4 n Year 2022, Vol. 69, No. 1 ActaChimicaSlovenica ActaChimicaSlovenica ActaChimicaSlovenica ActaChimicaSlovenica SlovenicaActaChim A cta C him ica Slovenica 69/2022 Pages 1–250 Pages 1–250 n Year 2022, Vol. 69, No. 1 http://acta.chem-soc.si 1 69/2022 1 ISSN 1580-3155 Glucose-sensitive biosensors are known as glucose-oxidase, protein, and phenyl boronic acid based systems. Sugar-sensitive polymeric particles are produced via crosslinker and monomer in one step and surfactant-free emulsion polymerization technique. Sensitivity of the polymer particles to sugar molecules is monitored in glucose/fructose rich media EDITOR-IN-CHIEF EDITORIAL BOARD ADVISORY EDITORIAL BOARD 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 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 David Šarlah, University of Illinois at Urbana-Champaign, USA; Università degli Studi di Pavia, Italy Ivan Švancara, University of Pardubice, Czech Republic Jiri Pinkas, Masaryk University Brno, Czech Republic Gašper Tavčar, Jožef Stefan Institute, Slovenia Ennio Zangrando, University of Trieste, Italy Chairman Branko Stanovnik, Slovenia Members Udo A. Th. Brinkman, The Netherlands Attilio Cesaro, Italy Vida Hudnik, Slovenia Venčeslav Kaučič, Slovenia Željko Knez, Slovenia Radovan Komel, Slovenia Stane Pejovnik, Slovenia Anton Perdih, Slovenia Slavko Pečar, Slovenia Andrej Petrič, Slovenia Boris Pihlar, Slovenia Milan Randić, Des Moines, USA Jože Škerjanc, Slovenia Đurđa Vasić-Rački, Croatia Marjan Veber, Slovenia Gorazd Vesnaver, Slovenia Jure Zupan, Slovenia Boris Žemva, Slovenia Majda Žigon, Slovenia 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 Izdaja – Published by: SLOVENSKO KEMIJSKO DRUŠTVO – SLOVENIAN CHEMICAL SOCIETY Naslov redakcije in uprave – Address of the Editorial Board and Administration Hajdrihova 19, SI-1000 Ljubljana, Slovenija Tel.: (+386)-1-476-0252; Fax: (+386)-1-476-0300; E-mail: chem.soc@ki.si Izdajanje sofinancirajo – Financially supported by: National Institute of Chemistry, Ljubljana, Slovenia Jožef Stefan Institute, Ljubljana, Slovenia Faculty of Chemistry and Chemical Technology, University of Ljubljana, Slovenia Faculty of Chemistry and Chemical Engineering, University of Maribor, Slovenia Acta Chimica Slovenica izhaja štirikrat letno v elektronski obliki na spletni strani http://acta.chem-soc.si. 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Articles in this journal are published under the   Creative Commons Attribution 4.0 International License – Graphical Contents Graphical Contents ActaChimicaSlovenica ActaChimicaSlovenica SlovenicaActaChimica Year 2022, Vol. 69, No. 1 1–12 Analytical chemistry Fabrication of Zirconium Silicate Reinforced Superhydrophobic Coating for the Evaluation of Corrosion-Resistance Mubarak Ali Muhamath Basha and Sathiya Srinivasan 13–29 Organic chemistry Synthesis of Fused Quinoline Derivatives with Antiproliferative Activities and Tyrosine Kinases, Pim-1 Kinase Inhibitions Rafat Milad Mohareb, Rehab Ali Ibrahim, Amira Mohamed Elmetwally and Marwa Soliman Gamaan 30–38 Organic chemistry Glucose-Decorated Silica-Molybdate Complex: A novel Catalyst for Facile Synthesis of Pyrano[2,3-d]- Pyrimidine Derivatives Arezoo Pourkazemi, Negin Asaadi, Mahnaz Farahi, Ali Zarnegaryan and Bahador Karami Scientific pAper Graphical Contents 60–72 General chemistry The Effect of Heuristics on the Reasoning of the Pre- Service Science Teachers on the Topic of Melting and Boiling Point Gülen Önal Karakoyun and Erol Asiltürk 49–59 Analytical chemistry Proposal of an HPLC/UV/FLD Screening Method for the Simultaneous Determination of Ten Antibiotics in Environmental Waters Idalia Francisca Carmona-Alvarado, María de la Luz Salazar-Cavazos, Noemí Waksman de Torres, Aurora de Jesús Garza-Juárez, Lidia Naccha-Torres, Jose Francisco Islas and Norma Cavazos-Rocha 39–48 Organic chemistry Synthesis of Glucose/Fructose Sensitive Poly(ethylene glycol) Methyl Ether Methacrylate Particles with novel Boronate Ester Bridge Crosslinker and their Dye Release Applications Şeküre Yildirim, Hasan Akyildiz and Zeynep Çetinkaya 73–80 Organic chemistry Synthesis of the new 1-(7-Methoxy-1-benzofuran- 2-yl)-3-(4-methylphenyl)prop-2-en-1-one and Controlling of Spectroscopic, optical and Conductivity Properties by Concentration Demet Coskun, Mehmet Fatih Coskun and Bayram Gunduz 81–90 Biochemistry and molecular biology Effects of Individual and Co-exposure of Copper oxide nanoparticles and Copper Sulphate on nile Tilapia Oreochromis niloticus: nanoparticles Enhance Pesticide Biochemical Toxicity Özgür Fırat, Rabia Erol and Özge Fırat Graphical Contents 108–115 Analytical chemistry A novel Solid-State PVC-Membrane Potentiometric Dopamine-Selective Sensor Based on Molecular Imprinted Polymer Nurşen Dere, Zuhal Yolcu and Murat Yolcu 98–107 Applied chemistry Synthesis and Application of Silica Supported Calix[4] arene Derivative as a new Processing Aid Agent for Reducing Hysteresis of Tread Rubber Compounds Used in Low Rolling Resistance Tires Seyedeh Nazanin Sadat-Mansouri, Nasrin Hamrahjou, Saeed Taghvaei-Ganjali and Reza Zadmard 91–97 Organic chemistry A Facile Synthesis of Bioactive Five- and Six-membered N-heterocyclic Aromatic Compounds Using AlCoFe2o4 as a Green Catalyst Fatemeh Mostaghni, Homa Shafiekhani and Nosrat Madadi Mahani 116–124 Organic chemistry Combustion Synthesis of nano Fe2o3 and its Utilization as a Catalyst for the Synthesis of Nα-Protected Acyl Thioureas and Study of Anti-bacterial Activities Raghavendra Mahadevaiah, Lalithamba Haraluru Shankraiah and Latha Haraluru Kamalamma Eshwaraiah 125–132 Analytical chemistry Acetyl Cellulose Film with 18-crown-6 Ether for Colorimetric Phosgene Detection Martin Lobotka, Vladimír Pitschmann and Zbyněk Kobliha Graphical Contents 157–166 inorganic chemistry Synthesis, Characterization, X-Ray Crystal Structures and Antibacterial Activity of Zinc(II) and Vanadium(V) Complexes Derived from 5-Bromo-2-((2-(methylamino) ethylimino)methyl)phenol Cheng Liu 147–156 inorganic chemistry Synthesis, Structure, Thermal Decomposition and Computational Calculation of Heterodinuclear niII – ZnII Complexes Yaprak Gürsoy Tuncer, Hasan Nazır, Kübra Gürpınar, Ingrid Svoboda, Nurdane Yılmaz, Orhan Atakol and Emine Kübra İnal 133–146 inorganic chemistry Modulation of Cerium Carbonate Crystal Growth by Polyvinylpyrrolidone using Density Functional Theory Deyun Sun, Yanhong Hu, Mao Tang, Ze Hu, Peng Liu, Zhaogang Liu and Jinxiu Wu 167–186 chemical education The Students’ Perceptions Using 3DChemMol Molecular Editor for Construction and Editing of Molecular Models Danica Dolničar, Bojana Boh Podgornik and Vesna Ferk Savec 187–199 Organic chemistry Synthesis of new Regioisomers of 5-nitro-1,4- naphthoquinone, Evaluation of Antioxidant and Catalase Inhibition Activities Aesha F. SH. Abdassalam, Nahide Gulsah Deniz, Cigdem Sayil, Mustafa Ozyurek, Emin Ahmet Yesil and Huseyin Salihoglu Graphical Contents 227–234 General chemistry Synthesis, Crystal Structure and Separation Performance of p-tert-butyl(tetradecyloxy)calix[6]arene Wei Zhang, Zhi-qiang Cai, Xiao-min Shuai, Wei Li, Qiu-chen Huang, Ruo-nan Chen, Qi-qi Zang, Fei-fei Li and Tao Sun 217–226 Materials science Metal and non-Metal Modified Titania: the Effect of Phase Composition and Surface Area on Photocatalytic Activity Boštjan Žener, Lev Matoh, Martin Reli, Andrijana Sever Škapin and Romana Cerc Korošec 200–216 inorganic chemistry Metal Based Bioactive nitrogen and oxygen Donor Mono and Bis Schiff Bases: Design, Synthesis, Spectral Characterization, Computational Analysis and Antibacterial Screening Sajjad Hussain Sumrra, Wardha Zafar, Sabaahatul Ain Malik, Khalid Mahmood, Syed Salman Shafqat and Saira Arif 235–242 inorganic chemistry Two oxidovanadium(V) Complexes with Hydrazone Ligands: Synthesis, Crystal Structures and Catalytic oxidation Property Yan Lei 243–250 Organic chemistry Crystallography and DFT Studies of Synthesized Tetraketones Elma Veljović, Krešimir Molčanov, Mirsada Salihović, Una Glamočlija, Amar Osmanović, Nevzeta Ljubijankić and Selma Špirtović-Halilović Graphical Contents 1Acta Chim. Slov. 2022, 69, 1–12 Basha and Srinivasan: Fabrication of Zirconium Silicate Reinforced ... DOI: 10.17344/acsi.2021.6519 Scientific paper Fabrication of Zirconium Silicate Reinforced Superhydrophobic Coating for the Evaluation of Corrosion-Resistance Mubarak Ali Muhamath Basha1,* and Sathiya Srinivasan1 Department of Chemistry, Chikkaiah Naicker College, Erode-638004. Tamilnadu, India * Corresponding author: E-mail: mubarakscience@gmail.com Received: 07-19-2021 Abstract The present work investigates on anodisation of aluminium in 1.0 M sodium oxalate and methodically evaluates the in- fluence of zirconium silicate as an additive. The effect of additive upon structure, morphology, micro hardness and com- position of the coating formed under various anodising conditions has been examined comprehensively. The surface of the coating was modified by stearic acid and its immersion time was optimized. The dependence of surface morphology, kinetic parameters, and microstructural characteristics of the coating on electrolyte /additive concentration, anodising time, and the temperature has also been inspected. X-ray diffraction (XRD), Scanning Electron Microscopy (SEM) com- bined with EDAX studies indicates the beneficial role of zirconium silicate towards the formation of crystalline coating with improved corrosion-resistant characteristics. The static water contact angle on the surface-modified coatings was 122° ± 0.4°. This contact angle of super hydrophobic coating has been improved by KOH treatment (152.76o ± 0.4°) which is obtained under optimized conditions exhibit the corrosion resistance (1.68 × 108 Ω cm–1) which is nearly 8 times higher than that of bare aluminium (8.36 × 101 Ω cm–1). The efficacies of the surface-modified coatings against bacteria that are commonly encountered in marine (Desulfovibrio desulfuricans) and medical applications (Staphylococcus au- reus and Escherichia coli) are also demonstrated. Keywords: Anodisation; aluminium; sodium oxalate; corrosion resistance. 1. Introduction Aluminium is selected for their optimal combina- tion of physical and mechanical properties.1 Another benefit, which may be just as significant from an environ- mental standpoint, is that aluminium components may be recycled with relatively little energy use.2 In damp en- vironments, however, this thin layer is reactive and sus- ceptible to corrosion and contamination.3 Anodisation of aluminium is a well-established and easy method in com- parison with micro-arc discharge oxidation (MDO), gas flame spray, plasma thermal spray, physical vapour depo- sition, and high-temperature glass enamelling methods to progress the applicability of aluminium.4–5 Since, ano- disation is an electrochemical process, its conditions (voltage, time, temperature) and composition of electro- lytes together, play a central role in the characteristics of the coating.  In this connection, numerous investigations devoted to exploring the relationship between the composition of the electrolytes (e.g., sulfuric acid, chromic acid, phos- phoric acid, or oxalic acid) and anodic behaviour of alu- minium. However, because the acid molecules confined within the holes may cause more corrosion, the porous type oxide layer is not desirable for corrosion prevention.6 To resolve this issue, the weak electrolytes are preferred. To enhance further the corrosion resistance, abrasion resis- tance, and electrical insulation of the oxide coating and to increase the coating thickness, additive salts have been added into the electrolytic solution as investigated by nu- merous studies.7–9 Scarcely, reports are available upon the use of zirconium silicate as an additive for anodisation of aluminium, except for our earlier study on aluminium an- odisation with Lithium sulphate-zirconium silicate bath, 10 which illustrated that significant enhancement in corro- sion protection and micro hardness behaviour of nano- composite coatings. Super hydrophobicity, found on many natural sur- faces, the most classic example being the lotus leaf, has in- spired researchers around the world for its unique charac- 2 Acta Chim. Slov. 2022, 69, 1–12 Basha and Srinivasan: Fabrication of Zirconium Silicate Reinforced ... teristics such as self-cleaning,11 water repellence,12 an- ti-icing,13 anti-corrosion,14 and oil-water separation.15 The presence of a low energy layer over a rough hierarchi- cal structure is crucial for constructing a hydrophobic surface.16 So far, a large number of approaches have been successfully used to develop superhydrophobic surfaces, including chemical vapour deposition,17 chemical etch- ing,18 sol-gel,19 solution immersion,20 hydrothermal syn- thesis,21 and laser fabrication,22 etc., Anodisation is an effective technique to fine-tune the surface morphology trough constructing superhydrophobic surface.23 Prepa- ration of the superhydrophobic surface of the aluminium substrate by anodisation in phosphoric acid followed by low-temperature plasma treatment is reported.24 Investi- gation about a superhydrophobic surface on aluminium alloy via anodising in an electrolyte consisting of sulfuric acid, oxalic acid, and sodium chloride, followed by poly- propylene coating is also attempted successfully.25 Here- tofore, researchers coated an anodized aluminium alloy surface with RF-sputtered polytetrafluoroethylene to generate a superhydrophobic surface.26 In terms of re- al-world applications, fabricating hydrophobic alumini- um alloy surfaces with superior corrosion resistance and chemical stability is critical.27 In this regard, herein we report the anodic be- haviour of aluminium in the presence and absence of a zirconium silicate additive using sodium oxalate as an electrolyte. The parameters such as the concentration of the electrolyte/additive, voltage, temperature, etc., were studied to establish the correlation with growth kinetic parameters namely thickness and growth rate. The anod- ic coating formed which was immersed in stearic acid for surface modification and the immersion time was varied for optimization. We have also studied the mechanical properties, microstructural characterisation, chemical composition, and electrochemical corrosion behaviour of the coating which is prepared with and without the addition of zirconium silicate additive in non-conven- tional weak electrolyte namely sodium oxalate followed by surface modification of anodic coating. Further, we have also carried out the corrosion behaviour of anodic layer and surface modified anodic layer formed under various experimental conditions are determined by Tafel polarization and electrochemical impedance spectrosco- py (EIS) analyses. 2. Experimental Methods 2. 1. Materials Sodium oxalate, zirconium silicate, sulphuric acid, sodium chloride, chromic acid, phosphoric acid, per chlo- ric acid, ethanol, stearic acid, and acetone were purchased from Aldrich chemicals (Aldrich, India). All of the chemi- cals used were analytical grade and were used as received. Deionized water was used in all of the studies. 2. 2. Preparation of Anodic Coating High purity aluminium (99.999% pure, AL104, Met- tler-Toledo International. Inc.) The plate of thickness 0.5 mm was used for this investigation. Surface pre-treatment is a successive step involved in the process of anodisation. The surface pre-treatment process was accomplished by following the procedure reported elsewhere.28 Aluminium was cleaned and degreased with the use of ultrasonicator by using acetone, water and ethanol as a medium. The sur- face cleaned aluminium samples were subjected to the thermal annealing process at 450 °C for 30 min. The etching process is performed by immersing the samples in 5% sodium hydroxide for 2 min at room tem- perature (35° ± 1 °C) to cast off the native oxide layer. The resulted aluminium samples were subjected to electro pol- ishing in a mixture consists of perchloricacid:ethanol (1: 3) under an applied voltage of 20 V, which is maintained for 3 min. The pre-treated samples thus obtained were used for anodising studies in a two-electrode configuration using a direct current power supply (Aplab Model: 05 A/30 V and 0–1 A/120 V) and we determined the weight of every speci- men. Graphite sheet is used as a cathode and the sample to be anodised acts as an anode in an electrolytic bath com- prising 1.0 M sodium oxalate with and without zirconium silicate additive. The anodised coatings obtained from the bath containing zirconium silicate concentration of 0, 0.1 g/L, 0.2 g/L, 0.3 g/L to 0.4 g/L are designated as SO, Zr1, Zr2, Zr3 and Zr4 respectively. Anodised specimens were cleaned with de-ionized water pursued by drying in the N2 atmo- sphere and their weight is determined. 1 cm2 working sur- face is used for the study and the rest of the surface was in- sulated using an epoxy resin. All the experimental studies were carried out in aerated and stirred conditions. These experiments were repeated in triplicate to acquire accurate results. The anodic coating formed under an optimized con- dition was immersed in stearic acid for surface modification at various time intervals such as 15 and 30 min. These two samples are designated as SA1 and SA2. The steps involved in the process is schematically depicted in Scheme S1. 2. 3. Characterisation of Anodic Coating The crystalline nature of the coating was examined using a Philips X-Pert X-ray Diffractometer (XRD) with the use of CuKa radiation of wavelength, λ = 1.54 Å with Step Size [° 2θ] 0.0300. Scanning Electron Microscope (SEM, FEI - QUANTA-FEG 250, Japan) is used to examine the surface morphologies of the coating. Field emission scanning electron microscope and energy-dispersive X-ray spectroscopic studies were carried out to analyse the microstructure and elemental composition of the coatings. Vickers micro hardness indenter is used to quantify the micro hardness of the specimen. (Tester MH6, USA). Thickness tester (Touchstone1) is employed for measuring the coating thickness non-destructively. Contact Angle as- sociated with SA1 and SA2 surfaces was measured with a 3Acta Chim. Slov. 2022, 69, 1–12 Basha and Srinivasan: Fabrication of Zirconium Silicate Reinforced ... contact angle measuring system (OCA20, Data physics Corporation, Germany) under ambient temperature. All the measurements were conducted at five positions on each sample with 4 μL DI water droplets. To evaluate the durability of the Zr3, SA1 and SA2 samples, the influences of exposure to air at room temperature (35o ± 1 oC) were systematically investigated. 2. 4. Preparation of Superhydrophobic Coating To examine the corrosion resistance of the superhy- drophobic coatings were carried out for optimized Zr3 (Zir- conium silicate), SA1 (stearic acid) and SA2 (stearic acid & KOH) samples for comparison. The SA1 was prepared by using the Zr3 sample immersed in stearic acid 3g/L for 15 min. To further enhance the super hydrophobicity, KOH treatment was applied to prepare by using the SA1 im- mersed in stearic acid 10 g/L KOH for 30 min (SA2). 2. 5. Evaluation of Corrosion Resistance Tafel polarisation investigations and electrochemical impedance spectroscopy (EIS) in 3.5 percent NaCl (using an electrochemical workstation (600TM Potentiostat/ Gal- vanostat, Inc.) are used to examine the coatings’ electro- chemical corrosion behaviour. These studies were execut- ed in the three-electrode configuration that consists of Ag/ AgCl/saturated KCl as a reference electrode, a platinum wire as a counter electrode, and aluminium as a working electrode. Fully aerated conditions are maintained during the experiments. Tafel polarisation studies were performed by applying potential in the range of +2 V to –2 V with a scan rate of 1 mV/s. Using the method of extrapolation of anodic and cathodic sections, current density associated with corrosion (Icorr) and the resistance offered for charge transfer reaction (RP) is determined and tabulated as shown in Table. 1. Rp is determined with the use of Stern- Geary equation as follows,29 (1) Where in Icorr signifies corrosion current density, ca- thodic/anodic Tafel constants as (ba and bc). Icorr and Ecorr are obtained from the intercept of Tafel slopes. EIS plots are recorded at frequencies between 100 kHz and 0.01 Hz with 12 points per decade. The amplitude of the sinusoidal potential signal was set as 5 mV. The impedance spectra obtained were analysed by fitting with an appropriate Ran- del’s equivalent circuit. 2. 6. Marine Bacterial Studies The marine bacteria used in this study were the sul- phate reducing strains of Desulfovibrio desulfuricans (ATCC#14563). The bacterium was cultured at 37 °C in a modified Postgate’s C medium used for enrichment cul- ture, which contained 35 g NaCl, 0.5 g KH2PO4, 0.06 g CaCl2 2H2O, 2 g MgSO4 7H2O, 1 g yeast extract, 0.004 g FeSO4 7H2O, and 0.3 g sodium citrate in 1 L deionized wa- ter. The medium was autoclaved at 121°C and 20 psi for 15 min.30–31 after the stipulated period (7 days), the SA1 and SA2 were washed twice with phosphate-buffered saline (19 PBS, pH 7.4). The cells were detected by live/dead staining. 200 mL of dye mixture (100 mL acridine orange, green flu- orescence in live cells, and 100 mL ethidium bromide, red fluorescence in dead cells in distilled water) was mixed with 2 mL cell suspension in a well plate. The suspension was studied right away using an Olympus Ti-Eclipse in- verted fluorescence microscope to record the data under the magnification of 400 x of the instrument. 3. Results and Discussion 3. 1. Anodising Process Optimization Studies A methodical comprehensive evaluation of the influ- ence of the composition of the additive into the electrolyte, period of anodisation process, and anodising temperature upon morphology and kinetics of the coating has been carried out to comprehend the fundamental mechanism behind the formation of the highly corrosion-resistive lay- er. To recognize the role of additive towards tuning the mi- crostructural features, morphology, micro-hardness, and corrosion resistance of the coating was carried out in sodi- um oxalate in the presence and absence of zirconium sili- cate. The optimal concentration of sodium oxalate was kept as 1.0 M. 3. 1. 1. Effect of Zirconium Silicate Concentration Additive The formation and properties of the coating are greatly manipulated by the concentration of zirconium silicate as illustrated by Fig S1a, wherein the concentra- tion of zirconium silicate is varied from 0.1 g/L to 0.4 g/L in 1.0 M sodium oxalate at constant voltage of 55 V and temperature (35° ± 1° C). This behavior could be ex- plained due to the existence of two competing reactions, namely oxide coating growth and its dissolution. Based on an earlier study, a sequence of reactions occurs during the anodisation process together with the combination of metal ions with OH and SiO3 to form Al (OH)3 and Al2 (SiO3)3. In the current report, the presence of zirconium silicate (0.3 g/L) leads to develop the coating with a high thickness (82 µm). The coating obtained using sodium oxalate without additive results in poor growth performance (thickness: 70 µm; growth rate: 0.9 µm/min). From Fig. S1a, it is intended that the zirconium silicate concentration directly impacts the thickness (growth rate: 1.2 µm/min) of the coating. 4 Acta Chim. Slov. 2022, 69, 1–12 Basha and Srinivasan: Fabrication of Zirconium Silicate Reinforced ... Therefore, to throw the light about the kinetics of the coat- ing, zirconium silicate concentration is varied from 0.1 g/L to 0.4 g/L (0.1 g/L, 0.2 g/L, 0.3 and 0.4 g/L) and exploration is directed towards to develop the coating uniformly. When the concentration of zirconium silicate increases from 0.1 g/L to 0.4 g/L, the silicate ion concentration in- creases and these ions interact with Al2 (SiO3)3. The credi- ble rationale behind this performance is strongly related to the presence of zirconium silicate additive resulting in non-uniform cooling near the anode surface. The coating thickness has been increased when zir- conium silicate is added into the sodium oxalate solu- tion. At this juncture, anodic coating with more thick- ness is favoured rather than thin oxide coating and the incorporation of ZrO2 into the oxide layer is high. So the thickness of the coating also upsurges. Owing to pitting attack on pre-formed barrier oxide which is formed un- der the low zirconium silicate concentration (< 0.1 g/L), the surface becomes non-uniform. However, when the additive concentration exceeds optimum, the dissolution rate of the coating by anodisation dominates over its for- mation. When the concentration of zirconium silicate is in- creased beyond the optimal level (0.3 g/L) ease of access and inner movement of the oxygen ions (O2–) or hydrox- ide ions (OH−) towards the Al/Aluminium oxide inter- face improves, which react with Al3+ ions that are shifted around outwards from aluminium surfaces. This tenden- cy results in declined growth parameters because the en- tire specimen is being surrounded by an oxide layer. From the results, it is inferred that the rate of deposition, morphological properties, and thickness of the coating which strongly depends on zirconium silicate additive concentration. This observation is analogous to previous studies wherein aluminium anodisation is carried out in weak electrolyte with the addition of silicate materi- al.13–14 3. 1. 2. Effect of Anodisation Voltage Fig. S1b depicts the influence of anodisation voltage on growth kinetics which is evaluated by carrying out an- odisation at constant temperature and optimized zirconi- um silicate additive concentration on varying voltage from 45 V to 65 V. From the results, it can be construed that the thickness and growth rate characteristics go lin- early with voltage up to 55 V and started to decline after that. Since the oxidation occurs at a slow rate, the coating thickness (62 µm) at a lower voltage (45 V), has been formed with deprived mechanical properties. When volt- age maintained is optimal (55 V), the system can afford more driving force for the ions needed for the growth, and it makes it likely to increase the incorporation of ZrO2 into the oxide coating. However, at the higher volt- age (65 V), growth parameters are decreased significantly owing to both local joule heat effect and of the presence of more number of Zr2+ ions that competes with alumin- ium oxide deposition. In addition to this, dissociation of sodium oxalate has also been accelerated at higher ap- plied voltage. The pH of the solution is increased from 8 to 9 due to the release of Na+ ions that in sequence fa- vours the coating dissolution. When the anodising volt- age is 65 V, there is a high degree of hydration and ion incorporation into the anodic coating.  3. 1. 3. Effect of Temperature Fig. S1c  illustrates the influence of anodising tem- perature on the properties of the coating when anodisa- tion is carried out at a constant voltage of 55 V under zir- conium silicate concentration at various temperatures (20° to 35 °C). When anodisation is carried out at low temperature (at 20 °C), both rates of deposition of oxide coating and its dissolution are slowed down. Therefore, at this low temperature (≤ 20 °C), the desired thickness is not achieved. When the temperature of the electrolyte in- creased (35° ± 1 °C) the growth properties of the coating condition, the heat produced is not disseminated effi- ciently and distributed uniformly throughout the electro- lyte. The nucleation and the growth of the coating are not favoured and afterward, the coating starts to dissolve chemically. Ultimately, burning or breaking of the coat- ing in the highly heated solution which leads to a decrease in thickness. It is worth mentioning that unlike conven- tional sulphuric acid anodisation, the process employed here does not require ice-cold conditions, thereby mak- ing the process simple. Since anodisation temperature straightforwardly influences the rate of mass transfer of O2– and Al3+ ions and inward diffusion of O2– ions are assisted when the temperature is increased beyond room temperature.  3. 1. 4. Effect of Process Dduration Fig. S1d shows the effect of duration 30 min, 60 min, 90 min of anodising process on coating properties when the reaction is carried out under applied voltage of 55 V at 30 °C using electrolyte bath comprising 0.3 g/L zirconium silicate additive in 1.0 M sodium oxalate. At the beginning step, anodised layers formation prevail over the chemical dissolution of it and in compliance with Faradays’ law growth kinetics of the anodised layer which is a linear de- pendence on anodising period. As the duration of the an- odising process is prolonged beyond 30 min, aluminium reacted with oxide ions and there is a weight gain in the coating because the rate of deposition is directly propor- tional to process duration. This oxide coating continues to grow up to a definite period (60 min) to cover the entire surface. Accordingly, when anodisation is performed for 90 min, the thickness of the coating went down attribut- able for cracking or breaking of the coating. Inefficient dis- sipation of the amount of heat evolved during the process 5Acta Chim. Slov. 2022, 69, 1–12 Basha and Srinivasan: Fabrication of Zirconium Silicate Reinforced ... is the underlying reason for this behaviour. For example, the thickness of the coating is determined to be 60 µm, 70 µm and 68 µm for 30 min, 60 min, and 90 min respectively. These results exemplify the significance of the optimal du- ration of the anodising process. Based on the aforemen- tioned studies and observations, the anodisation process carried out at room temperature (35o ± 1 oC) using 1.0 M sodium oxalate and 0.3 g/L zirconium silicate bath under an applied voltage of 55V for 65 min is determined to be the optimal condition. 3. 2. Micro-structural Analysis 3. 2. 1. XRD Fig.1 shows the X-ray diffraction pattern of alumini- um without additive (SO) and with additives for Zr1, Zr2, Zr3 and Zr4 samples. The pristine SO shows peaks at 17o (101), 18o (102), 23o (106), 26o (113), 30o (204), 42o (109), 51o (204), 62o (325), 87o (204) and 95o (204) which con- firmed the presence of monoclinic structure. However, the addition of zirconium silicate at a constant current density exhibits the peaks located at 38o, 63o and 74o with pre- ferred orientation of (109), (326) and (337) respectively. In the case of other additive concentration, there is little vari- ability in peak position and depicted clearly in Fig. 2. The observed peaks are well-matched with the standard data (JCPDS card No: 88-1609). The δ-Al2O3 phase is observed for these compositions. Based on the anodising condi- tions, the coatings formed are mainly crystalline. It is found that the coatings after the addition of additives show some traces of zirconia and alumina. Using the De- bye-Scherer equation, the average crystalline size for SO, Zr1, Zr2, Zr3 and Zr4 were 26.8 nm, 22.3 nm, 20.18 nm, 18.7 nm and 21.55 nm respectively. 