, 2CH2), 3.66 (s, 3H, OCH3), 4.84 (s, 2H, D2O exchangeable NH2), 5.12 (s, 1H, pyran H-4), 7.247.59 (m, 8H, 2C6H4), 8.34 (s, 1H, D2O exchangeable, NH); 13C NMR (DMSO-d6, 75 MHz): 5 37.6, 42.4 (2CH2), 50.3 (OCH3), 51.4 (pyran C-4), 116.9 (CN), 120.1, 120.8, 121.6, 122.7, 123.4, 124.3, 124.9, 126.2 (2C6H4), 130.3, 133.7, 134.6, 136.0 (pyran C), 166.8 (C=N), 168.6 (C=O). Anal. Calcd. for C23H19ClN4O3: C, 63.52; H, 4.40; N, 12.88. Found: C, 63.71; H, 4.27; N, 12.73. MS: m/z 434 (M+, 50%). 8-(2-(4-Chlorophenyl)hydrazono)-2-hydroxy-4-(4-methoxyphenyl)-7-oxo-5,6,7,8-tetrahydro-4H-chro-mene-3-carbonitrile (6r) Pale yellow crystals from1,4-dioxane, yield 3.43 g (79%). Mp 85-87 °C. IR (KBr) vmax (cm-1): 3550-3329 (OH, NH), 3054 (CH, aromatic), 2223 (cN), 1689 (C=O), 1643 (C=N), 1633 (C=C); 1H NMR (DMSO-d6, 300 MHz): 6 2.80-2.96 (2t, 4H, 2CH2), 3.70 (s, 3H, OCH3), 5.11 (s, 1H, pyran H-4), 7.25-7.56 (m, 8H, 2 C6H4), 8.29 (s, 1H, D2O exchangeable, NH), 10.31 (s, 1H, D2O exchangeable, OH); 13C NMR (DMSO-d6, 75 MHz): 6 38.5, 42.0 (2CH2), 50.1 (OCH3), 51.6 (pyran C-4), 118.0 (CN), 120.1, 120.6, 121.8, 122.7, 123.2, 124.3, 125.5, 126.8 (2C6H4), 130.7, 132.8, 134.8, 136.1 (pyran C), 166.6 (C=N), 168.9 (C=O). Anal. Calcd. for C23H18ClN3O4: C, 63.38; H, 4.16; N, 9.64. Found: C, 63.47; H, 3.93; N, 9.83. MS: m/z 435 (M+, 58%). 2. 3. 9-Phenyl-3,4,5,6,7,9-hexahydro-1H-xanthene-1,8(2H)-dione (7) Benzaldehyde (1.06 g, 0.01 mol) was added to a solution of compound 1 (2.24 g, 0.02 mol) in absolute ethanol (40 mL) containing piperidine (1.0 mL). The whole reaction mixture was heated under reflux for 3 h then poured onto ice/water mixture containing a few drops of hydro -chloric acid and the formed solid product was collected by filtration. White crystals from ethanol, yield 2.35 g (80%). Mp 214-217 °C. IR (KBr) vmax (cm-1): 3056 (CH, aromatic), 1702, 1689 (C=O), 1631 (C=C); 1H NMR (DMSO-d6, 300 MHz): 6 1.59-1.80 (m, 8H, 4CH2), 2.58-2.73 (m, 4H, 2CH2), 5.09 (s, 1H, pyran H-4), 7.25-7.41 (m, 5H, C6H5); 13C NMR (DMSO-d6, 75 MHz): 6 26.3, 28.4, 32.6 (6CH2), 50.9 (pyran C-4), 120.6, 121.4, 123.6, 125.8 (C6H5), 168.9 (2C=o). Anal. Calcd. for C19H18O3: C, 77.53; H, 6.16. Found: C, 77.80; H, 6.29. MS: m/z 294 (M+, 100%). 2. 4. General Procedure for the Synthesis of the Dithieno[3,2-a:2',3'-_/']xanthenes Derivatives 8a,b Each of elemental sulfur (0.64 g, 0.02 mol) and either malononitrile (1.32 g, 0.02 mol) or ethyl cyanoacetate (2.26 g, 0.02 mol) were added to a solution of compound 7 (2.94 g, 0.01 mol) in 1,4-dioxane (40 mL) containing tri-ethylamine (1.00 mL). The reaction mixture was heated under reflux for 3 h then poured onto ice/water mixture and the precipitated product was collected by filtration. 12,10-Diamino-12-phenyl-5,7,8,12-tetrahydro-4H-dithieno[3,2-a:2',3'-/]xanthene-1,11-dicarbonitrile (8a) Orange crystals from 1,4-dioxane, yield 2.81 g (62%). Mp 144-146 °C. IR (KBr) vmax (cm-1): 3468-3369 (NH2), 3055 (CH, aromatic), 2223, 2220 (2CN), 1630 (C=C); 1H NMR (DMSO-d6, 300 MHz): 6 2.84-3.39 (m, 8H, 4CH2), 4.89, 5.13 (2s, 4H, D2O exchangeable, 2NH2), 5.12 (s, 1H, Mohareb et al.: Multi-component Reactions ... 56 Acta Chim. Slov. 2021, 68, 51-64 pyran H-4), 7.26-7.43 (m, 5H, C6H5); 13C NMR (DM-SO-d6, 75 MHz): 6 39.2, 45.8 (4CH2), 116.8, 116.9 (2CN), 120.3, 120.5, 123.9, 125.3 (C6H5), 130.6, 131.6, 132.7, 134.6, 137.8, 139.2, 140.5, 141.2 (pyran, two thiophene C). Anal. Calcd. for C25H18N4OS2: C, 66.06; H, 3.99; N, 12.33; S, 14.11. Found: C, 65.93; H, 4.13; N, 12.29; S, 14.30. MS: m/z 454 (M+, 58%). Diethyl 2,10-Diamino- 12-phenyl-5,7,8,12-tetrahydro-4H-dithieno[3,2-a:2',3'--/]xanthene-1,11-dicarboxylate (8b) Orange crystals from 1,4-dioxane, yield 3.61 g (66%). Mp 118-121 °C. IR (KBr) vmax (cm-1): 3479-3339 (NH2), 3055 (CH, aromatic), 1633 (C=C); 1H NMR (DM-SO-d6, 300 MHz): 6 1.12, 1.14 (2t, 6H, J1 = 5.90 Hz, J2 = 6.48 Hz, two OCH2CH3), 2.87-3.42 (m, 8H, 4CH2), 4.22, 4.24 (2q, 4H, J1 = 5.90 Hz, J2 = 6.48 Hz, two OCH2CH3),4.82, 5.14 (2s, 4H, D2O exchangeable, 2NH2), 5.11 (s, 1H, pyran H-4), 7.25-7.42 (m, 5H, C6H5); 13C NMR (DM-SO-d6, 75 MHz): 6 16.5, 16.8 (two OCH2CH3), 39.6, 45.5 (4CH2), 50.8 (pyran C-4), 52.6, 52.9 (two OCH2CH3), 120.5, 120.3, 124.7, 125.8 (C6H5), 130.2, 131.6, 132.4, 132.8, 133.8, 137.2, 138.7, 140.3 (pyran, thiophene C). Anal. Calcd. for C29H28N2O5S2: C, 63.48; H, 5.14; N, 5.14; S, 11.69. Found: C, 63.62; H, 5.41; N, 5.08; 11.73. MS: m/z 548 (M+, 76%). 2,12-Diamino-4,10,14-triphenyl-5,6,8,9,10,14-hexahy-dro-4H-dipyrano[2,3-a:3',2'-_/]xanthene-3,11-dicarbo- nitrile (9) Each of benzaldehyde (2.12 g, 0.02 mol) and malon-onitrile (1.32 g, 0.02 mol) were added to a solution of compound 7 (2.94 g, 0.01 mol) in 1,4-dioxane (40 mL) containing triethylamine (1.00 mL). The reaction mixture was heated under reflux for 3 h then poured onto ice/water mixture and the precipitated product was collected by filtration. Pale yellow crystals from 1,4-dioxane, yield 4.69 g (78%). Mp 189-202 °C. IR (KBr) vmax (cm-1): 3453-3326 (NH2), 3055 (CH, aromatic), 2223, 2220 (2CN), 1630 (C=C); 1H NMR (DMSO-d6, 300 MHz): 6 2.82-3.42 (m, 8H, 4CH2), 4.95, 5.16 (2s, 4H, D2O exchangeable, 2NH2), 5.08, 5.12, 5.14 (3s, 3H, pyran H-4), 7.22-7.58 (m, 15H, 3C6H5); 13C NMR (DMSO-d6, 75 MHz): 6 39.2, 45.7 (4CH2), 51.2, 51.6 (threepyran C-4), 116.6, 117.2 (2CN), 120.1, 120.8, 122.1, 122.5, 123.0, 123.5, 123.8, 124.2, 124.6, 125.3, 125.8, 126.8 (3C6H5), 130.3, 133.5, 134.6, 135.0, 136.7, 137.0, 137.6, 139.8 (three pyran C). Anal. Calcd. for C39H30N4O3: C, 77.72; H, 5.02; N, 9.30. Found: C, 77.90; H, 4.79; N, 9.42. MS: m/z 602 (M+, 42%). I,11,12-Triphenyl-4,5,7,8-tetrahydro-1H-xantheno [1,2-d:8,7-d']bis(thiazole)-2,10(11H,12H)-dithione (11) Each of elemental sulfur (0.64 g, 0.02 mol) and phe-nylisothiocyanate (2.60 g, 0.02 mol) were added to a solution of compound 7 (2.94 g, 0.01 mol) in 1,4-dioxane (40 mL) containing triethylamine (1.00 mL). The reaction mixture was heated under reflux for 2 h then poured onto ice/water mixture and the precipitated product was collected by filtration. Orange crystals from1,4-dioxane, yield 3.96 g (67%). Mp > 300 °C. IR (KBr) vmax (cm-1): 3055 (CH, aromatic), 1632 (C=C), 1208 (C=S); 1H NMR (DMSO-d6, 300 MHz): 5 2.96-3.41 (m, 8H, 4CH2), 5.08 (s, 1H, pyran H-4), 7.23-7.49 (m, 15H, 3C6H5); 13C NMR (DMSO-d6, 75 MHz): 5 37.6, 42.5 (2CH2), 51.1 (pyran C-4), 120.2, 120.8, 121.2, 121.6, 122.0, 122.3, 123.1, 123.9, 124.8, 125.1, 125.5, 126.8 (3C6H5), 130.2, 131.3, 132.6, 136.5, 139.4, 140.8 (pyran, two thiazole C), 180.3 (2C=S). Anal. Calcd. for C33H24N2OS4: C, 66.86; H, 4.08; N, 4.73; S, 21.64. Found: C, 66.93; H, 4.19; N, 4.90; S, 21.47. MS: m/z 592 (M+, 18%). 3,3-Dimethyl-9-phenyl-3,4,5,6,7,9-hexahydro-1H-xan-thene-1,8(2H)-dione (13) Each of benzaldehyde (1.06 g, 0.01 mol) and dime-done (1.40 g, 0.01 mol) was added to a solution of compound 1 (1.12 g, 0.01 mol) in absolute ethanol (40 mL) containing piperidine (1.0 mL). The whole reaction mixture was heated under reflux for 1 h then poured onto ice/ water mixture containing a few drops of hydrochloric acid and the formed solid product was collected by filtration. White crystals from1,4-dioxane, yield 2.25 g (70%). Mp 149-152 °C. IR (KBr) vmax (cm-1): 3054 (CH, aromatic), 1704, 1689 (2C=O), 1635 (C=C); 1H NMR (DMSO-d6, 300 MHz): 5 1.06, 1.08 (2s, 6H, 2CH3), 1.68-1.96 (m, 6H, 3CH2), 2.79, 2.83 (2s, 4H, 2CH2), 5.13 (s, 1H, pyran H-4), 7.26-7.46 (m, 5H, C6H5); 13C NMR (DMSO-d6, 75 MHz): 5 24.4 (2CH3), 26.5, 28.8, 32.9, 36.5, 42.1 (5CH2), 50.8 (pyran C-4), 120.3, 121.8, 122.4, 124.2 (C6H5), 168.8, 170.3 (2C=O). Anal. Calcd. for C21H22O3: C, 78.23; H, 6.88. Found: C, 78.40; H, 6.68. MS: m/z 322 (M+, 60%). 2. 5. General Procedure for the Synthesis of the Dithieno[3,2-a:2',3'-_/']xanthenes Derivatives 14a,b Each of elemental sulfur (0.64 g, 0.02 mol) and either malononitrile (1.32 g, 0.02 mol) or ethyl cyanoacetate (2.26 g, 0.02 mol) were added to a solution of compound 13 (3.22 g, 0.01 mol) in 1,4-dioxane (40 mL) containing triethylamine (1.00 mL). The reaction mixture was heated under reflux for 3 h then poured onto ice/water mixture and the precipitated product was collected by filtration. 2,10-Diamino-4,4-dimethyl- 12-phenyl-5,7,8,12-tet-rahydro-4H-dithieno-[3,2-a:2',3'-_/]xanthene-1,11-di-carbonitrile (14a) Yellow crystals from 1,4-dioxane, yield 2.89 g (60%). Mp 138-141 °C. IR (KBr) vmax (cm-1): 3476-3337 (NH2), 3055 (CH, aromatic), 2224, 2220 (2CN), 1633 (C=C); 1H NMR (DMSO-d6, 300 MHz): 5 1.06, 1.09 (2s, 6H, 2CH3), Mohareb et al.: Multi-component Reactions ... 57 Acta Chim. Slov. 2021, 68, 51-64 2.86-3.42 (m, 4H, 2CH2), 3.62 (s, 2H, CH2), 4.87, 5.15 (2s, 4H, D2O exchangeable, 2NH2), 5.14 (s, 1H, pyran H-4), 7.28-7.40 (m, 5H, C6H5); 13C NMR (DMSO-d6, 75 MHz): 5 24.7 (2CH3), 39.8, 44.6, 48.3 (3CH2), 51.2 (pyran C-4),116.6, 117.3 (2CN), 120.2, 120.8, 123.3, 124.1 (C6H5), 132.3, 134.2, 135.1, 135.6, 136.3, 138.3, 138.7, 139.4, 140.2, 141.2, 142.0, 142.6 (pyran, two thiophene C). Anal. Calcd. for C27H22N4OS2: C, 67.19; H, 4.59; N, 11.61; S, 13.29. Found: C, 66.93; H, 4.75; N, 11.82; 13.05. MS: m/z 482 (M+, 58%). Diethyl 2,10-Diamino- 12-phenyl-5,7,8,12-tetrahydro-4H-dithieno[3,2-a:2',3'-/]xanthene-1,11-dicarboxylate (14b) Pale white crystals from 1,4-dioxane, yield 4.32 g (75%). Mp 175-179 °C. IR (KBr) vmax (cm-1): 3449-3326 (NH2), 3055 (CH, aromatic), 11695, 1689 (2CO), 633 (C=C); 1H NMR (DMSO-d6, 300 MHz): 5 1.07, 1.09 (2s, 6H, 2CH3), 1.12, 1.13 (2t, 6H, J1 = 6.77 Hz, J2 = 6.92 Hz, two OCH2CH3), 2.89-3.48 (2t, 4H, 2CH2), 3.70 (s, 2H, CH2), 4.22, 4.23 (2q, 4H, J1 = 6.77 Hz, J2 = 6.92 Hz, two OCH2CH3), 4.82, 5.14 (2s, 4H, D2O exchangeable, 2NH2), 5.11 (s, 1H, pyran H-4), 7.25-7.42 (m, 5H, C6H5); 13C NMR (DMSO-d6, 75 MHz): 5 16.5, 16.8 (two OCH2CH3),24.4 (2CH3), 39.4, 45.8, 47.2 (3CH2), 51.3 (pyran C-4), 52.6, 52.7(two OCH2CH3),120.3, 122.8, 124.6, 125.7 (C6H5), 130.1, 131.8, 132.6, 131.1, 133.5, 136.6, 137.2, 138.9, 139.4, 140.3, 141.2, 142.6 (pyran, two thiophene C). Anal. Calcd. for C31H32N2O5S2: C, 64.56; H, 5.59; N, 4.86; S, 11.12. Found: C, 64.41.; H, 5.79; N, 5.16; 11.30. MS: m/z 576 (M+, 76%). 2,12-Diamino-5,5-dimethyl-4,10,14-triphenyl-5,6,8,9, 10,14-hexahydro-4H-dipyrano[2,3-a:3',2'-_/]xanthene-3,11-dicarbonitrile (15) Each of benzaldehyde (2.12 g, 0.02 mol) and malon-onitrile (1.32 g, 0.02 mol) were added to a solution of compound 13 (3.22 g, 0.01 mol) in 1,4-dioxane (40 mL) containing triethylamine (1.00 mL). The reaction mixture was heated under reflux for 3 h then poured onto ice/water mixture and the precipitated product was collected by filtration. Pale yellow crystals from1,4-dioxane, yield 4.69 g (78%). Mp 136-138 °C. IR (KBr) vmax (cm-1): 3453-3326 (NH2), 3055 (CH, aromatic), 2223, 2220 (2CN), 1630 (C=C); 1H NMR (DMSO-d6, 300 MHz): 5 1.07, 1.08 (2s, 6H, 2CH3), 2.82-2.96 (2t, 4H, 2CH2), 3.11-3.42 (s, 2H, CH2), 4.95, 5.16 (2s, 4H, D2O exchangeable, 2NH2), 5.09, 5.11, 5.14 (3s, 3H, threepyran H-4), 7.22-7.58 (m, 15H, 3C6H5); 13C NMR (DMSO-d6, 75 MHz): 5 24.5 (2CH3), 39.2, 45.7 (4CH2), 51.6 (pyran C-4), 116.6, 117.2 (2Cn), 120.8, 122.5, 123.5, 123.8, 124.6,125.3, 125.8, 126.8 (3C6H5), 130.3, 131.2, 131.9, 132.3, 133.5, 134.6, 135.0, 136.7, 137.0, 137.6, 138.4, 139.8 (three pyran C). Anal. Calcd. for C41H34N4O3: C, 78.07; H, 5.43; N, 8.88. Found: C, 77.86; H, 5.60; N, 9.02. MS: m/z 630 (M+, 32%). 4,4-Dimethyl-1,11,12-triphenyl-4,5,7,8-tetrahydro-1H-xantheno[1,2-d:8,7-d']bis(thiazole)-2,10(11H,12H)-dithione (16) Each of elemental sulfur (0.64 g, 0.02 mol) and phenyl-isothiocyanate (2.60 g, 0.02 mol) were added to a solution of compound 13 (3.22 g, 0.01 mol) in 1,4-dioxane (40 mL) containing triethylamine (1.00 mL). The reaction mixture was heated under reflux for 2 h then poured onto ice/water mixture and the precipitated product was collected by filtration. Yellowish white crystals from 1,4-dioxane, yield 4.24 g (68%). Mp 147-149 °C. IR (KBr) vmax (cm-1): 3054 (CH, aromatic), 1632 (C=C), 1209 (C=S); 1H NMR (DMSO-d6, 300 MHz): 5 1.05, 1.08 (2s, 6H, 2CH3), 2.82-2.98 (2t, 4H, 2CH2), 3.07-3.40 (s, 2H, CH2), 5.12 (s, 1H, pyran H-4), 7.23-7.57 (m, 15H, 3C6H5); 13C NMR (DMSO-d6, 75 MHz): 5 24.6 (2CH3), 37.8, 42.7, 44.2 (3CH2), 51.5 (pyran C-4), 120.1, 120.9, 121.1, 121.8, 122.3, 122.7, 123.5, 123.8, 124.3, 125.6, 126.0, 126.5 (3C6H5), 130.2, 131.3, 132.6, 136.5, 138.0, 139.4, 141.3, 142.6 (pyran, two thiazole C), 179.2, 180.1 (2C=S). Anal. Calcd. for C35H28N2OS4: C, 67.71; H, 4.55; N, 4.51; S, 20.66. Found: C, 67.80; H, 4.48; N, 4.72; S, 20.82. MS: m/z 620 (M+, 32%). 2. 6. Biology Section 2. 6. 1. Cell Proliferation Assay Most of the newly synthesized compounds were screened against the six cancer cell lines namely A549, HT-29, MKN-45, U87MG, SMMC-7721, and H460 using the standard MTT assay in vitro, with foretinib as the positive control.26-28 Their anti-proliferative activities against the six cancer cell lines and the mean values of three independent experiments, expressed as IC50 values, are presented in Table 1. Most of the synthesized compounds exhibited potent anti-proliferative activity with IC50 values less than 30 ^M. Generally, the variations of substituents within the thienopyridine moiety together with the heter-ocyclic ring being attached have a notable influence on the anti-proliferative activity. 2. 6. 2. Structure-Activity Relationship Table 1 shows the cytotoxicity of most of the synthesized compounds toward the six cancer cell lines A549, H460, HT-29, MKN-45, U87MG, and SMMC-7721. The reaction of cyclohexan-1,3-dione with the aryldiazonium salts 2a-c produced the arylhydrazono derivatives 3a-c, respectively. The two compounds 3b (X = CH3) and 3c (X = Cl) showed the highest cytotoxicity among these three compounds toward the six cancer cell lines. The multi-component reactions of either of 3a-c with either of the arylaldehydes 4a-c and either malononitrile or ethyl cy-anoacetate gave the 4H-chromenone derivatives 6a-r, respectively. Eleven compounds of this series were selected for screening against the six cancer cell lines and their ac- Mohareb et al.: Multi-component Reactions ... 58 Acta Chim. Slov. 2021, 68, 51-64 Table 1. In vitro growth inhibitory effects IC50 ± SEM (|iM) of selected compounds of the synthesized compounds against cancer cell lines Compound No A549 H460 IC50 ± HT29 SEM (^M) MKN-45 U87MG SMMC-7721 3a 6.26 ± 2.86 8.36 ± 3.24 5.69 ± 1.39 6.58 ± 1.37 9.62 ± 3.15 6.43 ± 2.25 3b 0.28 ± 0.12 0.33 ± 0.18 0.53 ± 0.13 0.33 ± 0.17 0.61 ± 0.28 0.52 ± 0.16 3c 0.43 ± 0.31 0.51 ± 0.25 0.49 ± 0.28 0.63 ± 0.39 0.82 ± 0.27 0.93 ± 0.39 6b 1.38 ± 0.91 2.46 ± 1.16 1.52 ± 0.92 1.63 ± 0.78 1.54 ± 0.85 2.53 ± 1.06 6c 1.34 ± 0.79 2.41 ± 1.20 1.35 ± 0.84 1.52 ± 0.71 2.58 ± 1.23 2.63 ± 1.17 6d 0.56 ± 0.32 0.29 ± 0.26 0.48 ± 0.22 0.41 ± 0.26 0.35 ± 0.12 0.53 ± 0.23 6e 2.16 ± 1.02 3.27 ± 1.38 3.38 ± 1.80 2.80 ± 1.38 2.32 ± 1.09 4.64 ± 1.42 6f 1.64 ± 0.36 1.52 ± 0.85 1.43 ± 0.75 2.60 ± 0.89 1.46 ± 0.63 1.63 ± 0.45 6g 4.36 ± 1.20 3.45 ± 1.81 2.61 ± 1.59 6.83 ± 2.28 4.60 ± 1.52 6.50 ± 2.63 6h 3.28 ± 1.48 5.83 ± 1.39 4.60 ± 1.24 6.80 ± 1.79 5.53 ± 1.61 6.45 ± 2.23 6i 1.25 ± 1.06 2.34 ± 1.13 2.32 ± 1.16 2.29 ± 1.71 1.29 ± 0.47 1.36 ± 0.95 6m 1.37 ± 0.71 0.50 ± 0.29 1.96 ± 1.19 1.80 ± 0.88 1.69 ± 0.82 1.33 ± 0.86 6n 0.46 ± 0.09 0.73 ± 0.44 0.85 ± 0.34 0.63 ± 0.41 0.52 ± 0.24 0.30 ± 0.26 6o 4.41 ± 1.49 6.72 ± 1.53 6.41 ± 2.49 6.29 ± 2.17 8.09 ± 2.17 5.19 ± 1.29 6q 2.34 ± 1.21 3.63 ± 1.32 4.58 ± 1.56 6.28 ± 2.39 5.32 ± 2.43 3.36 ± 1.28 8a 3.48 ± 1.09 2.63 ± 1.19 3.64 ± 1.26 4.38 ± 0.84 2.48 ± 0.89 2.23 ± 1.27 8b 1.13 ± 0.59 2.28 ± 0.72 3.29 ± 1.85 2.26 ± 1.79 3.62 ± 1.29 1.81 ± 0.84 9 4.13 ± 1.29 5.09 ± 1.36 6.16 ± 2.93 6.92 ± 1.37 5.82 ± 1.39 7.27 ± 1.92 11 2.32 ± 1.18 2.35 ± 1.08 3.42 ± 1.26 2.46 ± 0.98 1.26 ± 0.63 2.39 ± 0.98 14a 1.29 ± 0.59 1.39 ± 0.79 2.42 ± 1.08 1.36 ± 0.62 1.72 ± 0.98 1.42 ± 0.63 14b 4.69 ± 1.22 5.48 ± 2.21 6.42 ± 2.20 5.37 ± 1.19 4.49 ± 1.28 6.52 ± 1.28 15 0.35 ± 0.22 0.44 ± 0.16 0.62 ± 0.26 0.35 ± 0.16 0.62 ± 0.45 0.38 ± 0.16 16 0.29 ± 0.03 0.46 ± 0.23 0.46 ± 0.26 0.33 ± 0.20 0.59 ± 0.29 0.48 ± 0.21 Foretinib 0.08 ± 0.01 0.18 ± 0.03 0.15 ± 0.023 0.03 ± 0.0055 0.90 ± 0.13 0.44 ± 0.062 tivites varied from moderate to high. Compounds 6b (X = Y = H, R' = OH), 6c (X = H, Y = Cl, R' = NH2), 6f (X = H, Y = OCH3, R' = OH) and 6i (X = CH3, Y = Cl, R' = NH2) showed moderate inhibitions. However, compounds 6d (X = H, Y = Cl, R' = OH) and 6n (X = Cl, Y = H, R' = OH) showed the highest inhibitions among the eleven compounds. On the other hand, compounds 6e, 6g, 6h, 6o and 6q had declining inhibition activities. The reaction of compound 7 with two folds of elemental sulfur and either malononitrile or ethyl cyanoacetate gave the dithieno[3,2-fl:2',3'-j]xanthene derivatives 8a,b. It is obvious from Table 1 that compound 8b (R = COOEt) was more cytotoxic than compound 8a (R = CN); it seemsd that the oxygen content in 8b was responsible for its high inhibition activity. Surprisingly, the dipy-rano[2,3-fl:3',2'-j]xanthene derivative 9 and the xanthe-no[1,2-d:8,7-d']bis(thiazole) derivative 11 exhibited low inhibition values. Considering the dithieno[3,2-a:2',3'-j] xanthene-2,10-diamine derivatives 14a and 14b, it is clear that compound 14a (R = CN) showed higher inhibitions than 14b (R = COOEt). Finally both of the 5,6,8,9,10,14-hexahydro-4H-dipyrano[2,3-fl:3',2'-j]xan-thene derivative 15 and the 4,5,7,8-tetrahydro-1H-xan-theno[1,2-d:8,7-d']bis(thiazole)-2,10(11H,12H)-dithione derivative 16 exhibted high inhibitions against the six cancer cell lines. It is of great importance to note from Table 1 that compounds 3b, 3c, 6d, 6n, 15 and 16 showed the highest cytotoxicity among the tested compounds against the six cancer cell lines, while compounds 6b, 6c, 6f, 6i, 6m, 8b, and 14a exhibited moderate inhibitions. The high inhibition compounds together with those of moderate inhibitions were selected to be tested against tyrosine kinases. 2. 6. 3. Inhibitions of the Most Active Compounds Against Tyrosine Kinases Compounds 3b, 3c, 6b, 6c, 6d, 6f, 6m, 6n, 14a, 15 and 16 that showed from moderate to high inhibitions against the six cancer cell lines were further evaluated Table 2. Inhibitions of tyrosine kinases [Enzyme IC50 (nM)] by compounds 3b, 3c, 6b, 6c, 6d, 6f, 6m, 6n, 14a, 15 and 16 Compound c-Kit Flt-3 VEGFR-2 EGFR PDGFR 3b 2.83 3.25 2.16 0.73 0.52 3c 0.21 0.34 0.23 0.46 0.29 6b 1.32 1.08 1.69 0.43 1.02 6c 0.82 0.63 0.36 0.69 0.42 6d 0.25 0.31 0.47 0.24 0.29 6f 0.53 0.21 0.53 0.39 0.22 6m 1.16 0.29 0.42 1.80 2.01 6n 0.46 0.25 0.31 0.37 0.27 14a 0.82 0.29 0.37 0.44 0.29 15 0.33 0.21 0.47 0.35 0.26 16 2.09 1.28 1.62 1.59 2.42 Mohareb et al.: Multi-component Reactions ... 59 Acta Chim. Slov. 2021, 68, 51-64 against other five tyrosine kinases (c-Kit, Flt-3, VEGFR-2, EGFR, and PDGFR) using the same screening method (Table 2). These receptor tyrosine kinases (RTKs) have been implicated in vascular development by affecting the proliferation and migration of endothelial cells or pericytes. It is clear from Table 2 that compounds 3c, 6c, 6d, 6f, 6n, 14a and 15 were the most potent against the five tyrosine kinases. Compound 6b showed high inhibitions towards EGFR kinase with IC50 0.43 nM, while it showed moderate inhibition towards c-Kit, Flt-3, VEGFR-2 and PDGFR with IC50 1.32, 1.08, 1.69 and 1.02 nM, respectively. Compound 6m showed high inhibitions toward Flt-3 and VEGFR-2 tyrosine kinases with IC50 0.29 and 0.42 nM, respectively. On the other hand, compound 3b showed high inhibitions against EGFR and PDGFR kinas-es with IC50 0.73 and 0.52 nM, respectively. Compounds 3c, 6d and 15 were the most active toward c-Kit tyrosine kinase with IC50 0.21, 0.25 and 0.33 nM, respectively. Compounds 3b and 16 showed the lowest potency among the tested compounds. 2. 6. 4. Inhibitions of Selected Compounds Against Pim-1 Kinase Compounds 3c, 6c, 6d, 6f, 6n, 14a and 15 were selected to examine their Pim-1 kinase inhibition activity (Table 3) as these compounds showed high inhibition against the tested cancer cell lines at a range of 10 concentrations and the IC50 values were calculated. Compounds 3c, 6c, 6d, 6n and 15 most potently inhibited Pim-1 kinase with IC50 values of 0.24, 0.27, 0.24, 0.28 and 0.32 ^M, respectively. On the other hand, compounds 6f and 14a were less effective (IC50 > 10 ^M). These profiles in combination with cell growth inhibition data of compounds 3c, 6c, 6d, 6f, 6n, 14a and 15 are listed in Table 3, indicating that Pim- Table 3. The inhibitions of compounds 3c, 6c, 6d, 6f, 6n, 14a and 15 against Pim-1 kinase. Compound Inhibition ratio at 10 mM IC50 (mm) 3c 94 0.24 6c 90 0.27 6d 94 0.24 6f 20 > 10 6n 90 0.28 14a 31 >10 15 86 0.32 SGI-1776 - 0.048 1 is a potential target of these compounds where SGI-1776 was used as the positive control with IC50 0.048 ^M in the assay. 2. 6. 5. Pan Assay Interference Compounds (PAINS) Good antitumor drugs should give false positive results when evaluated within Pan Assay Interference Compounds (PAINS).29,30 Compounds can be regarded as false positives due their binding interactions by forming aggre-gates31-33 by being protein-reactive entities34-36 or by directly interfering with assay signaling. Pan Assay Interference Compounds (PAINS) are chemical entities that are frequently false positive in HTS. PAINS have a tendency to non-specifically react with several biological targets moderately, then specifically disturbing one preferred target.37 A number of disorderly functional groups are collected by numerous PAINS.38 Unwanted compounds may negatively influence not only enzyme assays but also phenotypic Table 4. Drug-like character of different compounds and standard drugs foretinib and SGI-1776 Compound Drug-likeness Rule Medicinal Chemistry Rules Lvio.a/No. Vvio.b/No. Gvio.c/No. Lead likeliness /No. PAINS of vio.a of vio.b of vio.c alertd of vio. 3b None None None None 0 3c None None None None 0 6b None None None 3 1 6c None None None 2 0 6d None None None 3 0 6f None None None None 0 6m None None None 2 0 6n None None None 3 0 14a None None None 3 0 15 None None None 2 0 16 None None None None 1 Foretinib None None None None 0 SGI-1776 None None None None 0 a Lvio. = Lipinski's rule. b Vvio. = Veber Rules. c Gvio. = Ghose filter. d PAINS = Pan Assay Interference Compounds Analysis. Mohareb et al.: Multi-component Reactions ... 60 Acta Chim. Slov. 2021, 68, 51-64 screens and show biological activity for the wrong reason.39 PAINS violations of proposed compounds and reference drugs are given in Table 4. Almost all the compounds showed zero PAINS alert and can be used as good anticancer agents in the future without side effects. 3. Results and Discussion Initially 2-arylhydrazonocyclohexan-1,3-dione was chosen as the model substrate for the synthesis of fused het- erocyclic compounds through studying its multi-component reactions with aromatic aldehydes and cyanomethyl-ene reagents to give biologically active fused pyran derivatives. The arylhydrazone derivatives 3a-c were obtained through the coupling reaction between cyclohex-ane-1,3-dione (1) and either benzenediazonium chloride (2a), 4-methylbenzenediazonium chloride (2b) or 4-chlorobenzenediazonium chloride (2c) in ethanol solution containing the appropriate amount of sodium acetate. The multi-component reactions of either 3a, 3b or 3c with either of benzaldehyde (4a), 4-chlorobenzaldehyde (4b) or Sheme 1. Synthesis of compounds 3a-c and 6a-r. Mohareb et al.: Multi-component Reactions ... 61 Acta Chim. Slov. 2021, 68, 51-64 4-methoxybenzaldehyde (4c) and either malononitrile (5a) or ethyl cyanoacetate (5b) in 1,4-dioxane solution containing a catalytic amount of triethylamine gave the 5,6,7,8-tet-rahydro-4H-chromene derivatives 6a-r, respectively (Scheme 1). The chemical structures of new compounds were assured by spectral data (IR, 1H, 13C NMR, MS). Thus, the 1H NMR spectrum of compound 6a (as an example) showed (beside the expected signals) signals at 5 4.82 ppm (D2O exchangeable) indicating the presence of the NH2 group, a multiplet at 5 7.23-7.48 ppm for the two phenyl groups. In addition, the 13C NMR spectrum revealed the presence of a signal at 51.2 due to the pyran C-4, one signalat 5 117.3 for CN groups, signals at 5 130.2, 131.6, 134.8, 136.1 for the pyran carbons and two signals at 5 166.8 and 167.2 for the C=N and C=O groups. Next, we studied the reaction of two-fold amount of cyclohexan-1,3-dione with benzaldehyde in ethanol containing a catalytic amount of triethylamine to give the 9-phenyl-3,4,5,6,7,9-hexahydro-1H-xanthene-1,8(2H)-di-one (7). The analytical and spectral data of compound 7 Sheme 2. Synthesis of compounds 7; 8a,b; 9 and 11. Mohareb et al.: Multi-component Reactions ... 62 Acta Chim. Slov. 2021, 68, 51-64 were in agreement with the proposed structure. Thus, the NMR spectrum showed the presence of two multiplets at 5 1.59-1.80 and 2.58-2.73 ppm equivalent to the six CH2 groups, a singlet at 5 5.09 ppm for the pyran H-4 and a multiplet at 5 7.25-7.41 ppm corresponding to the C6H5 group. In addition, the 13C NMR spectrum showed signals at 5 26.3, 28.4, 32.6 equivalent to the six CH2 groups, signal at 50.9 due to the pyran C-4, four signals at 5 120.6, 121.4, 123.6 and 125.8 for the phenyl carbons and a signal at 5 168.9 for the two symmetric C=O groups. Compound 7 showed interesting reactivity toward heterocyclization reactions through its reactions with some reagents. It was ready to undergo Gewald's thiophene40-42 reaction to produce biologically active fused thiophene derivatives. Thus, the reaction of compound 7 with two folds of either malo-nonitrile (5a) or ethyl cyanoacetate (5b) and elemental sulfur gave the dithieno[3,2-a:2',3'-j]xanthene derivatives 8a and 8b, respectively. On the other hand, compound 7 Sheme 3. Synthesis of compounds 13, 14a,b; 15 and 16. Mohareb et al.: Multi-component Reactions ... 63 Acta Chim. Slov. 2021, 68, 51-64 underwent multi-component reactions with two folds of benzaldehyde (4a) and malononitrile (5a) affording the hexahydro-4H-dipyrano[2,3-a:3',2'-j]xanthene derivative 9. Its structure was established on the basis of its analytical and spectral data (see experimental section). In addition, the reaction of compound 7 with two folds of elemental sulfur and phenylisothiocyanate (10) in 1,4-dioxane solution containing a catalytic amount of triethylamine gave the xantheno[1,2-d:8,7-d']bis(thiazole)-dithione derivative 11 (Scheme 2). Compounds 8, 9 and 11 were obtained in pure state and high yields and promising structure identification were obtained thus encouraging us to carry similar reactions using cyclohexan-1,3-dione in one side and dimedone in the other side. Therefore, the multi-component reactions of cyclohexan-1,3-dione with benzaldehyde (4a) and dimedone gave the 3,3-dimethyl-9-phenyl-3,4,5,6,7,9-hexahydro-1H-xanthene-1,8(2H)-dione (13). Compound 13 showed interesting reactivity toward heterocyclization reactions through its reactions with some reagents. Thus, the reaction of compound 13 with two folds of either malononitrile (5a) or ethyl cyanoace-tate (5b) and elemental sulfur gave the 4,4-dime-thyl-5,7,8,12-tetrahydro-4H-dithieno[3,2-a:2',3'-;]xan-thenes derivatives 14a and 14b, respectively. On the other hand, compound 13 underwent multi-component reactions with two folds of benzaldehyde (4a) and malononitrile (5a) affording the 2,12-diamino-5,5-dimethyl-4,10,14-triphenyl-5,6,8,9,10,14-hexahydro-4H-dipy-rano[2,3-fl:3',2'-j]xanthene derivative 15. In addition, the reaction of compound 13 with two folds of elemental sulfur and phenylisothiocyanates (10) in 1,4-dioxane solution containing a catalytic amount of triethylamine gave the 4,4-dimethyl-4,5,7,8-tetrahydro-1H-xantheno[1,2-d: 8,7-d']bis(thiazole)-dithione derivative 16 (Scheme 3). 4. Conclusion The main result of these studies is the synthesis of a series of novel heterocyclic derivatives synthesized from arylhydrazonocyclohexan-1,3-dione followed by screening of the newly synthesized compounds against six cancer cell lines. Compounds 3b, 3c, 6b, 6c, 6d, 6f, 6m, 6n, 14a, 15 and 16 were the most cytotoxic. Screening against the five tyrosine kinases c-Kit, Flt-3, VEGFR-2, EGFR, and PDGFR showed that compounds 3c, 6c, 6d, 6f, 6n, 14a and 15 were the most active compounds. On the other hand, inhibition against Pim-1 kinase indicated that compounds 3c, 6c, 6d, 6n and 15 were of the highest inhibitions. 5. References 1. T. Moriguchi, H. Matsuura, Y. Itakura, H. Katsuki, H. Saito, N. Nishiyama, Life Sci. 1997, 61, 1413-1420. DOI:10.1016/S0024-3205(97)00687-5 2. G. H. Abou El-Fotooh, O. I. Abd El-Salam, M. M. Ashraf, A. H. Nagla, Ind. J. Chem. 2005, 44B, 1887-1893. 3. D. Armesto, W M. Horspool, N. Martin, A. Ramos, C. Seoane, J. Org. Chem. 1989,54, 3069-3072. 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Jha, S. Kumar, I. Tomer, A review Der Pharma Chemica 2011, 3, 38-54. 42. K. Wang, D. Kim, A. Dömling, J. Comb.Chem. 2010, 12, 111118. DOI:10.1021/cc9001586 Povzetek V prispevku predstavljamo večkomponentne reakcije med arilhidrazocikloheksan-1,3-dionskimi derivati 3a-c in ben-zaldehidom (4a), 4-klorobenzaldehidom (4b) ali 4-metoksibenzaldehidom (4c) ter malononitrilom (5a) ali etil ciano-acetatom (5b), ki vodijo do nastanka 5,6,7,8-tetrahidro-4ff-kromenskih derivatov 6a-r. Pri reakciji dveh ekvivalentov cikloheksan-1,3-diona z benzaldehidom je nastal heksahidro-1H-ksanten-1,8(2H)-dionski derivat 7. Po drugi strani pa večkomponentna reakcija med spojino 1 in dimedonom ali benzaldehidom daje produkt 13. Obe spojini 7 in 13 lahko sodelujeta v reakcijah heterociklizacij, pri katerih nastanejo pripojeni tiofenski, piranski in tiazolski derivati. Izmed pripravljenih spojin smo nekatere uporabili za določevanje potencialne inhibitorne aktivnosti proti šestim rakavim celičnim linijam; rezultati so bili obetavni, spojine 3b, 3c, 6b, 6c, 6d, 6f, 6i, 6m, 6n, 8b, 14a, 15 in 16 so se izkazale kot še posebej citotoksične. Nadaljnje testiranje na petih tirozin kinazah (c-Kit, Flt-3, VEGFR-2, EGFR in PDGFR) ter Pim-1 kinazi je pokazalo, da so spojine 3c, 6c, 6d, 6f, 6n, 14a in 15 najbolj aktivne proti prvim petim tirozin kinazam, medtem ko so spojine 3c, 6c, 6d, 6n in 15 najbolj aktivne proti Pim-1 kinazi. © 0 Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Mohareb et al.: Multi-component Reactions ... DOI: 10.17344/acsi.2020.6138 Acta Chim. Slov. 2021, 68, 65-71 /^creative ^commons Scientific paper 6-Bromo-2'-(2-chlorobenzylidene)mcotinohydrazide and 6-Bromo-2'-(3-bromo-5-chloro-2-hydroxybenzylidene) nicotinohydrazide Methanol Solvate: Synthesis, Characterization, Crystal Structures and Antimicrobial Activities Hai-Yun Zhu College of Energy and Chemical Engineering, Ningxia Vocational Technical College of Industry and Commerce, Yinchuan 750021, P. R. China * Corresponding author: E-mail: zhuhaiyun76@126.com Received: 05-25-2020 Abstract Two newly synthesized nicotinohydrazones, 6-bromo-2'-(2-chlorobenzylidene)nicotinohydrazide (1) and 6-bromo-2'-(3-bromo-5-chloro-2-hydroxybenzylidene)nicotinohydrazide methanol solvate (2), have been obtained and structurally characterized by spectroscopic method and single crystal X-ray determination. The molecules in both compounds are in E configuration regarding to the azomethine groups. The molecules of compound 1 are linked via hydrogen bonds of N-H—O, generating one dimensional chains running along the c-axis direction. The hydrazone molecules of compound 2 are linked by methanol molecules via hydrogen bonds of N-H—O and O-H—N, generating dimers. The in vitro antimicrobial activities of these compounds indicate that they are interesting antibacterial agents. Keywords: Hydrazone; synthesis; hydrogen bonding; X-Ray crystal structure; antimicrobial activity 1. Introduction Hydrazones with the central group -CH=N-NH-are of great importance in biological fields, especially for the new drug investigation.1 These compounds have been reported to show interesting biological activities like antimicrobial, antifungal, anticonvulsant, analgesic, antiplatelet, antitubercular, antiinflammatory, as well as antitumor.2 Hydrazones are also a kind of interesting ligands in coordination chemistry.3 The metal complexes with hydrazones are reported to have interesting biological activities.4 Isoniazide is a front-line antituberculotic drug. The derivatives of isoniazide have been widely used as attractive drugs in the treatment of various deseases.5 To date, a number of hydrazones derived from benzohydra-zides were reported.6 However, those derived from nico-tinohydrazide are relatively rare. Moreover, the compounds bearing halide substituent such as F, Cl and Br usually possess effective antimicrobial activities.7 We have reported on some hydrazone compounds with antimicrobial activities.8 In pursuit of new antimicrobial agents, in this paper, two nicotinohydrazones, 6-bro-mo-2'-(2-chlorobenzylidene)nicotinohydrazide (1) and 6-bromo-2'-(3-bromo-5-chloro-2-hydroxybenzylidene) ? " fN1 "Y^A-'Y^i V-O" ° 1 2 Scheme 1. The nicotinohydrazones 1 and 2 Zhu: 6-Bromo-2'-(2-chlorobenzylidene)nicotinohydrazide 66 Acta Chim. Slov. 2021, 68, 65-71 nicotinohydrazide methanol solvate (2), possessing simultaneously Cl, Br and isoniazide skeleton are presented (Scheme 1). 2. Experimental 2. 1. Materials and Methods 5-Bromonicotinohydrazide, 2-chlorobenzaldehyde and 3-bromo-5-chloro-2-hydroxybenzaldehyde were purchased from Bide Chemical Reagent Co. Ltd. The other chemicals with AR grade were obtained commercially and used as received. CHN elemental analyses were performed on a Perkin-Elmer 240C elemental analyzer. IR spectra were measured with a FT-IR 170-SX (Nicolet) spectrophotometer. 1H NMR and 13C NMR data were measured with a Bruker 500 MHz instrument. 2. 2. Synthesis of 6-Bromo-2'-(2- chlorobenzylidene)nicotinohydrazide (1) 5-Bromonicotinohydrazide (0.216 g, 1.0 mmol) and 2-chlorobenzaldehyde (0.140 g, 1.0 mmol) were mixed and stirred in methanol (50 mL) for 1 h at ambient temperature to give a colourless solution. The solution was left to slow evaporation of the methanol for a week, yielding colourless needle-shaped single crystals. The crystals were filtered out and washed with methanol. Yiled 0.28 g (83%). M.p. 173.2-174.5 °C. Analysis calculated for C13H9BrCl-N3O: C, 46.1; H, 2.7; N, 12.4; found: C, 45.9; H, 2.7; N, 12.5. IR data (KBr, cm-1): 3178 (w), 1654 (s), 1598 (m), 1561 (m), 1471 (w), 1437 (w), 1369 (w), 1303 (s), 1158 (w), 1031 (m), 963 (w), 927 (w), 745 (m). 1H NMR (500 MHz, DMSO-d6): 5 12.24 (s, 1H, NH), 9.05 (s, 1H, PyH), 8.92 (s, 1H, CH=N), 8.83 (s, 1H, PyH), 8.52 (s, 1H, PyH), 8.03 (d, 1H, ArH), 7.55 (d, 1H, ArH), 7.46 (m, 2H, ArH). 13C NMR (126 MHz, DMSO-d6): 8 160.26, 153.01, 147.34, 144.71, 137.64, 133.36, 131.78, 131.23, 130.48, 129.95, 127.66, 126.97, 120.05. 2. 3. Synthesis of 6-Bromo-2'-(3-bromo-5-chloro-2-hydroxybenzylidene) nicotinohydrazide methanol solvate (2) 5-Bromonicotinohydrazide (0.216 g, 1.0 mmol) and 3-bromo-5-chloro-2-hydroxybenzaldehyde (0.235 g, 1.0 mmol) were mixed and stirred in methanol (50 mL) for 1 h at ambient temperature to give a slight yellow solution. The solution was left to slow evaporation of the methanol for 2 days, yielding light yellow block-shaped single crys- tals. The crystals were filtered out and washed with methanol. Yiled 0.39 g (84%). M.p. 210.5-211.3 °C. Analysis cal- culated for C14H12Br2ClN3O3: C, 36.1; H, 2.6; N, 9.0; found: C, 35.9; H, 2.7; N, 9.1. IR data (KBr, cm-1): 3457 (w), 3190 (w), 1666 (s), 1600 (w), 1550 (w), 1443 (s), 1344 (m), 1294 (w), 1164 (s), 1078 (s), 955 (s), 861 (s), 734 (m). 1H NMR (500 MHz, DMSO-d6): 8 12.68 (s, 1H, OH), 12.39 (s, 1H, NH), 9.05 (s, 1H, PyH), 8.92 (s, 1H, CH=N), 8.52 (s, 1H, PyH), 8.50 (s, 1H, PyH), 7.72 (s, 2H, ArH). 13C NMR (126 MHz, DMSO-d6): 8 160.23, 153.31, 153.25, 147.98, 147.36, 137.71, 133.30, 129.63, 129.28, 123.39, 120.25, 120.07, 110.94. 2. 4. X-Ray Structure Analysis X-Ray diffraction intensities were collected using a Bruker SMART 1000 CCD area detector equipped with graphite-monochromated Mo-Ka radiation (A = 0.71073 A) at 298(2) K. Absorption corrections were applied by SADABS.9 The structures of the compounds were solved by direct methods and refined on F2 by full-matrix least-squares methods with SHELXTL.10 All non-hydrogen atoms were refined anisotropically. The amino and methanol H atoms in both compounds were located in difference Fourier maps and refined isotropically, with N-H and O-H distances restrained to 0.90(1) A and 0.85(1) A, respectively, and with (7iso(H) values fixed at 1.2^eq(N) and 1.5Ueq(O). The other H atoms were placed in idealized positions and constrained to ride on their parent atoms. The Cl atoms in 1 is disordered over two sites, with occupancies of 0.84(2) and 0.16(2). The details of the crystallographic data are summarized in Table 1. Supplementary crystallographic data have been deposited at the Cambridge Crystallographic Data Center (CCDC 850161 and 2022935). 2. 5. Antimicrobial Test Qualitative determination of antimicrobial activity was done using the disk diffusion method. Suspensions in sterile peptone water from 24 hour cultures of microorganisms were adjusted to 0.5 McFarland. Muller-Hinton Petri dishes of 90 mm were inoculated using these suspensions. Paper disks (6 mm in diameter) containing 10 ^L of the substance to be tested (at a concentration of 2048 ^g/mL in DMSO) were placed in a circular pattern in each inoculated plate. Incubation of the plates was done at 37 °C for 18-24 h. DMSO impregnated discs were used as negative controls. Toxicity tests of the solvent, DMSO, showed that the concentrations used in antimicrobial activity assays did not interfere with the growth of the microorganisms. Reading of the results was done by measuring the diameters of the inhibition zones generated by the test substance. Penicillin was used as a reference. Determination of MIC was done using the serial dilutions in liquid broth method. The materials used were 96-well plates, suspensions of microorganism, Muller-Hinton broth and stock solutions of each substance to be tested (2048 ^g/mL in DMSO). The following concentrations of the substances to be tested were obtained in the 96-well plates: 1024, 512, 256, 128, 64, 32, 16, 8, 4, 2, and Zhu: 6-Bromo-2'-(2-chlorobenzylidene)nicotinohydrazide ... 67 Acta Chim. Slov. 2021, 68, 65-71 Table 1. Crystal data, data collection and structure refinement for the compounds Compound 1 2 Molecular formula C13H9BrClN3O C14H12Br2ClN3O3 Molecular weight 338.6 465.5 Crystal system Monoclinic Monoclinic Space group P21/c C2/c Temperature (K) 298(2) 298(2) a (À) 11.482(2) 11.862(1) b (À) 14.034(3) 13.494(1) c (À) 8.443(2) 19.860(2) ß (°) 90.05(3) 95.485(1) V (À3) 1360.5(5) 1562.1(5) Z 4 8 Dcalc (g cm-3) 1.653 1.954 Crystal dimensions (mm) 0.23 x 0.20 x 0.20 0.27 x 0.27 x 0.27 Absorption coefficient (mm-1) 3.212 5.309 Reflections measured 11289 7887 Total no. of unique data 2963 [Rint = 0.0409] 2890 [Rint = 0.0434] No. of observed data, I > 2a(T) 1825 1714 No. of variables 184 216 No. of restraints 4 2 Goodness of fit on F2 1.001 0.961 R1, wR2 [I 3 2a(I)]a 0.0486, 0.1097 0.0324, 0.0639 R1, wR2 (all data)a 0.0877, 0.1271 0.0754, 0.0757 aRi = 2||F0| - \FC\\/Z\F0\, WR2 = [2w(F02 - Fc2)2/Zw(F02)2]1/2 1 pg/mL. After incubation at 37 °C for 18-24 h, the MIC for each tested substance was determined by microscopic observation of microbial growth. It corresponds to the well with the lowest concentration of the tested substance where microbial growth was clearly inhibited. 3. Results and Discussion 3. 1. Chemistry The nicotinohydrazones 1 and 2 were facile prepared by the reaction of 1:1 molar ratio of 5-bromonicotinohy-drazide with 2-chlorobenzaldehyde and 3-bromo-5-chloro-2-hydroxybenzaldehyde, respectively in methanol. The elemental analyses are in good agreement with the formulae proposed for the compounds determined by single crystal X-ray diffraction. The crystals of the compounds are stable in air at room temperature, and easily soluble in DMF, DMSO, methanol, ethanol, chloroform, dichloromethane, and acetonitrile. Synthesis of the compounds was indicated in their IR spectra by the presence of bands for imine bonds, i.e. 1654 cm-1 for 1 and 1666 cm-1 for 2. In 1H NMR, the absence of NH2 signals and the appearance of peaks for NH protons in the region S 12.24-12.39 ppm and imine CH proton in the region S 8.92 ppm confirmed the synthesis of the compounds. The aromatic proton signals were found in their respective regions with different multiplicities, confirming their relevant substitution pattern. 3. 2. Crystal Structure Description of 1 and 2 The molecular structures of compounds 1 and 2 are shown in Figures 1 and 2, respectively. Compound 2 contains a methanol molecule of crystallization. All the related bond lengths and angles (Table 2) in the compounds are similar, and within the ranges of the bond values observed in reported hydrazone compounds.81,11 The C7-N1 bond lengths of 1.278(5) A in 1 and 1.243(4) A in 2 indicate the double bond nature. The C8-N2 bond lengths of 1.339(4) A in 1 and 1.335(4) A in 2, and the N1-N2 bonds (1.388(4) A in 1 and 1.340(4) A in 2) are shorter than normal, suggesting the existence of delocalization in the molecules. Table 2. Selected bond lengths (A) and bond angles (°) for the compounds 1 and 2 1 C7- N1 1.278(5) N1- N2 1.388(4) N2 -C8 1.339(4) C8- O1 1.226(4) C1- -C7- N1 122.3(3) C7- -N1- N2 113.3(3) N1 -N2- -C8 118.8(3) N2 -C8- C9 115.9(3) N2 -C8- O1 123.8(3) O1 -C8- C9 120.3(3) 2 C7- N1 1.243(4) N1 N2 1.340(4) N2 -C8 1.335(4) C8- O1 1.185(4) C1- -C7- N1 118.6(3) C7- -N1- N2 119.3(3) N1 -N2- -C8 115.2(3) N2 -C8- C9 115.8(4) N2 -C8- O1 122.7(4) O1 -C8- C9 121.5(3) Zhu: 6-Bromo-2'-(2-chlorobenzylidene)nicotinohydrazide ... 68 Acta Chim. Slov. 2021, 68, 65-71 Table 3. Distances (A) and angles (°) involving hydrogen bonding of the compounds 1 and 2 D-H-A d(D-H) (A) d(H-A) (A) d(D"A) (A) Angle(D-H-A) (°) 1 N2-H2-OP C7-H7-O11 2 02-H2A—N1 N2-H2—O3 03-H3-N3U 0.90(1) 0.93 0.82 0.90(1) 0.85(1) 2.02(2) 2.32(2) 1.81 1.93(1) 1.96(1) 2.859(4) 3.133(4) 2.518(4) 2.817(4) 2.799(4) 157(4) 146(4) 144(3) 168(4) 172(4) Symmetry code for i: x, 1/2 - y, 1/2 + z; ii: 1 - x, - y, 1 - z. Table 4. n-n interactions Cg Distance between ring centroids (A) Dihedral Perpendicular distance angle (°) of Cg(I) on Cg(J) (A) Perpendicular distance of Cg(J) on Cg(I) (A) 1 Cg1-Cg1a Cg1-Cg2h Cg2^"Cg2 2 Cg1—Cg2v 3.9762 4.1626 3.8892 3.6959 4.8932 0 7.201 0 3.876 0 3.5858 3.4858 -3.4367 -3.4413 3.3600 3.5859 -3.7417 -3.4367 -3.5234 3.3600 Cg1 and Cg2 are the centroids of the N3-C12-C11-C10-C9-C13 and C1-C2-C3-C4-C5-C6 rings, respectively. Symmetry codes: iii: 1 - x, 1 - y, 1 - z; iv: x, 1/2 - y, 1/2 + z; v: - x, - y, 1 - z; vi: 1/2 - x, 1/2 - y, 1 - z. Figure 1. Molecular structure of 1 at 30% probability displacement. Only the major component of the disordered group is shown. Figure 2. Molecular structure of 2 at 30% probability displacement. Intramolecular O-H—N and N-H—O hydrogen bonds are drawn as dashed lines. Zhu: 6-Bromo-2'-(2-chlorobenzylidene)nicotinohydrazide ... 69 Acta Chim. Slov. 2021, 68, 65-71 Figure 3. Molecular packing of 1, viewed along the b axis. Hydrogen bonds are drawn as dashed lines. Figure 4. Molecular packing of 2, viewed along the b axis. Hydrogen bonds are drawn as dashed lines. The benzene ring and the pyridine ring form a dihedral angle of 6.4(4)° in 1 and 3.8(4)° in 2. In the crystal structure of 1, the molecules are linked through hydrogen bonds of N2-H2—O1 and C7-H7—O1 (Table 3), generating one dimensional chains running along the c-axis direction (Figure 3). In the crystal structure of 2, the adjacent two hydrazone molecules are linked by two methanol molecules through hydrogen bonds of N2-H2—O3 and O3-H3-N3 (Table 3), generating a di-mer (Figure 4). In addition, in both compounds the presence of short ^-electron ring - ^-electron ring interactions with Cg-Cg distances < 6.0 Â and fi < 60.0° that are specified in Table 4 was detected.12 3. 3. Antimicrobial Activity of the Compounds The antimicrobial activities of the compounds against the organisms Streptococcus pyogenes (S. pyogenes), Streptococcus agalactiae (S. agalactiae), Staphylococcus aureus (S. aureus), Bacillus anthracis (B. anthracis), Klebsiella pneumonia (K. pneumonia) and Pseudomonas aeruginosa (P. aeruginosa) are summarized in Table 5. The results show that both compounds have effective antimicrobial activities against S. pyogenes, S. agalactiae, and B. anthracis, and have relatively poor or negative activities against other bacteria when compared to the Penicillin. Compounds 1 and 2 have similar activities against S. agalactiae and B. Zhu: 6-Bromo-2'-(2-chlorobenzylidene)nicotinohydrazide ... 70 Acta Chim. Slov. 2021, 68, 65-71 anthracis. Interestingly, compound 2 has stronger activities against S. pyogenes, K. pneumonia and P. aeruginosa than compound 1. This indicates that the Br and Cl substi-tunts are a good choice in the search for new antimicrobial agents. The activities of the nicotinohydrazone compounds in this work are stronger than the benzohydrazones with Br as substituent.6a The compounds are more active against S. pyogenes, S. agalactiae, B. anthracis and P aeruginosa than the benzohydrazone compound with Br, NO2 and Cl as the substituent.13 Thus, the present compounds show promising activity against S. pyogenes, S. agalactiae and B. anthracis, which deserves further investigation for developing new antimicrobial drugs. Table 5. 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Son-timuang, H.-K. Fun, Crystallogr. Rep. 2017, 62, 1104-1108. D0I:10.1134/S1063774517070069 12. A. L. Spek, Acta Crystallogr. 2009, D65, 148-155. DOI: 10.1107/S090744490804362X 13. H.-Y. Zhu, Chinese J. Struct. Chem. 2011, 30, 724-730. Povzetek Pripravili smo dva nova predstavnika nikotinohidrazonov: 6-bromo-2'-(2-klorobenziliden)nikotinohidrazid (1) in 6-bromo-2'-(3-bromo-5-kloro-2-hidroksibenziliden)nikotinohidrazid metanolni solvat (2). Strukturi obeh produktov smo določili s spektroskopskimi metodami in z rentgensko difrakcijsko analizo monokristalov. Molekule obeh spojin imajo v azometinski skupini E konfiguracijo. Molekule v spojini 1 so povezane v enodimenzionalne verige vzdolž c osi z vodikovimi vezmi N-H-O. Hidrazonske molekule spojine 2 so z metanolnimi povezane v dimere preko vodikovih vezi N-H—O in O-H—N. Določitev in vitro antimikrobnih aktivnosti za ti dve spojini je pokazala, da bi lahko bili potencialno zanimivi antibakterijski učinkovini. © 0 Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Zhu: 6-Bromo-2'-(2-chlorobenzylidene)nicotinohydrazide ... DOI: 10.17344/acsi.2020.6182 Acta Chim. Slov. 2021, 68, 72-87 /^creative ^commons Scientific paper New Approaches for the Synthesis of Heterocyclic Compounds Derived from Cyclohexan-1,3-dione with Anti-proliferative Activities Rafat Milad Mohareb,1 Yara Raafat Milad2,3 and Ayat Ali Masoud1 1 Department of Chemistry, Faculty of Science, Cairo University, A. R. Egypt 2 Jean Coutu, 531 Jarry Est, Montréal, Québec, Canada 3 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Cairo, A. R. Egypt * Corresponding author: E-mail: raafat_mohareb@yahoo.com Received: 06-11-2020 Abstract In the present work a series of heterocyclization reactions were adopted using cyclohexan-1,3-dione through its reaction with either furan-2-carbaldehyde or thiophene-2-carbaldehyde to give the corresponding ylidene derivatives 3a,b. The latter compounds underwent heterocyclization reactions to give thiophene and pyran derivatives 5a-d and 6a-d, respectively. Moreover, compounds 3a,b reacted with elemental sulfur and phenyl isothiocyanate to give the fused thiazole derivatives 8a,b. In addition, the reaction with either of hydrazine hydrate or phenylhydrazine has given the 4-hydrazo-no-4,5,6,7-tetrahydro-2ff-indazole derivatives 10a-d, respectively. Similarly, the reaction of either 3a or 3b with hydrox-ylamine hydrochloride gave the 6,7-dihydrobenzo[c]isoxazol-4(5H)-one oxime derivatives 12a and 12b, respectively. Other fused heterocyclic compounds were produced and their structures were elucidated. Evaluation of the synthesized compounds against selected cancer cell lines was performed. The most active compounds were further evaluated against tyrosine kinases and Pim-1 kinase inhibitions. Keyword: Cyclohexan-1,3-dione, thiophene, pyrazole, isoxazole, cytotoxicity 1. Introduction Within the last few years the synthesis of heterocyclic compounds attracted the attention due to the wide spectrum of their high biological activities. In addition, many compounds were considered as good synthons for fused systems that were characterized by different pharmaceutical applications.1-10 Therefore, organic chemists have been making extensive efforts to produce heterocyclic compounds by developing new and efficient synthetic transformations. Within the field of pharmaceutical chemistry, many pyrazoles, thiophenes and thiazoles were reported with a wide spectrum of biological activities that included potent analgesic, anti-convulsant, antiinflammatory and anti-bacterial, anti-pyretic, anti-tumor, anti-parasitic, anti-microbial, anti-histaminic (H1), anti-anexiety activities in tests in mice, anti-arrhythmic and as serotonin antagonists.11-23 The present work is dealing with the current application of pyrazole, thio- phene, pyrimidine and oxazine cores in the designing of anticancer agents within tumor progression. In our research it was possible to verify that such compounds are readily applicable to provide new insights and valuable inspiration in the research of new drugs and in their development as well as to contribute to the management of cancer. This encouraged our group to be attracted toward the synthesis of pyran derivatives research through the uses of b-diketones. The produced heterocyclic compounds showed high anti-proliferative activities against cancer cell lines together with high inhibitions toward tyrosine kinases.24-26 Based on such importance of heterocyclic compounds, therefore, we studied the reaction of cyclohexane-1,3-dione with some heterocyclic aldehydes and cyanomethylene reagents followed by heterocycliza-tion of the products. Additionally, the anti-tumor evaluations of the resulting compounds towards cancer cell lines are reported. Mohareb et al.: New Approaches for the Synthesis of Heterocyclic Acta Chim. Slov. 2021, 68, 72-87 73 2. Experimental 2. 1. General The melting points of the obtained compounds were determined using Electrothermal digital melting point apparatus and are uncorrected. IR spectra (KBr discs) were measured on an FTIR plus 460 or PyeUnicam SP-1000 spectrophotometer (PyeUnicam, UK, Cambridge). NMR spectra were obtained using Varian Gemini-300 (300 MHz, Varian UK) using DMSO-d6 as the solvent and tetramethylsilane (TMS) as the internal standard; chemical shifts are expressed as 5 ppm. The mass spectra were measured with Hewlett Packard 5988 A GC/MS system (Hewlett-Packard, Agilent, USA) instrument. 2. 1. 1. General Procedure for the Synthesis of the 2-Methylenecyclohexane-1,3-dione Derivatives 3a,b Either of furan-2-carbaldehyde (0.96 g, 0.01 mol) of thiophene-2-carbaldehyde (1.12 g, 0.01 mol) was added to a solution of cyclohexane-1,3-dione (1) (1.12 g, 0.01 mol) in absolute ethanol (40 mL) containing piperdine (0.50 mL). The reaction mixture, in each case, was heated under reflux for 3 h then poured onto ice/water containing a few drops of hydrochloric acid and the formed solid product was collected by filtration. 2-(Furan-2-ylmethylene)cyclohexane-1,3-dione (3a) Yellow crystals from ethanol, yield 1.46 g (77%), m.p. 185-188 °C. IR (KBr) vmax (cm-1) 3053 (CH, aromatic), 1704, 1687 (2 CO), 1632 (C=C); 1H NMR (DMSO-d6, 300 MHz) 5 1.45-1.69 (m, 2H, CH2), 2.63-2.76 (m, 4H, 2CH2), 6.82 (s, 1H, CH), 6.80-7.83 (m, 3H, furan H); 13C NMR (DMSO-d6, 75 MHz) 5 16.4, 36.5, 39.2 (3CH2), 112.4, 158.1 (C=CH), 135.8, 140.2, 142.6, 146.1 (furan C), 177.1, 179.4 (2CO). Anal. Calcd for C11H10O3: C, 69.46; H, 5.30. Found: C, 69.31; H, 5.52. MS: m/z 190 (M+, 28%). 2-(Thiophen-2-ylmethylene)cyclohexane-1,3-dione (3b) Orange crystals from ethanol, yield 1.44 g (70%), m.p. 201-204 °C. IR (KBr) vmax (cm-1) 3055 (CH, aromatic), 1703, 1689 (2 CO), 1630 (C=C); 1H NMR (DMSO-d6, 200 MHz) 5 I.43-1.68 (m, 2H, CH2), 2.61-2.74 (m, 4H, 2CH2), 6.80 (s, 1H, CH), 6.82-7.88 (m, 3H, thiophene H); 13C NMR (DMSO-d6, 75 MHz) 5 16.8, 36.2, 39.1 (3CH2), 112.6, 158.8 (C=CH), 135.6, 140.5, 142.3, 146.2 (thiophene C), 177.6, 179.2 (2Co). Anal. Calcd for C11H10O2S: C, 64.05; H, 4.89; S, 15.55. Found: C, 63.80; H, 4.73; 15.38. MS: m/z 206 (M+, 32%). 2. 1. 2. General Procedure for the Synthesis of the 6,7-Dihydrobenzo[fo]thiophen-5(4H)-one Derivatives 5a-d Either malononitrile (0.66 g, 0.01 mol) or ethyl cyanoacetate (1.07 g, 0.01 mol) was added to a solution of either compound 3a (1.90 g, 0.01 mol) or 3b (2.06 g, 0.01 mol) in ethanol (40 mL) containing triethylamine (0.50 mL). The reaction mixture was heated under reflux for 3 h then poured onto ice/water mixture containing a few drops of hydrochloric acid and the formed solid product was collected by filtration. 2-Amino-4-(furan-2-ylmethylene)-5-oxo-4,5,6,7-tetrahydrobenzo[fo]thiophene-3-carbonitrile (5a) Pale yellow crystals from ethanol, yield 1.94 g (70%), m.p. 130-132 °C. IR (KBr) vmax (cm-1) 3482-3353 (NH2), 3056 (CH, aromatic), 2220 (CN), 1686 (CO), 1630 (C=C); 1H NMR (DMSO-d6, 300 MHz) 5 2.62-2.78 (2t, 4H, 2CH2), 4.73 (s, 2H, D2O exchangeable, NH2), 6.84 (s, 1H, CH), 6.82-7.86 (m, 3H, furan H); 13C NMR (DMSO-d6, 75 MHz) 5 16.6, 36.3, 39.5 (3CH2), 112.6, 158.4 (C=CH), 116.8 (CN), 135.4, 140.6, 141.4, 142.2, 142.7, 144.8, 145.6, 146.5 (thiophene, furan C), 179.3 (CO). Anal. Calcd for C14H10N2O2S: C, 62.21; H, 3.73; N, 10.36; S, 11.86. Found: C, 62.36; H, 3.80; N, 10.41; S, 12.04. MS: m/z 270 (M+, 32%). Ethyl 2-Amino-4-(furan-2-ylmethylene)-5-oxo-4,5,6,7-tetrahydrobenzo[fo]thiophene-3-carboxylate (5b) Yellow crystals from ethanol, yield 2.21 g (70%), m.p.125-127 °C. IR (KBr) vmax (cm-1) 3494-3368 (NH2), 3058 (CH, aromatic), 2931, 2972 (CH2, CH3), 1689, 1688 (2 CO), 1630 (C=C); 1H NMR (DMSO-d6, 300 MHz) 5 1.12 (t, 3H, J = 7.28 Hz, CH3), 2.68-2.74 (2t, 4H, 2CH2), 4.21 (q, 2H, J = 7.28 Hz, CH2), 4.76 (s, 2H, D2O exchangeable, NH2), 6.86 (s, 1H, CH), 6.84-7.92 (m, 3H, furan H); 13C NMR (DMSO-d6, 75 MHz) 5 16.3 (OCH2CH3), 16.4, 36.8, 39.3 (3CH2), 52.8 (OCH2CH3), 112.3, 158.6 (C=CH), 135.6, 18.0, 140.8, 141.6, 142.3, 143.5, 144.2, 146.7 (thiophene, furan C), 164.8, 178.3 (2CO). Anal. Calcd for C16H15NO4S: C, 60.55; H, 4.76; N, 4.41; S, 10.10. Found: C, 60.80; H, 4.83; N, 4.60; S, 10.26. MS: m/z 317 (M+, 38%). 2-Amino-5-oxo-4-(thiophen-2-ylmethylene)-4,5,6,7-tetrahydrobenzo[fo]thiophene-3-carbonitrile (5c) Orange crystals from ethanol, yield 1.94 g (68%), m.p. 202-205 °C. IR (KBr) vmax (cm-1) 3459-3342 (NH2), 3058 (CH, aromatic), 2221 (CN), 1688 (CO), 1630 (C=C); 1H NMR (DMSO-d6, 300 MHz) 5 2.64-2.75 (2t, 4H, 2CH2), 4.74 (s, 2H, D2O exchangeable, NH2), 6.88 (s, 1H, CH), 6.80-7.87 (m, 3H, thiophene H); 13C NMR (DM-SO-d6, 75 MHz) 5 16.3, 39.8 (2CH2), 112.3, 158.8 (C=CH), 116.9 (CN), 134.2, 138.6, 140.3, 142.4, 143.1, 143.6, 145.2, 146.7 (two thiophene C), 179.8 (CO). Anal. Calcd for C14H10N2OS2: C, 58.72; H, 3.52; N, 9.78; S, 22.39. Found: C, 58.36; H, 3.80; N, 9.68; S, 22.41. MS: m/z 286 (M+, 46%). Ethyl 2-Amino-5-oxo-4-(thiophen-2-ylmethylene)-4,5,6,7-tetrahydrobenzo[fo]thiophene-3-carboxylate (5d) Pale yellow crystals from ethanol, yield 2.13 g (66%), m.p. 189-192 °C. IR (KBr) vmax (cm-1) 3486-3342 (NH2), 3054 (CH, aromatic), 2938, 2893 (cH2, CH3), 1689, 1686 (2 Mohareb et al.: New Approaches for the Synthesis of Heterocyclic ... 74 Acta Chim. Slov. 2021, 68, 72-87 74 CO), 1630 (C=C); XH NMR (DMSO-d6, 300 MHz) 5 1.13 (t, 3H, J = 7.59 Hz, CH3), 2.64-2.78 (2t, 4H, 2CH2), 4.23 (q, 2H, J = 7.59 Hz, CH2), 4.79 (s, 2H, D2O exchangeable, NH2), 6.84 (s, 1H, CH), 7.29-7.85 (m, 3H, thiophene H); 13C NMR (DMSO-d6, 75 MHz) 5 16.1 (OCH2CH3), 16.5, 36.4, 39.7 (3CH2), 52.3 (OCH2CH3), 112.1, 158.4 (C=CH), 135.3, 136.7, 138.3, 140.5, 141.5, 142.0, 143.8, 144.6, 146.8 (two thiophene C), 164.5, 178.9 (2CO). Anal. Calcd for C16H15NO3S2: C, 57.64; H, 4.53; N, 4.20; S, 19.23. Found: C, 57.80; H, 4.71; N, 4.38; S, 19.46. MS: m/z 333 (M+, 26%). 2. 1. 3. General Procedure for the Synthesis of the 2.ff-Chromen-5-one Derivatives 6a-d Either malononitrile (0.66 g, 0.01 mol) or ethyl cyano-acetate (1.13 g, 0.01 mol) was added to a solution of either compound 3a (1.90 g, 0.01 mol) or 3b (2.06 g, 0.01 mol) in absolute ethanol (40 mL) containing triethylamine (1.0 mL). The reaction mixture, in each case, was heated under reflux for 3 h then the excess solvent was removed under vacuum. The remaining product was triturated with diethyl ether and the formed solid product was collected by filtration. 2-Amino-4-(furan-2-yl)-5-oxo-5,6,7,8-tetrahydro-2H-chromene-3-carbonitrile (6a) Pale yellow crystals from ethanol, yield 1.66 g (69%), m.p. 194-196 °C. IR (KBr) vmax (cm-1) 3470-3328 (NH2), 3055 (CH, aromatic), 2220 (cN), 1632 (C=C); 1H NMR (DMSO-d6, 300 MHz) 5 1.41-1.66 (m, 4H, 2CH2), 2.612.76 (m, 4H, 2CH2), 4.71 (s, 2H, D2O exchangeable, NH2), 6.04 (s, 1H, pyran H-4), 6.87-7.83 (m, 3H, furan H); 13C NMR (DMSO-d6, 75 MHz) 5 16.1, 36.3, 37.2, 39.5 (4CH2), 116.8 (CN), 134.3, 141.5, 141.8, 142.6, 1431.4, 143.8, 145.2, 146.4 (pyran, furan C), 178.4 (CO). Anal. Calcd for C14H14N2O2: C, 69.41; H, 5.82; N, 11.56. Found: C, 69.38; H, 5.92; N, 11.29. MS: m/z 242 (M+, 28%). 4-(Furan-2-yl)-2-hydroxy-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (6b) Pale brown crystals from ethanol, yield 1.79 g (74%), m.p. 198-200 °C. IR (KBr) vmax (cm-1) 3568-3339 (Oh), 3055 (CH, aromatic), 2222 (CN), 1630 (C=C); 1H NMR (DMSO-d6, 300 MHz) 5 1.32-1.68 (m, 4H, 2CH2), 2.632.74 (m, 4H, 2CH2), 6.02 (s, 1H, pyran H-4), 6.84-7.85 (m, 3H, furan H), 9.80 (s, 1H, D2O exchangeable, OH); 13C NMR (DMSO-d6, 75 MHz) 5 16.3, 36.5, 37.1, 39.3 (4CH2), 116.7 (CN), 134.1, 138.0, 140.2, 141.7, 142.3, 142.9, 143.3, 146.6 (furan, pyran C). Anal. Calcd for C14H13NO3: C, 69.12; H, 5.39; N, 5.76. Found: C, 68.92; H, 5.42; N, 5.58. MS: m/z 243 (M+, 36%). 2-Amino-4-(thiophen-2-yl)-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (6c) Brown crystals from ethanol, yield 1.85 g (72%), m.p. 211-214 °C. IR (KBr) vmax (cm-1) 3445-3372 (NH2), 3055 (CH, aromatic), 2220 (Cn), 1630 (C=C); 1H NMR (DMSO-d6, 200 MHz) 8 1.41-1.64 (m, 4H, 2CH2), 2.612.77 (m, 4H, 2CH2), 4.73 (s, 2H, D2O exchangeable, NH2), 6.07 (s, 1H, thiophene H-4), 6.91-7.73 (m, 3H, thiophene H); 13C NMR (DMSO-d6, 75 MHz) 8 16.3, 36.6, 37.2, 39.8 (4CH2), 116.4 (CN), 134.6, 138.7, 140.2, 141.6, 142.3, 142.8, 143.6, 146.2 (thiophene, pyran C). Anal. Calcd for C14H14N2O2S: C, 65.09; H, 5.46; N, 10.84; S, 12.41. Found: C, 64.90; H, 5.62; N, 10.68; S, 12.58. MS: m/z 258 (M+, 40%). 2-Hydroxy-4-(thiophen-2-yl)-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (6d) Orange crystals from ethanol, yield 1.96 g (76%), m.p. 177-179 °C. IR (KBr) vmax (cm-1) 3542-3329 (Oh), 3055 (CH, aromatic), 2220 (CN), 1688 (CO), 1630 (C=C); 1H NMR (DMSO-d6, 300 MHz) 8 1.34-1.66 (m, 4H, 2CH2), 2.61-2.76 (m, 4H, 2CH2), 6.04 (s, 1H, pyran H-4), 6.76-7.89 (m, 3H, furan H), 9.83 (s, 1H, D2O exchangeable, OH); 13C NMR (DMSO-d6, 75 MHz) 8 16.1, 36.3, 37.4, 39.6 (4CH2), 116.9 (CN), 134.4, 139.2, 140.8, 141.5, 143.7, 144.3, 145.2, 146.8 (furan, pyran C), 178.6 (CO). Anal. Calcd for C14H13NO2S: C, 64.84; H, 5.05; N, 5.40; S, 12.36. Found: C, 64.72; H, 5.24; N, 12.59. MS: m/z 259 (M+, 42%). 2. 1. 4. General Procedure for the Synthesis of 2-Thioxohexahydrobenzo[d]thiazole Derivatives 8a,b Each of elemental sulfur (0.32 g, 0.01 mol) and phenyl isothiocyanate (1.30 g, 0.01 mol) were added to a solution of either compound 3a (1.90 g, 0.01 mol) or 3b (2.06 g, 0.01 mol) in 1,4-dioxane (40 mL) containing triethylamine (1.0 mL). The reaction mixture was heated under reflux for 2 h then was left to cool and the formed solid product, in each case, was collected by filtration. 4-(Furan-2-ylmethylene)-3-phenyl-2-thioxo-2,3,6,7-tetrahydrobenzo[ d]thiazol-5(4H)-one (8a) Yellow crystals from ethanol, yield 2.50 g (74%), m.p. 168-169 °C. IR (KBr) vmax (cm-1) 3055 (CH, aromatic), 1688 (CO), 1630 (C=C), 1205 (C=S); 1H NMR (DMSO-d6, 300 MHz) 8 2.63-2.78 (2t, 4H, 2CH2), 6.87 (s, 1H, CH), 6.82-7.85 (m, 8H, C6H5, furan H); 13C NMR (DMSO-d6, 75 MHz) 8 16.1, 36.6, 39.5 (3CH2), 112.1, 158.4 (C=CH), 120.3, 122.6, 124.8, 127.2, 134.6, 140.8, 142.2, 143.2, 143.8, 146.9 (C6H5, furan, thiazole C), 179.6 (CO), 181.3 (C=S). Anal. Calcd for C18H13NO2S2: C, 63.69; H, 3.86; N, 4.13; S, 18.89. Found: C, 63.80; H, 3.69; N, 4.32; S, 19.18. MS: m/z 339 (M+, 48%). 3-Phenyl-4-(thiophen-2-ylmethylene)-2-thioxo-2,3,6,7-tetrahydrobenzo[ d]thiazol-5(4H)-one (8b) Pale yellow crystals from 1,4-dioxane, yield 2.84 g (80%), m.p. 222-225 °C. IR (KBr) vmax (cm-1) 3055 (CH, aromatic), 1689 (CO), 1630 (C=C), 1205 (C=S); 1H NMR (DMSO-d6, 300 MHz) 8 2.61-2.79 (2t, 4H, 2CH2), 6.88 (s, 1H, CH), 6.96-7.89 (m, 8H, C6H5, thiophene H); 13C Mohareb et al.: New Approaches for the Synthesis of Heterocyclic ... Acta Chim. Slov. 2021, 68, 72-87 75 NMR (DMSO-d6, 75 MHz) 8 16.5, 39.7 (2CH2), 112.4, 158.6 (C=CH), 120.7, 122.5, 124.3, 126.8, 136.2, 140.6, 141.3, 142.6, 143.4, 146.6 (C6H5, thiophene, thiazole C), 179.3 (CO), 181.3 (C=S). Anal. Calcd for C18H13NOS3: C, 60.81; H, 3.69; N, 3.94; S, 27.06. Found: C, 60.69; H, 3.74; N, 4.12; S, 26.86. MS: m/z 355 (M+, 59%). 2. 1. 5. General Procedure for the Synthesis of the 4-Hydrazono-4,5,6,7-tetrahydro-2H-indazole Derivatives 10a-d Either hydrazine hydrate (0.1 mL, 0.02 mol) or phe-nylhydrazine (2.16 g, 0.02 mol) was added to a solution of either of compound 3a (1.90 g, 0.01 mol) or 3b (2.06 g, 0.01 mol) in 1,4-dioxane (40 mL). The reaction mixture was heated under reflux for 3 h then poured onto ice/water mixture containing a few drops of hydrochloric acid and the formed solid product was collected by filtration. 3-(Furan-2-yl)-4-hydrazono-4,5,6,7-tetrahydro-2H-indazole (10a) Yellow crystals from ethanol, yield 1.55 g (72%), m.p. 158-161 °C. IR (KBr) vmax (cm-1) 3472-3328 (NH, NH2), 3055 (CH, aromatic), 1658 (exocyclic C=N), 1630 (C=C); 1H NMR (DMSO-d6, 300 MHz) 8 1.32-1.64 (m, 2H, CH2), 2.60-2.78 (m, 4H, 2CH2), 4.86 (s, 2H, D2O exchangeable, NH2), 7.14-7.84 (m, 3H, furan H), 8.28 (s, 1H, D2O exchangeable, NH); 13C NMR (DMSO-d6, 75 MHz) 8 16.5, 36.3, 39.7 (3CH2), 112.4, 158.6 (C=CH), 136.4, 138.0, 139.2, 140.3, 143.8, 146.2 (furan, pyrazole C), 168.3, 172.8 (2C=N). Anal. Calcd for C11H12N4O: C, 61.10; H, 5.59; N, 25.91. Found: C, 60.93; H, 5.29; N, 25.77. MS: m/z 216 (M+, 36%). 3-(Furan-2-yl)-2-phenyl-4-(2-phenylhydrazono)-4,5,6,7-tetrahydro-2H-indazole (10b) Pale yellow crystals from 1,4-dioxane, yield 2.50 g (68%), m.p. 177-180 °C. IR (KBr) vmax (cm-1) 3463-3341 (NH), 3055 (CH, aromatic), 1652 (exocyclic C=N), 1630 (C=C); 1H NMR (DMSO-d6, 300 MHz) 8 1.31-1.67 (m, 2H, CH2), 2.63-2.77 (m, 4H, 2CH2), 7.26-7.89 (m, 13H, 2C6H5, furan H), 8.30 (s, 1H, D2O exchangeable, NH); 13C NMR (DMSO-d6, 75 MHz) 8 16.2, 36.1, 39.4 (3CH2), 120.2, 121.6, 123.2, 123.8, 124.0, 126.3, 128.3, 1290.1, 136.4, 140.3, 140.8, 141.2, 143.4, 146.8 (2C6H5, pyrazole, furan C), 176.2, 178.3 (2C=N). Anal. Calcd for C23H20N4O: C, 74.98; H, 5.47; N, 15.21. Found: C, 75.19; H, 5.31; N, 15.02. MS: m/z 368 (M+, 41%). 4-Hydrazono-3-(thiophen-2-yl)-4,5,6,7-tetrahydro-2H-indazole (10c) Pale yellow crystals from 1,4-dioxane, yield 1.76 g (76%), m.p. 233-236 °C. IR (KBr) vmax (cm-1) 3458-3342 (NH, NH2), 3055 (CH, aromatic), 1656 (exocyclic C=N), 1630 (C=C); 1H NMR (DMSO-d6, 300 MHz) 8 1.33-1.67 (m, 2H, CH2), 2.62-2.75 (m, 4H, 2CH2), 4.80 (s, 2H, D2O exchangeable, NH2), 6.96-7.88 (m, 3H, thiophene H), 8.28 (s, 1H, D2O exchangeable, NH); 13C NMR (DMSO-d6, 75 MHz) 8 16.5, 36.3, 39.7 (3CH2), 112.4, 158.6 (C=CH), 136.6, 138.4, 139.2, 140.1, 143.5, 146.4 (thiphene, pyrazole C), 176.4, 178.6 (2C=N). Anal. Calcd for C11H12N4S: C, 56.87; H, 5.21; N, 24.12; S, 13.80. Found: C, 56.93; H, 5.30; N, 24.31; S, 13.98. MS: m/z 232 (M+, 28%). 2-Phenyl-4-(2-phenylhydrazono)-3-(thiophen-2-yl)-4,5,6,7-tetrahydro-2H-indazole (10d) Orange crystals from ethanol, yield 2.61 g (68%), m.p. 180-184 °C. IR (KBr) vmax (cm-1) 3484-3326 (Nh), 3055 (CH, aromatic), 1655 (exocyclic C=N), 1630 (C=C); 1H NMR (DMSO-d6, 300 MHz) 8 1.32-1.69 (m, 2H, CH2), 2.60-2.78 (m, 4H, 2CH2), 7.23-7.85 (m, 13H, 2C6H5, thiophene H), 8.29 (s, 1H, D2O exchangeable, NH); 13C NMR (DMSO-d6, 75 MHz) 8 16.4, 36.6, 39.4 (3CH2), 120.8, 122.2, 123.6, 123.9, 125.8, 126.6, 128.1, 129.7, 135.8, 140.6, 142.6, 142.9, 143.1, 146.5 (2C6H5, pyrazole, thiophene C), 176.4, 178.6 (2C=N). Anal. Calcd for C23H20N4S: C, 71.85; H, 5.24; N, 14.57; S, 8.34. Found: C, 72.13; H, 5.42; N, 14.38; S, 8.62. MS: m/z 384 (M+, 26%). 2. 1. 6. General Procedure for the Synthesis of the 6,7-Dihydrobenzo[c]isoxazol-4(5H)-one Oxime Derivatives 12a,b Hydroxylamine hydrochloride (1.40 g, 0.02 mol) was added to a solution of either compound 3a (1.90 g, 0.01 mol) or 3b (2.06 g, 0.01 mol) in 1,4-dioxane (40 mL) containing sodium acetate (2.0 g). The reaction mixture was heated under reflux for 3 h then poured onto ice/water mixture and the formed solid product, in each case, was collected by filtration. 3-(Furan-2-yl)-6,7-dihydrobenzo[c]isoxazol-4(5H)-one Oxime (12a) Yellow crystals from 1,4-dioxane, yield 1.52 g (70%), m.p. 177-180 °C. IR (KBr) vmax (cm-1) 3524-3376 (OH), 3055 (CH, aromatic), 1655 (exocyclic C=N), 1630 (C=C); 1H NMR (DMSO-d6, 300 MHz) 8 1.31-1.68 (m, 2H, CH2), 2.63-2.77 (m, 4H, 2CH2), 6.93-7.81 (m, 3H, furan h), 10.29 (s, 1H, D2O exchangeable, OH); 13C NMR (DM-SO-d6, 75 MHz) 8 16.2, 36.3, 39.2 (3CH2), 132.3, 135.1, 140.6, 141.5, 142.8, 143.6 (isoxazole, furan C), 176.1, 178.4 (2C=N). Anal. Calcd for C11H10N2O3: C, 60.55; H, 4.62; N, 12.84. Found: C, 60.39; H, 4.80; N, 12.93. MS: m/z 218 (M+, 32%). 3-(Thiophen-2-yl)-6,7-dihydrobenzo[c]isoxazol-4(5H)-one Oxime (12b) Pale yellow crystals from 1,4-dioxane, yield 1.52 g (65%), m.p. 222-225 °C. IR (KBr) vmax (cm-1) 3551-3349 (OH), 3055 (CH, aromatic), 1653 (exocyclic C=N), 1630 (C=C); 1H NMR (DMSO-d6, 300 MHz) 8 1.32-1.65 (m, 2H, CH2), 2.61-2.74 (m, 4H, 2CH2), 7.22-7.80 (m, 3H, Mohareb et al.: New Approaches for the Synthesis of Heterocyclic ... 76 Acta Chim. Slov. 2021, 68, 72-87 76 thiophene H), 10.29 (s, 1H, D2O exchangeable, OH); 13C NMR (DMSO-d6, 75 MHz) 5 16.4, 36.6, 39.8 (3CH2), 132.1, 134.3, 138.8, 140.9, 141.6, 144.2 (isoxazole, thiophene C), 176.0, 178.6 (2C=N). Anal. Calcd for C11H10N2O2S: C, 56.39; H, 4.30; N, 11.96; S, 13.69. Found: C, 56.42; H, 4.49; N, 12.05; S, 13.83 MS: m/z 234 (M+, 42%). 2. 1. 7. 2-(Ethoxymethylene)cyclohexane-1,3-dione (14) Ethyl orthoformate (1.68 g, 0.01 mol) was added to a solution of cyclohexan-1,3-dione (1.12 g, 0.01 mol) in acetic acid (40 mL). The reaction mixture was heated under reflux for 2 h then evaporated in vacuum and the remaining product was triturated with ethanol and the formed solid product was collected by filtration. Yellow crystals from ethanol, yield 1.27 g (76%), m.p. 282-258 °C. IR (KBr) vmax (cm-1) 2980 (CH2), 1689, 1686 (CO), 1632 (C=C); 1H NMR (DMSO-d6, 300 MHz) 5 1.28 (t, 3H, J = 6.83 Hz, CH3), 1.49-1.67 (m, 2H, CH2), 2.65-2.73 (m, 4H, 2CH2), 3.89 (q, 2H, J = 6.83 Hz, CH2), 6.79 (s, 1H, CH); 13C NMR (DMSO-d6, 75 MHz) 5 16.3 (OCH2CH3), 16.2, 36.8, 39.0 (3CH2), 62.8 (OCH2CH3), 112.3, 158.2 (C=CH), 177.3, 179.2 (2CO). Analysis Calcd for C9H12O3 (168.19): C, 64.27; H, 7.19%. Found: C, 64.08; H, 7.33%. MS: m/z 168 (M+, 22%). 2. 1. 8. General Procedure for the Synthesis of the 2-(Aminomethylene)cyclohexane-1,3-dione Derivatives 16a-c Equimolar amounts of aniline (0.93 g, 0.01 mol), 4-methylaniline (1.08 g, 0.01 mol) or 4-methoxyaniline (1.34 g, 0.01 mol) and compound 14 (1.68 g, 0.01 mol) in I,4-dioxane (50 mL) were refluxed for 4 h. The reaction mixture was evaporated under vacuum and the remaining product was triturated with ethanol and the solid product formed, in each case, was collected by filtration. 2-((Phenylamino)methylene)cyclohexane-1,3-dione (16a) Yellow crystals from ethanol, yield 1.24 g (58%), m.p. 165-167 °C. IR (KBr) vmax (cm-1) 3480-3363 (Nh), 3055 (CH aromatic), 2980 (CH2), 1688, 1686 (2CO), 1632 (C=C); 1H NMR (DMSO-d6, 300 MHz) 5 1.42-1.69 (m, 2H, CH2), 2.63-2.75 (m, 4H, 2CH2), 6.05 (s, 1H, CH), 7.28-7.39 (s, 5H, C6H5), 8.28 (s, 1H, D2O exchangeable, NH); 13C NMR (DMSO-d6, 75 MHz) 5 16.5, 36.8, 39.4 (3CH2), 94.3 (CH), 120.3, 123.1, 124.8, 126.4 (C6H5), 177.1, 179.8 (2CO). Anal. Calcd for C13H13NO2 (215.25): C, 72.54; H, 6.09; N, 6.51%. Found: C, 72.31; H, 6.29; N, 6.38%.%. MS: m/z 215 (M+, 35%). 2-((Phenylamino)methylene)cyclohexane-1,3-dione (16b) Yellow crystals from ethanol, yield 1.67 g (73%), m.p. 214-217 °C. IR (KBr) vmax (cm-1) 3459-3341 (NH), 3054 (CH aromatic), 2982, 2886 (CH3, CH2), 1688, 1686 (2CO), 1631 (C=C); 1H NMR (DMSO-d6, 300 MHz) 5 1.43-1.67 (m, 2H, CH2), 2.61-2.76 (m, 4H, 2CH2), 2.88 (s, 3H, CH3), 6.08 (s, 1H, CH), 7.24-7.45 (s, 4H, C6H4), 8.26 (s, 1H, D2O exchangeable, NH); 13C NMR (DMSO-d6, 75 MHz) 5 16.3, 36.6, 39.5 (3CH2), 23.8 (CH3), 90.6 (CH), 120.8, 122.9, 123.4, 127.9 (C6H4), 177.3, 179.5 (2CO). Analysis Calcd for C14H15NO2 (229.27): C, 73.34; H, 6.59; N, 6.11%. Found: C, 73.29; H, 6.41; N, 6.26%. MS: m/z 229 (M+, 40%). 2-(((4-Methoxyphenyl)amino)methylene)cyclohexane-1,3-dione (16c) Yellow crystals from 1,4-dioxane, yield 1.47 g (60%), m.p. 193-196 °C. IR (KBr) vmax (cm-1) 3463-3329 (NH), 3055 (CH aromatic), 2982, 2886 (CH3, CH2), 1689, 1686 (2CO), 1630 (C=C); 1H NMR (DMSO-d6, 300 MHz) 5 1.41-1.69 (m, 2H, CH2), 2.61-2.78 (m, 4H, 2CH2), 3.68 (s, 3H, OCH3), 6.06 (s, 1H, CH), 7.27-7.48 (s, 4H, C6H4), 8.28 (s, 1H, D2O exchangeable, NH); 13C NMR (DMSO-d6, 75 MHz) 5 16.3, 36.6, 39.5 (3CH2), 50.3 (OCH3), 90.2 (CH), 120.3, 122.4, 125.6, 128.7 (C6H4), 177.1, 179.3 (2co). Analysis Calcd for C14H15NO3 (245.27): C, 68.56; H, 6.16; N, 5.71%. Found: C, 68.80; H, 6.24; N, 5.93%. MS: m/z 245 (M+, 38%). 2. 1. 9. General Procedure for the Synthesis of the 6,7-Dihydrobenzo[fo]thiophen-5(4H)-one Derivatives 17a-f Either malononitrile (0.66 g, 0.01 mol) or ethyl cy-anoacetate (1.07 g, 0.01 mol) was added to a solution of either compound 16a (2.15 g, 0.01 mol), 16b (2.29 g, 0.01 mol) or 16c (2.45 g, 0.01 mol) in ethanol (40 mL) containing triethylamine (0.50 mL). The reaction mixture was heated under reflux for 3 h then poured onto ice/water mixture containing a few drops of hydrochloric acid and the formed solid product was collected by filtration. 2-Amino-5-oxo-4-((phenylamino)methylene)-4,5,6,7-tetrahydrobenzo[fo]thiophene-3-carbonitrile (17a) Pale yellow crystals from ethanol, yield 2.06 g (70%), m.p. 127-129 °C. IR (KBr) vmax (cm-1) 3472-3353 (NH2, NH), 3055 (CH, aromatic), 2220 (Cn), 1686 (CO), 1630 (C=C); 1H NMR (DMSO-d6, 300 MHz) 5 2.60-2.79 (2t, 4H, 2CH2), 4.78 (s, 2H, D2O exchangeable, NH2), 6.89 (s, 1H, CH), 7.26-7.42 (m, 5H, C6H5), 8.39 (s, 1H, D2O exchangeable, NH); 13C NMR (DMSO-d6, 75 MHz) 5 36.0, 39.8 (2CH2), 112.2, 158.4 (C=CH), 116.8 (CN), 121.6, 122.4, 124.8, 127.2, 132.7, 134.2, 138.0, 142.6 (C6H5, thiophene C), 179.6 (CO). Anal. Calcd for C16H13N3OS: C, 65.06; H, 4.44; N, 14.23; S, 10.86. Found: C, 65.18; H, 4.60; N, 14.19; S, 11.17. MS: m/z 295 (M+, 38%). Ethyl 2-Amino-5-oxo-4-((phenylamino)methylene)-4,5,6,7-tetrahydrobenzo[fo]thiophene-3-carboxylate (17b) Yellow crystals from ethanol, yield 2.18 g (64%), m.p. 96-98 °C. IR (KBr) vmax (cm-1) 3486-3351 (NH2), 3058 Mohareb et al.: New Approaches for the Synthesis of Heterocyclic ... Acta Chim. Slov. 2021, 68, 72-87 77 (CH, aromatic), 2930, 2972 (CH2, CH3), 1689, 1686 (2 CO), 1630 (C=C); 1H NMR (DMSO-d6, 300 MHz) 5 1.13 (t, 3H, J = 7.08 Hz, CH3), 2.68-2.76 (2t, 4H, 2CH2), 4.22 (q, 2H, J = 7.08 Hz, CH2), 4.77 (s, 2H, D2O exchangeable, NH2), 6.86 (s, 1H, CH), 7.22-7.40 (m, 5H, C6H5), 8.33 (s, 1H, D2O exchangeable, NH); 13C NMR (DMSO-d6, 75 MHz) 5 16.3 (OCH2CH3), 36.8, 39.6 (2CH2), 52.5 (OCH2CH3), 112.4, 158.8 (C=CH), 121.3, 123.6, 125.2, 126.8, 132.9, 133.3, 138.0, 142.7 (C6H5, thiohene C), 164.2, 179.8 (2CO). Anal. Calcd for C18H18N2O3S: C, 63.14; H, 5.30; N, 8.18; S, 9.36. Found: C, 62.91; H, 5.49; N, 8.25; S, 9.60. MS: m/z 342 (M+, 27%). 2-Amino-5-oxo-4-((p-tolylamino)methylene)-4,5,6,7-tetrahydrobenzo-[c]thiophene-3-carbonitrile (17c) Orange crystals from 1,4-dioxane, yield 2.31 g (75%), m.p. 177-179 °C. IR (KBr) vmax (cm-1) 3493-3323 (NH2, NH), 3055 (CH, aromatic), 2220 (Cn), 1688 (CO), 1630 (C=C); 1H NMR (DMSO-d6, 300 MHz) 5 2.62-2.77 (2t, 4H, 2CH2), 2.79 (s, 3H, CH3), 4.77 (s, 2H, D2O exchangeable, NH2), 6.82 (s, 1H, CH), 7.28-7.46 (m, 4H, C6H4), 8.37 (s, 1H, D2O exchangeable, NH); 13C NMR (DMSO-d6, 75 MHz) 5 36.0, 39.6 (2CH2), 24.3 (CH3), 112.0, 158.6 (C=CH), 116.9 (CN), 120.3, 123.7, 124.8, 128.9, 132.9, 134.7, 138.3, 142.2 (C6H4, thiophene C), 179.3 (CO). Anal. Calcd for C17H15N3OS: C, 66.00; H, 4.89; N, 13.58; S, 10.36. Found: C, 65.85; H, 4.91; N, 13.70; S, 10.42. MS: m/z 309 (M+, 24%). Ethyl 2-Amino-5-oxo-4-((p-tolylamino)methylene)-4,5,6,7-tetrahydrobenzo[fo]thiophene-3-carboxylate (17d) Brown crystals from 1,4-dioxane, yield 2.45 g (69%), m.p. 203-206 °C. IR (KBr) vmax (cm-1) 3459-3337 (NH2), 3055 (CH, aromatic), 2930, 2970 (CH2, CH3), 1689, 1687 (2 CO), 1630 (C=C); 1H NMR (DMSO-d6, 300 MHz) 5 1.12 (t, 3H, J = 6.59 Hz, CH3), 2.65-2.78 (2t, 4H, 2CH2), 2.80 (s, 3H, CH3), 4.24 (q, 2H, J = 6.59 Hz, CH2), 4.79 (s, 2H, D2O exchangeable, NH2), 6.83 (s, 1H, CH), 7.21-7.45 (m, 4H, C6H4), 8.36 (s, 1H, D2O exchangeable, NH); 13C NMR (DMSO-d6, 75 MHz) 5 16.1 (OCH2CH3), 36.3, 39.7 (3CH2), 23.9 (CH3), 52.1 (OCH2CH3), 112.2, 158.7 (C=CH), 120.1, 123.8, 124.9, 128.3, 134.8, 137.2, 140.2, 142.6 (C6H5, thiophene C), 164.3, 179.5 (2CO). Anal. Cal-cd for C19H20N2O3S: C, 64.02; H, 5.66; N, 7.86; S, 9.00. Found: C, 63.91; H, 5.53; N, 8.01; S, 9.26. MS: m/z 356 (M+, 32%). 2-Amino-4-(((4-methoxyphenyl)amino)methylene)-5-oxo-4,5,6,7-tetrahydrobenzo[fo]thiophene-3-carbonitrile (17e) Yellow crystals from ethanol, yield 2.30 g (71%), m.p. 166-169 °C. IR (KBr) vmax (cm-1) 3474-3329 (NH2, NH), 3055 (CH, aromatic), 2220 (CN), 1688 (CO), 1630 (C=C); 1H NMR (DMSO-d6, 300 MHz) 5 2.61-2.77 (2t, 4H, 2CH2), 3.68 (s, 3H, OCH3), 4.75 (s, 2H, D2O exchangeable, NH2), 6.87 (s, 1H, CH), 7.24-7.46 (m, 4H, C6H4), 8.35 (s, 1H, D2O exchangeable, NH); 13C NMR (DMSO-d6, 75 MHz) 5 16.1, 36.2, 39.4 (3CH2), 50.2 (OCH3), 112.5, 158.4 (C=CH), 116.7 (CN), 120.1, 122.9, 125.3, 127.6, 132.2, 134.7, 137.6, 143.3 (C6H4, thiophene C), 179.6 (CO). Anal. Calcd for C17H15N3O2S: C, 62.75; H, 4.65; N, 12.91; S, 9.85. Found: C, 62.53; H, 4.79; N, 12.83; S, 10.01. MS: m/z 325 (M+, 32%). Ethyl 2-Amino-4-(((4-methoxyphenyl)amino) methylene)-5-oxo-4,5,6,7-tetrahydrobenzo[fo]-thiophene-3-carboxylate (17f) Yellow crystals from ethanol, yield 2.40 g (65%), m.p. 203-206 °C. IR (KBr) vmax (cm-1) 3459-3337 (NH2), 3055 (CH, aromatic), 2930, 2970 (CH2, CH3), 1689, 1687 (2 CO), 1630 (C=C); 1H NMR (DMSO-d6, 300 MHz) 5 1.13 (t, 3H, J = 7.59 Hz, CH3), 2.62-2.75 (2t, 4H, 2CH2), 3.61 (s, 3H, OCH3), 4.22 (q, 2H, J = 7.59 Hz, CH2), 4.79 (s, 2H, D2O exchangeable, NH2), 6.83 (s, 1H, CH), 7.247.48 (m, 4H, C6H4), 8.34 (s, 1H, D2O exchangeable, NH); 13C NMR (DMSO-d6, 75 MHz) 5 16.1 (OCH2CH3), 36.3, 39.7 (3CH2), 50.2 (OCH3), 52.2 (OCH2CH3), 112.2, 158.7 (C=CH), 120.2, 122.2, 124.3, 128.1, 134.6, 136.7, 138.1, 140.8 (C6H5, thiophene C), 164.1, 179.7 (2CO). Anal. Calcd for C19H20N2O4S: C, 61.27; H, 5.41; N, 7.52; S, 8.61. Found: C, 60.91; H, 5.23; N, 7.39; S, 8.59. MS: m/z 372 (M+, 36%). 2. 1. 10. General Procedure for the Synthesis of the 6,7-Dihydrobenzo[fc]thiophen-3-carboxamide Derivatives 19a-c Either of compounds 18a (1.12 g, 0.01 mol), 18b (1.74 g, 0.01 mol) or 18c (1.94 g, 0.01 mol) was added to a solution of cyclohexane-1,3-dione (1) (1.12 g, 0.01 mol) in 1,4-dioxane (40 mL) containing triethylamine (0.50 mL). The reaction mixture was heated under reflux for 3 h then poured onto ice/water mixture containing a few drops of hydrochloric acid and the formed solid product was collected by filtration. 2-Amino-5-oxo-AT-phenyl-4,5,6,7-tetrahydrobenzo[fo] thiophene-3-carboxamide (19a) Pale yellow crystals from ethanol, yield 2.14 g (75%), m.p. 120-123 °C. IR (KBr) vmax (cm-1) 3484-3337 (NH2, NH), 3055 (CH, aromatic), 2220 (CN), 1689, 1686 (2 CO), 1630 (C=C); 1H NMR (DMSO-d6, 300 MHz) 5 2.62-2.79 (2t, 4H, 2CH2), 3.02 (s, 2H, CH2), 4.75 (s, 2H, D2O exchangeable, NH2), 7.28-7.40 (m, 5H, C6H5), 8.38 (s, 1H, D2O exchangeable, NH); 13C NMR (DMSO-d6, 75 MHz) 5 16.3, 36.6, 40.3 (3CH2), 116.6 (CN), 120.8, 122.2, 125.3, 127.3, 132.2, 134.5, 138.0, 142.9 (C6H5, thiophene C), 164.3, 179.6 (2CO). Anal. Calcd for C15H14N2O2S: C, 62.92; H, 4.93; N, 9.78; S, 11.20. Found: C, 63.22; H, 4.75; N, 9.59; S, 11.36. MS: m/z 286 (M+, 42%). 