3. 2. 2. Microstructure and Composition of the Coating Microstructures related to SO, Zr1, Zr2, Zr3 and Zr4 coating are represented by Fig. S2 that depicts the dif- ferences in the morphological features. Fig. S2 of SO por- trays distinctly dissimilar morphological characteristics from that of Fig S2 (b-d) which are associated with SO, Zr1, Zr2, Zr3 and Zr4 coating. SO microstructure exhib- ited the pores of different dimension that is formed as a result of hydrogen evolution during the process in addi- tion to the many cracks that are visible which are formed due to the drying shrinkage. When zirconium silicate is introduced into the electrolyte, the coating becomes smoother and denser. The pores of different shapes are distributed all over the surface of the specimen after in- troducing the additive. Noticeably dissimilar surface morphologies prove that the addition of zirconium sili- cate into the electrolyte has a greater control over deter- mining the surface morphology of the coatings. As the zirconium silicate concentration becomes higher (0.4 g/L), aluminium will be consumed at a higher rate near the bottom of the pore which is allowing continued growth of the porous layer. The surface of Zr1, Zr2, Zr3 and Zr4 coating is smooth which is beneficial to resist the corrosion. Change in the morphology has been observed when the concen- tration of zirconium silicate was increased (0.3 g/L) even beyond the optimal level. Micro-sized fissures are detected with Zr4, which are created as a result of the local heating effect on the surface produced because of uneven electric field distribution and detained breakdown of the oxide layer. At lower zirconium silicate concentrations (0.1 g/L and 0.2 g/L) pores are formed along with the surfaces of micro-cracks. Only under the optimal concentration (0.3 g/L), the compact homogenous microstructure is devel- oped. These results illustrate that the presence of zirconi- um silicate and its concentration are influential compo- nents that determine the morphology and properties of the coating. 3. 3. SEM Cross-sectional Studies The cross-sectional SEM images of the SO and Zr3 are represented in Fig. 2. The thickness was measured from the cross-sectional images. The average thickness of the SO and Zr3 is 5.1 μm and 8.2 μm, respectively. It could be visibly inferred that the quality and thickness of the coating has been improved by adding zirconium silicate into the electrolyte bath. Closer examination of images re- veals that a thin deposited layer of about 1 to 1.2 µm thick- ness comprising three distinct regions such as a dense bar- rier section at the Al /oxide interface, centre porous sec- tion followed by a columnar part of the outer surface. The outward growth of layer and retarded dissolution rate are the possible reasons for enhanced thickness value (82 µm) exhibited by Zr3. Figure 1. X- ray diffraction pattern of anodized aluminium coating comprising SO, Zr1, Zr2, Zr3 and Zr4. 6 Acta Chim. Slov. 2022, 69, 1–12 Basha and Srinivasan: Fabrication of Zirconium Silicate Reinforced ... 3. 4. EDS Analysis The elements present in the various specimens are analysed by EDS spectra. Fig. S3 shows EDS spectrum of the SO and Zr3 in which both are fabricated in sodium oxalate electrolyte with the absence of additive and with the presence of optimal content (0.3g/L) of zirconium sil- icate additive. Aluminium and oxygen are the main con- stituents that are present in SO and Zr3 at 1.486 Kev (71.4 wt. % for SO and 55.8 wt. % for Zr3) and 2.307 Kev (27.2 wt. % that for SO and 39 wt. % for Zr3). In contrast, the EDS spectrum of Zr3 shows well-defined peaks for Zr and Si at 4.508 Kev and 0.525 Kev respectively. This reveals the incorporation of the additive into the coating. Similarly, the weight percentage of aluminium and oxygen in sam- ple SO are found to be 71.4% and 86.4% respectively. Whereas, in the case of Zr3 sample, the weight percentage of both elements decreases to 55.8% and 39%. The exis- tence of Zr and Si (0.6 wt. % and 0.3 wt. %), which evi- dences the role of additive towards the facilitating forma- tion of the compact and mechanically stable coating. 3. 5. Studies on Micro Hardness Micro hardness associated with bare aluminium, SO, Zr1, Zr2, Zr3 and Zr4 samples are measured using Vickers micro hardness tester and are represented in the Fig S4. The average micro hardness values associated with Zr1 (0.1 g/L), Zr2 (0.2 g/L), Zr3 (0.3g/L) and Zr4 (0.4g/L) are 372 HV, 381 HV, 410 HV and 393 HV respectively. The micro hardness value increases with the concentration of zirconium silicate from 0.1g/L to 0.4g/L and then decreas- es. The maximum micro hardness value (410 HV) is ob- tained for 0.3 g/L concentration. This is related to the change in surface morphology of the coating when zirco- nium silicate is incorporated into the electrolyte. These silica particles are helpful in preventing the generation of dislocations, the spread of cracks and in restraining grain boundary slides, which eventually resulted in improved mechanical properties. In addition, the increased driving force experiences at the metal / oxide interface, which pro- motes the reactions and on this account, the defect is formed over the surface. 3. 6. Studies on Corrosion Resistance of Anodised Layers Electrochemical corrosion characteristics of SO, Zr1, Zr2, Zr3, Zr4 samples under investigation are relatively as- sessed by Tafel polarisation and electrochemical imped- ance spectroscopy (EIS) techniques in 3.5% NaCl solution. 3. 6. 1. Tafel Polarisation Studies In a 3.5% NaCl solution, the potentiodynamic polar- isation behaviour of different coatings and bare alumini- um is shown in Fig. 3. Table. S1 summarizes the parame- ters obtained by fitting the potentiodynamic curves. It can be deduced from the Fig.3 that the corrosion potential of in anodic region associated with SO, Zr1, Zr2, Zr3 and Zr4 coating display a drastic shift in the direction of the posi- tive potential as compared to bare aluminium (–0.699V) illustrating the enhanced corrosion resistance of the coat- ing. Icorr is associated with bare aluminium is highest. (27 × 10–4 µA cm–2) but due to passivation of the anodised samples which exhibit low Icorr values as evident from Ta- ble. S1. Icorr is determined to be low for the Zr3 specimen demonstrating its better corrosion-resistance. The ob- served values are consistent with earlier literature re- ports.31–33 The corrosion resistance of the Zr3 (6.70 × 104 Ω cm–2) is high compared to SO (1.51 × 104 Ω cm–2), Zr1 (2.19 × 104 Ω cm–2), Zr2 (2.35 × 104 Ω cm–2) and Zr4 (6.44 × 104 KΩ cm–2). The corrosion resistance of the coating Figure 2. Cross-sectional SEM images of SO, and Zr3 coating 7Acta Chim. Slov. 2022, 69, 1–12 Basha and Srinivasan: Fabrication of Zirconium Silicate Reinforced ... increases with the concentration of zirconium silicate up to 0.3 g/L and after that begins to decline. These results are compliant with the open circuit potential values. This shows that the incorporation of zirconium silicate into the electrolyte bath imparts mechanical strength to the coat- ing rather porous with smaller thickness is resulted when sodium oxalate alone is used as an electrolyte that produc- es a detrimental effect on its corrosion resistance. The dif- ference in the resistance and polarisation behaviour is mainly owing to the structure and morphology of the coating. Since the corrosion rate depends on the coating thickness, the coating with lower thickness and high po- rous nature will undergo the corrosion readily due to un- complicated accessibility of destructive ions. However when zirconium silicate is incorporated with sodium oxa- late electrolyte, both pores size and the number of pores decreased. By increasing the concentration of zirconium silicate additive, highly thick non-porous morphology has been formed that helps to acquire more corrosion-resis- tant property due to the restricted penetrations of corro- sive ions into the coatings. This type of coating is generated when the optimal zirconium silicate concentration (0.3 g/L) is maintained during anodisation at appropriate temperature and volt- age. However, when zirconium silicate concentration is exceeded above 0.3 g/L, the thickness is reduced through modified rate of deposition and perceptibly and the corro- sion potential decreases. As a result of their lower corro- sion current densities, thicker coatings with less porous nature reflect higher corrosion resistance. On the other hand, shape and porosity, not just thickness, have a signif- icant effect in determining the specimen’s corrosion resis- tance. substrate interface of aluminium specimens Fig. 4 and Fig. 5 show an electrical equivalent circuit model used in this study for the fitting analysis, which is a simplified electro- chemical model that has been consistently reported for the comparative Nyquist of SO, Zr1, Zr2, Zr3 and Zr4 samples in 3.5% of NaCl obtained at open circuit potential after im- mersion of the samples for 500s. Based on the equivalent circuit model proposed, these EIS curves were best fitted and the resulting parameters are tabulated in Table. 1. Rp represents a measure of corrosion resistance that indicates the extent of protection against corrosion and is inversely proportional to Icorr. Like the Tafel polarisation studies, EIS data also indicate that corrosion resistance improved, sub- stantially after anodising as compared to the bare alumini- um (Rf: 3011 Ω cm–2; Cdl: 11.25μFcm–2). Fascinatingly, the coating generated on the surface that plays a dual role in preventing corrosion both through decreasing rate of charge transfer process and diffusion across the surface layer. From Table.1 it is apparent that Zr4 (1607 Ω cm–2) samples display high resistance compared to other samples under study, which demonstrates a role of silicate additive towards protecting surface corrosion. It is implied that wa- ter and electrolytic solution penetrate through the anodic layer which composed of larger pores to initiate the corro- sive attack in case of the thin layered SO sample, but Zr1, Zr2, Zr3 and Zr4 samples comprising dense, close-packed network restricts the penetration of corrosive ions through them. Rp and Cdl are noted to be minimised for Zr3 layer, which is due to a diminution in local dielectric constant and/or to an increase in electrical double layer thickness. On comparing passivation current densities, it is in- ferred that the anodic coating obtained using sodium oxa- late-zirconium silicate electrolyte provides an excellent bar- rier for defending against corrosion. The features of the coating are reflected by the high-frequency section of the Figure 3. Tafel polarization plots for bare aluminium and various anodized coating immersed in 3.5% NaCl solution (a) bare Al, (b) SO, (c) Zr1, Zr2, Zr3 and Zr4. 3. 6. 2. Electrochemical Impedance Studies EIS measurements were performed with bare alu- minium, SO, Zr1, Zr2, Zr3 and Zr4 coating to distinguish the corrosion kinetics across the modified coating and Figure 4. Nyquist plot curves associated with bare aluminium and various anodized aluminium coating immersed in 3.5% NaCl solu- tion Zr1, Zr2, Zr3 and Zr4 anodic layers. Inset of figure shows that of bare Al and SO. 8 Acta Chim. Slov. 2022, 69, 1–12 Basha and Srinivasan: Fabrication of Zirconium Silicate Reinforced ... spectrum, while the lower portion of the spectrum is con- nected to the Faradic reaction occurring on the aluminium surface, according to the literature.24–25 EIS of SO is differ- ent from that of all other samples which are consistent with the formation of a stable passive layer on these surfaces. Low capacitance that arises owing to strong interaction of small organic molecules on the surface that results in poor dissolution reaction. The surface homogeneity of coating also helps to develop good corrosion resistance, which higher resistance (Rf) compared to bare aluminium. By re- stricting the access of aggressive ions causing corrosion.26–30 3. 6. 3. Wettability Studies The aforementioned studies confirm that Zr3 exhibits suitable surface and further it is subjected to hydrophobic treatment. It can be seen from Fig. 6 that contact angle (CA) associated with the anodised aluminium surface. After Stea- ric acid 3 g/L immersion, the contact angle of Zr3 is in- creased to 122o ± 0.4° for 15 min (SA1) treatment. When the specimen is further subjected to KOH 10 g/L treatment the contact angle is increased to 152.76o ± 0.4° (SA2). Among SA1 and SA2, the SA2 sample shows a higher contact angle. The wettability of a solid surface is strongly affected by both surface structure and chemical composition.34 3. 6. 4. Studies on Corrosion Behaviour of Hydrophobic Surface SA1 and Superhydrophobic Surface SA2 To examine the corrosion resistance of the hydro- phobic coating corrosion studies were carried out for Zr3, SA1 and SA2 samples for comparison. The results are shown in Fig. S5 and Fig. S6. Fig. S5 shows the Tafel polar- Table 1. Corrosion parameters derived from EIS Nyquist plot analysis associated with bare aluminium, SO and various Zr3 anodic layers under applied voltage of 45 V for 65 min at room temperature. Sample CPEf RF CPEdl Rct R1 (µF cm–2) (Ohm cm2) (µF cm–2) (Ohm cm2) (Ohm cm2) Bare Al 11.25 3011 198 1765 1298 SO 8.21 1023 209 1613 1267 Zr1 7.43 879 221 1658 1109 Zr2 6.02 674 227 1654 979 Zr3 5.45 476 238 1607 875 Zr4 5.77 489 241 1605 889 Figure 5. An electrochemical equivalent circuit model fitted for im- pedance data analysis of anodized aluminium layers in this study. Figure 6. Effect of various treatment duration on contact angle of the coating. Table 2. Corrosion parameters derived from EIS Nyquist plot analysis associated with SA1 and SA2. Sample CPEf RF CPEd Rct R1 (µF cm–2) (Ohm cm2) (µF cm–2) (Ohm cm2) (Ohm cm2) SA1 8.5 438 217 1564 778 SA2 7.8 341 252 1245 651 9Acta Chim. Slov. 2022, 69, 1–12 Basha and Srinivasan: Fabrication of Zirconium Silicate Reinforced ... isation curves for SA1 and SA2. Among all samples SA2 shows low Icorr (6.04 × 10–8 µA/cm2) and high Ecorr (–0.02 V) shifts towards a more positive direction compared to SA1 (Icorr: 7.01 × 10–7 µA/cm2; Ecorr: –0.321 V) and Zr3 (Icorr: 6.14 × 10–7 µA/cm2; Ecorr: –0.428V) as shown by Ta- ble S2. The obtained result shows that in the instance of SA2, chloride ion (Cl–) transport may have been severely hindered. This demonstrates that a covering with a larger Figure 7. Marine antibacterial activities of Zr3 specimens tested with S.Aureus, E.Coli and D.desulfuricans. 10 Acta Chim. Slov. 2022, 69, 1–12 Basha and Srinivasan: Fabrication of Zirconium Silicate Reinforced ... contact angle inhibits corrosion better. Here SA2 has a higher contact angle than SA1 and Zr3. Two key elements might be responsible for the increased corrosion resistance mechanisms. For starters, the SA2 is made up of hierarchi- cal micro-nanostructures that may readily trap significant volumes of air within the coating’s micro-/nano-pores. The trapped air acts as a passivation layer, protecting the alu- minium substrate from Cl attachment. Water transport against gravity is simple in a porous surface structure with a CA greater than 150°. Therefore, the NaCl solution can be pushed out from the pores of the SA1 by the pressure and the aluminium substrate could be effectively protected. Hence, the structure and properties are influence directly to its corrosion behaviour as exemplified by Tafel studies. Fig. S6 shows the impedance plots of Zr3, SA1, and SA2. Like Tafel studies, the SA2 sample shows higher corrosion resistance than other sample SA1 and Zr3. This model has been proposed for analysis of superhydrobhobic alumini- um surface previously by recent researchers.35–39 3. 6. 5. Marine Bacterial Activity The bacterial activity of Zr3 coating was tested against E.coli, S. aureus, and marine bacterial strain name- ly, D. desulfuricans for 24 h, which is shown in Fig. 7. From the results, it is shown that the zone of inhibition for Zr3 against S.aureus strains and D. desulfuricans is 10 mm (40 min), 12 mm (90 min). The zone of inhibition against D. desulfuricans is increased from 12 mm (40 min) to 15 mm (90 mm) respectively. The results show that the D. desulfu- ricans bacteria demonstrated higher activity than E.coli and S. aureus due to the minimal solid-liquid contact at the surface, weak surface interactions with bacteria, and low surface energy. The key to the antibacterial qualities is the self-cleaning concept, which allows for easy washing and eliminates the need for antibacterial chemicals. Due to the fact that this antibacterial design is simply structural, a product with permanent characteristics may be created for everyday use with minimum customer maintenance. 4. Conclusions In conclusion, this work reports on the fabrication of the coatings by anodisation of aluminium in 0.1M sodium oxalate with and without the addition of zirconium sili- cate. The results revealed that experimental conditions such as additive concentration, applied voltage, process time and temperature play a crucial role in coating forma- tion and tailoring of their surface properties which in- creases the thickness, growth rate, and micro hardness up to a certain concentration (0.3 g/L; Zr3) and then decreas- es. The maximum thickness (82 µm), growth rate (1.2 µm/ min), and micro hardness (410HV) are obtained at the voltage of 55V in room temperature (35 ±1 °C) for 60 min. The SEM and EDX results demonstrate that the addition of zirconium silicate into the electrolyte has an impact on the morphology as it favours the formation of dense and uni- form coating with fewer structure imperfections. The XRD studies confirmed the presence of δ alumina. Our studies further show that the coating formed with the addition of zirconium silicate has more corrosion resistance (6.70×104 Ω cm–2) when compared to the sodium oxalate electrolyte (1.0 M) alone as an electrolyte. To extend its applications in the marine field, the surface of the Zr3 sample is modi- fied to superhydrophobic (SA2). The superhydrophobic (SA2) sample shows higher corrosion resistance (1.68 x108 Ω cm-1), which is higher than the Zr3 sample (6.8 × 103 Ω cm-1). The contact angle of the coating for the SA1 and SA2 sample is found to be 122o ± 0.4o and 152.76o ± 0.4o. This contact angle of super hydrophobic coating is im- proved owing to base (KOH) treatment. The marine appli- cations and microbial activity were investigated against S. aureus (40 min: 10 mm; 90 min: 12 mm) and D.desulfuri- cans (40 min: 12 mm; 90 min: 15 mm). Thus the fabricated coating is compatible with industrial, biological, biomedi- cal, optical, and aerospace applications with the specifical- ly focused utility to mitigate the marine corrosion.  5. References 1. E. W. Lee, T. Oppenheim, K. Robinson, B. Aridkahari, N. Neylan, D. Gebreyesus, M. Richardson, M. Arzate, C. Bove, M. Iskandar, C. Sanchez, E. Toss, I. Martinez, D. Arenas, J. Ogren, J. Mclennan, R. Clark, W. E. Frazier, O. S. Es-Said, Eng. Fail. Anal., 2007, 14, 1538–1549. DOI:10.1016/j.engfailanal.2006.12.008 2. M. G. Mueller, M. Fornabaio, G. Zagar, A. Mortensen, Acta Ma- ter., 2016, 105, 165–175. 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Slov. 2022, 69, 1–12 Basha and Srinivasan: Fabrication of Zirconium Silicate Reinforced ... Povzetek V tem delu smo preučili anodizacijo aluminija v 1.0 M raztopini natrijevega oksalata in metodološko ovrednotili vpliv cirkonijevega silikata kot aditiva. Podrobneje smo preučili vpliv aditiva na strukturo, morfologijo, mikrotrdoto in sestavo prevleke pod različnimi pogoji anodizacije. Površina prevleke je bila obdelana s stearinsko kislino, čas stika prevleke s kislino pa je bil optimiziran. Poleg že navedenega smo preučili tudi odvisnost morfologije površine, kinetskih paramet- rov in mikrostrukturnih lastnosti prevleke od elektrolita oziroma koncentracije aditiva, časa anodizacije in temperature. Analize z rentgensko praškovno difrakcijo (XRD) in vrstično elektronsko mikroskopijo (SEM), kombinirano z energij- skodisperzijsko spektroskopijo (EDS), so pokazale ugoden vpliv aditiva na nastanek kristalinične prevleke z izboljšanimi protikorozijskimi lastnostmi. Prevleke z aditivom so superhidrofobne. Z namenom povečanja statičnega kontaktnega kota vode, ki je znašal 122° ± 0.4°, so bile prevleke z aditivom obdelane s KOH, kar je pod najugodnejšimi pogoji kot povečalo na 152.76° ± 0.4°. Pri tem kotu je korozijska upornost sistema znašala 1.68 × 10 Ω cm−1, kar je skoraj osemkrat več kot pri čistem aluminiju (8.36 × 101 Ω cm−1). Prikazana je tudi učinkovitost tovrstnih površinsko modificiranih prevlek proti trem različnim vrstam bakterij; morski bakteriji Desulfovibrio desulfuricans in medicinsko relevantnima bakterijama Staphylococcus aureus ter Escherichia coli. Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License 13Acta Chim. Slov. 2022, 69, 13–29 Mohareb et al.: Synthesis of Fused Quinoline Derivatives with ... DOI: 10.17344/acsi.2021.6733 Scientific paper Synthesis of Fused Quinoline Derivatives with Antiproliferative Activities and Tyrosine Kinases, Pim-1 Kinase Inhibitions Rafat Milad Mohareb,1,* Rehab Ali Ibrahim,2 Amira Mohamed Elmetwally,3 and Marwa Soliman Gamaan1 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 Egyptian Drug Authority (NODCAR), P.O. 29, Cairo AR Egypt * Corresponding author: E-mail: raafat_mohareb@yahoo.com Received: 02-05-2021 Abstract Cyclohexan-1,3-dione (1) reacted with either 2-aminoprop-1-ene-1,1,3-tricarbonitrile (2a) or diethyl 3-amino-2-cy- anopent-2-enedioate (2b) to give the 5,6,7,8-tetrahydronaphthalene derivatives 3a and 3b, respectively. The latter com- pounds underwent further heterocyclization reactions to give the thieno[2’,3’:5,6]benzo[1,2-e][1,3]oxazine derivatives. On the other hand, the reaction of compound 1 with trichloroacetonitrile afforded the (2,2,2-trichloroethylidene)cy- clohexane derivative 14. The latter underwent a series of reactions to produce 2,3,6,7-tetrahydroquinazoline, dihydroth- ieno[2,3-h]isoquinoline, octahydrobenzo[h]quinazoline and dihydrothieno[2,3-h]isoquinoline derivatives. The synthe- sized compounds were tested toward six cancer cell lines where most of them gave high inhibitions with c-Met enzymatic activity, with tyrosine kinases and Pim-1 inhibitions. The results obtained will encourage further work through such compounds to produce optimized anticancer agents. Keywords: Cyclohexan-1,3-dione, trichloroacetonitrile, quinoline, isoquinoline, cytotoxicity 1. Introduction With its origins rooted in organic synthesis and me- dicinal chemistry, heterocyclic compounds present them- selves as a fundamental division of organic chemistry. De- fined by IUPAC as “cyclic compounds having as ring members atoms of at least two different elements” (IUPAC Gold Book 2015),1 heterocycles’ ring structures are in es- sence composed by elements other than carbon, where the most frequent substituents are oxygen, nitrogen and sul- fur.2,3 According to the heteroatom(s) present in the ring structures, heterocycles can be classified as oxygen, nitro- gen or sulfur based and, within each class, compounds are organized based on the size of the ring structure deter- mined by the total number of atoms.4 The type and size of ring structures, together with the substituent groups of the core scaffold, impact strongly on the physicochemical properties.2,5 Among the various clinical applications, het- erocyclic compounds have a considerable active role as an- ti-bacterial,6,7 anti-viral,8 anti-fungal,9 anti-inflmmatory10 and anti-tumor drugs.11–13 The engineering and rationale behind drug design are closely related to the strategic in- corporation of heterocyclic fragments with specific physic- ochemical properties. Potency and selectivity achieved through bioisosteric replacements, lipophilicity, polarity, and aqueous solubility can ultimately be fine-tuned to the point of altering and conditioning the possible mechanisms of action of pharmaceutical drugs in an attempt to obtain molecularly targeted agents.14 Despite their versatility and potential, as for any other pharmaceutical, there are several issues hindering wider application and further develop- ment of such compounds into market drugs. Oncology is one of the areas where this is perhaps most noticeable, par- tially due to the intrinsic limitations regarding main thera- peutic routes of chemotherapy, concomitant side effects and toxicity to healthy tissues. Such deleterious effects may be circumvented via selective targeting of delivery, passive- ly or actively into cancerous cells.15 It should be noted that 14 Acta Chim. Slov. 2022, 69, 13–29 Mohareb et al.: Synthesis of Fused Quinoline Derivatives with ... for some playmakers within the chemotherapy field, the success of “molecularly targeted agents”, such as imatinib are merely fortunate exceptions and that the number of success in this area is considerably low.16 Recent advances in interdisciplinary field of nanobiotechnology have led to the development of new inventive therapeutic strategies and drug delivery alternatives taking advantage of the ar- chitectural geniality of systems based on nanoscale devices particularly tailored to deliver drugs to a selected tissue.17–19 Recently our research group reported several reac- tions of cyclic β-diketones to produce thiazoles and thio- phene derivatives. The produced compounds showed high anti-proliferative activities against cancer cell lines togeth- er with high inhibitions toward tyrosine kinases.20–22 This encouraged us to continue this goal through the reaction of cyclohexan-1,3-dione with dimeric cyanomethylene and trichloroacetonitrile reagents together with using the produced molecule as a suitable starting material for sub- sequent heterocyclization to produce a variety of fused de- rivatives. The antiproliferative activities of the synthesized compounds and their inhibitions toward tyrosine kinases were determined. 2. Experimental 2. 1. General All melting points are uncorrected and were record- ed using an Electrothermal digital melting point appara- tus. IR spectra (KBr discs) were measured using a FTIR plus 460 or PyeUnicam SP-1000 spectrophotometer. 1H NMR spectra were measured using a Varian Gemini-300 (300 MHz) and Jeol AS 500 MHz instruments; spectra were recorded in DMSO-d6 as the solvent using TMS as the internal standard and chemical shifts are expressed as δ ppm. MS (EI) spectra were measured using Hewlett Packard 5988 A GC/MS system and GCMS-QP 1000 Ex Shimadzu instruments. Analytical data were obtained from the Micro-analytical Data Unit at Cairo University and were performed on Vario EL III Elemental analyzer. The anti-tumor evaluation has been carried out through the National Cancer Research Centerat Cairo, Egypt where the IC50 values were calculated. 2. 1. 1. General Procedure for the Synthesis of the 5,6,7,8-Tetrahydronaphthalene 3a,b Equimolar amounts of dry solids of compound 1 (1.12 g, 0.01 mol) and either of 2a (1.32 g, 0.01 mol) or 2b (2.14 g, 0.01 mol) and ammonium acetate (1.50 g) were heated in an oil bath at 120 °C for 1 h then were left to cool. The remain- ing product was triturated with diethyl ether and the formed solid product, in each case, was collected by filtration. 2,4-Diamino-5-oxo-5,6,7,8-tetrahydronaphthalene-1,3- dicarbonitrile (3a) Yellow crystals from 1,4-dioxane, yield 1.58 g (70%). Mp 256–258 °C. IR (KBr) νmax 3488–3352 (NH2), 3055 (CH, aromatic), 2223, 2220 (2CN), 1703 (CO), 1632 cm–1 (C=C); 1H NMR (DMSO-d6, 300 MHz) δ 4.93, 4.53 (s, 4H, D2O exchangeable, 2NH2), 2.93–2.80 (m, 4H, 2CH2), 1.98–1.28 (m, 2H, CH2); 13C NMR (DMSO-d6, 75 MHz) δ 174.2 (C-5), 127.9, 125.6, 124.9, 123.5, 121.8, 120.4 (C-1, C-2, C-3, C-4, C-5, C-6), 116.8, 116.3 (2CN), 40.6, 38.9, 17.4 (C-6, C-7, C-8); MS m/z 226 (M+, 36%). Anal. Cal- cd for C12H10N4O: C, 63.71; H, 4.46; N, 24.76. Found: C, 63.92; H, 4.79; N, 24.80. Ethyl 2-Amino-3-cyano-4-hydroxy-5-oxo-5,6,7,8-tet- rahydronaphthalene-1-carboxylate (3b) Orange crystals from ethanol, yield 1.89 g (69%). Mp 180–183 °C. IR (KBr) νmax 3554–3338 (OH, NH2), 3055 (CH, aromatic), 2220 (CN), 1708, 1689 (2CO), 1636 cm–1 (C=C); 1H NMR (DMSO-d6, 300 MHz) δ 10.26 (s, 1H, D2O exchangeable, OH), 4.92 (s, 2H, D2O exchangea- ble, NH2), 4.22 (q, 2H, J = 7.31 Hz, OCH2CH3), 2.80–2.96 (m, 4H, 2CH2), 1.98–1.28 (m, 2H, CH2), 1.12 (t, 3H, J = 7.31 Hz, OCH2CH3); 13C NMR (DMSO-d6, 75 MHz) δ 165.2, 164.3 (C-5, ester CO), 125.4, 123.0, 122.8, 122.5, 121.9, 120.5, 119.2 (C-1, C-2, C-3, C-4, C-5, C-6), 116.9 (CN), 50.3 (OCH2CH3), 40.1, 38.5, 17.1 (C-6, C-7, C-8), 16.2 (OCH2CH3); MS m/z 274 (M+, 28%). Anal. Calcd for C14H14N2O4: C, 61.31; H, 5.14; N, 10.21. Found: C, 61.26; H, 5.39; N, 10.36. 2. 1. 2. General Procedure for the Synthesis of the 5,6,7,8-Tetrahydronaphthalene Derivatives 4a,b A solution of either compound 3a (2.26 g, 0.01 mol) or 3b (2.74 g, 0.01 mol) in acetic acid (40 mL) and acetic anhydride (15 mL) was heated under reflux for 3 h then left to cool. The reaction mixture, in each case was evaporated under vacuum and the remaining product was triturated with ethanol and the formed solid product was collected by filtration. N-(3-Amino-2,4-dicyano-8-oxo-5,6,7,8-tetrahydron- aphthalen-1-yl)acetamide (4a) Pale yellow crystals from 1,4-dioxane, yield 1.82 g (68%). Mp 236–239 °C. IR (KBr) νmax 3464–3342 (NH2, NH), 3055 (CH, aromatic), 2223, 2220 (2CN), 1702, 1688 (2CO), 1630 cm–1 (C=C); 1H NMR (DMSO-d6, 300 MHz) δ 8.26 (s, 1H, D2O exchangeable, NH), 4.56 (s, 2H, D2O exchangeable, CH2), 3.02 (s, 3H CH3), 2.93–2.85 (m, 4H, 2CH2), 1.96–1.84 (m, 2H, CH2); 13C NMR (DMSO-d6, 75 MHz) δ 174.3, 166.2 (C-8, CO amide), 125.9, 123.9, 123.7, 122.5, 122.0, 121.6, 119.8 (C-1, C-2, C-3, C-4, C-5, C-6), 116.5, 116.4 (2CN), 40.6, 38.5, 17.6 (C-6, C-7, C-8), 24.8 (CH3); MS m/z 268 (M+, 44%). Anal. Calcd for C14H12N4O2: C, 62.68; H, 4.51; N, 20.88. Found: C, 62.93; H, 4.63; N, 20.68. 15Acta Chim. Slov. 2022, 69, 13–29 Mohareb et al.: Synthesis of Fused Quinoline Derivatives with ... Ethyl 4-Acetoxy-2-amino-3-cyano-5-oxo-5,6,7,8-tetrah ydronaphthalene-1-carboxylate (4b) Pale brown crystals from ethanol, yield 2.17 g (60%). Mp 158–161 °C. IR (KBr) νmax 3473–3328 (NH), 3055 (CH, aromatic), 2220 (CN), 1705, 1688 (2CO), 1630 cm–1 (C=C); 1H NMR (DMSO-d6, 300 MHz) δ 4.72 (s, 2H, D2O exchangeable, NH2), 4.23 (q, 2H, J = 6.56 Hz, OCH2CH3), 3.01 (s, 3H CH3), 2.96–2.82 (m, 4H, 2CH2), 1.96–1.81 (m, 2H, CH2), 1.12 (t, 3H, J = 6.56 Hz, OCH2CH3); 13C NMR (DMSO-d6, 75 MHz) δ 174.3, 166.1 (C-8, CO ester), 120.3, 121.8, 122.6, 123.2, 124.1, 125.1, 125.2 (C-1, C-2, C-3, C-4, C-5, C-6), 117.0 (CN), 50.2 (OCH2CH3), 40.1, 38.5, 17.3 (C-6, C-7, C-8), 24.8 (CH3), 16.6, 16.3 (two OCH2CH3); MS m/z 316 (M+, 30%). Anal. Calcd for C16H16N2O5: C, 60.75; H, 5.10; N, 8.86. Found: C, 60.43; H, 5.28; N, 8.90. 2. 1. 3. General Procedure for the Synthesis of the 3,4,7,8,9,10-Hexahydro-2H-naphtho[2,1-e][1,3] azine Derivatives 6a,b To a solution of either of compound 4a (2.68 g, 0.01 mol) or 4b (3.16 g, 0.01 mol) in ethanol (40 mL) contain- ing triethylamine (1.0 mL), phenyl isothiocyanate (1.30 g, 0.01 mol) was added and heated under reflux for 3 h then left to cool. The formed solid crystals, in each case, were collected by filtration. 5-Amino-4-imino-10-oxo-3-phenyl-2-thioxo-1,2,3,4,7, 8,9,10-octahydrobenzo[h]-quinazoline-6-carbonitrile (6a) Yellowish white crystals from 1,4-dioxane, yield 2.64 g (73%). Mp 212–215 °C. IR (KBr) νmax 3480–3329 (NH), 3055 (CH, aromatic), 2220 (CN), 1689 (CO), 1630 (C=C), 1209 cm–1 (C=S); 1H NMR (DMSO-d6, 300 MHz) δ 8.35, 8.28 (2s, 2H, D2O exchangeable, 2NH), 7.42–7.26 (m, 5H, C6H5), 4.52 (s, 2H, D2O exchangeable, NH2), 2.96– 2.83 (m, 4H, 2CH2), 1.96-1.82 (m, 2H, CH2); 13C NMR (DMSO-d6, 75 MHz) δ 179.8 (C-2), 173.6 (C-10), 126.8, 126.1, 125.2, 125.1, 124.6, 124.1, 123.8, 123.2, 122.6, 121.8, 120.3 (C-1, C-2, C-3, C-4, C-5, C-6, C6H5), 117.0, 116.3, 116.1 (3CN), 46.8, 40.2, 38.5 (C-7, C-8, C-9); MS m/z 361 (M+, 28%). Anal. Calcd for C19H15N5OS: C, 63.14; H, 4.18; N, 19.38; S, 8.87. Found: C, 63.28; H, 4.25; N, 19.26; S, 8.69. Diethyl 2-Cyano-4-(3-oxocyclohexylidene)-3-(3-phen- ylthioureido)pent-2-enedioate (6b) Orange crystals from ethanol, yield 3.09 g (67%). Mp 211–214 °C. IR (KBr) νmax 3468–3347 (NH), 3055 (CH, aro- matic), 1689, 1687 (2CO), 1630 (C=C), 1209 cm–1 (C=S); 1H NMR (DMSO-d6, 200 MHz) δ 8.32 (s, 1H, D2O exchange- able, NH), 7.40–7.23 (m, 5H, C6H5), 4.53 (s, 2H, D2O ex- changeable, NH2), 4.22 (2q, 2H, J = 7.03 Hz, OCH2CH3), 2.96–2.83 (m, 4H, 2CH2), 1.96–1.82 (m, 2H, CH2), 1.12 (t, 3H, J = 7.03 Hz OCH2CH3); 13C NMR (DMSO-d6, 75 MHz) δ 179.7 (C-2), 174.6, 166.1 (C-3, ester CO), 126.9, 126.5, 125.3, 125.0, 124.9, 124.6, 123.4, 123.1, 122.9, 122.3, 120.1 (C-1, C-2, C-3, C-4, C-5, C-6, C6H5), 50.1 (OCH2CH3), 40.5, 38.5, 17.1 (C-7, C-8, C-9), 16.3 (OCH2CH3); MS m/z 409 (M+, 38%). Anal. Calcd for C21H19N3O4S: C, 61.60; H, 4.68; N, 10.26; S, 7.83. Found: C, 61.39; H, 4.78; N, 10.58; S, 7.57. 2. 1. 4. General Procedure for the Synthesis of the 3,4,7,8,9,10-Hexahydro-2H-naphtho[2,1-e] [1,3]azinone Derivatives 7a,b A suspension of either compound 6a (3.61 g, 0.01 mol) or 6b (4.09 g, 0.01 mol) in sodium ethoxide [pre- pared 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 then triturated with hydrochloric acid (till pH 7) and the formed solid product was collected by filtration. 5-Amino-4,10-dioxo-3-phenyl-2-thioxo-1,2,3,4,7,8,9, 10-octahydrobenzo[h]-quinazoline-6-carbonitrile (7a) Yellow crystals from ethanol, yield 1.99 g (55%). Mp 210–212 °C. IR (KBr) νmax 3472–3346 (NH2), 3055 (CH, aromatic), 2220 (CN), 1688 (CO), 1630 cm–1 (C=C); 1H NMR (DMSO-d6, 200 MHz) δ 8.31 (s, 1H, D2O exchange- able, NH), 7.24–7.48 (m, 5H, C6H5), 4.80 (s, 2H, D2O ex- changeable, NH2), 2.98–2.81 (m, 4H, 2CH2), 1.94–1.80 (m, 2H, CH2); 13C NMR (DMSO-d6, 75 MHz) δ 179.5 (C-2), 173.4, 168.2 (C-4, C-10), 126.9, 126.1, 125.7, 125.2, 124.4, 124.0, 123.8, 123.4, 122.3, 122.6, 120.0 (C-1, C-2, C-3, C-4, C-5, C-6, C6H5), 116.6 (CN), 40.6, 38.2, 17.3 (C-7, C-8, C-9); MS m/z 362 (M+, 38%). Anal. Calcd for C19H14N4O2S: C, 62.97; H, 3.89; N, 15.46; S, 8.85. Found: C, 62.77; H, 4.19; N, 15.52; S, 8.59. Ethyl 5-Amino-4,10-dioxo-3-phenyl-2-thioxo-3,4,7,8,9, 10-hexahydro-2H-naphtho-[2,1-e][1,3]oxazine-6-car- boxylate (7b) Pale brown crystals from ethanol, yield 2.70 g (66%). Mp 177–179 °C. IR (KBr) νmax 3462, 3330 (NH2), 3055 (CH, aromatic), 1689–1687 (3CO), 1630 (C=C), 1209 cm–1 (C=S); 1H NMR (DMSO-d6, 300 MHz) δ 7.25–7.42 (m, 5H, C6H5), 4.83 (s, 2H, D2O exchangeable, NH2), 4.23 (q, 2H, J = 7.43 Hz, OCH2CH3), 2.98–2.83 (m, 4H, 2CH2), 1.93–1.80 (m, 2H, CH2), 1.12 (t, 3H, J = 7.43 Hz, OCH2CH3); 13C NMR (DMSO-d6, 75 MHz) δ 179.8 (C-2), 174.2, 166.5 (C-4, C-10), 127.1, 126.4, 125.9, 125.0, 124.6, 124.3, 123.5, 123.1, 122.5, 122.6, 120.3 (C-1, C-2, C-3, C-4, C-5, C-6, C6H5), 50.3 (OCH2CH3), 40.8, 38.5, 17.0 (C-7, C-8, C-9), 16.1 (OCH2CH3); MS m/z 410 (M+, 18%). Anal. Calcd for C21H18N2O5S: C, 61.45; H, 4.42; N, 6.83; S, 7.81. Found: C, 61.50; H, 4.38; N, 4.40; S, 7.14. 2. 1. 5. 2-Phenyl-4-thioxo-7,8-dihydro-4H- benzo[e][1,3]oxazin-5(6H)-one (10) To a solution of compound 1 (1.12 g, 0.01 mol) in 1,4-dioxane (40 mL) benzoyl isothiocyanate (1.63 g, 0.01 16 Acta Chim. Slov. 2022, 69, 13–29 Mohareb et al.: Synthesis of Fused Quinoline Derivatives with ... mol) [prepared by adding benzoyl chloride (1.40 g, 0.01 mol) to ammonium thiocyanate (0.76 g, 0.01 mol) in 1,4-dioxane (20 mL) with gentle heating for 5 min followed by filtration of the produced ammonium chloride] was heated under reflux for 3 h then left to cool. The formed solid crystals were collected by filtration. White crystals from ethanol, yield 1.74 g (67%). Mp 188–191 °C. IR (KBr) νmax 3055 (CH, aromatic), 1689 (CO), 1630 (C=C), 1208 cm–1 (C=S); 1H NMR (DMSO-d6, 300 MHz) δ 7.43–7.22 (m, 5H, C6H5), 2.78–2.68 (m, 2H, CH2), 1.93–1.67 (m, 4H, 2CH2); 13C NMR (DMSO-d6, 75 MHz) δ 180.4 (C-4), 174.2 (C-2), 168.2 (C-5), 142.7, 133.3, 126.3, 125.2, 123.6, 121.1 (C6H5, C-2, C-4a, C-8a), 39.8, 36.8, 16.0 (C-6, C-7, C-8); MS m/z 259 (M+ +2, 36%). Anal. Calcd for C14H11NO2S: C, 65.35; H, 4.31; N, 5.44; S, 12.46. Found: C, 65.26; H, 5.28; N, 5.60; S, 12.46. 2. 1. 6. General Procedure for the Synthesis of the Thieno[2’,3’:5,6]benzo[1,2-e][1,3]oxazine Derivatives 12a,b To a solution of compound 10 (2.57 g, 0.01 mol) in ethanol (40 mL), containing triethylamine (0.50 mL), ei- ther malononitrile (0.66 g, 0.01 mol) or ethyl cyanoacetate (1.07 g, 0.01 mol) was added. 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. 8-Amino-3-phenyl-1-thioxo-5,6-dihydro-1H-thieno[2’, 3’:5,6]benzo[1,2-e][1,3]oxazine-9-carbonitrile (12a) Orange crystals from ethanol, yield 2.35 g (70%). Mp 180–183 °C. IR (KBr) νmax 3472–3353 (NH2), 3055 (CH, aromatic), 2220 (CN), 1630 (C=C), 1208 cm–1 (C=S); 1H NMR (DMSO-d6, 300 MHz) δ 7.42–7.26 (m, 5H, C6H5), 4.78 (s, 2H, D2O exchangeable, NH2), 2.82–2.60 (2t, 4H, 2CH2); 13C NMR (DMSO-d6, 75 MHz) δ 179.6 (C-1), 176.3 (C-3), 142.6, 140.7, 134.2, 132.6, 132.7, 132.3, 127.2, 124.8, 122.4, 121.6 (C6H5, C-5, C-6, C-6a, C-9a, C-4a, C-9b), 116.8 (CN); MS m/z 339 (M+ + 2, 28%). Anal. Cal- cd for C17H11N3OS2: C, 60.51; H, 3.29; N, 12.45; S, 19.01. Found: C, 60.37; H, 3.63; N, 12.52; S, 18.93. Ethyl 8-Amino-3-phenyl-1-thioxo-5,6-dihydro-1H-thieno [2’,3’:5,6]benzo[1,2-e][1,3]oxazine-9-carboxylate (12b) Grey crystals from acetic acid, yield 2.84 g (74%). Mp 177–180 °C. IR (KBr) νmax 3459–3337 (NH2), 3055 (CH, aromatic), 2930, 2970 (CH2, CH3), 1689 (CO), 1630 cm–1 (C=C); 1H NMR (DMSO-d6, 300 MHz) δ 1.12 (t, 3H, J = 6.59 Hz, CH3), 2.78–2.65 (2t, 4H, 2CH2), 4.26 (q, 2H, J = 6.59 Hz, CH2), 4.80 (s, 2H, D2O exchangeable, NH2), 7.45–7.21 (m, 5H, C6H6); 13C NMR (DMSO-d6, 75 MHz) δ 180.4 (C-1), 176.2 (C-3), 168.4 (ester CO), 142.3, 141.3, 134.1, 132.6, 132.9, 132.3, 128.3, 124.9, 123.3, 120.6, (C6H5, C-5, C-6, C-6a, C-9a, C-4a, C-9b), 16.1 (OCH2CH3), 52.3 (OCH2CH3); MS m/z 386 (M+ +2, 28%). Anal. Calcd for C19H16N2O3S2: C, 59.35; H, 4.19; N, 7.29; S, 16.68. Found: C, 59.28; H, 4.47; N, 7.37; S, 16.39. 2. 1. 7. 2-(1-Amino-2,2,2-trichloroethylidene) cyclohexane-1,3-dione (14) Equimolar amounts of cyclohexan-1,3-dione (1.12 g, 0.01 mol) and trichloroacetonitrile (1.42 g, 0.01 mol) in absolute ethanol (40 mL) containing triethylamine (0.50 mL) was heated under reflux for 3 h. The solid product formed upon evaporation of the excess alcohol was col- lected by filtration. Yellow crystals from ethanol, yield 1.99 g (78%). Mp 204–207 °C. IR (KBr) νmax 3472–3346 (NH2), 1702, 1688 (2CO), 1630 cm–1 (C=C); 1H NMR (DMSO-d6, 300 MHz) δ 4.85 (s, 2H, D2O exchangeable, NH2), 1.96–1.82 (m, 2H, CH2), 2.95–2.80 (m, 4H, 2CH2); 13C NMR (DMSO-d6, 75 MHz) δ 173.4, 168.0 (C-1, C-3), 112.3, 90.8 (C-2, C-1 ethylidene), 94.8 (CCl3), 40.8, 38.2, 17.1 (C-4, C-5, C-6); MS m/z 256 (M+ + 2, 28%). Anal. Calcd for C8H8Cl3NO2: C, 37.46; H, 3.14; N, 5.46. Found: C, 37.80; H, 3.39; N, 5.52. 2. 1. 8. 1-Phenyl-2-thioxo-4-(trichloromethyl)-2,3,6,7- tetrahydroquinazolin-5(1H)-one (16) Equimolar amounts of compound 14 (2.56 g, 0.01 mol) and phenyl isothiocyanate (1.30 g, 0.01 mol) in 1,4-di- oxane (40 mL) containing triethylamine (0.50 mL) was heated under reflux for 2 h. The solid product formed upon pouring onto ice/water mxture was collected by filtration. Yellow crystals from ethanol, yield 2.61 g (70%). Mp 168–170 °C. IR (KBr) νmax 3470–3380 (NH), 3050 (CH aromatic), 1689 (CO), 1630 (C=C), 1208 cm–1 (C=S); 1H NMR (DMSO-d6, 300 MHz) δ 8.28 (s, 1H, D2O exchange- able, NH), 7.42–7.29 (m, 5H, C6H5), 5.21 (t, 1H, CH), 2.95–2.80 (m, 4H, 2CH2); 13C NMR (DMSO-d6, 75 MHz) δ 178.8 (C-2), 168.2 (C-5), 135.2, 133.6, 130.3, 129.0, 123.9, 123.6, 121.5, 120.5 (C6H5, C-8, C-9, C-3, C-4), 94.4 (CCl3), 40.8, 38.2 (C-6, C-7); MS m/z 373 (M+, 42%). Anal. Calcd for C15H11Cl3N2OS: C, 48.21; H, 2.97; N, 7.50. Found: C, 48.45; H, 3.19; N, 7.28. 2. 1. 9. General Procedure for the Synthesis of the 3,5,6,7-Tetrahydroquinazoline Derivatives 18a,b To a solution of compound 16 (3.73 g, 0.01 mol) in absoute ethanol (60 mL) either hydrazine hydrate (1.0 mL, 0.02 mol) or phenylhydrazine (2.16 g, 0.02 mol) was add- ed. The reaction mixture, in each case, was heated under reflux for 2 h then poured onto ice/water containing a few drops of hydrochloric acid and the formed solid product was collected by filtration. 4-Hydrazinyl-5-hydrazono-1-phenyl-3,5,6,7-tetrahy- droquinazoline-2(1H)-thione (18a) 17Acta Chim. Slov. 2022, 69, 13–29 Mohareb et al.: Synthesis of Fused Quinoline Derivatives with ... Orange crystals from ethanol, yield 2.04 g (68%). Mp 210–212 °C. IR (KBr) νmax 3489–3329 (NH2), 3054 (CH, aromatic), 1630 (C=C), 1210 cm–1 (C=S); 1H NMR (DMSO-d6, 300 MHz) δ 8.41, 8.29 (2s, 2H, D2O ex- changeable, 2NH), 7.45–7.28 (m, 5H, C6H5), 5.62 (t, 1H, CH), 2.89–2.64 (m, 4H, 2CH2), 4.90, 4.78 (2s, 4H, D2O exchangeable, 2NH2), 13C NMR (DMSO-d6, 75 MHz) δ 179.3 (C-2), 168.6 (C-5), 142.6, 140.7, 134.2, 132.6, 132.7, 132.3, 127.2, 124.8, 122.4, 121.6 (C6H5, C-4, C-4a, C-8, C-8), 39.8, 36.7 (C-6, C-7); MS m/z 300 (M+, 40%). Anal. Calcd for C14H16N6S: C, 55.98; H, 5.37; N, 27.98; S, 10.67. Found: C, 56.26; H, 5.49; N, 27.73; S, 10.88. 1-Phenyl-4-(2-phenylhydrazinyl)-5-(2-phenylhydrazo- no)-3,5,6,7-tetrahydroquinazoline-2(1H)-thione (18b) Orange crystals from methanol, yield 2.71 g (60%). Mp 177–180 °C. IR (KBr) νmax 3449–3352 (NH), 3055 (CH, aromatic), 1630 (C=C), 1208 cm–1 (C=S); 1H NMR (DMSO- d6, 300 MHz) δ 8.44–8.29 (4s, 4H, D2O ex- changeable, 4NH), 7.49–7.29 (m, 15H, 3C6H5), 5.60 (t, 1H, CH), 2.93–2.64 (m, 4H, 2CH2); 13C NMR (DMSO-d6, 75 MHz) δ 179.6 (C-2), 168.4 (C-5), 141.8, 140.7, 133.0, 132.7, 132.1, 131.8, 127.2, 126.7, 126.5, 124.8, 123.8, 123.6, 123.3, 122.4, 120.9, 120.6, 120.3 (3C6H5, C-4, C-4a, C-8, C-8), 39.9, 36.5 (C-6, C-7); MS m/z 452 (M+, 36%). Anal. Calcd for C26H24N6S: C, 69.00; H, 5.35; N, 18.57; S, 7.09. Found: C, 69.21; H, 5.58; N, 18.80; S, 7.26. 2. 1. 10. General Procedure for the Synthesis of the 6,7-Dihydroisoquinoline Derivatives 20a,b To a solution of compound 14 (2.65 g, 0.01 mol) in 1,4-dioxane containing ammonium acetate (2.00 g) either malononitrile (0.66 g, 0.01 mol) or ethyl cyanoacetate (1.13 g, 0.01 mol) was added. The whole reaction mixture was heated under reflux for 3 h and the solid product formed, in each case, upon pouring onto ice/water containing a few drops of hydrochloric acid, was collected by filtration. 3-Amino-8-oxo-1-(trichloromethyl)-5,6,7,8-tetrahy- droisoquinoline-4-carbonitrile (20a) Orange crystals from 1,4-dioxane, yield 1.97 g (65%). Mp 211–214 °C. IR (KBr) νmax 3458, 3332 (NH2), 3050 (CH aromatic), 2220 (CN), 1689 (CO), 1630 cm–1 (C=C); 1H NMR (DMSO-d6, 300 MHz) δ 4.80 (s, 2H, D2O exchangeable, NH2), 2.93–2.82 (m, 4H, 2CH2), 1.86–1.62 (m, 2H, CH2); 13C NMR (DMSO-d6, 75 MHz) δ 168.2 (C-8), 164.2 (C-3), 124.2, 123.8, 121.5, 120.3, 119.6 (C-1, C-4, C-4a, C-8a), 117.2 (CN), 94.6 (CCl3), 40.8, 38.2, 24.8 (C-5, C-6, C-7); MS m/z 304 (M+, 28%). Anal. Calcd for C11H8Cl3N3O: C, 43.38; H, 2.65; N, 13.80. Found: C, 43.52; H, 2.80; N, 13.68. Ethyl 3-Amino-8-oxo-1-(trichloromethyl)-5,6,7,8-tet- rahydroisoquinoline-4-carboxylate (20b) Pale brown crystals from 1,4-dioxane, yield 2.52 g (72%). Mp 180–180 °C. IR (KBr) νmax 3468, 3329 (NH2), 3045 (CH aromatic), 1702, 1689 (2CO), 1630 cm–1 (C=C); 1H NMR (DMSO-d6, 300 MHz) δ 4.85 (s, 2H, D2O ex- changeable, NH2), 4.23 (q, 2H, J = 6.80 Hz, OCH2CH3), 2.96–2.80 (m, 4H, 2CH2), 1.86–1.61 (m, 2H, CH2), 1.12 (t, 3H, J = 6.80 Hz, OCH2CH3); 13C NMR (DMSO-d6, 75 MHz) δ 168.2 (C-8), 164.8 (C=N), 124.9, 123.5, 122.8, 120.1, 119.8 (C-1, C-4, C-4a, C-8a), 94.5 (CCl3), 50.3 (OCH2CH3), 40.8, 38.7, 24.3 (C-5, C-6, C-7), 16.5 (OCH2CH3); MS m/z 350 (M+, 28%). Anal. Calcd for C13H13Cl3N2O3: C, 44.41; H, 3.73; N, 7.97. Found: C, 44.60; H, 3.84; N, 18.26. 2. 1. 11. General Procedure for the Synthesis of the 5,6-Dihydrothieno[2,3-h]isoquinoline Derivatives 21a–d To a solution of either compound 20a (3.04 g, 0.01 mol) or 20b (3.50 g, 0.01 mol) in 1,4-dioxane (40 mL) con- taining triethylamine (1.00 mL) either malononitrile (0.66 g, 0.01 mol) or ethyl cyanoacetate (1.07 g, 0.01 mol) was added. The reaction mixture, in each case, was heated un- der reflux for 1 h then poured onto ice/water containing a few drops of hydrochloric acid and the formed solid prod- uct was collected by filtration. 3,8-Diamino-1-(trichloromethyl)-5,6-dihydrothieno [2,3-h]isoquinoline-4,9-dicarbonitrile (21a) Pale brown crystals from 1,4-dioxane, yield 2.95 g (77%). Mp > 300 °C. IR (KBr) νmax 3493–3362 (NH2), 3050 (CH aromatic), 2223, 2220 (2CN), 1630 cm–1 (C=C); 1H NMR (DMSO-d6, 300 MHz) δ 2.98–2.86 (m, 4H, 2CH2), 4.87, 4.84 (2s, 4H, D2O exchangeable, 2NH2); 13C NMR (DMSO-d6, 75 MHz) δ 164.7 (C-3), 134.5, 132.4, 130.2, 129.8, 124.9, 122.6, 120.8, 120.6, 119.8 (C-1, C-4, C-4a, C-9, C-8, C-8a, C-6a, C-9a), 117.1, 116.8 (2CN), 94.8 (CCl3), 40.9, 38.6 (C-5, C-6); MS m/z 384 (M+, 62%). Anal. Calcd for C14H8Cl3N5S: C, 43.71; H, 2.10; N, 18.21; S, 8.34. Found: C, 43.52; H, 1.89; N, 17.82; S, 8.08. Ethyl 3,8-Diamino-9-cyano-1-(trichloromethyl)-5,6-di- hydrothieno[2,3-h]isoquinoline-4-carboxylate (21b) Pale brown crystals from 1,4-dioxane, yield 3.17 g (73%). Mp 284–287 °C. IR (KBr) νmax 3482–3339 (NH2), 3050 (CH aromatic), 2220 (CN), 1688 (CO), 1630 cm–1 (C=C); 1H NMR (DMSO-d6, 300 MHz) δ 1.12 (t, 3H, J = 6.47 Hz, OCH2CH3), 2.84–2.96 (m, 4H, 2CH2), 4.22 (q, 2H, J = 6.47 Hz, OCH2CH3), 4.86, 4.86 (2s, 4H, D2O ex- changeable, 2NH2); 13C NMR (DMSO-d6, 75 MHz) δ 168.2 (CO ester), 164.8 (C-3), 133.6, 130.3, 128.0, 127.2, 124.7, 123.7, 122.7, 120.8, 119.6 (C-1, C-4, C-4a, C-9, C-8, C-8a, C-6a, C-9a), 116.9 (CN), 94.5 (CCl3), 50.6 (OCH2CH3), 40.6, 38.8 (C-5, C-6), 16.8 (OCH2CH3); MS m/z 431 (M+, 54%). Anal. Calcd for C16H13Cl3N4O2S: C, 44.51; H, 3.04; N, 12.98; S, 7.43. Found: C, 44.72; H, 3.29; N, 13.18; S, 7.72. 18 Acta Chim. Slov. 2022, 69, 13–29 Mohareb et al.: Synthesis of Fused Quinoline Derivatives with ... Ethyl 3,8-Diamino-4-cyano-1-(trichloromethyl)-5,6-di- hydrothieno[2,3-h]isoquinoline-9-carboxylate (21c) Pale brown crystals from 1,4-dioxane, yield 2.58 g (60%). Mp 179–182 °C. IR (KBr) νmax 3459–3321 (NH2), 3050 (CH aromatic), 2220 (CN), 1689 (CO), 1630 cm–1 (C=C); 1H NMR (DMSO-d6, 300 MHz) δ 4.88, 4.84 (2s, 4H, D2O exchangeable, 2NH2), 4.21 (q, 2H, J = 7.25 Hz, OCH2CH3), 2.98–2.82 (m, 4H, 2CH2), 1.13 (t, 3H, J = 7.25 Hz, OCH2CH3); 13C NMR (DMSO-d6, 75 MHz) δ 168.6 (CO ester), 164.5 (C-3), 116.8 (CN), 133.9, 131.2, 128.5, 127.6, 125.2, 123.9, 122.8, 120.6, 120.3 (C-1, C-4, C-4a, C-9, C-8, C-8a, C-6a, C-9a), 94.7 (CCl3), 50.3 (OCH2CH3), 40.8, 38.6 (C-5, C-6), 16.9 (OCH2CH3); MS m/z 431 (M+, 54%). Anal. Calcd for C16H13Cl3N4O2S: C, 44.51; H, 3.04; N, 12.98; S, 7.43. Found: C, 44.72; H, 3.29; N, 13.18; S, 7.72. Diethyl 3,8-Diamino-1-(trichloromethyl)-5,6-dihydro- thieno[2,3-h]isoquinoline-4,9-dicarboxylate (21d) Pale brown crystals from 1,4-dioxane, yield 2.58 g (60%). Mp 179–182 °C. IR (KBr) νmax 3459–3321 (NH2), 3050 (CH aromatic), 2220 (CN), 1689 (CO), 1630 cm–1 (C=C); 1H NMR (DMSO-d6, 300 MHz) δ 4.88, 4.84 (2s, 4H, D2O exchangeable, 2NH2), 4.23, 4.21 (2q, 4H, J1 = 5.80 Hz, J2 = 7.25 Hz, two OCH2CH3), 2.98–2.82 (m, 4H, 2CH2), 1.13, 1.12 (2t, 6H, J1 = 5.80 Hz, J2 = 7.25 Hz, two OCH2CH3); 13C NMR (DMSO-d6, 75 MHz) δ 168.6, 167.2 (2CO ester), 164.5 (C-3), 133.9, 131.2, 128.5, 127.6, 125.2, 123.9, 122.8, 120.6, 120.3 (C-1, C-4, C-4a, C-9, C-8, C-8a, C-6a, C-9a), 116.8 (CN), 94.7 (CCl3), 50.6, 50.3 (two OCH2CH3), 40.8, 38.6 (C-5, C-6), 16.9, 16.7 (two OCH2CH3); MS m/z 478 (M+, 54%). Anal. Calcd for C18H18Cl3N3O4S: C, 45.16; H, 3.79; N, 8.78; S, 6.70. Found: C, 44.92; H, 3.59; N, 8.92; S, 6.82. 2. 1. 12. General Procedure for the Synthesis of the 1-Hydroxy-5,6-dihydrothieno[2,3-h] isoquinoline derivatives 22a–d A solution of either 21a (3.86 g, 0.01 mol), 21b (4.29 g, 0.01 mol), 21c (4.31 g, 0.01 mol) or 21d (4.31 g, 0.01 mol) in ethanol (60 mL) containing sodium hydroxide solution (10%, 5 mL) was heated under reflux for 4 h till ammonia gas evaluation cease. The solid product formed, in each case, upon pouring onto ice/water containing a few drops of hy- drochloric acid (till pH 6) was collected by filtration. 3,8-Diamino-1-hydroxy-5,6-dihydrothieno[2,3-h]iso- quinoline-4,9-dicarbonitrile (22a) Pale yellow crystals from 1,4-dioxane, yield 1.98 g (66%). Mp 220–223 °C. IR (KBr) νmax 3563–3362 (OH, NH2), 3050 (CH aromatic), 2224, 2220 (2CN), 1630 cm–1 (C=C); 1H NMR (DMSO-d6, 300 MHz) δ 10.27 (s, 1H, D2O exchangeable, OH), 4.89, 4.81 (2s, 4H, D2O exchangeable, 2NH2), 2.96–2.83 (m, 4H, 2CH2); 13C NMR (DMSO-d6, 75 MHz) δ 164.8 (C-2), 133.8, 131.4, 130.6, 129.3, 125.3, 124.6, 123.8, 121.6, 120.7, 120.2 (C-1, C-4, C-4a, C-9, C-8, C-8a, C-6a, C-9a), 117.0, 116.5 (2CN), 40.7, 38.4 (C-5, C-6); MS m/z 283 (M+, 55%). Anal. Calcd for C13H9N5OS: C, 55.11; H, 3.20; N, 24.72; S, 11.32. Found: C, 54.85; H, 3.59; N, 24.83; S, 11.48. Ethyl 3,8-Diamino-9-cyano-1-hydroxy-5,6-dihydroth- ieno[2,3-h]isoquinoline-4-carboxylate (22b) Pale brown crystals from 1,4-dioxane, yield 2.17 g (66%). Mp 189–192 °C. IR (KBr) νmax 3542–3359 (OH, NH2), 3050 (CH aromatic), 2220 (CN), 1689 (CO), 1630 cm–1 (C=C); 1H NMR (DMSO-d6, 300 MHz) δ 10.22 (s, 1H, D2O exchangeable, OH), 4.89, 4.84 (2s, 4H, D2O ex- changeable, 2NH2), 4.24 (q, 2H, J = 7.02 Hz, OCH2CH3), 2.98–2.85 (m, 4H, 2CH2), 1.12 (t, 3H, J = 7.02 Hz, OCH2CH3); 13C NMR (DMSO-d6, 75 MHz) δ 168.6 (CO ester), 164.3 (C-3), 133.8, 130.1, 128.2, 126.5, 124.1, 122.8, 122.2, 120.9, 120.6 (C-1, C-4, C-4a, C-9, C-8, C-8a, C-6a, C-9a), 116.7 (CN), 50.3 (OCH2CH3), 40.8, 38.5 (C-5, C-6), 16.9 (OCH2CH3); MS m/z 330 (M+, 28%). Anal. Calcd for C15H14N4O3S: C, 54.53; H, 4.27; N, 16.96; S, 9.71. Found: C, 54.66; H, 4.30; N, 17.16; S, 9.89. Ethyl 3,8-Diamino-4-cyano-1-hydroxy-5,6-dihydroth- ieno[2,3-h]isoquinoline-9-carboxylate (22c) Pale brown crystals from 1,4-dioxane, yield 1.98 g (60%). Mp 201–204 °C. IR (KBr) νmax 3539–3345 (OH, NH2), 3050 (CH aromatic), 2220 (CN), 1689 (CO), 1630 cm–1 (C=C); 1H NMR (DMSO-d6, 300 MHz) δ 10.22 (s, 1H, D2O exchangeable, OH), 5.01, 4.86 (2s, 4H, D2O ex- changeable, 2NH2), 4.23 (q, 2H, J = 6.85 Hz, OCH2CH3), 2.96–2.80 (m, 4H, 2CH2), 1.12 (t, 3H, J = 6.85 Hz, OCH2CH3); 13C NMR (DMSO-d6, 75 MHz) δ 168.4 (CO), 164.6 (C=N), 133.7, 132.5, 129.7, 127.9, 124.8, 123.3, 122.5, 120.4, 120.1 (C-1, C-4, C-4a, C-9, C-8, C-8a, C-6a, C-9a), 116.8 (CN), 50.5 (OCH2CH3), 40.9, 38.2 (C-5, C-6), 16.7 (OCH2CH3); MS m/z 330 (M+, 36%). Anal. Calcd for C15H14N4O3S: C, 54.53; H, 4.27; N, 16.96; S, 9.71. Found: C, 54.82; H, 4.08; N, 17.26; S, 9.87. Diethyl 3,8-Diamino-1-hydroxy-5,6-dihydrothieno[2,3 -h]isoquinoline-4,9-dicarboxylate (22d) Pale brown crystals from 1,4-dioxane, yield 2.58 g (60%). Mp 179–182 °C. IR (KBr) νmax 3559–3321 (NH2), 3050 (CH aromatic), 2220 (CN), 1689 (CO), 1630 cm–1 (C=C); 1H NMR (DMSO-d6, 300 MHz) δ 10.21 (s, 1H, D2O exchangeable, OH), 4.84, 4.80 (2s, 4H, D2O exchange- able, 2NH2), 4.24, 4.21 (q, 4H, J1 = 6.39 Hz, J2 = 7.25 Hz, two OCH2CH3), 2.98–2.82 (m, 4H, 2CH2), 1.13, 1.12 (2t, 6H, J1 = 6.39 Hz, J2 = 7.25 Hz, two OCH2CH3); 13C NMR (DMSO-d6, 75 MHz) δ 169.0, 168.6 (two CO ester), 164.5 (C-3), 133.9, 131.2, 128.5, 127.6, 125.2, 123.9, 122.8, 120.6, 120.3 (C-1, C-4, C-4a, C-9, C-8, C-8a, C-6a, C-9a), 116.8 (CN), 50.3, 50.1 (two OCH2CH3), 40.8, 38.6 (C-5, C-6), 16.9, 16.6 (two OCH2CH3); MS m/z 377 (M+, 68%). Anal. Calcd for C17H19N3O5S: C, 54.10; H, 5.07; N, 11.13; S, 8.50. Found: C, 54.26; H, 4.85; N, 11.26; S, 8.79. 19Acta Chim. Slov. 2022, 69, 13–29 Mohareb et al.: Synthesis of Fused Quinoline Derivatives with ... 2. 1. 13. General Procedure for the Synthesis of the 5,6-Dihydrothieno[2,3-h]isoquinoline derivatives 24a–h To a solution of either 21a (3.86 g, 0.01 mol), 21b (4.29 g, 0.01 mol), 21c (4.31 g, 0.01 mol) or 21d (4.31 g, 0.01 mol) in ethanol (60 mL) either potassium cyanide (1.28 g, 0.02 mol) or potassium thiocyanide (1.94 g, 0.01 mol) dissolved in water (10 mL) was added drop-wise. Af- ter complete addition, the whole mixture, in each case, was heated in a water bath at 60 °C for 2 h then was poured onto ice/water mixture containing a few drops of hydro- chloric acid and the formed solid product was collected by filtration. 3,8-Diamino-5,6-dihydrothieno[2,3-h]isoquinoline- 1,4,9-tricarbonitrile (24a) Pale brown crystals from 1,4-dioxane, yield 1.69 g (58%). Mp 266–268 °C. IR (KBr) νmax 3469–3341 (NH2), 3045 (CH aromatic), 2223–2220 (3CN), 1630 cm–1 (C=C); 1H NMR (DMSO-d6, 300 MHz) δ 2.86–2.98 (m, 4H, 2CH2), 4.84, 4.87 (2s, 4H, D2O exchangeable, 2NH2); 13C NMR (DMSO-d6, 75 MHz) δ 164.7 (C-3), 133.5, 132.6, 129.4, 127.8, 124.8, 122.7, 121.5, 120.9, 120.6 (C-1, C-4, C-4a, C-9, C-8, C-8a, C-6a, C-9a), 117.2, 117.1, 116.5 (3CN), 40.8, 38.4 (C-5, C-6); MS m/z 292 (M+, 58%). Anal. Calcd for C14H8N6S: C, 57.52; H, 2.76; N, 28.75; S, 10.97. Found: C, 57.69; H, 2.80; N, 28.66; S, 10.57. Ethyl 3,8-Diamino-1,9-dicyano-5,6-dihydrothieno[2,3 -h]isoquinoline-4-carboxylate (24b) Pale yellow crystals from 1,4-dioxane, yield 2.10 g (62%). Mp 180–184 °C. IR (KBr) νmax 3488–3331 (NH2), 3050 (CH aromatic), 2224, 2220 (2CN), 1689 (CO), 1630 cm–1 (C=C); 1H NMR (DMSO-d6, 300 MHz) δ 4.97, 4.84 (2s, 4H, D2O exchangeable, 2NH2), 4.22 (q, 2H, J = 6.41 Hz, OCH2CH3), 2.98–2.83 (m, 4H, 2CH2), 1.14 (t, 3H, J = 6.41 Hz, OCH2CH3); 13C NMR (DMSO-d6, 75 MHz) δ 168.8 (CO ester), 164.8 (C-3), 133.9, 132.1, 128.3, 126.8, 124.3, 123.6, 121.9, 120.8, 120.4 (C-1, C-4, C-4a, C-9, C-8, C-8a, C-6a, C-9a), 117.0, 116.3 (2CN), 50.3 (OCH2CH3), 40.7, 38.4 (C-5, C-6), 16.8 (OCH2CH3); MS m/z 339 (M+, 63%). Anal. Calcd for C16H13N5O2S: C, 56.63; H, 3.86; N, 20.64; S, 9.45. Found: C, 56.80; H, 3.96; N, 20.80; S, 9.62. Ethyl 3,8-diamino-1,4-dicyano-5,6-dihydrothieno[2,3 -h]isoquinoline-9-carboxylate (24c) Pale yellow crystals from 1,4-dioxane, yield 2.03 g (60%). Mp 222–225 °C. IR (KBr) νmax 3488–3331 (NH2), 3050 (CH aromatic), 2223, 2220 (2CN), 1689 (CO), 1630 cm–1 (C=C); 1H NMR (DMSO-d6, 300 MHz) δ 4.99, 4.86 (2s, 4H, D2O exchangeable, 2NH2), 4.23 (q, 2H, J = 7.22 Hz, OCH2CH3), 2.96–2.81 (m, 4H, 2CH2), 1.13 (t, 3H, J = 7.22 Hz, OCH2CH3); 13C NMR (DMSO-d6, 75 MHz) δ 168.8 (CO ester), 164.9 (C-3), 133.9, 132.1, 128.1, 126.2, 124.1, 123.8, 121.9, 120.8, 120.1 (C-1, C-4, C-4a, C-9, C-8, C-8a, C-6a, C-9a), 117.0, 116.3 (2CN), 50.4 (OCH2CH3), 40.8, 38.4 (C-5, C-6), 16.2 (OCH2CH3); MS m/z 339 (M+, 44%). Anal. Calcd for C16H13N5O2S: C, 56.63; H, 3.86; N, 20.64; S, 9.45. Found: C, 56.49; H, 3.77; N, 20.41; S, 9.53. Diethyl 3,8-Diamino-1-cyano-5,6-dihydrothieno[2,3-h] isoquinoline-4,9-dicarboxylate (24d) Yellow crystals from 1,4-dioxane, yield 2.70 g (70%). Mp 177–180 °C. IR (KBr) νmax 3493–3352 (NH2), 3050 (CH aromatic), 2220 (CN), 1689 (CO), 1630 cm–1 (C=C); 1H NMR (DMSO-d6, 300 MHz) δ 4.97, 4.84 (2s, 4H, D2O exchangeable, 2NH2), 4.24, 4.22 (2q, 4H, J1 = 6.80 Hz, J2 = 7.51 Hz, two OCH2CH3), 2.97–2.83 (m, 4H, 2CH2), 1.14, 1.12 (2t, 6H, J1 = 6.80 Hz, J2 = 7.51 Hz, two OCH2CH3); 13C NMR (DMSO-d6, 75 MHz) δ 170.1, 168.9 (two CO ester), 164.7 (C-3), 134.2, 131.7, 128.5, 125.8, 124.2, 123.4, 121.6, 120.6, 120.3 (C-1, C-4, C-4a, C-9, C-8, C-8a, C-6a, C-9a), 117.1 (CN), 50.4, 50.1 (two OCH2CH3), 40.9, 38.2 (C-5, C-6), 16.5, 16.2 (two OCH2CH3); MS m/z 386 (M+, 36%). Anal. Calcd for C18H18N4O4S: C, 55.95; H, 4.70; N, 14.50; S, 8.30. Found: C, 56.25; H, 4.59; N, 14.73; S, 8.62. 3,8-Diamino-1-thiocyanato-5,6-dihydrothieno[2,3-h] isoquinoline-4,9-dicarbonitrile (24e) Pale brown crystals from 1,4-dioxane, yield 2.52 g (78%). Mp 243–247 °C. IR (KBr) νmax 3482–3326 (NH2), 3045 (CH aromatic), 2224–2220 (3CN), 1630 cm–1 (C=C); 1H NMR (DMSO-d6, 300 MHz) δ 4.86, 4.82 (2s, 4H, D2O exchangeable, 2NH2), 2.96–2.84 (m, 4H, 2CH2); 13C NMR (DMSO-d6, 75 MHz) δ 164.8 (C-3), 133.9, 132.8, 129.6, 127.3, 123.2, 122.1, 121.8, 120.7, 120.4 (C-1, C-4, C-4a, C-9, C-8, C-8a, C-6a, C-9a), 40.6, 38.1 (C-5, C-6), 117.1, 116.4 110.8 (3CN); MS m/z 324 (M+, 40%). Anal. Calcd for C14H8N6S2: C, 51.84; H, 2.49; N, 25.91; S, 19.77. Found: C, 51.69; H, 2.63; N, 26.25; S, 19.80. Ethyl 3,8-Diamino-9-cyano-1-thiocyanato-5,6-dihy- drothieno[2,3-h]isoquinoline-4-carboxylate (24f) Yellow crystals from 1,4-dioxane, yield 2.74 g (74%). Mp 170–172 °C. IR (KBr) νmax 3479–3343 (NH2), 3050 (CH aromatic), 2221, 2220 (2CN), 1689 (CO), 1630 cm–1 (C=C); 1H NMR (DMSO-d6, 300 MHz) δ 4.97, 4.83 (2s, 4H, D2O exchangeable, 2NH2), 4.22 (q, 2H, J = 6.84 Hz, OCH2CH3), 2.98–2.81 (m, 4H, 2CH2), 1.13 (t, 3H, J = 6.84 Hz, OCH2CH3); 13C NMR (DMSO-d6, 75 MHz) δ 168.6 (CO ester), 164.8 (C-3), 116.8, 112.6 (2CN), 133.5, 132.4, 128.8, 126.2, 124.4, 123.9, 122.3, 120.6, 120.0 (C-1, C-4, C-4a, C-9, C-8, C-8a, C-6a, C-9a), 50.2 (OCH2CH3), 40.6, 38.1 (C-5, C-6), 16.5 (OCH2CH3); MS m/z 371 (M+, 32%). Anal. Calcd for C16H13N5O2S2: C, 51.74; H, 3.53; N, 18.85; S, 17.27. Found: C, 52.01; H, 3.49; N, 18.63; S, 17.08. Ethyl 3,8-Diamino-4-cyano-1-thiocyanato-5,6-dihy- drothieno[2,3-h]isoquinoline-9-carboxylate (24g) Yellow crystals from 1,4-dioxane, yield 2.04 g (55%). Mp 193–196 °C. IR (KBr) νmax 3493–3329 (NH2), 3050 (CH aromatic), 2224, 2220 (2CN), 1688 (CO), 1630 cm–1 20 Acta Chim. Slov. 2022, 69, 13–29 Mohareb et al.: Synthesis of Fused Quinoline Derivatives with ... (C=C); 1H NMR (DMSO-d6, 300 MHz) δ 4.97, 4.82 (2s, 4H, D2O exchangeable, 2NH2), 4.23 (q, 2H, J = 7.12 Hz, OCH2CH3), 2.96–2.80 (m, 4H, 2CH2), 1.12 (t, 3H, J = 7.12 Hz, OCH2CH3); 13C NMR (DMSO-d6, 75 MHz) δ 168.4 (CO ester), 164.7 (C-3), 132.8, 131.6, 120.3, 129.2, 126.2, 124.1, 123.9, 122.6, 121.8 (C-1, C-4, C-4a, C-9, C-8, C-8a, C-6a, C-9a), 116.6, 111.5 (2CN), 50.2 (OCH2CH3), 40.6, 38.4 (C-5, C-6), 16.3 (OCH2CH3); MS m/z 371 (M+, 36%). Anal. Calcd for C16H13N5O2S2: C, 51.74; H, 3.53; N, 18.85; S, 17.27. Found: C, 51.91; H, 3.42; N, 18.43; S, 17.33. Diethyl 3,8-Diamino-1-thiocyanato-5,6-dihydroth- ieno[2,3-h]isoquinoline-4,9-dicarboxylate (24h) Pale yellow crystals from 1,4-dioxane, yield 2.71 g (52%). Mp 166–169 °C. IR (KBr) νmax 3470–3332 (NH2), 3050 (CH aromatic), 2221, 2220 (CN), 1689 (CO), 1630 cm–1 (C=C); 1H NMR (DMSO-d6, 300 MHz) δ 4.97, 4.84 (2s, 4H, D2O exchangeable, 2NH2), 4.23, 4.22 (2q, 4H, J1 = 6.49 Hz, J2 = 6.21 Hz, two OCH2CH3), 2.98–2.81 (m, 4H, 2CH2), 1.13, 1.12 (2t, 6H, J1 = 6.49 Hz, J2 = 6.21 Hz, two OCH2CH3); 13C NMR (DMSO-d6, 75 MHz) δ 168.8 (CO ester), 164.5 (C-3), 133.8, 131.3, 128.2, 124.9, 124.7, 122.6, 121.9, 120.8, 120.1, (C- 1, C-4, C-4a, C-9, C-8, C-8a, C-6a, C-9a), 112.3, 117.0 (2CN), 50.3, 50.6 (two OCH2CH3), 40.8, 38.3 (C-5, C-6), 16.1, 16.4 (two OCH2CH3); MS m/z 418 (M+, 24%). Anal. Calcd for C18H18N4O4S2: C, 51.66; H, 4.34; N, 13.39; S, 15.32. Found: C, 51.49; H, 4.60; N, 13.26; S, 15.53. 2. 1. Biology Section 2. 2. 1. Cell Proliferation Assay The anti-proliferative activities of the newly syn- thesized compounds (Table 1) were evaluated against the six cancer cell lines A549, HT-29, MKN-45, U87MG, SMMC-7721, and H460 using the standard MTT assay in vitro, with foretinib as the positive control.23 The cancer cell lines were cultured in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS). Table 1. In vitro growth inhibitory effects IC50 ± SEM (µM) of the newly synthesized compounds against cancer cell lines Compound IC50 ± SEM (µM) No A549 H460 HT29 MKN-45 U87MG SMMC-7721 3a 8.72 ± 2.62 6.25 ± 3.06 7.83 ± 2.54 8.01 ± 2.41 8.72 ± 2.63 8.08 ± 3.19 3b 3.46 ± 1.29 4.53 ± 1.44 3.65 ± 1.64 2.43 ± 0.86 3.82 ± 1.06 2.63 ± 1.16 4a 5.83 ± 1.43 6.73 ± 2.54 3.29 ± 1.13 2.62 ± 0.74 4.80 ± 2.43 3.78 ± 0.62 4b 7.72 ± 2.63 8.69 ± 2.36 6.73 ± 2.33 8.62 ± 1.43 7.25 ± 2.49 8.30 ± 3.59 6a 9.29 ± 2.59 8.17 ± 2.89 5.08 ± 1.69 4.32 ± 2.41 6.50 ± 1.52 6.30 ± 2.83 6b 0.32 ± 0.20 0.34 ± 0.13 0.52 ± 0.24 0.45 ± 0.12 0.53 ± 0.25 0.39 ± 0.14 7a 5.63 ± 1.28 3.49 ± 1.28 5.46 ± 2.36 6.05 ± 2.47 4.29 ± 1.59 6.07 ± 2.62 7b 1.26 ± 0.85 0.99 ± 0.63 0.86 ± 0.49 0.32 ± 0.19 0.68 ± 0.19 0.80 ± 0.38 10 8.24 ± 3.68 6.26 ± 2.34 5.29 ± 2.89 6.27 ± 1.29 3.83 ± 1.53 5.59 ± 2.32 12a 6.28 ± 1.78 4.83 ± 1.23 4.70 ± 1.20 6.73 ± 2.30 5.82 ± 2.69 6.49 ± 2.28 12b 0.34 ± 0.21 0.69 ± 0.30 0.53 ± 0.32 0.28 ± 2.39 0.42 ± 0.29 0.52 ± 0.26 14 0.52 ± 0.13 0.83 ± 0.20 0.71 ± 1.82 0.26 ± 0.12 0.60 ± 0.21 0.15 ± 0.02 16 0.31 ± 0.22 0.13 ± 0.07 0.22 ± 0.16 0.32 ± 0.17 0.42 ± 0.19 0.36 ± 0.15 18a 1.63 ± 0.23 1.63 ± 0.34 1.08 ± 0.81 0.92 ± 0.63 0.90 ± 0.71 1.31 ± 0.80 18b 0.62 ± 0.39 0.72 ± 0.53 0.39 ± 0.26 0.49 ± 0.26 0.58 ± 0.19 0.64 ± 0.28 20a 1.26 ± 0.69 1.38 ± 0.99 1.79 ± 0.82 0.96 ± 0.42 0.86 ± 0.26 0.57 ± 0.30 20b 0.30 ± 0.19 0.24 ± 0.10 0.43 ± 0.27 0.52 ± 0.18 0.23 ± 0.0.8 0.32 ± 0.17 21a 1.16 ± 0.75 1.80 ± 0.69 1.25 ± 0.48 2.04 ± 0.38 1.90 ± 0.58 1.49 ± 0.78 21b 0.31 ± 0.20 0.39 ± 0.12 0.23 ± 0.06 0.23 ± 0.06 0.28 ± 0.16 0.56 ± 0.23 21c 0.58 ± 0.17 0.52 ± 0.23 0.62 ± 0.22 0.42 ± 0.19 0.53 ± 0.25 0.72 ± 0.19 21d 0.49 ± 0.21 0.52 ± 0.15 0.46 ± 0.19 0.50 ± 0.27 0.70 ± 0.25 0.38 ± 0.18 22a 1.02 ± 0.72 1.26 ± 0.59 1.42 ± 0.69 1.26 ± 0.82 0.86 ± 0.31 1.63 ± 0.82 22b 1.12 ± 0.69 1.04 ± 0.80 1.36 ± 0.88 1.26 ± 0.73 2.13 ± 1.79 0.85 ± 0.41 22c 0.26 ± 0.19 0.35 ± 0.16 0.42 ± 0.27 0.19 ± 0.02 0.36 ± 0.18 0.28 ± 0.06 22d 0.32 ± 0.18 0.26 ± 0.13 0.51 ± 0.29 0.35 ± 0.29 0.28 ± 0.06 0.18 ± 0.07 24a 1.44 ± 0.86 1.22 ± 0.76 0.89 ± 0.54 0.95 ± 0.63 0.86 ± 0.39 1.39 ± 2.28 24b 1.05 ± 0.61 1.70 ± 0.72 0.96 ± 0.26 0.83 ± 0.39 1.46 ± 0.79 1.36 ± 0.87 24c 2.82 ± 0.93 1.69 ± 0.93 1.38 ± 0.62 0.79 ± 0.42 0.89 ± 0.32 1.67 ± 0.58 24d 0.27 ± 0.12 0.38 ± 0.09 0.52 ± 0.28 0.38 ± 0.19 0.46 ± 0.21 0.39 ± 0.18 24e 1.66 ± 0.26 1.42 ± 0.73 1.39 ± 0.86 0.72 ± 0.63 0.68 ± 0.31 1.41 ± 0.89 24f 0.87 ± 0.32 0.69 ± 0.28 0.39 ± 0.21 0.62 ± 0.28 0.72 ± 0.39 0.83 ± 0.36 24g 0.77 ± 0.26 0.82 ± 0.31 0.59 ± 0.82 0.32 ± 0.17 0.32 ± 0.18 0.21 ± 0.05 24h 0.53 ± 0.14 0.61 ± 0.28 0.42 ± 0.21 0.62 ± 0.18 0.60 ± 0.32 0.59 ± 0.15 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 21Acta Chim. Slov. 2022, 69, 13–29 Mohareb et al.: Synthesis of Fused Quinoline Derivatives with ... Approximate 4 × 103 cells, suspended in MEM medium, were plated onto each well of a 96-well plate and incubat- ed in 5% CO2 at 37 °C for 24 h. The compounds tested at the indicated final concentrations were added to the cul- ture medium and the cell cultures were continued for 72 h. Fresh MTT was added to each well at a terminal con- centration of 5 mg/mL, and incubated with cells at 37 °C for 4 h. The formazan crystals were dissolved in 100 µL of DMSO each well, and the absorbency at 492 nM (for absorbance of MTT formazan) and 630 nM (for the ref- erence wavelength) was measured with an ELISA reader. All of the compounds were tested three times in each cell line. The results expressed as IC50 (inhibitory concentra- tion 50%) were the averages of three determinations and calculated by using the Bacus Laboratories Incorporated Slide Scanner (Bliss) software. The mean values of three independent experiments, expressed as IC50 values, are presented in Table 1. Most of the synthesized compounds exhibited potent anti-prolif- erative activity with IC50 values less than 30 µM. Gener- ally, the variations of substituents within the aryl moiety together with the heterocycle ring being attached have a notable influence on the anti-proliferative activity. 