2-Amino-5-oxo-AT-(p-tolyl)-4,5,6,7-tetrahydrobenzo[fo] thiophene-3-carboxamide (19b) Mohareb et al.: New Approaches for the Synthesis of Heterocyclic ... 78 Acta Chim. Slov. 2021, 68, 72-87 78 Yellow crystals from ethanol, yield 1.98 g (66%), m.p. 194-196 °C. IR (KBr) vmax (cm-1) 3449-3327 (NH2, NH), 3055 (CH, aromatic), 2220 (CN), 1689, 1687 (2 CO), 1630 (C=C); 1H NMR (DMSO-d6, 300 MHz) 6 2.63-2.77 (2t, 4H, 2CH2), 2.80 (s, 3H, CH3), 3.06 (s, 2H, CH2), 4.78 (s, 2H, D2O exchangeable, NH2), 7.26-7.46 (m, 4H, C6H4), 8.38 (s, 1H, D2O exchangeable, NH); 13C NMR (DMSO-d6, 75 MHz) 6 16.3, 36.6, 40.3 (3CH2), 116.6 (CN), 120.8, 122.2, 125.3, 127.3, 132.7, 134.7, 138.4, 142.6 (C6H4, thiophene C), 164.3, 179.6 (2CO). Anal. Calcd for C16H16N2O2S: C, 63.98; H, 5.37; N, 9.33; S, 10.67. Found: C, 63.72; H, 5.75; N, 9.59; S, 10.36. MS: m/z 300 (M+, 36%). 2-Amino-AT-(4-chlorophenyl)-5-oxo-4,5,6,7-tetrahydrobenzo[fo]thiophene-3-carboxamide (19c) Brown crystals from 1,4-dioxane, yield 2.49 g (78%), m.p. 205-208 °C. IR (KBr) vmax (cm-1) 34733351 (NH2, NH), 3055 (CH, aromatic), 2220 (CN), 1689, 1686 (2 CO), 1630 (C=C); 1H NMR (DMSO-d6, 300 MHz) 6 2.64-2.76 (2t, 4H, 2CH2), 3.06 (s, 2H, CH2), 4.79 (s, 2H, D2O exchangeable, NH2), 7.23-7.48 (m, 4H, C6H4), 8.36 (s, 1H, D2O exchangeable, NH); 13C NMR (DMSO-d6, 75 MHz) 6 16.1, 36.4, 40.6 (3CH2), 116.9 (CN), 120.2, 123.5, 125.8, 128.6, 132.3, 134.7, 138.0, 142.7 (C6H4, thiophene C), 164.3, 179.8 (2CO). Anal. Calcd for C15H13ClN2O2S: C, 56.16; H, 4.08; N, 8.73; S, 10.00. Found: C, 56.29; H, 4.26; N, 8.93; S, 10.26. MS: m/z 320 (M+, 28%). 2. 1. 11. General Procedure for the Synthesis of the Tetrahydrobenzo[fc]thiophene-3-carboxamide Derivatives 20a-c A mixture of either 19a (2.86 g, 0.01 mol), 19b (3.00 g, 0.01 mol) or 19c (3.20 g, 0.01 mol) in N,N-dimethylfor-mamide (30 mL) and ethyl cyanoacetate (1.07 g, 0.01 mL) was heated under reflux for 3 h. The solid product, formed in each case, produced upon pouring onto ice/water mixture, was collected by filtration. 2-(2-Cyanoacetamido)-5-oxo-AT-phenyl-4,5,6,7-tetrahydrobenzo[fo]thiophene-3-carboxamide (20a) Dark brown crystals from ethanol, yield 3.28 g (78%), m.p 118-120 °C. IR (KBr) vmax (cm-1) 3469-3329 (NH), 3054 (CH, aromatic), 2256 (CN), 1690-1686 (3 CO), 1630 (C=C); 1H NMR (DMSO-d6, 300 MHz) 6 2.61-2.82 (2t, 4H, 2CH2), 3.08 (s, 2H, CH2), 3.68 (s, 2H, CH2), 7.29-7.42 (m, 5H, C6H5), 8.24, 8.34 (2s, 2H, D2O exchangeable, 2NH); 13C NMR (DMSO-d6, 75 MHz) 6 16.6, 36.2, 40.5 (3CH2), 60.3 (CH2), 170.1 (CN), 120.3, 123.5, 125.9, 126.5, 132.8, 133.3, 138.6, 142.8 (C6H5, thiophene C), 164.3, 166.1, 179.6 (3CO). Anal. Calcd for C18H15N3O3S: C, 61.18; H, 4.28; N, 11.89; S, 9.07. Found: C, 61.31; H, 4.29; N, 11.68; S, 8.85. MS: m/z 353 (M+, 20%). 2-(2-Cyanoacetamido)-5-oxo-N-phenyl-4,5,6,7-tetrahydrobenzo[fo]thiophene-3-carboxamide (20b) Brown crystals from ethanol, yield 2.27 g (62%), m.p 211-214 °C. IR (KBr) vmax (cm-1) 3453-3340 (Nh), 3055 (CH, aromatic), 2256 (CN), 1693-1685 (3 CO), 1630 (C=C); 1H NMR (DMSO-d6, 300 MHz) 6 2.63-2.80 (2t, 4H, 2CH2), 2.70 (s, 3H, CH3), 3.10 (s, 2H, CH2), 3.62 (s, 2H, CH2), 7.25-7.38 (m, 4H, C6H4), 8.23, 8.38 (2s, 2H, D2O exchangeable, 2NH); 13C NMR (DMSO-d6, 75 MHz) 6 16.3, 36.4, 40.8 (3CH2), 60.5 (CH2), 168.3 (CN), 120.6, 122.1, 126.4, 127.1, 132.8, 134.2, 138.6, 142.9 (C6H4, thiophene C), 164.2, 166.1, 179.8 (3CO). Anal. Calcd for C19H17N3O3S: C, 62.11; H, 4.66; N, 11.44; S, 8.73. Found: C, 62.26; H, 4.48; N, 11.52; S, 8.90. MS: m/z 367 (M+, 28%). AT-(4-Chlorophenyl)-2-(2-cyanoacetamido)-5-oxo- 4,5,6,7-tetrahydrobenzo[fo]thiophene-3-carboxamide (20c) White crystals from ethanol, yield 2.12 g (55%), m.p 118-121 °C. IR (KBr) vmax (cm-1) 3470-3338 (Nh), 3055 (CH, aromatic), 2256 (CN), 1691-1685 (3 CO), 1630 (C=C); 1H NMR (DMSO-d6, 300 MHz) 6 2.62-2.82 (2t, 4H, 2CH2), 3.12 (s, 2H, CH2), 3.62 (s, 2H, CH2), 7.22-7.49 (m, 4H, C6H4), 8.26, 8.32 (2s, 2H, D2O exchangeable, 2NH); 13C NMR (DMSO-d6, 75 MHz) 6 16.1, 36.6, 40.4 (3CH2), 60.2 (CH2), 168.9 (CN), 120.4, 124.2, 125.1, 128.6, 132.5, 134.9, 138.4, 142.6 (C6H4, thiophene C), 164.4, 166.3, 179.6 (3CO). Anal. Calcd for C18H14ClN3O3S: C, 55.74; H, 3.64; N, 10.83; S, 8.27. Found: C, 55.80; H, 3.80; N, 11.01; S, 8.38. MS: m/z 387 (M+, 30%). 2. 1. 12. General Procedure for the Synthesis of the Phenylthioureido Derivatives 21a-c Equimolar amounts of either 19a (2.86 g, 0.01 mol), 19b (3.00 g, 0.01 mol) or 19c (3.20 g, 0.01 mol) and phenyl isothiocyanate (1.30 g, 0.01 mol) in ethanol (40 mL) containing triethylamine (1.0 mL) were heated under reflux for 3 h then left to cool. The formed solid crystals, in each case, were collected by filtration. 5-Oxo-AT-phenyl-2-(3-phenylthioureido)-4,5,6,7-tetrahydrobenzo[fo]thiophene-3-carboxamide (21a) Yellow crystals from ethanol, yield 2.52 g (60%), m.p. 115-117 °C. IR (KBr) vmax (cm-1) 3464-3331 (Nh), 3054 (CH, aromatic), 1689, 1687 (2 CO), 1632 (C=C), 1205 (C=S); 1H NMR (DMSO-d6, 300 MHz) 6 2.632.84 (2t, 4H, 2CH2), 3.66 (s, 2H, CH2), 7.28-7.40 (m, 10H, 2C6H5), 8.21, 8.23, 8.38 (3s, 3H, D2O exchangeable, 3NH); 13C NMR (DMSO-d6, 75 MHz) 6 16.1, 36.6, 40.6 (3CH2), 120.8, 122.4, 125.8, 126.0, 126.3, 127.5, 127.9, 128.2, 131.9, 133.8, 139.3, 142.9 (2C6H5, thiophene C), 164.2, 179.2 (2CO),180.2 (C=S). Anal. Calcd for C22H19N3O2S2: C, 62.68; H, 4.54; N, 9.97; S, 15.21. Found: C, 62.77; H, 4.49; N, 10.21; S, 15.49. MS: m/z 421 (M+, 32%). Mohareb et al.: New Approaches for the Synthesis of Heterocyclic ... Acta Chim. Slov. 2021, 68, 72-87 79 5-Oxo-2-(3-phenylthioureido)-N-(p-tolyl)-4,5,6,7-tetrahydrobenzo[fo]thiophene-3-carboxamide (21b) Pale yellow crystals from 1,4-dioxane, yield 2.70 g (60%), m.p. 205-208 °C. IR (KBr) vmax (cm-1) 3485-3329 (NH), 3055 (CH, aromatic), 1689, 1687 (2 CO), 1638 (C=C), 1208 (C=S); 1H NMR (DMSO-d6, 300 MHz) 5 2.61-2.89 (2t, 4H, 2CH2), 2.80 (s, 3H, CH3), 3.67 (s, 2H, CH2), 7.23-7.48 (m, 9H, C6H5, C6H4), 8.26, 8.21, 8.36 (3s, 3H, D2O exchangeable, 3NH); 13C NMR (DMSO-d6, 75 MHz) 5 16.5, 36.6, 40.6 (3CH2), 30.6 (CH3), 120.2, 121.6, 124.5, 126.7, 126.8, 128.1, 128.7, 129.3, 133.5, 135.2, 138.6, 142.6 (C6H5, C6H4, thiophene C), 164.6, 179.4 (2CO), 180.6 (C=S). Anal. Calcd for C23H21N3O2S2: C, 63.42; H, 4.86; N, 9.65; S, 14.72. Found: C, 63.52; H, 4.68; N, 9.74; S, 14.80. MS: m/z 435 (M+, 28%). AT-(4-Chlorophenyl)-5-oxo-2-(3-phenylthioureido)- 4,5,6,7-tetrahydrobenzo[fo]thiophene-3-carboxamide (21c) Yellow crystals from ethanol, yield 2.95 g (65%), m.p. 200-203 °C. IR (KBr) vmax (cm-1) 3442-3338 (NH), 3054 (CH, aromatic), 1689, 1685 (2 CO), 1630 (C=C), 1208 (C=S); 1H NMR (DMSO-d6, 300 MHz) 5 2.62-2.86 (2t, 4H, 2CH2), 3.68 (s, 2H, CH2), 7.22-7.48 (m, 9H, C6H5, C6H4), 8.22, 8.25, 8.41 (3s, 3H, D2O exchangeable, 3NH); 13C NMR (DMSO-d6, 75 MHz) 5 16.0, 36.6, 40.9 (3CH2), 120.3, 124.6, 125.5, 126.6, 127.2, 128.3, 128.7, 128.9, 132.5, 134.7, 138.0, 142.6 (C6H5, C6H4, thiophene C), 164.5, 179.8 (2CO), 180.5 (C=S). Anal. Calcd for C22H18ClN3O2S2: C, 57.95; H, 3.98; N,9.22; S, 14.06. Found: C, 57.63; H, 3.77; N, 9.39; S, 14.26. MS: m/z 455 (M+, 28%). 2. 1. 13. General Procedure for the Synthesis of the Hexahydrobenzo[4,5]thieno[2,3-d] pyrimidin-6(5H)-one Derivatives 22a-c A suspension of either compound 21a (4.21 g, 0.01 mol), 21b (4.35 g, 0.01 mol), 21c (4.55 g, 0.01 mol) in sodium ethoxide [prepared through dissolving metallic sodium (0.46 g, 0.02 mol) in absolute ethanol (50 mL)] was heated in a boiling water bath for 6 h. The reaction mixture was poured onto ice/water mixture then triturated with hydrochloric acid (till pH 7) and the formed solid product was collected by filtration. 3-Phenyl-4-(phenylimino)-2-thioxo-1,2,3,4,7,8-hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-6(5H)-one (22a) Yellow crystals from ethanol, yield 3.14 g (78%), m.p. 111-113 °C. IR (KBr) vmax (cm-1) 3458-3324 (NH), 3055 (CH, aromatic), 1688 (CO), 1632 (C=C), 1205 (C=S); 1H NMR (DMSO-d6, 300 MHz) 5 2.61-2.86 (2t, 4H, 2CH2), 3.68 (s, 2H, CH2), 7.28-7.40 (m, 10H, 2C6H5), 8.21 (s, 1H, D2O exchangeable, NH); 13C NMR (DMSO-d6, 75 MHz) 5 16.1, 36.6, 40.6 (3CH2), 120.8, 122.4, 125.8, 126.0, 126.3, 127.5, 127.9, 128.2, 132.2, 134.7, 137.2, 142.9 (2C6H5, thiophene C), 179.1 (CO), 180.3 (C=S). Anal. Calcd for C22H17N3OS2: C, 65.48; H, 4.25; N, 10.41; S, 15.89. Found: C, 65.63; H, 4.19; N, 10.32; S, 15.74. MS: m/z 403 (M+, 38%). 3-Phenyl-2-thioxo-4-(p-tolylimino)-1,2,3,4,7,8-hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-6(5H)-one (22b) Brown crystals from ethanol, yield 2.46 g (59%), m.p. 273-275 °C. IR (KBr) vmax (cm-1) 3479-3336 (Nh), 3055 (CH, aromatic), 1688 (CO), 1631 (C=C), 1209 (C=S); 1H NMR (DMSO-d6, 300 MHz) 5 2.60-2.88 (2t, 4H, 2CH2), 2.93 (s, 3H, CH3), 3.65 (s, 2H, CH2), 7.227.45 (m, 9H, C6H5, C6H4), 8.25 (s, 1H, D2O exchangeable, NH); 13C NMR (DMSO-d6, 75 MHz) 5 16.4, 36.8, 40.9 (3CH2), 32.6 (CH3), 120.2, 122.6, 125.5, 125.9, 126.9, 127.7, 127.3, 128.1, 132.2, 134.9, 137.6, 142.3 (C6H5, C6H4, thiophene C), 179.6 (CO), 180.8 (C=S). Anal. Calcd for C23H19N3OS2: C, 66.16; H, 4.59; N, 10.06; S, 15.36. Found: C, 66.39; H, 4.42; N, 10.15; S, 15.80. MS: m/z 417 (M+, 42%). 4-((4-Chlorophenyl)imino)-3-phenyl-2-thioxo-1,2,3,4,7,8-hexahydrobenzo[4,5]thieno[2,3-d] pyrimidin-6(5H)-one (22c) Pale yellow crystals from ethanol, yield 3.49 g (80%), m.p. 119-122 °C. IR (KBr) vmax (cm-1) 3459-3328 (NH), 3054 (CH, aromatic), 1688, 1685 (2CO), 1630 (C=C), 1210 (C=S); 1H NMR (DMSO-d6, 200 MHz) 5 2.62-2.88 (2t, 4H, 2CH2), 3.68 (s, 2H, CH2), 7.22-7.48 (m, 9H, C6H5, C6H4), 8.22 (s, 1H, D2O exchangeable, NH); 13C NMR (DMSO-d6, 75 MHz) 5 16.0, 36.6, 40.9 (3CH2), 120.3, 124.6, 125.5, 126.6, 127.2, 128.3, 128.7, 128.9, 132.8, 134.3, 137.2, 142.9 (C6H5, C6H4, thiophene C), 164.8, 179.4 (2CO), 180.2 (C=S). Anal. Calcd for C22H16ClN3OS2: C, 60.33; H, 3.68; N, 9.59; S, 14.64. Found: C, 60.26; H, 3.49; N, 9.41; S, 14.52. MS: m/z 437 (M+, 32%). 2. 2. Biology Section 2. 2. 1. Cell Proliferation Assay Foretinib was used as the positive control27-29 during measuring the anti-proliferative activities of the newly synthesized compounds (Table 1). The newly synthesized compounds were evaluated against the six cancer cell lines A549, HT-29, MKN-45, U87MG, and SMMC-7721 and H460 using the standard MTT assay in vitro. The IC50 values were measured through three independent experiments and the data are shown in Table 1. It is clear that many of the tested compounds showed potent anti-proliferative activity with IC50 values less than 6.00 ^M. Generally, the variations of substituents within the aryl moiety together with the heterocyclic ring being attached had a notable effect and a positive impact on the anti-proliferative activity. Mohareb et al.: New Approaches for the Synthesis of Heterocyclic ... 80 Acta Chim. Slov. 2021, 68, 72-87 80 Table 1. In vitro growth inhibitory effects IC50 ± SEM (|iM) of the newly synthesized compounds against cancer cell lines Compound IC50 ± SEM (^M) A549 H460 HT29 MKN-45 U87MG SMMC-7721 3a 6.62 ± 2.34 5.42 ± 2.31 7.73 ± 2.59 6.72 ± 2.21 6.82 ± 1.73 5.01 ± 2.32 3b 0.40 ± 0.25 0.39 ± 0.17 0.29 ± 0.16 0.24 ± 0.16 0.19 ± 0.17 0.42 ± 0.23 5a 4.16 ± 1.49 4.18 ± 1.26 5.43 ± 2.71 5.32 ± 1.41 6.53 ± 251 6.80 ± 1.49 5b 1.07 ± 0.68 1.17 ± 0.63 2.04 ± 0.95 2.27 ± 1.33 1.95 ± 0.49 2.17 ± 0.52 5c 0.19 ± 0.07 0.26 ± 0.11 0.37 ± 0.21 0.26 ± 0.08 0.52 ± 0.23 0.62 ± 0.25 5d 0.39 ± 0.15 0.36 ± 0.21 0.27 ± 0.18 0.29 ± 0.17 0.36 ± 0.22 0.35 ± 0.18 6a 6.41 ± 2.26 5.73 ± 2.17 6.42 ± 2.31 8.92 ± 3.41 6.22 ± 2.73 5.82 ± 1.39 6b 1.08 ± 0.69 0.82 ± 0.26 0.63 ± 0.37 0.38 ± 0.26 1.82 ± 0.79 0.63 ± 0.31 6c 0.32 ± 0.21 0.37 ± 0.19 0.40 ± 0.15 0.25 ± 0.07 0.62 ± 0.14 0.51 ± 0.23 6d 0.32 ± 0.15 0.42 ± 0.26 0.16 ± 0.09 0.26 ± 0.14 0.37 ± 0.17 0.28 ± 0.08 8a 0.33 ± 0.12 0.28 ± 0.15 0.28 ± 3.19 6.28 ± 1.08 7.89 ± 2.63 9.39 ± 2.37 8b 0.65 ± 0.18 0.29 ± 0.12 0.43 ± 0.25 0.36 ± 0.19 0.26 ± 0.18 0.56 ± 0.18 10a 6.48 ± 1.54 7.60 ± 2.42 6.63 ± 2.29 6.16 ± 2.59 5.26 ± 1.83 6.29 ± 2.28 10b 2.48 ± 1.01 3.80 ± 1.18 2.47 ± 1.14 4.52 ± 2.16 0.93 ± 0.42 2.83 ± 1.02 10c 0.26 ± 0.18 0.43 ± 0.18 0.41 ± 0.20 0.38 ± 0.16 0.46 ± 0.26 0.38 ± 0.13 10d 0.65 ± 0.32 0.58 ± 0.21 1.08 ± 0.62 0.73 ± 0.32 0.46 ± 0.29 0.53 ± 0.25 12a 1.82 ± 0.78 1.06 ± 0.62 0.84 ± 0.38 0.61 ± 0.19 0.72 ± 0.36 0.59 ± 0.16 12b 0.22 ± 0.15 0.36 ± 0.12 0.43 ± 0.19 0.29 ± 0.16 0.42 ± 0.25 0.58 ± 0.24 14 8.68 ± 2.59 6.70 ± 2.63 7.28 ± 1.62 5.63 ± 1.26 6.92 ± 2.37 7.29 ± 2.62 16a 4.91 ± 1.56 5.41 ± 1.28 3.52 ± 1.15 3.30 ± 1.86 5.02 ± 2.80 4.69 ± 1.38 16b 6.53 ± 2.38 7.24 ± 2.49 6.80 ± 1.92 7.49 ± 2.63 5.60 ± 1.32 8.05 ± 3.26 16c 0.76 ± 0.39 0.68 ± 0.27 0.43 ± 0.26 0.51 ± 0.23 0.60 ± 0.29 0.39 ± 0.24 17a 4.83 ± 1.53 6.28 ± 2.54 6.22 ± 2.26 3.42 ± 1.60 5.23 ± 1.21 4.72 ± 1.38 17b 3.41 ± 1.25 4.61 ± 1.28 4.82 ± 1.52 2.16 ± 0.73 2.37 ± 1.29 3.42 ± 1.53 17c 3.42 ± 1.80 2.63 ± 1.03 4.81 ± 1.68 3.68 ± 1.29 3.62 ± 1.26 2.46 ± 1.82 17d 1.02 ± 0.71 1.65 ± 0.83 0.93 ± 0.42 0.85 ± 0.31 0.72 ± 0.29 0.69 ± 0.23 17e 0.82 ± 0.18 0.51 ± 0.23 0.42 ± 0.19 0.31 ± 0.25 0.39 ± 0.13 0.58 ± 0.13 17f 0.21 ± 0.09 0.33 ± 0.17 0.48 ± 0.31 0.35 ± 0.12 0.59 ± 0.23 0.23 ± 0.17 19a 7.28 ± 2.62 8.29 ± 2.17 6.39 ± 1.02 5.86 ± 2.23 7.43 ± 2.49 6.32 ± 2.34 19b 2.34 ± 1.14 2.28 ± 0.82 3.51 ± 1.50 3.46 ± 1.63 3.68 ± 1.23 2.18 ± 1.02 19c 0.24 ± 0.13 0.32 ± 0.17 0.48 ± 0.23 0.29 ± 0.36 0.26 ± 0.15 0.43 ± 0.29 20a 8.52 ± 3.50 7.62 ± 2.40 6.39 ± 3.60 7.80 ± 2.68 5.18 ± 2.70 6.48 ± 2.37 20b 4.26 ± 1.05 5.72 ± 2.83 6.93 ± 2.40 4.70 ± 1.52 5.94 ± 1.29 1.04 ± 0.89 20c 0.37 ± 0.16 0.35 ± 0.18 0.46 ± 0.261 0.51 ± 0.28 0.24 ± 0.63 0.28 ± 0.18 21a 8.24 ± 3.51 6.39 ± 2.73 4.46 ± 1.28 5.34 ± 1.60 6.70 ± 2.93 5.45 ± 1.69 21b 1.42 ± 0.98 1.64 ± 0.52 0.68 ± 0.31 1.25 ± 0.79 2.35 ± 1.06 3.36 ± 1.20 21c 6.43 ± 2.53 8.69 ± 2.64 7.16 ± 1.69 8.69 ± 2.26 8.53 ± 2.38 8.76 ± 2.49 22a 6.42 ± 2.26 8.25 ± 2.13 5.29 ± 2.30 3.27 ± 1.04 5.52 ± 1.31 4.50 ± 1.46 22b 2.43 ± 1.16 2.37 ± 1.28 3.45 ± 1.19 2.26 ± 1.16 4.58 ± 1.14 3.27 ± 3.16 22c 0.42 ± 0.23 0.29 ± 0.15 0.54 ± 0.29 0.19 ± 0.04 0.59 ± 0.31 0.42 ± 0.63 Foretinib 0.08 ± 0.01 0.18 ± 0.03 0.15 ± 0.023 0.03 ± 0.0055 0.90 ± 0.13 0.44 ± 0.062 2. 2. 2. Structure Activity Relationship It is clear from Table 1 that most of the compounds exhibited high cyctotoxicities against the six cancer cell lines A549, HT-29, MKN-45, U87MG, and SMMC-7721 and H460. Where in the most of the cases the presence of electronegative substituents is responsible for the high inhibitions. Considering the 2-ylidene-cyclohexane-1,3-dione derivatives 3a,b it is clear that compound 3b (X = S) showed higher cytotoxicities than compound 3a (X = O). For the 4,5,6,7-tetrahydroben-zo[fr]thiophene derivatives 5a-d, compound 5a (X = O, R = CN) exhibited relatively low inhibitions while compound 5b (X = O, R = COOEt) showed moderate inhibitions. On the other side, compounds 5c (X = S, R = CN) and 5d (X = S, R = COOEt) showed extensively high inhibitions toward the six cancer cell lines. Similarly, for the 5,6,7,8-tetrahydro-4H-chromene derivatives 6a-d, it is obvious that compound 6a (X = O, Y = NH2) showed low inhibitions, compound 6b (X = O, Y = OH) displayed moderate inhibitions and compounds 6c (X = S, Y = NH2) and 6d (X = S, Y = NH2) showed high inhibitions. Considering the 2,3,6,7-tetrahyd-robenzo[d]thiazole derivatives 8a,b, where both of the two compounds exhibited high cytotoxicities, this is at- Mohareb et al.: New Approaches for the Synthesis of Heterocyclic ... Acta Chim. Slov. 2021, 68, 72-87 81 tributed to the presence of the thiazole moiety in both compounds. For the 4,5,6,7-tetrahydro-2H-indazole derivatives 10a-d it is clear that compound 10a (X = O, R = H) showed low inhibitions, compound 10b with high inhibition only toward U87MG cell line with IC50 = 0.93 ^M and moderate inhibitions toward the other five cell lines A549, H460, HT29, MKN-45 and SMMC-7721. Compounds 10c (X = S, R = H) and 10d (X = S, R = Ph) revealed high inhibitions toward the six cancer cell lines. On the other hand, the 6,7-dihydrobenzo[c] isoxazole derivatives 12a and 12b showed high inhibitions. Compound 14 the 2-(ethoxymethylene)cyclohex-ane-1,3-dione showed low inhibitions toward the six cancer cell lines. Considering the 2-(arylamino)meth-ylene)cyclohexane-1,3-dione derivatives 16a-c where compound 16c (X = OCH3) showed the highest inhibitions among the three compounds although compound 16a (X = H) showed relatively higher inhibitions than compound 16b (X = CH3). For the 6,7-dihydroben-zo[fo]thiophene derivatives 17a-f, it is clear from Table 1 that compounds 17a, 17b and 17c showed low inhibitions while compounds 17d (X = CH3, R = COOEt), 17f (X = OCH3, R = CN) and 17d (X = OCH3, R = COOEt) showed high inhibitions. Within the 6,7-di-hydrobenzo[fr]thiophene derivatives 19a-c and 20a-c, compounds 19c (Y = Cl) and 20c (X= Cl) showed the highest inhibitions among the six compounds. Surprisingly, for compounds 21a-c where compound 21b (Y = CH3) showed higher inhibitions than 21a (Y = H) and 21c (y = Cl). Finally for the 1,2,3,4,7,8-hexahydroben-zo[4,5]thien[2,3-d]pyrimidine derivatives 22a-c, where compound 22a (Y = H) showed low inhibitions, compound 22b (Y = CH3) displayed moderate inhibitions and compound 22c (Y = Cl) showed high inhibitions. It is of great value to mention that compounds 3b, 5c, 5d, 6b, 6c, 6d, 8a, 8b, 10c, 10d, 12a, 12b, 16c, 17d, 17e, 17f, 19c, 20c and 22c were the most cytotoxic compounds among the tested compounds. 2. 2. 3. Inhibition of Tyrosine Kinases Compounds 3b, 5c, 5d, 6b, 6c, 6d, 8a, 8b, 10c, 10d, 12a, 12b, 16c, 17d, 17e, 17f, 19c, 20c and 22c were selected for inhibition of the five tyrosine kinases c-Kit, Flt-3, VEGFR-2, EGFR, and PDGFR. It is clear from Table 2 that compounds 3b, 5d, 6b, 6d, 8a, 8b, 10c, 12a, 12b, 16c, 17e, 17f and 19c were the most potent of the tested compounds towards the five tyrosine kinases. Compound 5c showed high potency towards the two ki-nases c-EGFR and PDGFR with IC50 0.96 and 0.68 nM, respectively. In addition the 2-(((4-methoxyphenyl)ami-no)methylene)cyclohexane-1,3-dione (16c) showed activity towards the five kinases with IC50 0.14, 0.32, 0.21, 0.36 and 0.40 nM, respectively. Compounds 6c, 10d, 20c and 22c showed the lowest potency among the tested compounds. Table 2. Inhibition of tyrosine kinases (enzyme IC50 in nM) by compounds 3b, 5c, 5d, 6b, 6c, 6d, 8a, 8b, 10c, 10d, 12a, 12b, 16c, 17d, 17e, 17f, 19c, 20c and 22c Compound c-Kit 3b 5c 5d 6b 6c 6d 8a 8b 10c 10d 12a 12b 16c 17d 17e 17f 19c 20c 22c 0.80 1.03 0.23 0.48 1.42 0.16 0.58 0.29 0.16 2.07 0.36 0.18 0.14 1.85 0.26 0.55 0.26 1.27 2.49 Flt-3 VEGFR-2 EGFR PDGFR 0.37 0.42 0.58 0.38 2.63 1.82 0.96 0.68 0.26 0.42 0.69 0.72 0.27 0.62 0.49 0.52 2.58 1.61 1.80 2.31 0.24 0.57 0.34 0.28 0.42 0.38 0.27 0.19 0.48 0.68 0.52 0.40 0.13 0.28 0.31 0.28 1.24 1.30 1.28 1.72 0.24 0.62 0.18 0.24 0.53 0.61 0.53 0.42 0.32 0.21 0.36 0.40 1.64 1.52 2.83 1.18 0.23 0.37 0.28 0.46 0.80 0.92 0.16 0.27 0.42 0.31 0.50 0.62 1.43 2.60 2.88 1.69 2.61 1.96 2.37 3.39 2. 2. 4. Inhibition of Selected Compounds Towards Pim-1 Kinase Compounds 3b, 5d, 6b, 6d, 8a, 8b, 10c, 12a, 12b, 16c, 17e, 17f and 19c were selected to examine their Pim-1 kinase inhibition activity (Table 3) as these compounds showed high inhibition towards the tested cancer cell lines at a range of 10 concentrations and the IC50 values were calculated. Compounds 5d, 6b, 6d, 10c, 12a, 17e and 17f were the most potent to inhibit Pim-1 activity with IC50 values of 0.24, 0.41, 0.30, 0.28, 0.45, 0.23 and 0.25 ^M, respectively. On the other hand, compounds 3b, 8a, 8b, 12b, 16c, and 19c were less effective (IC50 > 10 ^M). SGI-1776 was used as the positive control with IC50 0.048 ^M in the assay. These profiles in combination with cell growth inhibition data of compounds 3b, 5d, 6b, 6d, 8a, 8b, 10c, 12a, 12b, 16c, 17e, 17f and 19c are listed in Table 3 indicating that Pim-1 is a potential target of these compounds. 3. Results and Discussion The synthesis of the 2-(hetero-2-ylmethylene)cy-clohexane-1,3-dione derivatives 3a,b has been accomplished as outlined in Scheme 1 starting from cyclohex-an-1,3-dione (1). Compounds 3a and 3b were obtained through the reaction of 1 with either of furan-2-carbalde-hyde or thiophene-2-carbaldehyde. The reaction of either of compound 3a or 3b with elemental sulfur and either of malononitrile (4a) or ethyl cyanoacetate (4b) gave the 6,7-dihydrobenzo[fr]thiophen-5(4H)-one derivatives 5a- Mohareb et al.: New Approaches for the Synthesis of Heterocyclic ... 82 Acta Chim. Slov. 2021, 68, 72-87 82 Table 3. The inhibitory activity of compounds 3b, 5d, 6b, 6d, 8a, 8b, 10c, 12a, 12b, 16c, 17e, 17f and 19c toward Pim-1 kinase. Compound Inhibition ratio at 10 mM IC50 (MM) 3b 16 > 10 5d 96 0.24 6b 90 0.41 6d 89 0.30 8a 26 > 10 8b 24 > 10 10c 92 0.28 12a 0.88 0.45 12b 28 > 10 16c 26 > 10 17e 92 0.23 17f 90 0.25 19c 18 > 10 SGI-1776 - 0.048 d, respectively. The structures of the latter products were established on the analytical and spectral data. Thus, the 1H NMR spectrum of compound 5a (as an example) showed the presence of two triplets at 5 2.62-2.78 ppm for the two CH2 groups, a singlet at 5 4.73 ppm (D2O exchangeable) indicating the presence of the NH2 group, a singlet at 5 6.84 for the pyran H-4 and a multiplet at 5 6.82-7.86 ppm for the furan protons. In addition, the 13C NMR spectrum revealed three signals at 5 16.6, 36.3 and 39.5 equivalent to the three CH2 groups, two signals at 5 112.6 and 158.4 for the C=CH group, a signal at 5 116.8 for the CN group, eight signals at 5 135.4, 140.6, 141.4, 142.2, 142.7, 144.8, 145.6, 146.5 for the thiophene and furan carbons and a signal at 5 179.3 indicating the CO group. The reaction of either compound 3a or 3b with either of malononitrle (4a) or ethyl cyanoacetate (4b) in ethanol containing a catalytic amount of triethylamine gave the 2H-chromen-5-one derivatives 6a-d, respectively (Scheme 1). Scheme 1. Synthesis of compounds 3a,b; 5a-d and 6a-d. Mohareb et al.: New Approaches for the Synthesis of Heterocyclic ... Acta Chim. Slov. 2021, 68, 72-87 83 The reactivity of compounds 3a and 3b toward thi-azole synthesis was studied. Thus, the reaction of either compound 3a or 3b with elemental sulfur and phenyl iso-thiocyanate (7) gave the 2-thioxohexahydrobenzo[d]thi-azole derivatives 8a and 8b, respectively. The reaction of either of compound 3a or 3b with either hydrazine hydrate (9a) or phenylhydrazine (9b) gave the 4-hydrazono-4,5,6,7-tetrahydro-2H-indazole derivatives 10a-d, respectively. Similarly, the reaction of either 3a or 3b with hydroxylamine hydrochloride (11) gave the 6,7-dihydrobenzo[c]isoxazol-4(5H)-one oxime derivatives 12a and 12b, respectively (Scheme 2). Next, we moved toward studying the use of 2-(ethox-ymethylene)cyclohexane-1,3-dione (14), obtained according to the reported work30 via the reaction of cyclohex-ane-1,3-dione (1) with ethyl orthoformate in acetic acid solution, through different heterocyclization reactions. Thus, the reaction of compound 3 with any of the aromatic amines namely aniline (15a), 4-methylaniline (15b) or 4-methoxyaniline (15c) gave the 2-(aminomethylene) cyclohexane-1,3-dione derivatives 16a-c, respectively. Structures of compounds 16a-c were confirmed on the basis of their respective analytical and spectral data (see experimental section). Compounds 16a-c were used to synthesize thiophene derivatives using the Gewald's thi-ophene synthesis.31-33 Thus, the reaction of any of compounds 16a, 16b or 16c with elemental sulfur and either of malononitrile (4a) or ethyl cyanoacetate (4b) gave the 6,7-dihydrobenzo[fo]thiophene derivatives 17a-f, respectively. The reaction of cycohexane-1,3-dione (1) with elemental sulfur and any of cyanoacetanilide (19a), cy-ano-4-methylacetanilide (19b) or cyano-4-methoxyacet-anilide (19c) gave the 6,7-dihydrobenzo[fo]thiophene HC X O. .o Ss + PhNCS 7 1,4-dioxane 3a, X = O b, X = S 8a, X = () h, X = S 3a, b R-NHNH2 9a, R = H b, R = Ph 1,4-dioxanc NNHR 10a, X = O, R = H b, X " O, R " Ph c, X = S, R = H d, x = s ,R = Ph 3a, Ii NH2OH.HCl 11 1,4-dioxane NaOAc NOH Scheme 2. Synthesis of compounds 8a,b; 10a-d and 12a,b. 12a, X = 0 b, X=S Mohareb et al.: New Approaches for the Synthesis of Heterocyclic ... 84 Acta Chim. Slov. 2021, 68, 72-87 84 Scheme 3. Synthesis of compounds 14, 16a-c; 17a-f and 19a-c. derivatives 19a-c, respectively (Scheme 3). The analytical and spectral data of the latter compounds were consistent with their respective structures. Thus, the JH NMR of compound 19a showed the presence of two triplets at 8 2.62-2.79 ppm equivalent to the two CH2 groups, a singlet for the third CH2 group, a singlet at 8 4.75 ppm (D2O exchangeable) for the NH2 group, a multiplet at 8 7.28-7.40 ppm for the phenyl protons and a singlet at 8 8.38 ppm (D2O exchangeable) indicating the NH group. Moreover, the 13C NMR spectrum showed the presence of three sig- nals at 5 16.3, 36.6, 40.3 corresponding to the three CH2 groups, a signal at 5 116.6 indicating the presence of the CN group, signals at 5 120.8, 122.2, 125.3, 127.3, 132.2, 134.5, 138.0, 142.9 for the C6H5 and thiophene carbons and two signals at 5 164.3, 179.6 confirming the presence of two CO groups. Compounds 19a-c were ready to form amide derivatives through their reactions with cyanomethylene esters. Thus, the reaction of either compounds 19a-c with ethyl cyanoacetate in N,N-dimethylformamide solution Mohareb et al.: New Approaches for the Synthesis of Heterocyclic ... Acta Chim. Slov. 2021, 68, 72-87 85 COOEt 19a, Y = H b. Y = CH3 c, Y = C! Scheme 4. Synthesis of compounds 20a-c; 21a-c and 22a-c. NCH2C-C~HN Ö 20a, Y = H b, Y = CHj c, Y = Cl -HNOC PhHN—C-HN 11 \ s s took place to form the 4,5,6,7-tetrahydrobenzo[b]thio-phen-2-yl)acetamide derivatives 20a-c, respectively. On the other hand, the reaction of either compounds 19a, 19b or 9c with phenyl isothiocyanate gave the corresponding N-phenylthiourea derivatives 21a-c, respectively. The analytical and spectral data of compounds 21a-c were consistent with their respective structures. Compounds 20a-c were ready for further cyclization to form biologically active annulated compounds. Thus, heating of either compound 21a, 21b or 21c in sodium ethoxide solution in a boiling water bath afforded the hexahydrobenzo[4,5] thieno[2,3-d]pyrimidine derivatives 22a-c, respectively (Scheme 4). Their structures were based on the analytical and spectral data (see experimental section). 21a, Y = H b, Y = CHj c, Y = Cl NaOEt 4. Conclusion Forty novel heterocyclic compounds bearing cy-clohexanone moiety were designed and synthesized. Their structures were confirmed by multiple techniques. The synthesized compounds were screened for cytotoxic activity against a panel of six human cancer cell lines using MTT assay. Some intriguing structure-activity relationships were found and discussed and the most active compounds were selected for further screening against tyrosine kinases, Pim-1 kinase and the results indicated that these compounds are good condidates as anti-cancer agents that will encourange further work in the future. 22a, Y = H b, Y = CHj c, Y = Cl Consent for Publication This work is consent for publication through the Journal formats. Conflict of Interest The authors declare no conflict of interest, financial or otherwise. Human and Animal Rights No Animals/Humans were used for studies that are basis of this research. 5. References 1. W. Pfau, H. Marquardt, Toxicology 2001, 166, 25-30. DOI:10.1016/S0300-483X(01)00435-8 Mohareb et al.: New Approaches for the Synthesis of Heterocyclic ... 86 Acta Chim. Slov. 2021, 68, 72-87 86 2. V. P. Boyarskiy, K. V. Luzyanin, V. Y. Kukushkin, Coord. Chem. Rev. 2012, 256, 2029-2056. D01:10.1016/j.ccr.2012.04.022 3. R. K. Singh, N. Sinha, S. Jain, M. 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D0I:10.1016/j.ejmech.2009.06.007 24. R. M. Mohareb, R. A. Ibrahim, E. S. Alwan, Acta Chim. Slov. 2020, in press. D0I:10.17344/acsi.2020.6090 25. R. M. Mohareb, F. M. Manhi, A. Abdelwahab, Acta Chim. Slov. 2020, 67, 83-95. D0I:10.17344/acsi.2019.5224 26. R. M. Mohareb, P. A. Halim, Acta Chim. Slov. 2018, 65, 554568. D0I:10.17344/acsi.2017.4146 27. L. Liu, A. Siegmund, N. Xi, P. Kaplan-Lefko, K. Rex, A. Chen. J. Lin, J. Moriguchi, L. Berry, L. Y. Huang, Y. Teffera, Y. J. Yang, Y. H. Zhang, S. F. Bellon, M. Lee, R. Shimanovich, A. Bak, C. Dominguez, M. H. Norman, J. C. Harmange, I. Dussault, T. S. Kim, J. Med. Chem. 2008, 51, 3688-3691. D0I:10.1021/jm800401t 28. M. L. Peach, N. Tan, S. J. Choyke, A. Giubellino, G. Athauda, T. R. Burke, M. C. Nicklaus, D. P. Bottaro, J. Med. Chem. 2009, 52, 943-951. D0I:10.1021/jm800791f 29. F. D. Bacco, P. Luraghi, E. Medico, G. Reato, F. Girolami, T. Perera, P. Gabriele, P. M. Comoglio, C. Boccaccio, J. Natl. Cancer Inst. 2011, 103, 645-661. D0I:10.1093/jnci/djr093 30. R. M. Mohareb, K. M. H. Hilmy, Y. A. Elshehawy, Bull. Chem. Soc. Ethiop. 2018, 32, 285-308. D0I:10.4314/bcse.v32i2.9 31. B. P. McKibben, C. H. Cartwright, A. L. Castelhano, Tetrahedron Lett. 1999, 40, 5471-5474. D0I:10.1016/S0040-4039(99)01108-9 32. K. Wang, D. Kim, A. Dömling, J. Comb. Chem. 2010, 12, 111118. D0I:10.1021/cc9001586 33. R. Mishra, K. K. Jha, S. Kumar, I. Tomer, Der Pharma Chem. 2011, 3, 38-54. Mohareb et al.: New Approaches for the Synthesis of Heterocyclic ... Acta Chim. Slov. 2021, 68, 72- 87 87 Povzetek V prispevku opisujemo serijo heterociklizacijskih reakcij, ki smo jih izvedli na cikloheksan-1,3-dionu s furan-2-karbal-dehidom ali tiofen-2-karbaldehidom pri čemer sta nastala ustrezna ilidenska derivata 3a,b; ti dve spojini sta bili izhodišče za nadaljnje heterociklizacijske reakcije, ki so vodile do tiofenskih in piranskih derivatov 5a-d oz. 6a-d. Ob reakciji spojin 3a,b z elementarnim žveplom in fenil izotiocianatom sta nastala pripojena tiazolska derivata 8a,b. Pri reakciji s hi-drazin hidratom ali fenilhidrazinom pa so nastali 4-hidrazono-4,5,6,7-tetrahidro-2ff-indazolski derivati 10a-d. Podobno sta pri reakciji med 3a ali 3b s hidroksilamin hidrokloridom nastala 6,7-dihidrobenzo[c]izoksazol-4(5H)-on oksima 12a in 12b. Pripravili smo še več drugih heterocikličnih spojin in določili njihove strukture. Pripravljenim spojinam smo določili citotoksične aktivnosti na izbrane celične linije raka. Za najbolj aktivne spojine smo v nadaljevanju določili še inhibitorne lastnosti proti tirozin kinazam in Pim-1 kinazi. Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Mohareb et al.: New Approaches for the Synthesis of Heterocyclic ... 88 DOi: I0.i7344/acsi.2020.6i83 Acta Chim. Slov. 2021, 68, 88-101 ©cwnrfrons Scientific paper Synthesis, Crystallographic Structure, Hirshfeld Surface Analysis, Drug-likeness Properties and Molecular Docking Studies of New Oxime-pyridine Compounds Tufan Topal* Department of Chemistry, Pamukkale University, 20020, Denizli, Turkey, * Corresponding author: E-mail: tufantopal@hotmail.com Phone: +90 258 2963457, fax: +90 258 2963535 Received: 06-11-2020 Abstract A detailed description of the two new pyridine ligands, (2E,3Z)-3-[2-(3-chloropyridin-2-yl)hydrazinylidene]-N-hy-droxybutan-2-imine and 3-chloro-2-{(2Z)-2-[1-(4 nitrophenyl)ethylidene]hydrazinyl}, is reported. The synthesized compounds were characterized by spectroscopic studies, spectral features were performed by TD-DFT calculations. New-generation pyridine ligand of HL2 was also determinate by single-crystal X-ray diffraction and Hirshfeld surface analysis with two-dimensional fingerprint plots was used to analyze intermolecular interactions in crystals. Molecular-docking was performed to investigate the binding areas of chemical compounds, and the results showed the inhibitory activity of the studied HL1 and HL2 against E. coli. The results of the current study revealed the drug-likeness and bioactive properties of the ligands. Keywords: Pyridine-oxime; molecular electrostatic potential (MEP); Drug-likeness; E. Coli; Hirshfeld surface analysis; X-ray diffraction 1. Introduction For biological activities, pyridine compounds are widely used as antibacterial, antifungal, and anticancer agents.1-3 Several studies have been conducted on biological compounds in health-related journals and books. Accordingly, pyridine derivatives cause interactions with high binding capacity by targeting enzymes, proteins, and deoxyribonucleic acid (DNA) that create biological problems.4 With the discovery of new compounds, several studies were performed in the last decade to inhibit antibacterial drug resistance and reduce associated adverse effects on human health.5 It is clear that new types of viruses and bacteria affect the lives of humans worldwide in a variety of ways. Accordingly, this places immense responsibility on researchers and chemists who work to develop new materials to decrease the effects of viruses and bacteria as well as biologists and physicians who test the new compounds on animals and humans. Requiring extensive research, conducting in vitro studies is costly and time-consuming; accordingly, one of the most important advantages of the current study was the contribution to perform in silico studies inactivating viruses, bacteria, and cancer cells by the production of ligands with medicinal potential. Pyridine and oxime compounds have a high interference of hydrogen bond in electrostatic potential capacity. Intramolecular and intermolecular hydrogen bonds play a major role in the interacting and binding of biological molecules. In addition, pyridine and oxime compounds have been selected particularly for their ability to easily transfer the electrons of nitrogen atoms participating in the aromatic ring and C=N groups into the donor-acceptor system.6-11 Due to their high levels of antibacterial properties and bioactive multizones, nitrogenous organic or inorganic compounds are reported to have positive effects on Escherichia coli.12 The reason for the investigation of E. coli pathogens in molecular-docking studies is this bacterium's resistance against some medications and its high binding ca-pacity.5 Furthermore, E. coli, also known as the most-common human pathogen, causes different types of infections, such as kidney, gallbladder, skin, and respiratory infections in addition to meningitis in neonates.13,14 The physicochem-ical properties of compounds affect bioavailability, including electrostatic potential, molar absorptivity, stability, solubility, structure, intracellular absorption, hydrogen bonds, and bonding energy.15,16 The theoretical method such as Density Functional Theory (DFT) in the computational chemistry is Topal: Synthesis, Crystallographic Structure, Hirshfeld Surface 89 Acta Chim. Slov. 2021, 68, 88-101 important tool to predict the assignment of specific electronic transitions in the UV-Vis spectra. In the present study, oxime and pyridine derivative compounds were synthesized and characterized by X-ray and spectroscopic methods. In addition, the surface analysis was performed to analyze their chemical properties, and molecular electrostatic potential was calculated to determine the nucleophilic and electro-philic zones. Molecular-docking was conducted to investigate the hydrogen-binding interactions of E. coli DNA gy-rase subunit B (GyrB) and E. coli beta-ketoacyl-acyl carrier protein synthase III (FabH) and determined pharmacoki-netic and pharmacological properties. 2. Materials and Methods 2. 1. Materials and Physical Measurements The chemicals and solvents: methanol, acetonitrile, 3-chloro-2-hydrazinopyridine, 2,3 butanedione monox-ime, 4'-nitroacetophenone were obtained from Sigma-Al-drich. BRUKER BIOSPIN NMR AVANCE Spectrometer III 400MHz model spectrometer was used for 'H-NMR and 13C-NMR analysis. Elemental analysis were determined using a Costech Elemental analysis device ECS 4010 Model analyzer. IR spectrums were recorded in the 400-4000 cm-1 on Perkin Elmer FTIR-Spectrometer Spectrum Two Model and Mass Spectra (ESI) on TSQ Fortis™ Triple Quadrupole Mass Spectrometer. Melting points of ligands were determined by Stuart SMP10. The mains water was passed through the Thermo Scientific Smart2pure device to make it pure water. Absorption spectra was carried out using Shimadzu UV-1800 UV-VIS spectrophotometer. Single-crystal X-ray structure was determined using an Agilent SuperNova Dual CCD detector diffrac-tometer eguipped with graphite-monochromated MoKa radiation (X = 0.71073 °A) at room temperature. 2. 2. Synthesis of Ligands HL1, HL2 Chemical preparation of (2£,3Z)-3-[2-(3-chloropyr-idin-2-yl)hydrazinylidene]-N-hydroxybutan-2-imine HL1 and 3-chloro-2-{(2Z)-2-[1-(4-nitrophenyl)ethylidene]hy-drazinyljpyridine HL2. 3-chloro-2-hydrazinopyridine (1 mmol, 0.1435 g) in acetonitrile solution (10 ml) was added 2,3 butanedione monoxime (1 mmol, 0.1011 g) HL1 and 4'-Nitroacetophe-none (1 mmol, 0.1651 g) HL2 in 10 ml of acetonitrile respectively (Fig.1). The both solution stirred for 24 h at room temperature and were kept aside for slow evaporation of solvent for about 5 days. The HL2 crystal was obtained from slow evaporation technique by dissolving the product in acetonitrile. C9H11ClN4O (HL1), Crem; Yield 87%. m.p.:194 oC. 1H-NMR (400 MHz, Chloroform-d6, ppm): 5 9.83 (s, 1H, O-H), 5 8.45 (s, 1H, -NH), 5 8.37 (d, 1H, Ar-H), 5 7.62 (d, 1H, Ar-H), 5 6.84 (t, 1H, Ar-H), 5 2.34 (s, 3H, -CH3), 5 2.23 (s, 3H, -CH3). 13C-NMR (100 MHz, Chloroform-d6, ppm): 5 157.39 (-C=N-OH), 5 157.32 (-C=N-), 5 150.00, 147.12, 137.32, 116.50, 115.35 (C-Arpyridine), 5 10.03 (-CH3), 5 9.53 (-CH3).17 LC/MS-MS, (ESI) m/z= 226.66284 [m+1]+ (100%). Calcd. for C9H11ClN4O: C, 47.69; H, 4.89; N, 24.72%; found: C, 47.71; H, 4.86; N, 24.69%. IR (KBr) cm-1: 3357 (N-H), 3112 (O-H), 2981 (C-HAr), 1596 (C=n) pyridine, 1561 (C=N)imtae, 1516 (C=N)oxime, 935 (N-O). C13H11ClN4O2 (HL2), Orange; Yield 86%. m.p.:169 oC. 1H-NMR (400 MHz, Chloroform-d6, ppm): 5 8.48 (s, 1H -NH), 5 8.30 (d, 1H, Ar-H), 5 8.20 (d, 2H, Ar-H), 5 8.01 (d: 2H, Ar-H), 5 7.61 (d, 1H, Ar-H), 6.84 (t, 1H, Ar-H), 5 2.38 (s, 3H, -CH3), 13C-NMR (100 MHz, Chloroform-d6, ppm) 5 149.92, 5 147.61 (C-Arpyridine), 5 147.17 (-C=N-), 5 144.49, 144.42 (C-Ar), 5 137.33 (C-Arpyridine), 5 126.89: 126.89, 123.64, 123.64 (C-Ar), 5 116.97, 115.49 (C-Arpyri-dine), 5 12.53 (-CH3). LC/MS-MS, (ESI) m/z= 290.70558 Topal: Synthesis, Crystallographic Structure, Hirshfeld Surface ... 90 Acta Chim. Slov. 2021, 68, 88-101 [M+1]+ (100%). Calcd. for C13HnClN4O2: C, 53.71; H, 3.81; N, 19.27%; found: C, 53.73; H, 3.79; N, 19.22%. IR (KBr) cm-1: 3378 (N-H), 2922 (C-HAr), 1585 (C=N) pyridine, 1557 (C=N)imine, 1392 (NO2).181H NMR and UV-Vis spectra. Reaction of 1:1 stoichiometric proportion of HL with Na2[PdCl4] in methanol affords a mononuclear pal-ladium(II 2. 3. X-ray Crystallography Analysis Orange crystal of the C13H11ClN4O2 compound was obtained in acetonitrile solution through slow evaporation for 5 days at room temperature. The data set of reflections were collected using an Agilent SuperNova X-Ray diffrac-tometer with MoKa (X = 0.71073) at 293 K. The data reduction and data correction were performed by Olex2 software (version 1.3).19,20 Refinements were obtained by the Full-Matrix method on F2 using the Olex2 software, and crystal packing diagrams were created by Mercury 4.3.0 software. All the nonhydrogen atoms were anisotropically refined using the riding model approximation.21 Tables 1 and 2, a summary of the experimental details of HL2.22-24 CCDC DOI: 10.5517/ccdc.csd.cc24rlzh and number 1988897 contains the supplementary rystallographic data for this work. This data can be obtained from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_ request/cif. 3. Results and Discussion 3. 1. Description of the Crystal Structures and Hydrogen Bonding Slow evaporation technique was used to make the sample suitable for X-ray structure analysis. The HL2 crys- Table 1. Crystal data and structure refinement for HL2. CCDC number 1988897 Empirical formula C^HnClNA Formula weight 290.71 Temperature/K 293(2) Crystal system monoclinic Space group P2i/c a/Â 11.7767(7) b/Â 14.4529(7) c/Â 7.9073(4) a/° 90 91.381(5) Y/o 90 Volume/Â3 1345.49(12) Z 4 Pcalcg/cm3 1.435 |i/mm' 0.291 F(000) 600.0 Crystal size/mm3 0.14 x 0.13 x 0.12 Radiation Mo Ka (X = 0.71073) 20 range for data collection/° 6.756 to 49.996 Index ranges -8 < h < 14, -16 < k < 16, -8 < l < 9 Reflections collected 4286 Independent reflections 2343 [Rint=0.0161, Rsigma = 0.0302] Data/restraints/parameters 2343/0/182 Goodness-of-fit on F2 1.047 Final R indexes [I>=2a (I)] Rj = 0.0548, wR2 = 0.1338 Final R indexes [all data] Rj = 0.0790, wR2 = 0.1491 Largest diff. peak/hole / e Â-3 0.21/-0.41 tallizes in the centrosymmetric space group P21/c of monoclinic system with a unit cell volume of 1345.49(12) A3, the cell dimensions are: a = 11.7767(7) A, b = 14.4529(7) Table 2. Selected Bond Lengths/Â, Angles/0 and Torsion/0 for HL2. Atom Atom Length Atom Atom Atom Angle Atom Atom Atom Atom Torsion N7 N8 1.355(3) C9 N8 N7 117.57(19) N7 N8 C9 C10 -1.6(3) N8 C9 1.287(3) N8 N7 C2 121.4(2) N8 N7 C2 N1 -1.4(4) N1 C2 1.329(3) C2 N1 C6 117.2(3) C2 N1 C6 H6 -179.5(4) N1 C6 1.333(4) C13 C12 C9 120.2(2) C13 C12 C9 N8 8.3(3) C9 C12 1.474(3) C17 C12 C9 122.1(2) C9 N8 N7 H7 -4.3(4) C12 C13 1.397(3) C17 C12 C13 117.7(2) C2 N7 N8 C9 175.7(2) C12 C17 1.391(3) N1 C2 C3 122.5(2) C6 N1 C2 N7 -177.6(3) C3 C2 1.394(4) C4 C3 C2 118.7(3) C6 N1 C2 C3 2.4(4) C9 C10 1.505(3) C12 C9 C10 120.8(2) C10 C9 C12 C13 -170.9(2) C14 C13 1.375(3) N8 C9 C12 115.0(2) N8 C9 C12 C17 -172.1(2) C17 C16 1.369(4) N8 C9 C10 124.2(2) C10 C9 C12 C17 8.7(3) C15 C14 1.379(4) N7 C2 C3 118.7(2) N8 C9 C10 10HA -55.3 C4 C3 1.375(4) C16 C17 C12 122.0(2) N8 C9 C10 10HB -175.3 C6 C5 1.368(5) N1 C2 N7 118.9(2) N8 C9 C10 10HC 64.7 C5 C4 1.371(5) N1 C6 C5 124.2(3) C12 C9 C10 10HA 123.9 N7 C2 1.371(3) C14 C15 N18 119.1(3) C12 C9 C10 10HB 3.9 C16 C15 1.361(4) C14 C13 C12 120.6(2) C12 C9 C10 10HC -116.1 Topal: Synthesis, Crystallographic Structure, Hirshfeld Surface ... 91 Acta Chim. Slov. 2021, 68, 88-101 Á, c = 7.9073(4) Á, p = 91.381(5)° and Z = 4. The full data collection was done for indices h, k and l with ranges of -8 < h < 14, -16 < k < 16, -8 < l < 9 and R value of R1 = 0.0548, wR2 = 0.1338. Carbon and hydrogen atoms have been geometrically positioned. The bond lengths between the atoms N8=C9 1.287(3) Á, N7-N8 1.355(3) Á, C9-C12 1.474(3) Á, C12-C13 1.397(3) Á and C12-C17 1.391(3) Á were found. In literature 1.28(14) Á, 1.35(3) Á, 1.49(14) Á, 1.39(15) Á and 1.38(16) Á are similar with our values of atomic lengths.25-27 The N8=C9 bond was shorter than the N8-N7 bond and it confirms that N8=C9 shows a double bond character. In HL2, pyridyl N1-C2 and N1-C6 bond distances were 1.329(3) and 1.333(4) Á, bond angle C2-N1-C6 117.2(3)° and torsion angle C2-N1-C6-H6 -179.5(4)CI were found and this torsion value is competible with the expected 180o.11,28 Torsion angles of atoms between pyridyl and benzene ring have been observed C13-C12-C9-N8 8.3(3)o N8-N7-C2-N1 -1.4(4)o and N7-N8-C9-C10 -1.6(3)° The ORTEP-3 drawn with 35% probability is given in Fig. 2. Basically 3 pairs of hydrogen bond interactions are observed in the structure, these are C-H—O, C-H—N and C-H—Cl intramolecular and intermolecular hydrogen bonds.29,30 Selected hydrogen bond distances and angles are listed in Table 3. In HL2, carbon atom C10 acts as a donor to N1 atom at C(10)-H(10A)-N(1) (x,1.5-y,-1/2+z), Fig. 2. Molecular structure of the HL2. Thermal ellipsoids are shown at 35% probability level developing the capped stick style and two-dimensional chain along the crystallographic axis as depicted in Fig. 3a. As illustrated in Fig. 3b, in HL2, C-H—O intermolecular hydrogen bonding interactions were used to generate a three-dimensional (3D) supramolecular network along the c axis. All the figures were drawn in Mercury software. Other hidrogen bonds were C(10)-H(10A)-Cl(11) (1-x,2-y,1-z), C(5)-H(5)-O(20) (1-x,1-y,1-z), C(10)-H(10B)-O(19) (2-x,-1/2 + y,1/2-z), C(17)-H(17) -O(20) (2-x,-1/2 + y,1/2-z), C(5)-H(5)—O(19) (-1 + x,y,1 + z), C(10)-H(10B)—O(20) (2-x,-1/2 + y,1/2-z), C(13)- H(13)-Cl(11) (1-x,2-y,1-z).31 Table 3. Selected hydrogen bond distances (A), symmetry and angles (o) for HL2. D-H-À d (D-H) d (H-À) d(D-H-À) Symmetry codes ^D-H-À C(10)-H(10A)—Cl(11) 0.960 3.174 3.882 1-x,2-y,1-z 131.90 C(10)-H(10A) —N(1) 0.960 2.768 3.602 x,1.5-y, -1/2+z 145.68 C(5)-H(5)—0(20) 0.930 3.019 3.606 1-x,1-y,1-z 122.57 C(10)-H(10B)—0(19) 0.960 2.730 3.417 2-x, -1/2+y,1/2-z 129.08 C(17)-H(17)—0(20) 0.930 2.460 3.322 2-x, -1/2+y,1/2-z 154.19 C(13)-H(13) •••Cl(ll) 0.930 2.941 3.764 1-x,-1/2+y,1.5-z 148.37 C(5)-H(5)—0(19) 0.930 2.909 3.747 -1+x,y,1+z 150.54 C(10)-H(10B)—0(20) 0.960 3.057 3.978 2-x, -1/2+y,1/2-z 161.22 Topal: Synthesis, Crystallographic Structure, Hirshfeld Surface ... 92 Acta Chim. Slov. 2021, 68, 88-101 3. 2. 1H NMR Studies 1H-NMR spectrum of HL1 is shown in Fig. 4. 1H-NMR spectrum of the HL1 ligand oxime (N-OH) group of the proton (H13) was observed at 9.83 ppm as a singlet peak. The NH proton (H8) generated a signal at 8.45 ppm. The spectrum of the (2£,3Z)-3-[2-(3-chloropy-ridin-2-yl)hydrazinylidene]-N-hydroxybutan-2-imine ligand observed at singlet peaks 2.23 ppm (H14A, H14B, H14C) and 2.34 ppm (H15A, H15B, H15C) methylene group of protons. While H1 proton signal appeared at d 8.37 ppm, the signals at d 7.62 and d 6.84 ppm were due to H3 and H2 of pyridinium moiety, respectively.32 In HL2, the NH proton (H7) generated a sharp signal at 8.48 ppm, as a singlet peak. The peaks were observed at range 8.306.84 ppm were assignable to the protons of aromatic rings as multiplet peaks. The spectrum of the 3-chloro-2-{(2Z)-2-[1-(4-nitrophenyl)ethylidene]hydrazinyl}pyridine ligand observed at singlet peaks 2.38 ppm (H10A, H10B, H10C) methylene group of protons. The signal at d 8.30 ppm was attributed at to (H6) aromatic proton of pyridine. The proton signal appearing at d 8.20 and d 8.01 ppm were due to (H13, H17, H16, H14) of aromatic moiety respectively. Aromatic protons of pyridine moiety produced a broad signal at d 7.61 and t 6.84 ppm (H5, H4).11,18,33 It was observed that the obtained results were exactly compatible with the structure. 3. 3. 13C NMR Studies 13C-NMR spectrum of HL1 is shown in Fig. 5. 13C-NMR spectrum HL1 observed a single resonance at 157.39 and 157.32 ppm, respectively which showed that the oxime (C=NOH) and hydrazone (-NHN=CH) (C11,C10) carbon atoms. All the signals were assigned to the aromatic carbons (C1-C5) of the pyridine at the range of 150.00115.35 ppm. The signals observed at 10.03 and 9.53 ppm are attributable to the carbon atom of methyl group (C15,C14). For HL2, aromatic carbons of pyridine and benzene rings gave different signals in their resonance. Hence the signals at 149.92, 147.61, 137.33, 116.97, 115.49 ppm were due to carbon (C2-C6) while (C12-C17) presented in different signals at 144.49, 144.42, 126.89, 126.89, 123.64, 123.64 ppm in the aromatic moiety of ligand. Two equivalent para carbons (C13,C17) and (C14,C16) brought out a signals at d 126.89 and 123.64 ppm in the spectrum. The signal at 147.17 ppm was due to the (C9) carbon of the imine group of moiety. The signal observed at 12.53 ppm is attributable to the carbon atom ofmethyl group (C10).25,32,33 3. 4. UV-Vis Absorption Spectra and TD-DFT Calculations UV-Vis calculations were performed by TD-DFT/ B3LYP method with 6-31G basis set using Gaussian 09 Fig. 4. 'H-NMR spectrum of HLj Topal: Synthesis, Crystallographic Structure, Hirshfeld Surface ... 93 Acta Chim. Slov. 2021, 68, 88-101 Fig. 5. 13C-NMR spectrum of HL1 program.34,35 The electronic absorption spectra of the li-gands, along with the molar extinction coefficient, were obtained in a 5 x 10-5 mol L-1 chloroform solution in the wavelength zone (240-480 nm) using the spectroscopic method.23 The electron transition possibilities of different compounds were compared using ultraviolet-visible spectroscopy. For HL1 and HL2, slightly different absorption peaks centered at 322, 285 nm and 362, 312 nm (e = 42800, 41100 and 34520, 26300 mol-1 L cm-1), respectively. The experimental UV-Vis spectra of the HL1 compund and corresponding theoretical calculations are plotted in Fig. 6. The- oretical calculations predicted two peaks at 307, 287 nm and 364, 331 nm which indicated formation of the HL1 and HL2.36 The calculated excitation energy, excitation wavelength, oscillator strength with the aid of TD-DFT/B3LYP method are given in Table 4. The electronic absorption spectra of the ligands were defined with two sharp absorption bands. These two bands were observed at 285-312 and 322-362 nm indicative of the n-n* and n-n* transitions, respectively.37 While the n-n* transitions of the ligands originated from the electrons of the pyridine ring, the n-n* band occurred due to hydrazone groups (-NHN=CH) of atoms.38 240 270 300 330 360 390 420 450 480 240 270 300 3S0 360 390 420 450 4B0 Wavelength Wavelength Fig. 6. The (left) experimental and (right) calculated spectrum and observed UltravioleteVisible spectra of the HLj in CH2Cl2 solution at room temperature Topal: Synthesis, Crystallographic Structure, Hirshfeld Surface ... 94 Acta Chim. Slov. 2021, 68, 88-101 Table 4. The experimental and calculated UV-Vis spectral parameters for HL1 and HL2 ligand with its assignments. Compounds Experimental Calculated Assignment \(nm) \(nm) E (eV) f HLj 285 287 4.36 0.18 n-n* 322 307 4.03 0.03 n-n* HL2 312 331 3.73 0.02 n-n* 362 364 2.56 0.06 n-n* 3. 5. Mass and FT-IR Spectra Mass spectral datas of ligands were obtained by elec-trospray ionization (ESI) method. The mass exhibited the molecular ion at m/z 226.66284 [M+1]+ and 290.70558 [M+1]+ which indicated formation of the HL1 and HL2 (Fig. S1). The moleculer peak of both ligands have a 100% relative abundance.32 Generally compunds are characterized by three IR absorption bands such as v(O-H), v(C=N) and v(N-O) stretching vibrations. FT-IR spectrum of the HL1 ligand showed (C=N) imine (C=N) oxime peaks at 1596 cm-1 and 1516 cm-1 (Fig. S2).39 The bands 3361 cm-1 and 3377 cm-1 were due to (N-H) vibrations for HL1 and HL2 respectively. Also (O-H) band of oxime group peak was observed at 3111 cm-1.33 But this peak was not seen at HL2 ligand. At the same time, the bands at 935 cm-1 and 1392 cm-1 assignable to (N-O) and (NO2) vibrations, respectively. The FT-IR spectrum of ligands displayed bands at 2981 cm-1 and 2972 cm-1 which assignable to (C-HAr) protons. The medium bands observed at 553 cm-1 and 546 cm-1 assigned to pyridyl rings. For HL1 and HL2, the bands 1454-1451 cm-1, 1394-1392 cm-1, 1044-1032* cm-1 and 1012-1032 cm-1 assignable to the aromatic pyridine ring.40 FT-IR analysis give us the preliminary information about whether this structure is formed or not. Our datas are in agreement with similar oxime and pyridine ligands in the literature.41 3. 6. Hirshfeld Surface Analysis The surface analysis method is the best way to identify crystal packing and intermolecular interacting in a structure. For this reason, the Hirshfeld surface analysis was performed using CrystalExplorer software (version 17.5).42,43 Accordingly, the close correlations between the fragments were quantitatively analyzed. Furthermore, it is one of the computer calculation programs to investigate the mechanism of molecular interactions in proteins and with which intermolecular interactions they could bond to a receptor. In addition, it helps to identify the intermolecular hydrogen bond as well as n-n, C-H—X (X=halogens) interactions with great importance in crystal package arrangement and stabilization of the molecule. Therefore, C(17)-H(17)-O(20) and C(13)-H(13)-Cl(11) interactions are illustrated in Fig. 7. Intermolecular interactions of HL2 ligand is presented in Fig. 8 as 2D fingerprints plot.44 Blue zone shows intermolecular interaction areas whereas grey zone shows outside of the this interaction area. According to fingerplots studies results of for HL2; Cl-H 11,4%, H-H 28,7%, N-C 6,4%, N-H 6,5%, O-H 18,3%, Cl-N 2,5%, C-C 3,8%, C—H 15,2%.22-24 Other bondings constituted all interactions by making small contributions on the surface. The highest H—H interaction rate (28.7%) was shown to be derived from the abundance of the H—H interactions in aromatic rings (Fig. S3).4 Fig. 7. Close contact of C(17)-H(17)™O(20) and C(13)-H(13)—Cl(11) interactions determined by Hirshfeld surface analysis over dnorm of HL2 Topal: Synthesis, Crystallographic Structure, Hirshfeld Surface ... 95 Acta Chim. Slov. 2021, 68, 88-101 Fig. 8. The 2D fingerprint plos of the HL2 3. 7. Molecular Electrostatic Potential Analysis Molecular electrostatic potential mapping is a method for the observation of the interactions of molecules with each other based on their charge distribution on 3D diagrams. This is an auxiliary method that estimates elec-trophilic and nucleophilic reactive sites of ligands leading to investigate protein binding and medicine developing by defining hydrogen bond interactions.4,45 Electrostatic po- a) b) Fig. 9. Electrostatic potential maps of a) C9H11ClN4O and b) C13H11ClN4O2 Topal: Synthesis, Crystallographic Structure, Hirshfeld Surface ... 96 Acta Chim. Slov. 2021, 68, 88-101 tential (3D) diagrams of ligands were mapped using the Hartree-Fock theory Slater type orbital-3G base set of Hirshfeld surface analysis by CrystalExplorer software as depicted in Fig. 9. The input file of the geometry was obtained using Tonto.46,47 Surface qualification values were set -0,025-0,025 au and high resolution was selected. When it was performed, the molecule was taken to transparency mode and atoms were made clear. The red color represents the negative electrostatic potential regions and acceptor hydrogen bonds. Moreover, the blue color represents the positive electrostatic potential regions and donor hydrogen bonds.48,49 In addition, blue-colored zones are the preferred regions for the nucleophilic attack, while red-colored negative zones are susceptible to electrophilic attack.11 Furthermore, red-colored regions have electron-rich atoms or atom groups; therefore, they can easily interact with amino acid residues. Based on the findings of molecular docking studies, hydrogen bond interactions between donor-acceptor confirmed the results of the electrostatic potential analysis as depicted in Fig. 11. 3. 8. Molecular Docking Studies In silico docking calculations are of great importance in drug design and medical chemistry fields. Molecular docking is commonly used in the studies carried out on target medicine designing by estimating the binding mechanisms of small molecules on target biologic pro-teins.50,51 Donor-acceptor binding mechanisms create complexations with hydrophobic hydrogen bond and electrostatic interactions.52 The current study examined the ligands creating intramolecular hydrogen bonds by targeting the active zones of E. coli DNA GyrB (PDB Code:4WUB) and E. coli FabH (PDB Code:1HNJ) amino acid residues. The protein-related data were obtained from Research Collaboratory for Structural Bioinformatics Protein Data Bank https://www.pdb.org. Molecular docking studies analyzed binding energy, hydrogen bonding, and interactions between the ligands and bacteria.53-56 Furthermore, by the calculation of the ligands' lowest binding energy to aminoacids residues, it was determined that which structure has stronger hydrogen bonding and higher binding energy score.57 Ligands SMILES formats were created at page https://www.cheminfo.org; it is C/C(=N\ O)/C(C)=N/Nc1ncccc1Cl for HL1 and C/C(=N/Nc1nc-ccc1Cl)c2ccc(N(=O)=O)cc2 for HL2. Molecular docking studies were performed Autodock vina program (https://www.vina.scripps.edu). Ligands were converted into mol2 format and prepared for molecular docking at Chimera software program with receptors (E.coli FabH and GyrB) (https://www.cgl.ucsf.edu/chime-ra/).58 In the Dock Prep method, firstly, all nonstandarts and solvents were selected and refined from receptors. Then, protein models were added by the selection of all hydrogen atoms (also considered H-bonds) and Gasteiger charges. Docking studies were conducted by targeting li-gand receptors and binding the most convenient coordi-nates.59 While defining an approximate donor and acceptor binding zone in in-silico studies, docking parameters are of great importance. Different types of grid box values were applied for the best and most accurate binding. The application of target protein binding zones (as a great scale) in a cubic box resulted in the best binding; however, this method requires a long computer calculation.60,61 The grid box values of ligands applied to E. coli GyrB receptor were 10, 25, -10 at the center with a grad spacing of 0.375 A including default the sizes of 40, 40, 40 for HL1 and HL2. The same method was employed to E. coli FabH receptor reporting the values of 30, 15, 30 at the center with the sizes of 30, 30, 30 for HL1 and HL2. In addition, default values were used for other parameters. Root-mean-square deviation were selected as minumum value. The binding energy values of -7.9 and -8.8 kcal/mol were applied by HL1 and HL2 ligands to the GyrB receptor, respectively. Fig. 10 illustrates the hydrophobicity surface area of HL1 inside GyrB and FabH proteins. The binding energy values a) b) Fig. 10. Illustration of hydrophobicity surface area of HLj inside a) gyrase subunit B and b) beta-ketoacyl-acyl carrier protein synthase III proteins Topal: Synthesis, Crystallographic Structure, Hirshfeld Surface ... 97 Acta Chim. Slov. 2021, 68, 88-101 Fig. 11. Results of molecular docking for a) HLj-gyrase subunit B (Gyr] synthase III (FabH) (1HNJ), and d) HL2-FabH (1HNJ) of -6.1 and -6.8 kcal/mol were applied by HL1 and HL2 ligands to the FabH receptor, respectively. The binding interactions and docking poses are depicted in Fig. 11. Since the ligands bind to the GyrB receptor with higher binding energy than that reported for the FabH receptor, there was a better docking to the ligands-GyrB complex. While there were two hydrogen bonds in the complex that ligands made with 4WUB protein, 1HNJ protein had one hydrogen bond. This finding proved that this effect increases the binding energy value. As the binding energy value enhances, the binding score increases indicating a better dock-ing.41,62 Therefore, the binding energy of HL2 was higher than that reported for HL1 which is considered a better docking. The other reason behind that is believed to be the electron density and unpaired electron couples of two oxygen atoms that bond to the nitrogen atom of HL2.63 In a study carried out by Fathima et al. (2018), docking results were observed to be -8.4 and -8.5 kcal/mol for 2AB-P2C-1HNJ and 2ABHB-1HNJ, respectively.54 The HL1 created a hydrogen bond to GLY 117 and GLN 335.A HE22 amino acid residues of 4WUB protein with N12 and O13 atoms and binding lengths were observed as 1.992 A and 1.838 A, respectively. The HL2 bonded to GLY 119 and VAL 120 amino acid residues of 4WUB proteins with O19 and O20 atoms and binding lengths were observed as 2.252 A and 2.404 A. In another study conducted by Metelytsia et al. (2020), the binding of ligands to the regions of amino acid residues were reported as GLY 119 and His 116. This finding is similar to the results of docking investigations in the current study in terms of attachment regions.52 The HL1 and HL2 created a hydrogen bond to GLY 209, ARG 36.A HH22 amino acid residues of 1HNJ receptor with N8 and O19 atoms and binding lengths were observed as 2.481 A and 2.171 A, respectively. Donor and acceptor hydrogen binding interactions are shown as Table 5.64 The findings of docking studies confirmed that the results and interactions of molecular electrostatic potential generally occur in red regions. The Table 5. AutoDock results showing Compound-Protein name, Binding site of protein, Binding site of Ligand, Type of interactions, Bond length, Binding energy. Compound- Binding site of Binding site of Type of Bond Binding energy Protein name protein Ligand interaction length (Â) (Kcal/Mol) HLj-4WUB GLY 117 UNK1-N12 atom Hydrogen bond 1.992 -7.9 GLN 335.A HE22 UNK1-O13 atom Hydrogen bond 1.838 HL2-4WUB GLY 119 UNK1-O19 atom Hydrogen bond 2.252 -8.8 VAL 120 UNK1-020 atom Hydrogen bond 2.404 HLj- 1HNJ GLY 209 UNK1-N8 atom Hydrogen bond 2.481 -6.1 HL2- 1HNJ ARG 36.A HH22 UNK1-O19 atom Hydrogen bond 2.171 -6.8 Topal: Synthesis, Crystallographic Structure, Hirshfeld Surface ... 98 Acta Chim. Slov. 2021, 68, 88-101 findings of docking studies also revealed that ligands are potential inhibitors against E. coli DNA GyrB and E. coli FabH.65 3. 9 Drug-Likeness and Biological Activity Drug-likeness and bioactivity of ligands were obtained from molinspiration (https://www.molinspiration. com/cgi-bin/properties). Although the parameters of druglikeness cannot estimate the biological activity of each compound, it is one of the most successful and efficient methods for the analysis of compounds with medicinal potential through the determination of pharmacokinetic characteristics. Based on Table 6, the parameters of drug-likeness and biologic activity for the compounds in the present study (miLogP, TPSA, nAtoms, MW, nON, nOHNH, nviolations, and rotb) and (enzyme inhibitor, protease inhibitor, nuclear receptor ligand, kinase inhibitor, ion channel modulator, and GPRC ligand). The aforementioned findings have been the first obtained data regarding the medicinal potential of the compounds.66 The values of drug-likeness were determined in this study, and the ligands were examined regarding midicinal potential with the consideration of these values. In this sense, the miLogP parameter, which is the capacity of penetrating the cell membrane, is expected to be under 5. In this regard, the values of miLogP parameter were reported as 1.81 and 3.37 for HL1 and HL2, respectively. The polar surface area (TPSA) represents the hydrogen bonding potential of a compound. Accordingly, the TPSA values were observed as 69.88 and 83.11 A2 for HL1 and HL2, respectively. These values were below the 160 A2 limit defined for TPSA and at a good performance.67-70 Although miLogP and TPSA are not sufficient criteria for the investigation of druglikeness, they are two important parameters to represent oral absorption in cells.71 The number of acceptor hydrogen bonds was set to nON < 10, and the number of donor hydrogen bonds was set to nOHNH < 5. In this study, the obtained results were below the aforementioned values.72 Low molecule weight is important in terms of Table 6. Calculated drug-likeness parametres and Bioactivity Score of ligands. HLj HL2 miLogP 1.81 3.37 TPSA 69.88 83.11 natoms 15 20 MW 226,67 290.71 nON 5 6 nOHNH 2 1 nviolations 0 0 nrotb 3 4 GPCR ligand -0.78 -0.70 Ion channel modulator -0.62 -0.70 Kinase inhibitor -0.74 -0.55 Nuclear receptor ligand -1.32 -0.94 Protease inhibitor -1.32 -1.02 Enzyme inhibitor -0.43 -0.49 easy transport, diffusion, and absorption of the molecule. The values of molecular weight are expected to be < 500 Da. In this study, the molecular weights of the ligands were lower than the aforementioned value. The ligands were reported with successful results according to Lipinski's rule of five. Based on Fig. 12, the red column shows Lipinski's rule of five, and the green and red columns depict the druglikeness score of the ligands.68 If the value of violations equals 0, it shows that crystallized compounds can easily bond to the receptor. This value was reported as 0 for the compounds of the present study. The number of rotatable bonds is a simple topological value and measurement of flexibility.73 If the bioactivity results of the compounds are > 0, -5.0-0.0, and < -5.0, they are considered active, medium active, and not active, respectively. All the results of biological activity parameters were within the range of -5.0-0.0; therefore, the ligand-swere regarded as medium active.74 As a result, it was concluded that the ligands of the current study obtained satisfactory druglikeness scores and properties to be considered medicine potential agents. 10 LdJ I miLogP nON nOHNH [HL1] [HL2] ■ Five Lipinski's Rule 500 ■ 400 300 200 ■ 100 .■1 0 ■ I ■ TPSA MW ■ [HL1] ■ [HL2] ■ Five Lipinski's Rule Fig. 12. Druglikeness scores of ligands according to Lipinski's rule of five Topal: Synthesis, Crystallographic Structure, Hirshfeld Surface ... 99 Acta Chim. Slov. 2021, 68, 88-101 4. Conclusion In this work, the new (2£,3Z)-3-[2-(3-chloropyri-din-2-yl)hydrazinylidene]-N-hydroxybutan-2-imine and 3-chloro-2-{(2Z)-2-[1-(4-nitrophenyl)ethylidene]hydraz-inyljpyridine ligands were synthesized and characterized by elemental analysis, LC/MS-MS, FT-IR, 1H-NMR, 13C-NMR and UV-Vis. HL2 was also determinated by single-crystal X-ray diffraction (XRD) and crystallized in the space group P21/c of with Z=4 and was linked into (3-D) network by C-H- • -O intermoleculer hydrogen bonding interactions. Additionally, Cl-H 11,4%, H-H 28,7%, N-C 6,4%, N—H 6,5%, O—H 18,3%, Cl-N 2,5%, C-C 3,8%, G"H 15,2% reciprocal influence were revealed by Hirsh-feld Surface Analysis. The mass spectra of the ligands showed the main peaks that corresponding to [M+1]+. UV-Vis studies demonsrated that the n-n* and n-n* transitions appearing at 285, 312 nm and 322, 362 nm, respectively. The obtained experimental results of the present study were fully compatible with the theoretical results. The binding energy values of -7.9 and -8.8 kcal/mol were applied by HL1 and HL2 ligands to the GyrB receptor, respectively. Moreover, the binding energy values of -6.1 and -6.8 kcal/mol were applied by HL1 and HL2 ligands to the FabH receptor. 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D0I:10.1016/j.cdc.2019.100320 Povzetek A detailed description of the two new pyridine ligands, (2E,3Z)-3-[2-(3-chloropyridin-2-yl)hydrazinylidene]-N-hy-droxybutan-2-imine and 3-chloro-2-{(2Z)-2-[1-(4 nitrophenyl)ethylidene]hydrazinyl}, is reported. The synthesized compounds were characterized by spectroscopic studies, spectral features were performed by TD-DFT calculations. New-generation pyridine ligand of HL2 was also determinate by single-crystal X-ray diffraction and Hirshfeld surface analysis with two-dimensional fingerprint plots was used to analyze intermolecular interactions in crystals. Molecular-docking was performed to investigate the binding areas of chemical compounds, and the results showed the inhibitory activity of the studied HLj and HL2 against E. coli. The results of the current study revealed the drug-likeness and bioactive properties of the ligands. Podan je podroben opis dveh novih piridinskih ligandov, (2E,3Z)-3-[2-(3-kloropiridin-2-il) hidraziniliden]-N-hidrok-sibutan-2-imina in 3-kloro-2-{(2Z)-2-[1-(4nitrofenil)etiliden] hidrazinila}. Sintetizirane spojine so bile okarakterizirane s spektroskopskimi študijami, spektralne značilnosti pa so bile ovrednotene z izračuni TD-DFT. Nova generacija piridinskih ligandov HL2 je bila določena tudi z žarkovno rentgensko difrakcijo, za analizo medmolekularnih interakcij v kristalih pa je bila uporabljena Hirshfeldova površinska analiza s specifičnimi dvodimenzionalnimi prikazi. Za proučitev vezavnih površin kemijskih spojin smo izvedli molekularno sidranje, pri čemer so rezultati pokazali inhibitorno aktivnost proučevanih HLj in HL2 spojin napram E. coli. Rezultati sedanje študije kažejo na potencialne zdravilne in bio-aktivne lastnosti ligandov. © 0 Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Topal: Synthesis, Crystallographic Structure, Hirshfeld Surface ... DOI: 10.17344/acsi.2020.6205 Acta Chim. Slov. 2021, 68, 102-108 /^creative ^commons Scientific paper Copper(II) and Zinc(II) Complexes Derived from N,N,-Bis(4-bromosalicylidene)propane-1,3-diamine: Syntheses, Crystal Structures and Antimicrobial Activity Yu-Mei Hao Department of Chemistry, Baicheng Normal University, Baicheng 137000, P.R. China * Corresponding author: E-mail: jyxygzb@163.com Received: 06-19-2020 Abstract A mononuclear copper(II) complex, [CuL] (1), and a phenolato-bridged trinuclear zinc(II) complex, [Zn3Cl2L2(DMF)2] (2), where L is the deprotonated form of N,N'-bis(4-bromosalicylidene)propane-1,3-diamine (H2L), have been prepared and characterized by elemental analyses, IR and UV-Vis spectroscopy, and single crystal X-ray diffraction. The Cu atom in complex 1 is in square planar coordination, while the terminal and central Zn atoms in complex 2 are in square pyramidal and octahedral coordination, respectively. The antibacterial activities of the complexes have been tested on the bacteria Staphylococcus aureus and Escherichia coli, and the yeast Candida parapsilosis. Keywords: Schiff base; copper complex; zinc complex; crystal structure; antibacterial property 1. Introduction Schiff bases have been extensively used as multi-dentate ligands to construct metal complexes with versatile structures due to their easy formation and strong metal-binding ability.1 The increasing interest in the synthesis and structural studies of Schiff bases is due to their bioactivity and coordination properties. Schiff base with donors (N, O, S, etc.) have structure similarities with neutral biological systems. Most of the Schiff bases and their complexes with transition metals have a broad range of applications in biological and pharmaceutical fields.2 A number of Schiff bases have been reported for their remarkable biological activities, such as antibacterial, antifungal, antimalarial, anti-proliferative, anti-inflammatory, antiviral and antipyretic activities.3 Copper(II) and zinc(II) Schiff base complexes have been studied extensively and are considered as excellent alternatives for classic organic antibacterial agents.4 Despite the presence of considerable research on the antibacterial properties of such complexes, it is still necessary to search for new complexes to find more effective agents as well as to better understand the mechanism of the action of this class of compounds. With an interest in the chemistry of biologically active Schiff bases and their metal complexes, this study aimed to synthesize copper(II) and zinc(II) complexes. The newly synthesized complexes, [CuL] (1) and [Zn3Cl2L2(DMF)2] (2), where L is the deprotonated form of N,N'-bis(4-bromosalicylidene)propane-1,3-di-amine (H2L), were structurally characterized, and examined for their antimicrobial activities. 2. Experimental 2. 1. Materials and Methods 1,3-Diaminopropane and 4-bromosalicylaldehyde were purchased from TCI. CuCl2 ■ 2H2O and ZnCl2 were purchased from Aladdin Chemical Reagent Co. Ltd. The solvents methanol and DMF were purchased from Kemi-ou Chemical Reagent Co. Ltd. Elemental analyses were performed on a Perkin-Elmer 240C elemental analyzer. IR spectra were recorded on a Jasco FT/IR-4000 spectrometer as KBr pellets in the 4000-400 cm-1 region. UV-Vis spectrum was recorded on a Lambda 900 spectrometer. 1H and 13C NMR were determined with a Bruker 500 MHz instrument. Single crystal X-ray diffraction was carried out on a Bruker SMART 1000 CCD diffractometer. 2. 2. Syntheis of H2L 1,3-Diaminopropane (0.15 g, 2.0 mmol) was diluted by methanol (20 mL). Then, it was added dropwise to the methanol solution (30 mL) of 4-bromosalicylalde- Hao: Copper(II) and Zinc(II) Complexes Derived Acta Chim. Slov. 2021, 68, 102-108 103 Scheme 1. The Schiff base H2L, showing the H atoms. hyde (0.84 g, 4.0 mmol). The reaction mixture was stirred and heated to reflux for 30 min. The solvent was removed by evaporation under reduced pressure. The yellow solid was recrystallized from ethanol to give yellowish crystalline product. Yield: 0.72 g (82%). :H NMR (500 MHz, DMSO-d6; Scheme 1) 8 /ppm: 8.58 (s, 2H, CHd=N), 7.50 (s, 2H, ArHc), 7.33 (t, 2H, ArHa), 7.14 (d, 2H, ArHb), 3.72 (t, 4H, CHe2), 2.07 (t, 2H, CHf2). 13C NMR (126 MHz, DMSO-d6) 8 /ppm: 165.12, 153.13, 133.27, 124.54, 123.27, 122.02, 115.57, 54.78, 30.73. IR (KBr, cm-1): 3449, 2980, 2921, 1632, 1471, 1385, 1287, 1231, 1096, 930, 845, 646, 451. UV-Vis Amax/nm (1.13 x 10-5 mol L-1, MeOH; e, L mol-1 cm-1): 230 (27,300), 270 (13,800), 330 (7,120), 420 (4,520). Anal. Calcd. (%) for C17H16Br2N2O2: C, 46.39; H, 3.66; N, 6.36. Found (%): C, 46.22; H, 3.75; N, 6.43. 2. 3. Synthesis of [CuL] (1) H2L (44 mg, 0.10 mmol) and CuCl2-2H2O (34 mg, 0.20 mmol) were mixed in methanol (30 mL). The mixture was stirred at room temperature for 30 min to give a blue solution. Single crystals of the complex, suitable for X-ray diffraction, were obtained after 8 days. Yield: 31 mg (62%). IR data (cm-1): 2925, 2853, 1608, 1514, 1407, 1289, 1192, 1126, 1060, 973, 913, 853, 777, 600, 543, 451. UV-Vis (1.03 x 10-5 mol L-1, MeOH; e, L mol-1 cm-1): 230 (23,500), 247 (24,100), 280 (16,320), 355 (7,135). Anal. Calcd. (%) for C17H14Br2CuN2O2: C, 40.70; H, 2.81; N, 5.58. Found (%): C, 40.86; H, 2.92; N, 5.47. 2. 4. Synthesis of [Zn3Cl2L2(DMF)2] (2) H2L (44 mg, 0.10 mmol) and ZnCl2 (27 mg, 0.20 mmol) were mixed in methanol (30 mL). The mixture was stirred at room temperature for 30 min to give a white precipitate. DMF (5 mL) was added to the mixture until the precipitate dissolved. Single crystals of the complex, suitable for X-ray diffraction, were obtained after 17 days. Yield: 27 mg (42%). IR data (cm-1): 2917, 2849, 1632, 1615, 1581, 1533, 1460, 1390, 1276, 1198, 1112, 1071, 978, 911, 863, 800, 677, 602, 539, 464. UV-Vis (1.30 x 10-5 mol L-1, MeOH; e, L mol-1 cm-1): 230 (22,370), 245 (23,920), 272 (12,100), 350 (7,630). Anal. Calcd. (%) for C40H42Br4Cl2 N6O6Zn3: C, 37.26; H, 3.28; N, 6.52. Found (%): C, 37.43; H, 3.35; N, 6.44. 2. 5. X-ray Crystallography Single crystal X-ray data for the complexes were collected on a Bruker SMART 1000 CCD diffractometer using the SMART/SAINT software.5 Intensity data were collected using graphite-monochromatized Mo^a radiation (0.71073 Â) at 298(2) K. The structures were solved by direct methods using SHELX.6 Empirical absorption corrections were applied with SADABS.7 All non-hydrogen atoms were refined with anisotropic displacement coefficients. The hydrogen atoms bonded to carbon were included in geometric positions and given thermal parameters equivalent to 1.2 and 1.5 times those of the atom to which they were attached. The C8-C9-C10 group of the Schiff base ligand in complex 1 is disordered over two sites, with occupancies of 0.5 and 0.5 due to the symmetry. The distances of C8-C9 and C9-C10 are restrained to 1.51(1) Â. The atoms C8, C9 and C10 were refined using ISOR instruction. The thermal factors of atoms C8 and C10 were constrained to be equal. Crystallographic data and refinement parameters are given in Table 1, and important interatomic distances and angles are given in Table 2. Table 1. Crystallographic data and refinement parameters for the complexes 1 2 Chemical Formula Ci7Hi4Br2CuN2O2 C4öH42Br4Cl2N6O6Zn3 Fw 501.66 1289.45 T (K) 298(2) 298(2) Crystal system Monoclinic Monoclinic Space group C2/c P2/c a (A) 23.0385(16) 11.9976(12) b (A) 8.0706(11) 12.9584(12) c (A) 9.0741(12) 16.0855(13) « (°) 90 90 P (°) 93.0960(10) 107.2880(10) Y (°) 90 90 V (A3) 1684.7(3) 2387.8(4) Z 4 2 p (Mo Ka) (cm-1) 6.052 5.003 Dc (g cm-3) 1.978 1.793 Reflections/ parameters 1543/117 4457/278 Unique reflections 955 3272 Restraints 21 0 Goodness of fit on F2 1.039 1.043 Rint 0.0597 0.0360 Ru wR2 [I > 2a(I)] 0.0617, 0.1442 0.0517, 0.1302 Ru wR2 (all data) 0.1064, 0.1641 0.0744, 0.1436 2. 6. Biological Assay The antibacterial property of the complexes was evaluated by a macro-dilution method using Staphylococcus aureus, Escherichia coli, and the yeast Candida parapsi- Hao: Copper(II) and Zinc(II) Complexes Derived 104 Acta Chim. Slov. 2021, 68, 102-108 Table 2. Selected bond distances (A) and angles (°) for the complexes 1 Cu(1)-O(1) 1.902(5) Cu(1)-N(1) 1.964(7) O(1)-Cu(1)-O(1A) 87.4(3) O(1)-Cu(1)-N(1)A 154.1(2) O(1)-Cu(1)-N(1) 92.5(3) N(1)-Cu(1)-N(1)A 98.6(4) 2 Zn(2)-O(2) 2.075(4) Zn(2)-O(1) 2.101(4) Zn(2)-O(3) 2.109(4) Zn(1)-O(2) 2.059(4) Zn(1)-O(1) 2.064(4) Zn(1)-N(1) 2.078(5) Zn(1)-N(2) 2.080(5) Zn(1)-Cl(1) 2.2902(18) O(2)-Zn(2)-O(2A) 169.0(2) O(2)-Zn(2)-O(1A) 95.80(14) O(2)-Zn(2)-O(1) 76.08(14) O(1)-Zn(2)-O(1A) 86.5(2) O(2)-Zn(2)-O(3) 91.16(15) O(2)-Zn(2)-O(3A) 96.71(15) O(1)-Zn(2)-O(3A) 172.65(15) O(1)-Zn(2)-O(3) 92.91(16) O(3)-Zn(2)-O(3A) 88.6(2) O(2)-Zn(1)-O(1) 77.24(14) O(2)-Zn(1)-N(1) 142.30(17) O(1)-Zn(1)-N(1) 87.16(17) O(2)-Zn(1)-N(2) 86.45(18) O(1)-Zn(1)-N(2) 155.07(18) N(1)-Zn(1)-N(2) 94.7(2) O(2)-Zn(1)-Cl(1) 107.19(12) O(1)-Zn(1)-Cl(1) 100.25(12) N(1)-Zn(1)-Cl(1) 109.28(14) N(2)-Zn(1)-Cl(1) 102.55(15) Symmetry code for A: 1 - x, y, V - z. losis. The cultures of bacteria and yeasts were incubated under vigorous shaking. The compounds were dissolved in small amount of DMSO. Concentration of the tested compounds ranging from 0.010 to 2.5 mmol L-1 for the bacteria and yeasts was used in all experiments. The antibacterial activity was characterized by IC50 and MIC values. MIC experiments on subculture dishes were used to assess the minimal microbicidal concentration (MMC). Subcultures were prepared separately in Petri dishes containing competent agar medium and incubated at 30 °C for 48 h. The MMC value was taken as the lowest concentration, which showed no visible growth of microbial colonies in the subculture dishes. 