2. 2. 3. Structure Activity Relationship It is clear from Table 1 that most of the tested com- pounds have high inhibitions toward the six cancer cell lines. Considering the 5,6,7,8-tetrahydronaphthalene de- rivatives 3a and 3b, it is clear that compound 3b (X = OH, R = COOEt) that is an oxygen-rich compound has higher inhibition than 3a (X = NH2, R = CN). Reaction of either compound 3a or 3b with acetic acid and acetic anhydride gave the acetylated derivatives 4a and 4b, respectively where both of two compounds showed moderate inhibi- tion, surprisingly, compound 3a exhibited higher inhibi- tion than 4b. For the 1,2,3,4,7,8,9,10-octahydrobenzo[h] quinazoline 6a and the 3,4,7,8,9,10-hexahydro-2H-naph- tho[2,1-e][1,3]oxazine 6b, it is obvious that compound 6b (Y = O, R = COOEt) showed higher inhibitions toward the six cancer cell lines than 6a (Y = NH, R = CN). The same findings were noticed after hydrolysis of the exocy- clic C=NH group present in 6a and 6b into C=O where compound 7b exhibited stronger inhibitions than 7a. The 7,8-dihydro-4H-benzo[e][1,3]oxazine derivative 10 exhib- ited low inhibitions. The reaction of compound 10 with ei- ther malononitrile or ethyl cynoacetate and elemental sul- fur produces the thieno[2’,3’:5,6]benzo[1,2-e][1,3]oxazine derivatives 12a and 12b, respectively. It is obvious from Table 1 that compound 12b (R = COOEt) displayed higher inhibitions than compound 12a (R = CN). The reaction of cyclohexan-1,3-dione with trichloroacetonitrile gave the (2,2,2-trichloroethylidene)cyclohexane-1,3-dione deriv- ative 14 which exhibited high inhibitions toward the six cancer cell lines. Its conversion into the 2,3,6,7-tetrahydro- quinazoline derivative 16 through its reaction with phe- nyl isothiocyanate support the inhibition of the molecule where compound 16 showed high inhibitions. Increasing the nitrogen content of 16 through its reaction with either hydrazine hydrate or phenylhydrazine to give either 18a or 18b, respectively resulting in high cytotoxicities as well. Moreover, compound 18b (R = Ph) was more cytotoxic than 18a (R = H). The same argument appeared in the case of 20a (R = CN) and 20b (R = COOEt) where the latter showed higher cytotoxicities than the former. Consider- ing the 5,6-dihydrothieno[2,3-h]isoquinoline derivatives 21a–d, for which 21b (R1 = CN, R2 = COOEt), 21c (R1 = COOEt, R2 = CN) and 21d (R1 = R2 = COOEt) were of high inhibitions toward the six cancer cell lines. However, in the case of the hydroxyl derivatives 22a–d only com- pounds 22c and 22d were the most cytotoxic compounds. Finally, for the nucleophilic substituted compounds with the CN or the SCN moieties to give the eight compounds 24a–h, all of them exihibited high inhibitions. However, compounds 24a, 24b, 24c and 24e showed from moderate to high inhibitions together with compounds 24d, 24f, 24g and 24h exhibiting high inhibitions. It is of great value to mention that compounds 6b, 7b, 12b, 14, 16, 18b, 20b, 21b, 21c, 21d, 22c, 22d, 24d, 24f, 24g and 24h were the most cytotoxic compounds among the tested compounds. On the other hand, compounds 3b, 7a, 18a, 20a, 21a, 22a, 22b, 24a, 24b, 24c and 24e have moderate inhibitions. With special attention to compounds bearing the COOEt group within their structures there were some of them 6b, 12b, 20b, 21b, 21c and 21d showing high inhibitions while other compounds with other subtituents have lower inhi- bitions. In most cases compounds with the electronegative COOEt and/or CN groups exhibited high inhibitions al- though in some cases the nature of heterocyclic ring was in some cases a controlling factor.24 For example considering the inhibitions of compounds 5,6-dihydrothieno[2,3-h] isoquinoline derivatives 21a–d we found that the presence of isoquinoline moiety enhanced the inhibitions25 of com- pounds 21b, 21c and 21d. While the presence of pyridine moiety, like in 24a–h, enabled compounds 24d, 24f, 24g and 24h to exhibit high inhibitions. In fact the difference in anti-proliferative activities between fused heterocyclic compounds of the same substituents was reported before.26 2. 2. 4. HTRF Kinase Assay c-Met (mesenchymal epithelial transition factor) is a multifunctional transmembrane tyrosine kinase and acts as a receptor for hepatocyte growth factor/scatter factor (HGF/SF).27 It is expressed during embryogenesis in multi- ple epithelial tissues (liver, pancreas, prostate, kidney, mus- cle, bone-marrow) and was also discovered in numerous tumour cell communities on the cell surface.28 Multiple oncogenetic characteristics of c-Met were outlined shortly after its discovery, including cell dissociation stimulation, migration, motility, and extracellular matrix invasion.29,30 Moreover, the c-Met kinase activity has been revealed to be 22 Acta Chim. Slov. 2022, 69, 13–29 Mohareb et al.: Synthesis of Fused Quinoline Derivatives with ... correlated with prostate cancer where c-Met played a key role in the conversion of prostate cancer from the prima- ry androgen-sensitive to androgen-insensitive status along with the increase in radio resistance. First, an inverse rela- tionship between the expression of androgen receptor (AR) and c-Met has been observed in prostate epithelium and prostate cancer cells.31 Second, AR signaling suppressed c-Met transcription while androgen removal improved the expression of c-Met.32 Third, it is observed that c-Met ex- pression is high in late stage bone metastatic prostate can- cer.33 Furthermore, the latest research has shown that c-Met expression is closely related to cellular radiosensitivity.33 Based on these reported observations, the c-Met kinase activity of all compounds was evaluated using ho- mogeneous time-resolved fluorescence (HTRF) assay as previously reported.34 Taking foretinib as the positive con- trol, the results expressed as IC50 are summarized in Table 2. The anti-proliferative activity of all target compounds against the human prostatic cancer PC-3 cell line was measured by MTT assay using anibamine as the reference drug. The mean values of three independent experiments, expressed as IC50 values, are presented in Table 2. Gener- ally, the variations of substituents within the aryl moiety together with the heterocyclic ring being attached have a notable influence on the anti-proliferative activity. HTRF assay utilizes the signal generated by the fluo- rescence resonance energy transfer between donor and ac- ceptor molecules in close proximity. Dual-wavelength de- Table 2. c-Met enzymatic activity and PC-3 inhibition of the newly synthesized com- pounds. Compound IC50 (nM) IC50(µΜ) VEROa SI PC-3b No c-Met PC-3 (µM) 3a 1.42 ± 0.80 1.73 ± 0.73 58.41 ± 6.32 33.76 3b 0.31 ± 0.16 0.25 ± 0.17 55.61 ± 6.24 > 100 4a 0.54 ± 0.16 2.31 ± 0.92 36.22 ± 6.27 15.68 4b 0.32 ± 0.21 0.28 ± 0.23 50.68 ± 6.14 > 100 6a 4.16 ± 1.83 0.26 ± 0.10 39.56 ± 6.31 > 100 6b 4.28 ± 1.80 0.30 ± 2.53 58.23 ± 5.16 > 100 7a 6.27 ± 2.19 8.46 ± 2.24 36.69 ± 8.12 4.37 7b 2.47 ± 0.88 4.05 ± 1.82 58.36 ± 6.27 14.41 10 4.72 ± 1.83 1.26 ± 0.97 32.28 ± 5.71 25.62 12a 8.41 ± 2.53 2.82 ± 1.03 58.27 ± 5.80 20.66 12b 1.33 ± 0.78 0.29 ± 0.06 60.81 ± 7.26 > 100 14 0.29 ± 0.09 0.26 ± 0.18 58.32 ± 6.93 > 100 16 0.63 ± 0.42 0.29 ± 0.13 60.35 ± 6.56 > 100 18a 4.51 ± 1.86 6.41 ± 2.20 63.40 ± 8.27 9.89 18b 0.82 ± 0.32 0.42 ± 0.23 60.22 ± 7.32 > 100 20a 2.46 ± 1.30 2.80 ± 1.01 56.32 ± 6.57 20.11 20b 0.49 ± 0.21 0.53 ± 0.12 65.43 ± 6.81 > 100 21a 3.65 ± 1.83 4.82 ± 1.26 40.41 ± 8.32 8.38 21b 4.82 ± 1.16 3.20 ± 1.68 30.23 ± 7.19 9.44 21c 0.22 ± 0.13 0.38 ± 0.16 42.53 ± 6.63 > 100 21d 1.18 ± 0.92 0.24 ± 0.07 40.53 ± 5.63 > 100 22a 5.28 ± 1.47 2.79 ± 1.01 60.29 ± 8.20 21.61 22b 0.36 ± 0.14 0.42 ± 0.09 42.49 ± 6.53 > 100 22c 1.81 ± 0.96 1.48 ± 0.79 56.27 ± 8.93 38.02 22d 0.36 ± 0.18 0.63 ± 0.17 36.58 ± 5.30 58.06 24a 6.36 ± 2.31 5.57 ± 1.29 60.47 ± 6.93 10.86 24b 1.42 ± 0.80 1.73 ± 0.73 58.41 ± 6.32 33.76 24c 0.21 ± 0.05 0.39 ± 0.15 58.37 ± 6.19 > 100 24d 2.58 ± 0.80 4.18 ± 1.48 48.26 ± 5.39 11.54 24e 2.68 ± 1.72 3.80 ± 1.49 58.01 ± 5.77 15.26 24f 1.18 ± 0.89 0.24 ± 0.11 54.52 ± 6.70 > 100 24g 2.37 ± 1.16 4.93 ± 1.77 38.73 ± 4.83 7.85 24h 1.32 ± 0.93 0.28 ± 0.17 60.72 ± 8.19 > 100 Foretinib Anibamine 1.16 ± 0.17 3.26 ± 0.35 – – a VERO, monkey kidney cell line (Cat No-11095–080). b Selectivity index (SI) were calculated by IC50 values in normal cell line divided by IC50 values in PC-3 cancer cell line. 23Acta Chim. Slov. 2022, 69, 13–29 Mohareb et al.: Synthesis of Fused Quinoline Derivatives with ... tection helps to eliminate media interference, and the final signal is proportional to the extent of product formation. Thus far, the reported applications of this technology for in vitro kinase assays have mainly focused on high-through- put screening. The MTT assay is a colorimetric assay for assessing cell metabolic activity. NAD(P)H-dependent cellular oxidoreductase enzymes may, under defined con- ditions, reflect the number of viable cells present. These enzymes are capable of reducing the tetrazolium dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to its insoluble formazan, which has a purple col- our. Other closely related tetrazolium dyes including XTT, MTS and the WSTs, are used in conjunction with the inter- mediate electron acceptor, 1-methoxyphenazine metho- sulfate (PMS). With WST-1, which is cell-impermeable, reduction occurs outside the cell via plasma membrane electron transport. However, this traditionally assumed explanation is currently contended as proof has also been found of MTT reduction to formazan in lipidic cellular structures without apparent involvement of oxido reduc- tases. Tetrazolium dye assays can also be used to measure cytotoxicity (loss of viable cells) or cytostatic activity (shift from proliferation to quiescence) of potential medicinal agents and toxic materials. MTT assays are done in the dark since the MTT reagent is sensitive to light. Within this protocol, replacing the serum-containing media with serum-free media and MTT reagent in cell cultures incu- bated for 3 h at 37 °C adding MTT solvent and incubating for 15 min to be analyzed with microplate reader. As shown in Table 2, all the tested compounds dis- played potent c-Met enzymatic activity with IC50 values ranging from 0.21 to 8.41 nM and potent prostate PC-3 cell line inhibition with IC50 values ranging from 0.26 to 8.46 µM. Compared with foretinib (IC50 = 1.16 nM), the ten compounds (3b, 4a, 4b, 14, 16, 18b, 20b, 21c, 22b, 22d and 24c) exhibited higher potency with IC50 values less than 1.00 nM. Remarkably, among the synthesized compounds, 3a, 3b, 4a, 4b, 6a, 6b, 10, 2a, 12b, 14, 16, 18b, 20a, 20b, 21c, 21d, 22a, 22b, 22c, 24c, 24d, 24f and 24h displayed much higher anti-proliferation activities against PC-3 cell line than the standard anibamine (IC50 = 3.26 µM). All synthesized compounds were tested against the VERO, monkey kidney normal cell line, where they showed low activity against the normal cell line. Interestingly, from Table 2 compounds 3a, 22c, 22d and 24b showed SI > 30 while the fourteen compounds 3b, 4b, 6a, 6b, 12b, 14, 16, 18b, 20b, 21d, 22b, 24c, 24f and 24h exhibited SI > 100, while the rest of compounds showed SI < 30. 2. 2. 5. Inhibitory Effect of the Most Active Compounds Towards Tyrosine Kinases The most active compounds that showed the high- est inhibitions toward the six cancer cell lines were further evaluated against other five tyrosine kinases (c-Kit, Flt-3, VEGFR-2, EGFR, and PDGFR) using the same screening method (Table 3). These receptor tyrosine kinases (RTKs) have been implicated in vascular development by affecting the proliferation and migration of endothelial cells or per- icytes. It is clear from Table 3 that compounds 7b, 12b, 16, 20b, 21b, 22c, 22d, 24d, 24f and 24h were the most potent towards the five tyrosine kinases. Compound 25g showed high inhibitions towards the four kinases Flt-3 and VEG- FR-2 with IC50 values of 0.32 and 0.29 nM, respectively while it showed moderate inhibition towards c-Kit and EGFR with IC50 1.52 and 1.93 nM, respectively. Compound 24d was the most active compound against Flt-3 kinase with IC50 0.17 nM. Compounds 12b, 16 and 24f were the most active toward PDGFR with IC50 values og 0.28, 0.26 and 0.27 nM, respectively. Compounds 6b, 18b and 21c showed the lowest potency among the tested compounds. Table 3. Inhibition of tyrosine kinases [enzyme IC50 (nM)] by com- pounds 6b, 7b, 12b, 14, 16, 18b, 20b, 21b, 21c, 21d, 22c, 22d, 24d, 24f, 24g and 24h Com- c-Kit Flt-3 VEGFR-2 EGFR PDGFR pound 6b 1.80 2.43 1.72 2.93 1.05 7b 0.21 0.17 0.23 0.26 0.42 12b 0.30 0.51 0.29 0.33 0.28 14 1.08 2.62 1.17 2.39 1.52 16 0.28 0.16 0.52 0.74 0.26 18b 1.16 2.39 1.12 2.83 1.29 20b 0.31 0.46 0.35 0.29 0.33 21b 0.41 0.28 0.26 0.42 0.50 21c 1.22 2.96 1.53 2.72 1.38 22c 0.38 0.29 0.52 0.41 0.70 22d 0.27 0.25 0.41 0.66 0.37 24d 0.17 0.26 0.50 0.61 0.39 24f 0.25 0.36 0.42 0.36 0.27 24g 1.52 0.32 0.29 1.93 2.53 24h 0.22 0.26 0.36 0.28 0.37 2. 2. 6. Inhibition of Selected Compounds Towards Pim-1 Kinase Compounds 7b, 12b, 16, 20b, 21b, 22c, 22d, 24d, 24f and 24h were selected to examine their Pim-1 kinase inhibition activity (Table 4) as these compounds showed high inhibition towards the tested cancer cell lines at a range of ten concentrations and the IC50 values were calcu- lated. Compounds 7b, 12b, 16, 22c and 24f were the most potent to inhibit Pim-1 kinase with IC50 value of 0.22, 0.28, 0.24, 0.30 and 0.26 µM, respectively. On the other hand, compounds 20b, 21b, 22d, 24d and 24h were less effec- tive (IC50 > 10 µM). These profiles in combination with cell growth inhibition data of compounds 7b, 12b, 16, 20b, 21b, 22c, 22d, 24d, 24f and 24h listed in Table 3 indi- cate that Pim-1 was a potential target of these compounds where SGI-1776 was used as the positive control with IC50 0.048 µM in the assay. 24 Acta Chim. Slov. 2022, 69, 13–29 Mohareb et al.: Synthesis of Fused Quinoline Derivatives with ... Table 4. The inhibitor activity of compounds 7b, 12b, 16, 20b, 21b, 22c, 22d, 24d, 24f and 24h toward Pim-1 Kinase. Compound Inhibition ratio IC50 (µM) at 10 µM 7b 96 0.22 12b 90 0.28 16 94 0.24 20b 22 > 10 21b 32 >10 22c 89 0.30 22d 24 > 10 24d 20 > 10 24f 92 0.26 24h 30 > 10 SGI-1776 – 0.048 3. Results and Discussion Initially cyclohexan-1,3-dione was chosen as the model substrate for the synthesis of fused heterocyclic compounds through studying its reactivity toward some dimeric compounds and nitrile reagents to produce bi- ologically active products. Thus, we studied the conden- sation of cyclohexane-1,3-dione (1) with some dimeric compounds. Thus, the reaction of cyclohexane-1,3-dione (1) with either of 2-aminoprop-1-ene-1,1,3-tricarbonitrile (2a) or diethyl 3-amino-2-cyanopent-2-enedioate (2b) in the presence of a catalytic amount of ammonium acetate in an oil bath at 120 °C gave the 5,6,7,8-tetrahydronaphtha- lene derivatives 3a and 3b, respectively. The structures of the latter products were confirmed through their respec- Schema 1. Synthesis of compounds 3a,b; 4a,b; 6a,b and 7a,b. 25Acta Chim. Slov. 2022, 69, 13–29 Mohareb et al.: Synthesis of Fused Quinoline Derivatives with ... tive analytical and spectral data. Thus, the 1H NMR spec- trum of compound 3a revealed beside the expected sig- nals, two singlets, D2O exchangeable, indicating two NH2 groups and the 13C NMR spectrum showed the presence of a signal at δ 174.2 due to the C=N bonding, signals at δ 127.9, 125.6, 124.9, 123.5, 121.8, 120.4 indicating the aro- matic carbons and two signals at d116.8, 116.3 confirming the presence of the two CN groups. The latter compounds when heated in acetic acid/acetic anhydride solution gave the N-acetamido derivative 4a and the acetate ester deriv- ative 4b, respectively. On the other hand the reaction of either compound 4a or 4b with phenyl isothiocyanate (5) in 1,4-dioxane solution containing a catalytic amount of triethylamine gave the 1,2,3,4,7,8,9,10-octahydrobenzo[h] quinazoline 6a and the 3,4,7,8,9,10-hexahydro-2H-naph- tho[2,1-e][1,3]oxazine 6b, respectively. Compounds 6a and 6b underwent ready hydrolysis of the exocyclic C=N when heated in ethanol containing hydrochloric acid to Schema 2. Synthesis of compounds 10, 12a,b and 16. 26 Acta Chim. Slov. 2022, 69, 13–29 Mohareb et al.: Synthesis of Fused Quinoline Derivatives with ... afford the corresponding 4,10-dione derivatives 7a and 7b, respectively via ammonia liberation (Scheme 1). The chemical structures of new compounds were assured by spectral data (IR, 1H and 13C NMR, MS). The reaction of cyclohexane-1,3-dione (1) with ben- zoyl isothiocyanate (8) in 1,4-dioxane gave the benzo[e] [1,3]oxazin-5(6H)-one derivative 10. Formation of the latter product was explained through the first addition of the methyleno group of compound 1 to the isothiocy- anate moiety of 8 to give the intermediate 9 followed by the elimination of one molecule of water. The structure of compound 10 was based on the obtained analytical and spectral data. Thus, its mass spectrum revealed m/z 257 corresponding to its molecular mass. The 1H NMR spec- trum showed the presence of a multiplet at δ 7.43–7.22 due to the C6H5 group and the 13C NMR spectrum showed three signals at δ 180.4, 174.2, 168.2 due to the presence of C-4, C-2 and C-5, respectively and signals at δ 142.7, 133.3, 126.3, 125.2, 123.6, 121.1 indicating the C6H5, C-2, C-4a and C-8a carbons. Compound 10 is capable of forming fused thiophene derivatives through Gewald’s thiophene reaction.35,36 Thus, the reaction of compound 10 with elemental sul- fur and either malononitrile (11a) or ethyl cyanoace- tate (11b) in ethanol containing triethylamine gave the thieno[2’,3’:5,6]benzo[1,2-e][1,3]oxazine derivatives 12a Schema 3. Synthesis of compounds 18a,b; 20a,b and 21a-d. 27Acta Chim. Slov. 2022, 69, 13–29 Mohareb et al.: Synthesis of Fused Quinoline Derivatives with ... Schema 4. Synthesis of compounds 22a-d and 24a-h. and 12b, respectively. Next we studied the reaction of cy- clohexan-1,3-dione with trichloroacetonitrile followed by heterocyclization of the product in the aim of producing halogen-rich compounds that are characterized by high inhibitions toward cancer cell lines. Therefore, the reac- tion of cyclohexan-1,3-dione (1) with trichloroacetonitrile (13) in ethanol solution containing triethylamine gave the (2,2,2-trichloroethylidene)cyclohexane derivative 14. The latter compound showed interesting reactivity toward a variety of chemical reagents. Thus, the reaction of com- pound 14 with phenyl isothiocyanate (5) in 1,4-dioxane solution containing a catalytic amount of triethylamine gave the 2,3,6,7-tetrahydroquinazoline derivatives 16 via the intermediate formation of Michael addition adduct 15 followed by the cyclization through water elimination (Scheme 2). The analytical and spectral data of compound 16 were in agreement with its proposed structure. Thus, the 1H NMR spectrum showed the presence of a multiplet at δ 7.29–7.42 due to the presence of C6H5 group and a sin- glet at δ 8.28, D2O exchangeable, due to the NH group and the 13C NMR spectrum showed a signal at 94.4 indicating the CCl3 group, signals at δ 120.5, 121.5, 123.6, 123.9, 19.0, 130.3, 133.6, 135.2 for the C6H5, C-8, C-9, C-3 and C-4 carbons and two signals at δ 168.2, 178.8 for the C-5 and C-2 carbons. The reaction of compound 16 with two fold of either hydrazine hydrate (17a) or phenylhydrazine (17b) gave the 5-hydrazono-1-phenyl-3,5,6,7-tetrahydroquinazoline derivatives 18a and 18b, respectively. On the other hand, the reaction of compound 14 with either malononitrile (11a) or ethyl cyanoacetate (11b) in 1,4-dioxane solution containing a catalytic amount of triethylamine gave the 6,7-dihydroisoquinoline derivatives 20a and 20b, respec- tively. Formation of the latter products was assumed to took place via first Knoevenagel condensation of the cy- anomethylene reagent to give the intermediates 19a,b fol- lowed by Michael addition to produce 21a,b. The Gewald’s thiophene reactions of either 20a or 20b with elemental sulfur and either malononitrile (11a) or ethyl cyanoace- tate (11b) gave the 5,6-dihydrothieno[2,3-h]isoquinoline derivatives 21a–d, respectively (Scheme 3). The trichloromethyl moiety present in compounds 21a–d showed interesting reactivity toward nucleophilic displacement reactions. Thus, the heating of either 21a, 21b, 21c or 21d in ethanolic sodium hydroxide solution gave the 1-hydroxy-5,6-dihydrothieno[2,3-h]isoquinoline 28 Acta Chim. Slov. 2022, 69, 13–29 Mohareb et al.: Synthesis of Fused Quinoline Derivatives with ... derivatives 22a–d, respectively. On the other hand, the re- action of either 21a, 21b, 21c or 21d with either potassi- um cyanide (23a) or potassium thiocyanate (23b) gave the corresponding nucleophlic displacement products 24a–h, respectively (Scheme 4). All new compounds were con- firmed by their correct spectral data and elemental analy- ses values (see the Experimental section). 4. Conclusion The main findings of these studies is the synthesis of a series of novel heterocyclic derivatives synthesized from cyclohexan-1,3-dione followed by screening of the new- ly synthesized compounds towards six cancer cell lines. Sixteen compounds exhibited high inhibitions toward the cancer cell lines and the c-Met enzymatic activity re- vealed that eleven compounds were more active than the reference foretinib. In addition, twenty three compounds displayed much higher anti-proliferation activities against PC-3 cell line than the standard anibamine. 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DOI:10.1016/j.bmc.2016.02.046 Povzetek Cikloheksan-1,3-dion (1) reagira bodisi z 2-aminoprop-1-en-1,1,3-trikarbonitrilom (2a) ali z dietil 3-amino-2-cian- opent-2-endioatom (2b) in daje 5,6,7,8-tetrahidronaftalenska derivata 3a in 3b. Ti dve spojini s heterociklizacijsko reak- cijo dajeta tieno[2’,3’:5,6]benzo[1,2-e][1,3]oksazinska derivata. Po drugi strani reakcija spojine 1 s trikloroacetonitrilom daje (2,2,2-trikloroetiliden)cikloheksanski derivat 14, ki je uporaben v seriji reakcij za sintezo 2,3,6,7-tetrahidrokinazo- linskih, dihidrotieno[2,3-h]izokinolinskih, oktahidrobenzo[h]kinazolinskih in dihidrotieno[2,3-h]izokinolinskih deri- vatov. Vse sintetizirane spojine smo testirali na šestih rakavih celičnih linijah, kjer se jih je večina izkazala z visokimi inhibitornimi lastnostmi; raziskali smo tudi c-Met encimsko aktivnost ter inhibitorno aktivnost na tirozin kinaze in Pim-1. Dobljeni rezultati kažejo, da je nadaljevanje raziskovanja sintez na področju teh spojin z namenom optimizacije protirakavih učinkovin zelo obetavno. Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License 34. W. Zhu, W. Wang, S. Xu, Q. Tang, C. Wu, Y. Zhao, P. Zheng, Bioorg. Med. Chem. 2016, 24, 1749–1756. DOI:10.1016/j.bmc.2016.02.046 35. R. Mishra, K. K. Jha, S. Kumar, I. Tomer, Der Pharma. Chem- ica 2011, 3, 38–54. 36. K. Wang, D. Kim, A. Dömling, J. Comb. Chem. 2010, 12, 111– 118. DOI:10.1021/cc9001586 30 Acta Chim. Slov. 2022, 69, 30–38 Pourkazemi et al.: Glucose-Decorated Silica-Molybdate Complex: ... DOI: 10.17344/acsi.2021.6819 Scientific paper Glucose-Decorated Silica-Molybdate Complex: A Novel Catalyst for Facile Synthesis of Pyrano[2,3-d]-Pyrimidine Derivatives Arezoo Pourkazemi, Negin Asaadi, Mahnaz Farahi,* Ali Zarnegaryan and Bahador Karami Department of Chemistry, Yasouj University, P. O. Box 353, Yasouj 75918-74831, Iran * Corresponding author: E-mail: farahimb@yu.ac.ir Received: 03-13-2021 Abstract This article describes the preparation and identification of SiO2@Glu/Si(OEt)2(CH2)3N = Mo[Mo5O18] as a new bifunc- tional acid-base catalyst (both acidic and basic Lewis sites). Aminopropyltriethoxysilane was first reacted with hexam- olybdate anions and then treated with glucose to prepare Glu/Si(OEt)2(CH2)3N = Mo[Mo5O18]. Nano-silica was then modified by the prepared glucose/molybdate complex to obtain SiO2@Glu/Si(OEt)2(CH2)3N = Mo[Mo5O18]. The devel- oped catalyst was characterized by FT-IR, EDX, XRD, FE-SEM and TGA analyzes. Its catalytic efficiency was investigated for the preparation of pyrano[2,3-d]pyrimidine derivatives by the reaction between various aldehydes, malononitrile and barbituric acid. The desired products were prepared in the presence of 0.004 g of the prepared catalyst in high to excellent yields. Keywords: Silica nanoparticle; glucose; hexamolybdate anions; pyrano[2,3-d]pyrimidine; nanocatalyst 1. Introduction Catalysis includes the variants of homogeneous, het- erogeneous, and biological catalysis. Homogeneous cata- lysts offer many distinct advantages over their heteroge- neous counterparts. For example, due to the high solubility of homogeneous catalysts, all catalytic sites are accessible. In addition, they often exhibit high chemoselectivity, regi- oselectivity, and/or enantioselectivity in organic transfor- mations.1 Despite these advantages, most homogeneous catalysts have not been used commercially because they have one major disadvantage compared to heterogeneous catalysts: They are difficult to separate from the reaction mixture and solvent. The usual separation method requires high temperatures, while most homogeneous catalysts are thermally sensitive and usually decompose below 150 °C.2 Attempts have been made to solve the problem by immobi- lizing the catalysts on various supports such as carbon, sil- ica, metal oxide, polymers, and nanocomposites.1 Over the past century, the development of recoverable supported catalysts with high efficiency has been the subject of much research. Immobilized catalysts have significant advantages such as ease of handling, low solubility, possibility of recov- ery, and low toxicity.3-8 In heterogeneous supported cataly- sis, the catalytic ability of materials usually depends on their microscopic structure, which directly affects the ac- tivity, selectivity, and thermal or chemical stability of the catalyst.9 Nanomaterials are widely used as solid support materials for the preparation of many heterogeneous cata- lytic systems to solve various economic and environmental problems.10 Nano-silica is widely used due to its unique properties, such as controllable particle size and non-toxic- ity. Nano-SiO2 has a high surface-to-volume ratio and a porous structure that enables high chemical reactivity.11 In addition, nano-silica has been used in various fields such as biomedicine, fillers, catalysis, and drug delivery systems. The size and uniformity of nano-silica particles have the greatest influence on their quality; therefore, SiO2 nanopar- ticles with narrow and monodisperse size distribution are increasingly in demand.12–14 Compared with the well- known methods for preparing nano-SiO2, the Stöber meth- od is considered to be the most effective method for pre- paring monodisperse silica spheres. This method provides a flexible chemical route to fabricate materials that are highly pure, chemically reactive, and well dispersed.15 The great attention given to nanoparticles is due to their exceptional properties: easy availability, chemical in- 31Acta Chim. Slov. 2022, 69, 30–38 Pourkazemi et al.: Glucose-Decorated Silica-Molybdate Complex: ... ertness, high surface-to-volume ratio, high activity and selectivity, thermal stability, and low toxicity. Moreover, nanoscale systems significantly increase the contact be- tween reactants and catalyst. They open new perspectives for mild catalysis of important reactions with lower envi- ronmental impact. Nanoparticles differ from their solid counterparts and exhibit special properties. Due to the above advantages, they have been developed as suitable re- placements for conventional heterogeneous catalysts.16 Recently, inorganic-organic hybrid materials have been widely used as catalysts for organic reactions because they are well suited for various processes of environmen- tally friendly chemical transformations.17 Several interest- ing new materials with novel properties are currently emerging in this rapidly growing field. They combine the typical advantages of organic components, such as flexibil- ity, low density, toughness, and malleability, with the ad- vantages of typical inorganic materials, such as hardness, chemical resistance, strength, and optical properties.18 The properties of these materials are not just the sum of the individual contributions of the two phases, but the role of the internal interfaces could also be important. Organ- ic-inorganic graft materials have emerged as surrogate ma- terials for the development of unique products and have become a new area of academic research. When the idea of a monomolecular bifunctional cat- alyst for helpful catalysis was first introduced in 2003, both homogeneous and heterogeneous catalysts with molecular design and their use in organic reactions became the focus of interest.19 In this context, polyoxometalates (POMs) are an important class of nanoscale polynuclear clusters with significant physical and chemical properties based on transition metals in their highest oxidation states and oxy- gen bridges.20 Polyoxometalate clusters, known for their enormous size and interesting properties for medicine and nonlinear optics, are a prominent class of linkers for the preparation of interpenetrating networks. The direct ap- plication of POM clusters as linkers promises an attractive route to the development of new entangled network struc- tures. The main properties of polyoxometallates and the variation in their structures give them great potential for applications in various fields of chemical processing.21 De- spite the above advantages, the solubility and non-recover- ability of POMs in various media limit their applications in some processes. Immobilization of these clusters on sol- id supports such as silica and magnetic nanoparticles could be an important way to overcome this problem. Hexamolybdates are a group of POMs used in various in- organic and organic reactions due to their thermal stability and radiation resistance. Lindqvist hexamolybdate cluster, [Mo6O19]2−, as a unique class of metal oxide clusters, is an ideal building block for the construction of organic–inor- ganic hybrid assemblies.22,23 Carbon-based materials have attracted much atten- tion from researchers because they are environmentally friendly, cheap, and nontoxic. Numerous studies have been conducted on these materials as catalyst supports, such as carbon nanotubes, carbon-polymer composites, mesoporous carbons, graphitized carbons, graphitized ni- tride carbons, carbides, and carbon aero-gels. In this con- text, cellulose has been used as a catalyst support due to its uniform shape, stability in aqueous solution, good me- chanical strength, high specific surface area, biocompati- bility and biodegradability. The development of cellu- lose-based composites with metal oxides such as silicon dioxide, titanium dioxide, zinc oxide, and iron oxides is of great interest for various high-technology applications.24-26 Due to its polyhydroxy structure, glucose has been used as the main green monosaccharide catalyst and showed ex- cellent catalytic activity in chemical reactions such as ep- oxidation and enantioselective Michael addition. It has also played the role of a green medium for carrying out reactions. Recently, Fe3O4 nanoparticles coated with glu- cose were prepared and used as a heterogeneous catalyst for the synthesis of pyrazole derivatives.27 Nowadays, several chemists have paid great atten- tion to the development of new approaches to the produc- tion of nitrogen-containing heterocycles, which play an important role in our lives. They are components of many natural products, fine chemicals, and biologically active drugs that are of great importance in improving the quality of life.28 Pyrano[2,3-d]pyrimidine derivatives represent a “privileged” structural motif that is widely used in natural- ly occurring compounds with a variety of important bio- logical properties. Recently, a number of synthetic pyra- no[2,3-d]pyrimidines have been investigated for their potent anticancer, antibacterial, antifungal, and antirheu- matic properties.29 They also exhibit anti-inflammatory,30 anti-HIV,31 cytotoxic,32 antimicrobial,33 antimalarial, and antihyperglycemic properties.34 It is worth noting that many drug molecules bearing the pyrano-pyrimidine moiety are used in the treatment of various diseases such as bronchitis, as hepatoprotective agents, and as cardioton- ic agents.33 With increasing public concern about environmen- tal degradation and future resources, it is critical for chem- ists to develop new approaches that are less hazardous to human health and the environment. Therefore, in con- junction with our previous research on new heterogeneous catalysts,35-41 we decided to introduce SiO2@Glu/ Si(OEt)2(CH2)3N = Mo[Mo5O18] nanocatalysts, whose catalytic activity was investigated in the one-pot synthesis of pyrano[2,3-d]pyrimidines. 