3. Results and Discussion 3. 1. Chemistry The complexes 1 and 2 were facile prepared by the reaction of the Schiff base H2L with copper chloride and Hao: Copper(II) and Zinc(II) Complexes Derived ... Acta Chim. Slov. 2021, 68, 102-108 105 zinc chloride, respectively, in methanol (Scheme 2). Single crystals of the complexes were formed by slow evaporation of the solvent at room temperature. 3. 2. Crystal Structure Description The molecular structure of complex 1 is shown in Fig. 1. The complex is a mononuclear copper(II) species. Molecule of the complex possesses a crystallographic twofold rotation axis symmetry. The Cu(1) atom is coordinated by the two imine nitrogen and two phenolate oxygen of Fig. 1. A perspective view of the molecular structure of complex 1 with the atom labeling scheme. Thermal ellipsoids are drawn at the 30% probability level. the Schiff base ligand, forming a tetrahedrally distorted square planar geometry, which is evidenced by the trans bond angles of 154.1(2)° and the cis angles of 87.4(3)-98.6(4)°. The distortion of the square planar coordination results in the deflection of the two benzene rings, which has a dihedral angle of 35.2(3)°. Similarly, the dihedral angle between the two six-membered chelate rings Cu(1)-N(1)-C(7)-C(1)-C(2)-O(1) and Cu(1)-N(1A)-C(7A)-C(1A)-C(2A)-O(1A) is 33.8(3)°. The coordinate bond lengths in the complex are comparable to those observed in the copper(II) complexes with Schiff bases.8 In the crystal structure, the molecules are linked via Br—Br interactions, with a distance of 3.659(4) A, to form zigzag chains along the x axis (Fig. 2). The molecular structure of complex 2 is shown in Fig. 3. The complex is a phenolate-bridged trinuclear zinc(II) species, with the two [ZnClL] units connected by the central Zn atom (Zn(2)). Molecule of the complex possesses a crystallographic two-fold rotation axis symmetry. The Zn—Zn distance is 3.227(2) A. The terminal Zn atom (Zn(1)) shows a distorted square pyramidal coordination geometry, with the two phenolate oxygen atoms (O(1) and O(2)) and the two imine nitrogen atoms (N(1) and N(2)) occupying the basal coordination site, and with a Cl atom occupies the apical position. The Zn(1) atom deviates from the best coordination plane defined by the atoms N(1), N(2), O(1) and O(2) by 0.532(2) A in direction of the apical Cl atom. The cis and trans bond angles in the basal plane are in the range of 77.24(14)-94.7(2)° and 142.30(17)-155.07(18)°, and those between the apical and basal donor atoms are in the range of 100.25(12)-109.28(14)°. Thus, the coordination is distorted from the ideal geometry of a square pyramid. The bond lengths related to Zn(1) atom Fig. 2. Packing structure for complex 1 viewed along the z axis. Dashed lines represent Br—Br interactions forming zigzag chains along the x axis. Fig. 3. A perspective view of the molecular structure of complex 2 with the atom labeling scheme. Thermal ellipsoids are drawn at the 30% probability level. The carbon hydrogens are omitted for clarity. Atoms labelled with the suffix A or unlabelled are at the symmetry position 1 - x, y, V - z. Hao: Copper(II) and Zinc(II) Complexes Derived 106 Acta Chim. Slov. 2021, 68, 102-108 are 2.06 Á for Zn-O, 2.08 Á for Zn-N and 2.29 Á for Zn-Cl, which are comparable to those observed in Schiff base zinc complexes with square pyramidal coordination.9 The central Zn atom (Zn(2)), located at the two-fold rotation axis, shows a distorted octahedral coordination geometry. The donor atoms come from two Schiff base ligands (O(1), O(2), O(1A), O(2A)), and two DMF ligands (O(3), O(3A)). The cis and trans bond angles related to Zn(2) are 76.08(14)-96.71(15)° and 169.0(2)-172.65(15)°, respectively. Thus, the coordination is distorted from the ideal geometry of an octahedron. The Zn-O bond lengths related to Zn(2) atom are 2.07-2.11 Á, which are comparable to those observed in Schiff base zinc complexes with octahedral coordination.10 The [ZnClL] unit is butterfly shaped, with the dihedral angle between the two benzene rings of 33.1(3)°. The bond lengths are also similar to the trinuclear zinc complexes with square pyramidal and octahedral co-ordination.11 In the crystal structure, the molecules are linked via Br—Br interactions,12 with a distance of 3.871(3) Á, to form zigzag chains along the z axis (Fig. 4). 3. 3. IR and UV-Vis Spectra The medium and broad absorption centered at 3449 cm-1 in the spectrum of H2L substantiates the presence of O-H groups. The intense band indicative of the C=O groups of the DMF ligands of complex 2 are observed at 1632 cm-1. The strong absorption band at 1632 cm-1 for H2L is assigned to the azomethine groups, v(C=N),13 which is shifted to lower wave numbers 1608 cm-1 for complexes 1 and 1615 cm-1 for complex 2.14 The weak bands in the low wave numbers 450-550 cm-1 are due to the vibration of the Cu-O and Cu-N bonds.15 In the electronic spectra of complexes 1 and 2 measured in methanol, the intense bands observed at about 230-280 nm for the complexes are assigned to intra-ligand n-n* transitions. The complexes displayed bands centered at 270-280 nm, which can be assigned to the n-n* transition.16 The charge transfer LMCT bands are located in the range of 350 nm.17 3. 4. Antibacterial Activity The antibacterial results are summarized in Table 3. The Schiff base H2L showed activity against E. coli, while no activity on S. aureus and C. parapsilosis. Both the copper and the zinc complexes have higher activities than the free Schiff base. The copper complex showed strong activity against S. aureus and E. coli, and weak activity against C. parapsilosis. The zinc complex showed medium activity against S. aureus, and weak activities against E. coli and C. parapsilosis. Obviously, the copper complex is more effective than the zinc complex. The copper complex has the most activity against S. aureus, with IC50 and MIC values of 0.16 and 0.27 mmol L-1, which deserves further study. As a comparison, the copper complex has similar antibacterial activities against S. aureus and E. coli to the Schiff base manganese(III) complex18 and the Schiff base copper(II) complexes.19 However, it has weaker activity against the the yeast C. para- Table 3. Antibacterial property of the Schiff base and the complexes Compound S. aureus E. coli C. parapsilosis IC5o* MIC* IC50 MIC IC50 MIC h2l >2.50 >2.50 1.63 2.50 >2.50 >2.50 1 0.27 0.16 0.56 0.32 2.17 1.25 2 1.15 0.62 1.83 1.25 3.39 2.50 * mmol L Hao: Copper(II) and Zinc(II) Complexes Derived ... Acta Chim. Slov. 2021, 68, 102-108 115 psilosis when compared with the manganese and copper complexes. 4. Conclusion In summary, with the bis-Schiff base N,N'-bis(4-bro-mosalicylidene)propane-1,3-diamine a mononuclear cop-per(II) complex and a phenolato-bridged trinuclear zinc(II) complex were synthesized. The complexes were characterized by physic-chemical methods, and their structures were confirmed by single crystal X-ray determination. The Schiff base ligand coordinates to the metal atoms through the phenolate oxygen and imine nitrogen. The complexes have significant antibacterial activities on the bacteria Staphylococcus aureus and Escherichia coli, and the yeast Candida parapsilosis. 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Cu atom v kompleksu 1 ima kvadratno planarno koordinacijo, medtem ko imata terminalna Zn atoma v kompleksu 2 kvadratno piramidalno koordinacijo in centralni Zn atom oktaedrično koordinacijo. Protimikrobno aktivnost kompleksov smo testirali na bakterijah Staphylococcus aureus in Escherichia coli ter na glivi Candida parapsilosis. © 0 Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Hao: Copper(II) and Zinc(II) Complexes Derived ... DOI: 10.17344/acsi.2020.6208 Acta Chim. Slov. 2021, 68, 109-117 /^creative ^commons Scientific paper Ultrasound-Assisted Synthesis, Antioxidant Activity and Computational Study of 1,3,4-Oxadiazol-2-amines Hamid Beyzaei,1* Soheila Sargazi,2 Ghodsieh Bagherzade,2 Ashraf Moradi,1 and Elahe Yarmohammadi1 1 Department of Chemistry, Faculty of Science, University of Zabol, Zabol, Iran 2 Department of Chemistry, Faculty of Science, University of Birjand, Birjand, Iran * Corresponding author: E-mail: hbeyzaei@yahoo.com and hbeyzaei@uoz.ac.ir Tel: +98 5431232186 / Fax: +98 5431232180 Received: 06-19-2020 Abstract Development of synthetic procedures for the preparation of 1,3,4-oxadiazole derivatives has always been in the interest of researchers as a result of their widespread biological activities. In this study, an ultrasound-assisted procedure was proposed for the synthesis of 1,3,4-oxadiazol-2-amines form the reaction of hydrazides and cyanogen bromide. They were efficiently produced in 81-93% yields in the presence of ethanol and potassium bicarbonate as the reaction media and the base, respectively. Their antioxidant properties were determined via DPPH free radical scavenging method as one of the most basic steps in identifying other related biological effects. IC50 values were in the range of from 0.237 to 0.863 mM. The synthesized 1,3,4-oxadiazoles are protective agents against oxidative stress, and can be used in the treatment of cancer, candidiasis, diabetes, neurodegenerative and inflammatory diseases. Furthermore, bond dissociation energies (BDEs) and electron densities based NCI (non-covalent interactions) were calculated using density-functional theory (DFT) to understand the observed reactivities. It was found that reversible dipole-dipole forces play a key role in most interactions. Keywords: 1,3,4-Oxadiazole; Antioxidant activity; DPPH; Ultrasound irradiation; NCI; DFT 1. Introduction Antioxidants are synthetic or natural compounds that inhibit the oxidation reactions via free radical scavenging. Free radicals are unstable and reactive atoms or molecules containing one unpaired electron that can begin a propagation sequence in the chain reactions leading to cell damage.1 Vitamins C and E as two essential nutrients are well known for being potent antioxidants. Ascorbic acid, known as vitamin C, is a water-soluble biologically active compound which should be provided to humans through food especially fresh vegetables and citrus fruits. Lack of vitamin C is accompanied by early symptoms of weakness and fatigue which can lead to anemia, hair loss, bleeding gum and skin, and scurvy disease in the acute cases.2 Vitamin C is required for the proper functioning of some enzymes and immune system, tissue regeneration and the enzymatic production.3-5 Oil-soluble vitamin E includes four tocopherols as well as four tocotrienols. It is found in cereals, vegetable oils, meat, poultry, fruits and eggs, and its deficiency damages the nervous system.6 It has been understood that there is a significant relationship between the antioxidant capacity of compounds and some of their biological activities. Mendonca et al. have evaluated antioxidant and antiproliferative potentials of muscadine grape extracts on breast cancer cell lines; a strong positive correlation was observed between total phenolic content of extracts and their inhibitory activities against African American breast cancer cells.7 There are complex and often positive connections between oxidative stress with the inflammatory response given as a result of tissue injury and infection.8 Guava leaves are used as traditional medicines for the treatment of diabetes; they lowered levels of cholesterol, sugar, triglycerides, malonal-dehyde and glycated serum protein in the blood of strep-tozotocin-induced diabetic mice according to their ABTS, OH and DPPH free radical scavenging capabilities.9 Reactive oxygen species are one of the main causes of Alzheimer disease, as a result, any agent that blocks their generation, can be useful in treating the disease.10 1,3,4-Oxadiazole derivatives have attracted a great deal of interest due to their diverse biological properties Beyzaei et al.: Ultrasound-Assisted Synthesis, Antioxidant Activity 110 Acta Chim. Slov. 2021, 68, 109-117 such as antioxidant, antiproliferative, anticonvulsant, antimicrobial and anti-Alzheimer.11-15 Synthetic approaches of 1,3,4-oxadiazoles were reviewed in several literatures.16-18 Hypervalent iodine mediated reaction of N'-arylidene acetohydrazides, intracyclocondensation of thiosemicar-bazides prepared from the reaction of aryl hydrazides with ammonium thiocyanate using N,N,N',N'-tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate (TBTU) or N,N'-diisopropylcarbodiimide (DIC), and simultaneous reaction of 1-chloroketones, N-isocyaniminotriphe-nylphosphorane and aryl carboxylic acids are examples of recent methods proposed to synthesize 1,3,4-oxadiazole derivatives.19-21 Theoretical calculations can be performed to justify experimental observations. However, it is possible that a logical relationship between experimental data and theoretical parameters cannot be found in all cases. The thermodynamics of free radical scavenge of some 1,3,4-oxadi-azole derivatives were studied by DFT method in gas and aqueous phases to predict their action mechanisms; it was found that the selected reaction pathway is completely dependent on the reaction medium.22 It has been shown that phenolic compounds act as antioxidants in three ways (Scheme 1): OH OH 0' Scheme 1. Antioxidant mechanism of phenolic compounds: (A) Single Electron Transfer Followed by Proton Transfer (SET-PT); (B) Hydrogen Atom Transfer (HAT); (C) Sequential Proton Loss Electron Transfer (SPLET). Computational studies can be applied to calculate values of bond dissociation enthalpy, ionization potential and proton dissociation enthalpy, proton affinity and electron transfer enthalpy to determine the predominant path.23 DPPH free radical scavenging activity of some synthesized 1,2,4-triazole-3-thiones containing phenolic sub-stituents was evaluated by Ivanovic et al.; DFT calculations proved synergistic effects of 1,2,4-triazole-3-thione rings and predicted SPLET pathway as the action mechanism in methanol.23 In order to expand library of small organic bio-molecules, some 5-alkyl/aryl/heteroaryl-1,3,4-oxadiazol-2-amine derivatives were synthesized via a new procedure under ultrasound irradiation. Antioxidant activities of the synthesized 1,3,4-oxadiazoles were assessed against DPPH to predict their other possible biological capabilities. Bond dissociation energy and electron density of all synthesized heterocycles were calculated to establish the probable relation to the observed antioxidant activities. 2. Experimental 2. 1. Chemicals All chemicals, solvents and aluminium thin-layer chromatography (TLC) plates pre-coated with silica gel containing fluorescent indicator F254 were purchased from Merck and Sigma-Aldrich companies. The melting points were determined with Kruss KSP1N melting point apparatus, and are uncorrected. Bruker Tensor-27 FT-IR spectrometer was applied to record the FT-IR spectra of compounds. 1H and 13C NMR spectra were registered using a Bruker 300 MHz NMR spectrometer. Chemical shifts are provided as 8 values (ppm) and coupling constants J (Hz). Elemental analyses for C, H, N and S atoms were performed on a Termo Finnigan Flash EA micro-analyzer. Ultrasonic irradiation was supplied by Backer vCLEAN1-L03 (40 kHz frequency and 100 W output power). 2. 1. 1. General Procedure for the Synthesis of 5-Substituted 1,3,4-Oxadiazol-2-amines 3a-h A 25 mL round-bottom flask containing 5 mmol of each hydrazides 1a-h, cyanogen bromide (2) (0.53 g) and potassium bicarbonate (0.50 g) in 10 mL absolute ethanol was subjected to an ultrasonic bath. The reaction progress was checked by TLC with different volumetric ratios of methanol and dichloromethane as the desired mobile phase. The reaction content was added to 20 g of crushed ice containing an excess of salt. The solid phase was filtered out, washed respectively with water (5 mL) and ethanol (5 mL), and oven-dried at 70 °C to give pure 1,3,4-oxadi-azoles 3a-h. 5-Methyl-1,3,4-oxadiazol-2-amine (3a). Yellow powder; yield 0.40 g (81%); m.p. 172-174 °C (lit. m.p. 176-180 °C24); IR (KBr) v 3420, 3352 (NH2), 3120, 2359, 1666 (C=N), 1595, 1383, 1254, 1123 (CO), 1005 (N-N), 781, 626 cm-1; 1H NMR (300 MHz, DMSO-d6) 5 6.85 (s, 2H, NH2), 2.27 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6) 5 10.9 (CH3), 156.6 (C-5 oxadiazole), 164.0 (C-2 oxadiazole). Anal. Calcd. for C3H5N3O: C, 36.36; H, 5.09; N, 42.41. Found: C, 36.31; H, 5.11; N, 42.43. Beyzaei et al.: Ultrasound-Assisted Synthesis, Antioxidant Activity Acta Chim. Slov. 2021, 68, 109-117 111 5-Phenyl-1,3,4-oxadiazol-2-amine (3b). White powder; yield 0.74 g (92%); m.p. 243-245 °C (lit. m.p. 239-242 °C25); IR (KBr) v 3476, 3413 (NH2), 2924, 2358, 1613 (C=N), 1452, 1113 (C-O), 1027 (N-N), 620, 478 cm-1; 1H NMR (300 MHz, DMSO-d6) 8 7.25 (s, 2H, NH2), 7.80-7.78 (m, 2H, H-2',6' Ph), 7.51-7.49 (m, 3H, H-3',4',5' Ph); 13C NMR (75 MHz, DMSO-d6) 8 124.8 (C-1' Ph), 125.4 (C-2',6' Ph), 129.6 (C-3',5' Ph), 130.7 (C-4' Ph), 157.8 (C-5 oxadiazole), 164.3 (C-2 oxa-diazole). Anal. Calcd. for C8H7N3O: C, 59.62; H, 4.38; N, 26.07. Found: C, 59.59; H, 4.35; N, 26.11. 5-(4-Nitrophenyl)-1,3,4-oxadiazol-2-amine (3c). Yellow powder; yield 0.91 g (88%); m.p. 265-267 °C (lit. m.p. 267-270 °C25); IR (KBr) v 3436, 3375 (NH2), 2924, 2358, 1652 (C=N), 1598 (N-O), 1527, 1389, 1344, 1116 (C-O), 1037 (N-N), 858, 620 cm-1; 1H NMR (300 MHz, DMSO-d6) 8 7.52 (brs, 2H, NH2), 8.32 (d, J = 8.7 Hz, 2H, H-3',5' Ar), 7.98 (d, J = 8.7 Hz, 2H, H-2',6' Ar); 13C NMR (75 MHz, DMSO-d6) 8 124.9 (C-3',5' Ar), 126.4 (C-2',6' Ar), 130.2 (C-1' Ar), 148.3 (C-4' Ar), 156.5 (C-5 oxadiazole), 165.0 (C-2 oxadiazole). Anal. Calcd. for C8H6N4O3: C, 46.61; H, 2.93; N, 27.18. Found: C, 46.66; H, 2.93; N, 27.15. 3109, 2359, 1654 (C=N), 1489, 1390, 1322, 1284, 1215, 1106 (C-O), 1040 (N-N), 875, 786, 748, 683, 621 cm-1; 1H NMR (300 MHz, DMSO-d6) 8 7.48-7.38 (m, 2H, H-5',6'), 7.31-7.30 (m, 3H, NH2, H-2' Ar), 7.10 (d, J = 7.0 Hz, 1H, H-4' Ar), 3.83 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6) 8 55.7 (CH3), 110.3 (C-2' Ar), 116.8 (C-4' Ar), 117.8 (C-6' Ar), 126.6 (C-1' Ar), 130.9 (C-5' Ar), 157.7 (C-3' Ar), 160.0 (C-5 oxadiazole), 164.3 (C-2 oxadiazole). Anal. Calcd. for C9H9N3O2: C, 56.54; H, 4.75; N, 21.98. Found: C, 54.59; H, 4.74; N, 21.95. 5-(3-Bromophenyl)-1,3,4-oxadiazol-2-amine (3g). White powder; yield 1.10 g (92%); m.p. 246-248 °C (lit. m.p. 240-243 °C27); IR (KBr) v 3451, 3413 (NH2), 3116, 2358, 1654 (C=N), 1604, 1557, 1392, 1112 (c-o), 1044 (N-N), 794, 681 cm-1; 1H NMR (300 MHz, DM-SO-d6) 8 7.91 (m, 1H, H-6'), 7.80 (d, J = 7.8 Hz, 1H, H-4' Ar), 7.71 (d, J = 7.7 Hz, 1H, H-2' Ar), 7.50 (t, J = 7.5 Hz, 1H, H-5' Ar), 7.38 (s, 2H, NH2); 13C NMR (75 MHz, DM-SO-d6) 8 122.7 (C-3' Ar), 124.4 (C-6' Ar), 126.9 (C-1' Ar), 127.7 (C-4' Ar), 131.9 (C-5' Ar), 133.4 (C-2' Ar), 156.4 (C-5 oxadiazole), 164.5 (C-2 oxadiazole). Anal. Calcd. for C8H6BrN3O: C, 40.03; H, 2.52; N, 17.50. Found: C, 40.01; H, 2.50; N, 17.47. 5-(4-(ieri-Butyl)phenyl)-1,3,4-oxadiazol-2-amine (3d). Pink powder; yield 1.01 g (93%); m.p. 262-264 °C (lit. m.p. 256-258 °C25); IR (KBr) v 3434, 3358 (NH2), 3127, 2963, 2358, 1655 (C=N), 1609, 1391, 1118 (C-O), 1042 (N-N), 836, 621, 561 cm-1; 1H NMR (300 MHz, DMSO-d6) 8 7.55 (brs, 2H, NH2), 7.83 (d, J = 8.5 Hz, 2H, H-2',6' Ar), 7.63 (d, J = 8.5 Hz, 2H, H-3',5' Ar), 1.34 (s, 9H, 3 x CH3); 13C NMR (75 MHz, DMSO-d6) 8 31.1 (C(CH3)3), 35.0 (C(CH3)3), 121.4 (C-3',5' Ar), 125.0 (C-2',6' Ar), 127.2 (C-1' Ar), 152.6 (C-4' Ar), 157.5 (C-5 oxadiazole), 164.3 (C-2 oxadiazole). Anal. Calcd. for C12H15N3O: C, 66.34; H, 6.96; N, 19.34. Found: C, 66.30; H, 6.98; N, 19.36. 3-(5-Amino-1,3,4-oxadiazol-2-yl)phenol (3e). White powder; yield 0.73 g (83%); m.p. 240-242 °C (lit. m.p. 244-245 °C26); IR (KBr) v 3554 (OH), 3458, 3413 (NH2), 3145, 2924, 2360, 2341, 1643 (C=N), 1601, 1568, 1493, 1384, 1320, 1309, 1221, 1126 (C-O), 1064 (N-N), 1035, 994, 876, 799, 739, 701, 688, 669, 617, 450 cm-1; 1H NMR (300 MHz, DMSO-d6) 8 9.90 (s, 1H, OH), 7.35-7.26 (m, 5H, NH2, H-2',5',6' Ar), 6.91 (d, J = 6.9 Hz, 1H, H-4' Ar); 13C NMR (75 MHz, DMSO-d6) 8 112.0 (C-2' Ar), 116.3 (C-4' Ar), 118.0 (C-6' Ar), 125.8 (C-1' Ar), 130.8 (C-5' Ar), 157.9 (C-3' Ar), 158.2 (C-5 oxadiazole), 164.2 (c-2 oxadiazole). Anal. Calcd. for C8H7N3O2: C, 54.24; H, 3.98; N, 23.72. Found: C, 54.19; H, 3.96; N, 23.75. 5-(3-Methoxyphenyl)-1,3,4-oxadiazol-2-amine (3f). Yellow powder; yield 0.86 g (90%); m.p. 193-194 °C (lit. m.p. 192-195 °C27); IR (KBr) v 3415, 3386 (NH2), 5-(Pyridin-4-yl)-1,3,4-oxadiazol-2-amine (3h). Brown powder; yield 0.68 g (84%); m.p. 260-261 °C (lit. m.p. 262-264 °C25); IR (KBr) v 3417, 3383 (NH2), 2358, 1665 (C=N), 1538, 1391, 1340, 1123 (C-O), 1043 (N-N), 837, 682, 620 cm-1; 1H NMR (300 MHz, DM-SO-d6) 8 8.74 (d, J = 7.7 Hz, 2H, H-2',6' Ar), 7.71 (d, J = 7.7 Hz, 2H, H-3',5' Ar), 7.58 (brs, 2H, NH2); 13C NMR (75 MHz, DMSO-d6) 8 119.2 (C-3',5' Ar), 131.6 (C-4' Ar), 151.1 (C-2',6' Ar), 156.2 (C-5 oxadiazole), 165.0 (C-2 oxadiazole). Anal. Calcd. for C7H6N4O: C, 51.85; H, 3.73; N, 34.55. Found: C, 51.89; H, 3.75; N, 34.51. 2. 2. Half Maximal Inhibitory Concentration (IC50) Identification DPPH free radical scavenging activities of prepared 1,3,4-oxadiazoles were evaluated and compared to those of ascorbic acid.28 1 mL of any oxadiazole at concentrations 25, 50, 75, and 100 ^g mL-1 in methanol was added to 4 mL of 0.004% (w/v) methanolic solution of DPPH. The mixed solutions were stored at room temperature for 30 min in darkness. Then, the absorbance of the solutions was read against the blank at Amax 517 nm. The inhibition percentage (I%) was calculated according to the following equation: Wher A(blank) and A(sample) are the absorbance of control and sample solutions, respectively. A graph of I% (y-axis) vs. concentration (x-axis) was plotted. The IC50 is Beyzaei et al.: Ultrasound-Assisted Synthesis, Antioxidant Activity 112 Acta Chim. Slov. 2021, 68, 109-117 x in the equation of a straight line y = mx + b, while y = 50. Finally, the ^g mL-1 units were converted to the mM units. 2. 3. Computational Details All the geometries were optimized in the gas phase and frequency calculations confirmed the nature of the stationary points. The methanol solvent was modelled using C-PCM as implemented in Gaussian 09 program package at the B3LYP level of density functional theory using the 6-311++G (d) basis set.29-32 NCI analysis was performed by the NCIPlot software.33 3. Results and Discussion 3. 1. Synthesis and Spectroscopic Characterization of 1,3,4-Oxadiazol-2-amines 3a-h 5-Substituted 1,3,4-oxadiazol-2-amine derivatives 3a-h were synthesized via reaction of hydrazides 1a-h and cyanogen bromide (2) under ultrasonic irradiation (Scheme 2). Potassium bicarbonate and absolute ethanol were applied as the base and the solvent, respectively. e: 3-HO-C6H4; f: 3-H3CO-C6H4; g: 3-Br-C6H4; h: 4-Pyridinyl Scheme 2. Reaction scheme of the synthesis of 1,3,4-oxadi-azol-2-amines 3a-h. The interaction of 1 mmol of both benzhydrazide (1b) and cyanogen bromide was selected as the model reaction for the preparation of 1,3,4-oxadiazoles (Table 1). Water and hydrous solvents were not used as a result of hydrolysis of cyanogen bromide to hypobromous acid and hydrogen cyanide. Absolute ethanol as a green, nontoxic, readily available, low-cost, water soluble, nonexplosive, easily removable, and recoverable solvent was selected and preferred to the toxic methanol. 2 mL of ethanol was used in all processes as the minimum solvent required to dissolve the most reactants and perform the reaction. Only nucleophilic attack of NH2 group of hydrazide to cyano group of cyanogen bromide occurred at room temperature (Table 1, entry 1). Intramolecular cyclization to 3b occurred at temperatures above 60 °C (Table 1, entry 2). The product yield and the reaction time were improved under refluxing conditions due to the increased solubility of the hydrazide and effective interactions between the reagents (Table 1, entry 3). Higher yields were achieved at similar times in the presence of bases such as potassium bicarbonate (1 mmol) and potassium carbonate (0.5 mmol); increasing basicities had no effect on the yield and time. They only neutralized hydrogen bromide produced during the process so that it would not react with the hydrazide. Relative cyanogen bromide cleavage was observed in the presence of potassium hydroxide. The reaction progress was checked in the presence of KHCO3 as a nontoxic and inexpensive base under ultrasound irradiation at 50 °C. 1,3,4-Oxadiazole 3b was afforded in the shortest time with the highest yield under these conditions (Table 1, entry 7). Table 1. Optimization of reaction conditions in the synthesis of 5-phenyl-1,3,4-oxadiazol-2-amine (3b). Entry Base T (°C) Time (h) Yield (%) 1 - r.t. 3 - 2 - 60 9 68 3 - reflux 7 72 4 khco3 reflux 5 81 5 k2co3 reflux 5 81 6 KOH reflux 5 75 7 khco3 ))), 50 3.5 92 1,3,4-Oxadiazoles 3a-h were synthesized under optimized conditions. The results are given in the Table 2. Table 2. The data of ultrasonic-assisted reactions of hydrazides 1a-h and cyanogen bromide in ethanol yielding 1,3,4-oxadiazoles 3a-h. Product R Time (h) Yield (%) 3a ch3 7 81 3b C6H5 3.5 92 3c 4-O2N-C6H4 4.5 88 3d 4-(H3C)3C-C6H4 5 93 3e 3-HO-C6H4 6 83 3f 3-H3CO-C6H4 6 90 3g 3-Br-C6H4 3.5 92 3h 4-Pyridinyl 2.5 84 The chemical structure of all synthesized 1,3,4-oxa-diazoles 3a-h was determined by spectral data. Recorded melting points are already in agreement with previously reported findings.24-27 The absorption bands around 1120 and 1660 cm-1 attributed respectively to C-O and C=N stretching vibrations confirmed the formation of 1,3,4-ox-adiazole rings. In 1H NMR spectra, singlet or broads peaks in the range of 6.85 to 7.58 ppm belong to amino groups. In 13C NMR spectra, signals corresponding to the asymmetric 2- and 5-carbons of 1,3,4-oxadiazole ring have appeared in the 156.2-165.0 ppm range. Beyzaei et al.: Ultrasound-Assisted Synthesis, Antioxidant Activity Acta Chim. Slov. 2021, 68, 109-117 113 The reaction between hydrazides 1 and cyanogen bromide is a straightforward method to synthesize 1,3,4-oxadiazole derivatives 3. Several procedures were developed for this purpose. Faizi et al. prepared 1,3,4-ox-adiazole 3b in 62% yield from the reaction of benzhy-drazide and a 1.22 molar excess of cyanogen bromide in boiling methanol for 4 h.34 Some 1,3,4-oxadiazol-2-amine derivatives were synthesized via the stirring of mixture including hydrazides and 1.5 molar excess of both cyanogen bromide and potassium bicarbonate in acetonitrile water mixture (v/v 6.25:93.75) for 1 day at room temperature.35 Katritzky et al. proposed a convenient method for the preparation of 1,3,4-oxadiazole 3b; the mixture of symmetric and asymmetric di(benzotriazolyl)methanimines prepared from 1,2,3-benzotriazole and cyanogen bromide was reacted with benzhydrazide in THF under reflux for 3 h to afford 3b in 94% yield.36 3. 2. Antioxidant Evaluation of the Synthesized Compounds Free radical scavenging activity of all 1,3,4-oxadi-azoles 3a-h was assessed against DPPH. The inhibitory effects were calculated as IC50 values and are reported in Table 3. 1,3,4-Oxadiazole derivatives 3a-h exhibited antioxidant activities in the following order: 3c > 3h > 3a > 3e > 3f > 3b > 3g > 3d which, were less than that of vitamin C. No antioxidant property was observed with derivative 3d containing 5-(4-(tert-butyl)phenyl) substituent. Unlike 3-(5-amino-1,3,4-oxadiazol-2-yl)phenol (3e), 1,3,4-oxadiazoles 3c and 3h with electron-withdrawing nitro and pyridinyl groups exhibited significant effects. Methylation of hydroxy group of derivative 3e did not significantly alter its antioxidant properties; this probably indicates a lack of hydrogen-atom donation of OH substituent in 3e. 3. 3. Non-Covalent Interactions (NCI) Analysis We have also used NCI index, while it is a visualization index based on the electron density (p) and the reduced density gradient (s). It is based on the empirical observation that NCI can be associated with the regions of small reduced density gradient at low electronic densities. In quantum chemistry, the NCI index is used to visualize non-covalent interactions in three-dimensional space. Its visual representation arises from the isosurfaces of the reduced density gradient colored by a scale of strength. The strength is usually estimated through the product of the electron density and the second eigenvalue (XH) of the Hessian of the electron density at each point of the isosurface, with the attractive or repulsive character being determined by the sign of XH. This allows for a direct representation and characterization of non-co-valent interactions in the three-dimensional space, including hydrogen bonds and steric clashes. Being based on the electron density and derived scalar fields, NCI indexes are invariant with respect to the transformation of molecular orbitals. Furthermore, the electron density of a system can be calculated by X-ray diffraction experiments as well as theoretical wavefunction calculations (Figure 1).37-39 Table 3. Antioxidant activity of 1,3,4-oxadiazole derivatives 3a-h. Products 3a 3b 3c 3d 3e 3f 3g 3h Vitamin C IC50 (mM) 0.237 0.306 0.114 0 0.273 0.284 0.863 0.222 0.022 °°°8D50 0.010 0.030 0020 -OOLO 0000 OOIO 0.020 00:30 0.0-40 00' $ign(Xx)p (a.u.) Beyzaei et al.: Ultrasound-Assisted Synthesis, Antioxidant Activity 114 Acta Chim. Slov. 2021, 68, 109-117 00-8050 -0.0+0 -0.030 -0 020 -O.OLO 0 000 0 010 0 020 0.030 0 040 0.050 signiX^p (a.u.) Beyzaei et al.: Ultrasound-Assisted Synthesis, Antioxidant Activity Acta Chim. Slov. 2021, 68, 109-117 115 Figure 1. Plots of the reduced density gradient (RDG) versus sign (X2 p and NCI isosurface (isovalue = 0.8 au) of compounds 3a-h. Beyzaei et al.: Ultrasound-Assisted Synthesis, Antioxidant Activity 116 Acta Chim. Slov. 2021, 68, 109-117 The reduced density gradient (s) is a scalar field of the electron density (p) that can be defined as: ' 1 2f3n2)V3 p(r)4/3 3. 4. Radicalization Energy and BDE Theory The BDE of radicals generated from compounds 3a-h were calculated and are reported in Table 4, they were arranged according to the increasing BDEs in moving from the top to the bottom on this table. Based on calculations and BDE theory, the stability of free radicals decreases as we go from left to right across the periodic table.40 Accordingly, the product 3e tends to form radical from nitrogen side; oxygen atom is more electronegative than nitrogen atom and its partially empty orbital is being held more closely to the positively charged nucleus. Thus, radical 3e(NH2) is more stable than radical 3e(OH) by about 4 kcal mol-1. Mesomeric and/or inductive electron-withdrawing groups such as nitro destabilize free radicals; as radical 3c is about 3 kcal mol-1 less stable than radical 3b. Radical 3g containing 3-bromophenyl substituent is about 1 kcal mol-1 more stable than radical 3b; this slight energy difference may be due to the dual nature of the halogen atoms (electron-withdrawing inductive effects versus electron-donating mesomeric effects). Unexpectedly, the replacement of C-4 in benzene with one nitrogen atom (4-pyridyl ring instead of a phenyl ring) in 3h can increase the stability of corresponding radical. Table 4. Calculation of energy opt + frq of compounds 3a-h using DFT method in 6-311++G (d) basis set. Com- Reactants Radicals BDE pounds (a.u.) (a.u) (kcal mol-1) 3h -564.5609986 -563.9221562 400.88 3e(OH) -623.7550404 -623.1156937 401.20 3d -705.7909084 -705.1461778 404.57 3b -548.5353695 -547.8896743 405.18 3e(NH2) -623.7550392 -623.1091857 405.28 3f -663.0593888 -662.4135282 405.28 3g -3119.639242 -3118.991964 406.17 3a -356.7947813 -356.1463463 406.90 3c -753.0392 -752.38958 407.64 4. Conclusions In this study, a new and efficient procedure was proposed for the synthesis of 1,3,4-oxadiazol-2-amines. They were prepared in good to excellent yields under ultrasound irradiation as promotion of the reaction between hydrazides and cyanogen bromide. Hydrogen-atom-donating abilities of all synthesized heterocycles were evaluated against DPPH radical. Acceptable to good antioxi- dant properties of the derivatives candidate them as potent antidiabetic, antiproliferative, anti-inflammatory and an-ti-neurodegenerative agents. 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J. Dannenberg, S. Dapprich, A. D. Daniels, O. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski, D. J. Fox, Gaussian 09, Revision A 02, Gaussian, Inc., Wallingford CT, USA, 2009. 30. S. J. A. Pople, J. Comput. Chem. 2004, 25, fmv-viii. DOI:10.1002/jcc.20049 31. M. J. S. Dewar, E. G. Zoebisch, E. F. Healy, J. J. P. Stewart, J. Am. Chem. Soc. 1985, 107, 3902-3909. DOI:10.1021/ja00299a024 32. J. J. P. Stewart, J. Comput. Chem. 1989, 10, 209-220. DOI:10.1002/jcc.540100208 33. J. Contreras-Garcia, E. R. Johnson, S. Keinan, R. Chaudret, J. P. Piquemal, D. N. Beratan, W. Yang, J. Chem. Theory Comput. 2011, 7, 625-632. DOI:10.1021/ct100641a 34. M. Faizi, S. Dabirian, H. Tajali, F. Ahmadi, E. Rezaee Zavareh, S. Shahhosseini, S. A. Tabatabai, Bioorg. Med. Chem. 2015, 23, 480-487. DOI:10.1016/j.bmc.2014.12.016 35. G. D. Fallon, C. L. Francis, K. Johansson, A. J. Liepa, R. C. J. Woodgate, Aust. J. Chem. 2005, 58, 891-900. DOI:10.1071/CH05070 36. A. R. Katritzky, B. V. Rogovoy, V. Y. Vvedensky, K. Kovalen-ko, P. J. Steel, V. I. Markov, B. Forood, Synthesis 2001, 2001, 897-903. DOI:10.1055/s-2001-13400 37. E. Pastorczak, C. Corminboeuf, J. Chem. Phys. 2017, 146, 120901. DOI:10.1063/1.4978951 38. E. R. Johnson, S. Keinan, P. Mori-Sanchez, J. Contreras-Garcia, A. J. Cohen, W. Yang, J. Am. Chem. Soc. 2010, 132, 6498-6506. DOI:10.1021/ja100936w 39. J. Contreras-Garcia, W. Yang, E. R. Johnson, J. Phys. Chem. A 2011, 115, 12983-12990. DOI:10.1021/jp204278k 40. I. Hamlaoui, R. Bencheraiet, R. Bensegueni, M. Bencharif, J. Mol. Struct. 2018, 1156, 385-389. DOI:10.1016/j.molstruc.2017.11.118 Povzetek Razvoj sinteznih postopkov za pripravo 1,3,4-oksadiazolskih derivatov je bil vedno v središču pozornosti raziskovalcev, saj tovrstni heterocikli izkazujejo množico uporabnih bioloških aktivnosti. V tej študiji smo z reakcijo med različnimi hidrazidi in cianogen bromidom s pomočjo ultrazvočnega valovanja, ob prisotnosti etanola kot topila in kalijevega hi-drogenkarbonata kot baze, uspešno sintetizirali serijo 1,3,4-oksadiazol-2-aminov, ki smo jih izolirali z 81-93 % izkoristki. Antioksidativne lastnosti pripravljenih spojin smo določili s pomočjo metode lovljenja DPPH prostih radikalov; to je ena izmed najbolj osnovnih stopenj pri identifikaciji povezanih bioloških učinkov. Izmerjene IC50 vrednosti so bile v območju 0.237 to 0.863 mM. Sintetizirani 1,3,4-oksadiazoli lahko služijo kot spojine, ki ščitijo pred oksidativnim stresom, in se lahko uporabljajo za zdravljenje raka, kandidiaze, sladkorne bolezni ter nevrodegenerativnih in inflamatornih obolenj. S pomočjo teorije gostotnega polja (DFT) smo izračunali energije disociacije vezi (BDE) in elektronske gostote, ki temeljijo na nekovalentnih interakcijah (NCI). Ugotovili smo, da reverzibilne sile dipol-dipol igrajo ključno vlogo pri večini interakcij. Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Beyzaei et al.: Ultrasound-Assisted Synthesis, Antioxidant Activity DOI: 10.17344/acsi.2020.6212 Acta Chim. Slov. 2021, 68, 118-127 /^creative ^commons Scientific paper Antidiabetic Potential of Stem Bark Extract of Enantia chlorantha and Lack of Modulation of Its Therapeutic Efficacy in Diabetic Rats Co-Administered with Lisinopril Latifat Bolanle Ibrahim, Patience Funmilayo Idowu, Opemipo Adekanye Moses, Mutiu Adewunmi Alabi* and Emmanuel Oladipo Ajani Department of Medical Biochemistry and Pharmacology, Faculty of Pure and Applied Sciences, Kwara State University, Malete, P. M. B. 1530, Ilorin, Nigeria * Corresponding author: E-mail: mutiu.alabi@kwasu.edu.ng Phone: +234-7030428661 Received: 08-18-2020 Abstract This study validates the antidiabetic efficacy of Enantia chlorantha stem bark and the possible therapeutic implications of the co-administration of lisinopril and E. chlorantha in type 2 diabetic rats. E. chlorantha stem bark was extracted by cold maceration. The inhibitory effect of the plant on carbohydrate metabolizing enzymes and its antioxidative potentials were assessed in vitro. The extract exhibited a-amylase and a-glucosidase inhibitory activities and also showed antioxidative properties in vitro. Administration of the extract normalized fasting hyperglycemia in vivo by showing 47.24% reduction in blood glucose levels relative to untreated diabetic rats. Co-administration of E. chlorantha and lisinopril restored serum glucose and serum lipid profile levels. E. chlorantha stem bark displayed antidiabetic potentials as compared with a standard antidiabetic drug (metformin). The study also showed that the plant contained some bioactive compounds which we hypothesize might be responsible for the observed activities. Co-administration of the plant with lisinopril conferred no significant therapeutic advantage on the serum glucose level and lipid profile. Keywords: Antidiabetic; Antioxidants; Co-administration; Enantia chlorantha; Hyperglycemia 1. Introduction Diabetes mellitus is a chronic metabolic disorder that affects the metabolism of carbohydrates, fats and proteins. It is characterized by hyperglycemia which can result from, the pancreas not producing enough insulin or cells of the body not responding properly to the insulin produced. Diabetes mellitus can cause long-term complications such as heart disease, stroke and dysfunction and failure of various organs.1 The three main types of diabetes are type 1, type 2 and gestational diabetes. Both women and men can develop diabetes at any age. Diabetes is associated with major abnormalities in fatty acid metabo-lism.2 The most common lipid pattern in type 2 diabetes consists of hypertriglyceridemia, low High-Density Lipoprotein Cholesterol (HDL-C) and normal plasma levels of Low-Density Lipoprotein Cholesterol (LDL-C).3,4 Type 2 diabetes is one of the primary threats to human health due to its increased prevalence and associated complications. Many and diverse therapeutic strategies for the treatment of type 2 diabetes are known. The conventional treatments for diabetes include the reduction of the body's demand for insulin, stimulation of endogenous insulin secretion, enhancement of the action of insulin at the target tissues and the inhibition of degradation of oligo and disaccharides by enzyme inhibitors.5,6 Currently, met-formin is considered the initial medication of choice for hyperglycemia in type 2 diabetes due to its effectiveness. Metformin is a biguanide class of antihyperglycemic drug which acts primarily by enhancing the action of insulin in the liver to reduce the rate of hepatic glucose production, to decrease glucose absorption and to increase target cell Ibrahim et al.: Antidiabetic Potential of Stem Bark Extract 119 Acta Chim. Slov. 2021, 68, 118-127 insulin sensitivity.7 Improvements in insulin action in skeletal muscle also contribute to the therapeutic actions of metformin, mainly resulting in increased non-oxidative glucose disposal.8 Together, these actions reduce blood glucose in the setting of hyperglycemia, with very little potential for inducing hypoglycaemia.9 Moreover, the enzymes alpha-glycosidase, is responsible for the breakdown of oligosaccharides, disaccharides and/or polysaccharides to monosaccharides and a-amylase degrades starch to more simple sugars (dextrin, maltotriose, maltose and glucose).10 The inhibitory action of these enzymes leads to a decrease of blood glucose level, because the monosaccha-rides are the form of carbohydrates which are absorbed through the mucosal border in the small intestine. However, many of the synthetic hypoglycemic agents have their limitations; are non-specific, produce serious side effects and fail to alleviate diabetic complications. The main side effects of these agents are gastrointestinal i.e. bloating, abdominal discomfort, diarrhea, and flatulence.11 There is need to develop effective, safe and cheap drugs for diabetes management because of the side effects associated with the present antidiabetic drugs. Drugs from medicinal plants are effective, safe and cheap for the management of diabetes. Many clinical studies have supported the view that utilization of herbal medicines could be a reliable alternative to manage diabetes effectively with little or no adverse effect.12,13 Enantia chlorantha belongs to Annonaceae family. This plant is commonly known as African yellow wood. Among the Yoruba in Nigeria it is known as Awopa.14 In traditional medicine, this plant has been used for a long time in many parts of the African continent to treat various ailments of the human body. Many of these uses are supported by several studies.15-18 For example, it was reported that a decoction of 500 g of stem bark in 3 l of water for 20 min may be used to treat malaria symptoms, aches, wounds, boils, vomiting, yellow fever, chills, sore, spleen in children and hepatitis.15 Lisinopril is an oral long-acting angiotensin converting enzyme (ACE) inhibitor. ACE inhibitors (ACE-Is) are a family of drugs commonly prescribed to combat hypertension. The primary vasodilatory action of ACE-Is is the blockage of ACE and thus preventing the formation of angiotensin II.19 With long-term administration, ACEIs lower blood pressure, even in patients with low renin hypertension. This thus suggests that effect of lisinopril may be independent of a decrease in angiotensin II. The appropriate blood pressure control in diabetes trial found that diabetic patients treated with ACE-Is had lower incidence of myocardial infarction (MI) and overall cardiac events. ACE-Is have been used for years to reduce the rate of diabetic nephropathy progression in patients with type 2 dia-betes.19 Thus, this drug may be efficacious in treatment of patients with type 2 diabetes. Herb-Drug interactions (HDIs) are either pharma-codynamic (PD) or pharmacokinetic (PK) in nature. For the former, this occurs when co-administered substances enhance or negate each other's effects as a result of similar or disparate pharmacological activities, respectively.20 Such interactions may render the drug less effective or change its activity and producing adverse effect. PK interactions on the other hand arise from the ability of the substance to modulate the absorption, distribution, metabolism and/or excretion (ADME) of the drug.20 Despite the fact that E. chlorantha is commonly used among the local communities in Nigeria in the management of diabetes, scientific data in support of this local medicinal use in diabetes is lacking in the literatures. Moreover, whereas, the general practice in the management of diabetes in Nigeria is to combine the use of antidi-abetic and antihypertensive agents, no study has reported on the therapeutic implications of combining E. chlorantha with any hypertensive agent. In view of the foregoing, the present study investigates the anti-diabetic properties of E. chlorantha stem bark extract and the thermodynamic implications of its co-administration with lisinopril. In addition, the study investigated the phytochemical constituents of the plant to identify the bioactive components that may be responsible for the pharmacological antidiabetic properties and the possible mechanism for its pharmacological action. 2. Materials and Methods 2. 1. Materials Streptozocin was a product of Sigma-Aldrich, USA. Metformin (Glucophage 500 mg) was manufactured by Merck Santé, France and Lisiofil (Lisinopril 5 mg) was manufactured by Fourrts India Laboratories Pvt Ltd., India. All the reagent kits used for bioassay were sourced from Randox Laboratories Ltd., Crumlin, UK. All other chemicals were of analytical grade. 2. 2. Methods 2. 2. 1. Plant Collection, Identification and Crude Extract Preparation The stem bark of E. chlorantha was collected in June 2018. The plant sample was identified and authenticated and a voucher number UIH/001/1356 was assigned. Thereafter, a sample specimen was deposited at the herbarium of the University of Ilorin, Ilorin, Nigeria. The stem bark was cleaned to remove adhering dirt, air-dried for two weeks and ground into powder using an electric blender. Extraction was carried out by cold maceration of 800 g of the coarse powder with 5 L of 70% ethanol for 72 h, with constant shaking. The resultant mixture was filtered using Whatman filter paper (No.1) and the filtrate was concentrated using a rotary evaporator at 40 °C. The extract was weighed, and the final yield was 12.5%. The dried extract was finally reconstituted in distilled water for use in the study. Ibrahim et al.: Antidiabetic Potential of Stem Bark Extract ... 120 Acta Chim. Slov. 2021, 68, 118-127 2. 2. 2. Qualitative and Quantitative Phytochemical Screening Qualitative phytochemical analysis of the extract was carried out using the method previously described21 to identify phytochemicals while quantitative phytochemical screening was carried out using different method previously described, for saponins,22 tannins,23 alkaloids24 and flavonoids.25 2. 3. Antioxidant Assay 2. 3. 1. 1,1-Diphenyl-2-picryl-hydrazyl Radical Inhibition The method previously described26 was used to evaluate the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging potential of the extract. One (1) ml of different concentrations (0.2-1.0 mg/ml) of the extract or vitamin C (reference) were added to 1 ml of 0.2 mM methanolic solution of DPPH. Similarly, sterile distilled water (1 ml) was mixed with an aliquot (1 ml) of 0.2 mM methanolic DPPH and used as control. Following incubation (30 min, 25 °C) in each case, the absorbance was read against blank at 516 nm using a spectrophotometer (Beckman, DU 7400, USA). The inhibitory effect (I%) of EOAE on DPPH radical was estimated as follow: (1) where A control is the absorbance of the control and A test is the absorbance of the test sample. Thereafter, the concentration of the extract eliciting 50% inhibitory (IC50) effect on the DPPH radical was calculated from a standard curve. 2. 3. 2. Hydrogen Peroxide Inhibition This was estimated using the adapted method previously described.27 Briefly 0.6 ml of 40 mM H2O2 was mixed with 3.4 ml of phosphate-buffered (pH 7.4) solution (0.21.0 mg/ml) concentrations of either the extract or vitamin C and incubated at 25 °C for 10 min. Sterile distilled water replaced the extract for the control sample. The absorbance was read spectrophotometrically at 230 nm. The H2O2 inhibitory potential of the extract was calculated as follow: % H2O2 scavenged = X 100 (2) Where, A control is the absorbance of the control; A test and A sample represent the absorbance of the mixture with the extract and that of the extract alone, respectively. The IC50 value was estimated from the standard curve. 2. 3. 3. Hydroxyl Radical (OH ) Inhibition The OH. inhibitory effect of the extract was determined as previously described.28 In brief, 2 ml at 0.2-1.0 mg/ml of the extract or vitamin C (reference) were mixed with 0.6 ml of ferrous sulfate (8 mM), 0.5 ml of H2O2 (20 mM), and 2 ml of salicylic acid (3 mM). After 30 min of incubation (37 °C), distilled water (0.9 ml) was added and the resulting mixture centrifuged (Beckman and Hirsch, Burlington, IO, USA) at 4472 g for 10 min. For the control, sterile distilled water was used. The absorbance was read at 510 nm, and the IC50 value was calculated subsequent to determination of inhibitory capacity of the extract against OH' using the expression: % hydroxyl radical scavenged _ A test - A sample A control x 100 (3) Where, A control, A test, and A sample represent the absorbance of the control, mixture with the extract, and that of the extract alone, respectively. 2. 3. 4. Reducing Power Activity The reducing power of extract was determined by previously described method.29 One (1) ml of extracts or gallic acid (reference) was mixed with 2.5 ml of phosphate buffer (200 mM, pH 6.6) and 2.5 ml of 1% potassium ferri-cyanide. The mixtures were incubated for 20 min at 50 °C. After incubation, 2.5 ml of 10% trichloroacetic acid were added to the mixtures, followed by centrifugation at 4000 rpm for 10 min. The upper layer (5 ml) was mixed with 5 ml of distilled water and 1 ml of 0.1% ferric chloride and the absorbance of the resultant solution were measured at 700 nm. 2. 4. In vitro Carbohydrate-Metabolizing Enzymes' Inhibitory Assay 2. 4. 1. Alpha-Amylase Inhibition Assay The alpha amylase inhibitory assay was performed as previously described method.30 Briefly, concentrations (50-200 mg/ml) of the extract or acarbose (standard) were prepared and 50 ^l of the extract or acarbose was added to 500 ^l of 20 mM sodium phosphate buffer (pH, 6.9, with 6 mM NaCl) containing porcine pancreatic alpha-amylase (0.5 mg/ml) and incubated at 25 oC for 10 min. One unit of the enzyme will liberate 1.0 mg of maltose from starch in 3 min. Then, 500 ^l of 1% starch solution in 20 mM sodium phosphate buffer (pH 6.9, with 6 mM NaCl) was added to each tube. The reaction mixture was incubated at 25 oC for 10 min and stopped with 1.0 ml of 3,5-dinitrosa-licylic acid colour reagent. Thereafter, the mixture was incubated in a boiling water bath for 5 min and cooled to room temperature. The reaction mixture was then diluted by adding 10 ml of distilled water, and absorbance was measured at 540 nm. The control experiment was performed without the test sample, and the a-amylase inhibi- Ibrahim et al.: Antidiabetic Potential of Stem Bark Extract ... Acta Chim. Slov. 2021, 68, 118-127 121 tory activity was expressed as percentage inhibition using the following equation: (4) Where AA and AA controi are the respective changes in absorbance of the extract sample and control. The IC50 of the extract against a-amylase activity was thereafter calculated from a standard calibration plot. 2. 4. 2. Alpha-Glucosidase Inhibition Assay The assay was performed as previously described.31 Briefly, known concentrations (50-200 mg/ml) of the extract or acarbose (standard) were prepared and 50 ^l of the extract or acarbose was added to 100 ^l of alpha-glucosi-dase solution (1.0 U/ml) in 0.1 M phosphate buffer (pH, 6.9) and incubated at 25 oC for 10 min. One unit of the enzyme will liberate 1.0 ^mol of D-glucose from p-nitro-phenyl-a-D-glucoside per min. Then, 50 ^l of 5 mM p-ni-trophenyl-a-D-glucopyranoside solution in 0.1M phosphate buffer (pH 6.9) was added. The mixture was incubated at 25 °C for 5 min, and the absorbance was measured at 405 nm. The control experiment was performed without the test sample, and the a-glucosidase inhibitory activity was expressed as percentage inhibition using the following equation: (5) Where AA extract and AA controi are the respective changes in absorbance of the extract sample and control. The IC50 of the extract against a- glucosidase activity was thereafter calculated from a standard calibration plot. 2. 5. Experimental Protocols 2. 5. 1. Experimental Animals The experiment was carried out on healthy forty-nine (49) male Wistar rats of about 10-12 weeks old and weighing an average of169 ± 6 g. The rats were housed in metallic cages at the animal house. The rats were acclimatized for fourteen days and fed with commercial diets and water ad libitum. They were all maintained at 25 ± 2 °C light and dark cycle of 12/12 hr, respectively. 2. 5. 2. Induction of Diabetes Type 2 diabetes was induced by the previously described method.32 The rats were first fed 15% fructose solution (w/v) for four weeks, after which they were fasted overnight and thereafter administered streptozotocin (40 mg/kg i.p.) freshly prepared in 0.1 M sodium citrate buffer. The diabetic state was confirmed 72 h after streptozotocin injection. Specifically, rats having fasting blood glucose levels greater than 200 mg/dl were considered diabetic. 2. 5. 3. Animal Grouping/Administration Forty-two (42) diabetic male Wistar rats were divided into six (6) groups consisting of seven (7) rats each: Diabetic groups consisted of DC - Diabetic Control group; treatment groups (T1 - E. chlorantha (200 mg/kg b.w.), T2 - Metformin (7.14 mg/kg b.w.), T3 - E. chlorantha + lisino-pril (200 mg/kg b.w. and 0.14 mg/kg b.w. respectively), T4 - Metformin + lisinopril (7.14 mg/kg b.w. and 0.14 mg/kg b.w. respectively) and T5 - lisinopril (0.14 mg/kg b.w.)) and another seven (7) non-diabetic male rats acted as the Normal control group. All administrations were carried out orally as a single dose daily for four weeks using a gavage needle. The rats were housed in cages in the Department Animal Facility Center maintained at 25 ± 2 °C light and dark cycles of 12/12 hr. The chosen dose of the extract and the route of administration were informed by both the results of our ethnobotanical survey on the use of the plant in the management of diabetic and the reported minimum effective (ME) and maximum safe (MS) dose of the plant.33,34 The rats were maintained in accordance with the principles of laboratory animal care guidelines.35 The weight of the rats was determined every week throughout the experiment period. The experiment was designed according to the Department Animal Ethics Committee guidelines and approval certificate (KSUMB/005/01/013) was given. 2. 5. 4. Monitoring of Blood Glucose Level during Treatment All blood samples for monitoring of blood glucose level in situ were taken from the tail vein of the rats using 24 gauge needles at intervals of 0, 5, 10, and 15 days. Blood glucose levels were determined by the glucose oxidase method using reactive strips and a single touch glucome-ter (Accu-Chek Active, Roche Diagnostics, Mannheim, Germany). Results were recorded in mg/dl. Percentage reduction in fasting blood glucose was calculated as: I Reduction of PB3 = Initiai value - Final value Initial value (6) 2. 6. Biochemical Assay Twenty-four hours after the last treatment, under mild diethylether anesthesia, the animals were sacrificed, and blood was obtained via jugular puncture and serum was obtained by centrifugation. Isolated serum was analyzed for total cholesterol, HDL- cholesterol (HDL-C), LDL-cholesterol (LDL-C) and total glycerides (TG) colori-metrically using Randox diagnostic kits. Ibrahim et al.: Antidiabetic Potential of Stem Bark Extract ... 122 Acta Chim. Slov. 2021, 68, 118-127 2. 7. Data Analysis All data were presented as mean ± standard error of mean (S.E.M) of seven replicates. One-way analysis of variance (ANOVA) using SPSS software package for windows (Version 16) for differences between means was used to detect differences between the treatment groups (a < 0.05) followed by the Tukey post hoc test using R statistical software. 3. Results and Discussion 3. 1. Screening and Detection of Phytoconstituents E. chlorantha stem bark gave positive results for some major constituents; alkaloids, saponins, flavonoids, coumarins, anthocyanins and phenolics (Table 1). Ter-penes, terpenoids, steroids and glycosides were not detected. The Table also showed that flavonoid concentration was the highest (114.92 ± 0.36 mg/kg) while saponin concentration was the lowest (40.50 ± 0.71 mg/kg). 3. 2. Antioxidant Assay Presented in Table 2 and Table 3 are the results of the in vitro antioxidant assay of the stem bark extract of E. chlorantha. The extract showed a significant inhibition of Table 1. Qualitative and quantitative phytochemical screening of E. chlorantha Phytochemical Reagent used Observations Result Concentrations group (mg/kg) Alkaloids Wagner's reagent A reddish-brown precipitate + 82.12 ± 0.02 Triterpenes Acetic anhydride No blue green colour - ND Glycosides Fehling's solution No brick red precipitate - ND Saponins Frothing test Frothing precipitate + 40.50 ± 0.71 Tannins KOH No dirty white precipitate - ND Phlobatannin HCl Absence of red precipitate - ND Steroids Salakowsti test No red colouration - ND Flavonoids Ferric chloride Yellow colour after HCl + 114.92±0.36 Coumarin + ND Anthocyanins + ND Terpenoids Liberman Burchard No reddish-brown boundary - ND Phenolics FeCl3 A greenish precipitate + ND + = detected, - = not detected; ND = not determined Table 3. Percentage OH , H2O2 and FRAP Scavenging Activity of E. chlorantha Extract Agent OH' (%) H2O2 (%) FRAP (%) E. chlorantha 102.650 ± 0.939a 42.347 ± 3.002a 148.240 ± 6.912a Reference 118.727 ± 0.000a 67.364 ± 0.000b 168.273 ± 0.000a Data are presented as mean ± SEM of 3 replicates. Value on the same row with similar superscripts are not significantly (P > 0.05) different from each other. Ibrahim et al.: Antidiabetic Potential of Stem Bark Extract ... DPPH radical with an IC50 of 62 mg/ml. The extract also showed a strong scavenging capacity of H2O2 and OH'. In addition, the extract demonstrated Ferric Reducing Antioxidant Power (FRAP). Table 2. % DPPH (2,2-diphenyl-1-picrylhydrazyl) inhibition of E. chlorantha extract Concentration E. Chlorantha Vitamin C (mg/ml) (%) (%) 20 31.781 ± 0.085a 35.474 ± 0.969a 40 61.791 ± 0.040a 67.540 ± 0.085a 60 62.025 ± 0.064a 70.590 ± 0.180a 80 69.990 ± 0.000a 80.720 ± 0.270b 100 79.850 ± 0.085a 90.590 ± 0.200b Data are presented as mean ± SEM of 3 replicates. Value on the same row with similar superscripts are not significantly (P>0.05) different from each other. 3. 3. Effect of Treatment with E. chlorantha on Blood Glucose and Total Body Weight Figure 1 is the result of the effect of treatment on the body weight changes. There was no significant difference in the body weight of all the rats between the groups before treatment. Body weights of rats in diabetic control group were observed to be lower than those in other groups after the treatment period. Acta Chim. Slov. 2021, 68, 118-127 123 On day 5, the fasting blood glucose of the E. chlorantha treated rat (183.6 ± 9.31 mg/dl) was observed to be significantly lower than that of the diabetic untreated group (299.20 ± 6.46 mg/dl). Similar results were obtained in the rats co -treated with E. chlorantha and lisinopril and the group treated with lisinopril only. At day 15, the blood glucose levels of all the treatment groups were observed not to be significantly different from each other but were significantly lower than that of the diabetic untreated group. They were also observed to be significantly higher than that of the normal control group (96.53 ± 0.73 mg/dl). The results also showed that the highest percentage reduction in blood glucose after 15th day of treatment relative to day 0 was observed in the group co-treated with metformin and lisinopril (60.64%). This was followed by the group co-treated with the E. chlorantha and lisinopril (52.70%). The percentage glucose reduction of the group treated with E. chlorantha alone was 47.24. There was no reduction in the glucose level of the normal and diabetic control groups. Table 5 shows the inhibitory potential of E. chlorantha extract on the specific activities of a-amylase and al-pha-glycosidase. The result revealed a dose-related inhibitory effect. The alpha-amylase IC50 values for E. chlorantha extract and acarbose were 90 and 65 mg/ml, respectively. The Table also showed an alpha-glucosidase IC50 of 145 and 125 mg/ml for the extract and acarbose respectively Group Treatment Day 0 Fasting Blood glucose level (mg/dl) Day 5 Day10 Day 15 Total % reduction Control Distilled water 95.12 ± 0.45a 95.56 ± 0.56a 95.08 ± 0.62a 96.53 ± 0.73a - Diabetic STZ-fructose + distilled 294.00 ± 2.31b 299.20 ± 6.46b 300.80 ± 6.67b 301.60 ± 6.93b - control water T1 Extract 235.80 ± 18.94b 183.6 ± 9.31c 135.80 ± 5.60c 124.40 ± 2.35c 47.24% T2 Metformin 305.40 ± 21.55b 287.60 ± 2.54b 207.00 ± 8.25d 152.80 ± 5.83c 50.09% T3 Extract + Lisinopril 272.80 ± 11.56b 200.60 ± 3.26c 151.40 ± 6.12c 129.60 ± 2.08c 52.70% T4 Metformin + lisinopril 271.80 ± 18.33b 268.60 ± 9.60b 182.60 ± 6.14d 146.80 ± 8.09c 60.64% T5 Lisinopril only 248.33 ± 14.52b 199.83 ± 9.06c 171.00 ± 4.80d 151.00 ± 7.55c 39.10% Data are presented as mean ± SEM of 7 determinations. Values in the same column with the same superscripts are not significantly (P > 0.05) different from each other. Table 5. Inhibitory potential of E. chlorantha ethanolic extract on the activity of a amylase and a glucosidase (n = 3, mean ± SD). Concentrations % Inhibition (mg/ml) a- Amylase a- Glucosidase Acarbose E. chlorantha Acarbose E. chlorantha 50 32.87 ± 3.00 44.52 ± 5.41 20.72 ± 0.18 18.67 ± 0.06 100 54.11 ± 4.01 60.89 ± 0.24 31.32 ± 0.25 34.96 ± 1.01 150 62.35 ± 2.91 74.22 ± 2.13* 54.70 ± 4.13 64.96 ± 3.09 200 67.21 ± 0.79 84.44 ± 2.14* 73.42 ± 0.13 87.93 ± 2.04* IC50 65.00 90.00* 125.00 145.00* Data are presented as mean ± SEM of 3 replicates. ""indicates that value differ significantly (P < 0.05) from the respective reference (acarbose). Ibrahim et al.: Antidiabetic Potential of Stem Bark Extract ... Fig. 1. Effect of treatment on Total Body Weight of Rats. NC - normal control group, DM - diabetic control group, T1 - E. chlorantha treated group, T2 - Metformin treated group, T3 - E. chlorantha + lisinopril treated group, T4 - Metformin + lisinopril treated group and T5 - Lisinopril treated group. Table 4 presents the result of the fasting blood glucose level. Fructose -STZ administration exhibited a significant increase in fasting blood glucose (235.80 ± 18.94 mg/ dl) as compared to the normal control group (95.12 ± 0.45 mg/dl). Before the commencement of treatment (Day 0), the blood glucose levels of all the treatment groups were not different from that of the diabetic non treated group. Table 4. Effect of treatments on fasting blood glucose levels 124 Acta Chim. Slov. 2021, 68, 118-127 3. 4. Effect of Treatment on Serum Lipid Profile The effect of treatments on serum lipid profile (Table 6) showed a significant increase in serum total cholesterol, total glycerides and LDL-C level and a decrease in the serum HDL-C level in the diabetic group when compared with the control group. Treatment with the extract restored the serum lipid levels. The serum levels of the lipids obtained in the rats treated with the extract alone were not significantly different from those of the diabetic rats treated with metformin but were different from diabetic rats treated with the metformin and lisinopril and the rats co-treated with the extract and lisinopril. The Table also showed that the concentration obtained in the diabetic rats treated with lisinopril alone was different from that of the control group and that of the diabetic control group. 3. 5. Discussion The currently available drugs for management of diabetes mellitus have certain drawbacks and therefore, there is a need to find safer and more effective antidiabetic drugs. Result from this study indicates that E. chlorantha stem bark extract is efficacious as an antidiabetic agent in rats and that the efficacy of the extract was not significantly altered when E. chlorantha was co-administered with lisinopril suggesting that combined administration of the plant with lisinopril does not increase its therapeutic indications. Medicinal plants received much attention due to presence of important bioactive secondary metabolites such as phenolics.36 Result of our phytochemical screening showed that E. chlorantha stem bark consist of a large proportion of phytochemicals which may play a role as antidiabetic. We reported in this study, that the bioactive compounds contained in the stem bark of the plant belong to the group of alkaloids, saponins, flavonoids, coumarins, anthocyanin and phenolics. The flavonoids were detected to be of highest concentration. Similar studies have also reported the presence of saponins, flavonoids, alkaloids and phenols in aqueous extract of E. chlorantha.37 However, it was reported that alkaloids had the highest content (46.26%).37 These phytochemicals could act in a number of potential mechanisms to show their antidiabetic activities. Some of the potential mechanisms include increase in insulin secretion and action, decreases in hepatic glucose output, regulation of certain enzymes involved in carbohydrate metabolism i.e. a-amylase and a-glucosidase, modulation of certain regulation molecules such as PPARy (Peroxisome Proliferator Activated Receptor-Y), hypolipidemic activities, antioxidant effects, enhancement of the expression of glucose transporters etc.38,39,40 Both flavonoids and alkaloids had been widely implicated in antidiabetic properties of plants. It was noted that some alkaloids e.g. nuciferine promotes glucose stimulated insulin secretion in rats' pancreatic islets, probably via a pathway involving hepatic nuclear factor 4a or by closing potassium-adenosine triphosphate channels.41 Some alkaloids (i.e. mescaline, pyrrole, pyridine, tropane, aporphine, and quercetin) have been reported to have an-tioxidant and antimicrobial properties.42,43 Studies on the antidiabetic potential of flavonoids from plants showed that flavonoids regenerate pancreatic islets and increase insulin secretion in streptozotocin (STZ)-induced diabetic rats.44 It also stimulates insulin release and enhance glucose uptake from isolated islet cells.45 Streptozotocin (STZ)-fructose type 2 diabetes model shares a number of features with human type 2 diabetes mellitus (TDM2) both histologically and metabolically and is characterized by moderate stable hyperglycemia.46,47 This is why in this study, STZ-fructose induced diabetes model was used. Streptozotocin injection caused ^ cells degeneration in rats, resulting in decrease in the release of insulin by the pancreas. Furthermore, high fructose ingestion causes insulin resistance (IR). This contributes negatively to blood glucose homeostasis thereby inducing hy-perinsulinemia which predispose to type 2 diabetes. Result of fasting blood glucose of > 200 mg/dl obtained in this study following STZ-fructose administration confirms induction of type 2 diabetes. This is similar to report from previous findings.48,49,50 Our study also showed that changes in body weight of fructose-streptozotocin-induced diabetes is associated with characteristic loss of body weight. This we hypothesized may due to increased muscle wasting and possibly Table 6. Effect of treatments on serum lipid profiles Group Treatment LDL-C (mg/dl) HDL-C(mg/dl) TG (mg/dl) TC (mg/dl) Control Distilled water 1.26 ± 0.02a 1.26 ± 0.82b 0.78 ± 0.82a 0.87 ± 0.97a Model STZ-fructose + distilled water 6.09 ± 0.95c 0.56 ± 0.07a 1.50 ± 0.47b 1.66 ± 0.48b T1 Extract 2.08 ± 0.41ab 0.97 ± 0.40b 0.89 ± 0.50a 0.98 ± 0.09a T2 Metformin 2.83 ± 0.06ab 1.10 ± 0.63b 0.93 ± 0.29a 1.06 ± 0.79a T3 Extract + Lisinopril 1.73 ± 1.00ab 1.07 ± 0.55b 0.77 ± 0.93a 0.85 ± 0.16a T4 Metformin + lisinopril 2.36 ± 0.71ab 1.18 ± 0.70b 0.81 ± 0.03a 0.96 ± 0.30a T5 Lisinopril only 3.08 ± 0.56b 0.97 ± 0.17b 1.07 ± 0.20a 1.08 ± 0.91a Data are presented as mean ± SEM of 7 determinations. Values in the same column with the same superscripts are not significantly different (p > 0.05) from each other. Ibrahim et al.: Antidiabetic Potential of Stem Bark Extract ... Acta Chim. Slov. 2021, 68, 118-127 125 loss of tissue proteins which is similar to the previous observations.50,51 As expected in the diabetic control group, the body weight of rats was progressively reduced whereas, in all the E. chlorantha treated rats and those co-administered with lisinopril there was a progressive improvement in the body weight. This indicates that treatment prevented muscle tissue damage associated with hyperglycemic condition. In the management of diabetes mellitus, alpha-gluco-sidase and alpha-amylase enzymes represent the most crucial of the pharmacological targets.52,53 These enzymes facilitate hydrolysis of starch to glucose with consequential increase in the systemic concentration of glucose. Hence, the inhibition of these enzymes activities delays glucose absorption and moderates postprandial blood sugar level.53 In this study, the in vitro alpha-amylase inhibitory activities of the ethanol extract of E. chlorantha were investigated. The extract (50-200 mg/ml) exhibited potent a-amylase inhibitory activity in a dose dependent manner. This was similar to what was obtained with acarbose. The IC50 obtained with acarbose was lower than that of E. chlorantha extract. Furthermore, the alpha-glycosidase inhibitory assay of the ethanolic extract of E. chlorantha stem bark revealed a significant inhibitory action of alpha-glucosidase enzyme. The percentage inhibition at 50-200 mg/ml concentrations of E. chlorantha extract also showed a dose dependent increase with an IC50 of 145 mg/ml. Similarly, acarbose showed alpha-glucosidase inhibitory activity with an IC50 value of 125 mg/ml. The result indicates that the ethanolic extract of E. chlorantha is a potent alpha-amylase and alpha-glucosidase inhibitor similar to acarbose. Previous reports had noted that alpha-amylase and alpha-gluco-sidase are the main pharmacological targets in the management of diabetes.52 These enzymes facilitate hydrolysis of starch to glucose with consequential increase in the systemic concentration of glucose in diabetes. This increased hyperglycemia may constitute a significant risk factor for diabetic complications. Our study suggests that E. chlorantha stem bark may be used as starch blockers indicating that the plant may prevent or slow the absorption of starch in to the body mainly by blocking the hydrolysis of 1,4-gly-cosidic linkages of starch and other oligosaccharides into maltose, maltriose and other simple sugars. Administration of E. chlorantha stem bark extract to diabetic rats caused significant reduction of blood glucose level compared to the control and diabetic untreated group. Many previous studies have provided evidence that oxidative stress resulting from increased reactive oxygen species (ROS) is a key factor in the pathogenesis of diabetes.53,54 Our study showed that E. chlorantha elicited marked antioxidant potentials suggesting that the plant has the capacity to regulate or stall free radicals chain reactions associated with diabetes complications, which is in agreement with previous report.53 Natural products are the major source of antioxidants which delay the development of diabetes.40 The role of dyslipidemia in the development of diabetes macrovascular complications has been reported.1,4 In this study, the STZ-fructose model of type 2 diabetes exhibited abnormalities in lipid metabolism as evidenced by the significant elevation of serum TC, TG, LDL-C and reduction of HDL-C levels. A previous study reported that treatment with metformin significantly reduced the TC, TG, LDL-C level and increased HDL-C levels in diabetic rats.55 The extract was shown to improve the condition of diabetic mellitus as indicated by the lipid profile monitored in the study, thus showing its good antidiabetic activity in STZ-fructose-induced hyperglycemic rats. Lisino-pril, an angiotensin converting enzyme inhibitor (ACE-I) acts by preventing the formation of Ang II, which has also been implicated in insulin resistance by inhibiting insulin receptor dependent PI3K signaling.56 Therefore, the blockade of this substance is important in affecting insulin sensitivity. The co-administration of lisinopril and the extract did not show any difference from that achieved with the extract alone. Serum total cholesterol, LDL cholesterol and triglycerides decreased in all the treated groups when compared with the model group. No significant difference was however observed in all these parameters when compared among the treatment groups. Serum HDL cholesterol was also observed to increase in all treatment groups compared with the model group, but no difference was observed when compared among the treatment groups. In previous study, inhibitors of the renin angiotensin system, such as angiotensin converting enzyme (ACE) inhibitors was reported to ameliorate the lipid abnormalities to a substantial extent.57 4. Conclusion The use of E. chlorantha in the management of diabetes is a common practice among some local communities in Nigeria. Data obtained from this study indicates that E. chlorantha is efficacious as antidiabetic agent and that combined administration of E. chlorantha and lisinopril does not in any way influence the efficacy of E. chlorantha stem bark as an antidiabetic agent. The study identified phytoconstituents belonging to the phenolics, flavonoids, saponins and alkaloids as some of the bioactive compounds which may be responsible for this pharmacological property. Study has just been concluded in our laboratory to evaluate the toxicological implication of the combined administration of these agents.58 Abbreviations HDL-C: High-Density Lipoprotein Cholesterol LDL-C: Low-Density Lipoprotein Cholesterol ACE: Angiotensin converting enzyme HDI: Herb-Drug interactions DPPH: 1,1-diphenyl-2-picryl-hydrazyl Ibrahim et al.: Antidiabetic Potential of Stem Bark Extract ... 126 Acta Chim. Slov. 2021, 68, 118-127 TDM2: Human type 2 diabetes mellitus ROS: Reactive Oxygen Species PPARy : Peroxisome Proliferator Activated Receptor-y 5. References 1. G. Keerthana, M. K. Kalaivani, A. Sumathy, Asian J. Pharm. Clin. Res., 2013, 6, 32-36. 2. G. H. Tomkin, Diabetes Care, 2008, 31, S241-S248. DOI:10.2337/dc08-s260 3. B. A. Salau, O. Osilesi, G. O. Idowu, S. Musa, E. O. 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Med., 1983, 118, 246-254. DOI: 10.7326/0003-4819-118-4-199302150-00002 58. E. O. Ajani, L. B. Ibrahim. Clin. Phytosci., 2020, 6, DOI:10.1186/s40816-020-00174-z Povzetek Ta študija potrjuje protidiabetično učinkovanje lubja Enantia chlorantha in morebitne terapevtske posledice sočasne uporabe lizinoprila in E. chlorantha pri podganah s sladkorno boleznijo tipa 2. Lubje E. chlorantha je bilo ekstrahirano s hladno maceracijo. Inhibitorni učinek rastline na encime, ki presnavljajo ogljikove hidrate, in njene antioksidativne potenciale so bili ovrednoteni in vitro. Ekstrakt je izkazoval inhibitorno delovanje na a-amilazo in a-glukozidazo ter antioksidativne lastnosti in vitro. Aplikacija ekstrakta in vivo je normalizirala hiperglikemijo na tešče, tako da je znižala raven glukoze v krvi v primerjavi z nezdravljenimi diabetičnimi podganami za 47,24 %. Sočasna uporaba E. chlorantha in lizinoprila je normalizirala nivo glukoze v serumu in nivo serumskega lipidnega profila. Lubje E. chlorantha je pokazalo antidiabetični potencial v primerjavi s standardnim antidiabetikom (metformin). Študija je pokazala tudi, da je rastlina vsebovala nekatere bioaktivne spojine, za katere domnevamo, da bi lahko bile odgovorne za opažene učinke. Sočasna uporaba rastline z lizinoprilom ni prinesla pomembnega terapevtskega izboljšanja ravni glukoze v serumu in lipidnega profila. © 0 Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Ibrahim et al.: Antidiabetic Potential of Stem Bark Extract ... DOI: 10.17344/acsi.2020.6225 Acta Chim. Slov. 2021, 68, 128-136 /^creative ^commons Scientific paper Application of Silica Supported Calix[4]arene Derivative as Anti-reversion Agent in Tire Tread Formulation Hediye Mohamadi,1 Fereshteh Motiee,1,* Saeed Taghvaei-Ganjali1 and Mandana Saber-Tehrani1 1 Chemistry Department, IA-University, North Tehran Branch, Tehran 1651153311, Iran * Corresponding author: E-mail: f_motiee@iau-tnb.ac.ir Phone: +9877009800 Received: 06-27-2020 Abstract In this research the influence of the silica supported calix[4]arene derivative (SS-Calix) on the reversion resistance, mechanical properties and thermal behavior of NR/BR tire tread formulation was investigated by the oscillating disc rheometer, FTIR, TGA and tensile testing. The results revealed that the reversion behavior of NR/BR vulcanizate is affected by SS-Calix. The data obtained from curing characteristics and thermal stability of test pieces indicate that, SS-Calix acts as an anti-reversion for rubbery materials that are exposed to thermal shock in the early stages of temperature rise. It's predicted that these results are due to the interaction between the OH groups present in the SS-Calix surface and the carbon of the polymer chains. The broad peak observed in the IR spectrum around 1824 cm-1 which is referred to C=O bond, confirms this prediction. In addition, the presence of SS-Calix in compound causes to increase modulus and hardness but reduce elongation and resilience. Keywords: Mechanical properties; rubber; thermal properties; tire tread formulation; silica supported calix[4]arene; perkalink900. 1. Introduction Reversion refers to the loss of crosslink density. It occurs especially in compound containing natural rubber (NR) and some synthetic rubber (Isoprene Rubber) when polysulphidic crosslinks are exposed to temperature -time procedure which causes breakdown of polysulphidic crosslinks to di-sulfides and mono-sulfides crosslinks. This occurrence leads to a reduction in crosslink density and consequently loss of functional properties of rubber products. The best method for evaluation of the reversion is based on rheometer torque curves. In this curves, the negative slope indicates the reversion phenomenon.1,2 Since reversion decline modulus and mechanical properties of the compound, there are some approaches in the industry to reduce it. One of these methods is the applying of efficient (EV) and semi-efficient (Semi-EV) vulcanization systems. The crosslinks created by these curing systems are usually mono-sulfide and disulfide type which has a higher thermal stability compared to polysulfide cross- links.3,4 Efficient (EV) and semi-efficient (Semi-EV) vulcanization systems improved mechanical properties and thermal aging characteristics, but because the length of the mono and disulfide bonds is less than the polysulfide, the flexibility and fatigue life of rubber compounds are reduced.5 Also in such curing systems due to faster cure rates and lower total sulphur levels the adhesion of rubber to metal and rubber to fabric is very low.6 One of the approaches used to control the reversion phenomenon is the employing of anti-reversion agent. Perkalink900 (1, 3-bis-citraconimidomethyl benzene) is the most commonly used anti reversion agent in the rubber industry. Perka-link900 prevents loss crosslink density by creating new crosslinks through the well-known Diels-Alder reaction (Figure 1).7,8 In recent years, there have been numerous studies on the reversion and its kinetic.9,10 In 2019, Nah et al. vulcanized chlorobutyl rubber and applied 4, 4'-bis (maleimi-do) diphenyl methane (BMDM) as a crosslinking agent. They examined rheometric curves and reported that Mohamadi et al.: Application of Silica Supported Calix[4]arene Acta Chim. Slov. 2021, 68, 128-136 129 Figure 1. Creating new crosslinks by Perkalink900 8 BMDM significantly enhanced the reversion resistance.11 In 2016, they applied BMDM in NR vulcanized with sulphur and described that the presence of 5 phr of BMDM significantly improves reversion resistance without adversely affecting the scorch time and cure rate. It also enhancement crosslink density and drops compression set value.12 In 2017, Sousa et al. revealed that the presence of devulcanized rubber chain in the compound has a deleterious effect on the mechanical properties of curing blends.13 In 2015, Milani et al. invented new approach to determine reversion kinetic constants.14 Calixarenes is a macromolecule with hydrophobic cavities which represent one of the most studied host systems known as host-guest chemistry. Calixarenes and their derivatives have many applications in membrane electrodes, construction of polyurethane foams and composites, remove ions, sensors, catalyst, adsorption, binders, detection and extraction.15-21 In rubber industry, they used as filler,22 coupling agent,23 tackifier resin,24 adsorbent of CBS, DPG25 and antioxidant.26 Prevent the reversion phenomenon using anti-reversion agents has been investigated by different researchers, while the possible influence of calixarenes as anti-reversion element have not been properly studied in the existing literature. In this project, attempt has been made to investigate the role of silica supported calix[4]arene (SS-Calix) as an anti-reversion agent in tire tread formulation based on NR/ BR blends. The SS-Calix was prepared according to available source22,27 and applied as an anti-reversion agent in rubber specimens. The anti-reversion value of vulcanized samples was assessed by comparing the rheometric curves of blends containing SS-Calix and Perkalink900. 2. Experimental 2. 1. Materials NR (SMR-20) was provided from Teh Ah Yau Rubber Factory (Malaysia). BR (High Cis-96%) was purchased by Shazand Petrochemical Company (Iran). Carbon Black 660 was supplied by Doodeh Sanati Pars Company (Iran). The commercial Silica (Ultrasil VN3) was supplied by Evonik Company (Germany). Aromatic oil was purchased from Iranol Co. (Tehran, Iran). Silane (Si69) was supplied by Shin-Etsu (japan). ZnO was provided from Shokohiye zinc oxid (Iran). Stearic Acid was supplied by AcidChem Company (Malaysia). Sulfur (OT-20) and other ingredients including accelerators were purchased from Taizhou Huangyan Donghai Chemical Co., Ltd. (China). Wax was provided from behran Factory (Iran). Perkalink 900 was supplied by RheinChemie Company (Germany). 25, 26, 27, 28 - tetrahydroxy - 5, 11, 17, 23 - tetrakis [cholorosul-fonyl] calix[4]arene (SS-Calix) was prepared according to the literature.22,27 Para tert-butyl phenol, hydrochloric acid, diethyl ether, sodium hydroxide, chloro sulfonic acid, di chloro methane, di phenyl ether were provided from Merck company (Germany) and formaldehyde, acetic acid, ethyl acetate, xylene and acetone were purchased from Mojalali company (Iran). 2. 2. Preparation of Rubber Compounds The SS-Calix was introduced to the blends as a novel anti-reversion agent, along with accelerated sulfur for the Semi-EV vulcanization of NR/BR compound. The tire tread formulation of the rubbery samples which has been used in this study is shown in Table 1. The compound that contains silica as filler, silane as coupling agent is designated as SP1 and it is used as control. The specimen that contains silica as filler, silane as coupling agent and Perkalink900 as anti-reversion agent is designated as SP2. SS-Calix is introduce to SP3 compound as filler, coupling agent and anti-reversion agent. Mixing was performed in a laboratory size two roll mixing according to ASTM D3184. SP1 sample preparation: Natural rubber was initially masticated for 2 minutes then Butadiene Rubber was loaded into the roll mixing. Silica, Si69, aromatic oil and carbon black were added and mixed. Subsequently, the other ingredients: ZnO, stearic acid, TMQ, 6PPD and wax were added and mixed. In the second stage, sulfur, TBBS were added to the compound. SP2 sample preparation: Preparation of SP2 sample is similar to SP1 sample. Only in the final stage, perkalink 900, in addition to sulfur and TBBS, were added to the blend. Although, in samples SP1 and SP2, the amount of carbon black (46 phr) is higher (almost 3 times) than silica, however, in order to ensure the salinization of the blend, the mixing operation was performed for a longer period of time and at the maximum possible temperature compared to sample SP3 Mohamadi et al.: Application of Silica Supported Calix[4]arene 130 Acta Chim. Slov. 2021, 68, 128-136 Table 1. Compound Formulations Ingredient Sample ( phr*) SP1 SP2 SP2 NR 70 70 70 BR 30 30 30 Carbon Black 660 46 46 46 Aromatic Oil 15 15 15 ZnO 3 3 3 Silica Ultrasil VN3 17 17 - Si69 0.5 0.5 - Perkalink900 - 1 - SS-Calix - - 17 Stearic Acid 1 1 1 TMQ 1 1 1 6PPD 1 1 1 Wax 2 2 2 TBBS 1.75 1.75 1.75 Sulphur 1.4 1.4 1.4 *phr represented the mass parts per 100 mass parts of NR/BR blend Where V1 is the molar volume of the solvent and V2 is the volume fraction of rubber in the swollen. X is an interaction parameter between the polymer and the swelling agent. 2. 3. 2. Cure Characteristics The curing behavior of specimens was investigated from the torque curves generated by a Monsanto Moving Die Rheometer HIWA900 MDR (HIWA, Iran) at 160 °C. 2. 3. 3. Mechanical Properties Specimens were cut from vulcanized blends with 2 mm thickness. The tensile strength and tear resistance test were carried out by using a tensile tester (Testometric M350) at room temperature according to ISO37. Hardness of the vulcanized was determined according to the ASTM D2240 method by ShoreA (Bareiss, Germany). Resilience was measured by Hiwa300 (HIWA, Iran) according to DIN 53512. SP3 sample preparation: In the first stage, natural rubber and butadiene rubber was masticated by laboratory two roll-mills of 8 inch diameter and 20 inch length. Following the compounding process, SS-Calix and carbon black added to the blend, then other components (aromatic oil, wax, stearic acid, zinc oxide, TMQ, 6PPD) except curing agents mixed with performing ingredients. Finally, the curatives (sulfur, TBBS) were added to the compound. The compounding of this sample at a maximum temperature of 75 °C lasted approximately 30 minutes. At all stages, the materials that falls on the tray under the rollers was collected by the brush and added again to the compounds. After mixing, in order to rest the compounds and control the expansion or shrinkage of polymer chain, samples were stored at room temperature for 24 h. 2. 3. Measurements 2. 3. 1. Determination of Crosslink Density The swelling tests were used to calculate the crosslink density of three different rubber vulcanisates according to ASTM D471. The rubber pieces were cut from the compression-molded rubber sample weighting about 0.2-0.25 g, and swollen in toluene to reach equilibrium, which took 72 hours at room temperature. The cured test pieces were taken out from the liquid, the toluene was removed from the sample surfaces wiped with tissue paper, the weight after swelling were measured. The samples were then dried in the oven until constant weights were obtained. The swelling results were used to calculate the crosslink density by applying the Flory-Rehner manner [equation (1)].28 2. 3. 4. Thermal Gravimetric Analysis (TGA) In order to understand the thermal stability of samples, thermo gravimetric analysis was performed on the TGA-SF1 (Mettler, Switzerland) apparatus using 15mg samples in the temperature range 25-800 °C and under nitrogen atmosphere, at a heating rate of 10 °C/min. 2. 3. 5. Fourier Transform Infrared Spectroscopy (FTIR) Fourier transform infrared spectroscopy (FTIR) was obtained by Bruker FTIR spectrometer (Germany). 3. Result and Discussion 3. 1. Crosslink Density The crosslink densities of the NR/BR composite were designated by the Flory-Rehner manner [equation (1)]. The results are presented in Table 2. As it can be seen the SP3 specimen have the most great crosslink density. This observation is due to the role of SS-Calix as filler and coupling agent and form more crosslinks. This is due to the interaction between the OH groups present in the SS-Calix structure and the rubber chains. The formation of C=O bonds observed in the IR spectrum of the samples is the evidence of this incident. Table 2. The results of crosslink density of three different samples V = SP1 Sample SP2 SP3 (1) Crosslink Density ( x 10-4 mol/cm3) 2.0812 2.596 2.9395 Mohamadi et al.: Application of Silica Supported Calix[4]arene Acta Chim. Slav. 2021, 68, 128-136 131 3. 2. Cure Characteristics The best well-known compounding method to minimize reversion is the application of anti-reversion agents, thereby diminishing the number of polysulphidic crosslinks and generating reversion resistant mono and disulphidic crosslinks. The curing behavior of three various resulting materials is displayed in Figure 2. The cure rate index (CRI), the difference between maximum and minimum torque (AM = MH-ML), scorch time (ts2) and optimum cure time (tc90) are listed in Table 3 and Figure 3. The start time of the reversion is reported in Table 4 and its value at different times is shown in Table 4 and Figure 4. The role of ZnO as activator depends on the type of rubber matrix and accelerator. The mechanism of zinc cat- Table 4. Time to start the reversion and the value of reversion at different times for three various samples 16 20 Time (mini Figure 2. The curing curve of compounds based on NR/BR for SP1, SP2, and SP3 Table 3. The results of curing behavior of three different samples SP1 Sample SP2 SP3 ts2(Min) 6.69 7.09 7.55 tc90 (Min) 9.32 10.59 10.13 CRI 35.52 28.58 7.59 AM 7.59 7.59 8.01 SP1 Sample SP2 SP3 TSR* (min) 14 15 15 Revt14 (dN.m) 0.14 0.00 0.00 Revt15 (dN.m) 0.28 0.14 0.14 Revt16 (dN.m) 0.28 0.14 0.14 Revt17 (dN.m) 0.42 0.28 0.14 Revt18 (dN.m) 0.42 0.28 0.28 Revt20 (dN.m) 0.69 0.28 0.42 Revt22 (dN.m) 0.83 0.28 0.42 Revt24 (dN.m) 0.97 0.41 0.56 Revt26 (dN.m) 1.11 0.55 0.69 Revt28 (dN.m) 1.11 0.55 0.69 Revt30 (dN.m) 1.24 0.69 0.83 *TSR: The start time of Rev (min) Figure 3. The curing characteristics of NR/BR blends Time (Min) Figure 4. The reversion value of NR/BR blends at different times alyzed sulfur vulcanization is very complex, but zinc-based complexes play a main role in determining the nature of the cross-linked products. Formation rate of zinc-accelerator complex determines scorch time of rubbery materials.1 As shown in Figure 3, the maximum scorch time value is related to SP3 compound which treated by SS-Calix as an anti-reversion agents. The reason for this increase could be the physical interact of accelerators with the SS-Calix cavities.22 Because of this, the formation of zinc-accelerator complex has delay and it caused to increases the scorch time. Another result of the rheometer's curve is 5% increase of AM in the SP3 specimen over the other two compounds and it indicates despite delayed, accelerator act its effect as good as before. AM indirectly indicates the crosslink density of rubber samples, an increase in AM and crosslink density in SP3 compound demonstrates an improvement in the curing properties of test pieces. As can be seen, the tc90 in SP3 and SP1 compounds are approximately equal but in SP2 (contain Perkalink900), tc90 has increased so the cure rate index (CRI) has drastically decreased. This decrement in curing rate can be attributed to the negative effect of Perkalink900 on the rate of vulcanization. Mohamadi et al.: Application af Silica Supported Calix[4]arene 132 Acta Chim. Slov. 2021, 68, 128-136 Another important and delicate point that can be considered from the torque curve of these three compounds is their reversion behavior which is significantly different at a certain time. The onset of reversion and its relationship with increasing curing time can be seen in Table 4 and Figure 4 for all samples. As the rheometer curves displayed the onset of reversion in SP1 sample is earlier than two other samples. In SP2 and SP3, the reversion begins simultaneously, but then it behaves differently. As can be seen from the results, at t17 of curing, the lowest amount of reversion (0.14 dN.m) is related to SP3. At the same time, the reversion of SP1 and SP2 is three times and twice that of SP3, respectively. However, at t18, reversion of SP1 and SP3 increases, but the value of this phenomenon in SP2 remains unchanged up to t22. At this point SP2 and SP3 samples have the same reversion resistant. The reversion of SP1 and SP3 will be increased after t20. The highest value of reversion is related to SP1 and the lowest is belonging to SP2 blends. This trend is continued and indicated the anti-reversion behavior of three samples as fallowed (Figure 4): SP2>SP3>SP1 As Figure 4 shows, the point to consider in this phenomenon is the performance of SS-Calix in SP3 blend as an anti-reversion agent in a certain curing temperature range (t15-t17). It seems that SS-Calix, in addition to being reinforcing filler that does not require a coupling agent 22, it can be act as anti-reversion agent in rubbery materials that exposed to thermal shock. In fact, it acts as an anti-reversion in the early stages of temperature rise for rubbery materials that are exposed to thermal shock. It is predicted that at the first 2 minutes of reversion, the polymer chains which crosslinks have been broken and radicalized by the reversion phenomenon form new positive interaction with the radicals created in the SS-Calix. As a result, it reduces the reversion. Because a limited number of calix moieties are bonded to the silica surface, over time, the number of calixarenes radicals on the silica surface are finished and the radical reaction of SS-Calix with elastomer chains are quenched. As a result, the anti-reversion effect of SS-Calix decreases through the time. In general, it can be suggested that the ability of Perka-link900 as an anti-reversion is slightly higher than the SS-Calix agent. In Table 5 the influence of the type of anti-reversion agents on the mechanical properties of resulting materials is shown. Figure 5a. Results of tensile strength, modulus100% and modulus 300% for SP1, SP2 and SP3 Figure 5b. Results of the elongation for SP1, SP2 and SP3 Hardness (shoreA) Figure 6. Results of hardness and resilience for SP1, SP2 and SP3 The results of the tensile strength, modulus100%, modulus 300% are displayed in Figure 5a and the result of the elongation is shown in Figure 5b. Hardness and resilience tests for all compounds are shown in Figure 6. As Figure 5a shows the tensile strength of both SP3 and SP2 compounds increase in comparison with SP1 and Table 5. The results of tensile, hardness and resilience properties of three different samples Sample Tensile Modulus100% Modulus 300% Elongation Hardness Resilience Strength (MPa) (MPa) (MPa) (%) (Shore A) (%) SP1 14.07 2.35 7.71 491 58 47 SP2 17.80 1.65 6.30 639 54 56 SP3 17.65 2.76 9.50 504 60 49 Mohamadi et al.: Application of Silica Supported Calix[4]arene ... Acta Chim. Slov. 2021, 68, 128-136 133 they are not significantly different from each other. So Per-kalink900 and SS-Calix have the same effect on tensile strength of rubber products. It seems that due to the mac-romolecule structure of the SS-Calix and the presence of many hydroxyl groups and cavities in this system, polymer chains have been interacted with SS-Calix. As a result, the compound structure has become tighter, therefore the tensile strength has increased in comparison with SP1 compound. Figures 5a and 6 show that the highest modulus and hardness is related to SP3 rubbery compound and the lowest modulus and hardness is related to SP2. In fact, Perkalink900 has a negative effect on modulus and hardness. The increase in modulus and hardness in SP3 blend is due to the increase in crosslink density in this compound. Increasing the density of crosslinks also reduces the elongation and resilience of SP3 (Figures 5b and 6). It should be noted that the crosslink density effect on the tensile strength, modulus, elongation at break, resilience and hardness.29-31 3. 3. The Thermal Gravimetric Analysis In order to investigate the thermal resistance of three different compounds thermal gravimetric was performed. Figure 7 show the weight loss behavior for all compounds. The data of thermal analysis for all samples are summarized in Table 6. The results show that the onset temperature of degradation of SP2 is lower than that of SP3 and this process continues up to 45% of degradation. After that, the destruction temperature of both samples up to 90% is almost the same. However, due to chemical and physical interactions between SS-Calix and SP3 blend it Figure 7. The weight loss behavior of SP1, SP2 and Sp3 specimens Mohamadi et al.: Application of Silica Supported Calix[4]arene 134 Acta Chim. Slav. 2021, 68, 128-136 Table 6. The TGA data for three different samples SP1 Sample SP2 SP3 Tin (°C) 320 290 320 Ts* (°C) 329 310 329 Tl5% (°C) 370 360 380 T25% (°C) 386 378 388 T35% (°C) 402 392 408 T45% (°C) 431 419 435 T50% (°C) 442 442 445 T550/0 (°C) 461 451 452 T65% (°C) 561 480 482 T75% (°C) 570 569 570 T85% (°C) 580 573 580 T95% (°C) 582 580 582 seems reasonable that the thermal resistance of SP3 is higher than that of SP2. 3. 4. Fourier Transform Infrared Spectroscopy (FTIR) The FTIR for all compounds were showed in Figure 8. The Fourier transform infrared spectroscopy was performed to evaluate the anti-reversion behavior of SP3 blend. Since the interpretation of the FTIR spectrum of rubber samples is so complex, only strong bands were considered in this study. The absorption curves of the SP3 blend showed that in addition to the bands characteristic for calix[4]arenes,27 there is a broad peak around 1824 cm-1 which is referred to C=O bond.32 As we know, the most important feature of SS-Calix is the position of the OH stretching bond in the 3100-3500 cm-1 region. The considerably weaker C=O bond for SP1 and SP2 blends is attributed to the fact that the oxygen of hydroxyl groups of the calixarene surface forms a C=O with the carbon in the polymer chain.33 3. 5. The Conversion Rate of Vulcanization By using equation (2), the conversion rate of vulcanization at various times had evaluated for all compounds (Table 7).34 (2) In this equation, Xt indicate the conversion rate at the given time t. Mt, MH, ML and MH-ML represent torque at the given time t, the maximum Torque, the Minimum Torque and the difference between maximum and minimum Torque, respectively. The value of X5 indicate the delay in scorch time for SP3 specimen compared to SP2. The conversion ratio of SP1 and SP3 at X13 equaled 1, which revealed that Mt=MH Figure 8. The FTIR for SP1, SP2 and Sp3 specimens Table 7. The conversion rate of vulcanization at various times for all compounds SP1 Sample SP2 SP3 X5 0.05 0.05 0.03 X10 0.95 0.85 0.86 X13 1.00 0.98 1.00 X14 0.98 1.00 1.00 X15 0.96 0.98 0.98 X16 0.96 0.98 0.98 X17 0.94 0.96 0.98 X18 0.94 0.96 0.96 X20 0.91 0.96 0.95 X22 0.89 0.96 0.95 X24 0.87 0.94 0.93 X26 0.85 0.93 0.91 X28 0.85 0.93 0.91 X30 0.84 0.91 0.90 Mohamadi et al.: Application af Silica Supported Calix[4]arene Acta Chim. Slov. 2021, 68, 128-136 135 and the 90% of curing is done, for SP2 sample it happened in the X14, While for SP3 sample in X14 still remains 1. The SP3 conversion rate up to X17 almost is more than SP2 (Table 7). These findings predicted that; I: The reversion resistance of SP3 up to about X17 is more than SP2 and SP1. II: The second important point is that after X17 up to X30, less reversion occurs for SP2. III: The SP3 rubber sample exhibits high thermal resistance in a short time. These outcomes confirm the result from curing behavior of three different samples. 4. Conclusion Since the reversion has an undesirable effect on the performance properties of the rubber compounds, this study investigated the influence of the presence of SS-Calix as a novel anti-reversion on the reversion resistance of rubber blend based on NR/BR. Its anti-reversion properties were also compared with perkalink900. Finally, the following results are obtained: • The presence of SS-Calix in compound based on NR/ BR increase the modulus and hardness also decrease the resilience and the elongation. • In a few minutes after the start of reversion the reversion resistance effect of the SS-Calix was much better than perkalink900. • The presence of SS-Calix increases the formation of C=O bonds in SP3 sample that cause to increase reversion resistance in the early minutes after the start of reversion. • Application of the SS-Calix to NR/BR rubber products subject to thermal shock is recommended. • In NR/BR rubber compound that contains SS-Calix the initial temperature degradation until T45% are more than perkalink900. Based on these findings, it can be predicted that this feature of calix[4]arene can be applied in the preparation of rubber products that must have high thermal resistance in a short time. 5. Reference 1. B. 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Feng, Polym. Advan. Technol. 2019, 30, 5. D0I:10.1002/pat.4555 33. C. D. Gutsche, in: Calixarenes, Royal Society of chemistry, Cambridge, 2008, p.70. 34. K. Chawla, A. P. S. Chauhan, A. Pandey, Plast. Rubber. Compos. 2016, 45, 6. DOI: 10.1080/14658011.2016.1178969 Povzetek Raziskali smo vpliv silikatnega nosilca ojačanega s kaliks [4] arenskim drivatom (SS-Calix) na zmanjševanje zamreženja, mehanske lastnosti in termično obnašanje NR/BR pnevmatik s pomočjo reometra z oscilacijskim diskom, FTIR, TGA in napetostnega testa. Rezultati so pokazali, da je zmanjševanje zamreženja NR/BR vulkanizata odvisno od dodatka SS-Ca-lix-a. Rezultati pridobljeni iz meritev značilnosti zamreževanja in termične stabilnosti testiranih vzorcev so pokazali, da SS-Calix deluje kot preprečevalec zmanjševanja zamreženja za gumijaste materiale izpostavljene termičnemu šoku v zgodnjih fazah naraščanja temperature. Predpostavljamo, da so opažene lastnosti posledica interakcij med OH skupinami prisotnimi na SS-Calix-u in ogljikom v polimernih verigah. Širok vrh opažen na IR spektru okoli 1824 cm-1, ki je značilen za C=O vez, potrjuje to predpostavko. Poleg tega prisotnost SS-Calix-a povečuje napetostni modul in trdoto a hkrati zmanjšuje raztegljivost in odpornost. Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Mohamadi et al.: Application of Silica Supported Calix[4]arene ... DOI: 10.17344/acsi.2020.6226 Acta Chim. Slov. 2021, 68, 137-143 /^creative ^commons Scientific paper Physicochemical Properties of Octane Isomers in View of the Structural Numbers Anton Perdih Faculty of Chemistry and Chemical Technology, University of Ljubljana (retired) Vecna pot 113, 1000 Ljubljana, Slovenia * Corresponding author: E-mail: a.perdih@gmail.com Received: 06-07-2020 Abstract The structural features of octane isomers were quantified with help of the Structural Numbers. A Mutually Optimized Contribution of the Structural Numbers (MOCSN) was used to calculate which parts of information regarding branching contribute the tested Structural Numbers. Besides the known Structural Numbers, two Asymmetry Numbers were developed in order to quantify the asymmetry of the octane isomers, one regarding the asymmetry along the main chain of the molecule and the other one regarding the asymmetry perpendicular to the main chain of the molecule. Their correlation to the values of 29 tested physicochemical properties of octanes was low, |R| < 0.6. After optimization of the Mutually Optimized Contribution of the Structural Numbers, the Information Content of the Mutually Optimized Contribution of the Structural Numbers ranged from 34.3% to 89.0% of the information contained in the physicochemical properties. The Structural Numbers enable the first step of the structural interpretation of physicochemical properties of octane isomers. In 17 out of 29 cases the most of information contained in the physicochemical properties is presented among the Structural Numbers by the Number of Branches, in 8 cases by the Peripheral Number, in 3 cases by an Asymmetry Number and in 1 case by the Distance Number. Keywords: Octanes; asymmetry number; distance number; number of branches; peripheral number; size of the largest branch; structural interpretation. 1. Introduction In order to estimate the unknown values of the physicochemical properties (PCP) of alkanes, Wiener1 started the development of the numerical measures of their structure, later named the topological indices.2,3 In the structure of alkanes, the branching of their molecules is obvious. Branching of molecules is easily apprehended. It comprises several structural variables, for example the number of branches, their valencies, their distances apart, their distance from the graph center, the lengths of branches, etc.4 However, any definition of branching must rest on an intuitive basis.5 Bonchev and Trinajstic6 presented several rules representing branching. Later, these rules were improved.7 In that time, branching was attempted to be defined using few topological indices,8 either the Wiener1 index or the largest eigenvalue of the adjacency matrix.9,10 As a new basis for the definition of branching the largest eigenvalue of the path matrix was provided.8 There was also stressed the dilemma, whether a branching index should correlate well with physico- chemical properties of alkanes or should it serve its own purpose to index branching regardless the correlation with the physicochemical properties. The attention was focused on the definition of branching, rather than to the application of it.8 Randic and Wilkins11 demonstrated the ordering of structures based on path indices. There have been presented also the intuitively derived sequences of octanes of increasing branching as well as some topolog-ical indices, which order the octane isomers into such sequences. Topological indices, which order the octane isomers into sequences of increasing branching, were, however, not good indices of the physicochemical properties of octanes.12 Later on,13 based on physicochemical properties of octanes, which correlated to R > 0.9 to an intuitively derived sequence of octanes of increasing branching,12 there was introduced the concept and the values of Branching Degrees of octane isomers. There were defined some Structural Numbers as well. Besides the previously known Number of Branches (Nbr), also the values of the Peripheral Number (Nper), the Distance Number (Nd), as well as the Perdih: Physicochemical Properties of Octane Isomers 138 Acta Chim. Slov. 2021, 68, 137-143 Size of the Largest Branch (Lbr) were defined and their correlation to a number of physicochemical properties of octanes was determined.13 In a previous trial, it has been noticed that the symmetry of molecules has some importance in the Principal Component Analysis of physicochemical properties of al-kanes, where the axis PC4 separates first of all the elongated and flat molecules from the spherical ones and among the former ones the symmetric from the asymmetric ones. However, the variance contained in the axis PC4 was low (3% or less). In the case of tested topological indices, it was hardly noticeable.14 To test the importance of symmetry of octanes regarding their physicochemical properties (PCP) in a different way, in present paper two measures of symmetry of octane molecules, i.e two Asymmetry Numbers were developed. They were tested alone as well as in combination with other Structural Numbers. The Structural Numbers are not another type of topological indices. They are the quantification of the structural features of alkanes. In present paper is performed the test, which part of information contained in the physicochemical properties of octanes can be explained using the mutually optimized contribution of the Structural Numbers. For other groups of alkanes, the Structural Numbers of them are to be derived and used using the approach described below. 2. Notation and Physicochemical Properties of Octane Isomers Notations and physicochemical properties of octane isomers were presented in a previous paper.15 As measures of the goodness of correlation were used the correlation coefficient, R, the standard error, S, and the information content regarding branching,15 IC. 4. Results The definitions of the Asymmetry Numbers Nasym^ and Nasyml are as follows: Nasym^ is the difference of distances of branches from the symmetry axis of the main chain of the octane molecule. Nasym± is the difference of the distances of vertices in branches perpendicular to the main chain of the octane molecule. The Asymmetry Numbers are the inverse measures of the symmetry of molecules. For symmetrical molecules any Nasym = 0, whereas for non-symmetrical ones any Nasym > 0. Their values are presented in Table 1. The Asymmetry Numbers were correlated to the physicochemical properties of octanes. At Nasym^, the absolute values of the correlation coefficient, |R|, range from 0.004 to 0.419, whereas at Nasyml the range is 0.016 < |R| < 0.602. This is in line with an earlier observation14 that on the Principal Component Analysis the variance contained in the axis PC4, which separates the symmetric molecules from the asymmetric ones, was low. The highest correlations with the Asymmetry Numbers were observed at the physicochemical properties Solubility Parameter, Cohesive Energy Density, the Antoine parameter B and Surface Tension. These are the physico-chemical properties of low correlation with other Structural Numbers. 15 They correlate negatively with the Asymmetry Numbers indicating that their values are higher among the more symmetrical molecules of octanes. 3. Calculations The software for statistics calculations included in the program package MS Excel was used. The definitions of the Structural Numbers are as follows.13 The Peripheral Number Nper was defined as Nper = Zdsy, where dsy is the distance of a branch from the axis of symmetry of the main chain of the molecular graph. The Distance Number Nd was defined as the distance between two branches. The Size of the Largest Branch Lbr was equal to the number of vertices in the branch. Here are defined as a new Structural Number two types of the Asymmetry Number: Nasym^ and Nasyml. Table 2. Correlation of the Asymmetry Numbers with the number of branches (Nbr), with the peripheral number (Nper), with the distance number (Nd), with the size of the largest branch (Lbr), as well as with the branching degrees of octane isomers (Br. Deg.VL). Struct. No. R Nasym^ D N ^asym± N 0.451 0.166 Lbr 0.031 0.385 Nd -0.296 -0.113 Nbr 0.000 0.067 Br. Deg.VL -0.017 0.087 Br. Deg.VL - Branching degrees derived from the values of the index Vl(-0.126, -0.139, -0.270).11 Table 1. Asymmetry Numbers of octane isomers. Oct 2M7 3M7 4M7 3Et6 25M6 24M6 23M6 34M6 3Et2M5 22M6 33M6 3Et3M5 234M5 224M5 223M5 233M5 2233M4 Nasym± 021010120 1 31 0 0 1 2 1 0 Nasym« 011120200 2 22 1 1 3 1 1 0 Perdih: Physicochemical Properties of Octane Isomers Acta Chim. Slov. 2021, 68, 137-143 139 The correlation of the Asymmetry Numbers with other Structural Numbers are presented in Table 2. In present stage, there is defined also the Mutually Optimized Contribution of the Structural Numbers (MOCsn) containing the Structural Numbers Nbr, Nper, Nd, Lbr, Nasym^, and Nasym±. MOCsn = k-Nbr X Nbr + kNper X Nper + kM X Nd + kLbr X Lbr + k^ X Nasym^ + k± X Nasyml In MOCsn there is I|kNi| = 1. MOCsn was optimized to each physicochemical property of octanes separately. The results are presented in Table 3 for the case of the tested physicochemical properties of octanes. The positive value of a coefficient kNi means that with increasing value of Ni the value of that physicochemical property increases, whereas the negative value of a coefficient kNi indicates the decrease. The optimized MOCSN represents 89.0% of the information regarding branching of octanes contained in Tc/Pc, whereas it represents only 34.3% of the information regarding branching of octanes contained in the critical property ac. The highest correlation with the optimized MOCSN have the physicochemical properties (R = 0.994, IC ~ 90) > Tc/Pc > Tc2/Pc > RON > (R = 0.990) > BON > BP/Tc > Pc > « > (R = 0.980, IC ~ 80). The lowest correlation have the critical properties (R = 0.86, IC ~ 50) > Vc > dc > Zc > ac > (R = 0.75, IC ~ 30). In slightly more than half of tested cases, i.e. in 16 out of 29 cases, |k±| > | k^| indicating that the asymmetry perpendicular to the main chain has a higher influence on the value of a physicochemical property of octanes then the asymmetry along the main chain. This can be observed for Solubility Parameter, the Cohesive Energy Density, the Antoine constant B and AHv. In other cases, especially at the critical properties ac, dc and Vc the reverse is true. As estimated by the absolute value of the factor |kNi|, the contribution of Nbr is higher than the contribution of other structural features at (|kNbr| = 0.7) > « > RON > BON > Tc2/Pc > C > (|kNbr| = 0.6) > MON > BP/Tc > Tc/Pc > R2 > AHv > (|kNbr| = 0.5) > S > Pc > (|kNbr| = 0.4) > d > Vm > (|kNbr| = 0.3) > nD > A > Zc > (|kmr| = 0.25). Table 3. Results of the optimization of the correlation between the MOCSN and the values of tested physicochemical properties of octanes sorted by the Information Content regarding branching,15 IC. PCP kNbr kNper kNd kLbr k« k± R S IC (%) Tc/Pc -0.541 0.231 0.003 -0.158 -0.043 0.024 0.9939 0.093 89.0 Tc2/Pc -0.635 0.088 -0.031 -0.138 -0.007 -0.101 0.9921 55.791 87.4 RON 0.694 -0.079 -0.006 0.150 0.059 -0.012 0.9916 4.753 87.1 BON 0.640 -0.090 0.023 0.171 0.052 0.024 0.9896 4.944 85.6 BP/Tc -0.596 0.201 0.066 -0.082 -0.028 0.027 0.9870 0.001 83.9 Pc 0.429 -0.263 -0.027 0.144 0.049 -0.088 0.9858 0.208 83.2 « -0.698 0.152 0.075 -0.031 -0.010 -0.034 0.9821 0.007 81.2 MON 0.598 -0.066 -0.017 0.262 0.048 0.009 0.9786 6.945 79.4 d 0.305 -0.256 -0.044 0.192 0.053 -0.150 0.9634 0.003 73.2 Vm -0.302 0.257 0.045 -0.191 -0.055 0.150 0.9627 0.716 72.9 nD 0.288 -0.225 -0.086 0.192 0.043 -0.166 0.9616 0.001 72.5 AHv -0.507 -0.086 -0.034 0.124 0.024 -0.225 0.9566 0.064 70.9 BP -0.240 -0.305 -0.011 0.109 0.086 -0.249 0.9554 1.862 70.5 ST 0.113 -0.343 -0.062 0.199 0.046 -0.237 0.9552 0.269 70.4 Tc 0.197 -0.350 -0.048 0.126 0.081 -0.198 0.9493 2.751 68.6 C 0.608 0.018 -0.180 0.033 -0.119 0.042 0.9488 1.300 68.4 S -0.498 -0.075 0.200 -0.067 0.106 0.054 0.9440 1.535 67.0 R2 -0.513 0.073 -0.127 -0.222 0.019 -0.046 0.9402 0.064 65.9 CED -0.072 -0.247 -0.098 0.252 0.033 -0.298 0.9398 0.001 65.8 Sol.par. -0.071 -0.246 -0.098 0.253 0.033 -0.299 0.9392 0.001 65.7 B -0.008 -0.218 -0.221 0.178 -0.068 -0.307 0.9371 9.183 65.1 MR -0.278 0.299 -0.083 -0.163 -0.090 0.087 0.9028 0.085 60.2 AHf°g 0.195 0.343 -0.148 -0.107 -0.124 0.083 0.9055 0.548 57.6 A -0.260 0.101 -0.189 0.120 -0.145 -0.185 0.9041 0.013 57.2 logVP 0.141 -0.101 0.261 -0.170 0.115 0.212 0.8840 0.083 53.3 Vc -0.264 0.284 -0.085 -0.157 -0.159 -0.051 0.8551 0.008 48.2 dc 0.262 -0.287 0.085 0.152 0.163 0.051 0.8460 0.005 46.7 Zc 0.257 0.189 -0.170 -0.051 -0.202 -0.131 0.7697 0.005 36.2 ac -0.243 0.250 -0.219 -0.056 -0.184 -0.048 0.7540 0.095 34.3 Perdih: Physicochemical Properties of Octane Isomers ... 140 Acta Chim. Slov. 2021, 68, 137-143 The contribution of Nper is higher than the contribution of other structural features at (|kNper| = 0.4) > Tc > AHf°g > ST > BP > (|kNper| = 0.3) > MR > dc > Vc > ac > (|kNper| = 0.2), thus at the most of the critical properties of octanes. The contribution of Nasyml is higher than the contribution of other structural features at B > Sol. par. > CED, all (|kNasym±| ~ 0.3). The contribution of Nd is higher than the contribution of other structural features at logVP only. The MOCsn was optimized also to some well-known topological indices of octanes in order to see the situation among them. The results are presented in Table 4. The optimized MOCSN represents 88.2% of the information regarding branching of octanes contained in the Branching degrees13 derived from the values of index VL(-0.126, -0.139, -0.270). It represents 87.9% of the information regarding branching of octanes contained in the Wiener1 index W, whereas it represents only 50.8% of the information regarding branching of octanes contained in the index p4. 5. Discussion Interestingly, the best correlation to the individually optimized MOCSN has the physicochemical property Tc/Pc (R = 0.9939, IC = 89.0%), closely followed by the Branching Degrees13 derived from the values of the index Vl(-0.126, -0.139, -0.270) (R = 0.9930, IC = 88.2%), the Wiener1 index W (R = 0.9926, IC = 87.9%), the index16 RW (R = 0.9924, IC = 87.7%), and index18 p2/w2 (R = 0.9910, IC = 86.6%), whereas the Randic17 index x correlates to R = 0.9764, IC = 78.4% and the Hosoya2 index Z to R = 0.9755, IC = 78.0% . Among the path indices,18 the correlation to the individually optimized MOCSN is p3 > p2 > p5 > p4, and p2/w2 > p3/w3 > p5/w5 > p4/w4 as well as p2/w2 > p2, p3 > p3/w3, p4/w4 > p4, p5 > p5/w5. Whereas at Tc/Pc the magnitude of the optimized factors |kNi| is |kNbr| > |kNper| > |kLbr| > |kNasym^| > |kNasyml| > |kNd|, at Vl(-0.126, -0.139, -0.270) it is |k™,r| > |kNper| > |kNasym-| > kM| > febr! > ^Nasyml!, at W it is |kmr| > febr! ~ |kNp er| > |kNasym±| > |kNasym^J > ^Nd^ at RW^ ft is |kNbr| > |kNper| > |kLbr| > kNd| > |kNasym±| > ^Nasy^ at the RandiC17 index X it is |kNbr| > |kLbr| ~ |kNper| > |kNd| > |kNasym±| > |kNasym^| and at the Hosoya2 index Z it is |kNbr| > |kLbr| > |kNper|| > |kNasym±| > |kNd> ^Nasym^. Information contribution of the Structural Numbers Nbr, Nper, Nd, Lbr, NaSym^, and NaSym± to the values of phys-icochemical properties of octanes as measured by the product ICx|kNi| (%) is presented in Table 4 and illustrated in Figure 1. In Table 4 and Figure 1 we can see that in the case of octanes the highest observed contributions of tested Structural Numbers are as follows. The Number of Branches contributes up to 60.4% of the Information Content (the case of RON). The Peripheral Number contributes up to 24.1% of the Information Content (the case of surface tension). The Size of the Largest Branch contributes up to 20.8% of the Information Content (the case of MON). The Asymmetry Number Nasyml contributes up to 20.0% of the Information Content (the case of the Antoine constant B). The Asymmetry number Nasym^, on the other hand, contributes only up to 8.3% of the Information Content (the case of the Antoine constant A). The maximum contributions are thus at Nbr >> Nper > Lbr > NaSymi > Nd > NaSym^. The contributions in particular physicochemical properties are quite varying. The Number of Branches in octanes contributes the most to the values of physico-chemical properties (in %; total IC = 100%): 61 > RON > w > Tc2/Pc > BON > BP/Tc > 50, and the least to the values of physicochemical properties: 10 > Zc > ac > Surface Tension > logVP > CED > Sol. par. > B > 0.5. The Peripheral number contributes in general much less, the most to the values of physicochemical properties (in %): 25 > Surface Tension > Tc > Pc > BP > Tc/Pc > 20, and the least to the Table 4. Results of optimization of the correlation between the MOCsn and the values of some topological indices of octanes. The values of topological indices of octanes were taken from Ref. W.13.16-18 Index kNbr kNper kNd kLbr kNasim ^ kNasim± R S IC (%) Br. Deg.VL 0.669 -0.108 -0.063 0.053 0.082 0.025 0.9930 0.062 88.2 W -0.554 0.181 0.003 -0.188 0.015 -0.059 0.9926 0.808 87.9 RW 0.634 -0.163 -0.053 0.085 0.021 0.044 0.9924 0.070 87.7 X -0.552 -0.141 0.118 0.145 0.007 0.037 0.9764 0.037 78.4 Z -0.459 -0.152 0.075 0.190 0.032 -0.092 0.9755 1.009 78.0 p2 0.689 0.005 -0.154 -0.102 -0.015 0.035 0.9704 0.365 75.8 p3 0.321 -0.273 0.015 0.194 0.082 -0.115 0.9765 0.334 78.5 p4 -0.363 0.083 0.168 -0.030 -0.060 0.296 0.8703 0.657 50.8 p5 -0.471 0.326 -0.021 0.021 -0.030 -0.131 0.9351 0.505 64.6 p2/w2 0.781 0.012 -0.098 -0.066 -0.008 -0.035 0.9910 0.007 86.6 p3/w3 0.232 -0.284 0.035 0.231 0.095 -0.123 0.9561 0.017 70.7 p4/w4 -0.356 -0.093 0.172 0.175 0.015 0.189 0.9024 0.014 56.9 p5/w5 -0.472 0.316 -0.032 0.027 -0.028 -0.125 0.9287 0.008 62.9 Perdih: Physicochemical Properties of Octane Isomers Acta Chim. Slov. 2021, 68, 137-143 141 Table 4. Information contribution of the Structural Numbers Nbr, Nper, Nd, Lbr, Nasym„, and Nasyml to the values of physicochemical properties of octanes as measured by the product ICx|kNi| (%) and sorted by IC. PCP JCxIkNbri ICx|kNper| ICx|kNd| ICx|kLbr| ICx|kNasym^| ICx|kNasym±| IC (%) Tc/Pc 48.2 20.6 0.3 14.1 3.8 2.1 89.0 Tc2/Pc 55.5 7.7 2.7 12.1 0.6 8.8 87.4 RON 60.4 6.9 0.5 13.1 5.1 1.0 87.1 BON 54.8 7.7 2.0 14.6 4.5 2.1 85.6 BP/Tc 50.0 16.9 5.5 6.9 2.3 2.3 83.9 Pc 35.7 21.9 2.2 12.0 4.1 7.3 83.2 « 56.7 12.3 6.1 2.5 0.8 2.8 81.2 MON 47.5 5.2 1.4 20.8 3.8 0.7 79.4 d 22.3 18.7 3.2 14.1 3.9 11.0 73.2 Vm 22.0 18.7 3.3 13.9 4.0 10.9 72.9 nD 20.9 16.3 6.2 13.9 3.1 12.0 72.5 AHv 35.9 6.1 2.4 8.8 1.7 15.9 70.9 BP 16.9 21.5 0.8 7.7 6.1 17.5 70.5 ST 8.0 24.1 4.4 14.0 3.2 16.7 70.4 Tc 13.5 24.0 3.3 8.6 5.6 13.6 68.6 C 41.6 1.2 12.3 2.3 8.1 2.9 68.4 S 33.4 5.0 13.4 4.5 7.1 3.6 67.0 R2 33.8 4.8 8.4 14.6 1.3 3.0 65.9 CED 4.7 16.3 6.5 16.6 2.2 19.6 65.8 Sol.par. 4.7 16.2 6.4 16.6 2.2 19.6 65.7 B 0.5 14.2 14.4 11.6 4.4 20.0 65.1 MR 16.7 18.0 5.0 9.8 5.4 5.2 60.2 AHf°g 11.2 19.7 8.5 6.2 7.1 4.8 57.6 A 14.9 5.8 10.8 6.9 8.3 10.6 57.2 logVP 7.5 5.4 13.9 9.1 6.1 11.3 53.3 Vc 12.7 13.7 4.1 7.6 7.7 2.5 48.2 dc 12.2 13.4 4.0 7.1 7.6 2.4 46.7 Zc 9.3 6.8 6.1 1.8 7.3 4.7 36.2 ac 8.3 8.6 7.5 1.9 6.3 1.6 34.3 Figure 1. Information contribution of the Structural Numbers Nbr, Nper, Nd, Lbr, Nasym„, and Nas)ml about the values of physicochemical properties of octanes as measured by the product ICx|kNi| (%) and sorted by the contribution of the Number of Branches. Perdih: Physicochemical Properties of Octane Isomers ... 142 Acta Chim. Slov. 2021, 68, 137-143 values of physicochemical properties: 6 > A > logVP > MON > S > R2 > C > 1. The Distance number contributes less, the most to the values of physicochemical properties (in %): 15 > B > logVP > S > C > A > 10, and the least to the values of physicochemical properties: 2 > BON > MON > BP > RON > Tc/Pc > 0.1. The Size of the Largest Branch contributes the most to the values of physicochemical properties (in %): 21 > MON > Sol. par. > CED > 15, and the least to the values of physicochemical properties: 7 > BP/Tc > A > AHf°g > S > « > C > ac > Zc > 1. The Asymmetry number Nasym^ contributes the most to the values of physicochemical properties (in %): 9 > A > C > Vc > dc > Zc > AHf°g > S > 7, and the least to the values of physicochemical properties: 3 > BP/Tc > CED > Sol. par. > AHv > R2 > « > Tc2/Pc > 0.5. The Asymmetry number Nasyml contributes the most to the values of physicochemical properties (in %): 20 > B > Sol. par. > CED > BP > Surface Tension > AHv > 15, and the least to the values of physicochemical properties: 2.5 > Vc > dc > BP/Tc > Tc/Pc > BON > ac > RON > MON > 0.5. In most cases (with exception of Tc/Pc, Tc2/Pc, and RON) the structural numbers in form of the optimized MOCsn do not suffice the criterion that for a useful correlation, R > 0.99 must be obtained.19 To obtain better correlations, the mutually optimized combinations of vertex-degree vertex-distance weighted elements of the Universal matrix and vertex-degree weighted path indices are to be used.15,20-22 As shown above, the values of the mutually optimized factors kNi as well as of products ICx|kNi| are useful for the structural interpretation of the values of physico-chemical properties of octanes, as well. Whereas in the best case, i.e. at Tc/Pc there they explain 89.0% of the information regarding branching of octanes (48.2% is explained by Nbr, 20.6% by Nper, 14.1% by Lbr, 3.8% by Nasym^, 2.1% by N asym±, and only 0.3% by Nd), in the worst observed case, i.e. at the critical property ac there is explained only 34.3% of the information regarding branching of octanes (8.6% is explained by Nper, 8.3% by Nbr, 7.5% by Nd, 6.3% by NaSym^, 1.9% by Lbr, and 1.6% by N asym±z • The mutually optimized factors kNi as well as the products 7Cx|kNi| contribute thus to the structural interpretation of the values of physicochemical properties of octanes to different extents at different physicochemical properties of octanes. After determining their contribution, in most cases additional factors are to be sought for. The situation for physicochemical properties of octanes Tc/Pc, d, MR, dc, and Tc is illustrated in Figure 2. It is presented as the difference between the experimental values of the physicochemical properties of octanes and the values calculated using mutually optimized contribution of all the tested Structural features reduced by the average of the experimental values of physicochemical properties of octanes. In the case of Tc/Pc, for example, where IC = 89.0%, there remains open the question why at the octane isomers 3M7, 3Et6, 24M6, 3Et2M5, 22M6, 234M5, and 2233M4 the calculated values almost fit the experimental ones, Figure 2. Reduced differences between the experimental values of the physicochemical properties of octanes and the values calculated using mutually optimized contribution of all the tested Structural features. Perdih: Physicochemical Properties of Octane Isomers Acta Chim. Slov. 2021, 68, 137- 143 143 whereas at 4M7, 25M6, 233M5 the experimental values are higher than the calculated ones, and at Oct, 34M6, 33M6, 3Et3M5, and 224M5 they are lower. Another example is e.g. dc, where IC = 46.7%. There remains open the question why at the octane isomers the calculated values almost fit the experimental ones only at Oct and 34M6, whereas at 3M7, 25M6, 2Et3M5, and especially at 33M6 and 223M5 the experimental values are higher than the calculated ones, and at 2M7, 4M7, 24M6, 22M6, 3Et3M5, 224M5, and 233M5 they are lower. This situation calls for additional study. 5. References 1. H. Wiener, J. Am. Chem. Soc. 1947, 69, 17-20. DOI:10.1021/ja01193a005 2. H. Hosoya, Bull. Chem. Soc. Jpn. 1971, 44, 2332-2339. DOI:10.1246/bcsj.44.2332 3. H. Hosoya, Internet Electron. J. Mol. Des. 2002, 1, 428-442. 4. E. C. Kirby, J. Chem. Inf. Comput. Sci. 1994, 34, 1030-1035. DOI:10.1021/ci00021a001 5. D. H. Rouvray, J. Comput. Chem. 1987, 8, 470-480. DOI:10.1002/jcc.540080427 6. D. Bonchev, N. Trinajstic, J. Chem. Phys. 1977, 67, 4517-4533. DOI:10.1063/1.434593 7. D. Bonchev, J. Mol. Struct. (Theochem) 1995, 336, 137-156. DOI:10.1063/1.434593 8. M. Randic, Acta Chim. Slov. 1997, 44, 57-77. 9. L. Lovasz, J. Pelikan, Period Math Hung. 1973, 3, 175-182. DOI:10.1007/BF02018473 10. D. M. Cvetkovic, I. Gutman, Croat. Chem. Acta 1977, 49, 115-121. 11. M. Randic, C. L. Wilkins, J. Phys. Chem. 1979, 83, 1525-1540. DOI:10.1021/j100474a032 12. A. Perdih, Indian J. Chem. 2003, 42A, 1246-1257. 13. A. Perdih, Acta Chim. Slov. 2016, 63, 411-415. DOI:10.17344/acsi.2015.1607 14. A. Perdih, M. Perdih, Acta Chim. Slov. 2000, 47, 231-259. DOI:10.17344/acsi.2015.1607 15. A. Perdih, Acta Chim. Slov. 2015, 62, 879-888. DOI:10.17344/acsi.2015.1607 16. M.V. Diudea, J. Chem. Inf. Comput. Sci. 1997, 37, 292-299. DOI:10.1021/ci960037w 17. M. Randic, J. Am. Chem. Soc. 1975, 97, 6609-6615. DOI:10.1021/ja00856a001 18. M. Randic, J. Chem. Inf. Comput. Sci. 2001, 41, 607-613. DOI:10.1021/ci0001031 19. Z. Mihalic, N. Trinajstic, J. Chem. Educ. 1992, 69, 701-712. DOI:10.1021/ed069p701 20. A. Perdih, Acta Chim. Slov. 2016, 63, 88-96. DOI:10.17344/acsi.2015.1975 21. A. Perdih, Acta Chim. Slov. 2016, 63, 411-415. DOI:10.17344/acsi.2016.2361 22. A. Perdih, Acta Chim. Slov. 2019, 66, 726-731. DOI:10.17344/acsi.2019.5346 Povzetek S strukturnimi števili so bile kvantificirane strukturne značilnosti izomer oktana. Za ugotovitev, kolikšen del vsebine podatkov o fizikokemijskih lastnosti oktanov nam dajejo strukturna števila, je bil razvit način, kako določiti hkratno optimiziran doprinos strukturnih števil (MOCSN). Poleg drugih strukturnih števil predstavljenih prej, sta uvedeni še števili, ki predstavljata merili za nesimetričnost izomer oktana. Eno predstavlja nesimetričnost vzdolž glavne osi molekule, drugo pa nesimetričnost pravokotno nanjo. Njuni korelaciji z vrednostmi 29 fizikokemijskih lastnosti oktanov sta nizki, |_R| < 0.6. Po optimizaciji vsebuje MOCSN od 34.3 % do 89.0 % informacije vsebovane v fizikokemijskih lastnostih oktanov. Strukturna števila omogočajo prvi korak k strukturni razlagi fizikokemijskih lastnosti oktanov. V 17 od 29 primerov največ informacij poda število vej, v 8 primerih periferno število, v 3 primerih eno od števil nesimetričnosti in v enem primeru število razdalj med vejami. © 0 Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Perdih: Physicochemical Properties of Octane Isomers ... DOI: 10.17344/acsi.2020.6237 Acta Chim. Slov. 2021, 68, 144-150 /^creative ^commons Scientific paper Structure of Biologically Active Benzoxazoles: Crystallography and DFT Studies Una Glamoclija,1,2'* Selma Spirtovic-Halilovic,3 Mirsada Salihovic,4 Iztok Turel,5 Jakob Kljun,5 Elma Veljovic,3 Selma Zukic3 and Davorka Zavrsnik3 1 Department for Biochemistry and Clinical Analysis, Faculty of Pharmacy, University of Sarajevo, Zmaja od Bosne 8, 71000 Sarajevo, Bosnia and Herzegovina 2 School of Medicine, University of Mostar, Zrinskog Frankopana 34, Mostar 88000, Bosnia and Herzegovina 3 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Sarajevo, Zmaja od Bosne 8, 71000 Sarajevo, Bosnia and Herzegovina 4 Department of Natural science, Faculty of Pharmacy, University of Sarajevo, Zmaja od Bosne 8, 71000 Sarajevo, Bosnia and Herzegovina 5 Faculty of Chemistry and Chemical Technology, University of Ljubljana, Vecna pot 113, 1000 Ljubljana, Slovenia * Corresponding author: E-mail: una.glamoclija@ffsa.unsa.ba, Received: 07-02-2020 Abstract Using X-ray single crystal diffraction, the crystal structures of biologically active benzoxazole derivatives were determined. DFT calculation was performed with standard 6-31G*(d), 6-31G** and 6-31+G* basis set to analyze the molecular geometry and compare with experimentally obtained X-ray crystal data of compounds. The calculated HOMO-LUMO energy gap in compound 2 (2-(2-hydroxynaphtalen-1-yl)-4-methyl-7-isopropyl-1,3-ben-zoxazol-5-ol) is 3.80 eV and this small gap value indicates that compound 2 is chemically more reactive compared to compounds 1 (4-methyl-2-phenyl-7-isopropyl-1,3-benzoxazol-5-ol) and 3 (2-(4-chlorophenyl)-4-methyl-7-isopro-pyl-1,3-benzoxazol-5-ol). The crystal structures are stabilized by both intra- and intermolecular hydrogen bonds in which an intermolecular O-H---N hydrogen bond generates N3 and O7 chain motif in compounds 1, 2, and 3, respectively. The calculated bond lengths and bond angles of all three compounds are remarkably close to the experimental values obtained by X-ray single crystal diffraction. Keywords: Benzoxazole; X-ray diffraction; DFT calculation 1. Introduction Benzoxazoles are compounds with a wide range of biological activities and represent a very important structural motif in medicinal chemistry. Benzoxazole ring can be found in natural and synthetic compounds used as pharmaceuticals.1 Caboxamycin (Figure 1a) is a new antibiotic of the benzoxazole family produced by the deep-sea strain Streptomyces sp. NTK 937.2 Figure 1b presents the synthetic compound with a two times better Pseudomonas aeruginosa inhibitory activity than ampicillin and streptomycin.3 Benzoxazoles have anti-inflammatory,4 antimicrobial,5-7 and antitumor6-10 activities. Figure 1. Structure of Caboxamycin (a) and (b) a synthetic derivative of benzoxazole. Benzoxazoles have a certain structural similarity with nucleic bases such as adenine and guanine, and they interact with biopolymers in living organisms.2 The mole- Glamoclija et al.: Structure of Biologically Active Benzoxazoles: Acta Chim. Slov. 2021, 68, 144-150 145 cule is planar with conjugated sextets of n electrons in the cyclic system.11 They have aromatic properties and are quite stable. Benzoxazoles are sensitive to hydrolysis which leads to ring-opening. Depending on substituents, different conditions of media and pH values lead to hydrolysis.11-14 While unsubstituted benzoxazole is resistant to hydrolysis in alkaline medium, rapid hydrolysis occurs in acidic medium, probably due to nucleophilic attack (Scheme 1).11 Hydrolysis can be seen in vivo, as Bray et al. (1952) have shown in experiments on rabbits. They evaluated the metabolism of benzoxazole, 2-methylbenzoxaz-ole, and 2-phenylbenzoxazole and found that the stability of the ring depends on substituents. Benzoxazole and 2-methylbenzoxazole mainly hydrolyzed in organisms while the ring in 2-phenylbenzoxazole is stable and mainly metabolized by hydroxylation of phenyl group at the position 2'.12 H Scheme 1. Hydrolysis of benzoxazole ring in acidic conditions. Hydrolysis of drugs containing benzoxazole rings can have pharmaceutical importance. It can result in the formation of active compounds that can be directly delivered to the site of interest.13 The stability and reactivity of benzoxazoles are crucial for their medicinal applications. Density functional theory (DFT) is a very common computational method used to solve Schrodinger and Dirac equations.15 In our previous paper,16 a library of thymoqui-none-derived benzoxazoles has been synthesized and their antiproliferative activities were reported. The starting compound for synthesis was thymoquinone that reacted with sodium azide to obtain aminothymoquinone (ATQ). ATQ reacted with aromatic aldehydes to obtain benzoxazoles. Out of the 10 novel compounds prepared,16 in this paper, the structures of three compounds: 1 (4-me-thyl-2-phenyl-7-isopropyl-1,3-benzoxazole-5-ol), 2 (2-(2-hydroxynaphtalen-1-yl)-4-methyl-7-isopro-pyl-1,3-benzoxazole-5-ol), and 3 (2-(4-chlorophe-nyl)-4-methyl-isopropyl-1,3-benzoxazole-5-ol) are in the focus. Synthetic details, analytical and spectroscopic data of compounds can be found in our previous paper.16 The simultaneous approach of X-ray crystallography and DFT calculation is used. This approach takes advantage of the great interpretive power of theoretical studies and the precision of the experimental method. We report the crystal structure of benzoxazole derivatives, as well as results of theoretical studies using the DFT(B3LYP) method and standards 6-31G*(d), 6-31G** and 6-31+G* basis set. The aim of the present work was to describe and characterize the molecular structure and some electronic struc- ture properties of the biologically active benzoxazole derivatives by using two approaches: experimental, using X-ray crystallography, and theoretical, using DFT calculation. Finally, the results of the two approaches are compared. 2. Experimental 2. 1. Crystallographic Data Collection X-ray diffraction data for all compounds was collected on an Oxford Diffraction SuperNova diffractometer with Cu microfocus X-ray source with mirror optics and an Atlas detector. The structures were solved by direct methods implemented in SHELXT17 and refined by a full-matrix least-squares procedure based on F2 using SHELXL18 within the Olex2 program pack.19 All non-hydrogen atoms were refined anisotropically. The hydrogen atoms were placed at calculated positions and treated using appropriate riding models. The programs Platon and Mercury were used for data analysis and figure preparation.20,21 The crystal structures have been submitted to the CCDC and have been allocated the deposition numbers 1586630, 1985495, 1985496. 2. 2. Computational Details The molecular structures of compounds 1, 2 and 3 were subjected to quantum chemical density functional calculation using the Becke-3Lee-Yang-Parr (B3LYP) hybrid functional with the standards 6-31G*(d), 6-31G** and 6-31+G* basis set. The computations were performed using Spartan 14. The structures are minima on potential energy surface. The calculated values were compared with the obtained experimental results. 3. Results and Discussion The crystal data and structure refinement of compounds 1, 2 and 3 are given in Table 1 and shown in Figure 2. The selected geometric parameters of the same compounds are given in Table 2. The structural parameters were calculated and are presented in Table 3. The crystal structure of compound 1 (4-methyl-2-phenyl-7-isopro-pyl-1,3-benzoxazole-5-ol) has been determined by X-ray diffraction in our previous paper.16 Previous crystallo-graphic results for this compound are used in this paper for comparison with the DFT results. The calculated bond lengths and bond angles of 1, 2 and 3 are remarkably close to the experimental values obtained by X-ray crystal diffraction. 3. 1. Geometrical Parameters Analysis Bond lengths of compounds 1, 2 and 3 are in the normal range.22 The experimentally obtained value of av- Glamoclija et al.: Structure of Biologically Active Benzoxazoles: 146 Acta Chim. Slov. 2021, 68, 144-150 a) b) c) CI17 Figure 2. Crystal structures of compounds 1 (a),16 2 (b), and 3 (c) with heteroatom labelling. Thermal ellipsoids are drawn at 50% probability level. erage mean bond distances were of C-C = 1.425 Á, C-O = 1.373 Á, C-N = 1.353 Á, for compound 1,16 C-C = 1.424 Á, C-O =1.366Á, C-N = 1.348 Á, for compound 2 and C-C = 1.424 Á, C-O =1.373Á, C-N = 1.355 Á, for compound 3 respectively, shown in Table 3. The bond distances of C2=N3 are 1.302 Á, 1.307 Á and 1.303 Á for compounds 1, 2 and 3, respectively. They are comparable with the reported double bond lengths.23 The theoretically obtained value of average mean bond distances were of C-C = 1.433 Á, C-O =1.373 Á, C-N = 1.347 Á, for compound 1, C-C = 1.433 Á, C-O =1.366 Á, C-N = 1.353 Á, for compound 2 and C-C = 1.432 Á, C-O =1.376 Á, C-N = 1.346 Á, for compound 3, respectively (Table 3). Experimentally obtained bond distances of C2=N3 show 1.302 Á (cal. 1.302 Á), 1.307 Á (cal. 1.318 Á) and 1.303 Á (cal. 1.301 Á), for compounds 1, 2 and 3, respectively. In addition to that, all three compounds display the electron delocalization over the atoms of O1-C2-N3. In all three compounds, the Figure 3. Hydrogen bond network in the crystal structure of 2. The hydroxyl group in position 5 forms intermolecular hydrogen bonds (green) with the O15 oxygen on the naphthyl group (O5-H5—O15') which in turn forms an intramolecular hydrogen bond (blue) with the neighboring benzoxazole N3 nitrogen atom (O15-H15---N3). Thermal ellipsoids are drawn at 50% probability level and non-relevant hydrogen atoms are omitted. sum of bond angles around the C2 atom of the benzoxazole ring (O1-C2-N3, O1-C2-C14, and N3-C2-C14 = 359.99° (cal. 360.01°) for compound 1, 359.98° (cal. 359.99°) for compound 2 and 359.98° (cal. 360.00°) for compound 3) indicates sp2 hybridization and the bond an- Figure 4. Hydrogen bond network in the crystal structures of compounds 1 (top) and 3 (bottom). The hydroxyl group in position 5 forms intermolecular hydrogen bonds (green) with the benzoxazole N3 nitrogen (O5'-H5,---N3). Thermal ellipsoids are drawn at 50% probability level and non-relevant hydrogen atoms are omitted. Glamočlija et al.: Structure of Biologically Active Benzoxazoles: ... Acta Chim. Slov. 2021, 68, 144-150 147 Table 1. Crystal data and structure refinement summary of compounds 2 and 3 Compound 2 3 Empirical formula C21H19NO3 C17H16ClNO2 Mw 333.37 301.76 T, K 150(2) 150(2) Crystal system monoclinic monoclinic Space group P 2i/n P 21/n a, A 6.7961(2) 8.3718(2) b, A 15.9766(4) 16.2112(1a) c, A 15.7268(4) 11.7098(1a) a, deg. 90 90 P, deg. 97.488(2) 110.472(2) Y, deg. 90 90 V, A3 1693.03(8) 1488.85(6) Z 4 4 Dcalc, g/cm3 1.308 1.346 mm-1 0.705 2.300 F(000) 704 632 Crystal size, mm 0.60x0.10x0.10 0.20x0.20x0.20 Color colorless yellow Data collected / unique 5939 / 3190 5382 / 5382 Rint/Rsigma 0.0359 / 0.0452 0.0195 / 0.0249 Restraints / parameters 0 / 231 0 / 194 S 1.059 1.043 Ri, wR2 [I > 2a(I)] 0.0595 / 0.1431 0.0371 / 0.1010 Rt, wR2 (all data) 0.0690 / 0.1558 0.0404 / 0.1047 Larg. diff. peak/hole (e-A-3) 0.35 / -0.58 0.28 / -0.33 gle of C2-N3-O1, C9-C8-C7, and C8-C9-C4 deviates from 120° due to the presence of substituents. 3. 2. Intra- and Intermolecular Interactions and Crystal Packing Analysis The crystal structures of compounds 1, 2 and 3 are stabilized via intramolecular O-H---N hydrogen bond and intermolecular O-H---N, O-H—O, and O-H---N hydrogen bonds (Table 2). The crystal structure of compound 2 is stabilized by intramolecular O15-H15--N3 hydrogen bond, in which the hydroxylic O15 acts as a donor and makes a hydrogen bond with imine N3 with the bond length of 2.477 Á (Figure 3). The crystal packing is stabilized by one intramolecular O15-H15--N3 hydrogen bond with the bond distances of 2.477 Á (cal. 2.640 Á), whereas in compounds 1, 2 and 3, three intermolecular hydrogen bonds contribute to crystal packing such as O5-H5--N3 with the bond distances of 2.811 Á (2.850 Á), O15-H15-O15 2.711 Á (cal. 2.900 Á) and O5-H5-N3 2.800 Á (2.850 Á), respectively. Atom N3 acts as a hydrogen bond acceptor for O-H---N [O5-H5-N3] contacts (Figure 4). Crystal data and structure refinement summary of compounds 2 and 3 are given in Table 1. The same data for compound 1 are presented in our previous paper.16 Table 2. Selected distances and angles Com- Cpd (D-H-A) type d(D-H) (A) d(D" •A) (A) d(H- •A) (A) ^(D-H- ••A) (°) pound Exp. Calc. Exp. Calc. Exp. Calc. Exp. Calc. 1 (O5-H5—N3) inter 0.84 0.90 2.8105 2.8500 1.97 1.90 175 172.9 2 (O5-H5—O15) inter 0.84 0.90 2.7108 2.9000 1.88 2.00 168 166.5 2 (O15-H15—N3) intra 0.84 0.90 2.4772 2.6400 1.72 1.74 149 147.0 3 (O5-H5—N3) inter 0.84 0.90 2.8004 2.8500 1.97 1.87 170 174.0 Table 3. Bond lengths [A] and angles [deg] of compounds 1, 2 and 3 Bond length and angles Compound 1 Compound 2 Compound 3 Exp. Cal. Exp. Cal. Exp. Cal. 6-31G(d) 6-31G** 6-31+G* 6-31G(d) 6-31G** 6-31+G* 6-31G(d) 6-31G** 6-31+G* O(1)-C(2) 1.364 1.3701.3711.369 1.363 1.3651.3621.363 1.367 1.3701.3681.371 O(5)-C(5) 1.368 1.3711.3721.371 1.361 1.3691.3701.368 1.366 1.3691.3671.369 C(9)-N(3) 1.403 1.3951.3941.396 1.388 1.3871.3881.389 1.406 1.4001.4021.404 C(2)-N(3) 1.302 1.3021.3031.301 1.307 1.3081.3071.309 1.303 1.3011.3021.304 C(2)-C(14) 1.461 1.4601.4611.360 1.458 1.4541.4551.457 1.457 1.4561.4571.459 O(5)-C(5)-C(4) 116.53 115.90115.92115.95 116.44 115.80115.83115.82 116.44 120.01119.98119.97 C(8)-C(9)-N(3) 108.29 108.89108.90108.91 107.76 107.95107.96107.97 108.22 108.80108.81108.83 C(4)-C(9)-N(3) 130.16 129.63129.65129.67 129.42 130.09130.10130.08 130.42 129.90129.94129.97 N(3)-C(2)-O(1) 114.97 114.89114.90114.91 113.79 113.53113.55113.53 114.83 114.87114.89114.92 N(3)-C(2)-C(14) 127.83 127.51127.53127.55 123.26 124.73124.74124.76 128.55 127.73127.98127.97 O(1)-C(2)-C(14) 117.19 117.60117.61117.59 122.93 121.99122.00122.02 116.60 117.56117.98117.97 Glamoclija et al.: Structure of Biologically Active Benzoxazoles: 148 Acta Chim. Slov. 2021, 68, 144-150 Table 4. Calculated HOMO and LUMO energy values in compounds 1, 2 and 3 Parameters 6-31G*(d); 1 6-31G**; 6-31+G* Compound 2 6-31G*(d); 6-31G**; 6-31+G* 6-31G*(d); 3 6-31G**; 6-31+G* Ehomo (eV) -5.61 -5.61 -5.62 -5.40 -5.42 -5.44 -5.71 -5.73 -5.74 Elumo (eV) -1.33 -1.34 -1.35 -1.61 -1.62 -1.63 -1.55 -1.57 -1.58 Energy gap (A) 4.28 4.27 4.27 3.79 3.80 3.81 4.16 4.16 4.16 HOMO LUMO HOMO LUMO homo 3 Figure 5. Atomic orbitals of HOMO to LUMO transition of the compounds 1, 2 and 3 3. 3. Molecular Orbital Analysis The HOMO-LUMO energy gap of a molecule will play a crucial role in deciding its bioactive properties and is a very important parameter for quantum chemistry. The HOMO energy distinguishes the capacity of electron donor, whereas LUMO energy characterizes the capacity of electron acceptor, and the gap distinguishes the chemical stabil-ity.24biologically active methylxanthines were investigated. All calculations were performed at B3LYP/6-31G* level of theory. The electronic chemical potential, highest occupied molecular orbital (HOMO The HOMO-LUMO energy gap for the compounds 1, 2 and 3 was calculated by 6-31G*(d) basis set and the values are 4.27 eV for compound 1, 3.80 eV for compound 2 and 4.15 eV for compound 3. The energies of HOMO and LUMO and the HOMO-LUMO energy gap are given in Table 4. The HOMO-LUMO orbital scheme of compounds 1, 2 and 3 are shown in Figure 5 (positive phases are red and the negatives ones are blue). The value of the HOMO and LUMO energy gap in compound 2 is the smallest, indicating that the molecule is more stable compared to 1 2 Glamočlija et al.: Structure of Biologically Active Benzoxazoles: ... Acta Chim. Slov. 2021, 68, 144-150 149 compounds 3 and 1. The lower value of the HOMO and LU-MO energy gap explains the eventual charge transfer interaction taking place within the molecules.25 The HOMO to LUMO transition indirectly explains the descriptor of electron donor and acceptor in order to understand their interacting ability with their target molecules. Compound 2, which is the most chemically reactive, showed the lowest antitumor activity in our previous study.16 4. Conclusions We presented the structural details of benzoxazole compounds, 1 (C17H17NO2), 2 (C21H19NO3) and 3 (C17H16ClNO2), by using single crystal X-ray diffraction data. DFT calculation was performed with a standards 6-31G*(d), 6-31G** and 6-31+G* basis set to analyze the molecular geometry and compare with experimentally available X-ray crystal data of investigated compounds. The calculated HOMO-LUMO energy gap in compound 2 for basis set 6-31G*(d) is 3.79, for 6-31G** is 3.8, and 6-31+G* is 3.8. This small gap value indicates that compound 2 is chemically more reactive compared to compounds 1 and 3. Chemical reactivity values, such as chemical hardness, chemical potential electronegativity and electrophilicity index and HO-MO-LUMO energy gap obtained theoretically, can be used to understand the biological activity of the title compound. Further, the crystal structure is stabilized by both intra- and intermolecular hydrogen bonds in which intermolecular N-H---O hydrogen bond generates N3 and O7 chain motif in compounds 1, 2 and 3, respectively. The calculated bond lengths and bond angles of 1, 2 and 3 show good agreement to the experimental values obtained by X-ray crystal diffraction. The values obtained theoretically show some correlations with previous results of biological activity testing. Acknowledgements The authors are grateful to financial support from Federal Ministry of education and science in Bosnia and Herzegovina (grant number: 05-39-3629-1/14, Most: Za-vrsnik D. "Modeliranje i doking studije novih potentnih azometinskih derivata timokinona i njihovih organome-talnih kompleksa". Ministarstvo za obrazovanje, nauku, kulturu i sport FBiH, 2014-2015. godine (Mostar, 22.12.2014. godine, Ugovor broj: 05-39-3629-1/14). We thank EN-FIST Centre of Excellence, Trg OF 13, 1000 Ljubljana, Slovenia for using SuperNova diffractome-ter and Slovenian Research Agency for financial support (P1-0175). 5. References 1. C. S. Demmer, L. Bunch, Eur. J. Med. Chem. 2015, 97, 778785. DOI:10.1016/j.ejmech.2014.11.064 2. S. Laeeq, A. K. Sirbaiya, H. H. Siddiqui, Indo. Am. J. Pharm. Res. 2013, 3, 1660-1682. 3. H. Song, C. Rao, Z. Deng, Y. Yi, J. H. Naismith, Angew. Chem. Int. Ed Engl., 2020, 59, 6054-6061. DOI:10.1002/anie.201915685 4. K. Seth, S. K. Garg, R. Kumar, P. Purohit, V. S. 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DOI: 10.17344/acsi.2020.6281 Acta Chim. Slav. 2021, 68, 151-158 /^creative ^commons Scientific paper Apoptotic Effect of Homobrassinin and Thiazino[6,5-b]indol is Associated with Downregulation of Heat Shock Proteins in Human Ovarian Adenocarcinoma Cells Zuzana Solarova,1 Martin Kello1 and Peter Solar2^ 1 Institute of Pharmacology, Faculty of Medicine, P. J. Safarik University in Kosice, SK-04154 Kosice, Slovak Republic 2 Institute of Medical Biology, Faculty of Medicine, P. J. Safarik University in Kosice, SK-04154 Kosice, Slovak Republic * Corresponding author: E-mail: peter.solar@upjs.sk Received: 09-04-2020 Abstract Phytoalexins are substances with antimicrobial properties produced by plants after being attacked by microorganisms, especially phytopathogenic fungi and viruses. They are also currently being studied for their antitumor effect. We aimed to study the apoptosis-stimulating effect of homobrassinin and thiazino[6,5-b]indol in human ovarian adenocarcinoma A2780 and A2780cis cells via flow cytometric analysis of annexin V/PI, caspase 3 and 9 activity, cytochrome C release, and smac-diablo accumulation. Using the western blot technique, we also monitored the effect of both indoles on the response of heat shock proteins in these cells. Thiazino[6,5-b]indol showed more pronounced sensitizing and/or pro-apoptotic effect compared to homobrassinin accompanied by increased smac-diablo accumulation at earlier time intervals and pronounced externalization of phosphatidylserine at 72 h in A2780cis compared to A2780 cells. The apoptosis stimulating effect of thi-azino[6,5-b]indol in A2780cis cells was associated with significant irreversible downregulation of HSP70 and HSP90 and partly with a decrease of HSP40. On the other hand, cisplatin-induced the apoptosis of sensitive A2780 cells with reversible downregulation of HSP40 and HSP57. In conclusion, the effect of thiazino[6,5-b]indol on resistant A2780cis cells could have a great utility in both the potential prevention and the treatment of other cisplatin-resistant tumor cells. Keywords: Homobrassinin; thiazino[6,5-b]indol; cisplatin resistance; apoptosis; heat shock proteins; human ovarian adenocarcinoma cells 1. Introduction Ovarian cancer has the highest mortality rate of all gynecologic neoplasms among women in Western Europe and the United States. Women who have one first-degree relative with ovarian cancer have an increased risk (5%) of developing ovarian cancer even without known genetic mutation (the average woman's lifetime risk is 1.4%).1 For most women, the standard of care remains surgery and platinum-based cytotoxic chemotherapy. Platinum-based drugs are the most active and effective treatment option for most patients with early-stage disease. Women with advanced disease will develop many episodes of recurrent disease with progressively shorter disease-free intervals. Eventually, the tumor is declared platinum resistant. Although paclitaxel chemotherapy has improved over the last two decades, the progression to free survival has remained fairly constant at about 18 months. Significant advances in the treatment over the last decade led to a significant prolongation of 5-year survival while the incidence of the disease has remained constant.2 Platinum resistance can occur in almost all patients with recurrent ovarian cancer. However, many patients with "platinum-resistant" disease respond to further platinum-based treatment. Platinum, either cisplatin or carbo-platin, is given weekly with the addition of etoposide or paclitaxel.3 or in combination with gemcitabine for two out of three weeks.4 Paclitaxel is one of the most active nonplatinum drugs used in this setting. Progression to free survival and overall survival increases significantly with paclitaxel administered weekly.5 In women with platinum-resistant ovarian cancer, antiangiogenic agents, like Solarova et al.: Apoptotic Effect of Homobrassinin 152 Acta Chim. Slav. 2021, 68, 151-158 bevacizumab, may also play an important role. Indeed, these agents have been used as a monotherapy or in combination with low dose cyclophosphamide.6 Other drugs, such as aflibercept (VEGF trap), have been found effective in controlling ascites.7 Other than anticancer chemotherapeutics, synthetic analogs of several natural substances with antitumor effects, such as indole phytoalexins, were also tested at our institute. Phytoalexins are substances with antimicrobial properties produced by plants after being attacked by microorganisms, especially phytopathogenic fungi and viruses. We tested indole phytoalexins on both cisplatin-sensi-tive human ovarian adenocarcinoma cell line A2780 (parental) as well as its cisplatin-resistant derivative A2780cis and compared their response to indoles to determine the rate of the resistance of A2780cis cells. Surprisingly, we found that A2780cis cells are only 3.2 times more resistant to thiazino[6,5-b]indol (K157) compared to the parental cell line, as opposed to cisplatin, where A2780cis cells revealed 18 times lower sensitivity.8 Thus, the fold resistance of A2780cis cells compared to parental ones was surprisingly low, suggesting that A2780cis cells have much lower resistance to K157 than to cisplatin. In the current work, we aimed to study the sensitizing effect of two indoles, homobrassinin (K1) and K157, on parental A2780 and cisplatin-resistant A2780cis cells. In addition to the apoptosis-stimulating effect of indoles, we were also interested in their effect on the response of selected heat shock proteins in these cells. 2. Experimental 2. 1. Cell Culture and Treatment Human ovarian adenocarcinoma cell lines A2780 and A2780cis were obtained from European Collection of Animal Culture (ECAC, Salisbury, UK). Cells were grown as monolayers in RPMI1640 medium with L-glutamine (GibcoBRL, Paisley, UK) supplemented with 10% fetal calf serum (GibcoBRL) and antibiotic/antimycotic solution (100 U/ml of penicillin, 100 ^g/ml of streptomycin and 0.25 ^g/ml of amphotericin B; GibcoBRL) and were maintained under standard tissue culture conditions at 37 °C and 5% humidified atmosphere of CO2. The acquired resistance of A2780cis cells was maintained by supplementation of media with 1mM of cisplatin (Sigma-Aldrich Co., St. Louis, MI, USA) every second passage. IC50 concentra- tions of homobrassinin (K1; N-[2-(indol-3-yl)ethyl] -S-methyldithiocarbamate), thiazino[6,5-b]indol (K157; 2-(4'-fluorphenylamino)-4H-1,3-tiazino[6,5-b]indol) (Fig. 1) and cisplatin determined in our previous stud-ies11,8 on resistant A2780cis cells were applied in all assays. 