2. Results and Discussion The desired catalyst was synthesized in a simple manner as shown in Scheme 1. To prepare SiO2@Glu/ Si(OEt)2(CH2)3NH2 grafted [Mo6O19]2− composite (3), 3-aminopropyltriethoxysilane was reacted with tetrabu- tylammonium hexamolybdate followed by glucose to give 32 Acta Chim. Slov. 2022, 69, 30–38 Pourkazemi et al.: Glucose-Decorated Silica-Molybdate Complex: ... Glu/Si(OEt)2(CH2)3N = Mo[Mo5O18] (2). SiO2 nanoparti- cles were also synthesized by the Stöber method.15 Finally, the OH groups on the silica surface can be grafted with Glu/Si(OEt)2(CH2)3N = Mo[Mo5O18] to obtain the de- sired nanocatalyst 3. The chemical and structural proper- ties of the catalyst were investigated by FT-IR, EDX, XRD, FE-SEM and TGA analyzes. The identification and determination of the organic functional groups were performed by FT-IR spectroscopy. Figure 1 shows the infrared spectra of glucose, SiO2, SiO2@ Glu/Si(OEt)2(CH2)3N = Mo[Mo5O18] and [n-Bu4N]2 [Mo6O19]. In Figure 1a, the peaks at 3662 and 3385 cm–1 are assigned to the OH groups. Also, the peaks at 2939 and 1458 cm–1 are related to the stretching of CH and the sym- metric bending stretching of CH2, respectively. The bands at 1160 cm–1 (antisymmetric C-O-C stretching), 1116 cm–1, and 1052 cm–1 (skeletal vibrations with C-O stretching) can be assigned.18 Figure 1b shows the bands at 1080, 948, and 797 cm–1 that are due to Si-O stretching, Si-OH stretching, and symmetric Si-O-Si stretching, respective- ly.15 In Figure 1c, the absorption peaks at 2934 and 2840 cm–1 correspond to the C-H vibrations of the alkyl chains.35 Moreover, the bands at 953, 804, and 798 cm–1 are attributed to N = Mo, Mo-O, and Mo-O-Mo, respec- tively, confirming the presence of [Mo6O18]-2 ions in the structure of the synthesized nanocatalyst.22 The XRD patterns of SiO2@Glu/Si(OEt)2(CH2)3N = Mo[Mo5O18] and SiO2 are shown in Figure 2, which was used to study the crystallographic features of the prepared catalyst. It can be seen that the reflection peaks are in the 2θ range of 0–70°. In Figure 2a, the broad peak in the 2θ 20°–32° range is consistent with an amorphous silica phase.36 The presence of sharp peaks proves the special status of the nanocatalyst structure. In Figure 2b, the XRD pattern shows that the amorphous structure of SiO2 parti- cles was preserved. The confirming peak indicating the Scheme 1. Schematic representation of the synthesis of SiO2@Glu/Si(OEt)2(CH2)3N = Mo[Mo5O18] (3). Figure 1. FT-IR Spectra of a) glucose, b) SiO2, c) SiO2@Glu/ Si(OEt)2(CH2)3N = Mo[Mo5O18] and d) [n-Bu4N]2[Mo6O19]. Figure 2. The XRD pattern of a) SiO2 and b) SiO2@Glu/ Si(OEt)2(CH2)3N = Mo[Mo5O18] nanocatalyst. 33Acta Chim. Slov. 2022, 69, 30–38 Pourkazemi et al.: Glucose-Decorated Silica-Molybdate Complex: ... presence of the molybdate group appeared in the range of 2θ = 20°–30°, which can be attributed to the amorphous molybdate on the composite.35,39 Energy-dispersive X-ray spectroscopy (EDS) was se- lected to provide the necessary information on elemental structure of the catalyst. According to the Fig. 3, the EDS pattern clearly indicated the existence expected the ele- mental composition of C, N, O, Si, and Mo in the nanocat- alyst structure. The surface morphology and particle size distribu- tion of the prepared nanocatalyst were observed using FE- SEM, and the corresponding image is shown in Figure 4. The results show that the particles are uniformly and regu- larly spherical with an average diameter of 22–42 nm. The results of the thermal stability of nanocatalyst 3 by thermogravimetric analysis (TG) from 0 to 900 °C are shown in Figure 5. The results show that the first weight loss at a temperature of 120 °C (about 12%) is related to the removal of H2O and other organic solvents left behind in the extraction process. The second weight loss at 220–320 °C (about 5%) is due to the removal of organic compo- nents located on the surface of the catalyst. The largest weight loss in a temperature range of 500–550 °C (about 12.5%) is related to the removal of propylamine groups at- tached to the catalyst framework.22 After characterization to study the catalytic activity and efficiency of the newly developed catalyst, it was used as a catalyst for the synthesis of pyrano[2,3-d]pyrimidines 7 via the three-component reaction of arylaldehydes 4, Figure 3. EDS analysis of SiO2@Glu/Si(OEt)2(CH2)3N = Mo[Mo5O18]. Figure 4. FE-SEM analysis of SiO2@Glu/Si(OEt)2(CH2)3N = Mo[Mo5O18]. Figure 5. TG analysis of SiO2@Glu/Si(OEt)2(CH2)3N = Mo[Mo5O18]. 34 Acta Chim. Slov. 2022, 69, 30–38 Pourkazemi et al.: Glucose-Decorated Silica-Molybdate Complex: ... malononitrile 5, and barbituric acid 6 under solvent-free conditions (Scheme 2). To find the optimum reaction conditions, a three-component reaction between benzaldehyde, malono- nitrile, and 1,3-dimethylbarbituric acid was selected as a model reaction, and the effect of various parameters such as temperature, catalyst loading, and solvent was evaluated. The reaction did not proceed well in the absence of the cat- alyst after a long reaction time. The model reaction was car- ried out at 25, 60, 80 and 100 °C in the presence of 0.002 g catalyst 3. The study showed that the reaction was affected by temperature, and the best result was observed at 80 °C. Next, the effect of the amount of catalyst was studied. It was found that the yield increased when the amount of catalyst was increased from 0.002 g to 0.004 g, and higher amount of catalyst did not have a good effect on the reac- tion process. Moreover, the model reaction was carried out with 0.004 g SiO2@Glu/Si(OEt)2(CH2)3N = Mo[Mo5O18] Table 1. Screening of various parameters in the synthesis of 7a cata- lyzed by nanocatalyst 3.a Entry Catalyst Solvent Temp. (°C) Yieldb (%) loading (g) 1 0.002 – 25 20 2 0.002 – 60 45 3 0.002 – 80 60 4 0.002 – 100 50 5 0.004 – 80 90 6 0.006 – 80 85 7 0.008 – 80 80 8 0.004 MeOH Reflux 60 9 0.004 EtOH Reflux 70 10 0.004 CH3CN Reflux 75 11 0.004 Toluene Reflux 65 a Reaction conditions: benzaldehyde (1 mmol), malononitrile (1 mmol), 1,3–dimethylbarbituric acid (1 mmol), time: 20 min. b Isolated yields. Scheme 1. Preparation of pyrano[2,3-d]pyrimidines 7 in the pres- ence of SiO2@Glu/Si(OEt)2(CH2)3N = Mo[Mo5O18] nanocatalyst. Table 2. Preparation of various pyrano[2,3-d]-pyrimidines by nanocatalyst 3. Entry R Aldehyde Tim (min) Yielda (%) Mp (Lit) (°C) 7a H C6H5CHO 30 90 212–214 (212–213)42 7b H 2,4-Cl C6H3CHO 15 93 242–244 (242–243)42 7c H 3-NO2 C6H4CHO 20 95 260–262 (262–263)43 7d H 4-NO2 C6H4CHO 20 93 237–239 (237–238)43 7e H 3-Br C6H4CHO 25 95 280–282 (279–280)43 7f H 4-Br C6H4CHO 20 90 228–230 (227–229)43 7g H 4-OCH3 C6H4CHO 25 95 280–282 (281–282)43 7h H 4-Cl C6H4 CHO 15 90 238–240 (239–240)44 7i CH3 C6H5CHO 20 95 228–230 (228–229)44 7j CH3 2-Cl C6H4 CHO 15 89 248–250 (250–251)44 7k CH3 4-OCH3 C6H4CHO 30 95 224–225 (225–227)44 7l CH3 4-Cl C6H4CHO 10 95 238–240 (239–241)44 7m CH3 3-NO2 C6H4CHO 20 93 212–214 (212–213)44 7n CH3 4-NO2 C6H4CHO 20 95 230–232 (231–232)44 7o CH3 3-Br C6H4CHO 15 93 218–220 (218–219)44 7p CH3 4-CH3 C6H4CHO 20 90 230–232 (229–230)44 7q CH3 2,4-Cl2 C6H3CHO 10 95 211–213 (211–212)30 7r CH3 1-Naphthaldehyde 40 93 360 (360–361)44 7s CH3 Biphenyl-4-carboxaldehyde 30 95 275–277b 7t CH3 Terephthalaldehyde 20 95 240–242b a Isolated yields. b Novel product. 35Acta Chim. Slov. 2022, 69, 30–38 Pourkazemi et al.: Glucose-Decorated Silica-Molybdate Complex: ... in some solvents such as methanol, ethanol, acetonitrile and toluene. As can be seen, considerable acceleration is observed especially for reactions carried out under sol- vent-free conditions. According to these results, the use of SiO2@Glu/Si(OEt)2(CH2)3N = Mo[Mo5O18] (0.004 g) as catalyst under solvent-free conditions at 80 °C would be the best choice (Table 1). After optimization of the reaction conditions, the re- action of various aromatic aldehydes, malononitrile and barbituric acid derivatives was studied in the presence of nanocatalyst 3 under optimal conditions, which showed the successful formation of the corresponding pyra- no[2,3-d]-pyrimidines (Table 2). Both aldehydes with electron-donating groups and aldehydes with electron-at- tracting groups were prepared in good to excellent yields. The proposed mechanism for the formation of pyra- no[2,3-d]pyrimidines 7 is shown in Scheme 3. According to the proposed mechanism, adduct 8 is first obtained by the condensation of aromatic aldehydes and malononitrile in the presence of the catalyst. Then, Michael addition of barbituric acid to this intermediate 9 is formed. The intra- molecular cyclization of 9 gives the adduct 10, which rear- ranges to give the pyrano[2,3-d]pyrimidinones 7. The main advantages of the presented protocol over existing methods become clear when our results are com- pared with those of some recent methods reported in arti- cles (see Table 3). To study the leaching of [Mo6O19]2− from SiO2@Glu/ Si(OEt)2(CH2)3N = Mo[Mo5O18], we performed an in situ filtration technique. When the model reaction reached 50%, warm EtOAc (5 ml) was added, and catalyst isolation was performed by simple filtration. After removal of the solvent, the process was continued with the catalyst-free residue under the previously optimized conditions. As ex- pected, the reaction stopped, confirming that no leaching of the supported catalytic centers occurred under the opti- mized conditions. The reusability of SiO2@Glu/Si(OEt)2 (CH2)3N = Mo[Mo5O18] was also investigated in the mod- el reaction. After completion of the reaction, EtOAc (5 mL) was added to the mixture, the catalyst was filtered, washed with EtOH (10 mL) and deionized water (10 mL), and then dried at 100 °C. The recovered catalyst was used ten times in the model reaction, and the yield was negligi- ble (Figure 6). These experiments indicate high stability and durability of this nanocatalyst under the applied con- ditions. To test the stability of the catalyst structure, the recycled nanocatalyst was examined using FT-IR spectra. The FT-IR spectra of the freshly prepared catalyst and the recycled catalyst are shown in Figure 7 and confirm the chemical stability of catalyst 3. Scheme 3. The proposed mechanism for the synthesis of pyrano[2,3-d]pyrimidinones catalyzed by nanocatalyst 3. Table 3. Comparison of results for the synthesis of 7I with other catalysts. Entry Catalyst Catalyst loading Condition Time (min) Yielda (%)a 1 Catalyst 3 0.004 g solvent-free, 80 °C 10 95b 2 Et3N 20 mol % EtOH, 50 °C 25 8728 3 Urea 10 mol % EtOH:H2O, r.t. 840 8629 4 Zn [(L) proline]2 17 mol % EtOH, Reflux 50 9045 5 {Fe3O4@SiO2@(CH2)3- Urea-SO3H/HCl} MNP 0.01 g solvent-free, 60 °C 30 9744 6 Nano-basic silica 25 mol % solvent-free 54 8946 a Isolated yields. b This study. 36 Acta Chim. Slov. 2022, 69, 30–38 Pourkazemi et al.: Glucose-Decorated Silica-Molybdate Complex: ... 3. Experimental All chemical materials were purchased from Merck and Aldrich. The reaction progress and purity of the com- pounds were monitored by TLC on silica gel SIL G/UV254 plates. Melting points were checked using a KSB1N elec- trothermal device and are correct. The IR spectra of all synthesized compounds were recorded in the KBr matrix using a spectrometer model JASCO FT-IR /680 plus. 1H NMR spectra were recorded in DMSO-d6 as solvent using a Bruker Avance Ultra Shield 400 MHz spectrometer, and 13C NMR spectra were registered at 100 MHz. A scanning electron microscope (FE-SEM) was used to measure the size of the particles and the shape of the catalyst. X-ray diffraction (XRD) patterns were recorded with a Philips X Pert Pro X diffractometer using Ni filtered Cu-Ka radia- tion. Energy dispersion spectroscopy (EDS) was recorded using a TESCAN Vega instrument. Preparation of the Glu/Si(OEt)2(CH2)3N = Mo[Mo5O18] First, the tetrabutylammonium hexamolybdate ([n-Bu4N]2 [Mo6O19]) was prepared according to the described procedure.20 The tetrabutylammonium hexam- olybdate (0.4 g) and DMSO (20 ml) were placed in a round bottom flask (50 ml) and dispersed for 20 minutes. Then 3-aminopropyltriethoxysilane (2.5 ml) was added dropwise to the mixture and stirred under reflux condi- tions for 24 hours under argon atmosphere. Dissolved glucose (0.26 g) in dry DMSO (5 mL) and H2SO4 (98%, 0.33 mL) were then added. The mixture was stirred at room temperature for 5 hours. Finally, the obtained prod- uct (compound 2) was washed with ethanol, distilled and dried at 80 °C. Protocol for the synthesis of nano-SiO2 Tetraethyl orthosilicate (TEOS) (6.2 ml) was added to ethanol (100 ml) and ammonium hydroxide (6.5 ml), and the mixture was stirred at room temperature for 15 hours. The mixture was then filtered by centrifugation (4000 rpm, 30 min), and the resulting white powder was washed three times with ethanol and dried for 12 h at 60 °C.15 Preparation of the SiO2@Glu/Si(OEt)2(CH2)3N = Mo[Mo5O18] To immobilize Glu/Si(OEt)2(CH2)3N = Mo[Mo5O18] on the surface of SiO2, the prepared SiO2 nanoparticles (1.0 g) were dispersed in dry toluene (30 mL) by ultrason- ication for 20 min. Compound 2 (0.5 g) was then added and the mixture was refluxed under argon atmosphere for 24 hours. Then the prepared SiO2@Glu/Si(OEt)2(CH2)3N = Mo[Mo5O18] was filtered and washed several times with ethanol and then with water. Finally, the brown powder was dried in vacuo at 80 °C for 24 hours. General procedure for the synthesis of pyrano[2,3-d]-py- rimidines derivatives 7 SiO2@Glu/Si(OEt)2(CH2)3N = Mo[Mo5O18] (0.004 g) was added to the mixture of arylaldehyde (1 mmol), malononitrile (1 mmol), and barbituric acid (1 mmol) at 80 °C under solvent-free conditions. The progress of the reaction was monitored by TLC. After completion of the reaction, ethyl acetate was added and the catalyst was separated by filtration. For further purification of the product, the obtained powder was recrystallized from EtOH. Spectral data 7-Amino-5-(2,4-dichlorophenyl)-1,3-dimethyl-2,4-di- oxo-1,3,4,5-tetrahydro-2H-pyrano[2,3-d]pyrimidine-6-car- bonitrile (7q). White solid; mp: 211–213 °C; IR (KBr) (vmax, cm–1) 3394, 3313, 3212, 3081, 2962, 2194, 1708, 1689, 1643, 1494, 1384, 1230, 1184, 1099, 1049, 844, 582, 755. 1H NMR (400 MHz, DMSO-d6) δ 3.09 (s, 3H), 3.39 (s, 3H), 4.88 (s, 1H), 6.93-7.55 (m, 5H) ppm. 13C NMR (100 MHz, DMSO-d6) δ 28.09, 29.63, 33.70, 57.19, 88.07, 118.86, 128.12, 129.01, 132.11, 132.44, 133.67, 140.85, 150.44, 151.05, 158.22, 160.78 ppm. Figure 7. FT-IR spectra for comparison of fresh catalyst and recov- ered catalyst. Figure 6. Reuse of SiO2@Glu/Si(OEt)2(CH2)3N = Mo[Mo5O18] na- nocatalyst. 37Acta Chim. Slov. 2022, 69, 30–38 Pourkazemi et al.: Glucose-Decorated Silica-Molybdate Complex: ... 5-([1,1’-Biphenyl]-4-yl)-7-amino-1,3-dimethyl-2,4-dioxo- 1,3,4,5-tetrahydro-2H-pyrano[2,3-d]pyrimidine-6-carboni- trile (7s). White solid; mp: 275–277 °C; IR (KBr) (vmax, cm–1) 3432, 3300, 3177, 3074, 2984, 2190, 1736, 1684, 1634, 1486, 1386, 1227, 1186, 1040, 1007, 850, 744, 751, 698, 571, 550. 1H NMR (400 MHz, DMSO-d6) δ 2.11 (s, 3H), 2.54 (s, 3H), 4.40 (s, 1H), 6.93-7.67 (m, 11H) ppm. 13C NMR (100 MHz, DMSO-d6) δ 28.17, 29.62, 36.70, 58.99, 81.25, 119.56, 127.39, 127.56, 127.56, 127.78, 128.02, 128.44, 129.37, 129.40, 129.59, 129.67, 138.72, 139.29, 140.47, 150.48, 158.20, 161.00, 161.27 ppm. 7-Amino-5-(4-formylphenyl)-1,3-dimethyl-2,4-dioxo- 1,3,4,5-tetrahydro-2H-pyrano[2,3-d]pyrimidine-6-carboni- trile (7t). White solid; mp: 240–242 °C; IR (KBr) (vmax, cm–1) 3380, 3316, 3192, 3074, 2984, 2195, 1708, 1685, 1638, 1487, 1386, 1226, 1185, 1040, 1115, 847, 751, 698, 571. 1H NMR (400 MHz, DMSO-d6) δ 3.116 (s, 3H), 3.386 (s, 3H), 4.403 (s, 1H), 7.341-7.672 (m,6H), 9.146, (s, 1H) ppm. 13C NMR (100 MHz, DMSO-d6) δ 28.25, 29.63, 36.70, 58.99, 81.25, 128.03, 128.44, 129.37, 129.67, 130.79, 131.81, 140.47, 150.49, 158.20, 161.01, 161.28 ppm. 4. Conclusions In this work, we have presented for the first time SiO2@Glu/Si(OEt)2(CH2)3N = Mo[Mo5O18] as a green and recyclable SiO2-based nanocatalyst. The efficiency of this catalyst was evaluated in the synthesis of pyrano[2,3-d] pyrimidinone derivatives. This new catalytic system showed the advantages of environmentally friendly char- acter, easy separation, non-toxicity, mild reaction condi- tions, short reaction times and good reusability. 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DOI:10.1515/mgmc-2016-0034 Povzetek Prispevek opisuje pripravo in identifikacijo SiO2@Glu/Si(OEt)2(CH2)3N = Mo[Mo5O18] kot novega bifunkcionalnega kislinsko-baznega katalizatorja (s kislimi in bazičimi Lewisovimi mesti). Najprej so aminopropiltrietoksisilan reagirali s heksamolibdatnimi anioni in nato obdelali z glukozo, ter tako pripravili Glu/Si(OEt)2(CH2)3N = Mo[Mo5O18]. Na- no-siliko so nato modificirali s pripravljenim kompleksom glukoza/molibdat in dobili SiO2@Glu/Si(OEt)2(CH2)3N = Mo[Mo5O18]. Razvit katalizator so okarakterizirali z FT-IR, EDX, XRD, FE-SEM in TGA analizo. Katalitsko učinkovi- tost novega katalizatorja so raziskali na primeru priprave derivatov pirano[2,3-d]pirimidina z reakcijo med različnimi aldehidi, malononitrilom in barbiturno kislino. Načrtovane produkte so pripravili v prisotnosti 0,004 g pripravljenega katalizatorja z visokim do odličnim izkoristkom. Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License 39Acta Chim. Slov. 2022, 69, 30–38 Yildirim et al.: Synthesis of glucose/fructose sensitive ... DOI: 10.17344/acsi.2021.6838 Scientific paper Synthesis of Glucose/Fructose Sensitive Poly(ethylene glycol) Methyl Ether Methacrylate Particles with Novel Boronate Ester Bridge Crosslinker and their Dye Release Applications Şeküre Yildirim,1 Hasan Akyildiz1,2 and Zeynep Çetinkaya1,3 1 Department of Metallurgical and Materials Engineering, Konya Technical University, Konya, Turkey 2 Nanotechnology and Advanced Materials Development, Application, and Research Center, Konya Technical University, Konya, Turkey 3 Advanced Technology Research and Application Center, Selçuk University, Konya, Turkey * Corresponding author: E-mail: zcetinkaya@ktun.edu.tr, + 90 332 205 1945 Received: 03-18-2021 Abstract In this study, it is aimed to develop glucose/fructose sensitive poly(ethylene glycol) methyl ether methacrylate (PEGMA) particles which can be employed in controlled drug delivery applications. For this purpose, a boric acid based crosslink- er was synthesized using 4-vinylphenylboronic acid (VPBA) and its formation was confirmed by 1H-NMR and FT-IR analyses. Sugar-sensitive polymeric particles were then achieved using this crosslinker and PEGMA monomer in single step and surfactant free emulsion polymerization technique. Polymeric particles were characterized by DLS, SEM, and TEM in terms of size and morphology. In order to determine the sensitivity of the particles to sugar molecules, first Rhodamine B dye (as a model drug) loading experiments were performed. Then, the particles were subjected to glucose/ fructose rich media and dye release was monitored as a function of time using UV-Vis spectrophotometry. The results of the current study revealed that the PEGMA particles were more sensitive to fructose (~39% release) compared to glucose (~25% release) at pH 7.4 and 310 K. Keywords: Controlled drug release, fructose sensitivity, phenylboronic acid, smart polymers, crosslinker 1. Introduction Smart polymers are a group of materials which can modify some of their physical/chemical properties upon exposure to external stimuli such as temperature,1 pH,2 light3, and magnetic/electric field.4 They may also show response to variety of organic compounds. Examples of these compounds include carbohydrates, enzymes, acids, and sugar molecules.5 Due to this unique behavior, these polymers are highly promising in various innovative ap- plications such as biomedical and bioengineering stud- ies.6 Among these applications, non-invasive biosensors have attracted great attention as these sensors provide the opportunity to detect the level of glucose or fructose molecules in the metabolism without disturbing the pa- tients.7 Glucose-sensitive biosensors can be classified into three types according to their chemical make-up and sens- ing mechanism.8 These are known as glucose-oxidase, pro- tein, and phenyl boronic acid (PBA) based systems. Glu- cose-oxidase sensors operate via the enzymatic oxidation of glucose molecules, whereas glucose-binding proteins are functioning through the binding of glucose molecules with glycol polymer-lectin complexes. These compounds are natural biological proteins which makes them intoler- ant to several environmental factors such as high tempera- ture and pH. Further, the instability of these materials lim- its their widely use as glucose-sensitive systems. On the contrary, PBA is known to be the synthetic derivative of boronic acid with good stability and easy preparation.7,9 Boronic acid can bind to diol and polyol species of saccha- rides with high affinity through reversible boronate ester 40 Acta Chim. Slov. 2022, 69, 30–38 Yildirim et al.: Synthesis of glucose/fructose sensitive ... formation.1a,7,10 For example, cis-1,2 and cis-1,3 diols of the sugar molecules are able to link reversibly with boron- ic acid. The details of this binding mechanism were well established in literature.6 PBA compounds exhibit an equilibrium between the charged and uncharged forms in aqueous media.11 These two forms of PBA can react with the cis-1,2 diols of sugar molecules. While the uncharged PBA is hydrophobic in nature, the complex of this form with glucose is not stable in aqueous media due to hydrolysis. On the other hand, the complex of charged PBA-glucose can form hydrophilic phenyl borate which is highly stable. Moreover, the indi- cated reaction can shift the equilibrium towards the hy- drophilic state12 which in principle increase the swelling ratio of the polymer network. This provides an opportuni- ty to use polymeric particles as sugar sensitive systems in glucose or fructose containing environments for con- trolled insulin release.10 However, PBA-based glucose sen- sitive systems cannot function effectively at physiological pH due to the high pKa value (~9) of this compound.13 This is due to low ionization of phenyl-boronic acid at pH = 7.4 which decrease the solubility of polymer in water and also its affinity to glucose. Among the proposed methods to reduce the pKa of PBA, using of alkaline solutions was suggested as a promising route which increase the binding between the boronic acid and sugar molecules.6,14 Up to now, many forms of PBA based system, such as hydrogels,15 multi-layered films,16 nanofibers,17 and nanoparticles (NPs)18 have been studied for insulin release applications. Chen et al., reported that a polymer network can be obtained by crosslinking the boronate ester of two separate polymers containing boronic acid functional PBA based system, such as hydrogels,15 multi-layered films,16 nanofibers,17 and nanoparticles (NPs)18 have been studied for insulin release applications. Chen et al., reported that a polymer network can be obtained by crosslinking the bor- onate ester of two separate polymers containing boronic acid functional groups and diols.14a With a similar ap- proach, it has been shown that polymeric NPs can be syn- thesized using a polymer containing PEG (polyethylene glycol)-based boronic acid and another polymer contain- ing diol groups.14b Further, the NPs synthesized with the surfactant free emulsion polymerization method were re- ported to exhibit smooth surface and high stability.6,19 In addition, these samples were successfully employed in drug delivery and glucose/fructose sensing applications. However, only a few studies have been reported in litera- ture which focused on the synthesis of a crosslinker with boronate ester bridge and using the surfactant free polym- erization method in the production of stimuli responsive polymeric particles.2a,14 Here, we have synthesized PEGMA particles which are sensitive to glucose/fructose molecules similar to those mentioned above albeit using a novel crosslinker contain- ing boronate ester bonds for the first time. The PEGMA particles were achieved using this novel crosslinker in one step and surfactant free emulsion polymerization method. These particles were loaded with a model drug (Rhodamine B dye) during the polymerization reaction and the amount of dye release was monitored carefully upon exposing the particles to glucose or fructose rich media and interpreted as the sensitivity level of the particles to sugar molecules. 2. Experimental 4-vinylphenyl boronic acid (VPBA, 97%, Sigma Al- drich), 4-allylatechol (%95, Sigma Aldrich), toluene (99.8%, Sigma Aldrich), and sodium hydroxide (NaOH, 97%, Sigma Aldrich) were used in the synthesis of the nov- el crosslinker. Poly(ethylene glycol) methyl ether methac- rylate (PEGMA, Mn of 300 g/mol, 97%, Sigma Aldrich), acetone (99.8%, Sigma Aldrich) and 2,2’-azobis 2-methyl- propinamide dihydrochloride (AMPDH, 97%, Sigma Al- drich) were employed in the production of PEGMA parti- cles. Toluene and deionized water (18.2 MΩ.cm) were used as catalyst and for cleaning purposes where neces- sary. 2. 1. Synthesis of Boranate Ester Bridge Containing Crosslinker The crosslinker was synthesized according to Scheme 1 given below.20 The samples were obtained by mixing 1 mmol (0.150 g) of 4-allylcatechol and 1 mmol (0.147 g) of VPBA in 10 mL of toluene in a glass beaker equipped with a reflux system. The solution pH was adjusted to 8.2 using NaOH and continuously stirred at 450 rpm for 72 h at a temperature of 383 K. After the reaction completed, the system was cooled down to room temperature and brown colored precipitates were collected and washed with fresh toluene for 3 times and then dried in a vacuum oven for 24 h at 348 K. Hereafter, this novel boronate ester bridge con- taining crosslinker is referred as CRX-3. Scheme 1. Schematic representation of boronate ester bridge containing crosslinker (CRX-3) synthesis 41Acta Chim. Slov. 2022, 69, 30–38 Yildirim et al.: Synthesis of glucose/fructose sensitive ... 2. 2. Synthesis of PEGMA Particles PEGMA particles were obtained by mixing poly(eth- ylene glycol) methyl ether methacrylate monomer (Mn of 300 g/mol) with various amounts of CRX-3, as summa- rized in Table 1. Scheme 2 shows the emulsion polymer- ization reaction between the two precursors. For the syn- thesis of the particles; 300 µL of PEGMA monomer and proper amount of crosslinker were dissolved in 30 mL of acetone:water mixture (1/29, V/V). The amount of the crosslinker was calculated based on the mole % of the monomer. The reaction flask was first purged with ultra- high purity N2 gas for 30 min to remove any dissolved ox- ygen. Polymerization process was carried out in this flask which was placed in a water bath on a magnetic stirrer at 343 K. 10 mg of AMPDH (free radical initiator) was added to the medium to initiate the polymerization reaction. The solution was stirred for 3 h at 400 rpm. After cooling to room temperature, pale orange color suspension was cen- trifuged at 7000 rpm for 10 min to collect the final PEG- MA particles. Any unreacted monomer or reactant were then removed by rinsing the product for 3 times with dis- tilled water. Finally, the particles were suspended in ul- tra-pure water for further use. actants at the initial stage of polymerization reaction. There- by, the dissolved dye molecules were forced to be trapped in the polymer network during polymerization. After the reac- tions completed, non-trapped dye molecules were removed by washing the particles in phosphate buffered saline (PBS) solution for 3 times. Then, certain amount of PEGMA par- ticles were dispersed in 30 mL of PBS and poured into 3 separate vials with identical volumes i.e., 10 mL. 10 mg of fructose was added to the first vial, 10 mg of glucose was added to the second vial and the third vial left as it is, as the control sample. The vials were placed on a magnetic stirrer and heated to a constant temperature of 310 K. Finally, the absorbance data were recorded using a UV-Vis spectropho- tometer after 0.5, 1, 2, 4, 6, 12, 24, and 48 h. 2. 