2. 2. Flow Cytometric Analysis (FCM) Human ovarian adenocarcinoma cells A2780 and A2780cis (1 x 106) were seeded for FCM analyses in Petri dishes and treated with homobrassinin, thiazino[6,5-b]in-dol and cisplatin at IC50 concentrations for 24, 48 or 72 h depending on experimental scheme. Floating and adherent cells were harvested, washed in PBS, divided for particular analysis and stained prior to analysis. Fluorescence was detected after 15-30 min incubation at room temperature in the dark using a BD FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA). A minimum of 1 x 104 cells were analysed per analysis. All experiments were performed in triplicate. 2. 3. Apoptosis Detection Via Annexin V/PI Staining For apoptosis detection, floating and adherent A2780 and A2780cis cells (1 x 106) were harvested 24, 48 and 72 h after homobrassinin, thiazino[6,5-b]indol and cisplatin treatment (IC50 concentrations). Complete cell population was washed in PBS and stained using annexin V Alexa Fluor® 647 conjugate (Thermo Fisher, Waltham, MA, USA) for 15 min at room temperature in the dark followed by incubation with propidium iodide (PI; Sigma-Aldrich, Saint-Louis, MO, USA) and analysed by flow cytometer (BD FACSCalibur). 2. 4. Detection of Active Caspase 3 Activation of executioner caspases (such as caspase 3) subsequently impacts the main structural proteins and activates other enzymes, leading to apoptosis. The changes in caspase 3 activation were analysed with FCM using active Caspase-3 PE Mab (Cell Signaling Technology, Danvers, MA, USA). The cells were harvested 24, 48 and 72 h after homobrassinin, thiazino[6,5-b]indol and cisplatin treatment (IC50 concentrations). The cell population was stained with phycoerythrin (PE) conjugated antibody and incubated for 30 min at room temperature in the dark. The cells homobrassinin 2-(4'-fluorphenylamino)-4H-l,3-tiazino[6,5-b](ndole Figure 1. Structure of homobrassinin (K1; N-[2-(indol-3-yl)ethyl]-S-methyldithiocarbamate) and thiazino[6,5-b]indol (K157; 2-(4'- fluorphenyl-amino)-4H-1,3-tiazino[6,5-b]indol). Solarova et al.: Apoptotic Effect of Homobrassinin ... Acta Chim. Slav. 2021, 68, 151-158 153 were then washed twice with PBS, resuspended in 500 ^M of the total volume, and analysed (1 x 104 cell per sample). 2. 5. Detection of Mitochondrial Apoptotic Pathway Associated Proteins Cytochrome c release, Smac/DIABLO accumulation and caspase-9 activity were analysed with FCM using Cytochrome c antibody (6H2) FITC conjugate; Smac/Diablo Rabbit mAb + goat anti-rabbit IgG (H + L) secondary antibody Alexa Fluor 488 and cleaved Caspase- 9 (Asp315) rabbit mAb PE conjugate. The A2780 and A2780cis cells were harvested 24, 48 and 72 h after homobrassinin, thiaz-ino[6,5-b]indol and cisplatin treatment (IC50 concentrations). Cell population was stained with conjugated antibody and incubated for 30 min at room temperature in the dark or stained with primary antibody (30 min), followed by secondary conjugated antibody staining (15 min in dark). The cells were then washed with PBS, resuspended in 500 ^L of the total volume in PBS, and analysed (1 x 104 cells per sample) by a BD FACSCalibur flow cytometer. 2. 6. Western Blotting Western blot analysis were carried out according to the standard protocol.12 The protein sample was separated on 10% SDS-PAGE, electroblotted onto Immobilon-P transfer membrane (Millipore Co., Billerica, MA, USA) and incubated using primary antibodies shown below: an-ti-HSP40 (#4868, 1:1000; Cell Signaling, Danvers, MA, USA), anti-protein disulfide isomerase (anti-HSP57; #3501, 1:1000; Cell Signaling Technology, Danvers, MA, USA), anti- HSP60 (#12165, 1:1000; Cell Signaling, Danvers, MA, USA), anti-HSP70 (#4873, 1:1000; Cell Signaling Technology, Danvers, MA, USA), anti-HSP90 (#4877, 1:1000; Cell Signaling Technology, Danvers, MA, USA) Figure 2. Effects of IC50 concentrations of homobrassinin (K1), thiazino[6,5-b]indol (K157), and cisplatin (Cis) on annexin V, caspase 3, caspase 9, smac-diablo, and cytochrome C in A2780 and A2780cis cell lines. The cells were grown in standard growth media for 24 h and then treated with mentioned above substances for 24, 48, and 72 h (untreated control - C). *p<0.05; **p<0.01***p<0.001 vs. C and +p<0.05; ++p<0.01; +++p<0.001 vs. Cis (three independent experiments) Solärovä et al.: Apoptotic Effect of Homobrassinin ... 154 Acta Chim. Slav. 2021, 68, 151-158 and anti-a-tubulin (sc-5286, 1:200; Santa Cruz Biotechnology, Dallas, Texas, USA). Subsequently, the membranes were incubated with secondary horseradish peroxi-dase-conjugated antibodies (PI-31461, 1:10.000, goat anti-rabbit IgG F(AB')2 or PI-31436, 1:10.000, goat anti-mouse IgG F(AB')2; Thermo Fisher Scientific, Waltham, MA, USA) for 1 h, and the antibody reactivity was visualized with ECL Western blotting substrate (PI-32106, Thermo Fisher Scientific) using Kodak Biomax films (#1788207, Sigma-Aldrich Co.). 2. 7. Statistical Analysis Data were processed using scientific graphing and ORIGIN analysis software (OriginLab Co., Northampton, MA, USA) and statistically analysed using one-way ANOVA followed by Tukey's multiple comparison tests. 3. Results and Discussion Both indoles induced strong apoptosis in A2780 and A2780cis cells at 24 h compared to cisplatin that showed a pro-apoptotic effect later (at 48 and 72 h) but only in A2780, not A2780cis cells (Fig. 2). Annexin V increased in A2780 and A2780cis cells 24 h after the addition of both indoles (Fig. 2). In this regard, K157 induced more pronounced ex-ternalization of phosphatidylserine in A2780cis cells compared to parental ones at 72 h time point. Changes in other selected markers of apoptosis, such as caspase 3 and 9, smac-diablo, and cytochrome C, confirmed the pro-apop- totic potential of both indoles tested, which was more pronounced in the case of K157 than in K1 (Fig. 2). Indeed, K157 increased both caspase 3 and 9 activities, increased the accumulation of smac-diablo, and induced the release of cytochrome C in both cell lines and at all time points monitored (Fig. 2). Interestingly, K157 increased smac-dia-blo accumulation in A2780cis cells at earlier time intervals (Fig. 2). On the other hand, cisplatin-induced the release of cytochrome C only in A2780 cells, not in A2780cis cells (Fig. 2). This effect of cisplatin correlated well with the mentioned externalization of phosphatidylserine (annexin V/PI staining) and apoptosis in A2780 cells (Fig. 2). Moreover, indoles reduced the level of HSP40, protein disulfide isomerase (HSP57), HSP70, and HSP90 in A2780 cells irreversibly and HSP40 and HSP57 in A2780cis cells reversibly (Fig. 3A, B; Tab. 1A, B). In this regard, indole K1 downregulated HSP70 in both cell lines at 24 h and HSP40 and HSP90 in A2780 cells at 48 h (Fig. 3A, B; Tab. 1A, B). On the other hand, indole K157 downregulat-ed HSP40 and HSP70 in both A2780 and A2780cis cells and HSP57 and HSP90 in A2780 cells at 24 h (Fig. 3A, B; Tab. 1A, B). Cisplatin did sufficiently modify the level of monitored HSPs in A2780 cells (Fig. 3A, B; Tab. 1A, B) and downregulated both HSP40 and HSP57 in A2780 cells re-versibly at 48 h and 24 h, respectively (Fig. 3A; Tab. 1A) and HSP57 in A2780cis cells at 48h (Fig. 3B; Tab. 1B). The level of only HSP60 did not change in A2780 or A2780cis cells after the indole or cisplatin administration (Fig. 3A, B; Tab. 1A, B). In general, the reduction in the level of HSPs in A2780 cells compared to A2780cis cells was more significant (Fig. 3A, B; Tab. 1A, B). A) B) Figure 3. Effects of IC50 concentrations of homobrassinin (K1), thiazino[6,5-b]indol (K157), and cisplatin (Cis) on HSP40, HSP57 (PDI), HSP60, HSP70, and HSP90 in A2780 (A) and A2780cis (B) cell lines. The cells were grown in standard growth media for 24 h and then treated with the substances mentioned above for 24, 48, and 72 h (untreated control - C). The detection of a-tubulin confirmed equal loading. A representative image of three independent experiments is shown. Solarova et al.: Apoptotic Effect of Homobrassinin ... Acta Chim. Slav. 2021, 68, 151-158 155 Table I. Effects of IC50 concentrations of homobrassinin (K1), thiazino[6,5-b]indol (K157), and cisplatin (Cis) on HSP40, HSP57 (PDI), HSP60, HSP70, and HSP90 in A2780 (A) and A2780cis (B) cell lines. Ratios ± standard deviation from quantitative densitometric analysis of HSP40, HSP57, HSP60, HSP70, and HSP90 were normalized to a-tubulin. Ratios at untreated control (C) were arbitrarily set to 1. *p<0.05; **p<0.01 vs. C (three independent experiments; one-way ANOVA tests). A Hsp40 Hsp57 Hsp60 Hsp70 Hsp90 24 h C K1 K157 Cis 1.0 ± 0.1 1.3 ± 0.3 0.0 ± 0.1** 1.6 ± 0.3 1.0 ± 0.1 0.7 ± 0.3 0.3 ± 0.1* 0.5 ± 0.1* 1.0 ± 0.2 1.3 ± 0.2 1.3 ± 0.3 1.1 ± 0.1 1.0 ± 0.1 0.1 ± 0.1** 0.0 ± 0.1** 0.8 ± 0.3 1.0 ± 0.1 0.7 ± 0.3 0.1 ± 0.1** 0.7 ± 0.3 Hsp40 Hsp57 Hsp60 Hsp70 Hsp90 48 h C K1 K157 Cis 1.0 ± 0.1 0.0 ± 0.1** 0.0 ± 0.2** 0.1 ± 0.1** 1.0 ± 0.2 1.0 ± 0.3 0.9 ± 0.1 1.2 ± 0.3 1.0 ± 0.3 1.1 ± 0.2 1.3 ± 0.2 1.0 ± 0.3 1.0 ± 0.2 0.0 ± 0.1** 0.5 ± 0.1* 0.7 ± 0.3 1.0 ± 0.1 0.0 ± 0.1** 0.3 ± 0.1* 1.1 ± 0.3 Hsp40 Hsp57 Hsp60 Hsp70 Hsp90 72 h C K1 K157 Cis 1.0 ± 0.1 0.0 ± 0.1** 0.0 ± 0.2** 1.1 ± 0.1 1.0 ± 0.1 0.9 ± 0.2 0.4 ± 0.1* 0.8 ± 0.3 1.0 ± 0.2 1.1 ± 0.3 0.8 ± 0.2 0.9 ± 0.2 1.0 ± 0.2 0.0 ± 0.1** 0.0 ± 0.1** 1.1 ± 0.3 1.0 ± 0.1 0.5 ± 0.1* 0.0 ± 0.1** 0.9 ± 0.3 B Hsp40 Hsp57 Hsp60 Hsp70 Hsp90 24 h C K1 K157 Cis 1.0 ± 0.1 0.8 ± 0.2 0.5 ± 0.1* 0.8 ± 0.3 1.0 ± 0.2 1.1 ± 0.3 1.1 ± 0.2 0.7 ± 0.2 1.0 ± 0.2 0.9 ± 0.2 1.3 ± 0.3 1.2 ± 0.2 1.0 ± 0.1 0.6 ± 0.3 0.0 ± 0.1** 0.7 ± 0.2 1.0 ± 0.2 0.8 ± 0.2 0.7 ± 0.2 0.7 ± 0.2 Hsp40 Hsp57 Hsp60 Hsp70 Hsp90 48 h C K1 K157 Cis 1.0 ± 0.1 0.6 ± 0.2 0.4 ± 0.1* 1.3 ± 0.3 1.0 ± 0.2 1.3 ± 0.3 0.6 ± 0.2 0.7 ± 0.2 1.0 ± 0.2 1.0 ± 0.3 0.8 ± 0.2 1.0 ± 0.3 1.0 ± 0.1 0.4 ± 0.1* 0.0 ± 0.1** 1.0 ± 0.2 1.0 ± 0.1 1.0 ± 0.2 0.5 ± 0.1* 0.9 ± 0.2 Hsp40 Hsp57 Hsp60 Hsp70 Hsp90 72 h C K1 K157 Cis 1.0 ± 0.2 1.0 ± 0.3 1.0 ± 0.2 1.0 ± 0.1 1.0 ± 0.3 1.0 ± 0.3 1.4 ± 0.3 1.0 ± 0.2 1.0 ± 0.2 1.0 ± 0.3 1.8 ± 0.4 0.7 ± 0.2 1.0 ± 0.1 0.0 ± 0.1** 0.1 ± 0.1** 1.4 ± 0.2 1.0 ± 0.1 0.8 ± 0.2 0.4 ± 0.1* 1.1 ± 0.3 Homobrassinin (K1) had the most pronounced antiproliferative activity of the seven tested brassinin derivatives.13 This compound accumulated human colorectal adenocarcinoma Caco2 cells in the G2/M phase of the cell cycle and inhibited microtubule formation through the dysregulation of a-tubulin, a 1-tubulin, and ^ 5 -tubulin expression.13 Moreover, the apoptosis of Caco2 cells induced by K1 was associated with DNA fragmentation, loss of mitochondrial membrane potential, and intracellular reactive oxygen species production.13 Our results demonstrated that K1 induced the apoptosis of cisplatin-sensitive human ovarian adenocarcinoma A2780 cells and cispla-tin-resistant derivative A2780cis cells, both associated with annexin V/PI positivity, increased caspase 3 activity, and released cytochrome C. K157 showed even more pronounced apoptosis-stimulating effect compared to K1, with increased annexin V/PI positivity, caspase 3 and 9 activities, the accumulation of smac-diablo, and the release of cytochrome C in both cisplatin-sensitive as well as cisplatin-resistant cells in a time-dependent manner. The K157-induced apoptosis of A2780cis cells has already been confirmed by a lower concentration than IC50 and through the detection of increased pro-apoptotic Bad protein.8 Cisplatin increased the release of cytochrome C and the exter-nalization of phosphatidylserine but only in the sensitive A2780 cells, not in the resistant ones. Hsp90 is one of the most abundant molecular chap-erones and highly conserved proteins, the association of which is required for the stability and function of multiple mutated, chimeric, and overexpressed signaling proteins that promote the growth and/or survival of cancer cells.14 Interestingly, antiproliferative and pro-apoptotic effects of Solarova et al.: Apoptotic Effect of Homobrassinin 156 Acta Chim. Slav. 2021, 68, 151-158 K157 were potentiated by HSP90 inhibitor 17-DMAG in A2780cis cells, which suggests a new strategy in cancer resistance therapy.8 Similarly, combined exposure of A2780cis cells to HSP90 inhibitor geldanamycin and cis-platin yielded greater than the additive cytotoxic effect.11 We have already shown that cisplatin-resistant CP70 and C200 cells (derivatives of parental A2780 cells) revealed significantly higher expression of HSPCA (HSP90a) and TRA1 (GRP94) proteins.15 Overexpressed GRP94 and HSP90a might protect CP70 and C200 cells from cisplatin toxicity and render them more resistant.15 Indeed, the role of HSPs in cisplatin resistance has been demonstrated in a previous study,16 where HSP27, mtHSP75, and HSP70 were upregulated in the cisplatin-resistant ovarian tumor cell line 2008/C13*5.25 in contrast to the sensitive 2008 one. Therefore, in the present study, we decided to test the effect of indoles on the level of selected HSPs, which can have significant cytoprotective effects in pathological conditions through the initiation of protein folding, repair, refolding of misfolded peptides, and possible degradation of irreparable proteins.17 Although some HSPs are produced constitutively, most are molecular chaperones that are normally overexpressed by cells in response to induc-ible signals that may lead to protein denaturation.18 HSPs appear to have a large number of functions in apoptosis itself, which, in most cases, leads to suppression of apop-totic pathways.17 In our case, the apoptosis-stimulating effects of indoles were associated with an irreversible decrease in HSP90 and HSP70 in both A2780 as well as A2780cis cells. At the same time, cisplatin did not cause any change in HSP90 and HSP70. Moreover, Beere et al.19 demonstrated that Hsp70, together with its co-chaperone Hsp40, inhibits nitric oxide-induced apoptosis in ATPase- and chaperoning-de-pendent manner by blocking the mitochondrial translocation of Bax, a pro-apoptotic member of the Bcl-2 family.19 HSP70 and HSP40 were irreversibly downregulated as a result of the effect of both K1 and K157 indoles on A2780 and A2780cis cells. On the other hand, HSP40 protein was reversibly downregulated as a result of cisplatin in A2780 cells. Indeed, downregulation of HSP70 and/or HSP40 correlated well with the apoptosis induced by either indole in both cell lines or cisplatin in A2780 cells. Despite all the negative functions of HSPs in attenuating apoptosis, Hsp60, found in mitochondria complexed with Hsp10, is involved in a signaling complex that leads to pro-caspase-3 activation and cytochrome-dependent apoptosis. In addition, several studies have shown that cytosolic Hsp60 is associated with the pro-apoptotic protein Bax, leading to its activation as well as to apoptosis mediated by this pro-tein.20,21 Indeed, neither indoles nor cisplatin caused a change in the level of HSP60 protein, and they are unlikely to be related to the process of apoptosis induced by indoles or cisplatin in A2780 or A2780cis cells. HSP57 (PDI) protein is highly expressed in many tumor cell types, including melanoma, prostate, lung, renal, brain, male germ cell tumors, and ovarian.22 Conversely, decreased PDI levels are also associated with higher survival rates in patients with breast cancer and glioblasto-ma.23 Although PDI promotes cancer cell survival, its silencing causes greater cytotoxicity in human breast cancer and neuroblastoma cell lines due to caspase activation.24 The suppression of apoptosis by PDI itself serves a mechanism to promote tumor growth and metastasis. In this regard, K157 induced both irreversible and reversible down-regulation of HSP57 in A2780 and A2780cis cells, respectively, while cisplatin caused a reversible decrease in HSP57 in both cell lines monitored. 4. Conclusions Our results signify more pronounced sensitizing and/or proapoptotic potential of thiazino[6,5-b]indol compared to homobrassinin accompanied by increased smac-diablo accumulation at earlier time intervals and pronounced externalization of phosphatidylserine at 72 h in A2780cis compared to A2780 cells. The apoptosis-stim-ulating effect of thiazino[6,5-b]indol in A2780cis cells was associated with significant irreversible downregulation of HSP70 and HSP90 and partly with a decrease of HSP40. On the other hand, cisplatin induced the apoptosis of sensitive A2780 cells with reversible downregulation of HSP40 and HSP57. In conclusion, the effect of thiazino[6,5-b]in-dol on resistant A2780cis cells could have a great utility in both the potential prevention and the treatment of other cisplatin-resistant tumor cells. Conflict of Interest There is no conflict of interest. Acknowledgments This study was supported (50%) by the project Medicinsky univerzitny park v Kosiciach (MediPark, Kosice) ITMS: 26220220185 (95%) supported by Operational Programme Research and Development (OPVaV-2012/2.2/08-R0) (Contract No. OPVaV/12/2013). We would like to thank also the Slovak Grant Agency for Science (Grant No. 1/0536/19) for financial support of this work. The authors are grateful to Mgr. Marian Curda for drawing the chemical structures of indoles. 5. References 1. Thomsen, L. H.; Schnack, T. H.; Buchardi, K.; Hummelshoj, L.; Missmer, S. A.; Forman, A.; Blaakaer, J., Risk factors of epithelial ovarian carcinomas among women with endometrio-sis: a systematic review. Acta Obstet Gynecol Scand 2017, 96 (6), 761-778. DOI:10.1111/aogs.13010 Solarova et al.: Apoptotic Effect of Homobrassinin ... Acta Chim. Slav. 2021, 68, 151-158 157 2. Luvero, D.; Milani, A.; Ledermann, J. A., Treatment options in recurrent ovarian cancer: latest evidence and clinical potential. Ther Adv Med Oncol 2014, 6 (5), 229-39. DOI: 10.1177/1758834014544121 3. Sharma, R.; Graham, J.; Mitchell, H.; Brooks, A.; Blagden, S.; Gabra, H., Extended weekly dose- dense paclitaxel/carbopla-tin is feasible and active in heavily pre-treated platinum-resistant recurrent ovarian cancer. Br J Cancer 2009, 100 (5), 707-12. D01:10.1038/sj.bjc.6604914 4. Rose, P. G.; Mossbruger, K.; Fusco, N.; Smrekar, M.; Eaton, S.; Rodriguez, M., Gemcitabine reverses cisplatin resistance: demonstration of activity in platinum- and multidrug-resist-ant ovarian and peritoneal carcinoma. Gynecol Oncol 2003, 88 (1), 17-21. D0I:10.1006/gyno.2002.6850 5. Katsumata, N.; Yasuda, M.; Isonishi, S.; Takahashi, F.; Michi-mae, H.; Kimura, E.; Aoki, D.; Jobo, T.; Kodama, S.; Terauchi, F.; Sugiyama, T.; Ochiai, K.; Japanese Gynecologic Oncology, G., Long-term results of dose-dense paclitaxel and carbopla-tin versus conventional paclitaxel and carboplatin for treatment of advanced epithelial ovarian, fallopian tube, or primary peritoneal cancer (JGOG 3016): a randomised, controlled, open-label trial. Lancet Oncol 2013, 14 (10), 1020-6. DOI:10.1016/S1470-2045(13)70363-2 6. Garcia, A. A.; Hirte, H.; Fleming, G.; Yang, D.; Tsao-Wei, D. D.; Roman, L.; Groshen, S.; Swenson, S.; Markland, F.; Gandara, D.; Scudder, S.; Morgan, R.; Chen, H.; Lenz, H. J.; Oza, A. M., Phase II clinical trial of bevacizumab and low-dose metronomic oral cyclophosphamide in recurrent ovarian cancer: a trial of the California, Chicago, and Princess Margaret Hospital phase II consortia. J Clin Oncol 2008, 26 (1), 76-82. DOI:10.1200/JCO.2007.12.1939 7. Gotlieb, W. H.; Amant, F.; Advani, S.; Goswami, C.; Hirte, H.; Provencher, D.; Somani, N.; Yamada, S. D.; Tamby, J. F.; Vergote, I., Intravenous aflibercept for treatment of recurrent symptomatic malignant ascites in patients with advanced ovarian cancer: a phase 2, randomised, double-blind, placebo-controlled study. Lancet Oncol 2012, 13 (2), 154-62. DOI:10.1016/S1470-2045(11)70338-2 8. Solarova, Z.; Kello, M.; Varinska, L.; Budovska, M.; Solar, P., Inhibition of heat shock protein (Hsp) 90 potentiates the antiproliferative and pro-apoptotic effects of 2-(4'fluoro-phe-nylamino)-4H- 1,3-thiazine[6,5-b]indole in A2780cis cells. Biomed Pharmacother 2017, 85, 463-471. D0I:10.1016/j.biopha.2016.11.052 9. Banerjee Mustafi, S.; Chakraborty, P. K.; Raha, S., Modulation of Akt and ERK1/2 pathways by resveratrol in chronic myelogenous leukemia (CML) cells results in the downregulation of Hsp70. PLoS One 2010, 5 (1), e8719. D0I:10.1371/journal.pone.0008719 10. Pilatova, M.; Sarissky, M.; Kutschy, P.; Mirossay, A.; Mezencev, R.; Curillova, Z.; Suchy, M.; Monde, K.; Mirossay, L.; Mojzis, J., Cruciferous phytoalexins: antiproliferative effects in T-Jur-kat leukemic cells. Leuk Res 2005, 29 (4), 415-21. D0I:10.1016/j.leukres.2004.09.003 11. Solar, P.; Horvath, V.; Kleban, J.; Koval, J.; Solarova, Z.; Kozubik, A.; Fedorocko, P., Hsp90 inhibitor geldanamycin increases the sensitivity of resistant ovarian adenocarcino-ma cell line A2780cis to cisplatin. Neoplasma 2007, 54 (2), 127-30. 12. Mahmood, T.; Yang, P. C., Western blot: technique, theory, and trouble shooting. N Am J Med Sci 2012, 4 (9), 429-34. D01:10.4103/1947-2714.100998 13. Kello, M.; Drutovic, D.; Chripkova, M.; Pilatova, M.; Budovska, M.; Kulikova, L.; Urdzik, P.; Mojzis, J., ROS-dependent antiproliferative effect of brassinin derivative homobrassinin in human colorectal cancer Caco2 cells. Molecules 2014, 19 (8), 10877-97. D0I:10.3390/molecules190810877 14. Neckers, L., Hsp90 inhibitors as novel cancer chemothera-peutic agents. Trends Mol Med 2002, 8 (4 Suppl), S55-61. D0I:10.1016/S1471-4914(02)02316-X 15. Solar, P.; Sytkowski, A. J., Differentially expressed genes associated with cisplatin resistance in human ovarian adenocarci-noma cell line A2780. Cancer Lett 2011, 309 (1), 11-8. D0I:10.1016/j.canlet.2011.05.008 16. Yamamoto, K.; Okamoto, A.; Isonishi, S.; Ochiai, K.; Ohtake, Y., Heat shock protein 27 was up- regulated in cisplatin resistant human ovarian tumor cell line and associated with the cisplatin resistance. Cancer Lett 2001, 168 (2), 173-81. D0I:10.1016/S0304-3835(01)00532-8 17. Ikwegbue, P. C.; Masamba, P.; Oyinloye, B. E.; Kappo, A. P., Roles of Heat Shock Proteins in Apoptosis, Oxidative Stress, Human Inflammatory Diseases, and Cancer. Pharmaceuticals (Basel) 2017, 11 (1). D0I:10.3390/ph11010002 18. Srivastava, P., Roles of heat-shock proteins in innate and adaptive immunity. Nat Rev Immunol 2002, 2 (3), 185-94. 19. Beere, H. M., "The stress of dying": the role of heat shock proteins in the regulation of apoptosis. J Cell Sci 2004, 117 (Pt 13), 2641-51. D0I:10.1242/jcs.01284 20. Samali, A.; Cai, J.; Zhivotovsky, B.; Jones, D. P.; Orrenius, S., Presence of a pre-apoptotic complex of pro-caspase-3, Hsp60 and Hsp10 in the mitochondrial fraction of jurkat cells. EMBO J 1999, 18 (8), 2040-8. D0I:10.1093/emboj/18.8.2040 21. Gupta, S.; Knowlton, A. A., Cytosolic heat shock protein 60, hypoxia, and apoptosis. Circulation 2002, 106 (21), 2727-33. 22. Xu, S.; Sankar, S.; Neamati, N., Protein disulfide isomerase: a promising target for cancer therapy. Drug Discov Today 2014, 19 (3), 222-40. D0I:10.1016/j.drudis.2013.10.017ww Solarova et al.: Apoptotic Effect of Homobrassinin 158 Acta Chim. Slov. 2021, 68, 151-158 Povzetek Fitoaleksini so snovi s protimikrobnimi lastnostmi, ki jih rastline proizvajajo po napadu mikroorganizmov, zlasti fito-patogenih gliv in virusov. Trenutno jih preučujejo tudi zaradi njihovih protitumorskih učinkov. Naš cilj je bil proučiti apoptozo-stimulirajoče delovanje homobrassinina and thiazino[6,5-b]indola v celicah človeškega ovarijskega adenokar-cinoma A2780 and A2780cis s pomočjo pretočne citometrične analize aneksina V/PI, aktivnosti kaspaz 3 in 9, sproščanja citokroma C in kopičenja smac-diablo. S tehniko prenosa po westernu smo spremljali tudi učinek obeh indolov na odziv proteinov toplotnega šoka v teh celicah. Thiazino [6,5-b]indol je v primerjavi s homobrassininom pokazal izrazitejši senzibilizacijski in/ali proapoptotični učinek, ki ga je spremljalo povečano kopičenje smac-diabla v zgodnejših časovnih intervalih in izrazita eksternalizacija fosfatidilserina po 72 urah v A2780cis celicah v primerjavi s celicami A2780. Učinek tiazino [6,5-b] indola, ki je stimuliral apoptozo, je bil v celicah A2780cis povezan z značilnim nepovratnim zmanjšanem HSP70 in HSP90, ter deloma z zmanjšanjem HSP40. Po drugi strani pa je cisplatin povzročil apoptozo občutljivih celic A2780 z reverzibilnim zmanjšanjem HSP40 in HSP57. Skratka, učinek tiazino [6,5-b]indola na odporne celice A2780cis bi bil lahko zelo koristen pri potencialnem preprečevanju in zdravljenju drugih tumorskih celic, odpornih na cisplatin. © 0 Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Solarova et al.: Apoptotic Effect of Homobrassinin ... DOI: 10.17344/acsi.2020.6298 Acta Chim. Slov. 2021, 68, 159-169 /^creative ^commons Scientific paper Characterization of Hydration Behaviour and Modeling of Film Formulation Arunima Pramanik,1 Rudra Narayan Sahoo,1,2 Souvik Nandi,1 Ashirbad Nanda1 and Subrata Mallick^* 1 Department of Pharmaceutics, School of Pharmaceutical Sciences, Siksha 'O' Anusandhan (Deemed to be University), Bhubaneswar 751003, Odisha, India. 2 Centurion University of Technology and Management, Odisha, India. * Corresponding author: E-mail: profsmallick@gmail.com (Telephone: +91-674-2386209; fax: +91-674 2386271 Received: 07-26-2020 Abstract Hydration behavior of hydrogel-based polymeric film possesses great importance in mucosal drug delivery. Modified Lag phase sigmoid model was used for the investigation of hydration of the film. Kaolin incorporated HPMC K100LVCR (Hl) and K100M (Hh) films containing dexamethasone as a model drug have been prepared for studying swelling kinetics. Swelling of HL and HH films was decreased with the gradual increase of kaolin content and HH of higher viscosity has shown higher value than HL matrix. Kaolin also inhibited the film erosion process. Mathematically modified lag phase sigmoid model demonstrated similarity of the predicted swelling content with the observed value. High R2 and small RMSE value confirmed the successful fitting of the modified lag phase sigmoid model to the experimental data of swelling content. t value similar to the observed one was obtained. This modified model could be reliable enough for estimating hydration process in food grains, food packaging films etc. Keywords: Hydration phenomenon; water diffusion; swelling kinetic model; modified lag phase. 1. Introduction Swelling behavior of polymer films possesses a significant importance in hydrogel-based transmucosal drug delivery systems and diverse application in biomolecular electronics and sensors, wound dressings, adsorption of chemical materials and contact lenses.1,2 Matrix film of hy-drophilic polymer is susceptible to environmental moisture and water due to the presence of hydrophilic groups in the macromolecule chains.3 Swelling of polymer network is affected by the cross-linking degree of polymer, solvent-polymer compatibility and polymer nature.4 In the process of swelling, the solvent molecules get in contact with the polymer and diffuse into the polymer materials. Diffusion of solvent molecules into the polymer network, expansion of network, and relaxation of polymer chain are the three steps of dynamic hydration process. 5 Thus swelling is responsible for the polymer chain relaxation and facilitates the patterned release of drug. Swelling develops bioadhesion through the intimate contact between polymer and mucosal tissue due to the entanglement of poly- mer and mucin chains of mucosal membrane lining.6 For transmucosal drug delivery amlodipine, budesonide, diclofenac film formulations were developed in our laboratory wherein dynamic hydration process has been de-scribed.6-8 The mucoadhesion between the mucus layer and the natural and synthetic polymers covered the muco-sal epithelial surface. Further, the controlled drug release is affected by the interaction capability of polymer with mucus layer.9 Swelling gets much attention as an important mechanism of controlled drug delivery system.10,11 For hydrogel film, the dynamic process of solvent penetration can be interpreted from swelling. Based on this knowledge, the drug release pattern can be adjusted by modifying the swelling of polymer matrix.5 The incorporation of clay minerals into the polymer matrix reduces the strain of polymer matrix due to their intercalation properties12 and increments significant property.13 Silicate clay, such as montmorrilonite, has been characterized for the improvement of the biodegradability and mechanical properties of polymeric film.14,15 Kaolin, a Pramanik et al.: Characterization of Hydration Behaviour 160 Acta Chim. Slov. 2021, 68, 159-169 phyllosilicate (1:1) crystalline clay has the cation exchange capacity.16 It consists of a tetrahedral silica sheet and an alumina octahedral sheet, and the layers are bonded together with hydrogen bonds.17 Incorporation of kaolin in drug delivery design is promoted due to its characteristic features such as chemical inertness, rheological properties, high specific area, swelling capacity and sorption capacity. The effect of kaolin incorporation in the film on the quality and functionality of swelling of film could be investigated by applying the mathematical model. This type of utilization of mathematical models is almost unavailable in the literature. The water diffusion and swelling behaviour of the polymeric film was previously studied by several researchers.18-21 In literature, the kinetic model for absorbent gels swelling was constructed by Tanaka and Filmore.22 After that, Li and Tanaka23 included some new approaches regarding swelling of gel and that was implemented by Chi Wu and Chui-Ying Yan24 for the gelatin of film swelling. Hans Schott described the swelling kinetics of gelatin film using second order kinetics.25 Peleg model has been utilized to describe the swelling kinetics of acrylamide-sodium acrylate hy-drogel26 and water sorption process of food packaging films as well.27 The cellulose polymer contains hydroxyl group and it is used in different fields due to their hydro-philic and hygroscopic nature.28 Hydroxypropyl methyl-cellulose (HPMC) contains many hydroxyl groups and is widely used in pharmaceutical industry and other fields.29 Kinetics of food grain hydration process has already been studied by several researchers earlier using lag phase sigmoid model and other kinetic models.30-32 In this study, lag phase sigmoid model and also the modified version of this model has been applied to investigate the swelling of the films. In our knowledge, no such modification of model was used to describe the swelling kinetics of any polymeric film. Mathematical modification of lag phase sigmoid model has been described as: = i+e,p(lVr)) Phase model) Modification: ^ = 1 + exp(—fc(t - t) 1 = exp(-fc(t - t) ■Jt SE is the equilibrium swelling, k (min-1) is the swelling rate constant and t is the time to obtain half of the swelling sat-ration (i. e. St = SE/2 at t = t). The swelling rate constant (k) can be determined from the slope of Ln f-^-r—vs. t linear plot. 2. Experimental 2.1. Materials Dexamethasone (DXM) was received as gift sample (Sigma Company). Kaolin, HPMC K100LVCR (HL) and HPMC K100M (Hh), triethanolamine were purchased from Qualikems, Burgoyne, Burbidges & co., Merck specialities Pvt Ltd (Mumbai) respectively. Ethanol was bought from MERCK (Germany). 2. 2. Preparation of Hydrogel Film The polymers (HL and HH) were swelled in distilled water overnight and stirred continuously for 24 hours at room temperature to prepare homogenous polymeric dispersion. Simultaneously, kaolin clay dispersion (10 wt. %) was prepared separately without any treatment by dispersing kaolin clay in distilled water and stirring for 24 h. This dispersion was centrifuged at 2500 rpm for 15 min and the thin nano-dispersion layer from the upper part was separated for film formulation. It was established that this process of dispersion in distilled water may be capable of reducing the average particle size to nano size clay particle.33 Kaolin dispersion in different amount (25, 50, 75 or 100 mg content) was gradually added into the HL or HH polymer dispersion (800 mg) and stirred continuously for 1 h with a magnetic stirrer. Dexamethasone (model drug, 100 mg) and triethanolamine (15 % of polymer as plasticizer) were dissolved in 5 ml of ethanol and incorporated in each polymeric kaolin dispersion with continuous stirring for 3 h. According to solvent casting and solvent evaporation method, final dispersion into the petri dish was spread and dried in an incubator for 24 h at 60 °C until constant weight. The prepared films were subjected to the following characterizations. At least three repetitions of the following experiments were done and mean ± SD values were calculated. 2. 3. Water Sorption Studies Films were accurately weighed (W1) at room temperature (30 °C) and placed in desiccators containing activated silica gel. After 24 h films were discarded from desiccators and weighed (W2) until constant weight achieved. Water content was estimated from the difference between initial and final weight with respect to final weight (W2). For water sorption analysis each film was weighed (W1) after removal from desiccators over silica gel and placed in the desiccators to maintain 65, 75, and 84 % relative humidity with 100 ml super saturated solution of sodium nitrite, sodium chloride and potassium chloride, respectively. After about 24 h or more films were weighed (W2) till three resulted weights of each film were same. Moisture uptake was determined from the difference between final and initial weight with respect to initial weight (W1) and expressed as percentage. Pramanik et al.: Characterization of Hydration Behaviour Acta Chim. Slov. 2021, 68, 159-169 161 2. 4. FTIR Spectroscopy The possible interaction between the components was interpreted from FTIR analysis. The FTIR characterization of pure drug and the film formulations (DHhK0, DHHK^ DHHK2, DHHK3, and DHHK4) was performed using JASCO FT/IR-4100 spectra. To prepare sample pellets, KBr pressed-disk method was used (ratio of sample to KBr is 1:20) and then sample pellet was placed in FTIR spectrometer. FTIR spectra were obtained in the range of 4004000 cm-1 and at a resolution of 4 cm-1 as transmission mode by accumulating 80 scans. 2. 5. Scanning Electron Microscopy (SEM) SEM images were utilized to visualize the morphology of pure drug, kaolin and film formulations (DHlK4, and DHHK4) of highest kaolin content. Samples were gold sputter-coated under argon atmosphere and placed in JEOL/EO $ CM_VERSION 1.0 Scanning Electron Microscope model no (JSM-6390) (operating at 5 kV) for imaging. 2. 6. Hydration and Swelling Studies Films were cut into small pieces and weighed (W0). Then the small pieces of film were immersed into each petri plate containing 25 ml of pH 7.4 phosphate buffer saline at room temperature. After predetermined time interval samples were taken out from the petri plate and excess buffer was soaked in filter paper. Then the final weight (Wt) was recorded and this process was continued up to 6 h. Swelling content was calculated by using the following equation,34 where, F is the fraction of swelling content, Smax i the maximum swelling content of the formulation, St the swelling content of the formulation at any time, K the gel network structure constant, and n the diffusion exponential of solvent. The 'n value of Peppas equation was utilized to determine the diffusion type. Coefficient of diffusion, an important parameter, was also evaluated from the swelling kinetics. It can be calculated for the square shape film from the following equation which was derived by re-arranging the Fick's II law. - hT (6) where D is the diffusion coefficient expressed in cm2s-1, and "a" is the side of square film in cm. In order to determine the swelling kinetics of films, lag phase sigmoid model was exploited in this study. The lag phase sigmoid equation has been modified to generalize the swelling kinetics study and also to compare it with the original lag phase sigmoid model. Kaptso et al also proposed a sigmoid model and described the swelling of film.36 The equation of this sigmoid model, comprised of an exponential rate of decay term, is accounted for an initial lag phase. The model is expressed as the following equation (7) SE is the equilibrium swelling, k (min-1) is the swelling rate constant and t is the time to obtain half of the swelling saturation (i. e. St = SE/2 at t = t). (3) S is the swelling content (g water/ g dry film), Wt is the weight of film after time t, and W0 is the weight of dry film. Erosion of film can also be analyzed from swelling studies. After 6 h of swelling study, films were dried at 40 °C in hot air oven. Films were removed from the oven after 24 h and reweighed (We) and erosion (E) was calculated as follow, (4) We is the weight of the eroded film after drying in oven. Swelling of polymer involved diffusion of water and necessitated the understanding of mechanism of solvent diffusion. The swelling kinetics and diffusion of polymer structure can be explained by basic law of Peppas law.24 (5) 2. 7. Statistical Analysis The model was statistically fitted with the experimental data and analyzed in Origin Pro 8.0 (OriginLab, Northampton, MA) software37 by the nonlinear regression analysis. Model fitness to the experimental data has been assessed using R2, x2, and root mean square error (RMSE) of statistical analysis data. x2 and RMSE can be calculated as (8) (9) 3. Results and Discussion 3. 1. Water Sorption Properties The moisture content and moisture uptake of DXM film composed of HPMC and kaolin were measured at Pramanik et al.: Characterization of Hydration Behaviour ... 162 Acta Chim. Slov. 2021, 68, 159-169 Table 1. Water sorption properties of the film formulations. Formulation DXM : kaolin % Water uptake at RH (mean ± SD, n = 3) Water content (%) 65 % 75 % 85 % (mean ± SD, n = 3) dhlko dhlki dhlk2 dhlk3 dhlk4 dhhko dhhki dhhk2 dhhk3 dhhk4 1 : 0.25 1 : 0.5 1 : 0.75 1 : 1 1 : 0.25 1 : 0.5 1 : 0.75 1 : 1 4.50 ± 0.02 4.31 ± 0.03 3.81 ± 0.02 3.42 ± 0.02 3.40 ± 0.03 6.76 ± 0.04 5.81 ± 0.05 5.22 ± 0.05 4.65 ± 0.05 4.57 ± 0.06 12.61 12.06 11.45 10.28 10.20 14.28 13.95 13.43 12.79 11.76 ± 0.03 ± 0.03 ± 0.03 ± 0.03 ± 0.04 ± 0.08 ± 0.09 ± 0.08 ± 0.06 ± 0.07 21.62 19.82 19.08 17.00 16.57 24.81 22.09 19.40 19.76 18.30 ± 0.03 ± 0.03 ± 0.02 ± 0.03 ± 0.03 ± 0.11 ± 0.10 ± 0.10 ± 0.06 ± 0.03 2.80 ± 0.01 2.58 ± 0.01 2.34 ± 0.01 2.06 ± 0.01 1.14 ± 0.02 3.87 ± 0.02 3.52 ± 0.02 3.22 ± 0.01 2.68 ± 0.02 2.40 ± 0.02 25 °C, and at 65, 75, and 85 % relative humidity. Table 1 displays the gradual depletion of both water content and water uptake while kaolin content increases in the film. It is also seen that moisture uptake of all respective formulations is progressively increasing with increasing RH from 65 to 85 %. The highest kaolin content in the film resulted in the decreased percent moisture uptake from 4.50 ± 0.02 to 3.40 ± 0.03 and 6.76 ± 0.04 to 4.57 ± 0.06 at 65 % RH for Hl and Hh matrices respectively in comparison with the absence of kaolin film. Percent moisture content of film has also been reduced from 2.80 ± 0.004 to 1.14 ± 0.02 and from 3.87 ± 0.02 to 2.40 ± 0.02 for HL and HH matrices respectively in the presence of kaolin (1:1) compared to the kaolin free films. The observed result indicated that the water resistance of both the matrix formulations was increased when kaolin was present in the film. In presence of triethanolamine as plasticizer, fine kaolin particles in the HPMC matrix enhanced the HPMC-kaolin interactions and left less free hydroxyl groups available for water binding. Thus, increased kaolin content gradually enhanced the barrier effect for the water uptake.38,39 3. 2. FTIR The analysis of the chemical constituent of pharmaceutical solids has been carried out by the FTIR spectroscopy. The drug-excipient interaction nature was also confirmed from the changes in the IR spectra. The changes in drug-excipient interactions are displayed as the disappearance of existing bands and appearance of new bands in the IR spectra, and also as intensity alteration and absorption band broadening. Figure 1 shows the IR spectra of kaolin, pure DXM and formulations. The characteristic absorption peaks of DXM in IR spectra that appear at 3000-2800 cm-1, 885 cm-1, 1718 cm-1, 1665 cm-1 and 1621 cm-1, are assigned to CH2 group, axial deformation of C-F group, Wavrnnmbfr [cm-1] Figure 1. FTIR spectra of pure DXM and of the prepared film formulations (DHhK0, DHhK1, DHhK2, DHhK3, and DHhKi). Pramanik et al.: Characterization of Hydration Behaviour Acta Chim. Slov. 2021, 68, 159-169 163 carbonyl group (C=O) of aliphatic ester and ketone,40 and C=C group, respectively.41 IR spectrum of pure kaolin demonstrated absorption peak at 1032 cm-1 with high intensity due to Si-O stretching in kaolin.42 Kaolin contains hydroxyl groups at different position, i.e. outer hydroxyl groups (OuOH, positioned in the upper unshared plan) and inner hydroxyl groups (InOH, positioned in the lower unshared plan of octahedral sheet). Kaolin spectra showed peaks at 3693, 3669, 3649 cm-1 and at 3619 cm-1 due to the stretching of OuOH and stretching of InOH in Al-OH.43 The broadening of absorption band at 3000-2800 cm-1 appeared due to the presence of polymer in the film. The successful incorporation of DXM in films has been confirmed by the presence of the main characteristic absorption peak ; f T- V fs - v 4 » * * « 10kV X2 000 10pm 0000 20 42 SEI 10kV X5.000 5pm 0000 19 41 SEI 10kV X10.000 1pm 0000 20 42 SEI P " ■ 10KV XS.000 „ M__ 5 (J m 0000 13 41 SEI Figure 2. Scanning electron micrograph of pure DXM (magnification 2,000) (i); kaolin (magnification 5,000) (ii); films with kaolin (magnification 10,000) (iii), (magnification 5,000) (iv) of HPMC K100 LVCR; and films with kaolin (magnification 10,000) (v), (magnification 5,000) (vi) of HPMC K100 M matrices. Pramanik et al.: Characterization of Hydration Behaviour ... 164 Acta Chim. Slov. 2021, 68, 159-169 at 1665 cm-1. This result suggested that the chemical properties of DXM have remained the same in the films. Interaction between DXM and kaolin in the film formulations has been confirmed by the shifting of the peak at 1032 cm-1. The formation of hydrogen bond can be concluded as a result of this band shifting. The broadening of the characteristic peak at 1032 cm-1 was observed in the wide range in IR spectra of films containing higher kaolin content. This result indicates stronger binding between DXM and kaolin with the corresponding increase of kaolin content in the films. 3. 3. Scanning Electron Microscopy The surface morphology of pure DXM, kaolin, and films of both HPMC K100 LVCR and HPMC K100 M matrices is demonstrated in Figure 2 by the scanning electron micrographs. The SEM images of Figure 2(i) demonstrate the geometric plate shaped microstructure of pure DXM and Figure 2(ii) that of nanosized kaolin. Figure 2(iii & iv) and (v & vi) represent the SEM images of films with higher kaolin content of both HL and HH matrices, respectively. The SEM images of films with added kaolin demonstrate the distinct distribution of nano-sized kaolin particles in the film. It can also be observed that the crystalline shape of DXM has almost disappeared in the films. In the presence of triethanolamine, the growth of drug crystal in HPMC film was not adequately noticed in the SEM micrographs due to the interfacial adhesion between the polymer phases and drug.44 The crystal growth is also inhibited by HPMC in the matrix.45 3. 4. Hydration Behavior The biological characteristic of hydrogel-based transmucosal drug delivery system significantly depended on the swelling of polymer present. Swelling capacity and swelling content of the film formulations were investigated in phosphate buffer saline (pH 7.4) at the laboratory ambient conditions. Swelling process included water uptake followed by the process of erosion of polymer matrix. The change of swelling content of film of both HL and Hh matrix with varied kaolin content is depicted in Figure 3 and Figure 4 respectively. Graphs display that film of HL and HH matrices exhibited maximum swelling at 60 min and 6 h respectively. Decreased swelling content was observed while kaolin content increased in the film. The water resistance ability of kaolin resulted in sustaining swelling of polymer-based film matrix. Water resistance ability has been improved by increasing the kaolin content and gradually sustained the swelling of the film. Water uptake, the main basis for swelling, depends on the hydrophilicity and other parameter such as morphology (macro voids), free volume, and crystal size. Hence, swelling may also be dependent on these parameters. Kaolin produced a tortuous pathway and also formed a denser cross-linking network due to its certain level of hydrophilicity.46,47 Thus kaolin had the capability of reducing the length of free pathway for water uptake into matrix, which led to sustained swelling of the film matrix. In film formulations (HL and Hh) swelling content was increased with the time of swelling. Higher swelling was found in the initial stage followed by a slower swelling at the later stages, known as hyperbolic form.25 The form of difference in swelling content in the graphs (between HL and HH) is related probably to the polymer viscosity and molecular weight, because high molecular weight polymers exhibit high viscosity in the swelling stage.48 The result of swelling content demonstrated higher value for the film of HPMC K100 M matrix compared with the film of HPMC K100LVCR matrix. This result suggests that the polymer viscosity and molecular weight play a role in the process of film swelling. The water uptake process did not easily empower through higher viscosity polymer of HPMC K100 M matrix. High molecular weight polymer increased the impact resistance due to higher degree of entanglement for rupturing more polymer bonds. In a fully hydrated state polymer chain disentanglement occured when there was no polymer-polymer interactions and higher viscosity induced greater chain entanglement than lower viscosity. Praveen et al.49 also described in their report that the polymer chain disentanglement is affected by the polymer viscosity. Film of Hh matrix with maximum kaolin content showed higher swelling index, which suggests decreased movement of DXM molecules from the film matrix to the medium. Erosion of film matrix is the process that follows after swelling and proceeds slowly. In this process, drug molecules diffuse through the micropores of polymer after Figure 3. Swelling content of film of HPMC K100 LVCR (HL) matrix with varied kaolin content. Pramanik et al.: Characterization of Hydration Behaviour Acta Chim. Slov. 2021, 68, 159-169 165 Figure 4. Swelling content of film of HPMC K100 M (Hh) matrix with varied kaolin content. water uptake of the matrix.6 After 6 h of the swelling study, erosion of films has been measured in pH 7.4 phosphate buffer saline. Figure 5 shows the progressive decrease in erosion with gradually increased kaolin content in the film. Presence of kaolin in the film hinders the process of erosion of film due to the entrapment of kaolin in the HPMC matrix networks. Higher kaolin content in the film has developed relatively stronger affinity between kaolin and polymer. Thus, the film with higher kaolin content shows more resistance for erosion. More resistance to the film erosion was also seen due to greater viscosity of HH compared to HL. 3. 5. Diffusion of Water The predicted swelling value is similar with the experimental swelling value, when the swelling kinetics of Figure 5. Erosion of film of HPMC K100 LVCR (HL) and HPMC K100 M (Hh) matrix with varied kaolin content. films has been described by the Peppas model (RMSE = 0.016-0.02 for HL matrix film and 0.016-0.02 for HH matrix film). The diffusion exponential (n), k, R2, reduced chi-square (x2) and RMSE values of all matrix films are demonstrated in Table 2. In order to investigate the water diffusion mechanism into hydrogel DXM film, Peppas equation was utilized. On the basis of the n value, the diffusion type of swelling was determined.50 Fickian and non-Fickian diffusion are identified by the n values of 0.5 and 1.0, respectively. The ranges of swelling exponent values of films of HL and HH matrix are 0.28-0.43 and 0.310.59 respectively. According to the calculated n value, the diffusion mechanism of swelling of films of HL and HH matrix was Fickian and anomalous diffusion, respectively. Diffusion coefficient (D) is an important factor that describes how quickly water can diffuse through the polymeric matrix film. The results revealed that the D value increased with the increased kaolin content in the film Table 2. Estimated swelling parameters of the film as per Peppas model and the diffusion coefficient of water. Formulation k n Parameter of Peppas model R2 x2 RMSE D (cm2s-1) dhlko 0.23 0.28 0.98 3.17E-04 0.016 1.32*10-4 dhlki 0.16 0.35 0.99 8.81E-05 0.008 4.04*10-4 dhlk2 0.14 0.37 0.99 3.61E-04 0.017 4.59*10-4 dhlk3 0.12 0.39 0.98 4.93E-04 0.020 5.54*10-4 dhlk4 0.10 0.43 0.99 4.44E-04 0.019 7.31*10-4 dhhko 0.16 0.31 0.98 3.19E-04 0.016 1.23*10-4 dhhki 0.12 0.36 0.99 2.98E-04 0.016 2.24*10-4 dhhk2 0.08 0.43 0.99 2.76E-04 0.015 4.12*10-4 dhhk3 0.06 0.49 0.98 0.00101 0.029 6.30*10-4 dhhk4 0.03 0.59 0.99 4.04E-04 0.018 10.89*10-4 Pramanik et al.: Characterization of Hydration Behaviour ... 166 Acta Chim. Slov. 2021, 68, 159-169 (Table 2). With the gradual increase in the kaolin content, water penetration becomes faster in both the polymers (Hl and Hh). The calculated diffusion coefficient increases from 1.32 *10-4 cm2s-1 to 7.31 *10-4 cm2s-1 for Hl matrix and 1.23 *10-4 to 10.89 *10-4 for HH matrix. 3. 6. Evaluation of Lag Phase Model and Modified Lag Phase Model The fitting of lag phase sigmoid model on the swelling data of film of both matrices (Hl and HH) is shown in Figure 6 & Figure 7, respectively. The swelling data fitting with modified lag phase sigmoid model is depicted in Figure 8 & Figure 9 of Hl and HH, respectively. The parameters (R2, k, t, Sobs eq, RMSE, x2) of this model are presented in Table 3 for all films. These parameters describe the kinetics of all films with lower RMSE and x2 value and high correlation coefficient indicating appropriate fitting of model on swelling data. The time to obtain half of the swelling saturation (t) is increased by higher kaolin content in the film. The predicted equilibrium swelling content and t show higher value for HH compared to Hl matrix film. Predicted equilibrium swelling content is similar to the experimentally obtained equilibrium moisture content. Modified lag phase sigmoid model demonstrates the increasing order of observed t (17.6 to 105.7 and 38.4 to 283.1) and decreasing or almost similar order of k (20.3 to 12.4 and 9.4 to 6.3) with increased kaolin content in the film of both matrices (Hl and HH matrix). Lag phase sig-moid model rate constant (k) decreases with increasing kaolin content in the film of both matrices supporting the effect of kaolin on prolonged swelling of the film. Kaptso et al 25 also described that the increased sigmoid model rate constant is due to increased mass transfer rate. However, Figure 6. Fitting of the Lag phase sigmoid model of films of HPMC K100LVCR (Hl) matrix with varied kaolin content. Figure 7. Fitting of the Lag phase sigmoid model of films of HPMC K100 M (Hh) matrix with varied kaolin swelling content. Table 3. Estimated parameters of the modified Lag phase model. Formulation Parameter of the modified lag phase model Ç ^obs.eq Obs t (min) Predicted t (min) k x 103 (min1) R2 RMSE X2 dhlko 6.32 17.6 24.3 20.3 0.951 0.174 0.03 dhlki 6.38 37.2 41.6 16.1 0.984 0.138 0.01 dhlk2 6.70 55.2 64.3 14.1 0.996 0.119 0.01 dhlk3 6.75 80.6 84.5 13.0 0.999 0.121 0.01 dhlk4 6.77 105.7 107.1 12.4 0.998 0.145 0.01 dhhko 10.62 38.4 57.7 9.4 0.964 0.338 0.03 dhhki 11.02 103.6 115.4 7.1 0.994 0.290 0.02 dhhk2 11.14 173.3 196.2 6.5 0.999 0.321 0.02 dhhk3 11.33 219.7 253.1 6.3 0.995 0.412 0.04 dhhk4 11.70 283.1 287.3 6.9 0.989 0.485 0.06 Pramanik et al.: Characterization of Hydration Behaviour Acta Chim. Slov. 2021, 68, 159-169 167 Figure 8. Fitting of the modified Lag phase sigmoid model of films of HPMC K100LVCR matrix with varied kaolin content. Figure 9. Fitting of the modified Lag phase sigmoid model of films of HPMC K100 M (Hh) matrix with varied kaolin swelling content. the predicted value of t increased from 24.26 to 107.10 and from 57.75 to 287.29 min for HL and HH, respectively. Correspondingly, k value decreased from 0.02 to 0.012 and from 0.009 to 0.006 min-1 for HL and HH, respectively. The fitting of this model is justified by the small RMSE and high R2 value. Hence, the proposed modified Lag phase sigmoid model may be acceptable in the hydration study. 4. Conclusion Kaolin incorporated HPMC films containing dexa-methasone as a model drug have been prepared for charac- terization of hydration behaviour utilizing modified lag phase model. In this study, the effect of kaolin on swelling of HL and HH hydrogel films was investigated. Gradually decreased water content and water uptake were observed with increased kaolin content in the film. Distribution of nano-sized kaolin particles in the films was noticed in the SEM images. Swelling of both the matrices (HL and HH) has been decreased with increasing the kaolin content in the film, while HH has shown higher value because of higher viscosity compared to the HL matrix. Presence of kaolin in the film inhibited the erosion process of the film due to the entrapment of kaolin in the HPMC matrix networks. Greater viscosity of HH also resulted in more resistance to the film erosion compared to HL. Increasing diffusion coefficient value indicated that water penetration became faster in presence of kaolin in the film. The calculated value of diffusion coefficient increased with increasing kaolin content in both the matrices. Swelling kinetics of the films with varied kaolin content was investigated by the lag phase sig-moid mathematical model. The predicted swelling content value from the modified lag phase sigmoid model was similar to the observed value. This modified lag phase sigmoid model was successfully fitted to the experimental data, as confirmed by R2 and lower RMSE value. The modified Lag phase sigmoid model resulted in t values almost equal to the observed t. Hence, we suggest that the modified version of the lag phase sigmoid model is also reliable enough to calculate kinetics in different food products. Acknowledgements We are very much grateful to the president, Siksha 'O' Anusandhan (Deemed to be University) Prof. (Dr) Manoj Ranjan Nayak for financial support and laboratory facility. Authors are also expressing our gratitude to the Pharmacia and Upjohn Company and Birla Institute of Technology, Mesra, Ranchi, for their gift sample and instrumental facility respectively. 5. References 1. A. S. Hoffman, Adv. Drug Deliv. Rev. 2002, 54, 3-12. DOI:10.1016/S0169-409X(01)00239-3 2. N. A. Peppas, K. B. Keys, M. Torres-Lugo, A. M. Lowman, J. Control. Release. 1999, 62, 81-87. DOI:10.1016/S0168-3659(99)00027-9 3. K. Xu, J. 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Shalabi, Saudi Pharm. J. 2015, 23, 167-176. D0I:10.1016/j.jsps.2014.06.006 45. R. Mohapatra, S. Mallick, A. Nanda, R. N. Sahoo, A. Praman-ik, A. Bose, D. Das, L. Pattnaik, RSC Adv. 2016, 6, 3197631987. D01:10.1039/C6RA03604J 46. A. Saada, B. Siffert, E. Papirer, J Colloid Interf. Sci. 1995, 174, 185-190. D0I:10.1006/jcis.1995.1381 47. A.Ghebaur, S. A. Garea, S.Cecoltan, H. Iovu, Mater. Plast. 2017, 54, 8-13. D0I:10.37358/MP. 17.1.4774 48. https://polymerdatabase.com/polymer%20physics/Solution_ Viscosity.html 49. S. P. Hiremath, N. R. Saha, AAPS Pharm. Sci. Tech. 2008, 9, 1171-1178. D0I:10.1208/s12249-008-9159-0 50. N. A. Peppas, L. Brannon-Peppas, J. Food Eng.1994, 22, 189210. D0I:10.1016/B978-1-85861-037-5.50015-1 Pramanik et al.: Characterization of Hydration Behaviour Acta Chim. Slov. 2021, 68, 159- 169 169 Povzetek Hidracijsko obnašanje polimernega filma na osnovi hidrogela ima velik pomen pri dostavi zdravil v sluznico. Za raziskovanje hidracije filma je bil uporabljen modificirani sigmoidni model na osnovi faznega zamika. Za preučevanje kinetike nabrekanja so bili pripravljeni filmi s kaolinom z vgrajenim HPMC K100LVCR (HL) in K100M (Hh), ki so vsebovali deksametazon kot modelno zdravilo. Nabrekanje HL in HH filmov se je s postopnim povečevanjem vsebnosti kaolina zmanjšalo, HH z višjo viskoznostjo pa je kazal večjo vrednost kot HL. Kaolin je zaviral tudi postopek erozije filma. Spremenjen matematični sigmoidni model faznega zamika je pokazal podobnost med predvideno in opaženo vsebnostjo nabrekanja. Visoka vrednost R2 in majhna vrednost RMSE sta potrdili uspešno prilagajanje modela eksperimentalnim podatkom za obseg nabrekanja. Tudi dobljena vrednost t je bila podobna opaženi. Ta spremenjeni model bi lahko bil dovolj zanesljiv za oceno postopka hidracije v zrnih živil, filmih za pakiranje hrane itd. © ® Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Pramanik et al.: Characterization of Hydration Behaviour ... DOI: 10.17344/acsi.2020.6299 Acta Chim. Slov. 2021, 68, 170-177 /^creative ^commons Scientific paper 4-(4,5-Diphenyl-1H-imidazole-2-yl)phenol: Synthesis and Estimation of Nonlinear Optical Properties using Z-Scan Technique and Quantum Mechanical Calculations Fatemeh Mostaghni Department of Chemistry, Payam Noor University, P.O. BOX 19395-4697 Tehran, Iran * Corresponding author: E-mail: mostaghnif@yahoo.com Received: 10-26-2020 Abstract In this study, 4-(4,5-Diphenyl-1H-imidazole-2-yl) phenol is successfully synthesized, and its nonlinear optical properties (NLO) are investigated both experimentally and theoretically. Theoretical investigations have been done by using TD-DFT and B3LYP functional with usual 6-31+G(d,p) basis set. The results of HOMO-LUMO and NBO analysis show the low energy gap, high total dipole moment, and hyperpolarizabilities (p, y) as well as the presence of dipolar excited states with relatively significant dipole-moment changes which are linked to the nonlinearity. The z-scan technique confirmed the NLO properties of title compound. The nonlinear absorption coefficient, refractive index, and third-order susceptibility were found to be 4.044 x 10-1 cmW-1, 2.89 x 10-6 cm2W-1 and 2.2627 x 10-6 esu, respectively. The negative sign of n2 indicated the occurrence of self-defocusing nonlinearity. The results show that the title compound can been used as potential NLO material. Keywords: Nonlinearity; z-scan technique; hyperpolarizabilities; triaryl imidazoles. 1. Introduction The development of photonic technology, nonlinear optical materials have received widespread attention both from the research as well as industrial point of view.1-3 In recent years, many studies have been reported by researchers to find new compounds with high nonlinear optical properties. The essential requirements of suitable photonic materials are their high nonlinearity, fast response time, chemical stability, and ease of molecular design.4-8 In this context, n-conjugated organic materials have received more attention due to their high nonlinearity and fast response times resulting from the ease of polarizability of the extended mobile n-electron clouds across the mole- cule.9-13 Unlike inorganic materials in which band structure phenomena cause nonlinear phenomena, in organic materials and polymers, these phenomena arise from the transition of an electron from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO) that caused a transition of the dipole moment from the ground state to the excited state.14-18 Different types of organic compounds with extensive conjugated n system are expected to exhibit nonlinear optical properties because of n-n interactions that allow an intramolecular charge transfer (ICT).19-29 Moreover, ICT is responsible for the broadening of the absorption spectrum, and the reduction of the optical bandgap.30-32 Over the past two decades, small organic molecules have been a subject of increasing research interest for their potential applications in organic electronics.33 Furthermore, it was shown that the n-conjugated bridges based on heterocyclic rings had improved stability relative to other polyenes.34-37 Among these, various types of imidazole derivatives have received widespread attention due to their piezoelectric, photochromic, and thermochromic properties. They are widely used in optoelectronics, superconductors, molecular photonics, sensors, and optical data storage devices.37-39 In this study, 4-(4,5-Diphenyl-1H-imidazole-2-yl) phenol was synthesized by one-pot three-component syn- Mostaghni: 4-(4,5-Diphenyl-1H-imidazole-2-yl)phenol: Acta Chim. Slov. 2021, 68, 170-177 171 thesis using CoFe2O4. Then for the first time, the nonlinear optical properties of this molecule are measured by the Z-scan technique and quantum mechanical methods. The results showed that the title compound could be a good candidate with the potential application in optoelectronic devices. 2. Experimental All chemicals were analytical grade and purchased from Sigma-Aldrich. Deionized water served as reacting medium. The melting point was measured by an Electro-thermal-9200 melting point apparatus. IR spectra were measured on the FTIR-6300 spectrometer (KBr). :HNMR spectra were recorded on Bruker ADVANC DRX 400 spectrometer, using DMSO as solvent. 2. 1. Synthesis of 4-(4,5-Diphenyl-1H-imidazole-2-yl) Phenol To the solution of benzoin (1 mM), 2-hydroxy benzaldehyde (1 mM), ammonium acetate (4mM) in ethanol (10 mL) was added 5 mol% CoFe2O4 nanocatalyst. The mixture was reflux in 50 °C, and the progress of the reaction was controlled by TLC using the mixture eluent (n-Hexane: Ethyl acetate 4:1). After completion of the reaction (30 minutes), the catalyst was separated by an external magnet, and the reaction mixture was allowed to cool. Then the precipitate was collected by filtration, washed with water, and recrystallized using ethanol. The product (figure 1) was characterized by FTIR and 1H NMR and Mass spectroscopy. Yield 87%; purity > 96%; mp: 278 °C (275-276 °C).40 Figure 1. Molecular structure of 4-(4,5-Diphenyl-1H- imidazo-le-2-yl) phenol IR (KBr, cm-1): v/cm-1 : 3598, 3445, 2996, 2468, 1643, 1613, 1546, 1506, 1490, 1240, 764, 698, 1H NMR: SH(ppm) (300 MHz, CDCl3): 8.03 (s, 1H, NH), 7.63-7.66 (m, 2H), 7.43-7.45 (m, 2H), 7.22-7.26 (m, 4H), 7.07-7.09 (d, 2H), 6.77-6.86 (m, 4H), GC-MS (IE, 70 eV) m/z (%) 312 (M+, 100.0), 208, 165, 89, 77, 51, 41. 2. 2. UV-Vis Absorption Spectrum The UV-Vis absorption spectrum of the sample was recorded using a spectrometer (Jenway model 6310). The Figure 2. UV-Vis absorption spectra of 4-(4,5-Diphenyl-1H- imi-dazole-2-yl) phenol sample was dissolved in DMF and measured in quartz cell in the range of 320 to 1000 nm (Figure 2). The spectrum showed absorption peaks at 340 nm, and 406 nm. 2. 3. Z-Scan Measurement The determining of the third-order nonlinear optical properties of the sample was carried out using the z-scan technique as a standard method. In this method, a very thin sample of matter is exposed to a laser beam while the sample was translated across the focal zone along the z-ax-is. The light passing through the sample is recorded as a function of the sample position relative to the beam focal point. The Z-Scan method is used to determine the nonlinear refractive index and absorption coefficient in close and open aperture configurations, respectively. 2. 4. Computational Method I performed density functional theory (DFT) and time-dependent DFT (TD-DFT) calculations on the title compound using the Gaussian 09 suite of software.41 The calculations were carried out by using TD-DFT and B3LYP functional.42,43 The usual 6-31G+(d,p) basis set was employed in the calculations. Numerous quantities including molecular structures, ionization potentials, electronegativity, HOMO-LUMO energies, and the HOMO-LUMO energy gap, dipole moments, polarizability, and hyperpo-larizability Have been measured and discussed. 3. Results and Discussion 3. 1. UV-Vis Absorption Spectrum As can be seen in Figure 2, this sample has shown the absorption bands in the UV-Vis region between 340-470 nm. The absorption observed at 406 nm due to the n^n* transition. The wide transparency of the sample in the vis- Mostaghni: 4-(4,5-Diphenyl-1H-imidazole-2-yl)phenol: ... 172 Acta Chim. Slov. 2021, 68, 170-177 ible region enables it for the second harmonic generation that required for all NLO materials. The band gap energy was determined using the Tauc relation.44 The band gap value obtained using the direct transition. Extrapolating the linear part of the curves to the X axis yield the bandgap equal to 2.54 eV (Figure 3). Figure 3. Plot (ahv)2 vs. photon energy 3. 2. Nonlinear Absorption Coefficient The nonlinear absorption coefficient, the nonlinear susceptibility, and imaginary part of x(3) were determined from open aperture z-scan data. For this purpose, the normalized transmittance of open aperture Z-scan at wavelengths 532 nm was plotted as a function of sample position (Figure 4). Figure 4. Open aperture Z-Scan data for 4-(4,5-Diphenyl- 1H-imi-dazole-2-yl) phenol solution. As can be seen from Figure 4, the curve shows a valley shape, indicating a positive nonlinear absorption coefficient resulting from the two-photon absorptions (TPA) whereby a molecule simultaneously absorbs two photons that are inherently weak at low intensities of light. The nonlinear absorption coefficient ^ was calculated by the following equations:45 (1) (2) (3) (4) Where I0 is peak on-axis irradiation at the focal point, Z is the sample position at the minimum transmit-tance, Z0 is diffraction length, T is the total transmittance, Leff is the effective thickness of the sample and ^ is NLA coefficient. The nonlinear absorption coefficient is tabulated in table 1. 3. 3. Nonlinear Refractive Index The sign and magnitude of the nonlinear refractive index, and real part of x(3) were determined from the closed aperture z-scan data. The normalized transmittance of the sample as a function of distance from the focus point is plotted in Figure 5. As figure 5 shows a peak-valley configuration, the nonlinear refractive index is negative and indicates self de-focusing nonlinearity. Figure 5. Closed aperture Z-Scan data for 4-(4,5-Diphenyl- 1H-im-idazole-2-yl) phenol solution Mostaghni: 4-(4,5-Diphenyl-1H-imidazole-2-yl)phenol: Acta Chim. Slov. 2021, 68, 170-177 173 However, AT„v is related to the nonlinear refractive index n2 by the following equations:46 AT* .„ = 0.406(1 - (y)n2/0L, err (5) (6) (7) In the above equation, ATp-v is the distance between the peak and valley transmittance which is obtained from figure 5, I0 is the intensity in the focused sample, ra is the radius of aperture, wa is the radius of the beam at the aperture, S is the linear transition, and n2 is the nonlinear refractive index. Besides, changes in the induction reflectance index were determined by the following equation. (8) (3) 3. 4. Third Order Susceptibility x' The real and imaginary parts of the third-order susceptibility are related to the nonlinear refractive index n2 and the nonlinear absorption coefficient p respectively. Where the imaginary part of the third-order susceptibility is calculated from the nonlinear absorption coefficient using the following equation. = inQ) (9) Moreover, the real part of the third order susceptibility can be obtained from the nonlinear refractive index through the relationship: (10) In the above equations, p is the nonlinear absorption coefficient, n2 is the nonlinear refractive index, n0 is the linear refractive index, c is the speed of light in the vacuum and £0 is the permeability coefficient in the vacuum. However, the absolute value of nonlinear third-order susceptibility can be obtained by the following equation. Table 1. Calculated third order nonlinear optical parameters of the title compound n2 x 10-6 p x 10-1 Re (x(3)) x 10-3 Im (x(3)) x 10-5 x(3) x 10-6 cm2/W cm/W esu esu esu -2.89 4.044 -1.5016 8.89 2.2627 |^h{[ffeCr(3))]2 + [/maC3))f} 2lV2 (11) The values of the third-order nonlinear susceptibil-itiies obtained for 4-(4,5-Diphenyl-1H-imidazole-2-yl) phenol are listed in table 1. The high values of negative nonlinear refractive index and the third-order susceptibility of the sample, which are associated with a 2PA resonance enhancement, indicate that it can be potentially used as an optical limiter to protect tools and human eyes. 3. 5. Electronic Structure and One-Photon Absorption Electronic properties such as ionization potential (IP), hardness (r|), softness (S), and electron affinity (EA), can be evaluated from HOMO and LUMO energies. HOMO and LUMO energies, Band gap energy, hardness, softness ionization potential (IP), and electron affinity (EA) are summarized in Table 2. Quantum mechanical calculations play an important role in the understanding of the relationship between the molecular structure and the nonlinear optical properties of the compounds. There are many factors contribute to enhancing the properties of NLO compounds such as: low bandgap energy, high dipole moment, reversing of ground-state charge distribution and the n-electronic cloud redistribution via the n-conjugated system.47 In conjugated organic materials, electrons in n bond are delocalized and have more motions rather than other electrons. In this case the n-bond electrons can easily move in the whole molecule space. Increasing the electron charge distribution will result in a larger hyperpolarizability, which is linked to the nonlinearity. The orbitals involved in the main transitions are shown in figure 6. As can be seen, the HOMO orbital is delocalized over the whole molecule. By contrast, the LUMO is mostly located over two phenyl rings 1 and 2. Table 2. Theoretically computed HOMO and LUMO energies, Bond gap energy (Egap), Hardness (r|), softness (S), Ionization potential (IP), Electron affinity (EA) ehomo elumo n S IP EA E gap eV eV eV eV KJ/mol KJ/mol eV -4.9980 -1.8098 1.594 0.314 482.225 174.622 3.18 Mostaghni: 4-(4,5-Diphenyl-1H-imidazole-2-yl)phenol: ... 174 Acta Chim. Slov. 2021, 68, 170-177 .JS LCMO-1.8098«V Eg=3.L881 eV HOMO=-4.99SO eV HOMO—4.9980 e\' H Figure 6. Frontier orbitals of 4-(4,5-Diphenyl-1H-imidazole-2-yl) phenol Moreover, LUMO+1 is based more on the phenyl ring 2 and phenolic ring while, LUMO+2 is mostly delo-calized on the two phenyl rings. Consequently, electron transition from ground to excited states facilitate an electron density transfer. The short-circuit current density is an important component of the photoelectric conversion efficiency (PCE) is determined by the light-harvesting efficiency (LHE). The light-harvesting efficiency (LHE) was approximately calculated from oscillator strength (f) using the following equation:48 LHE = 1-10-/ (12) As can be seen from table 3, both H^L and H^L+1 transitions dominantly dictate the electronic absorption profile of the studied molecule with sufficiently high f-val-ues. However, the value of excitation energy related to H^L transition is comparable to the band gap value obtained from the Tauc equation. By contrast, the oscillator strength value of H^L+2 transition is too low to contribute to the absorption spectra. As expected, the low energy electronic excitations have substantial ICT character. j UMO+2=-0.5464eV Eg=3.836 eV Ej=4.45 eY HOMO—4.9980 eV Both H^L and H^L+1 transitions with the high LHE will have a high short-circuit current density and so the high photoelectric conversion efficiency. The partially density of states (PDOS) spectra of the title compound was also obtained using quantum mechanical calculations. According to PDOS spectra diagram (Figure 7), band gap was low which is in good agreement with other results. Density of states / electrons Ha Energy / Ha Figure 7. Calculated DOS spectra of 4-(4,5-Diphenyl-1H-imida-zole-2-yl) phenol Table 3. Excitation energies (Eex), oscillator strengths (f), light harvesting efficiencies (LHE), and electronic transitions configurations of the title compound at TD-DFT-B3LYP/6-31+G(d,p) level in DMSO Excited State Eex f LHE Transition assignment 82 -> 83 (S) 2.9654 eV 418.10 nm 0.5678 0.7295 H^L (99.18%) 82 -> 84 (S) 3.3587 eV 369.14 nm 0.2601 0.4506 H^L+1 (96.24%) 82 -> 85 (S) 3.5015 eV 354.09 nm 0.0031 0.0071 H^L+2 (96.52%) Mostaghni: 4-(4,5-Diphenyl-1H-imidazole-2-yl)phenol: Acta Chim. Slov. 2021, 68, 170-177 175 3. 6. Second-Order Nonlinear Optical (NLO) Response The linear isotropic polarizability indicates the capacity of changing the charge density in the system under the influence of an external field. However, the magnitude of the isotropic polarizability a and anisotropy of polarizability (Aa) are calculated using the polarization components as follows.49,50 Cve = 3 (a** + ayy + aaIZ) (13) The first hyperpolarizability (P) , which is studied using second harmonic generation (SHG) is: Plot = (Pxxx + Pxyy + P xzz ) +(p yyy + Pyzz + Pyx*) + (3zzz + Pzxx + Pzyy)2 1/2 (15) Also, the direction of charge transfer in the title compound was determined by the ratio of Pvec and ptotal using the following equations: COS0 = ^ P tot (16) where pvec is the vector component of first hyperpolarizability.51 (17) The second hyperpolarizability (y), which is studied using third-harmonic generation (THG) is: Y — Y7,7,7,7. g |_Yxxxx Yyyyy Y7.7,7,7, Yzzzz xxyy Yxxzz Yyyzz)] (18) The isotropic polarizability (a), the anisotropy of the polarizability (Aa), the vector component of the first hyperpolarizability and hyperpolarizabilities (P, y) of the title compound are listed in the table 4. Table 4 dipole polarizability (a), first and second hyperpolarizability (P, y), the vector component of first hyperpolarizability (Pvec) and the anisotropy of the polarizability (Aa) of the title compound a (a.u) Aa (a.u) Ptotal (a.u) Pvec (a.u) -128.7264 255.5163 144.1682 133.6176 -4045.6719 As can be seen, 4-(4,5-Diphenyl-1H-imidazole-2-yl) phenol show the high total static dipole moment and hyperpolarizabilities (p, y) which can be attributed to the positive contribution of their conjugation. The ratio of pvec/ptotal in table 4 is equal to 0.93, which indicated the unidirectional charge transfer in the title compound. Therefore it is a good candidate for future studies of nonlinear optical properties. 4. Conclusion I have synthesized 4-(4,5-Diphenyl-1H-imidaz-ole-2-yl)phenol as an attractive material for potential application in nonlinear optics. The nonlinear optical properties of the title compound are investigated using the z-scan technique and quantum mechanical calculations. Both theoretical and experimental results reveal that the title compound exhibits large optical nonlinearity. The calculation of the HOMO-LUMO energy gap showed that the eventual charge transfer interactions occure within the molecule. Furthermore, the high value of total static dipole moment and hyperpolarizabilities (p, y) were found for the title compound, which was attributed to the positive contribution of their conjugation. The calculated transition dipole moments for ground and excited states indicated an electron density transfer. Besides, the ratio of pvec/ ptotal indicated the unidirectional charge transfer in the title compound. In summary, from all theoretical studies, it was concluded that the title compound can use as potential NLO molecule. The theoretical results are confirmed by the nonlinear refractive index, and the nonlinear absorption coefficient were determined by z-scan techniques. The magnitude and sign of the nonlinear refractive index (n2) determined using close aperture z-scan. n2 was in the range of 10-6 cm2/W. The negative sign of n2 indicated the occurrence of self-defocusing phenomena due to the local variation of the refractive index with temperature. The measured nonlinear absorption coefficient (P) by open aperture z-scan was in the range of 10-1 cm/W associated with the two-photon absorption (TPA) effect. Finally, the physico chemical studies on the title compound revealed the essential property of the title compound for application in the field of nonlinear optic. Acknowledgments The author are grateful to the Payame Noor University for encouragements. 5. References 1. M. H. Sadr, V. M. Mohammadi, B. Soltani, K. Jamshidi-Gha-leh, S. Z. Mousavi, Optik. 2016, 127, 6050-6055. D01:10.1016/j.ijleo.2016.04.051 2. C. Mallet, G. Savitha, M. Allain, V. Kozmik, J. Svoboda, P. Frère, J. Roncali, J. Org. Chem. 2012, 77, 2041-2046. D0I:10.1021/jo202412t 3. A. N. 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V. Perumal, Spect. Chim. Acta Part A, 2012, 86, 69-75. D0I:10.1016/j.saa.2011.09.067 47. J. Preat, D. Jacquemin, E. A. Perpete, Energy Environ. Sci. 2010, 3, 891-904. D0I:10.1039/c000474j Povzetek V prispevku opisujemo sintezo spojine 4-(4,5-difenil-1H-imidazol-2-il)fenol in eksperimentalne ter teoretične raziskave njenih nelinearnih optičnih lastnosti. Teoretične raziskave so bile izvedene z uporabo funkcij TD-DFT in B3LYP z običajno nastavitvijo 6-31 ++G(d,p). Rezultati analiz HOMO-LUMO in NBO kažejo nizko vrednost prepovedanega pasu, visok dipolni moment in hiperpolarizabilnost (p, y) kot tudi prisotnost dipolarnih vzbujenih stanj z razmeroma visokimi spremembami dipolnega momenta, povezanimi z nelinearnostjo. Metoda z-skeniranja je potrdila NLO lastnosti spojine. Nelinearni absorpcijski koeficient, refrakcijski indeks in susceptibilnost tretjega reda znašajo 4.044 x 10-1 cmW-1, 2.89 x 10-6 cm2W-1 in 2.2627 x 10-6 esu. Negativna vrednost n2 kaže na samo-defokusiranje in nelinearnost. Rezultati kažejo, da bi spojino lahko uporabljali kot potencialni NLO material. Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Mostaghni: 4-(4,5-Diphenyl-1H-imidazole-2-yl)phenol: ... 48. P. Günter, editor. Nonlinear optical effects and materials. 4rd ed. Berlin: Springer; 2012. 49. C. Valverde, S. A. L. Castro, G. R. Vaz, J. L. A. Ferreira, B. Baseia, F. A. P. Osorio, Acta Chim. Slov. 2018, 65, 739-749. D01:10.17344/acsi.2018.4462 50. Muhammad Ramzan Saeed Ashraf Janjua, Chun-Guan Liu, Wei Guan, Jia Zhuang, 51. S. Muhammad, L. K. Yan, Z. M. Su, J. Phys. Chem. A, 2009, 113, 3576-3587. D0I:10.1021/jp808707q 178 DOi: I0.i7344/acsi.2020.6305 Acta Chim. Slov. 2021, 68, 178-184 ©cwnmons Scientific paper On the Validity of Minimum Magnetizability Principle in Chemical Reactions Hiteshi Tandon,^* Tanmoy Chakraborty^* and Vandana Suhag3 1 Department of Chemistry, Manipal University Jaipur, Jaipur 303007, India 2 Department of Chemistry and Biochemistry, School of Basic Sciences and Research, Sharda University, Greater Noida 201310, India Department of Applied Sciences, BML Munjal University, Gurugram 122413, India Corresponding author: E-mail: tanmoychem@gmail.com, tanmoy.chakraborty@sharda.ac.in (T. Chakraborty) hiteshitandon@yahoo.co.in (H. Tandon) Received: 10-23-2020 3 Abstract A new principle known as Minimum Magnetizability Principle has recently been introduced in the context of Density Functional Theory. In order to validate this principle, changes in the magnetizability (A£) and its cube-root (A£1/3) are computed at B3LYP/LanL2DZ level of theory for some elementary chemical reactions. The principle is found to be valid for 77% of reactions under study. It is observed that the molecules with the lowest sum of £ or £1/3 are generally the most stable. The principle fails to work in the presence of hard species. A comparative study is also made with change in hardness (An), electrophilicity index (Aw), polarizability (Aa) and their cube-roots (An1/3, Aw1/3, Aa1/3). It is observed that the Minimum Magnetizability Principle is nearly as reliable as Minimum Electrophilicity Principle. It appears that this principle could be helpful in predicting the direction of diverse reactions as well as stable geometrical arrangements. Keywords: Density Functional Calculations; Magnetic Properties; Maximum Hardness Principle (MHP); Minimum Electrophilicity Principle (MEP); Minimum Polarizability Principle (MPP) 1. Introduction Theoretical Chemistry aims at unearthing novel concepts and principles to explain a broad range of chemical reactions. The most common questions that arise for any kind of reaction are about the pace and extent of the reaction. It is logical that thermodynamic data is required for providing an answer to the latter. Constructively, numerous reactivity descriptors have been established within the context of Conceptual Density Functional Theory (CDFT).1 These descriptors play a significant role in studying the changes taking place in a reacting system. This ultimately helps in understanding the reactivity and stability patterns of the reactants and products in a chemical reaction. Some of these descriptors are hardness (n),2 electrophilicity index (w),3-6 and polarizability (a)7 whose definitions are given as follows: (1) 2V U[( EL-E„)/2]i a (d2E(c)\ V )(f= O=o) (2) (3) Here E refers to the system's energy with respect to the number of electrons N at fixed external potential v(r). El and Eh stand for the frontier orbital energies while y is the chemical potential. [ represents the external electric field. Another reactivity descriptor is magnetizability (£) which is defined as the linear response of an atom, molecule or ion's electron cloud towards an external magnetic field.8 It is expressed as: (B=0) (4) where B signifies the external magnetic field. It is an important descriptor to study chemical reactivity, stability and aromaticity of different atoms and molecules.9-14 Dynamics of reactions have also been studied for molecules in Tandon et al.: On the Validity of Minimum Magnetizability Principle 179 Acta Chim. Slov. 2021, 68, 178-184 confinement using magnetizability.15 This property is of multidisciplinary interest and has extensive applications in the realm of physical, biological, engineering and materials science such as organic electronics,16 magnetically labelled cells, drugs and therapeutic agents,17 magnetic flux concentrators,18 magnetizable elastomers,19 bionanocom-posites,20 magnetic immunoadsorbents,21 magnetic nano-particles/ nanofibres17 and so on besides its use in general chemical science. Looking at the widespread relevance of this property, it is believed that further exploration on its behaviour and characteristics will assist in strengthening the fundamentals of the concept, thereby supporting its efficient and appropriate use in various applications. Based on above mentioned concepts and descriptors, some principles have been formulated in the framework of Density Functional Theory (DFT). The Maximum Hardness Principle (MHP) states that "there seems to be a rule of nature that molecules arrange themselves so as to be as hard as possible".22 Another principle known as Minimum Polarizability Principle (MPP) proposes that "the natural direction of evolution of any system is toward a state of mimimum polarizability".23 A Minimum Electrophilicity Principle (MEP) was also suggested according to which "the natural direction of a chemical reaction is toward a state of minimum electrophilicity".24 Numerous efforts have been made to verify the reliability of these principles in diverse reactions/species.25,26 However, a breakdown of these principles was also observed in many cases.27-30 Recently, one more principle, known as the Minimum Magnetizability Principle (MMP), has been introduced in the context of DFT based on the descriptor magnetizabili-ty. According to this principle, "a stable configuration/conformation of a molecule or a favourable chemical process is associated with a minimum value of the magnetizability".31 In case of MMP, unlike other principles, no study has been performed yet to test the validity of this principle for chemical reactions. As understandable, operational efficiency of a principle can only be determined through performing a validation procedure. Accordingly, in order to employ MMP for practical purposes, it is necessary to carry out its verification based on some criteria. Thus, in the present study we have tried to assess the potential and accuracy of this principle for the first time with respect to some chemical reactions. 2. Method of Computation In order to verify the validity of Minimum Magne-tizability Principle along with comparing it to the other principles, viz. MHP, MPP and MEP, 30 elementary reactions are selected. Geometry optimizations for each of the reactant and product molecules have been performed at B3LYP/LanL2DZ level of theory. This is a very common and powerful method employed in computations and incorporates the correlation effects. Hence, the energy values of the HOMO (Highest Occupied Molecular Orbital) (EH) and the LUMO (Lowest Unoccupied Molecular Orbital) (EL) as well as molecular polarizability are obtained for the molecules. The canonical molecular orbitals differ from Kohn-Sham orbitals, nevertheless their values for orbital energy are found to be comparable.32 As a result, the operational forms of hardness and electrophilicity presented in Eq. (1) and Eq. (2) are used to calculate the value for hardness and electrophilicity. Further, magnetizabilities are computed using the Keith and Bader's method33 at the same level of theory. All computations have been performed on the computational software Gaussian 03.34 Next, in view of the MMP, the reaction is favoured only when the products to be formed are of lower magne-tizability than the reactants. Thus, for a chemical reaction (Aj), represented as Zflj Aj = 0 in a condensed form, where aj corresponds to the stoichiometry of the jth atom/molecule, the corresponding deviation in a parameter X through the reaction can be clearly expressed as: In Eq. (5), Xj refers to the value of X for the jth atom/ molecule in the given chemical reaction. It follows that the direction in which the chemical reaction will progress may be indicated by the sign of AX. Thus in accordance with MMP, it appears that the sign of A£ provides an evidence of the direction in which any reaction proceeds. Hence, we have calculated the change in parameter X, viz. hardness (A^), electrophilicity (Aw), polarizability (Aa), and magnetizability (A£), for the selected reactions using Eq. (5). Change in the cube-roots of hardness (A^1/3), electrophilicity (Aw1/3), polarizability (Aa1/3) and magnetizability (A£1/3) have also been calculated. Enthalpy change (AfH0) for each molecule is also calculated using the atomization energy data from reference [35]. 3. Results and Discussion The study presents validity of Minimum Magnetiza-bility Principle using chosen 30 elementary reactions. A comparison of the results is also presented with respect to the other principles, viz. MHP, MPP and MEP. It must be noted that several of the chosen reactions are hypothetical. All the reactions are exothermic signifying that the products are thermodynamically stable. Also, the variation of chosen parameters is not considered along the reaction path, but merely the overall change in these parameters in the chemical reaction is evaluated, which can be simply computed as long the geometry can be. Further, it should be noted that the reactants in reaction 14 are the same as in reaction 19, however the type of products formed in both are different. The computed values for hardness (n), electrophilicity index (w), polarizability (a), magnetizability (£) and Tandon et al.: On the Validity of Minimum Magnetizability Principle ... 180 Acta Chim. Slov. 2021, 68, 178-184 HOMO (EH) and LUMO (EL) energy, along with magneti-zability (£t) data from the literature,36 for all the reactants and products of the selected reactions are presented in Table 1. A comparison of computed magnetizabilities (£) with tabulated magnetizabilities36 (£t) reveals a good similarity between the two datasets (R2 = 0.997) indicating the efficacy of our computed data. The changes in enthalpy (AfH0) and in parameter values with their reaction are provided in Table 2. As mentioned above, the formation of products is only favoured when the magnetizability of rea-ctant is more than that of the products to be formed according to the MMP. Moreover the sign of A£ gives an idea of the direction in which any reaction proceeds. A close look at Table 2 reveals that Minimum Magnetizability Principle is valid for chemical reactions since A£ < 0 for various reactions. Further, A£ is negative in numerous cases which undoubtedly demonstrates that A£ provides a sign for the most stable species. Thus, the favoured direction of a chemical reaction is towards less magnetizability. When the stability of products is more than the reactants, change in enthalpy is less than zero (AfH0 < 0). This condition is also clearly met by the reactions in the study. However, MMP is not valid always. It is reliable for approximately 77% of the selected reactions and fails in case of the remaining reactions. It is observed that the majority of the reactions where the principle fails contain a hard base such as OH, F, Cl, N, etc. It is further noted that MEP is as convincing as MMP and proves to be valid for 77% of the reactions tested. MPP performs slightly better than MHP; however it is less suitable than MEP and MMP. MPP works for 50% of the reactions while MHP is applicable for 47% of the considered reactions. It is observed that the minimum magnetizability Table 1. Computed frontier orbital energies (Eh and El), hardness (n), electrophilicity index (w), polarizability (a) and magnetizability (£) values of the reactant and product molecules of the selected reactions using B3LYP/LanL2DZ method and their tabulated magnetizabilities (£t) (in au) S. No. Molecules eH el n w a f ?T [a] 1 H2 -0.429 0.116 0.272 0.045 2.013 -0.588 -1.145 2 O2 -0.281 -0.212 0.034 0.887 5.834 719.683 718.631 3 H2O2 -0.266 -0.030 0.118 0.093 9.738 -2.523 -3.641 4 CO -0.382 -0.059 0.161 0.151 9.890 -1.030 -2.062 5 CO2 -0.298 -0.071 0.113 0.149 17.215 -0.120 -4.419 6 H2O -0.300 0.061 0.179 0.040 4.698 -1.911 -2.755 7 ch4 -0.177 -0.037 0.070 0.081 14.634 -3.408 -3.662 8 Cl2 -0.331 -0.199 0.066 0.529 13.712 -6.890 -8.502 9 HCl -0.331 0.003 0.167 0.080 4.751 -3.794 -4.756 10 H2S -0.264 0.026 0.145 0.049 11.533 -4.135 -5.366 11 N2 -0.420 -0.053 0.183 0.153 8.363 -2.448 -2.525 12 nh3 -0.227 0.098 0.162 0.013 7.050 -2.206 - 13 NO -0.246 -0.139 0.054 0.344 7.342 299.490 307.443 14 HCN -0.373 -0.007 0.183 0.098 11.917 -1.602 - 15 NO2 -0.312 -0.136 0.088 0.285 13.776 22.419 31.565 16 N2O4 -0.351 -0.139 0.106 0.283 32.779 -4.207 -4.839 17 OH -0.339 0.058 0.198 0.050 3.329 10.493 - 18 C2H6 -0.339 0.126 0.232 0.025 22.136 -5.338 -5.639 19 C3H8 -0.323 -0.125 0.099 0.254 32.538 -4.428 -8.122 20 C2H6O -0.249 0.081 0.165 0.021 25.055 -3.821 -7.070 21 (CH3)2S -0.219 0.038 0.129 0.032 36.373 -5.720 -9.448 22 C2H4 -0.273 -0.001 0.136 0.069 19.380 -1.850 -3.956 23 C2H2 -0.212 0.111 0.161 0.008 18.424 -2.029 -4.377 24 C2H5O -0.291 0.007 0.149 0.067 28.433 -5.145 - 25 Cl2CCH2 -0.279 -0.032 0.123 0.098 34.821 -8.833 -10.353 26 c2h5cn -0.332 0.020 0.176 0.069 33.637 -5.485 - 27 (CH2OH)2 -0.218 0.069 0.143 0.019 19.665 -4.571 -8.164 28 ch3f -0.343 0.060 0.201 0.050 11.571 -1.695 -3.745 29 chf3 -0.423 0.066 0.244 0.065 12.669 -3.148 - 30 cf4 -0.406 -0.195 0.106 0.427 17.824 -3.656 - 31 N2H4 -0.135 0.097 0.116 0.002 12.053 -3.145 -42.297 32 CH3CHO -0.262 -0.044 0.109 0.107 23.007 -1.859 -4.671 33 C6H6 -0.253 -0.009 0.122 0.070 54.838 -6.692 -11.531 34 CH3OH -0.269 0.065 0.167 0.031 14.780 -2.643 -4.503 35 C6H5OH -0.189 -0.110 0.039 0.283 62.282 -5.527 -12.752 36 C6H5NH2 -0.113 -0.006 0.053 0.033 79.933 -5.147 -13.131 [a] Data obtained from reference [36] and converted into atomic units (au). 1 au of £ = e2a02/me = 4.75209 cgs-ppm.37 Tandon et al.: On the Validity of Minimum Magnetizability Principle ... Acta Chim. Slov. 2021, 68, 178-184 181 Table 2. Computed changes in the enthalpy (AfH°) (at 298K in kJ mol '), hardness (An) (in au), electrophilicity index (Aw) (in au), polarizability (Aa) (in au) and magnetizability (A£) (in au) along with MHP, MEP, MPP and MMP validity (+) or invalidity (-) for selected reactions S. No. Reaction AfH° W An +/- Aw +/- Aa +/- A* +/- 1 CO2 + 4H2 ■ 2H2O + CH4 -148 -0.774 - -0.168 + 1.237 - -4.756 + 2 Cl2 + H2 ■ 2HCl -185 -0.004 - -0.414 + 6.223 - -0.111 + 3 3H2 + N2 ■ 2NH3 -71 -0.675 - -0.262 + 0.303 - -0.199 + 4 2NO ■ O2 + N2 -180 0.110 + 0.352 - -0.487 + 118.256 - 5 2NO + 2H2 ■ N2 + 2H2O -647 -0.111 - -0.545 + -0.951 + -604.072 + 6 CH4 + 2O2 ■ CO2 + 2H2O -786 0.333 + -1.625 + -0.308 + -1439.901 + 7 CO + 3H2 ■ CH4 + H2O -198 -0.729 - -0.164 + 3.403 - -2.523 + 8 2NO2 ■ N2O4 -47 -0.070 - -0.287 + -5.228 + -49.046 + 9 2C2H2 + 5O2 ■ 4CO2 + 2H2O -2503 0.318 + -3.771 + -12.235 + -3598.660 + 10 2OH ■ H2O2 -213 -0.279 - -0.006 + -3.080 + -23.509 + 11 H2O2 ■ O2 + H2O -98.5 0.078 + 0.390 - 2.123 - 360.454 - 12 H2O2 + Cl2 ■ O2 + 2HCl -50 0.185 + 0.424 - -8.114 + 721.507 - 13 4NH3 + 5O2 ■ 4NO + 6H2O -899 0.469 + -2.868 + -0.187 + -2403.099 + 14 C2H6 + O2 ■ (CH3)2O -108.5 -0.084 - -0.446 + -0.001 + -358.325 + 15 C2H2 + 2H2 ■ C2H6 -304 -0.473 - -0.074 + 0.314 - -2.132 + 16 C2H4 +HCl ■ C2H5Cl -72 -0.154 - -0.082 + 4.302 - 0.499 - 17 C2H2 + Cl2 ■ Cl2CCH2 -230 -0.104 - -0.440 + -2.685 + 0.085 - 18 HCN + C2H5Cl ■ C2H5CN + HCl -64 0.011 + -0.017 + -1.963 + -2.533 + 19 C2H6 + O2 ■ (CH2OH)2 -312 -0.123 - -0.892 + 8.306 - -718.916 + 20 C3H8 + 5O2 ■ 3CO2 + 4H2O -2015 0.787 + -4.078 + 8.727 - -3601.992 + 21 ch3f + CHF3 ■ CH4 + cf4 -88 -0.270 - 0.394 - -8.218 + -2.221 + 22 N2H4 + O2 ■ N2 + 2H2O -652 0.391 + -0.655 + 0.128 - -722.808 + 23 (CH3)2O + H2S ■ (CH3)2S + h2o -73 -0.003 - 0.002 - 4.484 - 0.325 - 24 2CH3OH + 3O2 ■ 2CO2 + 4H2O -1321 0.507 + -2.262 + 6.158 - -2161.648 + 25 C2H5OH + 3O2 ■ 2CO2 + 3H2O -1306 0.496 + -2.262 + -5.966 + -2161.202 + 26 C2H5OH + Cl2 ■ CH3CHO + 2HCl -53 0.212 + -0.283 + -6.258 + 1.263 - 27 CH3CHO ■ CH4 + CO -135 0.122 + 0.125 - 1.517 - -2.579 + 28 C6H5OH + H2 ■ C6H6 + H2O -234 -0.011 - -0.218 + 4.760 - -2.487 + 29 2C6H6 + 15O2 ■ 12CO2 + 6H2O -6309 1.679 + -11.406 + -37.574 + -10794.771 + 30 C6H5NH2 + H2 ■ C6H6 + NH3 -86 -0.041 - 0.005 - 20.058 - -3.162 + [a] Computed by taking energies of atomization from reference [35]. principle is almost as valuable as minimum electrophilicity principle in predicting the direction of a reaction. The sign of A£ can be used to provide an indication for higher stability of products thermodynamically. In order to understand the significance of the results, a brief statistical analysis is performed for each parameter with respect to AfH0. Our study is based on the stability criterion, i.e. minimum energy condition, thus it is important to validate the correlation of AfH0 with these descriptors. Regression analysis has been performed for this purpose. An attempt to explore the relationship of Aa with AfH0 is futile (R2 = 0.4871, R = +0.70, p = 0.001), although An is found to follow a satisfactory linear relation with AfH° (R2 = 0.6266, R = -0.79, p = 0.001). Lower values of R2 denote inferior correlation of Aa and An with AfH0 whereas p-values indicate that the result is significant. It can be inferred that MPP and MHP may not always follow the minimum energy criterion and may become invalid in several cases. An analysis of A£ and Aw with AfH0 presents an excellent relationship. For A£ and AfH0, R2 = 0.9792, R = +0.99 and p = 0.001 while R2 = 0.9679, R = +0.98 and p = 0.001 for Aw and AfH0. Such high values of R2 clearly signify the outstanding correlation between the parameters. Further, a perfect positive correlation is presented for A£ and AfH0. Significance of the correlation is highlighted by the p-values. It is concluded that MMP as well as MEP are highly related to AfH0 and thus follow the minimum energy and high stability condition. As the validity of minimum energy principle increases, MMP and MEP become valid as well. The cube-roots of exact polarizability a«1/3 = £y a^y3 have been proved to be more useful in comparison to Aa to predict the direction of a chemical reaction.35 Hence, following the above notion, we have determined the cube-roots for hardness, electrophilicity index, polarizability as well as magnetizability and these are presented in Table 3. It is apparent from the outcomes that the validity of MHP as well as MPP increases when their cube-roots are considered. In fact, the soundness of MHP increases drastically as compared to MPP. As expected, these results indicate efficiency of cube-roots of hardness and polarizability in predicting the path followed by a reaction. Although in Tandon et al.: On the Validity of Minimum Magnetizability Principle 182 Acta Chim. Slov. 2021, 68, 178-184 comparison to other principles, MHP and MPP still remain the least accurate for the reactions considered in the present study and have no predictive value. Conversely, the validity of MEP and MMP is found to be decreased in this case. As evident from Table 3, a decrease in the validity of MMP takes place when the cube-roots of magnetizabi-lity are considered. However MMP is still valid for several reactions (A£1/3 < 0) and performs remarkably in contrast to MHP and MPP. As a result, it appears that A£1/3 is more or less as reliable as A£. It is further observed that MEP is the most the convincing of all principles considering its high validity in both the cases. Next, AfH° demonstrates an excellent linear relationship with A£1/3 (R2 = 0.9818) and Aw1/3 (R2 = 0.8916) but poor connections are found with A^1/3 (R2 = 0.5530) or Aa1/3 (R2 = 0.5338). We have tried to accommodate reactions with different types of molecules, viz. inorganic, aliphatic and aromatic, in the present study to consider as many chemical effects as possible. Although for further studies it is suggested that the validity of this principle should be assessed in case of other reaction classes as well. 4. Conclusion We have tried to validate Minimum Magnetizabi-lity Principle employing 30 elementary chemical reactions. Change in the magnetizability (A£) and its cube-root (A£1/3) is computed in order to check the applicability of the principle in determining the direction of the reaction as well as stability of the products. We have also calculated change in hardness (An), electrophilicity index (Aw), polarizability (Aa) and their cube-roots (A^1/3, Aw1/3, Aa1/3) in order to make a comparative study. It is observed that the Minimum Magnetizability Principle is valid for chemical reactions however with some limitations. The principle fails to work in the presence of hard species. Minimum Electrophilicity Principle and Minimum Magnetizability Principle are found to have comparable reliability followed by Minimum Polarizability Principle, however Maximum Hardness Principle appears to be less valid for the chosen reactions. In conclusion, Minimum Magnetizability Principle is found to be fairly valid for reactions and can be successfully employed solely or in Table 3. Computed changes in the enthalpy (AfH°) (at 298K in kJ mol-1), cube-root of hardness (An1'3) (in au), cube-root of electrophilicity index (Aw1/3) (in au), cube-root of polarizability (Aa1'3) (in au) and cube-root of magnetizability (A£1/3) (in au) along with MHP, MEP, MPP and MMP validity (+) or invalidity (-) for selected reactions S. No. Reaction AfH° M An1/3 +/- A«1/3 +/- Aa1/3 +/- A£1/3 +/- 1 CO2 + 4H2 ■ 2H2O + CH4 -148 -1.537 - -0.836 + -1.837 + -0.142 + 2 Cl2 + H2 ■ 2HCl -185 0.049 + -0.302 + -0.294 + -0.379 + 3 3H2 + N2 ■ 2NH3 -71 -1.421 - -1.133 + -1.983 + 1.258 - 4 2NO ■ O2 + N2 -180 0.137 + 0.094 - -0.057 + -5.767 + 5 2NO + 2H2 ■ N2 + 2H2O -647 -0.356 - -0.894 + -1.033 + -15.535 + 6 CH4 + 2O2 ■ CO2 + 2H2O -786 0.550 + -1.139 + -0.115 + -19.393 + 7 CO + 3H2 ■ CH4 + H2O -198 -1.513 - -0.824 + -1.814 + 0.778 - 8 2NO2 ■ N2O4 -47 -0.417 - -0.659 + -1.594 + -7.254 + 9 2C2H2 + 5O2 ■ 4CO2 + 2H2O -2503 0.352 + -2.393 + -0.606 + -46.730 + 10 2OH ■ H2O2 -213 -0.676 - -0.282 + -0.851 + -5.740 + 11 h2o2 ■ O2 + H2O -98.5 0.236 + 0.369 - 0.439 - 4.601 - 12 H2O2 + Cl2 ■ O2 + 2HCl -50 0.532 + 0.561 - 0.633 - 9.106 - 13 4NH3 + 5O2 ■ 4NO + 6H2O -899 1.087 + -0.885 + 1.152 - -20.284 + 14 C2H6 + O2 ■ (CH3)2O -108.5 -0.229 - -0.493 + -0.782 + -4.296 + 15 C2H2 + 2H2 ■ C2H6 -304 -1.226 - -0.620 + -2.359 + 1.194 - 16 C2H4 +HCl ■ C2H5Cl -72 -0.535 - -0.434 + -1.315 + 1.061 - 17 C2H2 + Cl2 ■ Q2CCH2 -230 -0.451 - -0.547 + -1.769 + 1.102 - 18 HCN + C2H5Cl ■ C2H5CN +HCl -64 0.013 + -0.028 + -0.427 + -0.427 + 19 C2H6 + O2 ■ (CH2OH)2 -312 -0.416 - -0.983 + -2.583 + -8.873 + 20 C3H8 + 5O2 ■ 3CO2 + 4H2O -2015 1.622 + -2.475 + 2.252 - -49.609 - 21 ch3f + CHF3 ■ CH4 + cf4 -88 -0.327 - 0.416 - 0.465 - -0.387 + 22 N2H4 + O2 ■ N2 + 2H2O -652 0.883 + 0.144 - 1.286 - -11.326 + 23 (CH3)2O + H2S ■ (CH3)2S + H2O -73 -0.006 - 0.016 - -0.197 + 0.139 - 24 2CH3OH + 3O2 ■ 2CO2 + 4H2O -1321 1.148 + -1.080 + 1.555 - -30.069 + 25 C2H5OH + 3O2 ■ 2CO2 + 3H2O -1306 1.137 + -1.072 + 1.862 - -30.030 - 26 C2H5OH + Cl2 ■ CH3CHO + 2HCl -53 0.627 + 0.251 - 0.887 - -0.883 + 27 CH3CHO ■ CH4 + CO -135 0.478 + 0.490 - 1.748 - -1.285 + 28 c6h5oh + h2 ■ c6h6 + h2o -234 0.071 + -0.257 + 0.248 - -0.519 + 29 2C6H6 + 15O2 ■ 12CO2 + 6H2O -6309 3.330 + -6.815 + 6.432 - -144.017 + 30 C6H5NH2 + H2 ■ C6H6 + NH3 -86 0.018 + -0.030 + 0.146 - -0.622 + [a] Computed by taking energies of atomization from reference [35]. Tandon et al.: On the Validity of Minimum Magnetizability Principle ... Acta Chim. Slov. 2021, 68, 178-184 183 conjunction with other principles for theoretical as well as practical applications. Author Contributions Hiteshi Tandon: Conceptualization, Methodology, Software, Resources, Formal analysis, Validation, Investigation, Writing - Original Draft, Visualization. Tanmoy Chakraborty: Conceptualization, Supervision, Writing -Review & Editing. Vandana Suhag: Supervision, Writing - Review & Editing. Conflicts Of Interest The authors declare no conflicts of interest. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Acknowledgements Dr. Tanmoy Chakraborty is thankful to Sharda University and Dr. Hiteshi Tandon is thankful to Manipal University Jaipur for providing computational resources and research facility. 5. References 1. P. Geerlings, F. De Proft, W. Langenaeker, Chem. Rev. 2003, 103, 1793-1874. 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Scalmani, N. Rega, G. A. Petersson, H. Nakatsuji, M. Hada, M. Ehara, K. Toyota, Tandon et al.: On the Validity of Minimum Magnetizability Principle 184 Acta Chim. Slov. 2021, 68, 178-184 R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, M. Klene, X. Li, J. E. Knox, H. P. Hratchi-an, J. B. Cross, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, P. Y. Ayala, K. Morokuma, G. A. Voth, P. Salvador, J. J. Dannenberg, V. G. Zakrzewski, S. Dapprich, A. D. Daniels, M. C. Strain, O. Farkas, D. K. Malick, A. D. Rabuck, K. Raghavachari, J. B. Foresman, J. V. Ortiz, Q. Cui, A. G. Baboul, S. Clifford, J. Cioslowski, B. B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaromi, R. L. Martin, D. J. Fox, T. Keith, M. A. Al-Laham, C. Y. Peng, A. Nanayakkara, M. Challacombe, P. M. W. Gill, B. Johnson, W. Chen, M. W. Wong, C. Gonzalez, J. A. Pople, Gaussian 03, Revision C.01. Gaussian, Inc., Wallingford CT, 2004. 35. U. Hohm, J. Phys. Chem. A 2000, 104, 8418-8423. D01:10.1021/jp0014061 36. W. M. Haynes (Ed.), CRC Handbook of Chemistry and Physics, Vol. 97, CRC Press, Boca Raton, FL, 2017. 37. E. Steiner, P. W. Fowler, in: B. Grimm, R. J. Porra, W. Rüdiger, H. Scheer (Eds.): Chlorophylls and Bacteriochlorophylls: Biochemistry, Biophysics, Functions and Applications, Springer, Netherlands, 2006, pp. 337-347. Povzetek Novo načelo, znano kot načelo minimalne magnetizabilnosti, je bilo nedavno predstavljeno v okviru teorije gostotnega funkcionala (DFT). Da bi potrdili to načelo, smo na ravni teorije B3LYP/LanL2DZ izračunali spremembe v magnetizabilnosti (A£) in tretjem korenu iz A£ (A£1/3) za nekatere osnovne kemijske reakcije. Ugotovili smo, da načelo velja za 77% preučevanih reakcij. Opazili smo, da so molekule z najnižjo vsoto £ ali £1/3 na splošno najbolj stabilne. Načelo ne deluje v prisotnosti trdih zvrsti. Izvedli smo tudi primerjalno študijo s spreminjanjem trdote (An), indeksa elektrofil-nosti (A«), polarizabilnosti (Aa) in njihovih tretjih korenov (A^1/3, Aw1/3, Aa1/3). Opazili smo, da je načelo minimalne magnetizabilnosti skoraj tako zanesljivo kot načelo minimalne elektrofilnosti. Zdi se, da bi to načelo lahko pomagalo pri napovedovanju smeri različnih reakcij in stabilnih geometrijskih ureditev. © 0 Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Tandon et al.: On the Validity of Minimum Magnetizability Principle ... DOI: 10.17344/acsi.2020.6308 Acta Chim. Slov. 2021, 68, 185-192 /^.creative ^commons Scientific paper Electrodeposition and Growth of Iron from an Ethylene Glycol Solution Vusala Asim Majidzade,1,* Akif Shikhan Aliyev,1 Mahmoud Elrouby,2 Dunya Mahammad Babanly1,3 and Dilgam Babir Tagiyev1 1 Institute of Catalysis and Inorganic Chemistry named after M.Nagiyev, Azerbaijan National Academy of Sciences, AZ1143, H.Javid 113 2 Chemistry Department, Faculty of Science, Sohag University, 82524 Sohag, Egypt 3 French Azerbaijani University (UFAZ), AZ1010, Baku, Nizami 189 * Corresponding author: E-mail: vuska_80@mail.ru Received: 07-29-2020 Abstract The electrochemical reduction of iron (III) ions into zero-valent iron from a solution of ethylene glycol was accomplished. The kinetics and mechanism of the electroreduction process were investigated by cyclic and linear polarization techniques. The influence of temperature, potential sweep rate, and concentration of iron (III) ions on the electroreduction process was also studied. The observed values of effective activation energy revealed that the investigated electroreduction process is accompanied by mixed kinetics control. Moreover, the results of SEM and X-ray diffraction analysis confirmed the deposition of thin Fe films under the optimized conditions. Keywords: Polarization curves; chronoamperometry; iron ions; electroreduction; ethylene glycol. 1. Introduction Currently, due to the decrease in natural energy sources, the use of environmentally friendly solar energy is very important.1-4 Iron disulfide (FeS2) due to its non-toxicity and wide distribution on earth, it has the probability of becoming an inexpensive alternative material for fabricating highly efficient solar cells.5-10 Depending on its optical properties, it can be used in solar cells as a photoactive absorbing layer or as a frontal transparent layer in heterostructured cells.11 The preparation of these films has been carried out by various methods. The electrochemical deposition method has been used in this work, due to its simplicity to be optimized, and non-cost. For the co-electrodeposition of two or more components simultaneously, firstly the electrochemical reduction of each component should be studied individually. Therefore, this work aims to study the electrochemical reduction of iron ions, the kinetics, and the mechanism of the process in a non-aqueous solution of ethylene glycol. The electroreduction and electrodeposition of iron ions have been reported.12-18 The results presented in the previously published work12 showed that the electrodeposition of iron in an acidic sulfate medium occurred, at least, through three adsorbed intermediates. It was observed that on the nonlinear part of the polarization curves, the electrodeposition of iron occurred, but with very low efficiency. In this area, the main cathodic reaction is the reduction of hydrogen, and low-efficient electrode-position of iron was obtained through two intermediates. One of these adsorbed intermediates catalyzed the reduction of hydrogen, while the other blocked the process. The third intermediate appeared only at the potential region corresponding to the linear part of the polarization curves. It was also noted that the concentration of the adsorbed species at the surface strongly depends on the electrode potential. The metallic Fe films onto a copper substrate from a mixture of ChCl, urea, and FeCl3 has been previously elec-trodeposited.13 It was found that the use of direct current coating technology gives uniform, dense, gray, dull, and pure iron coatings. The electrodeposition at the ambient temperature produced iron films of high corrosion resistance and stable for several weeks. The surface morphology of the obtained iron films was also studied as a function Majidzade et al.: Electrodeposition and Growth of Iron 186 Acta Chim. Slov. 2021, 68, 185-192 of the applied current density. As a result, fine-grained and microcrystalline iron deposits without defects were obtained. The influence of ammonium ion on the electrodepo-sition of iron from the iron sulfate bath was studied by other authors.14 During the cathodic polarization process in a solution of 0.02 M FeSO4 at pH = 3 by using the quartz crystals microbalance technique, the presence of ammonium sulfate increased the mass of the electrodeposited Fe metal. The obtained pH (at the surface of the working electrode) - potential curves showed that in the presence of ammonium sulfate, the pH simultaneously increased to the alkaline levels during the electrolysis process. These results suggest that ammonium ion facilitates the formation of ammoniated ferrous iron (e.g., Fe(NH3)22+ and Fe(NH3)42+). This prevents the deposition of Fe(OH)2 on the electrode, which can lead to passivation of the surface and, therefore, to a limited deposition of Fe metal. Nanocrystalline iron was electrodeposited from 1-butyl-1-methylpyrrolidinium bis (trifluoromethylsulfo-nyl) amide ([Py1,4] TFSA) ionic liquid at 100 °C.15 The electrodeposition of iron from ([Py1,4] TFSA) was accomplished by using Fe (TfO)2 and FeCl2 as precursors. Cyclic voltammetry was used to investigate the electrochemical behavior of FeCl2, Fe(TfO)2, and (FeCl2+AlCl3) in the presence of ionic liquid as a supporting electrolyte. But, a thick iron film was obtained from FeCl2 / [Py1,4] TfO at these conditions. Iron metal films were also electrodeposited on copper substrates by the galvanostatic method at various pH, temperature, and current density from weak acidic (pH = 5.7) solutions containing iron sulfate and sodium gluconate.16 It was found that the optimal conditions for obtaining stable Fe films are as follows; a solution consists of 0.038 mol ■ dm3 Fe2(SO4)3.7H2O and 0.14 mol-dm3 sodium gluconate, at current density (j) = 0.33 A / cm2, pH = 5.7, and temperature 25 °C. The obtained Fe films from this electrolyte have a single-phase crystal structure. The polarization curves for the discharge of iron and hydrogen ions during the electrolysis of sulfate solutions with various concentrations of amino-acetic acid showed that the presence of glycine in the electrolyte inhibited both the discharge of iron and hydrogen ions.17 The obtained results showed that the current efficiency of the iron electrodeposition depends linearly upon the concentration of glycine passes. The influence of pH and temperature on the quality of the coating was also investigated. From the previous work, the optimum conditions for obtaining a high-quality deposit were as follows; sulfate electrolyte containing 0.1-0.15 M glycine, pH = 1.9-0.1, temperature 20 °C, and current density (j) = 20 mA / cm2 should be applied. The electrodeposition of iron metal was also studied from the ether solution.18 The electrodeposition bath consisted of iron (II) chloride (FeCl2), diglyme (G2) as a com-plexing agent, and aluminum chloride (AlCl3). The effect of hydrogen gas evolution on the morphology of iron deposits was investigated by using different aqueous electrolytes. A thin Fe film was obtained using FeCl2 -G2 - AlCl3 in the absence of the hydrogen gas evolution, and the nu-cleation of iron was explained by the instant nucleation mechanism. As a result, the surface morphology of the thin Fe film was found to be compact and smooth compared to that obtained from aqueous and nonaqueous electrolytes. M.A. Miller et. al.19 used ethylene glycol as an electrolyte but in the presence of choline chloride (ChCl) and FeCl2 that gave different behavior and results compared with the present work. They also used different ratios from ChCl:EG: FeCl2 and obtained different results at each ratio. But in the present work, EG and Fe(NO3)3 were used, where nitrates salts have a considerable solubility compared with chloride and other salts. Furthermore, the elec-trodeposition process was accomplished at room temperature and not at 80 oC. S. Higashino et. al.20 used FeCl3 as a precursor in presence of acetamide as a complexing agent that gave different results compared with our work. According to the published literature, the kinetics and mechanism of the electrochemical deposition of iron from different aqueous electrolytes were studied. Therefore, our work aims to study the kinetics and mechanism of the electroreduction of iron ions from non-aqueous electrolytes (using ethylene glycol). The current work is considered to be one from a series of our works on the electrochemical synthesis of chalcogenide compounds. 21-23 2. Experimental Part The electrolyte for the electrochemical reduction of iron (III) ions was prepared as follows: an appreciated amount of Fe(NO3)3 ■ 9H2O was dissolved in ethylene glycol by stirring in the temperature range of 313-323 K to give 0.1 M. Polarization and chrono-amperometric curves were accomplished by a potentiostat IVIUMSTAT electrochemical interface. An electrochemical three-electrode cell with a capacity of 100 ml was used. A Pt sheet with an area of 3 x 10-3 dm2 and a Ni sheet with an area of 2 cm2 were used as working electrodes. The silver chloride electrode was used as a reference electrode and the platinum sheet with an area of 4 cm2 as an auxiliary electrode. The UTU-4 universal ultra-thermostat was utilized to regulate the temperature in the bath of the electrolytic cell. The phase composition of the obtained thin films was analyzed using a Bruker D2 Phaser X-ray diffractom-eter (CuKa; Ni filter). The morphology and chemical elemental composition of the samples were determined by Carel Zeiss Sigma scanning electron microscopy (SEM). At the beginning of the experiments, the surface of Pt electrodes was cleaned in concentrated nitric acid and then washed with bidistilled water. Furthermore, after each experiment, the Pt electrodes were kept in boiling ni- Majidzade et al.: Electrodeposition and Growth of Iron ... Acta Chim. Slov. 2021, 68, 185-192 187 trie acid for 30 minutes. This is necessary to get rid of the adsorbed small amounts of ferric chloride on the electrode surface. After that, they were washed thoroughly with ordinary water, then with distilled water, and finally rinsed with alcohol or acetone. Ni electrode was subjected to electrochemical polishing in a solution consists of H2SO4, H3PO4, and citric acid (T = 293-303K, i = 50 A/dm2, t = 180 seconds), and was washed with bidistilled water. 3. Results and Discussion The study of the electroreduction of iron ions (III) from non-aqueous solutions was carried out by the poten-tiodynamic method. As can be seen from Figure 1, the electrochemical reduction of iron (III) ions at the cathode occurred in two stages within the potential range of 0.8 -(-1.2) V. The first stage (I) was observed at the potential range of 0.8 - (-0.36) V that shows the reduction of Fe (III) to Fe (II) as in the following reaction: Fe3+ + e- = Fe2+. ma os: oo. ■0.5. -10. I •1.5. if 11 ■10 -05 0.0 05 1 0 Potential Fig. 1. Cyclic voltammetric curves of the electroreduction of iron (III) ions on a Pt electrode in a non-aqueous medium. The electrolyte composition in (M): 0.1 M Fe(NO3)3 • 9H2O + CH2OH-CH2OH; at T = 293 K, and EV=0.02 V/s. The second stage corresponds to the reduction of Fe (II) to the atomic iron in the potential range of -0.36-(-0.8) V, which agreed with previously published work. 24 After the potential value of -0.8 V, iron is deposited on the substrate. To study the kinetics of the electroreduction of iron (III) ions, polarization curves of a linear nature were recorded depending on the temperature at a temperature range of 288-348 K. As can be seen from Fig. 2, the potential of the electroreduction of iron (III) ions moves towards a positive direction and the current increases due to the ion mobility which increases with increasing the temperature. With the aid of these polarizing curves, the dependence of lgik on 1/T in the potential interval of 0.0 - (-0.5) V was depicted as in Fig. 3(a), and the value of tga was calculated from the obtained curves. The value of the effective activation energy was calculated using the equation of Aeff. = 2.3Rtga. The values of Aeff showed that the electroreduction of iron ions (III) from non-aqueous electrolytes is accompanied by mixed kinetics. In the potential range of ma ■IS -1 0 -0 5 0 0 0 5 Potential v Fig. 2. The effect of temperature on the electroreduction of iron (III) ions. The electrolyte composition in (M): 0.1 Fe(NO3)3 • 9H2O + CH2OH-CH2OH; at different T (K): 1- 288; 2- 298; 3- 308; 4- 318; 5- 328; 6- 338; 7- 348, and scan rate EV= 0.02 V/s A/dm Fig. 3. (a) lgik - 1/T dependence, at different E(V) = 1- 0.0; 2- (-0.1); 3- (-0.2); 4- (-0.3); 5- (-0.4); 6- (-0.5). (b) Dependence of the activation energy upon the electrode potential. b a Majidzade et al.: Electrodeposition and Growth of Iron ... 188 Acta Chim. Slov. 2021, 68, 185-192 0.0 - (-0.2) V the process proceeds under electrochemical polarization control, and after -0.2 V under concentration polarization control (Fig. 3 b). Fig. 4. The influence of the concentration of iron (III) ions on the electroreduction process on the Pt electrode. The electrolyte composition in (M): Fe(NO3)3 • 9H2O + CH2OH-CH2OH; 1-0.005; 20.05; 3- 0.1; 4- 0.15; 5- 0.2. At T = 293 K, and EV=0.02 V/s The effect of concentration on the electroreduction of iron (III) ions has been also studied. Polarization curves at different concentrations of iron (III) ions are presented as in Fig. 4. As can be seen from the curves, the cathodic current of iron electrodeposition increases from -0.2 to -4.1 mA with the increase of the Fe3+concentra-tion in the electrolyte. Moreover, the reduction potential is shifted to the positive direction up to 0.05 V. This can be explained on the basis that, with an increase in the concentration of iron (III) ions in the electrolyte, the migration rate of these ions to the electrode increases, and hence electroreduction occurs faster than that of the previous concentration. 0.0. ■os: -1 o: c ai 5 -1.5. O •20. •2.5: —*— ^-yf / 1 3 \ /[ -10 0 5 0 0 Potential o.s Fig. 5. The effect of the scan rate on the electroreduction of iron (III) ions. Electrolyte composition in (M): 0,1 Fe(NO3)3 • 9H2O + CH2OH-CH2OH. Scan rate (V/s): 1-0,005; 2- 0,02; 3- 0,04; 4- 0,06; 5- 0,08; 6- 0,1. At T = 293 K. The influence of the scan rate on the electroreduction of iron (III) ions was also studied. Fig. 5 exhibits the cathodic potentiodynamic polarization curves of the process. As can be seen from Fig. 5, with an increase in the potential scan rate, the cathodic current of the electrore-duction of iron (III) ions increase:, the cathodic current was - 9.11 x 10-4 A at 0.005 V/s and - 27.60 x 10-4 A at 0.1 V/s. The chronoamperometric (CA) method was used for obtaining more precise information about the electrochemical deposition process, at which the potential can be fixed at the deposition potential. Mechanisms of the nucle-ation and growth of the electrodeposited particles can be investigated via CA method. Current-time curves were accomplished at different applied potentials; -0.40, -0.45, -0.50, -0.55, and -0.60 V at room temperature as shown in Fig. 6. It seems from the shown figure that the initial regime of the current-time curve is characterized by a sudden decrease in the current under application of the deposition potential. This can be attributed to the presence of the double-layer between the surface of the substrate and the ions of the solution, which lead to the formation of immediate nucleation of iron in all cases (Fig. 6). This sudden decrease is followed by a little increase in the resultant current. This is due to an increase in the electroactive surface area which correlated with the crystal growth. It can be noted that during the electrodeposition, in all cases, the current density increases by increasing the deposition potential. The mechanism of crystal nucleation and growth can be determined by the analysis of the obtained current-time curves. Fig. 6. Current-time curves of the electrodeposition of iron on Pt in the electrolyte of the 0.1 Fe(NO3)3 • 9H2O + CH2OH-CH2OH composition at room temperature and different deposition potentials: -0.4; -0.45; -0.5; -0.55; and -0.6 V vs. Ag/AgCl. The analysis of these curves can be achieved by applying the two equations of Scharifker-Hills 25 compared with the experimental calculated data as shown in Fig. 7 (a-e). The models of the theoretical transients for the in- Majidzade et al.: Electrodeposition and Growth of Iron ... Acta Chim. Slov. 2021, 68, 185-192 189 stantaneous and the progressive 3D nucleation are given by equations (1) and (2), respectively: (1) /(t)2 1.2254 ( --= -- 1 -exp 1max ___1, -2.3367 (2) Fig. 7 (a-e) shows the nondimensional I2/I2max vs. t/ tmax plots derived from the CA data at different conditions as in Fig. 6. The solid lines of black and red color are the theoretical transients of the instantaneous and the progressive nucleation, and blue lines for the experimental data. The nucleation and growth processes of iron at these conditions can be derived from Fig. 7 (a-e). At the early stage, the experimental curve fits well with the curve of the progressive nucleation model by which the iron nucleation occurred on many active sites of Pt substrates. Subse- Fig. 7. Comparison of the theoretical non-dimensional (I / Imax)2 vs (t / tmax) plots for instantaneous (red) and progressive (black) nucleation with experimental data (blue) of potentiostatic transients in the solution of 0.1 Fe(NO3)3 • 9H2O + CH2OH-CH2OH at different constant potentials: (a) -0.4, (b) -0.45, (c) -0.50, (d) -0.55 and (e)-0.6 V Majidzade et al.: Electrodeposition and Growth of Iron ... 190 Acta Chim. Slov. 2021, 68, 185-192 quently, the deposition deviates from the progressive nu-cleation as shown in Fig. 7 (a, b, c, d, and e). The deviation from the ideal assumption of the Scharifker model may be attributed to that the nuclei grow under diffusion control at these conditions. Bypassing time through the nuclei growth, the elec-trodeposition of iron will be under mixed control (diffusion and charge transfer). The deviation can be also interpreted as due to the hydrogen evolution reaction during the formation of nuclei which causes a morphology change of the nucleus. Further information for the growth mechanism can be obtained by calculating the density of actives sites for nucleation (N0) by using the following equation; ^o.oesf^V172 MM2 v P J \tmaxhnax' (3) where, C is the bulk concentration in mol cm-3, zf the molar charge of electrodepositing species, M and p are the molar mass and the density of the deposited material in g cm-3, respectively. The diffusion coefficient D of the active species in the electrolyte can be calculated via the chrono-amperometric method. According to the theoretical nu-cleation model, the D is related to the imax and the tm by the following equation; 25, 26 (4) The values of imax, tmax, D, and N0 at different deposition potentials are shown in Table 1. The D values of the electroactive species are very small at these conditions, which is due to the high relative density of the electrolyte and the high relative diameter of iron ions. Therefore, the diffusion of iron ions from the bulk of the electrolyte towards the polarized electrode will be very small. Accordingly, the process is controlled by the diffusion step. Also, the value of D is a function of the polarization potential as listed in Table 1. It is noted from the table that nuclei densities N0 decrease significantly with the increase of the deposition potential. This decrease is due to the decrease of the activation of the nucleation sites at higher potentials, which deviates from the classical nucleation models as confirmed by Fig. 6. This deviation can also be explained as, by in- Table 1. Experimental data on the electrodeposition of Fe on a Pt electrode E, V D, cm2 s-1 No, cm2 -0.40 1.429 x 10-17 5.00 x 1015 -0.45 6.556 x 10-18 10.09 x 1015 -0.50 3.619 x 10-17 1.98 x 1015 -0.55 1.468 x 10-16 0.49 x 1015 -0.60 5.240 x 10-16 0.14 x 1015 creasing the deposition potential the polarization of the working electrodes increases. But the diffusion of the active species is still slow because of the high density of surrounding media which hinders the diffusion of the active species. The data of the XRD pattern (Fig. 8) and SEM images (Fig. 9) confirmed the obtained films of the electrode-posited iron on the Ni electrode. Fig. 8. The results of the XRD analysis of electrodeposited iron from 0.1 M Fe (NO3)3 x 9H2O + CH2OH-CH2OH electrolyte on the Ni electrode. EV = 0.02 V / s, T = 293 K. Fig. 9. The image of SEM (a) and EDAX analysis (b) of electrodeposited iron from the 0.1 M Fe (NO3)3 x 9H2O + CH2OH- CH2OH electrolyte on the Ni electrode. At EV = 0.02 V / s, and T = 293 K. Majidzade et al.: Electrodeposition and Growth of Iron ... Acta Chim. Slov. 2021, 68, 185-192 191 The SEM image showed that the nickel substrate is well coated. It seems to be relatively homogeneous and has a few cracks (Fig. 9). However, judging by the obtained X-ray pattern, these coatings are very thin (Fig. 8). It is noted that nickel peaks are also shifted towards large diffraction angles (20). This is because of a slight contraction of the modified crystal lattice, which is due to the incorporation of iron atoms into the nickel lattice. All experimental results exhibit that, in order to achieve the process and obtain compact, smooth deposits, an optimal electrolyte composition of 0.1 M Fe (NO3) ■ 9H2O + CH2OH-CH2OH, at 293 K, and a potential range of 0.6 - (- 0.9) V should be applied. 4. Conclusion Electrochemical reduction of iron (III) ions on Pt electrode from ethylene glycol solution was studied by the potentiodynamic method. During the study of kinetics and mechanism of the process by the cyclic and linear polarization curves, it was revealed that the nature of polarization is accompanied by mixed kinetics. It is worth mentioning that, in the potential range of 0.0 - (-0.2) V, the process proceeds under electrochemical polarization control, whereas after -0.2 V it is controlled by concentration polarization. The obtained results show that the electrore-duction of iron (III) ions is affected by concentration, temperature, and the potential scan rate. From these results, the optimal mode and composition of the electrolyte for the preparation of the compact and smooth iron films have been detected. SEM and XRD data confirm the electrode-position of thin Fe films. 5. References 1. MR Palacin, P Simon, JM Tarascon, Acta Chim. Slov. 2016, 63, 417-423. DOI:10.17344/acsi.2016.2314 2. VN Fateev, OK Alexeeva, SV Korobtsev, EA Seregina, TV Fateeva, AS Grigoriev, ASh Aliyev, Chemical Problems. 2018, 4, 453-483. DOI:10.32737/2221-8688-2018-4-453-483 3. TL Kulova, II Nikolaev, VN Fateev, ASh Aliyev, Chemical Problems. 2018, 1, 9-34. DOI: 10.32737/2221-8688-2018-1-9-34 4. VN Fateev, TL Kulova, EA Seregina, NA Ivanova, DD Spasov, Chemical Problems. 2019, 3, 349-357. DOI:10.32737/2221-8688-2019-3-349-357 5. V Aluri, KTR Reddy, YM Reddy, Nanotechnol. Rev. 2015, 4, 469-472. DOI: 10.1515/ntrev-2014-0043 6. X Wang, G Wang, J Chen, X Zhu, J Tian, C Jiang, Y Zhang, X Liu, R Wang, Mater. Lett. 2013, 110, 144-147. DOI:10.1016/j.matlet.2013.07.107 7. 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DOI:10.1016/j.molstruc.2017.01.082 23. VA Majidzade, ASh Aliyev, DM Babanly, M Elrouby, DB Tagiyev, Acta Chim. Slov. 2019, 66, 155-162. DOI:10.17344/acsi.2018.4733 24. AA Sukhotin (Ed): Handbook of Electrochemistry, Chemistry, Leningrad, Russian, 1981, p. 488. 25. B Scharifker, G Hills, Electrochim. Acta. 1983, 28, 7, 879889. DOI:10.1016/0013-4686(83)85163-9 26. MR Khelladi, L Mentar, A Azizi, A Sahari, A Kahoul, Mater. Chem. Phys. 2009, 115, 385-390. DOI:10.1016/j.matchemphys.2008.12.017 Majidzade et al.: Electrodeposition and Growth of Iron ... 192 Acta Chim. Slov. 2021, 68, 185-192 Povzetek Dosegli smo elektrokemijsko redukcijo železovih (III) ionov v elementarno železo iz raztopine etilen glikola. Kinetiko in mehanizem procesa elektroredukcije smo raziskovali s cikličnimi in linearnimi polarizacijskimi tehnikami. Preučevali smo tudi vpliv temperature, hitrosti spreminjanja potenciala in koncentracije ionov železa (III) na postopek elektroredukcije. Opazovane vrednosti efektivne aktivacijske energije so pokazale, da je preiskovani postopek elektroredukcije kontroliran s procesi mešane kinetike. Poleg tega so rezultati analize SEM in rentgenske difrakcije potrdili, da lahko pod optimalnimi pogoji dosežemo odlaganje tankih filmov Fe. ©CD Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Majidzade et al.: Electrodeposition and Growth of Iron ... DOI: 10.17344/acsi.2020.6345 Acta Chim. Slov. 2021, 68, 193-204 /^creative ^commons Scientific paper Manganese(II) ^-Diketonate Complexes with Pyridin-4-one, 3-Hydroxypyridin-2-one and 1-Fluoropyridine Ligands: Molecular Structures and Hydrogen-bonded Networks Anze Cavic and Franc Perdih* Faculty of Chemistry and Chemical Technology, University of Ljubljana, Vecna pot 113, P. O. Box 537, SI-1000 Ljubljana, Slovenia * Corresponding author: E-mail: franc.perdih@fkkt.uni-lj.si Received: 08-21-2020 Abstract Manganese(II) bis(4,4,4-trifluoro-1-phenylbutane-1,3-dionate) complexes with pyridin-4-one (pyon), 3-hydroxypyri-din-2-one (hpyon), 1-fluoropyridine (pyF) and methanol were prepared and the solid-state structures were determined by single-crystal X-ray analysis. The coordination of the metal center in all complexes was found to be octahedral. In compounds [Mn(tfpb)2(pyon)2] (1) and [Mn(tfpb)2(hpyon)2] (2) extended hydrogen bonding is present facilitating the formation of a three-dimensional supramolecular structure in 1 and a layered structure in 2 through N-H—O hydrogen bonding enhanced by C-H—O interactions as well as C-F—n interactions. In [Mn(tfpb)2(pyF)2] (3) a layered structure is formed through C-H—O and C-H—F interactions as well as n—n and C-F—n interactions. In [Mn(tfpb)2(MeOH)2] (4) a layered structure is formed through a combination of O-H—O and C-F—n interactions. Keywords: p-Diketonates; manganese; pyridines; crystal structure; n—n interaction 1. Introduction Inorganic-organic hybrids, metal-organic coordination polymers and especially metal-organic frameworks (MOFs) are currently an extremely important topic and an active area of research because of their intriguing architectures and topologies,1,2 as well as due to their potential applications in catalysis, chemical separation processes, wastewater treatment, gas storage, magnetism and as sen-sors.3 Control of the solid-state arrangement of molecules within a crystal is the central challenge of materials chemistry. In metal-organic frameworks and coordination polymers, covalent bonding using bridging organic li-gands for creation of robust polymeric structures is of prime importance. Various kinds of these materials have been designed with special attention dedicated to the geometry of the metal ions as well as flexibility, bridging potential and coordination preferences of different organic linkers.1 On the other hand, in inorganic-organic hybrids non-covalent bonds adjust the dimensionality and enable new topologies to arise. Non-covalent forces, such as hydrogen bonding, C-H—n/F interactions, n—n stacking, and halogen bonding are much weaker compared to the covalent bonds, however, their multitude makes them a powerful tool in the crystal engineering. Also, a great variety of non-covalent donors-acceptors and their numbers, their unique directionality and simple introduction into structures make them a particularly good choice for the construction of self-assemblies. Here we report the influence of pyridin-4-one (4-pyridone; pyon), 3-hydroxypyridin-2-one (hpyon), and 1-fluoropyridine (pyF) ligands on the molecular and su-pramolecular structure in the cases of [Mn(tfpb)2(pyon)2] (1), [Mn(tfpb)2(hpyon)2] (2), [MnfbMpyF^] (3) complexes as well as the structure of [Mn(tfpb)2(MeOH)2] (4), where tfpb is the 4,4,4-trifluoro-1-phenylbutane-1,3-dio-nate (or 4,4,4-trifluoro-3-oxo-1-phenylbutan-1-olate) li-gand. The tfpb ligand was selected because it is not symmetric and possesses phenyl and trifluoromethyl groups enabling also the formation of C-H—F, F—F and C-F—n interactions besides the n—n and C-H—n interactions.4 Pyridin-4-on and 3-hydroxypyridin-2-one were selected since the tautomeric equilibrium between the lactam and lactim forms enables various coordination modes and also Cavic and Perdih: Manganese(II) fi-Diketonate Complexes 194 Acta Chim. Slov. 2021, 68, 193-204 due to their different hydrogen bond formation abilities when coordinated in lactam/lactim form. On the other hand, 1-fluoropyridine was selected in order to be able to study the influence of an additional fluorine substituent on the formation of supramolecular aggregation in the absence of the competing strong hydrogen bond donors. 2. Experimental 2. 1. Materials and Characterization Reagents and chemicals were obtained as reagent grade from commercial sources and were used as purchased without any further purification. [Mn(tfpb)2(H2O)2] was prepared according to the literature procedure.5 Infrared (IR) spectra (4000-600 cm-1) of the samples were recorded using a Perkin-Elmer Spectrum 100, equipped with a Specac Golden Gate Diamond ATR as a solid sample support. Elemental (C, H, N) analyses were obtained using a Perkin-Elmer 2400 Series II CHNS/O Elemental Analyzer. 2. 2. Synthesis Synthesis of [Mn(tfpb)2(pyon)2] (1) [Mn(tfpb)2(H2O)2] (0.065 g, 0.125 mmol) was dissolved in acetone (8 mL) and then pyon (0.024 g, 0.250 mmol) was added. The reaction mixture was stirred for 15 min at ~50 °C and then allowed to stand at room temperature. Orange crystals suitable for X-ray analysis were obtained after slow evaporation of the solvent over a few days. Yield: 0.036 g, 43%. Anal. Calcd. [Mn(tfpb)2(pyon)2] (C30H22F6MnN2O6) (MW = 675.44): C 53.35, H 3.28, N 4.15; Found C 52.92, H 2.90, N 4.06. IR (ATR, cm-1): 3244w, 3080w, 2663w, 1606s, 1597m, 1573m, 1527m, 1501m, 1471s, 1374m, 1315m, 1283s, 1248m, 1179s, 1127s, 1072m, 1025m, 996m, 939m, 831m, 763m, 717m, 697s, 635m. Synthesis of [Mn(tfpb)2(hpyon)2] (2) [Mn(tfpb)2(H2O)2] (0.065 g, 0.125 mmol) was dissolved in warm ethanol (12 mL) and then hpyon (0.028 g, 0.250 mmol) was added. The reaction mixture was stirred for 15 min at ~60 °C and then allowed to stand at room temperature. Orange crystals suitable for X-ray analysis were obtained after slow evaporation of the solvent over a few days. Yield: 0.058 g, 66%. Anal. Calcd. [Mn(tfpb)2(h-pyon)2] (C30H22F6MnN2O8) (MW = 707.44): C 50.91, H 3.14, N 3.96; Found C 50.72, H 3.14, N 3.90. IR (ATR, cm-1): 3251w, 3120w, 2958w, 1605m, 1596m, 1570s, 1543m, 1529m, 1491m, 1471m, 1456m, 1419m, 1377m, 1284s, 1251m, 1187s, 1134s, 1058m, 937m, 885m, 761s, 717m, 699s, 635m. Synthesis of [Mn(tfpb)2(pyF)2] (3) [Mn(tfpb)2(H2O)2] (0.065 g, 0.125 mmol) was dissolved in pyF (2 mL). The reaction mixture was stirred for 15 min at ~60 °C and then allowed to stand at room temperature. Orange crystals suitable for X-ray analysis were obtained after slow evaporation of the solvent over a few days. Yield: 0.045 g, 53%. Anal. Calcd. [Mn(tfpb)2(pyF)2] (C30H20F8MnN2O4) (MW = 679.42): C 53.02, H 2.97, N 4.12; Found C 52.55, H 2.81, N 3.99. IR (ATR, cm-1): 3381br, 1609s, 1597m, 1574s, 1532m, 1490m, 1458m, 1318m, 1281s, 1248m, 1182s, 1129s, 1096m, 1075m, 1025w, 941w, 798w, 768m, 718m, 699s, 635s. Synthesis of [Mn(fpb)2(MeOH)2] (4) [Mn(tfpb)2(H2O)2] (0.065 g, 0.125 mmol) was dissolved in warm methanol (12 mL) and then pyon (0.024 g, 0.250 mmol) was added. The reaction mixture was stirred for 15 min at ~60 °C and then allowed to stand at room temperature. Orange crystals suitable for X-ray analysis were obtained after slow evaporation of the solvent over a few days. Yield: 0.040 g, 58%. Anal. Calcd. [Mn(tfpb)2 (MeOH)2] (C22H20F6MnO6) (MW = 549.32): C 48.10, H 3.67; Found C 47.94, H 3.38. IR (ATR, cm-1): 2538br, 2421br, 1928w, 1876w, 1644m, 1602m, 1574m, 1350s, 1321m, 1259m, 1228m, 1191s, 1134m, 995s, 934s, 821s, 811s, 748w, 635s. 2. 3. X-ray Crystallography Single-crystal X-ray diffraction data were collected at room temperature (1, 2, 4) or 150 K (3) on a Nonius Kappa CCD diffractometer or an Agilent Technologies SuperNova Dual diffractometer with an Atlas detector using monochromated Mo-Ka radiation (X = 0.71073 A). The data were processed using DENZO6 or CrysAlis Pro.7 The structures were solved by direct methods implemented in SHELXS8 and SIR-979 and refined by a full-matrix least-squares procedure based on F2 with SHELXL.8 All non-hydrogen atoms were refined anisotropically. All H atoms were initially located in a difference Fourier maps. The hydrogen atoms on carbon atoms were treated as riding atoms in geometrically idealized positions. Hydrogen atoms attached to nitrogen and oxygen atoms were refined fixing the bond lengths and isotropic temperature factors as (7iso(H) = kUeq(N,O), where k = 1.5 for OH groups, and 1.2 for NH groups. In 1 and 4 the CF3 groups are disordered over two positions in 0.76(2):0.24(2) and 0.71(3):0.29(3) (in 1) and 0.66(3):0.34(3) (in 4) ratio. In 1 a possible pseudo-translation was detected, however, no additional space group could be found using the Platon program. The crys-tallographic data are listed in Table 1. 3. Results and Discussion Initial attempts to prepare 1 using methanol as a solvent gave 4 as the sole product. Thus, in the subsequent attempts of its synthesis other solvents were used instead. Compounds 1 and 2 were obtained by the reaction of Čavic and Perdih: Manganese(II) fi-Diketonate Complexes ... Acta Chim. Slov. 2021, 68, 193-204 195 Table 1. Crystallographic and refinement data for 1-4. Parameter [Mn(tfpb)2(pyon)2] (1) [Mn(tfpb)2(hpyon)2] (2) [Mn(tfpb)2(pyF)2] (3) [Mn(tfpb)2(MeOH)2] (4) Formula C3oH22F6MnN2O6 C3„H22F6MnN2O8 C30H20F8MnN2O4 C22H20F6MnO6 Mr 675.43 707.43 679.42 549.32 T (K) 293(2) 293(2) 150(2) 293(2) Crystal system Monoclinic Triclinic Monoclinic Triclinic Space group P21/n P-1 P2Jc P-1 a (A) 16.1805(3) 7.3146(2) 11.8720(3) 10.4921(4) b (A) 10.5318(2) 9.9367(2) 8.8105(2) 10.5763(4) c (A) 17.8217(3) 10.7440(2) 14.5507(4) 12.4197(5) a(°) 90 108.132(2) 90 70.893(2) P (°) 91.558(2) 100.589(2) 108.628(3) 66.685(2) Y (°) 90 91.833(2) 90 82.624(2) Volume (A3) 3035.87(10) 726.16(3) 1442.24(7) 1195.92(8) Z 4 1 2 2 DCalc (Mg/m3) 1.478 1.618 1.565 1.525 || (mm-1) 0.517 0.549 0.549 0.634 F(000) 1372.0 359.0 686.0 558.0 Crystal size (mm) 0.5 x 0.2 x 0.1 0.6 x 0.6 x 0.5 0.2 x 0.2 x 0.05 0.25 x 0.1 x 0.03 Reflections collected 28841 5959 13789 9395 Data/restraints/parameters 6949/2/471 3298/2/220 3311/0/205 5457/2/353 Rint 0.0322 0.0133 0.0333 0.0291 R, wR2 [I>2a(Z)]a 0.0416, 0.1040 0.0328, 0.0882 0.0399, 0.0961 0.0459, 0.1073 R, wR2 (all data)b 0.0650, 0.1162 0.0352, 0.0904 0.0529, 0.1037 0.0812, 0.1264 GOF, Sc 1.051 1.074 1.044 1.012 Max/min Ap (e/A3) 0.21/-0.21 0.31/-0.34 0.83/-0.27 0.32/-0.27 a R = Z||F0| - |Fc||/Z|F0|. b wR2 = number of parameters refined. {XMFo2 - Fc2)2]/Z[w(Fo2)2]} 1/2. c S = {X[(Fo2 - Fc2)2]/(n/p)}1/2 where n is the number of reflections and p is the total [Mn(tfpb)2(H2O)2] and the corresponding heteroaromatic ligands pyridine-4-on (pyon) and 3-hydroxypyridine-2-on (hpyon) in 1:2 molar ratio in warm ethanol or acetone, respectively. Compound 3 was prepared by the reaction of [Mn(tfpb)2(H2O)2] in warm 1-fluoropyridine (pyF) acting as a solvent and as a ligand. Crystals suitable for X-ray analyses were obtained by slow evaporation of the solvent at room temperature over a few days. The IR spectrum of 1 shows two bands at 3244 and 3080 cm-1 and the spectrum of 2 two bands at 3251 and 3120 cm-1 that suggest the involvement of the O-H and N-H groups of pyridone ligands in strong hydrogen bonding. The spectrum of 4 shows one broad band at 3381 cm-1 that suggests the involvement of the O-H groups of methanol ligands in strong hydrogen bonding. In all four compounds, there are bands in the frequency range 1609-1527 cm-1 characteristic for the v(C=O) and v(C=C) stretching of the tfpb li-gand. Compound 1 crystallizes in the monoclinic P21/n space group. Selected bond distances and angles of 1 are summarized in Table 2. The asymmetric unit contains two crystallographically independent half-molecules (A and B), with both independent Mn11 atoms sitting on the inversion centers. Both manganese(II) atoms are octahedrally coordinated (Fig. 1). In the equatorial plane, both metal centers are surrounded by four oxygen atoms of two che- lating tfpb ligands in a trans arrangement, with Mn-O distances 2.1365(14) and 2.1233(13) Â (for A) and 2.1245(13) and 2.1467(13) Â (for B). The Mn(tfpb)2 fragments deviate from planarity, the angle between the mean plane formed by the equatorial MnO4 core and that of the tfpb chelate C3O2 moiety being 14.48(6) and 16.47(6)°. In both complexes the axial positions are occupied by two pyon ligands bonded to the metal center through the O atom, with Mn1-O3 distance of 2.2358(12) Â and Mn1-O3-C23 angle of 131.10(11)° and Mn2-O6 distance of 2.2035(12) Â and Mn2-O6-C28 angle of 126.58(11)°. These distances are similar to those reported for the three known Mn complexes with tfpb.10 The pyon ligands are inclined toward the tfpb moiety. The angle between the plane of the pyon ring and the plane of the equatorial MnO4 core deviates from 90° being 78.60(5)° (for A) and only 44.51(5)° (for B). Superposition of both complexes shows that pyon ligands are oriented in the opposite direction (Fig. 2) with pyon ring in complex B inclined toward the phenyl ring of the tfpb ligand. Complex A is stabilized by an intramolecular C22-H22"-O2i interaction between pyon and tfpb ligand (Table 3) and C1-F3a/b—n interactions between -CF3 group and pyon ring with F—C^3 distances of 3.769(10) and 3.82(4) Â and C-F-C^3 angles of 130.4(5) and 128(3)°, respectively, where Cg3 is N1/C21-C25 ring cen-troid (Fig. 3). Complex B is stabilized by an intramolecular Cavic and Perdih: Manganese(II) fi-Diketonate Complexes Figure 1. Crystallographically independent molecules in 1. Disorder on CF3 groups has been omitted for clarity. Displacement ellipsoids are drawn at 30% probability level. C27-H27—O411 interaction between pyon and tfpb ligand. The NH groups of the pyon ligands of both Independent complexes act as hydrogen-bond donors interacting with the tfpb carbonyl oxygens of the adjacent complexes, facilitating the formation of a hydrogen-bonded tree-dimensional supramolecular structure (Fig. 3). Complex A inter- Figure 2. Superposition of crystallographically independent molecules A (green) and B (orange) in 1. Disorder on CF3 groups has been omitted for clarity. acts with two complexes B through N1-H1—O6m bonding enabling the formation of an ABAB chain. Complex B interacts with two complexes A through N2-H2—O3 bonding enhanced by C26-H26—O1i interaction with R22(7) ring motif11 enabling the formation of an ABAB chain in the second dimension. Furthermore, complex B interacts with two adjacent complexes B through the centrosym-metric C29-H29—F6aiv interactions with R22(18) ring motif forming a BBB chain in the third dimension (Table 3). Table 2. Selected bond distances and angles for 1. Distance (A) Mn1-O1 2.1365(14) Mn2-O4 2.1245(13) Mn1-O2 2.1233(13) Mn2-O5 2.1467(13) Mn1-O3 2.2358(12) Mn2-O6 2.2035(12) Angle (°) O1-Mn1- O2 84.10(5) O4-Mn2- O5 85.38(5) O1-Mn1- -O2i 95.90(5) O4-Mn2- -O5ii 94.62(5) O1-Mn1- O3 85.43(5) O4-Mn2- O6 85.82(5) O1-Mn1- -O3i 94.57(5) O4-Mn2- -O6ii 94.18(5) O2-Mn1- O3 86.91(5) O5-Mn2- O6 85.20(5) O2-Mn1- -O3i 93.09(5) O5-Mn2- -O6ii 94.80(5) Symmetry codes: (i) 1 - x, -y, -z; (ii) 1 - x, -y, 1 - z. Cavic and Perdih: Manganese(II) ß-Diketonate Complexes Acta Chim. Slov. 2021, 68, 193-204 197 a) b) Figure 3. Three-dimensional supramolecular structure in 1 is achieved by hydrogen bonding around a) molecule A and b) molecule B through a series of N1-H1—O6m, N2-H2---O3, C26-H26—O11 and C29-H29---F6alv interactions. Blue dashed lines indicate hydrogen bonds. For the sake of clarity, intramolecular interactions, disorder on CF3 groups and H atoms not involved in the motif shown have been omitted. For symmetry codes see Table 3. Table 3. Hydrogen bonds for 1-4 [Â and °] D-H-A d(D-H) d(H-A) d(D-A) <(DHA) 1 N1-H1-O6iu 0.872(17) 1.854(17) 2.720(2) 172(3) N2-H2-O3 0.888(16) 1.859(17) 2.723(2) 164(2) C22-H22-O2i 0.93 2.36 3.119(3) 139.1 C26-H26-OP 0.93 2.48 3.322(2) 151.1 C27-H27-O4U 0.93 2.47 3.206(2) 136.6 C29-H29-F6aiv 0.93 2.47 3.394(7) 175.7 2 O4-H4-OT 0.814(17) 1.952(18) 2.7495(16) 166(3) N1-H1-O3U 0.882(14) 2.009(15) 2.8829(15) 170.2(18) C13-H13-O4ia 0.93 2.58 3.4770(19) 162.9 C15-H15-O2iv 0.93 2.42 3.2428(19) 147.3 3 C13-H13-F2a 0.95 2.42 3.287(3) 151.6 C14-H14-O1ia A 0.95 2.58 3.493(3) 161.3 4 O5-H5-OT 0.822(10) 1.966(14) 2.772(2) 167(4) O6-H6-O3" 0.813(10) 2.001(13) 2.801(2) 168(4) Symmetry codes for 1: (i) 1 - x, -y, -z; (ii) 1 - x, -y, 1 - z; (iii) -/ + x, / - y, -/ + z; (iv) 1 - x, 1 - y, 1 - z; for 2: (i) 2 - x, 2 - y, -z; (ii) 1 - x, 2 - y, -z; (iii) 2 - x, 2 - y, 1 - z; (iv) -1 + x, y, z; for 3: (ii) -x, 1 - y, -z; (iii) x, / - y, / + z; for 4: (i) 2 - x, 1 - y, 1 - z; (ii) 2 - x, -y, 1 - z. The supramolecular structure is further stabilized also by C11-F4a—n interaction between -CF3 group of complex B and pyon ring of complex A with F—QgS distance of 3.806(11) A and C-F-C£3 angle of 139.1(5)°. Compound 2 crystallizes in the triclinic P-1 space group. Selected bond distances and angles are summarized in Table 4. The asymmetric unit contains one half of the complex, with the Mn11 atom sitting on the inversion center. Octahedrally coordinated manganese(II) atom is surrounded by four oxygen atoms positioned in the equatorial plane, stemming from two chelating tfpb ligands in a trans arrangement, with Mn-O distances 2.1132(10) and 2.1218(9) Â (Fig. 4). The Mn(tfpb)2 fragment deviates from planarity, the angle between the mean plane formed by the equatorial MnO4 core and that of the tfpb chelate C3O2 moiety being 15.91(4)°. The axial positions are occupied by two hpyon ligands bonded to the metal center through the O3 atom, with Mn1-O3 distance of 2.2768(10) Â and Mn1-O3-C11 angle of 128.19(9)°. The hpyon ligand is inclined toward the tfpb moiety, with the angle between the plane of the hpyon ring and that of the equatorial MnO4 core being 43.25(6)°. The hydroxy group of the hpyon ligand is involved in intramolecular hydrogen bonding with the tfpb ligand through O4-H4—O1i interaction (Table 3). The NH group of the hpyon ligand acts as a hydrogen bond donor, facilitating the formation of a centrosymmetric hydrogen-bonded motif via N1-H1-"O3ii interactions with the ligated carbonyl O3 atom enhanced by C15-H15--O2iv interactions with the graphset motifs R22(8) and R22(7), respectively (Fig. 5 and Table 3). This interaction is further stabilized by C1-F3—n interaction between CF3 group and the hpyon ring with d(F3-Cg3) = 3.2278(17) Â and <(C1-F3-Cg3) = 135.64(11)°, where Cg3 is N1/C11-C15 ring centroid. Consequently, a chain is formed along the a axis. The chains are further connected into layers along the ac plane via centrosymmetric C13-H13—O4m hydrogen bonding between hpyon CH moiety and the hydroxy group of the adjacent molecule (Fig. 5). There are no significant n—n interactions. Cavic and Perdih: Manganese(II) fi-Diketonate Complexes 198 Acta Chim. Slov. 2021, 68, 193-204 04 H4 il or Figure 4. Structure of 2. Displacement ellipsoids are drawn at the 30% probability level. Intramolecular hydrogen bonding is presented by dashed blue lines. Table 4. Selected bond distances and angles for 2. Distance (A) Mn1-O1 Mn1-O3 2.1132(10) 2.2768(10) Mn1-O2 2.1218(9) Angle (°) O1-Mn1- 01-Mn1- 02-Mn1- -O2 O3 O3 84.24(4) 92.67(4) 84.40(4) O1-Mn1- 01-Mn1- 02-Mn1- -O2i -O3i -O3i 95.76(4) 87.33(4) 95.60(4) Symmetry code: (i) 2 - x, 2 - y, -z. In the solid state, pyridin-4-one and 3-hydroxypyri-din-2-one are in the lactam form.12,13 Also in metal complexes the lactam form of both predominates. A search of the Cambridge Structural Database (CSD, Version 5.41, plus updates)14 has revealed 26 entries15 of metal complexes where pyridin-4-one, in its lactam form, is bonded via O atom also observed in complex 1. However, 9 entries with the lactim form (as 4-hydroxypyridine) bonded via N atom were found in the CSD with Re, Ir, Pt, and Ag16 as well as Cu and Fe.17 This observation can be explained by the Pearson HSAB (hard-soft acid-base) concept18 since soft acids, such as Re, Ir, Pt, and Ag, show a preference for bonding via pyridine N atom (an intermediate base) as opposed to the -OH group (a hard base). Additionally, 3 entries with the lactim form bonded via OH group were also found with Nd, Tb, Dy.19 In metal complexes with 3-hy-droxypyridin-2-on lactam form with monodentate ligation via O atom20,21 was found in 9 entries; the same type was also observed in complex 2. Additionally, 3 entries were found with 0,0'-chelating ligation.21,22 However, no entries were found with lactim form (as 2,3-dihydroxypyr-idine) bonded to the metal center. For comparison, metal complexes with pyridine-2-one were more often investigated than complexes with pyridin-4-one and 3-hydroxy-pyridin-2-one and a variety of coordination modes has been observed.21,23,24 Compound 3 crystallizes in the monoclinic P21lc space group. Selected bond distances and angles of 3 are summarized in Table 5. Initial attempts to collect XRD data at room temperature failed due to slow decomposition of the crystal when exposed to the air. Most probably 1-fluoropyridine molecule is eliminated from the complex and the crystal lattice is being thus destroyed. Similar loss of pyridine bonded in zinc picolinato complexes has been previously observed.61,62 The asymmetric unit contains one half of the complex, with the Mn11 atom sitting on the inversion center. The manganese(II) atom in compound 3 is octahedrally coordinated (Fig. 6). In the equatorial plane, MnII atom is surrounded by four oxygen atoms stemming from the two chelating tfpb ligands, being in a trans arrangement, with Mn-O distances 2.1415(14) and 2.1337(14) A. The Mn(tfpb)2 fragment deviates from pla-narity, the angle between the mean plane formed by the MnO4 core and that of the tfpb chelate C3O2 moiety being 18.00(7)°. The axial positions are occupied by two pyF li-gands bonded to the metal center through the N1 atom, with Mn1-N1 distance of 2.3425(17) A. PyF ligand plays the main role in the formation of a layered structure due to the absence of the competing strong hydrogen bond donors. As a hydrogen bond donor PyF is involved in C14-H14-"O1m interaction with carbonyl oxygen atom and in centrosymmetric C13-H13—F2" interactions with fluorine atom of -CF3 group of tfpb ligands of the adjacent complexes (Fig. 7 and Table 3). Thus, each complex is involved in eight hydrogen bonds with six adjacent complexes forming a layered structure. 