4. Sample Characterization Attenuated total reflectance/Fourier transform in- frared (ATR/FT-IR) spectra of the samples were collected using a Bruker VERTEX-70 spectroscopy over the range of 4000 – 400 cm‒1 at a resolution of 4 cm‒1 and averaging 10 scans for each measurement.  Solid-state 1H-NMR spec- trum was recorded on a Varian 400 MHz spectrometer with a 5 mm double-resonance probe, sample spinning rate of 8.0 kHz, contact time of 0.002 s, and a pulse delay of 5 s to verify the formation of the material. The following numbering scheme was used to determine the sample; 400 MHz, DMSO-d6, ppm, δ = 8.79–8.67 (Ar-OH), 8.07 (B- OH), 7.25–7.23 (d), 7.18–7.16 (c), 6.60 (b), 6.36–6.28 (f,e,g), 5.92 (a2), 5.88–5.85 (i), 5.04–5.00 (a1), 4.99–4.95 (j), 3.18–3.17 (h). The conversion efficiency for the cross- linker was estimated using an integration on the reduction of H peaks in 1H-NMR spectra of CRX-3 compared to the one belonging to the precursors. The calculations were performed using MestraNova software. The morphology of the PEGMA particles was inves- tigated in as-centrifuged state using SM Zeiss LS-10 scan- ning electron microscope (SEM). The surfaces of the par- ticles were coated with gold for 30 s prior to SEM analysis using a Cressington Sputter Coater system. For transmis- sion electron microscope (TEM) examinations, the parti- cles were first washed with DI-water for 3 times to remove any unreacted species and then mixed with 2 mL of fresh DI-water to obtain a dispersion in an ultrasonic bath. This dispersion was then dropped on a carbon-coated copper grid and dried for an overnight at room temperature. A JEOL 2100F model TEM was used to determine the size and morphology of the synthesized particles. The size of PEGMA particles were further verified using Malvern Ze- taSizer Nano ZS dynamic light scattering (DLS) system. The polydispersity index (PDI) and average hydrodynamic diameters (dH) of the particles were measured after dis- persing in 2 mL KCl solution (10 mM). The change in the absorbance of the samples during dye release was recorded via Biochrom Libra S22 UV-Vis spectrometer in the wave- length range of 450–650 nm. Table 1. Synthesis conditions for PEGMA particles. Sample PEGMA CRX-3 AMPDH Acetone DI water 300 (µL) (%)a (mg) (mL) (mL) P-P-1 1 P-P-2 3 P-P-3 300 5 10 1 29 P-P-4 7.5 P-P-5 10 a According to the mole of the monomer. Scheme 2. Schematic representation of PEGMA particle synthesis using poly(ethylene glycol) methyl ether methacrylate monomer (Mn of 300 g/mol) and boronate ester bridge containing crosslinker 2. 3. Synthesis of Dye Loaded PEGMA Particles In order to achieve dye loaded PEGMA particles, 1 mg of Rhodamine B (in powder form) was added to the re- 42 Acta Chim. Slov. 2022, 69, 30–38 Yildirim et al.: Synthesis of glucose/fructose sensitive ... 3. Results and Discussion 3. 1. Structural and Morphological Evaluation of the Prepared Materials 1H-NMR spectra of the synthesized crosslinker (Fig. 1a) and the precursors (4-allylcatechol (Fig. 1b) and VPBA (Fig. 1c)) are presented together for comparison. The spec- tra of VPBA and 4-allylcatechol exhibits (-OH) groups in Ar-OH, (δ= 8.79–8.67 ppm) and in B-OH, (δ= 8.07 ppm) peaks, respectively with high intensity. On the other hand, these peaks were almost disappeared in the spectrum of CRX-3. According to the integration calculations, the re- duction of H peaks of Ar-OH groups in 4-allylcatechol molecule and B-OH groups in VPBA is more than 80% which also suggests that the product was obtained with a yield of more than 80%. A detailed 1H-NMR spectrum for the crosslinker (Fig. S1) and the integration steps can be followed from the supplementary information. The formation of the crosslinker was further verified by comparing the FT-IR spectra of CRX-3 and VPBA. The results are presented in Fig. 2. In the FT-IR spectrum of boronate esters, bands at 1220 and 1250 cm‒1, 1000 -1090 cm‒1, and 500–750 cm‒1 are generally assigned to C−O stretching,21 B−C stretching,21b,21c,22 and out-of-plane vi- brations,21b,23 respectively. On the contrary, in some other studies, one can find that the assigned wavenumbers for C−O and B−C stretching were replaced; i.e., 1200 and 1270 cm‒1 for B-C stretching24 and 1100 and 1200 cm‒1 for C-O stretching.24a,25 In addition, bands around 1300 cm‒1 are mostly attributed to B-O stretching in boronate es- ters.26 The spectrum for our sample displayed 2 sharp peaks in this region at 1330 cm‒1 and 1366 cm‒1. However, boronic acid have also been reported to exhibit stretching Figure 1. 1H-NMR spectrum of a) CRX-3 b) 4-allylcatechol c) 4-vinylphenyl boronic acid. Figure 2. FT-IR spectrum of 4-vinylphenyl boronic acid (VPBA, top) and CRX-3 (bottom). 43Acta Chim. Slov. 2022, 69, 30–38 Yildirim et al.: Synthesis of glucose/fructose sensitive ... here which makes the distinction between the boranate es- ter and boronic acid disputable.27 As discussed above, peaks observed in the spectrum of the crosslinker at 1233 cm‒1 and 1050 cm‒1 are probably due to C−O and B-O stretching. The presence of these peaks was evaluated as the characteristic of boronate ester formation.21a Further, the vibrational mode at 662.8 cm‒1 also designates that the boronic acid was consumed during the reaction in order to form the boronate ester bridge containing crosslinker.27 These findings show that the 4-allylcatechol and 4-vinyl- phenyl boronic acid precursors almost reacted successfully to form the crosslinker under the experimental conditions applied in the current study. As discussed in the experimental section, PEGMA particles were obtained by mixing constant amount of PEGMA 300 monomers and different amounts of CRX-3 (1, 3, 5, 7.5, and 10% mole of the monomer) in the pres- ence of AMPDH. Further, the emulsion polymerization process yielded PEGMA including suspensions and the polymer content of these suspensions was collected by centrifugation. Morphological examination of these as-collected samples was carried out via SEM analysis. Fig. 3 (a and b) demonstrates SEM images of as-centrifuged P-P-3 sample. The product consists of uneven shaped and large sized (20–30 µm) individual agglomerates. Fig. 3a shows only a portion of the surface of a random agglomer- ate. As it is clear from this image, under the experimental conditions applied, the polymerization reaction ended-up with the formation of spherical shaped PEGMA (indicated by red arrows) embedded in an un-reacted matrix. Fig. 3b provides a closer view of the surface belonging to one of the PEGMA spheres. It is obvious that the large spheres are made-up of much smaller particles. As this image refer to unwashed state, it is not easy to determine the size and size distribution of the particles using the SEM micrograph. Yet, it can be stated that the particles exhibit a distinct spherical morphology, and the diameter of the largest par- ticles can reach up to ~194 nm. The size and morphology of cleaned PEGMA parti- cles were investigated using DLS and TEM techniques. The average hydrodynamic diameter (dH) and polydispersity index (PDI) of the particles were extracted from DLS mea- surements. The results are demonstrated in Fig. 4. In addi- tion, the numerical values can be followed from Table 2. No data are presented for P-P-1 and P-P-2 samples, since PEGMA formation could not be achieved when the amount of the crosslinker used was 1 or 3% mole of mono- mer probably due to insufficient linking. On the other hand, in case of CRX-3 additions at 5, 7.5, and 10% mole of monomer, PEGMA particles were successfully obtained. The values given in Table 2 states that the dH and PDI of the particles are directly proportional to the amount of the crosslinker used during synthesis. In consistent with the SEM examinations given above, the dH value of P-P-3 Figure 3. SEM images of P-P-3 sample in as-centrifuged state a) general view of the surface of a large sized agglomerate, 10k magnification, b) a closer view of the surface of a random sphere seen in (a), 100k magnification Figure 4. Polydispersity index and average hydrodynamic diameter values of the synthesized PEGMA particles as a function of the amount of CRX-3 used during synthesis 44 Acta Chim. Slov. 2022, 69, 30–38 Yildirim et al.: Synthesis of glucose/fructose sensitive ... sample was measured as 184.4 nm. In addition, dH values of 267.5 nm for 7.5% mole and 318 nm for 10% mole of monomer crosslinker additions were identified. Further, PDI values were obtained as 0.274, 0.336, and 0.393 for P-P-3, P-P-4, and P-P-5, respectively. Above-mentioned findings show that the amount of CRX-3 used during synthesis is a significant parameter such that the formation of particles directly depends on the amount of crosslinker present in the reaction environ- ment. Moreover, the amount of the crosslinker not only affects the size but also the size distribution of the PEGMA particles. In a similar study, the acetone/water ratio was reported as an another important parameter in terms of controlling the average hydrodynamic diameter of Poly methyl methacrylate (PMMA) particles.6 It was stated that increasing the fraction of acetone in the solvent could be used as an effective way to reduce the size of the particles. On the other hand, for the current study, a constant value of acetone to water ratio (1/29 V/V) was used for all exper- iments. Increasing the amount of acetone in the solvent mixture led to the formation of strong agglomerates which are not dispersible by washing/ultrasonic treatments. Therefore, the observed difference in the diameter of the particles here can be ascribed to the change in the amount of CRX-3 used during the synthesis reactions. As the size of the particles dictate a specific value for the surface area of the samples and further the reaction with sugar molecules was expected to proceed from boro- nate ester bridges exposed to the surface; the synthesis condition which provides the lowest hydrodynamic diam- eter for the particles was selected for further investigation. As seen from Fig. 4 and Table 2, the P-P-3 (synthesized by using CRX-3 at an amount of 5% mole of monomer) sam- ple exhibited the lowest size with dH value of 184.4 nm. In addition, the PDI (0.274) of this sample is the lowest among others which indicate that the synthesized particles exhibit acceptable narrow size distribution. Fig. 5 (a and b) shows low-resolution TEM images of P-P-3 sample. PEGMA particles are highly dispersed after the cleaning procedure and exhibit almost perfectly spher- ical morphology. Fig. 5a illustrates an array of PEGMA particles on holey carbon coated Cu grid with various siz- es. In addition, two random spherical PEGMA particle with similar diameters can be seen in Fig. 5b. According to the measurements conducted using TEM images, the par- ticles have sizes in the range of 90 to 186 nm. This result agrees well with SEM and DLS measurements given above. In addition, these observations reveal that the PEGMA particles were successfully formed by the surfactant free emulsion polymerization reaction using the boranate ester bridge containing crosslinker, exhibiting a particle size be- low 200 nm with a narrow size distribution. 3. 2. Dye Loading and Sugar Sensitivity Experiments Rhodamine B dye with specific absorbance in the visible region (λmax = 554 nm) was used as a model drug in this study. Scheme 3a summarizes the dye loading process into the network of a PEGMA particle. In addition, the proposed dye release mechanism is presented in Scheme 3b. According to this mechanism, in case of the presence of sugar molecules in the environment, the trapped dye is expected to be released due to the high sensitivity of CRX- 3 to these surrounding molecules. While the sugar mole- cules can bind to boronic acid, this simply breaks the bor- Table 2. Average hydrodynamic diameters and polydipersity index of the synthesized PEGMA particles Sample dH (nm) PDIa P-P-1 – – P-P-2 – – P-P-3 184.4 0.274 P-P-4 267.5 0.336 P-P-5 318 0.393 a Multi-distribution indicator, unitless. Figure 5. Low resolution TEM images of P-P-3 sample, a) an array of particles with different sizes b) two random PEGMA particles with similar diameters. 45Acta Chim. Slov. 2022, 69, 30–38 Yildirim et al.: Synthesis of glucose/fructose sensitive ... onate ester bridge between the monomers. Thus, the reaction will collapse the network of the polymer. There- fore, the amount of the liberated dye to the surrounding can be considered as a measure of drug released by the polymeric particles. Fig. 6a shows the absorbance spectra of P-P-3 sample (1.25 10–2 g) dispersed in PBS, PBS + glucose, and PBS + fructose environments. These spectra were recorded after 2 h of the dispersion process. Absorbance behavior of the pure PEGMA particles (synthesized without dye loading) in the range of 450 to 650 nm was also presented for com- parison. As seen, pure particles displayed no absorbance in this region. Therefore, any measured absorbance for other cases can be considered as due to the Rhodamine B dye molecules released from the network depending on the surrounding media. According to the measured values af- ter 2 h, the lowest amount of dye was released in PBS envi- ronment, which also means that the particles are releasing a certain amount of dye (16.7 %) even in sugar free envi- ronment. In case of PBS with 10 mg/mL glucose, the re- leased amount of dye (18.2 %) was only slightly increased compared to PBS environment, which suggests that the particles with boronate ester bridge containing crosslinker is only partially sensitive to glucose molecules. On the oth- er hand, in case of PBS + 10 mg/mL fructose environment, the amount of the released dye increased substantially and reached to 24.5 % after 120 min. Fig. 6b demonstrates time dependent increase of dye concentration in PBS+10 mg/mL fructose medium for a total holding duration of 48 h. The absorbance increases continuously as a function of time which means the quan- tity of the liberated dye is increasing in the environment Scheme 3. Schematic representation of a) dye loading into PEGMA particles, b) dye release in the presence of glucose/fructose. Figure 6. Absorbance spectra of P-P-3 sample after 2 h in a) PBS, PBS+10 mg/mL glucose, and PBS+10 mg/mL fructose environments, b) time de- pendent increase of dye release in PBS+10 mg/mL fructose environment (insets in (b) shows the digital images of the solution after releasing of Rhodamine B dye by the particles (left) and the collected particles with centrifuging (right)). 46 Acta Chim. Slov. 2022, 69, 30–38 Yildirim et al.: Synthesis of glucose/fructose sensitive ... with time. After 48 h, a total of 39 % dye release was achieved. The inset (a) shows the deep pink color of the solution because of Rhodamine B dye that was released to the environment in the presence of fructose after 48 h by the P-P-3 sample. In addition, the inset (b) presents these PEGMA particles collected from the pink colored solu- tion, which indicates that the particles can be separated from the solution via a simple centrifugation. The cumulative release of the dye (after 48 h) by the P-P-3 sample dispersed in PBS, PBS + glucose, and PBS + fructose environments are presented in a comparative base in Fig. 7. This figure implies that the dye release by the par- ticles reached a constant value of ~25 % in PBS or PBS + glucose environments within the first 6 h. After this peri- od, the recorded amount of dye lost by the network was ascended only slightly. On the other hand, in PBS + fruc- tose medium, dye release by the particles increased rapidly up to 28.52 % in 6 h. After this point, the concentration of Rhodamine B gradually continued to increase in the solu- tion, but a decline in the slope of the curve is obvious. At the end of 48 h, total amount of the released dye was re- corded as 39 %. As these dispersions were prepared by di- viding a single and homogenous 30 mL PBS-PEGMA par- ticles dispersion into three identical vials for dye release experiments (see section 2.3), the quantity of the particles in each vial and the amount of initially trapped dye by these particles can be assumed similar for each condition. As other parameters such as temperature, pH, time, etc. were all kept constant, the total amount of released dye for each case can be correlated to the presence of PBS, glucose, or fructose in these environments. The highest amount of dye was released in fructose environment. Therefore, it can be stated that the boranate ester bridge network is much more sensitive to external fructose molecules compared to glucose molecules. In a recent study, Wu et. al., discussed on an amphi- philic boronic acid glucose sensor, where the hydrophobic group in glucose and hydrophilic boronic acid attached to each other with a dynamic covalent linkage, preferably an imine bond.28 According to the proposed mechanism, the presence of glucose can induce aggregation of simple bo- ronic acids due to its ability to crosslink the two boronic acid molecules.29 On the other hand, experiments with fructose showed no sign of turbidity in the solutions which indicate little or no amphiphile aggregation.28 Therefore, they stated that the binding of glucose may lead to forma- tion of “Gemini-type” amphiphiles, which have a higher ability of aggregation compared to “single-tail” amphi- philes formed with boronic acid and fructose. Based on the dye release values for the current experiments, it is be- lieved that the boranate ester bridge in the network has opened in the presence of glucose, but due to a similar mechanism discussed above or a competition between the network disintegration and amphiphile aggregation, the dye release by the particles was inhibited. On the contrary, fructose molecules probably have disintegrated the net- work to form a single tail amphiphiles, which allowed the liberation of the trapped dye in the network. As a result, higher number of dye molecules were released to the envi- ronment for fructose containing experiment. Of course, this speculation needs to be verified by further experi- ments, which will be considered in future studies. In addi- tion, it is clear from the earlier studies that the affinity of samples to external molecules and the amount of the mod- el drug released by the particles may scatter widely de- pending on various parameters, such as temperature,1 time,30 pH,2 glucose/fructose concentration,5,31 and etc. On the other hand, the current study aimed to propose a simple preparation route for the polymeric particles using a novel crosslinker. Therefore, the pH, polymeric particle dosage or sugar molecule concentration were kept con- stant in all experiments. And the amount of the dye liber- ated by the samples were measured as a function of time only. This also implies that the dye release performance of the particles can be improved by applying the optimized conditions. 4. Conclusions In this study, PEGMA particles were synthesized successfully in one step with surfactant free emulsion po- lymerization method using a novel boronate ester bridge containing crosslinker. The characterization studies have revealed perfectly spherical morphology for the particles. Further, the study has shown the tunability of the process in terms of achievable particle sizes. It was observed that PEGMA particles could be synthesized in various sizes de- pending on the amount of the crosslinker added to the po- lymerization reaction. The particles with the lowest aver- age diameter and the narrower size distribution were then examined for controlled drug release application using their sensitivity against glucose or fructose molecules. The Figure 7. Time dependent Rhodamine B dye release % of P-P-3 sample dispersed in different environments 47Acta Chim. Slov. 2022, 69, 30–38 Yildirim et al.: Synthesis of glucose/fructose sensitive ... total amount of dye release in glucose and fructose envi- ronments were recorded as 25% and 39%, respectively. 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V ta namen smo z uporabo 4-vinilfenil- boronske kisline sintetizirali zamreževalec, osnovan na borovi kislini, ter njegovo tvorbo potrdili z 1H-NMR in FT-IR analizama. Polimerne delce, občutljive na izbrana sladkorja, smo nato pripravili z uporabo tega zamreževalca in PEGMA monomera v enostopenjski emulzijski polimerizaciji brez uporabe površinsko aktivnih snovi. Morfologijo in velikost polimernih delcev smo določili z DLS, SEM in TEM. Za analizo občutljivosti delcev na molekule sladkorja smo najprej izvedli poskuse polnjenja z barvilom rodamin B (kot vzorčno zdravilo). Nato smo delce izpostavili medijem, bogatim z glukozo/fruktozo, sproščanje barvila pa smo spremljali z UV-VIS spektrofotometrijo v odvisnosti od časa. Rezultati študije so pokazali, da so delci PEGMA pri pH 7,4 in 310 K bolj občutljivi na fruktozo (~39 % sproščanje) kot na glukozo (~25 % sproščanje). Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License 49Acta Chim. Slov. 2022, 69, 49–59 Carmona-Alvarado et al.: Proposal of an HPLC/UV/FLD Screening ... DOI: 10.17344/acsi.2021.6870 Scientific paper Proposal of an HPLC/UV/FLD Screening Method for the Simultaneous Determination of Ten Antibiotics in Environmental Waters Idalia Francisca Carmona-Alvarado,1 María de la Luz Salazar-Cavazos,1 Noemí Waksman de Torres,1 Aurora de Jesús Garza-Juárez,1,2 Lidia Naccha-Torres,1 Jose Francisco Islas2 and Norma Cavazos-Rocha1,* Universidad Autónoma de Nuevo León1. Departamento de Química Analítica2. Departamento de Bioquímica y Medicina Molecular, Facultad de Medicina, Ave. Madero y Dr. Eduardo Aguirre Pequeño S/N Col. Mitras Centro. 64460 Monterrey, Nuevo León, México. * Corresponding author: E-mail: norma.cavazosrc@uanl.edu.mx; nocavazos@yahoo.com Received: 04-05-2021 Abstract An HPLC-UV/FLD method for simultaneous detection of ten antibiotics in surface waters was developed. Antibiotics were extracted from water using solid phase extraction. An Atlantis T3 column was used with acetonitrile and 0.05% trifluoroacetic acid as a mobile phase for separation, with a total running time of 45 min. Signal detection was performed at 280 nm; fluoroquinolones were additionally quantified by fluorescence detection. Validation parameters such as line- arity, recovery and precision were evaluated. The limits of detection (LOD) in river waters were in the range 0.1–1.3 µg/L for antibiotics detected by UV, and 0.039 and 0.073 µg/L for fluoroquinolones detected by FLD. LOD are sufficiently low to consider this method as a first alternative for HPLC-MS methods that will allow alerting for the presence of antibiotics in surface waters. This screening method is rapid, sensitive, reproducible and economical. Keywords: Antibiotic analysis; emerging contaminants; simultaneous analysis by HPLC-UV/FLD; fluoroquinolones and sulfonamides; multiresidue analysis method; surface water analysis; antibiotic occurrence in environmental waters 1. Introduction During recent years there has been an increase in the global interest for determining new and potentially danger- ous compounds – contaminants, known as emerging con- taminants.1–3 These emerging contaminants include phar- maceuticals, pesticides, hormones, steroids, surfactants and surfactants’ metabolites, endocrine disrupting compounds and perchlorate.4 Particularly, antibiotics (as residues) are the pharmaceutical contaminants with the highest use worldwide; routinely used in both human and veterinary medicine. Administered antibiotics are partially metabo- lized in the body and the rest are excreted unchanged or as metabolite, into the ecosystems.5,6 Later, these waste resi- dues are further collected in the wastewaters and introduced to the wastewater treatment systems where antibiotics can- not be completely removed under current technology. Hence, they are continuously being discharged into the en- vironment.7,8 The presence of antibiotics in the aquatic envi- ronment can affect the evolution of the community struc- ture; however, the most negative effect attributed to the occurrence of antibiotics is the development of antibiotic resistant bacteria and antibiotic resistant genes, which com- prise health risks to humans and animals.9,10 Current re- search has determined certain community-minded antibi- otic hot-spots, which are deemed high risk, these include: hospital effluents, influents and effluents of municipal wastewater treatment plants, and to a lesser degree ground and seawater.11–19 This overall global phenomenon has been of much interest, as such “Antimicrobial resistance and its global spread” was the theme of World Health Day 2011, given by the World Health Organization (WHO).20 In addi- tion, the WHO has further developed medically relevant guidelines for antimicrobials in animal food production.21 Antibiotics have been grouped in specific families ac- cording to their use and chemical structure. These families include: macrolides, fluoroquinolones, penicillins, sulfon- amides, trimethoprim and cephalosporins. These emerg- 50 Acta Chim. Slov. 2022, 69, 49–59 Carmona-Alvarado et al.: Proposal of an HPLC/UV/FLD Screening ... ing contaminants have been detected in environmental waters within the range of μg/L down to ng/L. To detect these concentration levels the analytical methods currently used for the analysis of antibiotics consist of a purification and concentration step followed by a separation step by means of high performance liquid chromatography (HPLC) or ultra-high performance liquid chromatography (UHPLC) coupled to tandem mass spectrometry or hybrid tandem spectrometers such as quadrupole time of flight and/or quadrupole-linear ion trap.11,13,15-19,22-26 Also UV and fluorescence detection have been coupled to HPLC but only for determining one or two families or a few antibiot- ics.14, 27–32 On the other hand, the most widely used tech- nique for purification is by means of solid phase extraction (SPE), although some authors have used lyophilization18 or dispersive liquid-liquid microextraction.33 A variety of ad- sorbents have been used in SPE, but most authors agree that polymeric adsorbents have been more efficient in the extraction of antibiotics.6,8,13,15 Detection limits for mass spectrometry are in a few ng/L to µg/L, while in the case of fluorescence (FLD) and UV absorption are in µg/L. As mentioned above, the presence of antibiotics in the environment, especially in the aquifer, is a serious problem and has resulted in an increase in bacterial resis- tance, increasing health costs and mortality, causing a sig- nificant economic impact to private and governmental ser- vices. Thus, it is necessary to have rapid, simple, accessible, and reliable analytical methods for detecting and monitor- ing these emerging contaminants. Chromatographic meth- ods with mass spectrometry are highly sensitive and specif- ic, but high costs of acquisition and maintenance makes them inaccessible to routine laboratories. At a global scale there is an insufficient number of laboratories with this technology, to perform these monitoring tasks. A second burden is both the management of the equipment and in- terpretation of the results, which requires intense training and experience, further adding to the overall cost. There- fore, it is necessary to use analytical methods that are more cost-effective and accessible to analytical laboratories, es- pecially in developing countries, that can allow carrying out studies; monitoring and quantifying the occurrence of these emerging contaminants in the environment. Consid- ering aspects previously exposed, the aim of this work was to develop a sensitive and cost-effective multiresidue meth- od for the simultaneous analysis of antibiotics commonly used in human and veterinary medicine using high perfor- mance liquid chromatography with ultraviolet and fluores- cence detection (HPLC-UV/FLD) as an alternative to the HPLC-MS methods. To the best of our knowledge, it is the first method that includes nine antibiotics from 5 different families, and trimethoprim. The developed method would allow the continuous monitoring of environmental waters to detect the presence of these emerging pollutants. 2. Experimental 2. 1. Reagents For the mobile phase, acetonitrile (ACN) and meth- anol were HPLC grade (J. T. Baker). Trifluoroacetic acid Figure 1. Assayed antibiotic molecules: sulfamethoxazole (SMX), sulfadimethoxine (SDX), ciprofloxacin (CIP), tetracycline (TETRA), doxycycline (DOX), enrofloxacin (ENRO), oxytetracycline (OXI), tylosin (TYL), and amoxicillin (AMOXI). Structures developed using ACD/Labs ChemSketch ®. 51Acta Chim. Slov. 2022, 69, 49–59 Carmona-Alvarado et al.: Proposal of an HPLC/UV/FLD Screening ... (TFA) 99.0% was from Sigma Aldrich (Sigma Aldrich Corp, St Louis, USA). HCl was J.T. Baker (Center Valley, PA, USA). Deionized water was purified by a Milli-Q Sys- tem (Millipore Co., MA, USA). Sulfamethoxazole (SMX), sulfadimethoxine (SDX), ciprofloxacin (CIP), tetracycline (TETRA), doxycycline (DOXI), trimethoprim (TMP) were from Sigma Aldrich. Enrofloxacin (ENRO), oxytetra- cycline (OXI), tylosin (TYL) and amoxicillin (AMOXI) of MP Biomedicals (Irvine CA, U.S.A.) (Figure 1). All antibi- otics were 98% purity or greater. 2. 2. Equipment and Chromatographic Conditions. Chromatographic separation was achieved using an HP 1100 separation module (Agilent Technologies, Wald- bronn Germany) equipped with a quaternary pump, au- tosampler and temperature control, coupled with a vari- able wavelength UV detector and FLD detector. Three different chromatographic columns were tested: Synergi C18 (150 mm × 2 mm, 4 µm) and Luna C18 (250 mm x 4.6 mm, 5 µm) of Phenomenex (Torrance CA, USA), and At- lantis T3 (150 mm × 2.1 mm, 3 µm) of Waters (Waters Cor- poration, Milford, MA, USA). Samples were eluted in mo- bile phase (TFA 0.05%) and (ACN); gradient used is shown in supplementary Table S1. The flow rate was 0.2 mL/min and injection volume was 5 µL, column temperature was set at 35 °C. Detection by UV at λ 280 nm and fluorescence at λex 278 nm and λem 450 nm. The mobile phases and stan- dard solutions were filtered through a 0.45 µm Nylon filter (Waters Corporation, Milford, MA, USA) before use. 2. 