2D structure is supported by centrosymmetric n—n interactions between adjacent pyF rings with centroid-to-centroid distance of 3.9403(14) A, perpendicular distance between rings of 3.2624(10) A and ring slippage of 2.210 A. Layered structure is further supported also by C-F—n interactions between pyF fluorine atom and pyF aromatic ring with d(F4-Cg3) = 3.6714(19) A and <(C11-F4-Cg3) = 75.63(13)° as well as by interactions between -CF3 group and benzene ring of tfpb ligand with d(F2—Cg4) = 3.715(2) A and <(C1-F2-Cg4) = 126.72(15)°, where Cg3 and Cg4 are N1lC11-C15 and C5-C10 ring centroids, respectively (Fig. 7). The inclination of pyon and hpyon ligands toward the tfpb moiety in 1 and 2 is best compared with the compound 3 since the ligation of pyF via N atom cannot enable much deviation in comparison to the pyon and hpyon ligands bonded via O atom. Superposition of both crystallo-graphically independent molecules in 1 as well as mole- Čavic and Perdih: Manganese(II) fi-Diketonate Complexes ... Acta Chim. Slov. 2021, 68, 193-204 199 Figure 5. a) Hydrogen-bonded layer along the ac plane in 2 is formed by b) centrosymmetric N1-H1—O3u, C15-H15—O21T and C1-F3—Cg3u interactions and c) C13-H13—O4m interactions; d) packing of layers (arbitrary colors). Blue and green dashed lines indicate hydrogen bonds and C-F—n interactions, respectively. For the sake of clarity, H atoms not involved in the motif shown have been omitted. For symmetry codes see Table 3. Figure 6. Structure of 3. Displacement ellipsoids are drawn at 50% probability level. Cavic and Perdih: Manganese(II) fi-Diketonate Complexes 200 Acta Chim. Slov. 2021, 68, 193-204 Figure 7. a) Hydrogen-bonded layer along the ac plane in 3 is formed by C13-H13—O1m and centrosymmetric C14-H14—F211 interactions as well as centrosymmetric n—n interactions and C-F—n interactions; b) packing of layers (arbitrary colors). Blue and green dashed lines indicate hydrogen bonds and n—n and C-F—n interactions, respectively. For the sake of clarity, H atoms not involved in the motif shown have been omitted. For symmetry codes see Table 3. Table 5. Selected bond distances and angles for 3. Distance (A) Mn1-N1 Mn1-O2 2.3425(17) 2.1337(14) Mn1-O1 2.1415(14) Angle (°) N1-Mn1-N1-Mn1-O1-Mn1- O1 -O2 -O2 94.36(6) 88.31(6) 85.12(5) N1-Mn1-N1-Mn1 O1-Mn1 -O1i -O2i -O2i 85.64(6) 91.69(6) 94.88(5) Symmetry code: (i) -x, -y, -z. cules 2 and 3 is presented in Fig. 8. Pyon and hpyon ligands are inclined toward the tfpb moiety by 78.60(5)° (molecule A in 1), 44.51(5)° (molecule B in 1) and 43.25(6)° (2) representing a substantial deviation from 90°. However, in the case of molecule B in 1 and molecule 2 also the phenyl rings of tfpb are evidently inclined toward pyridone moieties. Compound 4 crystallizes in the triclinic P-1 space group. Selected bond distances and angles of 4 are summarized in Table 6. The asymmetric unit contains one complex molecule with cis-octahedral arrangement of methanol ligands on the manganese(II) central atom (Fig. 9). Two methanol ligands are bonded to the metal center with Mn1-O5 and Mn1-O6 distances of 2.1714(18) and 2.173(2) Â, respectively, and O5-Mn1-O6 angle of 88.54(8)°. The Mn1-O bond lengths with four oxygen atoms of the two chelating tfpb ligands are asymmetric with the longer ones of 2.1821(18) and 2.1751(17) Â at the triflu-oromethyl substituent and the shorter ones of 2.1266(18) and 2.1315(18) Â at the phenyl substituent. The Mn(tfpb) fragments deviate from planarity, the tfpb ligands being in- Čavic and Perdih: Manganese(II) fi-Diketonate Complexes ... Acta Chim. Slov. 2021, 68, 193-204 201 Figure 8. Two views on superposition of crystallographically independent molecules A (green) and B (orange) in 1, 2 (blue) and 3 (violet). Disorder on CF3 groups has been omitted for clarity. a) b) Figure 9. Structure of 4. Disorder on both CF3 groups has been omitted for clarity. Displacement ellipsoids are drawn at 30% probability level. Figure 10. a) Hydrogen-bonded chain along b axis in 4 formed by centrosymmetric O5-H5—O11 and O6-H6—O3U interactions; b) chains are linked into a layer through C-F—n interactions. Blue and green dashed lines indicate hydrogen bonds and C-F—n interactions, respectively. For the sake of clarity, H atoms not involved in the motif shown have been omitted. For symmetry codes see Table 3. Cavic and Perdih: Manganese(II) fi-Diketonate Complexes 202 Acta Chim. Slov. 2021, 68, 193-204 clined by 25.94(8) and 23.51(8)°. Each methanol ligand is involved in a centrosymmetric hydrogen-bonded motif via O5-H5-OL and O6-H6-O3ii interactions with the car-bonyl oxygen atom at the trifluoromethyl substituent of the adjacent complex. Both centrosymmetric hydrogen bonds have the graph-set motif R22(8) (Fig. 10 and Table 3) and enable the formation of a hydrogen-bonded chain along the b axis. A centrosymmetric C1-F3—n interaction between CF3 group and the benzene ring of tfpb ligand of the adjacent molecule is present with d(F3--Cg3) = 3.661(4) Â and <(C1-F3—Cg3) = 121.9(3)°, where Cg3 is C5-C10 ring centroid, connecting chains into a layer along the bc plane (Fig. 10). There are no significant n—n interactions. Table 6. Selected bond distances and angles for 4. Distance (A) Mn1-O1 2.1821(18) Mn1-O2 2.1266(18) Mn1-O3 2.1751(17) Mn1-O4 2.1315(18) Mn1-O5 2.1714(18) Mn1-O6 2.173(2) Angle (°) O1-Mn1- O2 82.45(7) O1-Mn1- O3 89.13(7) O1-Mn1- O4 91.80(7) O1-Mn1- O5 92.79(7) O1-Mn1- O6 171.30(7) O2-Mn1- O3 92.40(7) O2-Mn1- O4 172.98(7) O2-Mn1- O5 94.84(8) O2-Mn1- O6 88.87(7) O3-Mn1- O4 83.46(7) O3-Mn1- O5 172.69(7) O3-Mn1- O6 90.62(7) O4-Mn1- O5 89.43(7) O4-Mn1- O6 96.81(8) O5-Mn1- O6 88.54(8) 4. Conclusion We have prepared and structurally characterized four manganese(II) bis(4,4,4-trifluoro-1-phenylbutane-1,3-dionate) complexes with pyon, hpyon, pyF and methanol ligands. In all prepared compounds the coordination of the metal center is octahedral. Complexes 1-3 possess trans arrangement of ligands while in complex 4 the arrangement is cis. In 1-3 the Mn(tfpb)2 fragments deviate from planarity, the angles between the mean planes formed by the equatorial MnO4 cores and that of the tfpb chelate C3O2 moieties being in the range 14.48(6)-18.00(7)°. In 1 and 2 the axial positions are occupied by two pyon and hpyon ligands, respectively, bonded to the metal center through the O atom. Pyon and hpyon ligands are inclined toward the tfpb moiety by 78.60(5)° (molecule A in 1), 44.51(5)° (molecule B in l) and 43.25(6)° (2) representing a substantial deviation from 90°. Extended hydrogen bonding is present in 1 and 2 facilitating the formation of a three-dimensional supramolecular structure in 1 and a layered structure in 2 through N-H—O hydrogen bonding enhanced by C-H—O interactions as well as C-F—n interactions. In 3 pyF ligand plays the main role in the formation of crystal aggregation due to the absence of the competing strong hydrogen bond donors. A layered structure is formed through C-H—O and C-H—F interactions as well as n—n and C-F—n interactions. In 4 a layered structure is formed through a combination of O-H—O and C-F—n interactions. Supplementary Material CCDC 2024368-2024371 (1-4) 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. Acknowledgment Financial support from the Slovenian Research Agency through the grant P1-0230-0175 is gratefully acknowledged. We thank EN-FIST Centre of Excellence, Dunajska cesta 156, 1000 Ljubljana, Slovenia for using the Supernova diffractometer. 5. References 1. C. B. Aakeröy, N. R. Champness, C. Janiak, CrystEngComm 2010, 12, 22-43. D01:10.1039/B919819A 2. M. G. Goesten, F. Kapteijn, J. Gascon, CrystEngComm 2013, 15, 9249-9257. D0I:10.1039/c3ce41241e 3. (a) J. Liu, L. Chen, H. Cui, J. Zhang, L. Zhang, C.-Y. Su, Chem. Soc. Rev. 2014, 43, 6011-6061; D0I:10.1039/C4CS00094C (b) A. Dhakshinamoorthy, H. Garcia, Chem. Soc. Rev. 2014, 43, 5750-5765; D0I:10.1039/C3CS60442J (c) A. Herbst, C. 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DOI: 10.3390/molecules25040846 25. (a) T. Koleša-Dobravc, K. Maejima, Y. Yoshikawa, A. Meden, H. Yasui, F. Perdih, New J Chem 2017, 41, 735-746; DOI:10.1039/C6NJ02961B (b) T. Koleša-Dobravc, K. Maejima, Y. Yoshikawa, A. Meden, H. Yasui, F. Perdih, New J. Chem. 2018, 42, 3619-3632. DOI:10.1039/C7NJ04189F Cavic and Perdih: Manganese(II) fi-Diketonate Complexes 204 Acta Chim. Slov. 2021, 68, 193-204 Povzetek Sintetizirali smo manganove(II) bis(4,4,4-trifluoro-1-fenilbutan-1,3-dionato) komplekse s piridin-4-onom (pyon), 3-hi-droksipiridin-2-onom (hpyon), 1-fluoropiridinom (pyF) in metanolom ter določili strukture z monokristalno rentgensko difrakcijo. V vseh kompleksih je koordinacija kovinskega centra oktaedrična. Pri spojinah [Mn(tfpb)2(pyon)2] (1) in [Mn(tfpb)2(hpyon)2] (2) so prisotne številne vodikove vezi, ki omogočajo tvorbo tridimenzionalne supramolekularne strukture v 1 in plastovite strukture v 2 z N-H—O vodikovimi vezmi ojačane z C-H-O interakcijami ter tudi C-F—n interakcije. Pri [Mn(tfpb)2(pyF)2] (3) je prisotna plastovita struktura z C-H—O in C-H—F interakcijami ter tudi z n—n in C-F—n interakcijami. Pri [Mn(tfpb)2(MeOH)2] (4) je prisotna plastovita struktura s kombinacijo O-H—O in C-F—n interakcij. © 0 Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Čavic and Perdih: Manganese(II) fi-Diketonate Complexes ... DOI: 10.17344/acsi.2020.6359 Acta Chim. Slov. 2021, 68, 205-211 /^creative ^commons Scientific paper CO2 Improved Synthesis of Benzimidazole with the Catalysis of a New Calcium 4-Amino-3-hydroxybenzoate Ruo-Xuan Gao, Yuan-Yuan Gao, Ning Zhu and Li-Min Han* Chemical Engineering College, Inner Mongolia University of Technology, Hohhot, 010010, China * Corresponding author: E-mail: hanlimin@imut.edu.cn Received: 09-02-2020 Abstract In this paper, we explored the synthesis of benzimidazole by the reaction of DMF and o-phenylenediamine. In the process of catalyst screening, we found that 4-amino-3-hydroxybenzoic acid, benzoic acid, and benzene-1,3,5-tricarboxylic acid could catalyze the reaction. Moreover, the calcium 4-amino-3-hydroxybenzoate and CO2 could more effectively catalyze the reaction, the synergistic effect of CO2 and 4-amino-3-hydroxybenzoic acid calcium salt can increase the yield of benzimidazole from 28% to 94%. Keywords: Calcium 4-amino-3-hydroxybenzoate, CO2, benzimidazole, weak acid catalysis 1. Introduction Benzimidazole and its derivatives are fundamental building blocks in many kinds of functional compounds, especially in drugs and bioactive molecules,1-4 and widely used in anti-cancer,5 anti-inflammatory,6 anti-bacterial,7 anti-oxidant,8 anti-coagulant9 and so on.10 Therefore, it is of great significance to find a simple, economic and environmentally friendly method for the synthesis of ben-zimidazoles. Up to now, cycloaddition reaction of o-phe-nylenediamine and carbon source molecule seems to be an effective method for the synthesis of benzimidazoles. The carbon source generally involves a carboxylic acid,9,11 aldehyde,1,3,10 ketone,12 amide,11,13,14 and carbon dioxide2,15,16 (Scheme 1). As a special amide, N,N-dimethylformamide (DMF) is not only a carbon source but also an effective polar solvent for the synthesis of benzimidazoles.11,14,15,17,18 Scheme 1. Carbon sources in the synthesis of benzimidazoles with o-phenylenediamine. Catalysts, such as inorganic acids,19 organic acids,10 alkalis,18 Lewis acids,20 transition metals salts,21 organo-metallic complexes,22 play an important role in the synthesis of benzimidazoles. Hydrochloric acid is usually used in the synthesis of benzimidazoles, but it is corrosive and impossible to be reused.19 Then many catalysts had been prepared for this reaction, for instance, azole-anion-based aprotic ionic liquids with tetrabutylphosphonium hydroxide,23 ionic liquids of hydrochloric acid,24 glyoxylic acid,10 B(C6F5)316 and zinc acetate dehydrate.11 But they are usually used in combination with reducing substances such as hydrosilane, hydrogen, boron phosphorus and transition metal salts. In short, their application is undesirable under the green chemistry principles. It is of paramount importance to find an effective and pollution-free catalyst for the synthesis of benzimidazoles. Recently, Ru,25 Mn,26 Co,27,32 Cu,28 Zr,29 Fe30 and Ir31 organometallic complexes have been used to catalyze the synthesis of benzimidazoles, and the recycle times of catalysts were increased obviously. That provided a new idea for designing catalysts to catalyze the synthesis of benzimidazoles. When we investigated the catalytic mechanism of these organometallic salts, it was found that the main catalytic species was still hydrogen cation (i.e. proton).15,24 Hence, we planned to consider catalytic effects of several acids or salts especially 4-amino-3-hydroxybenzoic acid Gao et al.: CO2 Improved Synthesis of Benzimidazole with ... 206 Acta Chim. Slov. 2021, 68, 205-211 and its calcium salt on the synthesis of the benzimidazole and the results obtained are described in this paper. tails of the data collection and the structure refinements are given in Table S1. 2. Experimental 2. 1. Chemicals All of the reagents were purchased from commercial sources and used without further purification. Ben-zene-1,3,5-tricarboxylic acid, 4-amino-3-hydroxybenzoic acid, o-aminophenol, calcium benzoate, N,N-dimethyl-formamide and o-phenylenediamine were purchased from Shanghai Aladdin Biological Technology Co., Ltd. (Shanghai, China). Ca(NO3)2-4H2O, phenol and benzoic acid were purchased from Sinopharm Chemical Regagent Co., Ltd. (Shanghai, China). Calcium benzene-1,3,5-tri-carboxylate was synthesized according to the method in the literature.32 2. 2. Apparatus FT-IR spectra were measured on a Perkin-Elmer FT-IR spectrum. Fluorescence spectra were obtained by an Agilent Technologies Cary Fluorescence Spectrophotom-eter analyzer. NMR spectra in DMSO-d6 were recorded with an Agilent 500 MHz DD2 spectrometer. The molecular structure of calcium 4-amino-3-hydroxybenzoate was obtained on a Bruker D8 VENTURE diffractometer. X-ray powder diffraction (XRD) spectra were recorded on a Rig-aku (SmartLab 9KW) diffractometer for a Cu-target tube. 2. 3. Preparation of Calcium 4-Amino-3-hydroxybenzoate 4-Amino-3-hydroxybenzoic acid (91.8 mg, 0.6 mmol) and Ca(NO3)2 4H2O (141.7 mg, 0.6 mmol) were dissolved in MeOH (15 mL) in a flask, then the reaction mixture was stirred and refluxed for 3 hours. The resulting solution was cooled to room temperature and filtered. The filtrate was allowed to stand at room temperature for slow evaporation. IR: 3377.96 (m, Yo-H), 3307.00 (m, YN-H), 1609.45 (m, SN-H), 1532.81 (vs, Yas COO), 1413.58 (vs, Yas COO), 1223.39 (s, SC-O), 1024.68 (m, yC-N). 2. 4. Structure Determination Single-crystal X-ray diffraction measurements were carried out on a Bruker D8 VENTURE diffrac-tometer. The diffraction data were collected with MoKa radiation (A = 0.71073 A). The structures were solved by direct methods and refined against F2 by full-matrix least-squares methods with SHELXTL-20 1 4.33 All non-hydrogen atoms were refined with anisotropic thermal parameters. The hydrogen atoms were set in calculated positions and refined as riding atoms with a common fixed isotropic thermal parameter. Crystal data and de- 2. 5. The Synthesis of Benzimidazole o-Phenylenediamine (108.1 mg, 1 mmol) was catalyzed by calcium 4-amino-3-hydroxybenzoate (0.25 eq) with CO2 (3 bar) and DMF (2 mL) in a high-pressure reactor. Most of the o-phenylenediamine was consumed completely after the reaction mixture was stirred at 140 °C for 24 h. Then the reaction mixture was extracted with ethyl acetate, and the crude product (110.8 mg, 94%) was purified by column chromatography through a silica-gel column to afford the desired products eluted by CH3OH and CH2Cl2. Anal. Cal-cd. (%) for C7H4N2: C, 71.10; H, 5.12; N, 23.71. Found (%): C, 70.76; H, 5.51; N, 23.29. 1H NMR (500 MHz, DMSO-d6, 25 °C, TMS) 5 12.43 (s, 1H), 8.20 (s, 1H), 7.62-7.55 (m, 2H), 7.19 (dt, J1 = 6.0 Hz, J2 = 3.4 Hz, 2H). 13C NMR (500 MHz, DMSO-d6, 25 °C, TMS): 5 141.78, 121.56. IR: 3064.18 (m, Yc-h), 1592.49 (s, Yc-n), 1555.05 (w, Yc-n), 1480.17 (s, Yc-N), 1203.69 (m, YC-H), 742.89 (m, 5N-H). ESI-MS (CH3OH, m/z): 119.05 (M+). UV-Vis (CH3OH): 243, 278. 3. Results and Discussion Initially, the 4-amino-3-hydroxybenzoic acid-catalyzed cycloaddition reaction of o-phenylenediamine and DMF was investigated. The equivalents of the catalyst used, reaction temperatures and yields are listed in Table 1. As shown in Table 1, the reaction temperature (Table 1, entries 1-5) and the equivalent of 4-amino-3-hydroxy-benzoic acid used (Table 1, entries 5-9) could significantly influnce the reaction yield. The benzimidazole could be obtained in 84% yield after 24 hours of reaction at 140 °C when the equivalent of 4-amino-3-hydroxybenzoic acid was 0.5 (Table 1, entry 5). Table 1. The cyclization of o-phenylenediamine and DMF catalyzed by 4-amino-3-hydroxybenzoic acid Entry Cats [equiv.] T [°C] Yield [%] 1 0.5 100 26 2 0.5 110 40 3 0.5 120 57 4 0.5 130 71 5 0.5 140 84 6 0.25 140 67 7 0.125 140 37 8 0.05 140 15 9 0 140 - reaction condition: o-phenylenediamine (1 mmol), DMF (2 mL), reaction time 24 h, isolated yield Gao et al.: CO2 Improved Synthesis of Benzimidazole with ... Acta Chim. Slov. 2021, 68, 205-211 207 With the optimized reaction conditions, the catalytic performance of different weak organic acids was investigated (Table 2). Table 2. The cyclization of o-phenylenediamine and DMF catalyzed by an organic acid Entry Catalyst Cats Yield [equiv.] [%] 1 phenol 0.5 trace 2 o-aminophenol 0.5 trace 3 benzoic acid 0.5 85 4 4-amino-3-hydroxybenzoic acid 0.5 84 5 benzene-1,3,5-tricarboxylic acid 0.167 95 reaction condition: o-phenylenediamine (1 mmol), DMF (2 mL), 24 h at 140 °C, isolated yield As shown in Table 2, the yield of benzimidazole increased with the increasing of acidity of the catalyst. The phenol (p^a ~ 9.99) and o-aminophenol (p^a ~ 9.28) could not effectively catalyze the synthesis of benzimidazole. The benzoic acid (p^a ~ 4.20), benzene-1,3,5-tricar-boxylic acid (pK, ~ 2.12) and 4-amino-3-hydroxybenzoic acid (p^a ~ 4.74) could effectively catalyze the synthesis of benzimidazole.34 It is further proved that the catalytic species is proton.15,24 But when the reactions (presented in Tables 1 and 2) were performed, it was difficult to perform effective separation and recovery of the catalyst. Therefore, instead of the acids metal salts were used to catalyze this reaction. First, calcium 4-amino-3-hydroxybenzoate was prepared, its single crystal structure was determined by X-ray crystallography at 0 °C, showing that it crystallized in the monoclinic space group C2/c and showed an irregular dodecahedron. The molecular structure and coordination polyhedron of calcium 4-amino-3-hydroxybenzoate are shown in Figure 1. The calcium is in the center of eight oxygen atoms, four of them are from two polycarboxylic Figure 1. The molecular structure (left) and coordination polyhedron (right; ot calcium 4-amino-3-hydroxybenzoate. Figure 2. Structure of the calcium 4-amino-3-hydroxybenzoate showing the hydrogen bonding interactions (left) and packing diagram (right). Gao et al.: CO2 Improved Synthesis of Benzimidazole with ... 208 Acta Chim. Slov. 2021, 68, 205-211 groups, two of them are from CH3OH, and two of them are from two polycarboxylic groups coordinated with other calcium atoms. The bond length of Ca-O is in the range of 2.384-2.517 A, and there is a little difference in the distance between calcium atoms and eight oxygen atoms. The result of crystal structure analysis is consistent with the FT-IR spectrum (Figure S3). There are two kinds of hydrogen bonds in the structure of calcium 4-amino-3-hydroxybenzoate, shown in Figure 2 and Table S3. O(1)-H(1)—O(2) formed between the hydrogen atom on the methanol hydroxyl group coordinated with calcium ion and the oxygen atom on the carboxylic acid coordinated with the adjacent calcium ion. The distance between H(1) and acceptor O(2) was 1.92 A, and the angle between donor and acceptor was 176°. The other more weak interaction N(1)-H(1B)-O(4) formed between the hydrogen atom on the ligand amino group and the oxygen atom on the hydroxyl group of the adjacent ligand. The distance between H(1B) and acceptor O(4) was 1.98 A, and the angle between donor and acceptor was 167°. Moreover, the structure of the calcium 4-amino-3-hydroxybenzoate had some porous framework features, due to many uncoordinated amino and hydroxyl groups. The catalytic performance of the calcium 4-ami-no-3-hydroxybenzoate was tested, and compared with that of calcium benzoate, calcium benzene-1,3,5-tricarboxylate and 4-amino-3-hydroxybenzoic acid (Table 3.). Table 3. The cyclization of o-phenylenediamine and DMF catalyzed by various calcium salts and 4-amino-3-hydroxybenzoic acid EntryCatalyst Cats Yield [equiv.] [%] 1 calcium benzoate 0.5 10 2 calcium benzene-1,3,5-tricarboxylate 0.167 20 3 calcium 4-amino-3-hydroxybenzoate 0.25 28 4 4-amino-3-hydroxybenzoic acid 0.5 84 reaction condition: o-phenylenediamine (1 mmol), DMF (2 mL), 24 h, 140 °C, isolated yield As shown in Table 3, although the catalyst separation became easier, the calcium salts (i.e. calcium benzoate, calcium benzene-1,3,5-tricarboxylate and calcium 4-ami-no-3-hydroxybenzoate) did not exhibit the desired catalytic activity. Especially, with comparing corresponding carboxylic acid, the three calcium salts of thecarboxylic acids lost their catalytic activity seriously. Through exploration of their molecular structures, it was found that the benzoic acid exists in the crystal cell structure of calcium benzoate (Figure S5). And one of carboxyl groups of ben- zene-1,3,5-tricarboxylic acid was monodentate and might provide an active hydrogen (Figure S6). In the structure of calcium 4-amino-3-hydroxybenzoate, the active hydrogen might originate from an un-coordinated hydroxyl group. This small amount of the hydrogen in the crystal cell might be the catalytic species. In order to study the catalysis of weak acids further, CO2 was introduced into the reaction system of the cycli-zation of o-phenylenediamine and DMF. When we introduced CO2 at 3 bar into the reaction system (and no catalyst was added), nearly no benzimidazole was obtained (Table 4, entry 1). When the reaction was repeated with the addition of 0.5 equivalent of 4-amino-3-hydroxybenzoic acid, the yield of benzimidazole increased to 93% (Table 4, entry 2), which was higher than the previously obtained 84% (Table 3, entry 4). It was comfirmed that the presence of CO2 improved the reaction yield. Hence we introduced CO2 into other catalytic reaction systems of the cyclization of o-phenylenediamine and DMF (Table 4, entries 3-5). In calcium benzoate and calcium benzene-1,3,5-tricarbox-ylate catalytic reaction systems, no obvious improvement effect occurred (Table 4, entries 3 and 4). But in calcium 4-amino-3-hydroxybenzoate catalytic reaction system, the yield of benzimidazole (Table 4, entry 5) was almost the same as that of 4-amino-3-hydroxybenzoic acid catalytic reaction system. CO2 significantly improved the catalytic activity of calcium 4-amino-3-hydroxybenzoate. However, when the reaction mixture with separated calcium 4-ami-no-3-hydroxybenzoate catalyst was repeated, it lost its activity (Table 4, entry 6). By comparing the XRD patterns before and after its use (Figure 3), it was found that the structure of calcium 4-amino-3-hydroxybenzoate was destroyed during the reaction and can thus not be used again. Table 4. The cyclization of o-phenylenediamine and DMF catalyzed by the calcium salts in the presence of CO2 EntryCatalyst Cats Yield [equiv.] [%] 1 no 0 trace 2 4-amino-3-hydroxybenzoic acid 0.5 93 3 calcium benzoate 0.5 12 4 calcium benzene-1,3,5-tricarboxyate 0.167 23 5 calcium 4-amino-3-hydroxybenzoate 0.25 94 6 calcium 4-amino-3-hydroxybenzoate, 0.25 32 reused reaction condition: o-phenylenediamine (1 mmol), DMF (2 mL), 24 h, 140 °C, 3 bar CO2, isolated yield Although, the calcium 4-amino-3-hydroxybenzoate could not be reused, CO2 also could significantly improved Gao et al.: CO2 Improved Synthesis of Benzimidazole with ... Acta Chim. Slov. 2021, 68, 205-211 209 Figure 3. XRD pattern of the simulated and experimental calcium 4-amino-3-hydroxybenzoate and the catalyst used once. catalytic activity of calcium 4-amino-3-hydroxybenzoate whithout improved catalytic activity of the calcium ben-zoate and calcium benzene-1,3,5-tricarboxylate. That was still an interesting problem. 4-amino-3-hydroxybenzoate has the above group characteristics. On the basis of the experimental results and previous reports,14,20,24 a possible mechanism of carbon dioxide-assisted catalytic reaction was proposed (Scheme 2). First, the CO2 reacted with the un-coordinated amine of calcium 4-amino-3-hydroxybenzoate to form an intermediate A. Then the intermediate A activated DMF to form an intermediate B. The activated DMF reacted with the o-phenylenediamine to form an intermediate C, and the rest of the intermediate B returned to the intermediate A and participated in the further activation of DMF. The intermediate C lost a dimethylamine to form an intermediate D. Finally, the intermediate D fromed the product through the intermediate E. Because the intermediate A could not return to the original calcium 4-amino-3-hy-droxybenzoate. Hence, the XRD pattern of the used catalyst was no longer the original pattern of calcium 4-ami-no-3-hydroxybenzoate. Scheme 2. Possible reaction mechanism. Why CO2 could significantly improve catalytic activity of calcium 4-amino-3-hydroxybenzoate? According to the literature, CO2 easily interacts with amino groups forming carbamate,35 and many porous MOF materials with amino groups can adsorb CO2.36-38 In addition, more studies showed that a mixture of an alcohol and an amine was a good material for CO2 capture and enrichment.39,40 The un-coordinated amino and hydroxyl of calcium 4. Conclusions In this paper, the catalytic performance on the synthesis of the benzimidazole was compared. The catalysts investigated were phenol, o-aminophenol, benzoic acid, 4-amino-3-hydroxybenzoic acid and benzene-1,3,5-tri-carboxylic acid. We further verified that the proton was the key factor in the catalysis of this reaction. In order to improve the reusability of the catalyst, the catalytic perfor- Gao et al.: CO2 Improved Synthesis of Benzimidazole with ... 210 Acta Chim. Slov. 2021, 68, 205-211 mance of calcium benzoate, calcium benzene-1,3,5-tricar-boxylate and calcium 4-amino-3-hydroxybenzoate were evaluated, but they did not provide the desired results. When CO2 was added as weak acid to catalyze this reaction, the yield was very unsatisfactory. But an interesting result was obtained showing that CO2 could significantly improve the catalytic activity in the presence of calcium 4-amino-3-hydroxybenzoate. The cooperation of carbon dioxide and this salt increased the yield of product from 28% to 94%, and a possible mechanism was proposed to explain why cooperation of carbon dioxide and the salt could improved the catalytic activity. Acknowledgements We are grateful to the Program for New Century Excellent Talents in University (NCET-08-858) and the Natural Science Foundation of China (NSFC-21462029). Supplementary Material Supplementary (synthesis of the benzimidazole, IR, 1H and 13C NMR, UV-Vis) data associated with this article can be found, in the online version. Crystallographic data for structures reported in this paper in the form of CIF files have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication CCDC No.2011696. Copy of the data can be obtained free of charge from the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (Fax: +44-1223-336033; E-mail: de-posit@ccdc.cam,ac.uk). 5. References 1. F. P. Roudsari, M. Seddighi, F. Shirini, H. Tajik, ChemistrySe-lect 2019, 4, 6382-6389. DOI:10.1002/slct.201900806 2. I. H. Biswas, S. Biswas, M. S. Islam, S. Riyajuddin, P. Sarkar, K. Ghosh, S. M. Islam, New J. Chem. 2019, 43, 14643-14652. DOI:10.1039/C9NJ03015H 3. M. Schou, C. Halldin, J. Labelled Compd. Rad. 2012, 55, 460462. DOI:10.1002/jlcr.2972 4. M. Asif, Chem. Methodol. 2019, 3, 620-631. DOI: 10.33945/SAMI/ CHEMM.2019.6.1 5. S. 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Vaidhyanathan, S. S. Iremonger, K. W. Dawson, G. K. H. Shimizu, Chem. Commun 2009, 5230-5232. D0I:10.1039/b911481e 38. X. Wang, H. Li, X.-J. Hou, J. Phys. Chem. C 2012, 116, 1981419821. D0I:10.1021/jp3052938 39. B. Zhao, L. Liu, X. Yang, C. Liu, F. Sha, J. Zhang, Phys. Chem. Liq. 2016, 1-16. D0I:10.1080/00319104.2016.1269176 40. L. Zhao, Q. Li, L. Ma, C. Liu, F. Sha, J. Zhang, J. Mol. Liq. 2017, 241, 374-385. D0I:10.1016/j.molliq.2017.05.144 Povzetek V tem prispevku predstavljamo raziskavo sinteze benzimidazola z reakcijo DMF in orio-fenilendiamina. Med preučevanjem učinkovitosti različnih možnosti smo ugotovili, da so 4-amino-3-hidroksibenzojska kislina, benzojska kislina in benzen-1,3,5-trikarboksilna kislina uspešni katalizatorji za to reakcijo. Kalcijev 4-amino-3-hidroksibenzoat se je v prisotnosti CO2 izkazal kot še posebej učinkovita možnost, saj se je zaradi sinergističnega učinka med CO2 in kalcijevo soljo 4-amino-3-hidroksibenzojske kisline izkoristek benzimidazola povečal z 28 % na 94 %. © 0 Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Gao et al.: CO2 Improved Synthesis of Benzimidazole with ... DOI: 10.17344/acsi.2020.6379 Acta Chim. Slov. 2021, 68, 212-221 /^creative ^commons Scientific paper Catecholase-Like Activity and Theoretical Study in Solid State of a New Ru(III)-Schiff Base Complex Niladri Biswas,1 Sandeepta Saha,1,2 Ennio Zangrando,3 Antonio Frontera4 and Chirantan Roy Choudhury^* 1 Department of Chemistry, West Bengal State University, Barasat, Kolkata-700126, India 2 Sripur High School, Madhyamgram Bazar, Madhyamgram, Kolkata - 700130, India 3 Department of Chemical and Pharmaceutical Sciences, University of Trieste, Via L. Giorgieri 1, 34127 Trieste, Italy 4 Departament de Química, Universitat de les Illes Balears, Crta. De Valldemossa km 7.5, 07122 Palma de Mallorca, Baleares, Spain * Corresponding author: E-mail: crchoudhury2000@yahoo.com Tel: + 91-9836306502; Fax: +91-33-2524-1577 Received: 09-13-2020 Abstract A new ruthenium(III) complex of molecular formula [Ru(PPh3)Cl2(L)] (1) has been synthesized using the Schiff base ligand obtained from 5-chlorosalicylaldehyde and N,N-dimethylethylenediamine and characterized by FT-IR, UV-Vis, cyclic voltammetry and single crystal X-ray structural analysis. The metal ion exhibits a slightly distorted octahedral environment where the chelating Schiff base ligand contributes with its NNO donor set. The coordination geometry around the Ru(III) ion is completed by a PPh3 ligand and two chloride anions, and the charge balance is assured by the phenoxo oxygen of the Schiff base. With the aim to analyse the energy related to the halogen bonding interactions in solid state, a theoretical study has been performed on complex 1, by using the MEP and NCl plot computational tools. Furthermore, complex 1 shows catecholase-like activity in conversion of the model substrate 3,5-di-ferf-butylcatechol (3,5-DTBC) to the corresponding 3,5-di-ferf-butylquinone (3,5-DTBQ) under aerobic condition. The parameters regarding the enzymatic kinetics have been evaluated from the Lineweaver-Burk plot using the Michaelis-Menten approach of enzyme catalysis. A significant high T.O.N value (2.346 x 103 h-1) indicates that complex 1 has a very good catalytic efficiency towards 3,5-DTBC. Keywords: Ru-(III) complex • Schiff base • crystal structure • halogen interaction • catecholase-like activity 1. Introduction In last decade, coordination chemistry of transition metal ions with Schiff bases has evolved as an an area of active research.1,2 Schiff bases affect electronic factors of the metal centres, stabilizing different oxidation states, address the performance of complexes, which can acquire a variety of suitable properties like that to act as homogeneous and/or heterogeneous catalysts.3-6 Moreover, design of metal complexes having catalytic activity may be helpful to elucidate the mechanistic aspects of biochemically important metalloenzyme reactions. In fact, structurally simpler and more robust metal complexes can mimic catalytic oxidation of 3,5-di-tert-butylcatechol to quinone, as well as hydrolytic reactions (catecholase and phosphatise activity, respectively).7 In the last decade a variety of ruthenium complexes, that provide great interest specially for their catalytic activity, has been developed.8-10 Ruthenium metal complexes may be also relevant as therapeutic agents and one of these has successfully entered advanced clinical trials.11 In fact the energy barrier for the oxidation state change from Ru(III) to Ru(II) inside the cell is very low and due to the larger coordination number with respect to platinum-(II), ruthenium-(III/II) can form complexes with a number of elements having different electronegativity as well as chemical hardness.11-14 Till date, there are plenty of reports on Ru(II/III) compounds with bidentate ligands but design of such complexes with somewhat more rigid, tridentate Schiff bases ligands are rarely found.15 Intermolecular interactions, in addition to their structural role, influence the physical and chemical properties of crystalline solids.16,17 The advancement of these features has been one of the priorities of crystal engineering, a budding interdisciplinary field of research in modern Biswas et al.: Catecholase-Like Activity and Theoretical Study in Solid Acta Chim. Slov. 2021, 68, 212-221 213 Scheme 1. Synthesis of the Schiff base ligand (HL) and complex 1. chemistry with interest in the rational design of functional molecular solids.18 Even though hydrogen bonding and coordination bonds still remain at the forefront of crystal engineering strategies, Other interactions have received escalating interest over recent years, markedly, halogen bonds,19 non-classical hydrogen bonds,20 n-n interactions,21 lp-n interactions22 nitroso—nitroso interactions23 along with halogen—halogen contacts.20d-f Intermolecular interactions involving halogen substituents, mostly chlorine, have been observed to favour crystal formation, providing a tool for crystal engineering study.24 Keeping this in mind, and taking into account that triphenylphosphine transition metal complexes are very good candidate for catalytic organic transformations, we report here the synthesis (Scheme 1) of a ruthenium(III) complex, [Ru(PPh3)Cl2(L)] (1), which was characterized by different spectroscopic techniques, cyclic voltammetry. The single crystal X-ray structural analysis revealed interesting crystalline supra-molecular interactions. In addition, the complex has also been evaluated as model system for catecholase-like activity. 2. Experimental Section 2. 1. Materials All starting chemicals and solvents used in this study were of reagent grade and was used as purchased without further purification. Tris(triphenylphosphine)rutheni-um(II) dichloride [Ru(PPh3)3Cl2], 5-chlorosalicylalde-hyde, 3,5-di-tert-butylcatechol (3,5-DTBC) and tetrabu-tylammonium perchlorate (TBAP) were purchased from Sigma-Aldrich, USA and N,N-dimethylethylenediamine from Spectrochem. Spectroscopic grade methanol and dimethyl sulfoxide (DMSO) were obtained from E-Merck, India. 2. 2. Physical Measurements FT-IR spectrum of complex 1 was measured in the range 400-4000 cm-1 in solid KBr pellets by using a Perkin-Elmer SPECTRUM-2 FT-IR spectrophotometer. The UV-Vis spectra were recorded using a Perkin-Elmer Lambda-35 UV-Vis spectrophotometer in Tris-HCl buffer medium at 300K. Elemental analyses were performed with a Perkin-Elmer 2400 II elemental analyzer. Electrochemical experiment was carried out with three electrode configuration using a CH 660E cyclic voltammeter in Tris-HCl buffer medium. Saturated calomel electrode (SCE) as reference, Pt wire-electrode as counter electrode and glassy carbon electrode as working electrode were used as three electrode system with tetrabutylammonium per-chlorate (TBAP) as supporting electrolyte at a scan rate of 50 mV sec-1. Electrochemical data were recorded under a dry nitrogen environment. Nitrogen gas was passed into the sample solution at a constant rate for 1 minute. 2. 3. Synthetic Procedures 2. 3. 1. Synthesis of Schiff Base Ligand (HL) The Schiff base ligand was prepared by the standard procedure mentioned in the literature.25a-c 5-chlorosalicy-laldehyde (0.783 g, 5 mmol) in methanol medium was carefully added to a methanolic solution of N,N-dimethylethyl-enediamine (0.538 mL, 5 mmol). The colour of the solution turned light yellow and the reaction mixture was allowed to reflux for one hour and then cooled at room temperature (Scheme 1). The synthesized Schiff base ligand was used for complex preparation without further purification. 2. 3. 2. Synthesis of Complex [Ru(PPh3)Cl2(L)](1) Solid [Ru(PPh3)3Cl2] (1.92 g, 2 mmol) was added to 30 mL methanolic solution of the Schiff base ligand (2 Biswas et al.: Catecholase-Like Activity and Theoretical Study in Solid ... 214 Acta Chim. Slov. 2021, 68, 212-221 mmol) followed by continuous stirring. After 6 hours of continuous reflux, shiny green coloured crystals were separated out (Scheme 1), collected by filtration, washed with diethyl ether and dried in vacuo. These crystals were used for the X-ray structural determination. Yield: 67% (0.256 g). Anal. Calc. Ffor [C29H29Cl3N2OPRu]: C, 52.73; H, 4.39; N, 4.24 %. Found: C, 52.65; H, 4.32; N, 4.18%. IR (KBr cm-1) 3436 (b), 1631, 696 and 744 (s), 496, 481 and 1530 (m). Electronic spectrum in Tris-HCl buffer medium, X max (nm): 260 (n^n*), 360 (n^n*) and 683 (d^d). ESI-MS: (m/z) [found (calcd)]: 661.0511 (659.93). 2. 4. Single Crystal X-ray Diffraction Study Data collection of complex 1 was performed at the X-ray diffraction beamline (XRD1) of the Elettra Synchrotron of Trieste (Italy), with a Pilatus2M image plate detector. Complete dataset was collected at 100 K with a monochromatic wavelength of 0.700 A with the rotating crystal method. The crystal was dipped in N-paratone and mounted on the goniometer head with a nylon loop. The diffraction data were indexed, integrated and scaled using XDS.26 The structure was solved by direct methods27 and successive Fourier analysis and the refinement was performed by the full-matrix least-squares methods based on F2 implemented in SHELXL-2014.27 Anisotropic thermal motion was allowed for all non-hydrogen atoms, and H atoms, at calculated positions, were included in the final cycles of refinement. All calculations were done with the Wingxpackage Version 20 1 3.3,28 and the molecular graphics were prepared by using Cameron29 and DiamondVer 3.2k30 programs. Relevant crystallographic data and structure refinement parameters are summarized in Table T1 (Supplementary information). 2. 5. Theoretical Methods The geometry of the complex included in this study was computed at the M06-2X-D/def2-TZVP level of theory using the crystallographic coordinates. We have used the GAUSSIAN-09 program31 was used for the calculations of the interaction energies and the molecular electrostatic potential (MEP) surfaces (at the same level of theory). We have also used the Grimme's dispersion32 correction being this adequate for the evaluation of noncovalent interactions. The NCI plot33 isosurfaces have been used to characterize non-covalent interactions. These correspond to both favorable and unfavorable interactions, as differentiated by the sign of the second density Hessian eigen value and illustrated by the isosurface color. The color scheme is a red-yellow-green-blue scale, where red indicates p+cut (repulsive) and blue for p-cut (attractive). The Gaussian-09 M06-2X-D/def2-TZVP wave function has been used to generate the NCI plot. 2. 6. Catecholase Activity Study The catecholase-like activity of complex 1 has been investigated under aerobic condition at room temperature by using 3,5-di-tert-butylcatechol (3,5DTBC) as model substrate. Since complex 1 and the substrate are highly soluble in DMSO, the catalytic study was investigated in this particular solvent. The concentration of 3,5-DTBC was 100 times greater than that of the complex 1. The process was followed spectrophotometrically at intervals of 10 mins. in the range of 200-600 nm, and a significant increase of the 3,5-DTBQ concentration was observed by measuring its absorbance near 393 nm. For each set of catalytic reaction, initial rates were calculated and the rate versus concentration of substrate was determined according to the Michae-lis-Menten approach of enzymatic kinetics in order to get a Lineweaver-Burk plot.34-36 This methodology allowed to determine the Km, V_,ax and Lat values. 3. Results and Discussion 3. 1. Infrared Spectral Study IR spectrum of complex 1 (Fig. S1) showed a medium sharp band at 1631 cm-1, ascribed to the v(C=N) stretching, indicating the coordination of the azomethine nitrogen to the ruthenium(III) centre.37-39 A broad band found at 3436 cm-1 is due to the N-H stretching frequency. Well resolved bands, which appeared at 696, 744 and 1530 cm-1, are due to the stretching frequency of PPh3,40,41 while Ru-N and Ru-O stretching frequencies appeared at 496 and 581 cm-1, respectively. 3. 2. Electronic Spectral Study The electronic spectrum of complex 1, recorded in Tris-HCl buffer medium, displayed three absorption bands in the UV-Vis region (Fig. S2). The high intensity band at 260 nm, is assigned to intra ligand n—n*transition of the imine in coordinated Schiff base, while the low intensity band at 360 nm can be attributed to n—n transition of the azomethine group. In addition, low energy band has been observed at 683 nm, which can be assigned to d^d transition. In order to confirm the stability of complex 1, the UV-Vis spectral study was carried out for three successive days with same concentration of 1 in same medium, but no distinct change in the spectrum was observed. 3. 3. Cyclic Voltammetric Study The redox behaviour of complex 1 was studied by cyclic voltammetry by using a saturated calomel electrode (SCE) as reference. The ruthenium complex was found to be redox-active in the potential range from +2.0 to -2.0 V (Fig. S3), and the redox potential was examined by well-defined waves at 0.37 and 0.72 V for the oxidation, and at -0.64 V for the reduction process. Of these, the peak at +0.37 V (vs. SCE) can be attributed to the Ru(III)/Ru(IV) oxidation, while that at +0.72 V can be assigned to the Schiff base oxidation. The irreversible reduction peak at -0.86 V (vs. SCE) is associated to the Ru(III)/Ru(II) redox couple. Biswas et al.: Catecholase-Like Activity and Theoretical Study in Solid ... Acta Chim. Slov. 2021, 68, 212-221 215 3. 4. X-ray Crystal Structure Description The complex crystallizes in the monoclinic system, space group P21/c. The molecular structure of the complex is displayed in Fig. 1, while a packing diagram is shown in Fig. S5. (Supplementary information). All the relevant crystallographic data and structure refinement parameters for the complex reported are summarized in Table T1 (Supplementary information). A selection of bond distances and angles is collected in Table T2 (Supplementary information). The asymmetric unit consists of one complete complex molecule which is built by the tridentate Schiff base ligand meridionally coordinated, the triphenylphosphine molecule and two chlorides mutually located in trans position. The coordination geometry of the complex can be better described as distorted octahedral where the equatorial plane is formed by the donor atoms O1, N1, and N2 donors of the chelat-ing Schiff base (forming a six- and five-member ring) along with the P donor of the phosphine moiety. The coordination bond lengths Ru-O1, Ru-N1 and Ru-N2 of the chelating ligand are 1.9757(13), 2.0384(15) and 2.2141(16) Â, respectively, where the Ru-N bond values differ due to the different hybridization of the N atoms (sp2 vs. sp3). Finally the coordination geometry is completed by the phosphine with Ru-P distance is of 2.4058 (5) Â and two chlorides having comparable Ru-Cl bond length of 2.3485(5) and 2.3569(5) Â. Thus the +3 charge of the metal atom is satisfied by the chloride anions and the phenoxo oxygen (O1) of the Schiff base ligand. The distortion in the octahedron are well described by the bond angle values and the larger deviation from the ideal geometry is shown by the N2-Ru-P1 angle of 102.22(4)°, that appears induced by steric requirements. All the coordination distances (Table T2, Supplementary information) agree well with those reported for similar Ru(III) complexes.41-44 3. 5. Theoretical Study The crystal packing of 1 shows the complexes associated in pair through a symmetry center where C-Cl bond of the aromatic ring points towards one chloride ligand of the symmetry related complex. This halogen-halogen like-interaction can be inferred taking into consideration the anisotropy of the charge density around the Cl atom. The theoretical study, using the MEP and NCI plot computational tools, is devoted to analyse the energy associated to this halogen interaction and to characterize it. First the MEP surface of compound 1 was computed. It is worth mentioning that the more negative values of MEP are located at the chloro ligands (-45 kcal/mol). Since using the large scale given by the maximum and minimum MEP values the anisotropy around the chlo- Fig. 2. MEP surface (isodensity = 0.002 a.u.) of compound 1. The values are selected points of the surface are indicated. Negative values are in red and positive in blue colour. Fig. 1. ORTEP view of complex 1 with displacement ellipsoids drawn at 50% probability level (labels of C atoms not shown for clarity). Spin density Fig. 3. Spin density plot of compound 1. Isodensity = 0.004 a.u. rine cannot be appreciated, so the MEP around this atom using a reduced scale is represented in Fig. 2. As a result, the a-hole, of moderate energy (+5 kcal/mol), appears at the extension of the C-Cl bond, while the typical negative Biswas et al.: Catecholase-Like Activity and Theoretical Study in Solid ... 216 Acta Chim. Slov. 2021, 68, 212-221 belt around the Cl atom is larger in absolute value than the a-hole (-15 kcal/mol). So, the MEP analysis confirms that the Cl—Cl like-like interactions are electrostatically favoured. The spin density plot (Fig. 3) of compound 1 was also computed in order to analyse the location of the unpaired electron that is located, as expected, at the Ru metal centre with some little delocalization onto the atoms directly bonded to it. Fig. 4a shows a detail of the crystal packing of 1 with formation of the self-assembled dimer, where two symmetrically equivalent Cl—Cl interactions are established in addition to an antiparallel n-n interaction. This arrangement also designates a double Cl—n interaction, being the Cl located over one C atom of the aromatic ring at a distance (3.63 A)slightly longer than the sum of the Van der Waals radii (ZrvjW = 3.45 A). It should be mentioned that the Cl—Cl distance is also slightly longer (3.86 A) that the sum of Van der Waals radii (&vdW = 3.50 A). Therefore, by using DFT calculations, the dimerization energy of the pair of complexes were computed in solid state, and it was found to be moderately strong (AE1 = -12.0 kcal/mol) and accounts for the n-n, Cl—Cl halogen and other long range Van der Waals interactions (Fig. 4b). In an attempt to evaluate the contribution of the halogen bonding interactions, an additional model was used where the chloro ligands have been replaced by two hydrides (indicated by small arrows in Fig. 4c). As a consequence, the reduced interaction energy (AE2 = -11.1 kcal/mol) determines the contribution of the n-n interaction and indicates that both the Cl—Cl interactions are very week (-0.9 kcal/mol), as expected taking into consideration the small MEP value observed at the a-hole (Fig. 2). The "Non-Covalent Interaction (NCI) plot" was computed in order to characterize the interactions in the dimer of 1. The NCI plot is considered as an intuitive visualization index which enables the identification of non-covalent interactions easily and efficiently. In addition it is convenient for host-guest interaction analysis since it clearly shows the interacting molecular regions. Fig. 4d shows the representation of the NCI plot, where the colour scheme is shown in red-yellow-green-blue scale: red means repulsive and blue stands for attractive interactions. Yellow and green surfaces correspond to weak repulsive or weak attractive interactions, respectively. As noted, the halogen bonds are characterized by the presence of a small green isosurface that is located between the Cl atoms, confirming the existence of the interaction. The NCI plot also shows the presence of a green and more extended isosurface between the n-systems of the ligands, indicating the existence of n-interactions that are also main contributor to the formation of the self-assembled dimer. Finally, the analysis reveals that the green isosurface extends in between the pair of the Cl atom and the aromatic-system, thus unequivocally confirms the existence of the Cl—n interactions. 3. 6. Oxidation of 3,5-di-tert-butylcatechol (Kinetics Studies) The catalytic conversion of 3,5-DTBC to 3,5-DTBQ (Scheme 2) has already been investigated by a number of a) 1, X-Ray 3.86 AEi =-12.0 kcal/rnol Fig. 4. (a) Detail of the crystal packing of 1. (b,c) Theoretical models used to evaluate the interaction energies. Distances in A. (d) NCI surface of the assembly present in compound 1. The gradient cut-off is s = 0.35 a.u., and the color scale is -0.04 0.05). While the Al level of group A increased compared to all other groups (p < 0.001, p < 0.05), Al level of CM+A group decreased compared to A group (p 0.05). Mn levels of A and CM+A groups decreased compared to C and CM groups. Table 1: Liver mineral levels (ppm) Minerals C CM A CM+A Fe 103.20±3.49 100.55±3.00x 116.84±2.38a 102.25±2.2x Zn 25.79±1.66 25.95±0.66 24.43±1.26 29.16±1.22 Mg 238.06±6.09 230.34±5.11 232.74±9.28 240.56±1.93 Al 29.98±3.64 28.75±3.08z 94.14±5.44c 69.07±6.33cx Mn 2.90±0.07 2.94±0.06x 2.55±0.04a 2.65±0.09a Cu 5.37±0.76 4.69±0.23x 7.32±0.53a 5.29±0.40x Comparison with group C. a: p <0.05, b: p <0.01, c: p <0.001 Comparison with group A. x: p <0.05, y: p <0.01, z: p <0.001 Liver MDA, GSH and enzyme activity levels are shown in Table 2. Group A's MDA level was found to be increased compared to other groups (p <0.001; p <0.05). GSH level of group A decreased compared to other groups (p <0.05; p <0.001). Ces enzyme activity levels of A and CM + A groups decreased compared to C and CM groups (p <0.05). GST enzyme activity level of A group decreased compared to other groups (p <0.05). Table 2: MDA, GSH and enzyme activity levels of the liver. GST and Ces activities are expressed as nmol/min/mg protein ± mean standard error. GSH and MDA levels are expressed as nmol GSH/mg protein ± mean standard error, and nmol MDA/g wet weight tissue ± mean standard error, respectively. Parameters C CM A CM+A MDA 45.02±2.54 48.05±3.41z 78.75±5.85c 58.56±3.65ax GSH 88.45±3.02 96.41±4.23z 73.75±4.05a 86.32±5.45x Ces 1.78±0.06 1.81±0.05x 1.47±0.02a 1.48±0.09a GST 31.41±0.76 33.80±1.67x 24.34±1.04a 32.20±1.90x Comparison with group C. a: p <0.05, b: p <0.01, c: p <0.001 Comparison with group A. x: p <0.05, y: p <0.01, z: p <0.001 Ozkaya and Turkan: Protective Effects of 3-benzoyl-7-hydroxy Coumarin ... Acta Chim. Slov. 2021, 68, 222-228 225 4. Discussion Al is a metal commonly found in air, soil and water. This metal is widely used in industry, agriculture, transportation and daily lives of people. Al, absorbed by the gastrointestinal tract, accumulates in the liver and other organs. In many studies, Al has been reported to cause hepatotoxicity, nephrotoxicity and cardiotoxicity.30,31 In the present study, we observed that Al, Fe and Cu concentrations increased significantly in the liver of rats that were given AlCl3 intraperitoneally. In many experimental studies, it has been reported that the concentration of Al increases in rat liver exposed to Al.32,33 Al is a non-re-dox metal, and many studies have reported that Al affects concentrations of other metal ions.34,35 Yang et al. reported that the Al, Fe, Cu concentrations were increased in rat liver overloaded with Al.19 In addition, it has been reported that Fe levels increase in rat liver exposed to Al.36 Al is known to be linked to transfer protein carrying Fe3+, thereby reducing the binding of Fe2+. The increase in free intracellular Fe2+ causes peroxidation of membrane lipids, thereby causing membrane damage.37 Chronic Cu2+ and Fe3+ overload causes accumulation of Cu2+ and Fe3+ in the liver and kidney, which causes Fe2+ overload. Al has been reported to increase Fe-induced oxidative stress injuries.38-40 In this study, liver Mn concentration of the A and combination groups decreased compared to the C group. In some studies, it has been reported that the Mn concentration decreases in rat liver exposed to Al.41,42 In our study, we observed that the Zn and Mg concentration levels of group A decreased relatively compared to group C. Trace elements have important physiological functions in metabolism, and their abnormal usability causes serious negative effects. Trace elements, such as Cu, Zn, Fe, Mn, and Mg are essential co-factors for antioxidant enzymes. These enzymes are very important in preventing the oxidation of nucleic acids, lipids or proteins.43 In our study, the level of MDA increased in rat liver treated with AlCl3. Also, GSH levels and GST and Ces enzyme activity levels decreased in antioxidant defence system. Based on these results, we believe that increased oxidative stress with AlCl3 and reduction of enzymes involved in antioxidant defence could be caused by peroxidation damages. The glutamyl-cysteine-synthetase enzyme carries out the synthesis of the GSH molecule in the liver. In addition, it was found that enzymes, such as glucose-6-phosphate dehydrogenase and NADP-isocitrate dehydrogenase control the synthesis of the GSH molecule in the liver. In many studies, the inhibition effect of AlCl3 on enzymes controlling GSH biosynthesis has been reported. AlCl3 has pro-oxidant activity, and causes changes in the activity of antioxidant enzymes. In similar studies, while AlCl3 increased the level of MDA in rat liver, it decreased GSH level and the activity of Glutathione perox-idase (GSH-Px), superoxide dismutase (SOD) and cata-lase.44-46 In another study, it was reported that activities of catalase, GPx, SOD, and GST enzyme decreased in rat liver tissues with the toxic effects of Al.47 In the present study, it was observed that the Al, Fe and Cu levels increased in the liver of the rats exposed to oxidative stress with AlCl3, and the 3-benzoyl-7-hydroxy coumarin substance decreased the levels of these metals. In addition, it was determined that the level of MDA, which is the high lipid peroxidation product of AlCl3, was reduced by 3-benzoyl-7-hydroxy coumarin. It was observed that 3-benzoyl-7-hydroxy coumarin substance improved the GSH level and GST enzyme activity, which are effective in the antioxidant system. In this study, Ces enzyme activity decreased due to the toxic effect of AlCl3. Also, in the combination group, the activity of Ces enzyme decreased with the effect of 3-benzoyl-7-hydroxy coumarin. The results of the present study show that 3-ben-zoyl-7-hydroxy coumarin exerted antioxidant properties against the pro-oxidant effects of AlCl3. We found that 3-benzoyl-7-hydroxy coumarin improves both Al, Fe and Cu concentrations in the liver, and positively affects the antioxidant system. The antioxidant activity of coumarin is due to its ability to scavenge free radicals and chelate metal ions. The antioxidant capacity of many coumarin substances has been reported to be due to its molecular structure.48 The number and location of hydroxy, acetoxy and methoxy groups in the structure of coumarin affect the antioxidant capacity. These functional groups are very important for detoxification functions of coumarins.48 In a similar study, the hepatoprotective effect of four different coumarins (coumarin (1,2- benzopyran), esculetin (6,7-dihydroxycoumarin), scoparone (6,7-dimethoxycou-marin), and 4-methylumbelliferone (7-hyroxy-4- methyl) were investigated against hepatic damage caused by CCl4 in rat liver, and it was reported that two coumarins (escu-letin and scoparone) had hepatoprotective effects. It has been explained that antioxidant properties of coumarins are attributed to their chemical structure.49 In another study, the ameliorative effect of coumarin (1,2-benzopy-ron) was investigated in rats with ferric nitrilotriacetate (Fe-NTA)-induced renal oxidative stress. Fe-NTA increases the level of MDA with the effect of oxidative stress while decreasing the GSH level and GST enzyme activity. However, the ameliorative effect of the coumarin (1,2-ben-zopyrone) molecule has also been reported.48 Many studies have shown that coumarin has an antioxidative effect against various oxidative stress agents.50-52 Atmaca et al. (2011) suggested that coumarin and its derivatives showed antioxidative effects against rat liver damage induced by carbon tetrachloride by testing various antioxidant bio-markers.49 Various studies have shown that coumarin derivatives, such as fraxin, esculetin, grandivittin, agacyllin, aegolinol benzoate and osthol have free radical scavenging effects.8 In our study, we revealed the relationship between the toxic effect of Al on the liver and the detoxification enzymes such as Ces and GST. Al reduced GST and Ces en- Ozkaya and Turkan: Protective Effects of 3-benzoyl-7-hydroxy Coumarin 226 Acta Chim. Slov. 2021, 68, 222-228 zyme activities in rat liver. We could not find studies related to Ces enzyme activity to compare our results; however, there are reports indicating that Al decrease GST enzyme activity. In our previous study, we had found that Ces and GST enzyme activities decreased in liver of rats given lead acetate.53 In this study, although we saw the ameliorative effect of 3-benzoyl-7-hydroxy coumarin in GST enzyme activity, we also found that this had no ameliorative effect on Ces enzyme activity in the combination group. It may be considered that the ineffectiveness of coumarin on Ces activity depends on the dose applied. Perhaps Ces activity would have been affected if different doses of coumarin were tried. We believe that more in vitro and in vivo studies are needed regarding the effect of coumarin on Ces activity. In the study conducted by Khan et al., it was reported that coumarin applied in increasing doses against the oxidative stress induced by Fe-NTA has an ameliorative effect on GST enzyme activity at high dose.48 In advanced pharmacological studies, we believe that this study is important for the effects of coumarin-derived substances on Ces enzyme activities. 5. Conclusion In conclusion, we observed that AlCl3 induces oxi-dative stress in rat liver. We have determined ameliorative effects of 3-benzoyl-7-hydroxy coumarin on deleterious effects of aluminium on the antioxidant enzymes, Fe, Cu and Al levels. However, we observed that the 3-benzo-yl-7-hydroxy coumarin did not ameliorate the effect of Ces enzyme activity. In future studies, we believe that this study is important for investigating the effects of toxic substances and coumarin derivatives on Ces enzyme activity. 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Slov. 2021, 68, 222-228 Povzetek V tej raziskavi so so bili proučeni učinki molekule 3-benzoil-7-hidroksi kumarina na minerale in antioksidativne encime v jetrih podgan, izpostavljenih oksidativnemu stresu z aluminijevim kloridom (AlCl3). Odrasli samci podgan Wistar albino so bili razdeljeni v štiri skupine, poimenovane kontrola, kumarin, AlCl3 in kumarin + AlCl3. Kumarin v odmerku 10 mg/kg in AlCl3 v odmerku 8,3 mg/kg sta bila aplicirana 30 dni vsak drugi dan. V skupini AlCl3 so se v primerjavi s kontrolno skupino zvišale koncentracije malondialdehida (MDA), železa (Fe), aluminija (Al) in bakra (Cu), medtem ko so se ravni glutationa (GSH) in encimske aktivnosti glutation S-transferaze (GST) in karboksilesteraze (Ces) zmanjšala. V skupini kumarin + AlCl3 so se ravni MDA, Fe, Al in Cu z delovanjem kumarina zmanjšale v primerjavi s skupino Al-Cl3, medtem ko so se ravni GSH in aktivnost encima GST povečale. Glede na naše rezultate, AlCl3 povzroča oksidativni stres v jetrih podgan in verjamemo, da ima 3-benzoil-7-hidroksi kumarin blažilni učinek na antioksidativni encimski sistem in ravni Al, Fe ter Cu. © 0 Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Ozkaya and Turkan: Protective Effects of 3-benzoyl-7-hydroxy Coumarin ... DOI: 10.17344/acsi.2020.6419 Acta Chim. Slov. 2021, 68, 229-238 /^.creative o'commons Scientific paper Physico-Chemical Properties of the Pyrolytic Residue Obtained by Different Treatment Conditions of Meat and Bone Meal Marija Zupančič and Nataša Čelan Korošin* Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, 1000 Ljubljana, Slovenia * Corresponding author: E-mail: Natasa.Celan@fkkt.uni-lj.si Phone: +386 1 4798 524 Received: 09-25-2020 Abstract The depletion of phosphate rock reserves has led to the search for new, alternative and environmentally friendly products and processes. One of the safe and environmentally friendly sources of phosphate is animal bone char (ABC), the residue from the pyrolysis of meat and bone meal (MBM), a slaughterhouse waste material. The presented study investigated the physico-chemical properties of the residues (ABC) obtained from the pyrolysis of MBM under different treatment conditions. Two different end temperatures (600 °C and 1000 °C) and five different heating rates (5 °C min-1, 10 °C min-1, 20 °C min-1, 50 °C min-1 and 100 °C min-1) were used. The ABC samples obtained were characterised by X-ray powder diffraction, IR spectroscopy, elemental CHNS analysis and SEM/EDS analysis. The results showed the strong influence of both the pyrolysis end temperature and the heating rate on the morphology and chemical composition of the final products. Keywords: Bone char, meat and bone meal, natural hydroxyapatite, phosphate sources, pyrolysis, thermogravimetry 1. Introduction One of the main problems in many modern agricultural systems is the lack of sustainable development. The unauthorized use of chemicals, e.g. in phosphate fertilizers, has led to the eutrophication of waters, to the harming of many beneficial soil organisms and, as a result, to a negative impact on the level of biodiversity. The careful use of phosphate fertilizers leads to the better management of existing phosphorus sources and its continued circulation in nature. The existing fertilizer industry is based exclusively on non-renewable resources, i.e., a high grade phosphate ores that, according to one report, could be exhausted in as little as 60-80 years.1 Meat and bone meal (MBM) is a by-product of the rendering industry. Due to the medium-high heating value of MBM, the pyrolysis, gasification, combustion and co-combustion processes were studied as an environmentally friendly alternative to landfilling.2-12 During the controlled pyrolysis process raw MBM decompose into number of useful products i.e. gas, tar, oil and char, that make it more attractive than other thermal processes. The speed and the extent of the decomposition depend on process parameters such as the temperature in the reactor, the heating rate of the biomass, the pressure and the composition of the raw material.13 The solid residue of pyrolysed meat and bone meal, animal bone char (ABC), is a granular material that, in contrast to the biochar from plant biomass, contains a much smaller percentage of organic carbon (about 10 %). For this reason, it cannot be categorized as biochar, but as pyrogenic carbonated material.14,15 The carbon, which remains trapped in the structure after pyrolytic decomposition of MBM, increases the specific surface of ABC compared to crystalline hydroxyapatite (HA).16 Due to its high phosphate content, ABC can be used as a source for P-fertilizer production or as a stabilizing agent in the remediation of potentially toxic elements (PTE) contaminated sites.17-20 A few studies investigated the influence of treatment conditions of pyrolysis of bone meal on the properties of the bone char obtained.21-23 In contrast to the pyrolysis of bone meal, the pyrolysis of MBM poses a much greater challenge. Despite many studies that have investigated Zupančič and Čelan Korošin: Physico-Chemical Properties of the Pyrolytic Residue 230 Acta Chim. Slov. 2021, 68, 229-238 the pyrolysis process of MBM, data on the influence of experimental conditions on the properties of MBM pyrolysis products are seldom available. In order to improve the knowledge in this field, the aim of this work was to evaluate the influence of the MBM pyrolysis end temperature and the different heating rates on physico-chemical properties and the composition of the solid residue, ABC, focusing on a detailed analysis of the morphology and composition of the ABC samples. The pyrolysis of the MBM sample was performed at five different heating rates (from 5 °C min-1 to 100 °C min-1). Due to the large amount of organic matter in MBM the lowest pyrolysis end temperature examined was chosen at 600 °C. The characteristics of these residues were compared with residues obtained at MBM pyrolysis end temperature of 1000 °C, where the release of CO2 from carbonates is expected to be completed.24 2. Experimental MBM sample category 3 (low-risk material from the production of goods intended for human consumption using slaughtered animals not affected by any sign of diseases that are transmissible to humans or other animals) was obtained from a local slaughterhouse, where non-harmful animal remains are ground up, heated with steam for sterilization and with the animal fats squeezed out to obtain meat and bone meal. The sample was first dried for 24 hours at 100 °C, ground with a planetary mill (Retsch PM100, Germany) into fine powder and sieved through a 0.250 mm test sieve (ISO 3310-1, Retsch, Germany). The MBM was pyrolysed in a Mettler Toledo (CH) TGA/SDTA 851e LF1100 system for Thermal Analysis. Subsamples of 20 mg were inserted into the alumina crucible (150 ^L) and heat treated in an argon atmosphere (99.999 % high purity by Linde, 100 mL min-1). After an initial 30 min of isothermal conditioning at 25 °C the samples were dynamically heated up to 600 °C or 1000 °C to obtain the samples group T1ABC or group T2ABC (see Table 1 for sample abbreviations), respectively. We used five different heating rates (5 °C min-1, 10 °C min-1, 20 °C min-1, 50 °C min-1 and 100 °C min-1) for the samples for both end temperatures. The baseline measured with an empty alumina crucible was subtracted for all the measurements. In order to achieve reproducibility of the results, a small particle size, which is limited by a high content of fat components and a sufficiently high initial mass of the sample, was required, as reported elsewhere.6 To evaluate the repeatability, three replications of each thermal process, under the same conditions, were performed. The one-way analysis of variance (ANOVA) was used to determine the statistically significant differences between the samples. To determine the ash content in the MBM and T1ABC samples the TGA method, analogous to DIN 51719, was used.14 Approximately 5 mg of sample in a 150 ^L alumina crucible was measured in the TGA/DSC1 Mettler Toledo (CH) thermoanalyser using the following multi-segment thermal program: (1) heating with a rate of 5 °C min-1 to 106 °C in a nitrogen atmosphere (99.999 % high purity by Linde, 100 mL min-1), (2) hold in a nitrogen atmosphere at 106 °C for 30 min (100 mL min-1), (3) temperature increase with a 5 °C min-1 heating rate to 550 °C in an oxygen atmosphere (99.999 % high purity by Linde, 100 mL min-1), and (4) hold in an oxygen atmosphere at 550 °C for 60 min (100 mL min-1). The hygroscopic moisture content and the ash content were calculated from the mass loss up to the end of the second step and from the mass loss up to the end of the fourth step of the program run, respectively. The baseline measured with an empty alumina crucible was subtracted for all the measurements. For the evolved gases determination, the thermoanalyser was coupled to a Balzers Thermostar Mass Spectrometer (2.4 ■ 10-6 mbar vacuum) via a 75 cm long capillary heated at 190 °C. The initial sample mass was 2.9 mg. The semi-total concentrations of the elements in the MBM and T1H005 (bulk sample of replicates) were determined after aqua regia digestion. To an amount of 0.200 g ± 0.005 g of the sample in a PFA digestion vessel (561B, Savillex, Minnesota, USA) 3 mL of HNO3 (>69.0 %, TraceSelect, Fluka) and 9 mL of HCl (37 %, TraceSelect, Fluka, Sigma-Aldrich Chemie) were added, left in the loosely closed vessel in a fume hood overnight, then the vessel was tightly closed and the sample was digested at a temperature of reflux on a hot plate for 8 hours. The cold contents were filtered through a 0.45 ^m membrane filter and diluted to 30 mL with Milli-Q water. The samples were digested in triplicate, including three blank samples. The concentration of metals in the digested diluted samples was analysed with ICP-MS Agilent 7500ce (Cr, Ni, Cu, Zn, As, Mo, Cd and Pb) and Varian 240 AA system (Fe, Na, K, Ca and Mg). The limit of detection (LOD) was calculated as the concentration corresponding to three times the standard deviation (35, N = 3) of the blank determinations. Total P content in digested diluted samples were determined according to SIST EN ISO 6878:2004 (Water quality - Determination of phosphorus - Ammonium molybdate spectrometric method). The content of C, H, N and S in the raw MBM sample and the ABC samples obtained after the pyrolysis of the MBM sample up to 600 °C (T1ABC samples) was determined with a Perkin Elmer Elemental Analyser Series II CHNS/O. The inorganic carbon content was determined according to DIN 51726.14 The organic carbon content (Corg) in the samples was derived from the total carbon content minus the inorganic carbon content. A Perkin Elmer Spectrum 100 spectrometer was used for the FTIR spectroscopy of the MBM and ABC samples. The analyses of the samples were performed with a single reflection monolithic diamond ATR (Specac's Golden Gate ATR) in the wavenumber range from 600 cm-1 to Zupančič and Čelan Korošin: Physico-Chemical Properties of the Pyrolytic Residue ... Acta Chim. Slov. 2021, 68, 229-238 231 4000 cm-1 (background correction, resolution 2 cm-1, 10 scans for each spectrum). The mineralogical properties of the ABC samples were examined with a PANalytical X'Pert PRO MPD powder diffractometer using Cu Ka X-rays (wavelength of 1.5406 A). The measurements were made in the 20 range 5-80° with a step interval of 0.033°. The results were evaluated with the Crystallographica Search-Match program. SEM/EDS analyses of ground ABC samples on adhesive carbon tape were used to investigate the morphology and elemental composition of the samples. The Zeiss ULTRA Plus field emission scanning electron microscope was equipped with an energy dispersive spectrometer (EDS Oxford X-Max SDD 50 mm2 106 detector) and INCA 4.14 X-ray microanalysis software. Before analysis, the samples were coated with Au/Pd (80:20). SEM images were taken at an acceleration voltage of 5 kV and a working distance of 5.5 mm with the SE detector, while the elemental analysis of the particles was performed by EDS at 20 kV. 3. Results and Discussion 3. 1. Pyrolysis of Meat and Bone Meal The dynamic TGA measurements of the MBM pyro-lysed up to 600 °C and 1000 °C show (Figure 1) a similar course for the samples heated at all the heating rates for the observed time period. The mass loss of the samples pyrolysed up to 600 °C varied between 57.6 % and 59.7 % (Figure 1a, Table 1) with relative standard deviations of the replicates being between 0.76 % and 1.75 %. Although there are statistically significant differences (p < 0.05) in the mass losses of the samples with different heating rates, a weak correlation between the heating rates and the mass losses was observed. An additional step can be observed in the curves heated up to 1000 °C (Figure 1b), resulting in a total mass loss of at most 71.4 % and 71.5 % for the heating rates of 5 °C min-1 and 10 °C min-1, respectively. With the increased heating rate, the mass loss of the sample diminishes, so that at a heating rate of 100 °C min-1 it is only 66.6 % (Table 1). We can conclude that at higher heating rates there was not enough time for some of the thermal processes to be completed and therefore certain components were not released from the system. The same curves on a common temperature scale (Figure 2) indicate that the first step, i.e., dehydration, occurs up to 150 °C and is relatively small and similar for all the heating rates (2-4 %). The main mass loss after that temperature, for the curve with the lowest heating rate, can be distinguished in a few related steps. The weight loss up to 210 °C (1.6 %) is attributed to the evaporation of low-molecular-weight compounds and the decomposition reactions. The major event occurs in the temperature interval 210-450 °C and is due to the degradation reactions of the organic intermediates.25 A shoulder at 340-350 °C (Figure 1a, DTG curve-embedded graph) probably corre- Table 1. Sample abbreviations and comparison of the results of the dynamic pyrolysis measurements up to 600 °C and up to 1000 °C. Sample T Heating Group Total Inflection interval rate name mass point /°C /°C min-1 loss/% /°C T1H005 25-600 5 T1ABC 59.5 309.4 T1H010 10 57.7 320.2 T1H020 20 57.6 335.1 T1H050 50 58.4 351.1 T1H100 100 59.7 361.2 T2H005 25-1000 5 T2ABC 71.4 308.4 T2H010 10 71.5 323.1 T2H020 20 68.6 338.0 T2H050 50 68.1 352.3 T2H100 100 66.6 365.2 a) isothermal 'if i i ] \ i ...... .TGA ......-DTG Ï 1 1 ; i j i i \ \ \s.» 1 \ » ' 1 v! Temper atufer'C V> 200 2» j®0 350 400 1 i i j i, \ / Timetoiiri i j i i -T1H005 i 1 * t \ \ \ v. ----T1H010 1 ---T1H020 ..........T1H050 -----T1H100 Time/miri b) 20' 30. 2 40 70' isothermal' ^dynamic -T2H005 ----T2H010 ' •• \ ---T2H020 I ' \ ..........T2H050 i \ i > i 1 1 \ ' ' \ 1 1 \ -----T2H100 l ' \ ' ' \ \ * \ 1 1 \ l \ 1 \ \ X, \ \ ^ \ \ 30 50 100 Time/min 150 200 Figure 1. Dynamic TGA curves of the MBM pyrolysed up to 600 °C (a) and pyrolysed up to 1000 °C (b) measured with five different heating rates (5 °C min-1, 10 °C min-1, 20 °C min-1, 50 °C min-1 and 100 °C min-1). sponds to the start of the degradation of the bones2,4. The latter processes overlap at higher heating rates. In the last part of the decomposition, which takes place from 600 °C to 1000 °C, additional mass losses can be attributed to the Zupančič and Čelan Korošin: Physico-Chemical Properties of the Pyrolytic Residue ... 232 Acta Chim. Slov. 2021, 68, 229-238 ioo 70- 60- 40- dehydration p^j- _^ i ii or X11 100 ^ T2H005 ^^«Wieating rate -TIHOIO..... T2H010 -T1H020 ..... T2H020 V -T1H050 ..... T2H050 Vi -T1H100..... T2H100 inflection SntiiemDerature ' "".............. 25 100 200 400 500 600 Temperature/°C 700 800 900 1000 Figure 2. Inflection point determination for the dynamic pyrolysis curves up to 600 °C and up to 1000 °C at all heating rates (5 °C min-1, 10 °C min-1, 20 °C min-1, 50 °C min-1 and 100 °C min-1). decomposition of the carbonates and other mineral components. All the curves (in Figure 2), regardless of the final temperature, show a shift in the temperatures of the maximum rate of decomposition (inflection point) which expand between 309.4 °C and 361.2 °C for the samples heated up to 600 °C and between 308.4 °C and 365.2 °C for the samples heated up to 1000 °C to higher temperatures at even temperature intervals with the increasing heating rate (Table 1). These temperatures are in accordance with the data given by other authors for a heating rate of 10 °C min-1, for example, 310 °C by Chaala and Roy3, 335 °C by Conesa et al.4, and 346 °C by Ayllon et al.6 The inflection points occur in the range 72.3-74.6 % of the sample mass, as indicated by the position of the arrow on the graph. The appearance of an inflection point in the first part of the MBM decomposition indicates that a decomposition process above 600 °C seen on the curves measured up to 1000 °C, is slower and gradual. as can be seen for the DSC curve of sample T1H100 in Figure 4, associated with the formation and later decomposition of the intermediate fractions, which only occur in the presence of oxygen.4 The remainder of the isothermal part at 550 °C is the amount of ash in the sample. The results show that the ash quantity in the T1ABC samples increases with the heating rate of the MBM pyrolysis: from 57.4 % at 5 °C min-1 to 66.6 % at the highest heating rate. Figure 3. Ash-content determination for T1ABC curves at all five heating rates and MBM sample up to 550 °C. Figure 4 shows the result curves for the simultaneous TGA-DSC-MS analysis as a sample case, which was performed on the pyrolytic residue of the T1H100 sample using the ash-content determination method. The monotonic decline in the signal m/z 18 and m/z 44 for the ambient moisture (beside the hygroscopic moisture on the sample) and the carbon dioxide captured in the oven when opening, appears in the nitrogen segments. Switching to the oxygen atmosphere at 106 °C, gives rise to the CO2 gas with accompanying two consecutive exothermic peaks seen on DSC curve, which could be due to the combustion of light aliphatic hydrocarbons C4H4, C2H6 (m/z 52 and m/z 30) 3. 2. Determination of the Ash Content in MBM and T1ABC Samples The results of TGA determination of the ash content in the MBM and T1ABC samples are presented in Figure 3. For all the curves a similar course with several distinctive step losses was observed. The first one, the release of hygroscopic moisture in the temperature range up to 106 °C in a nitrogen atmosphere, for all the ABC samples and the MBM is expected to be small and similar, between 2.9 % and 3.8 % (Table S1 in Supplementary Material). In the temperature range between 106 °C and 550 °C in an oxygen atmosphere the major contributions to the weight loss of the samples appear; these are associated with the two-step exothermic decomposition of the organic matter 0 50 100 150 I ime/min Figure 4. Simultaneous TGA-DSC-MS analysis of the residue of the T1H100 sample up to 550 °C. Zupančič and Čelan Korošin: Physico-Chemical Properties of the Pyrolytic Residue ... Acta Chim. Slov. 2021, 68, 229-238 233 and in particular to the burned organic residue after the pyrolysis (char), in accordance with database of National Institute of Standards and Technology (NIST). 3. 3. Elemental Analyses of MBM and ABC Samples To evaluate the potential hazard posed by PTE in the MBM sample the semi-total concentrations of Cr, Ni, Cu, Zn, As, Mo, Cd and Pb were determined in the MBM and T1H005 sample and to evaluate the mineral-part composition of the samples also the concentrations of some macro elements. The elements contained in the original biomass, except elements that are volatile at the pyrolysis temperature, remain in the final products and are therefore more concentrated. The results (see Table S2 in Supplementary Material) showed that the concentrations of PTE in the MBM and T1H005 samples are very low, confirming the low potential environmental risk of both samples according to the PTE content. The concentrations of the PTE elements in both samples are far below the thresholds recommended by the EBC for biochar intended for agricultural use.14 The results of the CHNS analyses of the MBM and ABC samples, obtained after the pyrolysis of MBM up to 600 °C (T1ABC samples), are presented in Table 2. As a result of the heat treatment of the MBM, the mass content of the C, H, N and S in the residues decreased. The C, H, N and S contents in the MBM sample are comparable with the literature data, where broad concentration intervals are reported for these elements.2 This important variation in MBM composition strongly influences the thermochemi-cal treatment of MBM. Pyrolysed organic matter with a carbon content lower than 50 % is classified as pyrogenic carbonaceous material.14 The presence of C in the pyrolytic residues is the result of small organic remains, carbonized in argon, and the present of inorganic carbonate groups (CO32-), while H, N and S are found predominantly incorporated within the aromatic rings as hetero-atoms and thought to be a major contribution to the highly heterogeneous surface chemistry and reactivity of biochar.26 The content of sulphur in all the T1ABC samples (Table 2) is below the detection limit, while the concentrations of C, H and N decreased with increasing heating rate. The results are in accordance with the findings of previous studies, which showed that with increasing pyrolysis rate of biomass the yield of solid char decreases while the yields of gas and liquid phase in-creases.7,27 A lower heating rate allows most of the decomposed organic part of the MBM to be carbonized and thus remain in the solid residue, resulting in a higher carbon content in the samples prepared at a lower heating rate. At higher heating rates, the organic part of the MBM meal decomposes to a lesser extent, but the greater part of the decomposed organic matter is converted to the gas and liquid phase as it is carbonized and remains in the solid residue. The molar ratios H/Corg and N/Corg are carbonization degree parameters that characterize the degree of aromaticity of the biochar samples.14 The H/Corg as well as the N/Corg molar ratios in the T1ABC samples are comparable (on average 0.53 ± 0.04 and 0.162 ± 0.004, respectively) and represent nearly 30 % and 78 % of the H/Corg and N/ Corg molar ratios of the MBM samples (Table 2). The results indicate a greater loss of H-related than N-related functional groups during the heat treatment of MBM up to 600 °C. Although there are few studies on the characteristics of ABC obtained by pyrolysis of bone meal, the literature on the composition and characteristics of MBM pyrolysis products is sparse. Ayllón et al.7 investigated the influence of temperature (between 300 °C and 900 °C) and heating rate (from 2 °C to 14 °C) on the fixed bed pyrolysis of MBM. The MBM used in their study had a much higher organic matter content (higher content of CHNS) and consequently an almost 10 % lower ash content than MBM used in our study, making the composition and properties of char samples prepared under conditions similar to those used in our study difficult to compare. Table 2. Results of CHNS analyses of MBM and T1ABC samples and calculated quantities and ratios. Sample w(C) w(H) w(N) w(S) w(Corg) n(H) n(N) /% /% /% /% /% /«(Corg ) /n(Corg) MBM 38.23 5.52 9.16 0.62 37.71 1.74 0.21 T1H005 29.95 1.27 5.55 <0.1 28.40 0.53 0.17 T1H010 29.28 1.16 5.30 <0.1 27.80 0.50 0.16 T1H020 28.34 1.12 5.12 <0.1 26.95 0.49 0.16 T1H050 22.38 1.06 3.83 <0.1 20.97 0.53 0.16 T1H100 21.08 0.86 3.66 <0.1 19.54 0.60 0.16 3. 4. FTIR Spectroscopy FTIR spectroscopy was used to compare the presence of functional groups in the untreated MBM sample and the residues after pyrolysis of the MBM sample. The FTIR spectra of the original MBM sample and the commercially available hydroxyapatite (Merck, p.a.) are shown in Figure 5. The broad bands at 3671 cm-1 and 3281 cm-1 in the vibrations-rich FTIR spectrum of the MBM are attributed to the stretching vibrations of the O-H bond. The peak near 3700 cm-1 corresponded to vibrations of the OH groups in inorganic components of the MBM sample, while the O-H vibrations of the organic matter appeared at approximately 3000-3300 cm-1.28 The sharp bands at 2919 cm-1 and 2851 cm-1 belong to the asymmetric and symmetric stretching vibrations of the C-H bond. In the range from 1750 cm-1 to 1600 cm-1 the C=O stretching vibrations of amide (amide I vibra- Zupančič and Čelan Korošin: Physico-Chemical Properties of the Pyrolytic Residue ... 234 Acta Chim. Slov. 2021, 68, 229-238 tions) could be assigned, while C-N stretching vibrations strongly coupled with N-H bending vibrations (amide II) and N-H in plane bending vibrations coupled with C-N stretching vibrations (amide III) including C-H and N-H deformation vibrations are found in the range from 1600 cm-1 to 1500 cm-1 and 1350 cm-1 to 1200 cm-1, respectively.29 All these bands represent the organic phase of the MBM.23 The carbonate asymmetric stretching vibrations typically occur between 1410 cm-1 to 1530 cm-1.21,25,30 The presence of carbonate in our sample also indicates the observed weak band at 880 cm-1 which could be attributed to the carbonate out-of-plane bending vibrations. The bands in the range from 1100 cm-1 to 1000 cm-1 (asymmetric stretching vibrations) and from 600 cm-1 to 500 cm-1 belong to the relatively strong vibrations of the P-O bond of the phosphate group.31 The most prominent band in the FTIR spectrum of synthetic HA (Figure 5) and the main evidence for the presence of the phosphate group in the compounds is observed at 1026 cm-1. A small band at 892 cm-1 could belong to the vibrations of the HPO42- group and indicates the presence of the calcium-deficient apatite Ca10-x(H-PO4)x(PO4)6-x(OH)2-x.5,31 During the pyrolysis of MBM most of the organic substances decompose and remain in ABC as small carbonized residues or removed as gas products. The FTIR spectra of the residues of the pyrolysed MBM up to 600 °C (T1ABC) are present in Figure 6a. Weak vibrations of the O-H group in the mineral matter are observed near 3670 cm-1. The intense broad band near 3300 cm-1, observed in the FTIR spectra of the MBM, and which corresponded to the vibrations of the OH group in the organic matter, disappeared, suggesting that a large amount of free and associated hydroxyl groups and structural hydroxyl groups were decomposed during the MBM pyrolysis up to 600 °C.28 The weak split bands of the asymmetric and symmetric stretching vibrations of the C-H bond (2987 cm-1 and 2901 cm-1) were further observed. The group of bands with the maximum at 1407 cm-1 could be attributed to C-C stretching vibrations together with the asymmetric stretching vibrations of the C-O bond in the carbonate group.21,30 The presence of carbonate also indicates the absorption band at about 871 cm-1. The most intense band in the spectra of the T1ABC samples belongs to the stretching vibrations of the phosphate group where a slow shift of the band maxima from 1067 cm-1 at sample T1H100 to 1026 cm-1 at sample T1H005 was observed. The main phosphate-group vibration bands in the FTIR spectra of MBM pyrolysis residues during the thermal treatment up to 1000 °C (Figure 6b) are found at 1026 cm-1 for all the T2ABC samples. Small band shoulders near 957 cm-1 and 1100 cm-1 were observed which can be assigned to the ^-tricalcium phosphate phases.30 The presence of the ^-NaCaPO4 phase was also confirmed in XRD spectra of T2ABC samples (see Figure 7), prepared at lower heating rates. At 3670 cm-1 the weak band of stretching vibrations of the inorganic O-H group was still visible. We also observed the weak bands of the C-H stretching vibrations (2987 cm-1 and 2901 cm-1), Figure 5. FTIR spectra of untreated MBM sample and commercially available hydroxyapatite. Figure 6. FTIR spectra of residues after MBM pyrolysis up to 600 °C (a) and up to 1000 °C (b) at five different heating rates. Zupančič and Čelan Korošin: Physico-Chemical Properties of the Pyrolytic Residue ... Acta Chim. Slov. 2021, 68, 229-238 235 while the carbonate group vibrations (1407 cm-1 and 871 cm-1) disappeared. In all T2ABC samples, a new sharp band of unknown origin is observed at 2014 cm-1, which is also observed in FTIR spectra of animal bones pyrolysed at 450 °C and higher22 and in FTIR spectra of monolithic bone blocks pyrolysed at 800 °C.21 Since the band is not observed in the FTIR spectrum of synthetic hydroxyapatite (see Figure 5), we conclude that it probably belongs to an inorganic form of C that was trapped in the structure. 3. 5. X-ray Powder Diffraction Analysis The results of the X-ray powder-diffraction analyses showed that the peak intensities of all the T1ABC samples, regardless of the heating rate, indicate the presence of poorly crystalline phases (Figure 7). The weak broad peaks with no visible peak splitting observed can be attributed to Cl-bearing hydroxyapatite. A decrease in the peak width and a considerable increase in the peak intensity can be observed for the ABC prepared for the pyrolysis end temperature of 1000 °C (T2ABC samples) at lower heating rates. This indicates an increase in the size of the crystals, since crystallinity is a measure of the particle size.26 The crystallinity of the samples decreases with the higher heating rates, suggesting that some of the phases present are still not well crystallized. The presence of crystalline Cl-bearing hydroxyapatite (Ca5(PO4)3(-Cl,OH) and ^-sodium calcium phosphate (NaCaPO4) was confirmed. 3. 6. SEM/EDS Analysis The results of the SEM/EDS analyses revealed the strong influence of both, the pyrolysis end temperature and the heating rate on the morphology and the chemical composition of the final products. Figure 8 presents SEM images of the MBM pyrolysis residues obtained at pyrolysis end temperature 600 °C (upper row) and 1000 °C (bottom row) and at heating rates of 5 °C min-1 (a and d), 20 °C min-1 (b and e) and 100 °C min-1 (c and f). In the SEM images of the T1ABC samples (Figure 8, upper row) large amorphous agglomerates with varying rough to smooth surface texture were observed. In sample T1H005 the very beginning of small, poorly crystalline particles (up to few 100 nm), together with amorphous spilled formations (the EDS analysis shows us that these are amorphous KCl and NaCl - see Figure 9) are clearly visible. The SEM images of the T2ABC samples (Figure 8, bottom row) give us a completely different picture. The difference in particle size is clearly visible. The size of the well-crystallized particles of T1H005 ranges from less than 0.5 ^m to several tens of ^m. Also in the sample T2H020 the particles are already well crystallized, but in contrast to the particles in the sample T2H005 they are much smaller (from less than 1 ^m up to a few ^m) and stacked together into larger aggregates. The particles in the sample T2H100 exhibit very poor crystallinity. They are located in small aggregates and reach a size of up to 1 ^m. The distribution of elements over a particular area of the sample can be viewed using EDS elemental mapping. Element maps of the samples T1H005, T2H005, T1H100 Figure 7. XRD pattern of residues after pyrolytic MBM decomposition up to 600 °C (left) and 1000 °C (right) for different heating rates. Zupančič and Čelan Korošin: Physico-Chemical Properties of the Pyrolytic Residue ... 236 Acta Chim. Slov. 2021, 68, 229-238 Figure 8. SEM images of T1ABC (upper row) and T2ABC (bottom row) samples at heating rates 5 °C min 1 (a and d), 20 °C min 1 (b and e) and 100 °C min-1 (c and f). w ™ ■ Dftctror Inuge 1 O Ka1 CKi1_? j*-* k mm 4 *. * & M- "M r * , T t cam PKai CM" Afa m " ' -i ^S^ i is ■ a: • r4SKJ1_2 KK41 SlKal I Jyg P i 1 / - - flK" a ENma wnoe ■ OKU CKJ1_2 COKBI PKai a«ai * / isuKal ? K Kal Mg Ka1 2 * Jr . * ' i/ EiKirao imu* f OKai CkjU flft , | Ca Kal PKai OKai W H .. # ^^ I f«Kal_2 K Kal J*sKa1_2 mm v * -it i tlKlror 1 0 Kai CKa1j2 ■ " ^ f j caKai pk^i ciKai 1 Figure 9. EDS maps of elemental distribution in the samples T1H005 (a), T2H005 (b), T1H100 (c) and T2H100 (d). Distribution of the elements from left to right for all samples: upper line - electron image, oxygen, carbon; in the middle - calcium, phosphorus, chlorine; bottom line - sodium, potassium, magnesium or silicon (sample T1H005). Zupančič and Čelan Korošin: Physico-Chemical Properties of the Pyrolytic Residue ... Acta Chim. Slov. 2021, 68, 229-238 237 and T2H100 are shown in Figure 9. The EDS analyses revealed that carbon (grey images in the top right of the maps (a) and (c)) is still present in samples T1H005 and T1H100. The co-appearance of oxygen (green images in the first line), calcium (orange images) and phosphorus (pale-blue images) is clearly evident for all four samples, indicating the presence of apatite. For sample T1H005 a dispersed distribution of sodium is observed, although it is generally consistent with the distribution of Ca and P. The presence of KCl and a small particle containing silicon are also observed. For sample T1H100, sodium is observed in the form of amorphous NaCl, deposited onto the porous surface of the apatite. Compared to the sample T1H005, where the composition of the apatite particles is relatively uniform, a more diverse composition of apatite is observed in the T2H005 sample. The chlorine-bearing (red image) apatite particles, whose presence was confirmed by XRD analysis, contain the largest amount of calcium. In the apatite particles with a lower calcium content the higher sodium content was observed. The presence of ^-sodium calcium phosphate was also confirmed by XRD analysis. In the sample T2H100 the disperse distribution of the elements is observed, although the co-association of O, Ca and P is clearly visible. 4. Conclusions The results of the MBM pyrolysis study at various final temperatures of pyrolysis and various heating rates revealed the strong influence of both, not only the pyrolysis end temperature, but also the strong influence of the heating rate on the morphology and chemical composition of the final products. The TGA measurements showed a similar course of pyrolysis for the MBM samples heated at all the heating rates with the major pyrolytic event occurring in the temperature interval 210-450 °C being due to the degradation reactions of organic intermediates. An additional mass loss up to 12 % above 600 °C at low heating rates can be attributed to the decomposition of carbonates and other mineral components. Diminishing of the mass loss with increasing heating rate suggest a shortage of time for some thermal processes to be completed. The ash content in the T1ABC samples was in general increased with the increasing heating rate. The concentrations of PTE in the MBM are very low, which confirmed that the MBM pyrolytic residues are environmentally acceptable materials. The results of the CHNS analyses of the MBM and T1ABC samples showed that the concentrations of carbon, hydrogen and nitrogen decreased with increasing heating rate and indicated a greater loss of H-related than N-related functional groups. The results of the X-ray powder diffraction analyses showed that the peak intensity of all the residues of the pyrolysis end temperature at 600 °C, regardless of the rate of heating, indicates a low crystallinity, while the crystallinity of the samples obtained for the pyrolysis of the MBM to 1000 °C is reduced at higher heating rates. The presence of Cl-bearing hydroxyapatite and ^-sodium calcium phosphate was determined. The results of the SEM/EDS analyses revealed the strong influence of the pyrolysis end temperature and the heating rate on the morphology and chemical composition of the final products of the meat and MBM pyrolysis and confirmed the results of the XRD analyses. Although in practice raw MBM samples differ considerably in organic matter content, which can have a very strong influence on the pyrolysis process itself, our results have clearly shown that both, a pyrolysis end temperature of at least 600 °C or higher and lower heating rates are required to produce ABC samples with satisfactory properties. The results of this study imply the utilization of the pyrolysis residue of industrial by-products such as MBM as one of the attractive approaches that could improve the sustainable use of phosphate-bearing sources. Acknowledgements The authors would like to thank the Slovenian Research Agency (ARRS) for the financial support of the Network of Research Infrastructure Centres of the University of Ljubljana (MRIC UL), on whose equipment the part of the research was carried out, and for the financial support from the research programme P1-0134b: Chemistry for Sustainable Development. Thanks also to the native speaker Dr. Paul John McGuiness for his contribution to the improvement of the English language. Declaration of conflicting interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 5. References 1. N. Vassilev, E. Martos, G. Mendes, V. Martos and M. Vassile-va, J. Sci. Food Agric. 2013, 93, 1799-1804. DOI:10.1002/jsfa.6130 2. E. Cascarosa, G. Gea and J. Arauzo, Renewable Sustainable Energy Rev. 2012, 16, 942-957. DOI:10.1016/j.rser.2011.09.015 3. A. Chaala and C. Roy, Environ. Sci. Technol. 2003, 37, 4517-4522. DOI:10.1021/es026346m 4. J. A. Conesa, A. Fullana and R. Font, J. Anal. Appl. Pyrolysis 2003, 70, 619-630. DOI:10.1016/S0165-2370(03)00044-5 5. E. Deydier, R. Guilet, S. Sarda and P. Sharrock, J. Hazard. Mater. 2005, 121, 141-148. DOI:10.1016/j.jhazmat.2005.02.003 6. M. Ayllón, G. Gea, M. B. Murillo, J. L. Sánchez and J. Arauzo, J. 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DOI:10.1021/ie070415u 27. P. T. Williams and S. Besler, Renew. Energy 1996, 7, 233-250. DOI: 10.1016/0960-1481(96)00006-7 28. J. Jin, Y. Li, J. Zhang, S. Wu, Y. Cao, P. Liang, J. Zhang, M. H. Wong, M. Wang, S. Shan and P. Christie, J. Hazard. Mater. 2016, 320, 417-426. DOI:10.1016/j.jhazmat.2016.08.050 29. E. P. Paschalis, R. Mendelsohn and A. L. Boskey, Clin. Orthop. Relat. Res. 2011, 469, 2170-2178. DOI:10.1016/j.tca.2006.04.013 30. S. Meejoo, W. Maneeprakorn and P. Winotai, Thermochim. Acta 2006, 447, 115-120. DOI:10.1016/j.tca.2006.04.013 31. A. Destainville, E. Champion, D. Bernache-Assollant and E. Laborde, Mater. Chem. Phys. 2003, 80, 269-277. DOI:10.1016/S0254-0584(02)00466-2 Povzetek Izčrpavanje zalog fosfatnih rud kot glavnega vira fosforja je privedlo do iskanja novih, alternativnih in okolju prijaznejših produktov in procesov pridobivanja. Eden izmed varnih in okolju prijaznih fosfatnih virov je oglje iz živalskih kosti (ABC), pridobljeno s pirolizo mesno-kostne moke (MBM). V predstavljeni študiji smo raziskovali vpliv pogojev pirolize MBM na fizikalno-kemijske lastnosti pripravljenih produktov (ABC). Pirolizo MBM smo izvedli pri dveh končnih temperaturah (600 °C in 1000 °C) in petih različnih hitrosti segrevanja (5 °C min-1, 10 °C min-1, 20 °C min-1, 50 °C min-1 in 100 °C min-1). Pridobljene vzorce ABC smo okarakterizirali z rentgensko praškovno difrakcijo, IR spektroskopijo, elementno CHNS analizo in SEM/EDS analizo. Rezultati so pokazali močan vpliv tako končne temperature pirolize kot tudi hitrosti segrevanja na morfologijo in kemijsko sestavo pridobljenih produktov. © 0 Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Zupančič and Čelan Korošin: Physico-Chemical Properties of the Pyrolytic Residue ... DOI: 10.17344/acsi.2020.6438 Acta Chim. Slov. 2021, 68, 239-246 /^creative ^commons Scientific paper Hydrogen Bonds in Bis(1H-benzimidazole-KN3)cadmium(II) Dibenzoate: Hirshfeld Surface Analysis and AIM Perspective Jia-Jun Wang,1,2 Li-Nan Dun,1,2 Bao-Sheng Zhang,1,2 Zhong-Hui Wang,3 He Wang,1,2 Chuan-Bi Li1,2^ and Wei Liang1,2^ 1 Key Laboratory of Preparation and Application of Environmental Friendly Materials, Ministry of Education, Jilin Normal University, Changchun 130103, China 2 Chemistry Department of Jilin Normal University, Siping 136000, China 3 Sulfuric Acid Plant, Jilin Petrochemical Company Acrylonitrile Factory, Jilin 132021, China * Corresponding author: E-mail: li_c_b@163.com (Chuan-Bi Li); 16433576@qq.com (Wei Liang) Received: 10-09-2020 Abstract The coordination complex bis(1ff-benzimidazole-KN3)cadmium(II) dibenzoate has been synthesized and characterized by single crystal diffraction analysis. Cadmium center is six coordinated and formed a distorted octahedron coordinated geometry. The Hirshfeld analysis shows that in the dnorm-surface of the compound, there are dark red spots near the hydrogen-bonds acceptor and donor atoms, while intermolecular interactions result in faint-red spots. The AIM analysis was performed, there exist a BCP in each N(C)-H—O hydrogen bond, the bond paths also can be seen, the |V(b)|/G(b) < 1 and the H(b) > 0, the interaction is indicative of being a closed shell. The TG results are consistent with the X-ray diffraction structure. Keywords: Cd Complex, Crystal Structure, TG, Hirshfeld Surface Analysis, AIM Analysis 1. Introduction Crystal engineering based on coordination complexes1 have been fast developing in the last few years owing to their potential applications such as absorption,2 catalysis,3 luminescence,4 etc. Coordination complexes have already been applied in all aspects of life, and also used for removing the highly toxic heavy metal ions.5 The benzimidazoles (Bzim) possess a phenyl ring fused with imidazole ring.6 Many substances with benzim-idazole scaffold have been deeply participated in variety of life activities. For instance, mebendazole is used for treatment of parasitic diseases,7 and omeprazole is used for gastric ulcers,8 etc. Many other important properties of metal complexes containing benzimidazoles have also been investigated by researchers. The antibacterial activity of Bzim Zn(II) and Co(II) complexes have been investigated.9,10 For Bzim's Ir(III) N-heterocyclic carbene complexes' anticancer and antitumor properties were evaluated.11,12 Bzim derivate's Pt(II) and Cu(II) complexes have DNA binding and/or antioxidant activity.13,14 Even the Bzim-functional- ized ruthenium complexes can be used for dye-sensitized solar cells (DSSC) or energy-storage.15-17 Thus, exploring the benzimidazole metal coordination complexes is extremely important. The hydrogen bonds are critical in life science18 and chemistry.19 The Hirshfeld surface analysis20 and the Atom in Molecule (AIM) theory21 are two powerful tools to research hydrogen bonds from different perspectives. The Hirshfeld sufaces analysis is able to be utilized to identify a type and region of intermolecular interactions (including hydrogen bonds). Hirshfeld surface analyses comprises dnorm- and shape index surfaces, and 2D fingerprint plots (FP). While the AIM theory enables us to analyze the properties of hydrogen bonds. In AIM view, the main concept is based on the bond critical point (BCP), they are the evidence of hydrogen bond is the existence of a bond path and a BCP between the donor hydrogen and the acceptor. People also developed the criteria for the assessment of the existence of hydrogen bonds.22 In this article, we synthesized a new benzimidazole coordinated cadmium(II) dibenzoate (Cd(Bzim)2(BA)2 Wang et al.: Hydrogen Bonds in Bis(1H-benzimidazole-KN3)cadmium(II) 240 Acta Chim. Slov. 2021, 68, 239-246 (where Bzim is benzimidazole and BA is benzoate) through the low temperature hydrothermal method, obtained the crystal structure, calculated the molecular orbitals, carried out the Hirshfeld analysis and the AIM analysis, and we mainly focused on the analysis of the hydrogen bonds. 2. Experiments Cd(Ac)2 ■ 2H2O (0.235 g, 1.0 mmol), benzimidazole (Bzim, 0.236 g, 2.0 mmol), benzoic acid (BA, 0.244g, 2.0 mmol) and 15 mL water were mixed with stirring followed by adjusting the pH value to 6.5 with an aqueous solution of NaOH. Then the mixture was sealed in a 25 mL Teflon-lined stainless-steel reactor and heated at 100 °C for 96 h to give dark yellow crystals of the title compound after cooling. Yield: 32% (based on Cd). IR (cm-1): 3315(w), 3236(w), 1680(s), 1653(s), 1477(s), 1430(s), 1346(w), 877(w), 787(m), 449(m). Anal. Calcd. (%) for C28H22CdN4O4: C, 56.86; H, 3.72; N, 9.48; Found (%): C, 56.79; H, 3.81; N, 9.53. 3. Structure Determination and Physical Measurements A yellow block crystal for 1 was chosen for X-ray diffraction analysis. Crystal structure measurement was performed on a Bruker SMART APEX II CCD diffractom-eter equipped with a graphite-monochromatic Mo^a (X = 0.71073 A) radiation. Data integration and reduction were performed using SaintPlus 6.01.23 Absorption corrections were applied with a multi-scan mode.24 The structure was solved by direct methods with SHELXS25 and refined by full-matrix least-squares techniques using SHELXL-201826 within WINGX.27 All non-hydrogen atoms were refined anisotropically. All H atoms on C atoms were positioned Fig. 1. Molecular structure of [Cd(Bzim)2(BA)2], (ellipsoid probability at 30 %). geometrically and refined as riding, with C-H = 0.93 and ^iso(H) = 1.2Ueq(C). The molecular graphics was prepared using program Diamond 3.2.28 4. Results and Discussion 4. 1. Structure Description Table 1. Summary of data collection and structure refinement for the compound. CCDC 780823 Formula C28H22CdN4O4 Formula weight 590.89 Crystal system Orthorhombic Space group Pbca alA 14.0359(6) blA 17.7081(8) clA 21.8928(12) a, b, ylo 90 VIA3 5441.4(5) Z 8 Pcalc/g cm-3 1.443 Crystal size/mm 0.23x0.20x0.18 F(000) 2384 Reflections collected I unique 37904/6759 Rint 0.1203 Datalrestraintslparameters 6759 / 0 / 334 Goodness of fit on F2 0.971 R1, wR2 (I > 2a (I)) 0.0411, 0.0572 R1, wR2 (all data) 0.1336, 0.0672 Peak and holel e.A-3 0.670, -0.400 A perspective view of the compound, with the atomic numbering scheme, is shown in Fig. 1. The compound crystallizes in the space group Pbca (Table 1). Asymmetric unit consists of a cadmium, two benzimidazole molecules and two benzoic acid ions. The cadmium center is six coordinated and connected four oxygen from two ben-zoic acids and two nitrogen atoms from two Bzims. Bzim coordinates Cd center in kN coordinate mode and each benzoate ion is bonded to Cd center in a chelating mode. So, the compound can be described as bis(1H-benzimida-zole-KN3)cadmium(II) dibenzoate and possess an distorted octahedron geometry. The Cd-N distances are 2.258(3) and 2.279(3) A (Table 2), which are not only shorter than the average Cd-Nimidazole distance (2.302(3) A),29 but also shorter than the Cd-NN-alkylimidazoles (2.357(2) A).30 The Cd-O distances are in the range 2.263(2)-2.490(2) A, these distances are shorter than the Cd-O distances of the acetate (O2CMe in chelating mode, 2.294(4)-2.615(3) A).31, 32 The packing diagram is in Fig. 2. In crystal lattice, there are n—n and C-H—n interactions (Table SI). The distances between the center of gravity of the n rings (Cg—Cg distance) are from 3.68 to 3.97 A, and the distances between Wang et al.: Hydrogen Bonds in Bis(1H-benzimidazole-KN3)cadmium(II) ... Acta Chim. Slov. 2021, 68, 239-246 241 Table 2. Selected Bond Length and Angles for the Compound. Bond Length/Angle A/0 Bond Angle o Bond Angle o Cd-N3 2.258(3) N3-Cd-O1 112.92(10) O1-Cd-O4 151.39(10) Cd-O3 2.367(3) N3-Cd-N2 92.37(10) N2-Cd-O4 91.57(10) Cd-O1 2.263(2) O1-Cd-N2 96.80(10) O3-Cd-O4 54.82(9) Cd-O4 2.375(3) N3-Cd-O3 147.27(10) N3-Cd-O2 88.65(9) Cd-N2 2.279(3) O1-Cd-O3 96.86(9) O1-Cd-O2 54.60(8) Cd-O2 2.490(2) N2-Cd-O3 97.48(10) N2-Cd-O2 148.68(10) O4-Cd-O2 119.61(8) N3-Cd-O4 93.93(10) O3-Cd-O2 98.43(9) Fig. 2. The packing diagram of the compound. C atom and Cg of the n rings (C—Cg distance) are 3.49 and 3.63 Â. There also exist the N-H-O and C-H-O33 hydrogen bonds in crystal structure (Table 3), the N—O distances are 2.75 and 2.80 Â, while the C—O distances are 3.21and 3.35 Â. The D-H-A angle of N-H-O (161 and 176°) is larger than the angle of C-H-O (134 and 157°). Table 3. Hydrogen bonds (Â and Donor-H Acceptor D-H H-A D-A D-H-A N1-H1 O21 0.86 1.94 2.803(4) 176 N4-H4 O311 0.86 1.92 2.748(4) 161 C3-H3 O4111 0.93 2.47 3.350(4) 157 C22-H22 O111 0.93 2.49 3.211(5) 134 Symmetry codes: i = V - x, V + y, z; ii = V + x, y, V - z; iii = -V +x, y, V - z. 4. 2. Hirshfeld Surface Analysis In the last few years the analysis of molecular crystal structures using tools based on Hirshfeld surfaces has rapidly gained in popularity,34-36 and they were carried out and plotted using CrystalExplorer software.20,37-41 Hirshfeld surface analysis comprising dnorm- and shape index-surfaces, and 2D fingerprint plots (FP). In dnorm-surface, hydrogen-bonds result in dark red spots near the hydrogen-bonds acceptor and donor atoms while intermolecular interactions result in faint-red spots. Further, the presence of patterns of red- and blue triangles on the same region of the shape-index surfaces is characteristic of n—n stacking. The dnorm- and shape index-surfaces of the compound are shown in Fig. 3 and Fig. 4. Fig. 3. The dnorm surfaces of the compound. Wang et al.: Hydrogen Bonds in Bis(1H-benzimidazole-KN3)cadmium(II) 242 Acta Chim. Slov. 2021, 68, 239-246 The N-H—O hydrogen-bonds in title compound can be seen in the dnorm-surfaces as bright-red spots marked as a, b, c and g (the spots located near the atom each correspondence are: a — N1, b — N4, c — O2, g — O3) in Fig. 3, so that the a, b, c and g spots are due to N1-H1—O2 and N4-H4-O3 hydrogen bonds. The weak C-H—O hydrogen bond interactions (including intramolecular and intermolecular hydrogen bonds) in the compound are also demonstrated in the dnorm-surface as faint-red color spots (Fig. 3) and signed as e, f, h, i. (that is, each correspondence are: e — O4, f — C22, h — O1, i — C3). The faint-red spots above the center of the benzimidazole ring in the dnorm-surface of the compound (Fig. 3), all marked as d, arise from the C20-H20—n(Cg5) and C20-H20—n(Cg6) interaction, observed in crystal structure. The offset n—n interactions observed in the crystal structure are identified by distinctive patterns of red- and blue-triangles across the respective phenyl rings on the shape-index surface plots of the molecule (see the circle in Fig. 4). Fig. 4. The shape-index-surfaces. The quantitative analysis of the intermolecular interactions apparent in crystal structure was attempted by observing the 2D FP's (Fig. 5). The H—O intermolecular interactions in the compound, appeared as two distinct long sharp spikes in the FP, at di + de - 1.75 Â which is roughly close to the observed N1-H1-O2 and N4-H4-O3 distance of 1.92, 1.94 Â (Table 3). In one molecule, these two spikes appear at (di, de) - (0.70 Â, 1.05 Â) and (de, di) - (0.70 Â, 1.05 Â, Fig. 5d), and the spikes appear in the same manner. The C-H—n interactions appear as pair of blunt spikes at (de, di) - (1.78 Â, 1.0 Â) and (de, di) - (1.0 Â, 1.78 Â) in the FP, occurring at di + de - 2.78 Â, it appear as the spikes at di + de distances are close to the H-n(Cg) distances in crystal (2.57, 2.80 Â, Table SI, Fig.5 (c)). The C-H-n interactions (Table SI) are also supported by the 'two wings' characters,39-41 the wings are sites at the upper left and the bottom right. For the C—C close contacts (Fig. 5 (e)) with the shortest di + de (distances - 1.73 + 1.73 = 3.46 Â) is roughly accord with the C—C distances 3.68-3.97 Â in Table SI. It corresponds to the n—n stacking interactions. The relative contributions of the intermolecular interactions to the Hirshfeld surface for the compound was calculated (see the upper bar diagram in Fig. 5(g)). The greatest contribution (47.2%) is from H—H contacts, followed by H—C/C—H contacts (which is stands for the C-H—n intermolecular interactions, 27.4%), then is the H-O/O-H contacts (the C-H-O or N-H-O hydrogen bond interactions 14.0%), the contribution from the H—N/N—H contacts is 4.2%, other contacts is 1.1%. 4. 3. Atom in Molecules Analysis The topology analysis proposed by Bader was initially used for researching electron density in "atoms in Fig. 5a The full image of Fingerprint Plot. Fig. 5b The H—H contacts show the contribution of 47.2%. Fig. 5c The H—C contacts have a contribution of 24.7%. Fig. 5d The H—O contacts have a contribution of 14.0% which is manifested by the interactions due to hydrogen bonds of N-H—O and C-H—O type. Fig. 5e The C—C contacts display the contribution of 6.1%. Fig. 5f The H—N contacts exhibit the contribution of 4.2%. Fig. 5g The bar graph shown the proportion of each contacts. Wang et al.: Hydrogen Bonds in Bis(1H-benzimidazole-KN3)cadmium(II) ... Acta Chim. Slov. 2021, 68, 239-246 243 Fig. 6. The general view of hydrogen bonds BCP (pink ball) from the three coordinated unit model of the compound. molecules" (AIM) theory.21,42,43 In this theory, based on the bond critical point (BCP), Popelier22 proposed the hydrogen bond's electron density at the BCP (pBCP) should be in the scope of 0.002-0.035 a.u. and the electron density Laplacian value (v2pBCP) should be confined in the region from 0.024 to 0.139 a.u. For investigating the hydrogen bonds, we select three coordination units to form the model (it contain three molecules), in which it can exhibit the hydrogen bonds and the n—n interactions from the cif file (see supporting information) and calculate the single point to get the fchk file (see supporting information) from Gaussian 09 program44 at wB97XD45/GenECP (6-31+G** basis set for C, H, O, N and Lanl2dz46 basis set for Cd), finally, we use the Multiwfn program47 to study the topological properties of the hydrogen bonds in title compound (The Multiwfn produced CPs.pdb, Path.pdb and CPprop.txt are also in supporting information). Fig. 6 shows the existence of a BCP in each N-H—O and each C-H—O hydrogen bond. The bond paths associated with the hydrogen bonds can be seen in Fig. 5 (and also be seen in Fig. S1). The BCP electron density of the hydrogen bonds is listed in Table 4. The pBCP value (from 0.009 to 0.032 a.u.) for the N-H-O and C-H-O hydrogen bonds are all in the suggested interval of 0.002-0.035 a.u.48 for the hydrogen bond. The two negative Hessian matrix eigenvalues of electron density, X1 and X2, can measure the scope of contraction of pBCP which is perpendicular to the bond toward the critical point, while the positive A3 eigenvalue weighs the extent of contraction parallel to the bond and from the BCP toward each of the adjacent nuclei. The sum of eigenvalues A1, A2, and A3 is v2pBCP. In addition, the numeric value of v2pBCP (0.0322-0.1091 a.u.) for N(C)-H—O interactions are in the recommend region of 0.024-0.139 a.u. for hydrogen bonds. Bader et al. deemed that for the closed-shell interactions (including Table 4. The electron density (pbcp), the Laplacian of electron density (V2pBCp), and the eigenvalues of Hessian at BCP (Xj, X2 and X3)a. Computed at wB97XD/GenECP Level (6-31+G(d) for C, H, O, N and LanL2DZ for Cd). Interactions Pbcp V2Pbcp Xi X2 X3 N1-H1—O2 0.0324 0.1091 -0.0458 -0.0456 0.2006 N4-H4—O3 0.0254 0.0950 -0.0320 -0.0326 0.1596 C3-H3—O4 0.0093 0.0322 -0.0086 -0.0092 0.0500 C22-H22—O1 0.0091 0.0343 -0.0087 -0.0080 0.0510 a - The unit is atom unit. Wang et al.: Hydrogen Bonds in Bis(1H-benzimidazole-KN3)cadmium(II) 244 Acta Chim. Slov. 2021, 68, 239-246 the ionic bonds, hydrogen bonds, and van der Waals interactions) the v2pBCP value is positive.21,43,49-54 On the basis of Table 4, the N-H—O and C-H—O hydrogen bonds are typical closed-shell interactions. Clearly, the topological properties of the hydrogen bonds studied are closed-shell interactions. Table 5 The local properties at BCP: the potential energy density, V(b); the Lagrangian kinetic energy density, G(b); and the total energy density H(b)a. The calculations at wB97XD/GenECP level (6-31+G(d) for C, H, O, N and LanL2DZ for Cd). Interactions V(b) G(b) H(b) |V(b)|/G(b) N1-H1—O2 -0.02476 0.02602 0.00126 0.92158 N4-H4—O3 -0.02100 0.02237 0.00137 0.93876 C3-H3—O4 -0.00655 0.00731 0.00075 0.89603 C22-H22—O1 -0.00622 0.00740 0.00116 0.84054 a - The unit is atom unit (a.u.). Morrison,55 and Espinosa56 et al.57 suggested that bond interactions are sorted according to the |V(b)|/G(b) ratio, the ratio |V(b)|/G(b) < 1, the bonded interaction is taken for the closed shell, when |V(b)|/G(b) > 2, it is typically covalent interaction; and when 1 < |V(b)|/G(b) < 2, it is the intermediate character. As it is depicted in Table 5, the Lagrangian kinetic energy density is only slightly larger than the potential energy density for all the (N)C-H-O interactions. It brings about the total energy density H(b) is drawn near to 0 and the |V(b)|/G(b) ratio is a little less than 1.0. Consequently, the N(C)-H—O interactions are mainly the closed shell for the model of the compound. This conclusion is agreement with the v2pBCP > 0. 4. 4. Thermogravimetric Analysis The TG of the title compound at the heating rate of 10 °C/min in nitrogen atmosphere is shown in Fig. 7. Fig. 7. The TG Analysis of the Compound. There are two weight loss steps. The first weight loss stage mainly takes place from 301 to 393 °C, it may be related to the removal of the coordinated benzimidazole molecules (found: 41.14%; calcd: 39.99%). The second weight loss step mainly takes place from 405 to 636 °C, it corresponds to the release of the coordinated benzoate anion (found: 36.3%, calcd.: 40.99%). After 650 °C, nearly no weight loss is observed and the residue weight is 20.59%, which suggests it may be the cadmium oxide (CdO, calcd.: 21.73%). 5. Conclusion The coordination complex bis(1H-benzimida-zole-KN3)cadmium(II) dibenzoate has been synthesized, the crystal structures were characterized by the single crystal diffraction analysis, Hirshfeld analysis and the AIM analysis. The structure analysis reveals that the cadmium center is six coordinated and forms an distorted octahedron coordinated geometry, and the compound's formula is Cd(Bzim)2(C6H5COO)2. The Hirshfeld analysis shows that in the dnorm-surface, there are dark red spots near the hydrogen-bond acceptor and donor atoms, while intermolecular interactions result in faint-red spots, the offset n—n interactions observed are identified by distinctive patterns of red- and blue-triangles across the respective phenyl rings on the shape-index surface plots, and the 2D fingerprint plots were also investigated. The AIM analysis shows there exists a BCP in each N-H—O and each C-H—O hydrogen bond, the bond paths associated with the hydrogen bonds can be seen, and the |V(b)|/G(b) < 1.0 and the H(b) > 0, these results manifested that the interaction is a closed shell system. The TG plot shows two weight loss steps (corresponding to BA and Bzim), the residue may be the cadmium oxide (CdO). 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Morrison, Chem. Phys. Lett. 2000, 317, 97-102. D0I:10.1016/S0009-2614(99)01306-8 56. E. Espinosa, I. Alkorta, J. Elguero, E. Molins, J. Chem. Phys. 2002, 117, 5529-5542. D0I:10.1063/1.1501133 57. S. Dinda, A. G. Samuelson, Chem. Eur. J. 2012, 18, 3032-3042. D0I:10.1002/chem.201101219 Povzetek Sintetizirali smo koordinacijsko spojino bis(1ff-benzimidazol-KN3)kadmijev(II) dibenzoat in ga okarakterizirali z rentgensko monokristalno analizo. Kadmijev center je heksakoordiniran s popačeno oktaedrično geometrijo. Hirshfeldova analiza razkrije, da so na površini dnorm temno rdeče točke blizu akceptorjev in donorjev vodikovih vezi, medtem ko ble-dordeče točke predstavljajo intermolekularne interakcije. AIM analiza pokaže, da so BCP pri vsaki N(C)-H—O vodikovi vezi, |V(b)|/G(b) < 1 in H(b) > 0 pa nakazujeta na closed shell interakcijo. TG analiza potrjuje kristalno strukturo. © 0 Except when otherwise noted, articles in this journal are published under the terms and conditions of the Creative Commons Attribution 4.0 International License Wang et al.: Hydrogen Bonds in Bis(1H-benzimidazole-KN3)cadmium(II) ... S10 Acta Chim. Slov. 2021, 68, (1), Supplement DRUŠTVENE VESTI IN DRUGE AKTIVNOSTI SOCIETY NEWS, ANNOUNCEMENTS, ACTIVITIES Vsebina Doktorska in magistrska dela, diplome v letu 2020 .............................................................. S3 Navodila za avtorje.................................................................................................................. S32 Contents Doctoral theses, master degree theses, and diplomas in 2020............................................. S3 Instructions for authors.......................................................................................................... S32 Društvene vesti in druge aktivnosti S10 Acta Chim. 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Romana Cerc Korošec Somentorica: doc. dr. Andrijana Sever Škapin Datum zagovora: 8. 7. 2020 Tjaša LISAC KATIONSKA IZMENJAVA V STRUKTURAH KOVINSKO-ORGANSKIH MATERIALOV ZA ZAJEMANJE PLINOV Mentor: viš. znan. sod. dr. Matjaž Mazaj Somentorica: izr. prof. dr. Amalija Golobič Datum zagovora: 27. 8. 2020 Aljaž ŠKRJANC POSKUS ESTRENJA POD POGOJI FOTOKATALITSKE MITSUNOBU REAKCIJE Mentor: prof. dr. Janez Košmrlj Datum zagovora: 28. 8. 2020 Dominik JANKOVIČ SINTEZA MONOSUBSTITUIRANIH 'KLIK' TRIAZOLOV Mentor: doc. dr. Martin Gazvoda Datum zagovora: 31. 8. 2020 Ana SILJANOVSKA UČINKOVITA SINTEZA SUBSTITUIRANIH CINOLINOV PREKO VMESNIH DIAZONIJEVIH SOLI Mentor: prof. dr. Janez Košmrlj Datum zagovora: 31. 8. 2020 Jure GREGORC ORGANOKATALIZIRANA ASIMETRIČNA SINTEZA DERIVATOV DIHIDROPIRANOPIROLA Mentor: izr. prof. dr. Uroš Grošelj Datum zagovora: 31. 8. 2020 Tilen BERGLEZ MODELIRANJE ATAKTIČNE IN IZOTAKTIČNE POLIMETAKRILNE KISLINE S POUDARKOM NA ODVISNOSTI STRUKTURE OD TAKTIČNOSTI IN DIELEKTRIČNE KONSTANTE TOPILA Mentorica: prof. dr. Ksenija Kogej Datum zagovora: 1. 9. 2020 Društvene vesti in druge aktivnosti S10 Acta Chim. Slov. 2021, 68, (1), Supplement Žiga PONIKVAR UPORABA ELEKTROKEMIJSKIH METOD V ORGANSKI SINTEZI ZA TVORBO PINAKOLNE VEZI IZ SUBSTITUIRANIH ALDEHIDOV ALI KETONOV Mentor: doc. dr. Krištof Kranjc Datum zagovora: 3. 9. 2020 Brigita KRAJNC SPEKTROFOTOMETRIČNO DOLOČEVANJE VEZAVE MIKROELEMENTOV NA KOSTANJEV EKSTRAKT Mentor: izr. prof. dr. Bogdan Štefane Datum zagovora: 3. 9. 2020 Petra STARE SINTEZE KOORDINACIJSKIH SPOJIN CINKA(II) S KINALDINATOM IN IZBRANIMI ALKOHOLAMINI. Mentorica: doc. dr. Barbara Modec Datum zagovora: 7. 9. 2020 Katarina ŽIBERNA FEROELEKTRIČNA DOMENSKA STRUKTURA PIEZOELEKTRIČNE TRDNE RAZTOPINE BiFeO3-SrTiO3 Mentor: viš. znan. sod. dr. 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Aleš Ručigaj Datum zagovora: 10. 9. 2020 Gašper TEGELJ OPTIMIZACIJA AKTIVACIJE REOLOŠKEGA ADITIVA V POSTOPKU PROIZVODNJE 2K PUR HS PREMAZA Mentor: prof. dr. Matevž Dular Datum zagovora: 10. 9. 2020 Vid URBANČIČ VPLIV ANTIBIOTIKOV V ODPADNEM BLATU NA PROIZVODNJO BIOPLINA Mentorica: prof. dr. Andreja Zgajnar Gotvajn Datum zagovora: 10. 9. 2020 Anže KOVAČIČ VPLIV REOLOŠKIH LASTNOSTI TEKOČEGA PREMAZA NA LASTNOSTI SUHEGA FILMA NA SUBSTRATU Mentorica: doc. dr. Lidija Slemenik Perše Datum zagovora: 10. 9. 2020 Nanja BRELIH PROIZVODNJA BIOGORIV IZ LIGNOCELULOZNE BIOMASE Mentor: prof. dr. Igor Plazl Datum zagovora: 17. 9. 2020 Gaja MASTNAK SUBAN UPORABA AKRILNIH HIDROGELOV V BIOMEDICINI Mentorica: prof. dr. Urška Šebenik Datum zagovora: 29. 9. 2020 Jure VELKAVRH UČINKOVITOST KROMATOGRAFSKIH KOLON Mentor: prof. dr. Aleš Podgornik Datum zagovora: 2. 10. 2020 Samo STANKOVIČ PRIPRAVA IN UPORABA KOVINSKIH PEN Mentor: prof. dr. 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Bojan Kozlevčar Datum zagovora: 31. 1. 2020 David DRAGAN VPLIV SINTEZNIH PARAMETROV NA VELIKOST KRISTALOV ZEOLITA P: SINTEZA IN STRUKTURNA KARAKTERIZACIJA Mentor: prof. dr. Anton Meden Datum zagovora: 6. 2. 2020 Renata SKUŠEK KEMIJSKO RAVNOTEŽJE V VSAKDANJEM ŽIVLJENJU Mentor: prof. dr. Andrej Jamnik Datum zagovora: 7. 2. 2020 Domen IPAVEC PRIPRAVA 3-METOKSI-1H-INDOL-2-KARBOKSILNE KISLINE KOT KLJUČNEGA INTERMEDIATA ZA SINTEZO CEPHALANDOLA C Mentor: doc. dr. Martin Gazvoda Datum zagovora: 13. 2. 2020 Viljem VOLF MATERIALI ZA VARČEVANJE Z ENERGIJO V NOVEJŠIH GRADNJAH Mentorica: doc. dr. Klementina Zupan Datum zagovora: 9. 4. 2020 Uroš MEZEK SINTEZA ORGANOKATALIZATORJEV NA OSNOVI KINUKLIDINA Mentor: izr. prof. dr. Uroš Grošelj Datum zagovora: 10. 4. 2020 Blaž TOPLAK DOLOČANJE VITAMINA E V RASTLINSKIH OLJIH Z DIFERENČNO PULZNO VOLTAMETRIJO Mentor: izr. prof. dr. Mitja Kolar Datum zagovora: 5. 5. 2020 Nika MUZGA REOLOŠKE LASTNOSTI JOGURTA MASOVNE IN DOMAČE PROIZVODNJE Mentorica: doc. dr. Lidija Slemenik Perše Datum zagovora: 6. 5. 2020 Lara Eva RUDOLF RAZVOJ EKSTRAKCIJSKE METODE ZA DOLOČANJE BENZOTRIAZOLOV V TLEH Mentorica: prof. dr. Helena Prosen Datum zagovora: 6. 5. 2020 Luka SETNIKAR LUMINISCENČNE LASTNOSTI CINKOVIH KOORDINACIJSKIH SPOJIN Z INDOL-3-KARBOKSILATNIM LIGANDOM Mentor: izr. prof. dr. Franc Perdih Datum zagovora: 15. 5. 2020 Jan KOLER OPTIMIZACIJA SINTEZE BAKROVIH(I) KOORDINACIJSKIH SPOJIN S FOSFINSKIMI LIGANDI IN NEOKUPROINOM Mentor: doc. dr. Jakob Kljun Datum zagovora: 21. 5. 2020 Nika MARINČ KOBALTOVE SPOJINE Z BENZOJSKO KISLINO Mentor: doc. dr. Bojan Kozlevčar Datum zagovora: 22. 5. 2020 Uroš PAVLIN PRIPRAVA KLEŠČASTIH KOMPLEKSOV Mentor: izr. prof. dr. Janez Cerkovnik Datum zagovora: 8. 6. 2020 Matevž ŠTEFANČIČ VALIDACIJA HPLC METODE ZA SELEKTIVNO DOLOČEVANJE KANABINOIDOV Mentor: prof. dr. Matevž Pompe Datum zagovora: 9. 6. 2020 Jure JAKOŠ RAZVOJ PROGRAMSKE OPREME ZA DOLOČEVANJE STRUKTUR ORGANSKIH MOLEKUL NA PODLAGI IR IN 1H NMR SPEKTROSKOPIJE Mentor: doc. dr. Martin Gazvoda Datum zagovora: 24. 6. 2020 Hanno BRADEŠKO LUMINISCENČNE LASTNOSTI CINKOVIH KOORDINACIJSKIH SPOJIN Z DIKETONATNIMI IN KARBOKSILATNIMI LIGANDI Mentor: izr. prof. dr. Franc Perdih Datum zagovora: 10. 7. 2020 Ajda URANIČ ČIŠČENJE EKSTRAKTOV TRDNIH VZORCEV S HFME Mentorica: prof. dr. Helena Prosen Datum zagovora: 10. 7. 2020 Društvene vesti in druge aktivnosti S10 Acta Chim. Slov. 2021, 68, (1), Supplement Diana MARINKOVIC KARAKTERIZACIJA VZORCA NASTALEGA PRI REAKCIJI BAKROVEGA SULFATA IN KALIJEVEGA OKSALATA Mentorica: doc. dr. Nives Kitanovski Datum zagovora: 10. 7. 2020 Žiga VESEL DERIVATI OKSINDOLA IN ISATINA TER NJIHOVA UPORABA V ORGANOKATALIZIRANIH PRETVORBAH - SINTEZA IZOTIOCIANATOV NA OSNOVI 5-FLUOROINDOLIN-2,3-DIONA Mentor: izr. prof. dr. Uroš Grošelj Datum zagovora: 20. 8. 2020 Lucijan KOLENC IMOBILIZACIJA CuO FOTOKATALIZATORJA Mentorica: prof. dr. Urška Lavrenčič Štangar Datum zagovora: 27. 8. 2020 Mateja KLEMENIC IMOBILIZACIJA TiO2 NANOSTRUKTURIRANIH MIKRODELCEV Mentorica: prof. dr. Urška Lavrenčič Štangar Datum zagovora: 27. 8. 2020 Eva TERKAJ IDENTIFIKACIJA KRISTALINIČNIH SNOVI V KREMAH ZA SONČENJE Z RENTGENSKO PRAŠKOVNO DIFRAKCIJO Mentor: prof. dr. Anton Meden Datum zagovora: 27. 8. 2020 Jerneja MAČEK DOLOČANJE NAPAK PRI NEVTRALIZACIJSKIH TITRACIJAH Mentor: izr. prof. dr. Drago Kočar Datum zagovora: 27. 8. 2020 Martin NAGODE SINTEZA IN REAKTIVNOST PIRENMETILAMINSKEGA FLUORESCENČNEGA DERIVATIZACIJSKEGA REAGENTA Mentor: izr. prof. dr. Franc Požgan Datum zagovora: 27. 8. 2020 Klavdija GRANDLJIČ TEHNIKE TERMIČNE ANALIZE ZA PREUČEVANJE MATERIALOV KULTURNE DEDIŠČINE Mentorica: izr. prof. dr. Romana Cerc Korošec Datum zagovora: 27. 8. 2020 Uroš KASTELIC MERJENJE VLAŽNOSTI V FARMACIJI Mentor: viš. pred. dr. Andrej Godec Datum zagovora: 31. 8. 2020 Teja BELE RENTGENSKA PRAŠKOVNA DIFRAKCIJA PREHRANSKIH DOPOLNIL Mentor: prof. dr. Anton Meden Datum zagovora: 2. 9. 2020 Rene URH TEORIJSKA OBRAVNAVA FAZNEGA RAVNOTEŽJA TEKOČINA-PARA Mentor: prof. dr. Andrej Jamnik Datum zagovora: 2. 9. 2020 Klara METLIČAR KARAKTERIZACIJA KOORDINACIJSKE SPOJINE KROMA S PIRIDINSKIMI IN HALOGENIDNIMI LIGANDI Mentorica: doc. dr. Nives Kitanovski Datum zagovora: 2. 9. 2020 Matic KRIVEC DIDAKTIČNI MODUL ZA UČENJE UMERJANJA MERILNIKOV Mentor: izr. prof. dr. Janez Cerar Datum zagovora: 4. 9. 2020 Lara ŽIBERNA KATIONSKA MODIFIKACIJA A-CELULOZE IN KRISTALINIČNE NANOCELULOZE Z GIRARD REAGENTOM T Mentor: viš. pred. dr. Branko Alič Datum zagovora: 4. 9. 2020 Lucija PETRIČ VPLIV DODATKOV NA LASTNOSTI SULFONIRANIH MELAMINSKO FORMALDEHIDIH PEN Mentor: viš. pred. dr. Branko Alič Datum zagovora: 4. 9. 2020 Tina ZUPANČIČ PRETVORBE HALOGENIRANIH ORGANSKIH SPOJIN Mentor: prof. dr. Marjan Jereb Datum zagovora: 7. 9. 2020 Nataša ŠUKLJE SINTEZA NEKATERIH HALKOGENIRANIH DERIVATOV Mentor: prof. dr. Marjan Jereb Datum zagovora: 7. 9. 2020 Rok BRULC ADENIN KOT KATION V HEKSAFLUORIDOSILIKATNIH SOLEH Mentor: doc. dr. Andrej Pevec Datum zagovora: 7. 9. 2020 Urša JAMNIK FIZIKALNO KEMIJSKE LASTNOSTI MEDU Mentorica: prof. dr. Barbara Hribar Lee Datum zagovora: 8. 9. 2020 Patrik HORŽEN KOORDINACIJSKA KEMIJA CINKA(II) S PIPERAZINOM IN DERIVATI Mentorica: doc. dr. Barbara Modec Datum zagovora: 9. 9. 2020 Katja RUPARČIČ PREGLED ANALIZNIH METOD ZA DOLOČANJE KALCIJA V PREHRANSKIH DOPOLNILIH Mentor: izr. prof. dr. Drago Kočar Datum zagovora: 9. 9. 2020 Društvene vesti in druge aktivnosti S10 Acta Chim. Slov. 2021, 68, (1), Supplement Marko BUNARKIC ANALIZA SINTEZNIH POSTOPKOV IN STRUKTUR SREBROVIH KOORDINACIJSKIH SPOJIN Z IMIDAZOLIDIN-2-TIONI IN NAČRTOVANJE SINTEZ ANALOGNIH SREBROVIH KOORDINACIJSKIH SPOJIN S 1,2,4-TRIAZOLIDIN-3-TIONI. Mentor: doc. dr. Jakob Kljun Datum zagovora: 9. 9. 2020 Amela AVDIČEVIC UPORABA METOD TERMIČNE ANALIZE ZA PREUČEVANJE TERMIČNE STABILNOSTI OLJ Mentorica: izr. prof. dr. Romana Cerc Korošec Datum zagovora: 9. 9. 2020 David HOČEVAR MERJENJE TEMPERATURE V INDUSTRIJI Mentor: doc. dr. Bojan Šarac Datum zagovora: 9. 9. 2020 Andrej KUČIČ PRETOČNI SISTEMI Z DINAMIKO PRVEGA IN DRUGEGA REDA Mentor: izr. prof. dr. Janez Cerar Datum zagovora: 10. 9. 2020 Špela NOVAK MIKROINKAPSULACIJA TRDNEGA JEDRNEGA MATERIALA Z MELAMINSKO FORMALDEHIDNO SMOLO Mentor: viš. pred. dr. Branko Alič Datum zagovora: 14. 9. 2020 Sara MARINKOVIC PIPERAZIN IN DERIVATI KOT LIGANDI V KOORDINACIJSKIH SPOJINAH BAKRA(II) Mentorica: doc. dr. Barbara Modec Datum zagovora: 18. 9. 2020 Vesna DAVIDOVIC OKOLJU PRIJAZNA SINTEZA PROPILEN KARBONATA Z UPORABO KATALIZATORJEV NA OSNOVI CERIJEVEGA DIOKSIDA Mentorica: prof. dr. Urška Lavrenčič Štangar Datum zagovora: 18. 9. 2020 Monika LIŠTER SINTEZA IN HIDROGENIRANJE SUBSTITUIRANEGA METIL AKRILATA Mentor: izr. prof. dr. Bogdan Štefane Datum zagovora: 24. 9. 2020 Tanja JANKO ELEKTROKEMIJSKA PRETVORBA CO2 V GORIVA Mentor: doc. dr. Črtomir Podlipnik Datum zagovora: 24. 9. 2020 Gaja GERBEC PRIPRAVA N-ZAŠČITENIH 5-ARIL-, 5-HETEROARIL-IN SORODNIH DERIVATOV 2H-PIRAN-2-ONOV TER NADALJNJE PRETVORBE Z DIELS-ALDERJEVIMI REAKCIJAMI DO BICIKLO[2.2.2]OKTENOV Mentor: doc. dr. Krištof Kranjc Datum zagovora: 25. 9. 2020 Sanja ZRNIC IZRAČUN PORAZDELITVE ELEKTROLITA MED IZOTROPNO RAZTOPINO IN NABITO REŽO Mentor: prof. dr. Jurij Reščič Datum zagovora: 29. 9. 2020 Lavra KOS ADENIN KOT KATION V HEKSAFLUORIDOTITANATNIH SOLEH Mentor: doc. dr. Andrej Pevec Datum zagovora: 5. 10. 2020 Klemen MOVRIN ATOMI IN MOLEKULE V ELEKTRIČNEM IN MAGNETNEM POLJU Mentor: prof. dr. 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Slov. 2021, 68, (1), Supplement Klara Laura KONDA KOFEIN KOT INHIBITOR KOROZIJSKIH PROCESOV V AGRESIVNEM MEDIJU Datum zagovora: 9. 9. 2020 Sandra KOPŠE VEČSTOPENJSKO ODSTRANJEVANJE TEŽKIH KOVIN IZ REČNIH SEDIMENTOV Datum zagovora: 3. 9. 2020 Matic KOŠIR PRIMERJAVA BIOPROCESOV S CELIČNO LINIJO CHO V RAZLIČNIH BIOREAKTORJIH Datum zagovora: 9. 9. 2020 Marjetka KOUTER KONTROLIRANO SPROŠČANJE FEBUKSOSTATA IN DOLOČITEV KONCENTRACIJE V SERUMU IN URINU Datum zagovora: 7. 9. 2020 Adriana KRALJ CITOTOKSIČNOST IN UČINKOVITOST C87-INHIBITORJA TUMORJE - NEKROTIZIRAJOČEGA FAKTORJA ALFA NA HUMANIH CELIČNIH LINIJAH Datum zagovora: 9. 9. 2020 Analina KRALJ PRIMERJAVA ENERGETSKE UČINKOVITOSTI VEČSTANOVANJSKIH OBJEKTOV V RAZLIČNIH OBDOBJIH Datum zagovora: 9. 9. 2020 Špela Vivijana KRISTAN DOLOČEVANJE TEŽKIH KOVIN IN TOKSIČNIH ORGANSKIH SPOJIN V EKSTRAKTIH LISTOV LAWSONIA INERMIS L Datum zagovora: 28. 5. 2020 Eva KROPUŠEK POVRŠINSKA FUNKCIONALIZACIJA MAGNETNIH NANODELCEV (MND) ZA UPORABO V OSMOTSKIH PROCESIH ČIŠČENJA ODPADNIH VOD Datum zagovora: 3. 9. 2020 Mitja MACUR STABILNOST INKAPSULIRANE HRENOVE PEROKSIDAZE Datum zagovora: 21. 10. 2020 Kaja MAKOTER DOLOČEVANJE LAHKOHLAPNIH IN TEŽJEHLAPNIH SPOJIN V EKSTRAKTIH RASTLIN IZ DRUŽINE ZINGIBERACEAE Z UPORABO RAZLIČNIH KROMATOGRAFSKIH TEHNIK Datum zagovora: 3. 9. 2020 Ines MASTEN DETEKCIJA OHRANJENIH VOD NA MEDPROTEINSKIH POVRŠINAH Datum zagovora: 9. 9. 2020 Lara METLIKA BIOOZNAČEVALCI ZA NAPOVED ODZIVA NA VEDOLIZUMAB PRI BOLNIKIH S KRONIČNO VNETNO ČREVESNO BOLEZNIJO Datum zagovora: 17. 9. 2020 Erik MIHELIČ RAZVOJ METODE ZA DOLOČEVANJE EKVIVALENTNE TOČKE PRI OBARJANJU FLUORIDOV IZ EKSTRAKTA PO OBDELAVI IZRABLJENIH KATODNIH OSTANKOV ELEKTROLIZE ALUMINIJA Datum zagovora: 9. 9. 2020 Anže NOVAK VALIDACIJA METODE ZA DOLOČANJE Zn(II), Cd(II) IN Pb(II) Z UPORABO INTERNEGA STANDARDA Datum zagovora: 9. 9. 2020 Monika OSTROŠKO POVEZAVA EPIGENETSKIH IN EKSPRESIJSKIH MARKERJEV PRI RAKU GLAVE IN VRATU Datum zagovora: 9. 9. 2020 Nika OZIS VPLIV STRUKTURE TIOLA NA POLIMERIZACIJE V KOLOIDNIH MEDIJIH Datum zagovora: 9. 9. 2020 Janez PALČNIK VPLIV CINHONIDINA NA TIOL-EN POLIMERIZACIJO MULTIFUNKCIONALNIH TIOLOV Z ALKENI Datum zagovora: 9. 9. 2020 Nika PETELINŠEK PRIDOBIVANJE NANODELCEV Mg1+xFe2-2xTixO4 S TERMIČNIM RAZKROJEM Datum zagovora: 8. 7. 2020 Domen PETRIČ OPTIMIRANJE INAKTIVACIJE ENCIMOV V GRAHAM MOKI Datum zagovora: 23. 9. 2020 Vito PRŠA BIOLOŠKA AKTIVNOST NARAVNIH EKSTRAKTOV IZ ZAČIMB Datum zagovora: 9. 9. 2020 Matjaž RANTAŠA SINTEZA IN KARAKTERIZACIJA HIDROFILNIH MAGHEMITNIH NANODELCEV PREVLEČENIH S CITRATOM Datum zagovora: 3. 9. 2020 Simona SEDONJA INHIBICIJSKA UČINKOVITOST PROPOLISA V NAMEN ZAŠČITE KONSTRUKCIJSKIH MATERIALOV Datum zagovora: 9. 9. 2020 Tjaša SKARLOVNIK ANALIZA REZULTATOV ZA POVRŠINSKO ZAŠČITO Datum zagovora: 3. 9. 2020 Gal SLAČEK VPLIV EKSTRAKCIJSKE METODE NA VSEBNOST BIOAKTIVNIH KOMPONENT BRUSNICE (VACCINIUM VITIS-IDAEA) IN ANTIMIKROBNI POTENCIAL Datum zagovora: 9. 9. 2020 Društvene vesti in druge aktivnosti S10 Acta Chim. Slov. 2021, 68, (1), Supplement Saša STANKOVIC SINTEZA IN KARAKTERIZACIJA MEZOPOROZNIH KISLINSKIH KATALIZATORJEV Datum zagovora: 9. 9. 2020 Julija STRUNČNIK ODPRTOKODNI PROCESNI SIMULATOR DWSIM KOT ALTERNATIVA PROCESNEMU SIMULATORJU ASPEN PLUS Datum zagovora: 17. 8. 2020 Lucija ŠPES VSEBNOST ŠČITNIČNIH HORMONOV IN VITAMINA D3 V KRVNEM SERUMU Datum zagovora: 28. 9. 2020 Mihaela ŠVEC STABILNOST LAKAZE V SC CO2 Datum zagovora: 9. 9. 2020 Eva TRATNIK VPLIV POSTOPKA EKSTRAKCIJE NA KVALITETO EKSTRAKTA IZ YERBA MATE (ILEX PARAGUARIENSIS) Datum zagovora: 3. 9. 2020 Katarina TURK PROIZVODNJA AMONIJAKA IZ ODPADNIH PLINOV Datum zagovora: 3. 9. 2020 Staša VELCL ANALIZA KEMIJSKEGA PROSTORA, KI GA ZAVZEMAJO PROTIBAKTERIJSKE UČINKOVINE Datum zagovora: 9. 9. 2020 Andrej ZIDARIČ VPLIV TEMPERATURE NA IZOLACIJO KOMPONENT INDUSTRIJSKE KONOPLJE Datum zagovora: 9. 9. 2020 Matija ZIMŠEK PREVERJANJE LASTNOSTI SIROTKINEGA KONCENTRATA PO NANOFILTRACIJI Datum zagovora: 7. 9. 2020 Nika ŽURGA ANALIZA SLEDOV TEŽKIH KOVIN V KAPLJICI VZORCA S SISTEMOM LABORATORIJA NA ČIPU Datum zagovora: 28. 8. 2020 DIPLOME - VISOKOŠOLSKI STROKOVNI ŠTUDIJ VISOKOŠOLSKI STROKOVNI ŠTUDIJ - 1. 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Slov. 2021, 68, (1), Supplement Noemi SEP Kaja SMODIŠ ODSTRANJEVANJE TEŽKIH KOVIN IZ TRDNE UPORABA TEKOČE FRAKCIJE DIGESTATA BLATA FRAKCIJE DIGESTATA AKTIVNEGA BLATA S POMOČJO IZ ČISTILNIH NAPRAV KOT VIRA NUTRIENTOV ZA MAGNETNIH NANODELCEV IMOBILIZIRANE ALGE Datum zagovora: 9. 9. 2020 Datum zagovora: 3. 9. 2020 Društvene vesti in druge aktivnosti S10 Acta Chim. Slov. 2021, 68, (1), Supplement UNIVERZA V NOVI GORICI FAKULTETA ZA PODIPLOMSKI ŠTUDIJ 1. januar - 31. december 2020 DOKTORATI PODIPLOMSKI ŠTUDIJSKI PROGRAM ZNANOSTI O OKOLJU - 3. STOPNJA Klemen TERAN HOUSEHOLD AND ROAD DUST AS INDICATORS OF AIRBORNE PARTICULATE MATTER ELEMENTAL COMPOSITION Mentorja: dr. Gorazd Žibret in prof. dr. Mattia Fanetti Datum zagovora: 1. 12. 2020 FAKULTETA ZA ZNANOSTI O OKOLJU 1. januar - 31. december 2020 MAGISTERIJI ŠTUDIJSKI PROGRAM OKOLJE - 2. STOPNJA Mateja PEČNIK OSNUTEK STRATEGIJE ZA PREHOD MESTNE OBČINE NOVA GORICA DO OBČINE BREZ ODPADKOV Mentor: prof. dr. 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Typically, technical articles are not based on new experiments. Preparation of Submissions Text of the submitted articles must be prepared with Microsoft Word. Normal style set to single column, 1.5 line spacing, and 12 pt Times New Roman font is recommended. Line numbering (continuous, for the whole document) must be enabled to simplify the reviewing process. For any other format, please consult the editor. Articles should be written in English. Correct spelling and grammar are the sole responsibility of the author(s). Papers should be written in a concise and succinct manner. The authors shall respect the ISO 80000 standard [1], and IUPAC Green Book [2] rules on the names and symbols of quantities and units. The Système International d'Unités (SI) must be used for all dimensional quantities. Graphics (figures, graphs, illustrations, digital images, photographs) should be inserted in the text where appropriate. The captions should be self-explanatory. Lettering should be readable (suggested 8 point Arial font) with equal size in all figures. Use common programs such as MS Excel or similar to prepare figures (graphs) and ChemDraw to prepare structures in their final size. Width of graphs in the manuscript should be 8 cm. Only in special cases (in case of numerous data, visibility issues) graphs can be 17 cm wide. All graphs in the manuscript should be inserted in relevant places and aligned left. The same graphs should be provided separately as images of appropriate resolution (see below) and submitted together in a ZIP file (Graphics ZIP). Please do not submit figures as a Word file. In graphs, only the graph area determined by both axes should be in the frame, while a frame around the whole graph should be omitted. The graph area should be white. The legend should be inside the graph area. The style of all graphs should be the same. Figures and illustrations should be of sufficient quality for the printed version, i.e. 300 dpi minimum. Digital images and photographs should be of high quality (minimum 250 dpi resolution). On submission, figures should be of good enough resolution to be assessed by the referees, ideally as JPEGs. High-resolution figures (in JPEG, TIFF, or EPS format) might be required if the paper is accepted for publication. Tables should be prepared in the Word file of the paper as usual Word tables. The captions should appear above the table and should be self-explanatory. References should be numbered and ordered sequentially as they appear in the text, likewise methods, tables, figure captions. When cited in the text, reference numbers should be superscripted, following punctuation marks. It is the sole responsibility of authors to cite articles that have been submitted to a journal or were in print at the time of submission to ACSi. Formatting of references to published work should follow the journal style; please also consult a recent issue: 1. J. W. Smith, A. G. White, Acta Chim. Slov. 2008, 55, 1055-1059. 2. M. F. Kemmere, T. F. Keurentjes, in: S. P. Nunes, K. V. Peinemann (Ed.): Membrane Technology in the Chemical Industry, Wiley-VCH, Weinheim, Germany, 2008, pp. 229-255. 3. J. Levec, Arrangement and process for oxidizing an aqueous medium, US Patent Number 5,928,521, date of patent July 27, 1999. 4. L. A. Bursill, J. M. Thomas, in: R. Sersale, C. Coll e I a, R. Aiell o (Eds.), Recent Progress Report and Discussions: 5th International Zeo I ite Conference, Naples, Italy, 1980, Gianini, Naples, 1981, pp. 25-30. 5. J. Szegezdi, F. Csizmadia, Prediction of dissociation constant using microconstants, http://www. che-maxon.com/conf/Prediction_of_dissociation _con-stant_using_microco nstants.pdf, (assessed: March 31, 2008) Titles of journals should be abbreviated according to Chemical Abstracts Service Source Index (CASSI). Special Notes • Complete characterization, including crystal structure, should be given when the synthesis of new compounds in crystal form is reported. • Numerical data should be reported with the number of significant digits corresponding to the magnitude of experimental uncertainty. • The SI system of units and IUPAC recommendations for nomenclature, symbols and abbreviations should be followed closely. Additionally, the authors should follow the general guidelines when citing spectral and analytical data, and depositing crystallographic data. • Characters should be correctly represented throughout the manuscript: for example, 1 (one) and l (ell), 0 (zero) and O (oh), x (ex), D7 (times sign), B0 (degree sign). Use Symbol font for all Greek letters and mathematical symbols. • The rules and recommendations of the IUBMB and the International Union of Pure and Applied Chemistry (IUPAC) should be used for abbreviation of chemical names, nomenclature of chemical compounds, enzyme nomenclature, isotopic compounds, optically active isomers, and spectroscopic data. Društvene vesti in druge aktivnosti S10 Acta Chim. Slov. 2021, 68, (1), Supplement • A conflict of interest occurs when an individual (author, reviewer, editor) or its organization is involved in multiple interests, one of which could possibly corrupt the motivation for an act in the other. Financial relationships are the most easily identifiable conflicts of interest, while conflicts can occur also as personal relationships, academic competition, etc. The Editors will make effort to ensure that conflicts of interest will not compromise the evaluation process; potential editors and reviewers will be asked to exempt themselves from review process when such conflict of interest exists. When the manuscript is submitted for publication, the authors are expected to disclose any relationships that might pose potential conflict of interest with respect to results reported in that manuscript. In the Acknowledgement section the source of funding support should be mentioned. The statement of disclosure must be provided as Comments to Editor during the submission process. • Published statement of Informed Consent. Research described in papers submitted to ACSi must adhere to the principles of the Declaration of Helsinki (http://www.wma.net/e/policy/ b3.htm). These studies must be approved by an appropriate institutional review board or committee, and informed consent must be obtained from subjects. The Methods section of the paper must include: 1) a statement of protocol approval from an institutional review board or committee and 2), a statement that informed consent was obtained from the human subjects or their representatives. • Published Statement of Human and Animal Rights.When reporting experiments on human subjects, authors should indicate whether the procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008. If doubt exists whether the research was conducted in accordance with the Helsinki Declaration, the authors must explain the rationale for their approach and demonstrate that the institutional review body explicitly approved the doubtful aspects of the study. When reporting experiments on animals, authors should indicate whether the institutional and national guide for the care and use of laboratory animals was followed. • To avoid conflict of interest between authors and referees we expect that not more than one referee is from the same country as the corresponding au-thor(s), however, not from the same institution. • Contributions authored by Slovenian scientists are evaluated by non-Slovenian referees. • Papers describing microwave-assisted reactions performed in domestic microwave ovens are not considered for publication in Acta Chimica Slovenica. • Manuscripts that are not prepared and submitted in accord with the instructions for authors are not considered for publication. Appendices Authors are encouraged to make use of supporting information for publication, which is supplementary ma- terial (appendices) that is submitted at the same time as the manuscript. It is made available on the Journal's web site and is linked to the article in the Journal's Web edition. The use of supporting information is particularly appropriate for presenting additional graphs, spectra, tables and discussion and is more likely to be of interest to specialists than to general readers. When preparing supporting information, authors should keep in mind that the supporting information files will not be edited by the editorial staff. In addition, the files should be not too large (upper limit 10 MB) and should be provided in common widely known file formats to be accessible to readers without difficulty. All files of supplementary materials are loaded separately during the submission process as supplementary files. Proposed Cover Picture and Graphical Abstract Image Graphical content: an ideally full-colour illustration of resolution 300 dpi from the manuscript must be proposed with the submission. Graphical abstract pictures are printed in size 6.5 x 4 cm (hence minimal resolution of 770 x 470 pixels). Cover picture is printed in size 11 x 9.5 cm (hence minimal resolution of 1300 x 1130 pixels) Authors are encouraged to submit illustrations as candidates for the journal Cover Picture*. The illustration must be related to the subject matter of the paper. Usually both proposed cover picture and graphical abstract are the same, but authors may provide different pictures as well. * The authors will be asked to contribute to the costs of the cover picture production. Statement of novelty Statement of novelty is provided in a Word file and submitted as a supplementary file in step 4 of submission process. Authors should in no more than 100 words emphasize the scientific novelty of the presented research. Do not repeat for this purpose the content of your abstract. List of suggested reviewers List of suggested reviewers is a Word file submitted as a supplementary file in step 4 of submission process. Authors should propose the names, full affiliation (department, institution, city and country) and e-mail addresses of five potential referees. Field of expertise and at least two references relevant to the scientific field of the submitted manuscript must be provided for each of the suggested reviewers. The referees should be knowledgeable about the subject but have no close connection with any of the authors. In addition, referees should be from institutions other than (and countries other than) those of any of the authors. Authors declare no conflict of interest with suggested reviewers. Authors declare that suggested reviewers are experts in the field of submitted manuscript. How to Submit Users registered in the role of author can start submission by choosing USER HOME link on the top of the page, then choosing the role of the Author and follow the relevant link for starting the submission process. Prior to submission we strongly recommend that you familiarize yourself with the ACSi style by browsing the journal, particularly if you have not submitted to the ACSi before or recently. Društvene vesti in druge aktivnosti S10 Acta Chim. Slov. 2021, 68, (1), Supplement Correspondence All correspondence with the ACSi editor regarding the paper goes through this web site and emails. Emails are sent and recorded in the web site database. In the correspondence with the editorial office please provide ID number of your manuscript. All emails you receive from the system contain relevant links. Please do not answer the emails directly but use the embedded links in the emails for carrying out relevant actions. Alternatively, you can carry out all the actions and correspondence through the online system by logging in and selecting relevant options. Proofs Proofs will be dispatched via e-mail and corrections should be returned to the editor by e-mail as quickly as possible, normally within 48 hours of receipt. Typing errors should be corrected; other changes of contents will be treated as new submissions. Submission Preparation Checklist As part of the submission process, authors are required to check off their submission's compliance with all of the following items, and submissions may be returned to authors that do not adhere to these guidelines. 1. The submission has not been previously published, nor is it under consideration for publication in any other journal (or an explanation has been provided in Comments to the Editor). 2. All the listed authors have agreed on the content and the corresponding (submitting) author is responsible for having ensured that this agreement has been reached. 3. The submission files are in the correct format: manuscript is created in MS Word but will be submitted in PDF (for reviewers) as well as in original MS Word format (as a supplementary file for technical editing); diagrams and graphs are created in Excel and saved in one of the file formats: TIFF, EPS or JPG; illustrations are also saved in one of these formats. The preferred position of graphic files in a document is to embed them close to the place where they are mentioned in the text (See Author guidelines for details). 4. The manuscript has been examined for spelling and grammar (spell checked). 5. The title (maximum 150 characters) briefly explains the contents of the manuscript. 6. Full names (first and last) of all authors together with the affiliation address are provided. Name of author(s) denoted as the corresponding author(s), together with their e-mail address, full postal address and telephone/fax numbers are given. 7. The abstract states the objective and conc I usions of the research concisely in no mo re than 150 words. 8. Keywords (minimum three, maximum six) are provided. 9. Statement of novelty (maximum 100 words) clearly explaining new findings reported in the manuscript should be prepared as a separate Word file. 10. The text adheres to the stylistic and bibliographic requirements outlined in the Author guidelines. 11. Text in normal style is set to single column, 1.5 line spacing, and 12 pt. Times New Roman font is recommended. All tables, figures and illustrations have appropriate captions and are placed within the text at the appropriate points. 12. Mathematical and chemical equations are provided in separate lines and numbered (Arabic numbers) consecutively in parenthesis at the end of the line. All equation numbers are (if necessary) appropriately included in the text. Corresponding numbers are checked. 13. Tables, Figures, illustrations, are prepared in correct format and resolution (see Author guidelines). 14. The lettering used in the figures and graphs do not vary greatly in size. The recommended lettering size is 8 point Arial. 15. Separate files for each figure and illustration are prepared. The names (numbers) of the separate files are the same as they appear in the text. All the figure files are packed for uploading in a single ZIP file. 16. Authors have read special notes and have accordingly prepared their manuscript (if necessary). 17. References in the text and in the References are correctly cited. (see Author guidelines). All references mentioned in the Reference list are cited in the text, and vice versa. 18. Permission has been obtained for use of copyrighted material from other sources (including the Web). 19. The names, full affiliation (department, institution, city and country), e-mail addresses and references of five potential referees from institutions other than (and countries other than) those of any of the authors are prepared in the word file. At least two relevant references (important recent papers with high impact factor, head positions of departments, labs, research groups, etc.) for each suggested reviewer must be provided. Authors declare no conflict of interest with suggested reviewers. Authors declare that suggested reviewers are experts in the field of submitted manuscript. 20. Full-colour illustration or graph from the manuscript is proposed for graphical abstract. 21. Appendices (if appropriate) as supplementary material are prepared and will be submitted at the same time as the manuscript. 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Slov. 2021, 68, (1), Supplement Koristni naslovi Slovensko kemijsko druStva stovwifan chwnicaf society Slovensko kemijsko društvo www.chem-soc.si e-mail: chem.soc@ki.si Wessex Institute of Technology www.wessex .ac.uk SETAC www.setac.org European Water Association http://www.ewa-online.eu/ European Science Foundation www. esf .org O EFCE European Federation of Chemical Engineering https://efce.info/ International Union of Pure and Applied Chemistry https://iupac.org/ Novice europske zveze kemijskih društev EuChemS naj'dete na: t&r EuChemS Brussels News Updates i i.irupr'.sn, ChoiniLdl Snarly http://www.euchems.eu/newsletters/ Društvene vesti in druge aktivnosti Planetarni centrifugalni mikser ARM-310CE (M Brezkontaktno mešanje in disperzija Tudi za zelo viskozne materiale v Širok spekter uporabe Atraktivna cena Donau Lab d.o.o., Ljubljana Tbilisijska 85 SI-1000 Ljubljana www.donaulab.si office-si@donaulab.com VITAMIN DB Krka ZAGOTOVITE SI SONCE gi Edini kot zdravilo brez recepta Brez konzervansov in barvil PRED UPORABO NATANČNO PREBERITE NAVODILO! O TVEGANJU IN NEŽELENIH UČINKIH SE POSVETUJTE Z ZDRAVNIKOM ALI S FARMACEVTOM. ©(g)© l_J cu 1/1 cu ONAL INSTITUTE OF CHEMISTRY Hajdrihova 19 1000 Ljubljana Slovenia www.ki.si Basic and applied research on materials, life sciences, biotechnology, chemical engineering, structural and theoretical chemistry, analytical chemistry and environmental protection, In line with the priority areas of the EU Research and Innovation: nanotechnology, genomics and biotechnology for health, sustainable development, climate change, energy efficiency and quality and safety of food. We expand knowledge and technology transfer to the domestic and foreign pharmaceutical, chemical, automotive and nanobiotechnologycal industries. We are aware of the power of youth, so we transfer our knowledge on younger generations with providing many means of collaboration. contact: mladi@ki.si ActaChimica Slovenica Acta ChimicaSlovenica The use of continuous micro- and mesoscale systems offers several advantages for biotransformations with free biocatalysts, both in process development and in the realization of efficient sustainable productions (see page 1). Acta ChimicaSlc ActaOmmcdiSlc Slovenica Year 2021, Vol. 68, No. 1 ActaChimicaSlc ActaChimic'dSlc Slovenica ActaChimicaSlc ActaChlmkaSk Slovenica Acta ChimicaSlc ActaChumcaSk Slovenica Acta ChimicaSlc ActaChimica Slovenica Acta ChimicaSlc ActaChimicaSk Slovenica Acta ChimicaSlc ActaChimicaSk Slovenica Acta ChimicaSlc ActaChimicaSk Slovenica //?' my \I0 Coirosion f . Acta ChimicaSlc ActaChimicaSk Slovenica ActaChimicaSk ActaC himica Slovenica Acta ChimicaSlc ActaChimicaSk Slovenica Acta ChimicaSlc ActaChimicaSk Slovenica Acta ChimicaSlc ActaChimicaSk Slovenica