3. Standard Solutions Ten milliliters of stock solutions for each antibiotic were prepared at a concentration of 1000 µg/mL in metha- nol; only AMOXI was prepared in water. The solutions were stored in dark at –20 °C for up to 30 days. Stock solutions were further diluted for preparation of a standard mix, which contained the following concentrations: 4 µg/mL CIP and ENRO, 25 µg/mL TMP, OXI, TETRA, and SMX, and 50 µg/mL AMOXI, TYL, SDX, and DOX. From this standard mixture, and by appropriate dilutions, working aqueous standards were prepared and used to build calibra- tion curves. The concentrations of these working aqueous standard solutions were for CIP and ENRO in the range 0.08–0.24 μg/mL; for TMP, OXI, TETRA, and SMX in the range of 0.5–1.75 μg/mL and for AMOXI, TYL, SDX, and DOX 1–3.5 μg/mL (supplementary Figure 2S). These solu- tions were prepared fresh daily, filtered and placed in amber glass vials prior to analysis by HPLC-UV/FLD. 2. 4. Antibiotic Extraction SPE is the most employed technique for isolating and for the enrichment of antibiotics in the environmental waters, enabling concentrations of low concentrations.2,7,8,34 However, the procedure is highly dependent on the correct selection of the stationary phase, also of pH of the sample, composition, and volume of solvent used for elution.13 Due to the reasons stated above, the SPE was carried out consid- ering three important steps: first, STRATA X and OASIS HLB cartridges were compared for the stationary phase se- lection. Deionized water samples were spiked with a stan- dard mixture of antibiotics, and then extracted under ap- propriate conditions. Oasis HLB cartridges provided the best multiresidue retention with higher number of com- pounds and therefore were the one selected for further ex- periments. Once the most suitable adsorbent was selected, the effect of seven variables (pH of sample, flow rate of the sample load, composition, volume and flow rate of elution solvent, and washing solvent and drying time) for the ex- traction efficiency were evaluated through a series of exper- iments established with a Placket-Burman design. The re- sults indicated the most favorable conditions for multiresidue extraction, therefore, the extraction proce- dure was: Oasis HLB cartridges (6 mL 500 mg, Waters Co., Milford MA, USA) were conditioned with 5 mL of metha- nol and 5 mL of H2O pH 3. Next, 1000 mL of deionized water or sample was adjusted at pH 3 with HCl and passed through cartridge at a flow rate of 10 mL/min using a vacu- um manifold system connected to a vacuum pump. The loaded cartridges were rinsed with 5 mL of water and were dried under vacuum for 1 min. The elution was performed with 3 mL of methanol at 1 mL/min. The sample extract was evaporated to dryness under nitrogen stream at 40 °C in a Glas-Col evaporator and reconstituted with 1 mL of water, filtered and transferred to a dark vial for analysis. 2. 5. System Validation Calibration curves were constructed by triplicate at five concentration levels. The area values were plotted against the concentration. Deionized water was used as a blank. Limits of detection (LOD) and quantification (LOQ) were calculated from the calibration curve data ac- cording to equations LOD = 3.3 () and LOQ = 10 () where σ is the standard deviation of the calibration line intercept and s is the slope of the calibration line35 (supplementary Table S2). The precision of system was evaluated and re- ported as % relative standard deviations (%RSD). Intra and intermediate precision of the system were calculated by means of injection of three working aqueous standards each in triplicate, at three concentration levels and in three different days (supplementary Table S3). 2. 6. Method Validation River water (matrix) was collected and filtered through 0.45 µm pore size membranes. Accuracy was as- sessed as the (%) recovery as follows: Three samples of 1000 mL of matrix were spiked each with antibiotics at 52 Acta Chim. Slov. 2022, 69, 49–59 Carmona-Alvarado et al.: Proposal of an HPLC/UV/FLD Screening ... three levels of concentration (low, medium and high) in order to obtain final concentrations of 1, 2 and 3 µg/L for AMOXI, TYL, SDX, DOXI; 0.08, 0.16 and 0.24 µg/L for ENRO, CIPRO, and 0.5, 1 and 1.5 µg/L for TMP, OXI, TETRA, and SMX. After one hour the sample was ex- tracted with the SPE method previously described. Anal- yses were run in triplicate. Results were calculated from the calibration curves by means of external standard method. Intraday precision was determined by assaying by triplicate three samples spiked at three levels. The interme- diate precision was calculated by analyzing the samples fortified with the antibiotics at three levels of concentra- tion in triplicate on three different days. Selectivity was tested by examining different samples of river water free of antibiotics to confirm that the signals originating from the matrix did not interfere with the sig- nals from the analytes. Identification of each compound in the spiked samples was made by the retention times. 2. 7. Matrix Effect Calibration curves were constructed using river wa- ter, to take into consideration the matrix effect. Recoveries were compared with those of deionized water fortified with standard at the same concentration, using a paired t-test. Further, visual inspection of calibration curves was used to corroborate (supplementary Figure 3S). In addi- tion, to discard contaminants (within study range), river water as internal control was analyzed.35 2. 8. Sample Analysis Surface water samples from the city of Monterrey and metropolitan area were analyzed. The samples were collected on the following locations: Rio Pilon, Rio La Silla and the Rodrigo Gomez water dam. These samples were collected in plastic bottles, transported in cold and dark- ness, and stored at 4 oC until analysis. The samples were homogenized, filtered through a 0.45 µm Millipore filter and processed within 5 h after collection. 3. Results and Discussion This work presents a simple and inexpensive HPLC method for the first screening of the analysis of antibiot- ics in water. The HPLC conditions established and des- cribed in the experimental section enabled efficient se- paration of all antibiotics from different families in a single run. First, the chromatographic separation was optimized using an aqueous standard mix solution of the antibiotics. To obtain chromatograms with a good resolu- tion of the target compounds in a short time, various con- ditions (eluents, gradients) were tested. Three different chromatographic columns were used: Atlantis T3 (150 mm x 2.1 mm, 3 µm), Synergi C18 (150 mm x 2 mm, 4 µm) and Luna C18 (250 mm x 4.6 mm, 5 mm). Individual chromatograms are shown in supplementary Figure 1S. Finally, after testing the three columns with a 0.05% TFA-acetonitrile mobile phase,36 the best separation was achieved with the Atlantis T3 column. In the Synergy C18 Figure 2. Chromatogram of the mixture of antibiotics. (A) fluorescence detection (λex 278nm and λem 450 nm); (B) UV detection 280 nm. Elution order was amoxicillin (AMOXI), trimethoprim (TMP), oxitetracycline (OXI), ciprofloxacin (CIPRO), tetracycline (TETRA), enrofloxacin (ENRO), sulfamethoxazole (SMX), sulfadimethoxine (SDX), doxycycline (DOX) and tylosin (TYL). Separation conditions are described in text. 53Acta Chim. Slov. 2022, 69, 49–59 Carmona-Alvarado et al.: Proposal of an HPLC/UV/FLD Screening ... and Luna C18 columns, the pairs OXI-CIPRO, DOXI- SDX and TETRA-CIPRO, DOXI-SDX, respectively, did not present optimal resolution despite different tested elution gradients. Atlantis T3 has a trifunctional C18 bonding phase along with a special endcapping proce- dure; these characteristics allowed for the best separation of antibiotics. Additionally, T3 is a more stable column that is resistant to low pH and highly aqueous mobile phases.37 Chromatographic parameters efficiency (plate number, N), resolution (R) and selectivity (α) are shown in supplemental material (supplementary Table S4). Per- formance of Atlantis T3 column was better than Luna and Synergi, while resolution on Atlantis T3 column was >1.4 and the separation of all antibiotics was complete, in the case of Synergi and Luna chromatographic columns, re- sults showed selectivity values <1.2, the resolution values R for some antibiotics were below 1 and the compounds were not totally separated. The developed method includes antibiotics from five different families and trimethoprim (Figure 1) using HPLC-UV/FLD. Chromatogram of a standard mixture of ten antibiotics is shown in Figure 2. For FLD detection (Figure 2A) quinolones showed maximum excitation and emission at λ = 278 nm and λ = 450 nm, respectively.38 For UV detection (Figure 2B), the monitoring wavelength was at 280 nm. Complete elution of the antibiotics was ob- tained in the first 30 min. The established HPLC condi- tions allowed for efficient separation of all antibiotics from different families in a single run. 3. 1. Validation System validation was performed for the range of concentrations established for each antibiotic, coeffi- cients of determination for the calibration curves were over 0.99 with RSD of response factors (RFs) below 7%, and precision was 0.2 to 14%. Instrumental LOD and LOQ were assessed. The lowest LOD was 0.006 µg/mL for SMX and the highest was 0.225 µg/mL for DOX. Mean- while, LOQ for SMX was 0.020 and for DOX it was 0.750 µg/mL On the other hand, Table 1 shows the results for the validation of method. Evaluation of linearity was by means of the RFs and the coefficients of determination. The coef- ficients of variation of the response factors were between 6-15% and coefficients of determination were higher than 0.99 except for OXI, ENRO and TETRA. LOD and LOQ of the method were calculated from the matrix-matched cal- ibration curves and taking into consideration 3.3 and 10 times the standard deviation of the calibration line inter- cept (matrix water free of antibiotics), and the calibration line slopes (Table 1). Hence, low amounts of antibiotics were detectable due to the solid-phase extraction. LOD were between 1.29 µg/L for DOXI to 0.039 µg/L for ENRO, while LOQ from 4.13 to 0.13 µg/L for DOXI and ENRO, respectively. Thus, the lowest LOD and LOQ were 0.039 and 0.130 µg/L, respectively, for ENRO. Percentage recoveries were obtained from deionized water and matrix samples spiked with antibiotics at µg/L concentration. Table 2 shows the results of recoveries of the antibiotics in both deionized water and matrix (river water) in the experiments at low, medium, and high con- centrations. Recoveries achieved for all antibiotics in de- ionized water ranged from 44% (SDX) to 130% (CIPRO) with %RSD between 1 and 16%. According to AOAC Guidelines for Standard Method Performance Require- ments39 taking into account analyte concentrations, preci- sion and recovery results are acceptable. To determine the matrix effect, recovery experiments were carried out in river water spiked with antibiotics at the same concentra- tions (low, medium and high level) and the same extraction process was applied. Table 2 shows the recovery percentag- es, where it can be seen that the values obtained vary be- tween 54% (DOX) to 121% (TETRA) with RSD of of 2–12%. Data on recoveries in deionized water were com- pared with those obtained in matrix through a t-test. As observed in Table 2, statistically significant differences were found for CIPRO, ENRO, TETRA, and SDX. Al- though recoveries for CIPRO and ENRO were within the acceptable limits when the tests were assayed in deionized water, matrix effect caused recoveries up to 190%, so, for a correct quantification, it would be necessary to apply Table 1. Validation parameters of the method used for the analysis of the antibiotics in water. Antibiotic Linear equation Determination %RSD LOD LOQ y = mx + b coefficient (R2) of RF µg/L µg/L TMP 32069x –1.762 0.991 8 0.23 0.76 OXI 46188x – 8.811 0.989 15 0.56 1.88 TETRA 28873x + 8.06 0.970 10 0.10 0.35 SMX 61626x – 10.85 0.992 12 0.32 1.08 SDX 22898x – 2.847 0.990 6 0.45 1.52 DOX 56104x – 16.45 0.990 11 1.29 4.33 TYL 21441x – 1.001 0.993 6 0.45 1.49 CIPRO 15563x + 1.048 0.991 14 0.07 0.15 ENRO 42925x – 5.015 0.988 10 0.04 0.13 *Response factor 54 Acta Chim. Slov. 2022, 69, 49–59 Carmona-Alvarado et al.: Proposal of an HPLC/UV/FLD Screening ... methods such as standard addition or matrix-matched cal- ibration curves. 3. 2. Solid Phase Extraction Under normal circumstances, low concentrations of antibiotics in river waters are expected. Hence a precon- centration is necessary. Solid phase extraction has been shown over time to be the most widely used technique for this purpose. Antibiotics have different structure and charge depending on the pH. SPE extraction with different cartridges such as Oasis MX, Strata X, LiChrolut EN, C18 and OASIS HLB has been reported.40 The results have shown that in multiresidue methods the cartridges with mixed phase have given the best results obtaining the highest number of compounds in a single step and with reproducible recoveries. With this idea in mind, the pres- ent work tested the performance of OASIS HLB and STRATA X cartridges obtaining the best results with OASIS HLB. These cartridges have a sorbent with bal- anced mixture of two monomers: N-vinylpyrrolidone (hy- drophilic) and divinylbenzene (lipophilic). This combina- tion allows the retention of nonpolar and polar (with acidic or basic nature) compounds, through hydrophobic and hydrophilic interactions.15 The results obtained in our experiments showed that with OASIS HLB cartridges the recovery of all antibiotics was achieved, as shown in Figure 3. So the next step was the optimization of the extraction conditions. The sample volume was 1000 mL. Smaller vol- umes have been used in HPLC-mass spectrometry, where the detection capacity is ng/L. In this work it was consid- ered to concentrate water samples for a factor of 1000, to enable detection and quantitation with UV and fluores- cence detection. Cartridges with 500 mg of stationary phase were used. Since antibiotics in environmental waters can be detected at very low concentrations (ng/L), large volumes can be used, moreover, experiments were also carried out to confirm that the breakthrough volume was not exceeded. Several conditions that affect the perfor- mance of the SPE extraction were taken into account con- ducting a series of experiments in which factors involved during the process were combined in a Plackett Burman design. Seven factors at two levels of variation resulted in eight experiments. Table 3 shows the factors considered and the variation levels. The set of experiments to probe extraction efficiency are shown in supplementary Table S5. The results of the experiments indicated that the pH of the sample is very important in the extraction efficiency of an- tibiotics. It is in general adjusted between 2.5 to 4 to ex- tract the majority of antibiotics. Herein, we probed the values of pH 3 and 5. For elution we used methanol and a combination of solvents used as a mobile phase (0.05%TFA/ ACN 50:50) at a flow rate between 1 and 5 mL/min. Other factors considered are shown in Table 3. The selection of conditions was based on obtained recoveries of all antibiotics. The results showed that at pH Table 2. Recovery from antibiotics in deionized water (DW) and river water (RW). Data shown as percentage of recovery(%RSD). Antibiotic Low level Medium level High level Matrix effect* DW RW DW RW DW RW TMP 80(9) 91(9) 84(14) 104(3) 106(1) 90(5) No OXI 83(6) 82(2) 90(12) 90(7) 85(15) 92(10) No TETRA 61(8) 121(2) 66(13) 110(1) 68(3) 87(11) Yes SMX 64(8) 63(6) 66(13) 69(6) 68(3) 73(4) No SDX 44(16) 111(5) 65(15) 116(1) 65(12) 109(14) Yes DOX 57(12) 60(8) 68(16) 54(12) 65(13) 59(2) No TYL 60(4) 87(9) 84(15) 81(4) 91(6) 75(15) No CIPRO 115(8) 196(5) 130(16) 180(1) 126(12) 158(8) Yes ENRO 70(15) 139(12) 69(12) 143(1) 71(4) 123(12) Yes n = 3α = 0.05* = statistical difference due to matrix effect Table 3. Factors considered in the extraction process and levels of variations carried out in the experiments Factor Levels Low (-) High (+) pH 3 5 Flow rate of the sample load (mL/min) 3 10 Elution solvent MeOH TFA 0.05%: ACN (50:50) Elution flow rate (mL/min) 1 5 Elution volume (mL) 1 3 Washing solvent H2O H2O:MeOH 90:10 Drying time (min) 0.5 1 55Acta Chim. Slov. 2022, 69, 49–59 Carmona-Alvarado et al.: Proposal of an HPLC/UV/FLD Screening ... 3 and with an elution volume of 3 mL we were able to re- cover all antibiotics. Furthermore, it was observed that the variation of other factors (flow rate of sample loading, elu- tion flow rate, solvent washing and drying time) did not affect the process, and the recovery was dependent mainly on the pH of the solution and the elution volume. Lastly, we must mention that in multiresidue analysis methods, the main objective is to extract the greatest number of compounds so, with the final conditions all antibiotics were obtained with acceptable values of recovery.40 3. 3. Sample Analysis Figure 3 shows a chromatogram of river water forti- fied with antibiotics studied in this work. Next, Figure 4, shows chromatograms for the analyzed samples of the Ro- drigo Gomez water dam (sample 1), La Silla and Pilon riv- ers (samples 2 and 3, respectively). No signals were ob- served at the retention times corresponding to the antibiotics. It is recommended to continue monitoring environmental waters, to confirm that there is no presence of the antibiotics studied considering the limits of detec- tion established in this work for the target analytes. 3. 4. Applicability Recently, studies done in surface waters have report- ed TMP, ENRO, AMOXI, SMX and CIP at concentrations from 0.03 to 0.25 µg/L in five samples of the river Ter6 (North East of Spain). Meanwhile, in Taihu Lake, China,41 SMX was reported at a concentration up to 0.600 µg/L, Figure 3. Chromatogram of the mixture of added antibiotics in river water. (A) fluorescence detection (λex 278nm and λem 450 nm) (B) UV detection 280 nm. Conditions of analysis are described in the text. Figure 4. Chromatograms of river water samples 1, 2 and 3. Chro- matograms A correspond to fluorescence detection λex 278 nm and λem 450 nm. Chromatograms B correspond to UV detection at 280 nm. Conditions of analysis are described in the text. 56 Acta Chim. Slov. 2022, 69, 49–59 Carmona-Alvarado et al.: Proposal of an HPLC/UV/FLD Screening ... while in Kenya and Spain studies have reported SMX in concentrations of 0.95 µg/L and over 13.7 µg/L, respective- ly. Concentrations of 1.6 up to 1670 µg/L of ENRO were reported in water samples from Poland.42 Nonetheless, in Mexico, information about occurrence of antibiotics in en- vironmental waters is lacking.43,44 Thus, it is important to continue the analysis and monitoring of antibiotics in envi- ronmental waters in Monterrey and other major metropol- itan areas, as the intense use of these antibiotics in urban centers poses high health risks. Considering the levels of concentration of antibiotics that have been reported around the world, the LODs reached by our method allows to con- tinue with the monitoring and analysis of occurrence of these emerging contaminants in environmental waters. HPLC-MS methods have been around for close to 20 years, during this time analysis of antibiotics has been well studied.2,12,19,45 Through our work, previous HPLC-UV/ FLD studies that included several pharmaceuticals and an- tibiotics were reviewed.27–32 In that regard, in our research we included antibiotics of five different families. Moreover, our HPLC-UV/FLD method has detection limits compa- rable and, for some antibiotics, lower than other methods that also use UV and FLD detection.14,31,46 Particularly, for FLD detection, the obtained detection limits are compara- ble with other methods using the LC-MS/MS tech- nique.19,35 We should note that even though LOD and LOQ were optimized to a great extent, one drawback of our sys- tem was observed. In comparison with HPLC-MS our sys- tem is not capable of achieving sufficient specificity, as ul- trafine resolution is still a technological limitation. Nonetheless, our proposed method continues to be an op- timized, economical, and accessible system, which can be adopted by many laboratories worldwide, which may not have access to sufficient resources and highly trained per- sonnel, to continue rapid monitoring of environmental or other residual waters, in search to detect emerging con- taminants. 4. Conclusion The presence of antibiotics in different environmen- tal water matrices such as surface waters, groundwater, drinking water, hospital and urban wastewater, wastewater effluents and influents of treatment plants have been widely reported. Concentration and type of antibiotics may be in- fluenced by several key factors, such as source of sample, season of the year, prevalence of diseases, as well as facto- ries located in the vicinity or aquaculture activities. We be- lieve, based on our results, that our method can be the first option for antibiotic and contaminant screening studies. From our results, we demonstrate that our system is a simple and cost-efficient multiresidue analytical method for the determination of ten antibiotics of five different families. The method was validated and applied to samples of surface water. The method involved off-line solid phase extraction and quantification by HPLC in combination with UV and FLD detections. Complete separation was obtained in 30 min. This method is rapid, cost-efficient, reliable and reproducible, and for this reason represents an alternative for the analytical environmental laboratories that require investigation and monitoring of these emerg- ing contaminants. Acknowledgements The authors wish to thank to ICRA (Catalan Institute for Research of Water) for their kind welcome and for sharing their knowledge during the stay. Funding This work was funding by the CONACYT (Grant project 130997) and PAICYT (Grant Project CS780-11). Declaration of interest statement Authors have declared no conflict of interest. Authors’ contributions Research: I.F., C.-A., N. C-R, M.L., S.-C., N.W.-T., L.N.-T., A.J., G-J. Experiments and Analysis: I.F., C.-A., M.L., S.-C., N.C-R Writing: I.F., C.-A., M.L., S.-C., A.J.G.-J., J.F.I., N.C.C.-R. and Supervising: N.W.-T., A.J.,G-J, N.,C.-R. 5. References 1. B. González-Gaya, L. Cherta, L. Nozal and A. Rico. An opti- mized sample treatment method for the determination of an- tibiotics in seawater, marine sediments and biological samples using LC-TOF/MS, Sci. Total Environ. 2018, 643, 994–1004. DOI:10.1016/j.scitotenv.2018.06.079 2. C. Kim, H. D. Ryu, E. G. Chung, Y. Kim and J. kwan Lee. A review of analytical procedures for the simultaneous deter- mination of medically important veterinary antibiotics in en- vironmental water: Sample preparation, liquid chromatogra- phy, and mass spectrometry, J. Environ. Managem. 2018, 217, 629–645. DOI:10.1016/j.jenvman.2018.04.006 3. A. Gogoi, P. Mazumder, V. K. Tyagi, G. G. Tushara Chaminda, A. K. An and M. Kumar. 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Povzetek Razvili smo metodo HPLC-UV/FLD za hkratno detekcijo desetih antibiotikov v površinskih vodah. Antibiotike smo ekstrahirali iz vode z ekstrakcijo na trdno fazo. Za ločbo smo uporabljali kolono Atlantis T3 z mobilno fazo iz acetonitrila in 0,05% trifluoroocetne kisline, celotni čas analize je bil 45 min. Signal smo detektirali pri 280 nm; fluorokinolone smo dodatno kvantificirali s fluorescenčno detekcijo. Ovrednotili smo nekatere validacijske parametre, kot so linearnost, iz- koristek in natančnost. Meje zaznave (LOD) za rečno vodo so bile v območju 0,1–1,3 µg/L za antibiotike z UV detekcijo ter 0,039 oz. 0,073 µg/L za oba fluorokinolona, detektirana s FLD. LOD so dovolj nizke, da lahko to metodo označimo kot prvo alternativo metodam HPLC-MS. Omogočala bo opozarjanje na prisotnost antibiotikov v površinskih vodah. Ta presejalna metoda je hitra, občutljiva, ponovljiva in ekonomična. Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License 60 Acta Chim. Slov. 2022, 69, 60–72 Karakoyun and Asiltürk: The Effect of Heuristics on the Reasoning ... DOI: 10.17344/acsi.2021.6899 Scientific paper The Effect of Heuristics on the Reasoning of the Pre-Service Science Teachers on the Topic of Melting and Boiling Point Gülen Önal Karakoyun1,* and Erol Asiltürk2 1 Van Yuzuncu Yil University, Muradiye Vocational High School, Chemistry and Chemical Business Technologies Department, Chemical Technology Program, Van, Turkey 2 Firat University, Faculty of Education, Science Education Department, Elazig, Turkey * Corresponding author: E-mail: gulenonal@yyu.edu.tr Tel.: +905442930447 Received: 04-19-2021 Abstract The purpose of this study was to explore the effects of the heuristics on the reasoning processes of pre-service science teachers on the topic of melting and boiling point using the ten heuristic model proposed by Talanquer. In this phenom- enographic study carried out in the spring semester of the 2018–2019 academic year, interviews were conducted with 30 teacher candidates enrolled in the Science Teaching Program of Firat University Faculty of Education. Participants were asked to answer three different questions during the interviews. These questions were about the ranking of some compounds according to their melting or boiling points. Six different answer patterns for each question were obtained from the answers. The findings of this study showed that students generally used shortcut strategies instead of analytical/ scientific reasoning, as all ten heuristics affected participants’ reasoning. This study also revealed that although not in- cluded in the model proposed by Talanquer, periodic trends heuristic also influenced participants’ reasoning about the melting and boiling point. Keywords: Chemistry education; cognitive constraints; heuristics; melting and boiling point; reasoning; science educa- tion. 1. Introduction In order to make predictions about the melting and boiling points of compounds, it is necessary to know well the interactions between particles and the molecular structure-property relationships. The structure-property relationships and the effects of interactions between parti- cles on melting and boiling points have an important place in chemistry curricula. Because of this importance, there are many studies in the literature on students’ understand- ing of the structure-property relationships and the effects of interactions between particles on melting and boiling points.1–6 In these studies, students’ understanding of melting and boiling phenomena was examined from dif- ferent dimensions. The findings of these studies showed that students often had difficulties in understanding inter- actions between particles and structure-property relation- ships, and could not make accurate predictions or rank- ings about the melting and boiling points of compounds due to these difficulties. It was also reported in the findings of these studies that students generally relied on shortcut strategies instead of analytical/scientific reasoning, stu- dents had various misconceptions regarding the men- tioned subjects, and students’ reasoning, judgment and decision-making processes about melting points and boil- ing points were generally flawed. In order to understand the causes of students’ learn- ing difficulties, students’ reasoning and cognitive con- straints that constrain scientific reasoning should be ex- plored in detail. Reasoning is the act of thinking about something logically. Cognitive constraints are cognitive factors/elements that restrict individuals’ analytical/scien- tific reasoning.7–11 The best known of cognitive elements include core knowledge,12 intuitive rules,13 implicit as- sumptions,14 conceptual sources,15 basic hypotheses and ontological beliefs,16 inductive constraints,17 primitive phenomenologies18 and heuristics.19 61Acta Chim. Slov. 2022, 69, 60–72 Karakoyun and Asiltürk: The Effect of Heuristics on the Reasoning ... Heuristics are related to the “dual process” theory, which was developed to explain the judgment and deci- sion-making processes of individuals. According to the dual process theory, two different cognitive systems called System 1 and System 2 are used when individuals make judgments or decisions. System 1 includes cognitive pro- cesses that progress rapidly, automatically and uncon- sciously, while System 2 includes cognitive processes that progress slowly, prudently and consciously.20–23 Using pre- vious knowledge and beliefs, System 1 processes are con- textual, relational, holistic, automatic, and working mem- ory-independent processes. Slow and sequential System 2 processes are the processes that provide rule-based, ana- lytical, abstract reasoning and use working memory.23–26 No special effort is required to trigger System 1 process- es.23,27 System 1 processes are related to the intuitive rea- soning of individuals. System 2 processes require special cognitive effort and conscious interventions.27 The System 1 processes described in detail above are short-path rea- soning strategies and are called heuristics.20,23,28–30 In con- ditions where knowledge or motivation is lacking or when time is limited, heuristics play an extremely active role.23,31,32 Heuristics enable decision-making in a short time without cognitive effort since they evaluate fewer fac- tors and use fewer cues in the reasoning and judgment processes.33 However, heuristics are responsible for vari- ous cognitive biases observed in the reasoning process- es.10,23 There are many studies in the literature exploring the effects of heuristics on the judgment and decision-making processes of individuals’ daily lives.23,30,34 Research groups in different disciplines such as cognitive psychology, psy- chology, behavioral finance, and behavioral sciences gen- erally carried out these studies. The heuristics identified in these studies were generally named with different names specific to the studied field. The heuristics identified in these studies and named with different names actually use similar cognitive processes.23,30,31 For this reason, some scientists have started to study on collecting the heuristics that progress with the similar mechanism under a general heading. For example, Morewedge and Kahneman grouped the heuristics, which frequently affect the judg- ment and decision-making processes related to the daily lives of individuals, under three headings. These heuristics are representativeness, availability and recognition.23,30,34 Today, many researchers have used this model by More- wedge and Kahneman to explore the effects of heuristics on judgment and decision-making processes related to the daily lives of individuals. Thus, confusion such as naming the heuristics that progress with similar cognitive process- es with different names was prevented.23,30 Since the 2010s, science/chemistry educators have begun to explore in detail the roles of heuristics in stu- dents’ reasoning processes related to chemistry subjects, and the working mechanisms of heuristics in the field of chemistry. The intuitive reasoning and heuristic uses of students in some chemistry subjects have been studied in detail in some research until today. Chemistry subjects ex- plored in this context include chemical reactivity, bonding theories/molecular structures, addition reactions, IR and NMR spectra interpretation, chemical problem solving, elimination reactions, acidity/basicity strength of mole- cules, structure-property relations of molecules and classi- fication of chemical substances.2,11,23,26,29,35–37 The findings of these studies showed that due to the effects of intuitive judgments and heuristics, students generally answered the questions without using basic and significant chemistry knowledge. In addition to these studies mentioned above, in a theoretical study published in 2014, Talanquer ex- plained ten heuristics that are likely to be used in chemis- try subjects and the working mechanisms of these heuris- tics with examples specific to the field of chemistry.10 The ten heuristics model of Talanquer has the quality to be used as a model in studies exploring the role of heuristics in the chemistry topics. For example, in three different studies recently conducted to examine the heuristic rea- soning of students on “hydrogen bond” and “chemical structure – acidity/basicity relationship”, the researchers carried out their research using the ten heuristic mod- el.38–40 Except for these three studies, there is no other study in the literature that explores the heuristics that are effective in chemistry topics based on the ten heuristic model proposed by Talanquer. These ten heuristics sug- gested by Talanquer are:10 Associative activation: Using mental structures pres- ent in memory to fill in the blanks. Fluency: Using of easily accessible cues in the process of solving the problem. Attribute substitution: evaluation of other easily ac- cessible attributes instead of the target attribute / Substitution the original question with a simpler question. One reason decision making: Simplifying reasoning by using a single clue or factor in the process of prob- lem solving. Surface similarity: The assumption that chemical compounds that are similar to each other in struc- tural representation have similar properties and be- havior. Recognition: More value to recognized objects / less value to unrecognized. Generalization: Generalization of learned models or rules Rigidity: Reasoning in an inflexible or non-creative way. Overconfidence: Exceeding true accuracy due to self-confidence in decision-making processes. Affect: A positive or negative emotion towards an event, an object, or anything that affects learning. The purpose of this study is to explore the effect of ten heuristics on the pre-service science teachers’ reason- ing processes about melting and boiling points. Therefore, 62 Acta Chim. Slov. 2022, 69, 60–72 Karakoyun and Asiltürk: The Effect of Heuristics on the Reasoning ... the research problem of this study can be expressed as fol- lows: What is the role of the ten heuristics in the reasoning processes of the pre-service science teachers on the melt- ing and boiling points? The research questions of this study are as follows: • Which heuristics affect the reasoning of the stu- dents in the process of performing a task in which the compounds are ranked according to their melt- ing and boiling points? • How to explain the working mechanisms of these heuristics in a way specific to the field of chemis- try? 2. Method 2. 1. Participants This research was conducted at Firat University in the spring semester of the 2018–2019 academic year. The participants of the study were selected on a voluntary ba- sis, considering their successes in General Chemistry I and General Chemistry II courses from the students enrolled in the Science Education Program. Of the 30 teacher can- didates who voluntarily participated in the research, 16 were male and 14 were female. 1/3 of the participants were students who failed in the General Chemistry I and Gen- eral Chemistry II courses, 1/3 of them were moderately successful and 1/3 of them are highly successful. Partici- pants were students enrolled in the 2nd, 3rd and 4th grades. In the study, the real names of the participants were not used, instead the participants were named with a coding S1, S2, S3, S4 … S30. The grouping of the partici- pants according to their success in General chemistry I/II courses is as follows: Failed students; S1, S5, S7, S13, S14, S16, S18, S20, S23, S24; Moderately successful students; S2, S3, S4, S8, S10, S17, S21, S25, S26, S30; Highly successful students; S6, S9, S11, S12, S15, S19, S22, S27, S28, S29. 2. 2. Data Collection and the Interview Protocol In this qualitative study, the phenomenological re- search method was used. The interviews were conducted with the participants in order to properly examine the heuristic reasoning of the participants regarding “ranking chemical compounds according to their increasing melt- ing/boiling points”. The interviews were conducted ac- cording to the following eight-step interview protocol: I. How do you feel when talking about the ranking of compounds according to their increasing melting/ boiling points? Have you ever experienced any posi- tive or negative effects on this chemistry topic during your education? If so, does it still have any effect on you? II. If you are faced with questions about ranking compounds according to their increasing melting/ boiling points, what level of confidence do you have that you can answer the questions correctly? How would you score your confidence level between 1 and 10 points (1 is the lowest, 10 is the highest)? III. Do you have a constant judgment/bias about the ranking of compounds according to their increasing melting/boiling points? For example, do you have any approaches such as “I have judgments/reasoning regarding the order of compounds according to their increasing melting/boiling points, which I will not change regardless of the question, I always solve problems regarding the order of compounds accord- ing to their increasing melting/boiling points using my current judgments/reasoning”? IV. During the interviews, the following three ques- tions about melting/boiling points were asked/ showed to the participants: 1) Rank the HI, HCl, NaI, NaCl compounds accord- ing to their increasing boiling points. 2) Rank the HCl, HBr, NaI, NaBr compounds ac- cording to increasing melting points. 3) Rank the H2Se, H2S, PH3 compounds according to their increasing boiling points. Note: At this stage, the participants were given 2 minutes to answer each question. These chemistry ques- tions were taken from a different study previously done by Maeyer and Talanquer.6 V. You saw the questions, what do you feel? (This question was asked just before students started an- swering relevant chemistry questions) VI. What level of confidence do you have that you can answer these questions correctly? (This question was asked just before students started answering rel- evant chemistry questions) VII. What level of confidence do you have in yourself that you answered these questions correctly? (This question was asked after students answered relevant chemistry questions) VIII. Explain in detail the reasons for your answers to each chemistry question. (There was no time lim- itation at this stage.) Note: During the interviews, some additional ques- tions were asked in order to obtain more explanatory in- depth information. The third question in the interview protocol was pre- pared to explore the effects of rigidity heuristic. Partici- pants’ answers to this question were carefully examined. In addition, during the interviews, special attention was paid to whether the participants actually solved the questions using the strategies they were used to before, and whether they were flexible in solving the questions. The rigidity heuristic was coded when it was determined that the par- ticipants were not flexible. The second, sixth and seventh questions in the interview protocol were prepared to ex- plore the effects of overconfidence heuristic. In cases where 8, 9 or 10 points were given as an answer to the sec- 63Acta Chim. Slov. 2022, 69, 60–72 Karakoyun and Asiltürk: The Effect of Heuristics on the Reasoning ... ond, sixth and seventh questions, the overconfidence heu- ristic was coded. Students who gave such answers general- ly made the following statements: “I am confident; I defi- nitely solved /will solve the questions correctly”. The first and fifth questions in the interview protocol were prepared to explore the effects of affect heuristic. The affect heuristic was coded in cases where it was determined that the par- ticipant had negative or positive emotions due to experi- ences. 2. 3. Data Analysis The interviews that were recorded with audio and visuals later were turned into written documents. Thus, in- terview transcripts were created for each student. With the analysis of the data obtained from the interview tran- scripts, heuristic reasoning was detected and coded. While coding, other similar studies on students’ heuristic reason- ing in chemistry were also used.2,10,23,30,38 In order to en- sure the inter-rater reliability, eight interview transcripts related to acidity strength and eight interview transcripts related to basicity strength (approximately 25% of total in- terview transcripts) were selected and the selected inter- views were first evaluated and encoded separately by both the researcher and the consultant. The results of both eval- uators were compared with each other. The encodings were revised so that there was over 90% agreement be- tween the evaluators. After this compliance was achieved, all remaining interview transcripts were evaluated and coded by the researcher. Ten heuristics proposed by Talan- quer10 were used to create a coding scheme for heuristics. The heuristic encodings, except rigidity, overconfidence and affect, were carried out by associating the students’ specific statements about the solution of the questions with heuristics. Specific student statements that are the ba- sis of encodings were presented in the results and discus- sion section. 3. Results and Discussions During the interviews, the following three questions about melting/boiling points were asked to the partici- pants: 1. Rank the HI, HCl, NaI, NaCl compounds accord- ing to their increasing boiling points. 2. Rank the HCl, HBr, NaI, NaBr compounds ac- cording to increasing melting points. 3. Rank the H2Se, H2S, PH3 compounds according to their increasing boiling points. Table 1. Obtained Response Patterns Response Pattern Code name of students n % First question (Boiling point, HI, HCl, NaI and NaCl compounds) HI < HCl < NaI < NaCl S1(F), S7(F), S11(HS), S21(MS), S23(F) 5 16.66 HCl < HI < NaI < NaCl (Correct answer) S9(HS), S12(HS), S20(F), S25(MS), S26(MS), S28(HS), S29(HS) 7 23.33 HCl < HI < NaCl < NaI S2(MS), S17(MS), S19(HS) 3 10.00 NaCl < NaI < HCl < HI S6(HS), S15(HS), S27(HS) 3 10.00 NaI < NaCl < HI < HCl S4(MS), S14(F), S16(F) 3 10.00 HCl < NaCl < HI 3.0.CO;2-J 23. M. Shannigrahi, S. Bagchi, Spectrochim. Acta Part A Mol. Biomol. Spect. 2005, 61, 2131–2138. DOI:10.1016/j.saa.2004.08.012 24. L. Kamath, K. B. Manjunatha, S. Shettigar, G. Umesh, B. Narayana, S. Samshuddin, B. K. Sarojini, Opt. Laser Tech. 2014, 56, 425–429. DOI:10.1016/j.optlastec.2013.09.025 25. P. Günter: Nonlinear Optical Effects and Materials, Springer, Berlin, 2000. DOI:10.1007/978-3-540-49713-4 26. B. Ganapayya, A. Jayarama, R. Sankolli, V. R. Hathwar, S. M. Dharmaprakash, J. Mol. Struct. 2012, 1007, 175–178. DOI:10.1016/j.molstruc.2011.10.042 27. D. Coskun, B. Gunduz, M. F. Coskun, J. Mol. Struct. 2019, 1178, 261–267. DOI:10.1016/j.molstruc.2018.10.043 28. J. Tauc, A. Menth, J. Non-Cry. Solids. 1972, 8, 569–585. DOI:10.1016/0022-3093(72)90194-9 29. B. Gündüz, Poly. Bull. 2015, 72, 3241–3267. DOI:10.1007/s00289-015-1464-7 30. B. Gündüz, Opt. Mater. 2013, 36, 425–436. DOI:10.1016/j.optmat.2013.10.005 31. S. K. Tripathy, Opt. Mater. 2015, 46, 240–246. DOI:10.1016/j.optmat.2015.04.026 32. M. Cabuk, B. Gündüz, App. Surf. Sci. 2017, 424, 345–351. DOI:10.1016/j.apsusc.2017.03.010 33. F. Abeles: Optical properties of solids. North-Holland Publish- ing Company, Amsterdam, 1972. 34. M. M. Abd El-Raheem, J. Phys. Conden. Matt. 2007, 19, 216209–216215. DOI:10.1088/0953-8984/19/21/216209 Povzetek Halkonski derivati so pomembni v znanosti, saj njihove uporabe sežejo od polprevodniških lastnosti vse do bioloških učinkov. V tem delu smo s kondenzacijo 1-(7-metoksi-1-benzofuran-2-il)etanona s 4-metilbenzaldehidom v bazičnem mediju pripravili 1-(7-metoksi-1-benzofuran-2-il)-3-(4-metilfenil)prop-2-en-1-on (2). Kemijsko strukturo 2 smo potr- dili z elementno analizo, FT-IR, 1H NMR in 13C NMR. Za različne koncentracije 2 v raztopinah smo podrobno določili UV spektroskopske lastnosti, robove absorpcijskih trakov, optične pasovne vrzeli, lomne količnike, obnašanje v odvis- nosti od okolice in prevodnostne lastnosti. Preučili smo tudi, kako se spektroskopske, optične in prevodnostne lastnosti 2 spreminjajo v odvisnosti od koncentracije in kako lahko nanje vplivamo. Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License 81Acta Chim. Slov. 2022, 69, 81–90 Fırat et al.: Effects of Individual and Co-exposure ... DOI: 10.17344/acsi.2021.6995 Scientific paper Effects of Individual and Co-exposure of Copper Oxide Nanoparticles and Copper Sulphate on Nile Tilapia Oreochromis niloticus: Nanoparticles Enhance Pesticide Biochemical Toxicity Özgür Fırat1, Rabia Erol1 and Özge Fırat2,* 1 Adiyaman University, Science and Letters Faculty, Biology Department, Adiyaman, TURKEY 2 Adiyaman University, Kahta Vocational School, Veterinary Department, Adiyaman, TURKEY * Corresponding author: E-mail: ozfirat@adiyaman.edu.tr Phone: [+90] 4167258150 Received: 11-01-2021 Abstract Copper, like iron and zinc, is one of the most essential trace elements for organisms. Different forms of copper have distinctive and specific uses. For example, copper oxide nanoparticles (CuO-NP) are widely used in the world as a nanomaterial. Copper sulphate (CuSO4) is worldwide used as a fungicide in agriculture and as an algaecide in aqua- culture. Nowadays, the increasing use of these chemicals raises concerns regarding their potential effects on the health of aquatic organisms and ecological risks. Therefore, in the present research, toxic effects of CuSO4 and CuO-NP, alone and in combination, were evaluated using biochemical markers (plasma biochemical and gill and liver oxidative stress) in freshwater fish, Oreochromis niloticus. The fish were exposed to 0.05 mg/L CuSO4, CuO-NP, and CuSO4+CuO-NP for 4 and 21 days. Especially at 21 days, CuSO4 and CuO-NP, alone and combined, generally increased plasma alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, cortisol, glucose, creatinine, blood urea nitrogen, and tissue malondialdehyde while they decreased plasma total protein, and tissue superoxide dis- mutase, catalase, glutathione-S-transferase, glutathione reductase, and glutathione. Consequently, our results illustrate that CuSO4 and CuO-NP have similar toxic effects in fish, however, co-exposure of CuO-NP and CuSO4 is more toxic than effects of these chemicals alone. Keywords: Fish; metal; nanoparticles; blood; biomarkers 1. Introduction Most aquatic environments (e.g., seas and rivers) are contaminated by pollutants from natural and anthropo- genic sources. These ecosystems are considered to be the ultimate receiving medium for pesticides, metals, and nanoparticles.1 The entry of these dangerous substances into aquatic environments impairs the water quality to the extent that it is not suitable for aquatic organisms. Copper (Cu) is one of the most essential trace ele- ments for organisms like iron and zinc. The central role of copper in the cells is as a cofactor for many enzymes such as superoxide dismutase, monooxygenases, and cyto- chrome-c oxidase.2 Different forms of copper have distinc- tive and specific uses. For example, copper oxide nanopar- ticles (CuO-NPs) are widely used in the world as a nano- material. Copper sulphate (CuSO4), another form of cop- per, is worldwide used as an algaecide in aquaculture and as a fungicide in agriculture.3 Nowadays, the increasing use of these chemicals raises concerns regarding their po- tential health problems on aquatic organisms and ecologi- cal risks. Application, production, and use of nanoparticles (NPs) are increasing worldwide. While the global market for NPs reached $ 2.0 billion in 2017, it is estimated to reach approximately $ 7.0 billion by 2022.4 CuO-NPs glob- ally are one of the most widely used NPs and the fourth most commonly used metal oxide nanoparticle after tita- nium dioxide (TiO2), silicon dioxide (SiO2), and zinc oxide (ZnO). CuO-NPs are used in consumer products, medi- cine, and industrial applications. CuO-NPs are utilized in many different applications, including in gas sensors, cata- 82 Acta Chim. Slov. 2022, 69, 81–90 Fırat et al.: Effects of Individual and Co-exposure ... lytic processes, solar cells, and lithium batteries, as well as in face masks, wound dressings, and socks.5 These nanoparticles can also be toxic, which may be due to the particles themselves or the disintegration of ions from the particles.6 In the aquatic environment, CuO-NPs are con- sidered as a significant source of contamination due to their widespread applications in antifouling paints that used in boats and immersed structures, therefore, the po- tential toxicity CuO-NPs should not be ignored.7 CuSO4 is used in aquaculture applications as a thera- peutic agent for bacterial infections and various ectopara- sitic and is reducing the incidence of fish parasites (trema- todes, protozoa, and bacteria and external fungi, etc.).8 Another application area of CuSO4 is its usage as an effec- tive fungicide in agriculture. The blood indices, important biochemical indicators, provide valuable information to assess, monitor and quanti- fy the health of the organisms e.g., fish. Therefore, they are used to explain and diagnose the toxicological effects of var- ious stressors and chemicals. Plasma enzymes [alkaline phosphatase (ALP), aspartate aminotransferase (AST), ala- nine aminotransferase (ALT), lactate dehydrogenase (LDH)] activities and metabolite [cortisol, glucose, choles- terol, total protein, creatinine, blood urea nitrogen (BUN), etc.)] levels are often measured as sensitive indicators of the harmful effects of pesticides, metals, and nanoparticles on fish vital tissues (e.g., liver and kidney).9,10 The main disturbances occur in biological systems of organisms and are caused by pollutants released in aquatic ecosystems.11 Various aquatic pollutants, such as pesti- cides and nanoparticles induce reactive oxygen species (ROS), which may lead to oxidative stress, showing role of ROS in pesticide and nanoparticle toxicities.1,12 The oxida- tive stress induces as a result of unbalance between oxidat- ing and antioxidating compounds, which may be triggered by the predominance of ROS production, incapacity of defence or changes in antioxidant systems of organisms.13 Enzymatic [catalase (CAT), superoxide dismutase (SOD), glutathione-S-transferase (GST), glutathione peroxidase (GPX), glutathione reductase (GR)] and nonenzymatic [glutathione (GSH)] antioxidant defence systems play a vital role to neutralize the toxicity of oxidative stress on the biological functions/structures of the cells. Malondialde- hyde (MDA) is widely used as a biomarker of toxic effects of pollutants on the cell membrane. Fishes are consequential sources of proteins and lip- ids and the health of them is very paramount for human beings.14 Oreochromis niloticus (Nile tilapia) is an import- ant aquaculture species amongst cultivated freshwater fish in the world.10 These fishes are being the most farmed tropical fish species globally depending on their strong im- mune systems, high growth rates, and vigorous tolerance to a wide range of environmental conditions including aquatic pollutants.15 Some studies have documented the toxic effects of co-exposure of nanomaterials with classical pollutants (pesticides or heavy metals) on aquatic organisms. For ex- ample, deleterious effects of carbon nanotubes as nanoma- terial, carbofuran as pesticide, and the co-exposure of both on Astyanax ribeirae (fish),16 O. niloticus (Nile tilapia)17 and Palaemon pandaliformis (shrimp)18 were identified in detail. In other studies, it was reported that co-exposure of graphene oxide (carbon-based nanomaterial) with trace elements (Cd, Zn) impaired the routine metabolism of the freshwater fish Geophagus iporangensis19 and P. pandali- formis.20 In recent years, nanotoxicological researches show that nanoparticles are also dangerous for living organisms, just like pesticides and metals, which are more conven- tional pollutants.1,21,22 The increasing use of CuO-NPs and CuSO4 inevitably results in increased concentrations of their discharges into the aquatic environment, which in turn may then pose a potential risk to aquatic organisms. The effect of pesticides or heavy metals on fish has been the focus of extensive research for many years, however, the combined effect of these pollutants and nanomaterials is still a new subject that needs to be studied.23 In addition, the effects of CuSO4 and CuO-NPs on fish were individu- ally investigated, but no study was found on the combined effects of these chemical. Considering the constant expo- sure of fish to these chemicals in the natural water medi- um, the present investigation aimed to determine the acute and subchronic effects of CuO-NPs as a nanoparticle and CuSO4 as a pesticide, alone and in combination, on plasma biochemical indicators (ALP, ALT, AST, LDH, glucose, cortisol, cholesterol, total protein, creatinine, BUN) and tissue oxidative stress parameters (CAT, SOD, GR, GPX, GST, GSH, MDA) in freshwater fish, Oreochromis niloti- cus. The hypothesis of the present investigation was that CuSO4 and CuO-NPs interact synergistically on the O. ni- loticus, thus provoking alterations in biochemical indica- tors in its blood, gill, and liver tissues. 2. Materials and Methods Copper sulphate (CuSO4 . 5H2O) and CuO-NPs (form: nanopowder particle size: <50 nm; surface area: 29 m2/L) were purchased from Sigma–Aldrich Co. (USA). The morphology and size of CuO-NPs dispersed in dis- tilled water were determined by transmission electron mi- croscopy (TEM) (Hitachi High-Tech HT7700, Tokyo, Ja- pan). TEM measurements demonstrated that CuO-NPs were 55 ± 10 nm of average particle size and showed spherical and oval shapes (Figure 1). For measurements of zeta potential and hydrodynamic diameter of CuO-NPs’ suspension, Zetasizer instrument (Malvern Zetasizer Nano ZSP, UK) was used. The zeta size (328 nm), polydis- persity index (0.236), potential (22.7 mV), conductivity (0.00792 mS/cm), and mobility (1.8631 µmcm/Vs) of these nanoparticles were found. The stock dispersion (10 g/L) of CuO-NPs was prepared immediately in redistilled 83Acta Chim. Slov. 2022, 69, 81–90 Fırat et al.: Effects of Individual and Co-exposure ... water followed by sonication in an ultrasonic bath for 1 hour as previously described by Shahzad et al. (2018)24. 0.05 mg/L CuO-NPs (test concentration) was prepared daily by serial dilutions of this stock dispersion followed by sonication for 20 min to avoid aggregation before add- ing to the water of the experimental aquarium. Male O. niloticus specimens, two years old, were used as research material in our study. O. niloticus (52.71 ± 0.63 g weight and 14.33 ± 0.28 cm total length, as mean ± SEM) were commercially obtained from the Aquaculture Unit of Fisheries Faculty of Cukurova University (CU), where they have been cultured for more than 30 years, and transferred to the Animal Ecophysiology Laboratory of the Science and Letters Faculty of the same university and kept in the glass aquariums containing clean tap water de- chlorinated by intense aeration, static system for eight weeks to adapt to the ambient conditions (12-hour day- light /12-hour dark photoperiod, 25 ± 1 °C temperature, central ventilation system). The mean ± standard error of some physicochemical parameters of the waters was found as pH 7.98 ± 0.06, temperature 22.18 ± 0.42 °C, dissolved oxygen 7.65 ± 0.37 mg/L, and total hardness 318 ± 3.5 mg/L as CaCO3. During the acclimatization and experi- mentation period, the fish were fed once daily at the same hour with commercial fish feed (Pinar Yem, Turkey), in an amount equivalent to 2% of their body weight. All the experiments, including the controls, were set up in duplicate considering different exposure periods (4 and 21 days). In each repeat set the experiments were car- ried out in 4 glass aquariums sized 40 cm × 120 cm × 40 cm, each containing 120 L each of the experimental solu- tions and six fish. Solutions at the concentrations of 0.05 mg/L CuSO4, CuO-NPs, and CuSO4+CuO-NPs were add- ed to the first three aquariums, respectively. The fourth aquarium contained only 120 L of free Cu-tap water and constituted the control. The range of 96-h LC50 for Nile ti- lapia was 5.03-14.27 mg Cu/L.25 The 96 h LC50 value of CuO-NPs for O. niloticus was found as 100 mg/L.26 The 0.05 mg/L concentration of CuO-NPs and CuSO4 applied in the present investigation was therefore a sublethal con- centration and eco-relevant considering the contamina- tion levels of certain water resources.5 The solutions of CuSO4 and CuO-NPs in the treated groups were renewed every 24 hours.27 The bottoms of aquaria were mixed very well with air at an interval of three times a day to minimize aggregation of NPs.9 Test media were changed just after feeding, to prevent contamination of the environment with food remains. The control fish were maintained in the same manner. Fish were exposed to these chemicals for 4 and 21 days to determine their acute and subchronic expo- sures. At the end of each duration six fish were removed from each aquarium and used as replicates for biochemical testing. After 4 and 21 days, the fish in the control and the treatment groups were individually caught and placed in the anaesthetic bath containing 75 mg/L tricaine methane- sulfonate (MS222) for 1–2 min. Blood samples were taken from the caudal vein of each fish into tubes containing eth- ylene diamine tetra acetic acid (EDTA), anticlotting agent, and centrifuged at 3000 rpm over 10 min at 4 °C for the biochemical analyses of plasma. ALT, AST, ALP, LDH, cor- tisol, glucose, total protein, cholesterol, BUN and creati- nine in the plasma samples were immediately determined using biochemical otoanalyzers (Beckman Coulter DXC 800 and Beckman Coulter DXI 800, USA). ALT, AST, and LDH activities were determined by UV test technique.28,29 ALP activity was measured by use of the colorimetric as- say.30 Cortisol level was assayed using an electrochemilu- minometric technique.31 The enzymatic UV test was used for the determination of glucose level.32 The levels of cho- lesterol,33 total protein,34 BUN,35 and creatinine36 were de- termined by colorimetric test. Following blood sampling, fish were dissected. The gill and liver tissues were homog- enized in 0.05 M Na-P buffer (pH 7.4) containing 0.25 M sucrose with a ratio of 1/10 in using a steel homogenizer at 10000 rpm for 3 min. Thereafter, the homogenates were centrifuged at 10000 rpm for 30 min at +4 °C. The alter- ation in oxidative stress parameter in the gill and liver tis- sues determined using spectrophotometrically. The activi- ty of CAT was evaluated following the method based on measuring the rate constant of hydrogen peroxide (H2O2) degradation by the enzyme.37 The activity of SOD was de- termined by the inhibition of iodo-p-nitro tetrazolium vi- olet reduction by superoxide anion radical generated by xanthine–xanthine oxidase.38 The activity of GPX was measured according to Beutler (1984),39 using t-Butyl hy- droperoxide as the substrate. The activity was determined by calculating the difference in absorbance values during oxidation on nicotinamide adenine dinucleotide phos- phate (NADPH) to NADP+. The activity of GST was eval- uated by the method of Habig et al. (1974)40 who reported that activity of enzyme was calculated by monitoring the alterations in the absorbance at 340 nm. The GR activity was assayed by determination the oxidation of NADPH by oxidized glutathione at 340 nm.41 MDA forms a pink com- plex with thiobarbutiric acid and this complex is measured at 535 nm in spectrophotometer.42 Protein level was mea- sured according to the method described by Lowry et al. (1951).43 For statistical assessing, computer software pack- Figure 1. Transmission electron microscopy image of CuO-NPs. 84 Acta Chim. Slov. 2022, 69, 81–90 Fırat et al.: Effects of Individual and Co-exposure ... age SPPS 22 was used. Before the statistical analysis, the data were analysed regarding normality distribution using Shapiro-Wilk’s test, and Levene’s test was used for homo- geneity of variance (homoscedasticity). If the results were normal and homoscedastic, differences between means of experimental groups were evaluated using a variance anal- ysis (one-way ANOVA) followed by Student-New- man-Keuls (SNK) multiple comparisons test. Significant differences were statistically considered at p<0.05. All pro- cedures used in the animal experiment were carried out in accordance with the Animal Experiments Local Ethics Committee of the CU (Protocol 2/2018). 3. Results and Discussion In the investigation, no death was observed in O. ni- loticus exposed to CuO-NPs and CuSO4 and their combi- nation. Similarly, CuO-NPs (0.02 mg/L) did not cause mortality in O. niloticus.44 Aquatic ecosystems are the last ultimate receiving environment for almost all pollutants, and aquatic organisms are seriously threatened by toxic substances entering these environments. The ability of freshwater and marine fish to survive against both well- known pollutants such as metals and pesticides, and a new group of pollutants, nanoparticles, is primarily related to their adaptability and cellular defence mechanisms. It has been shown in many studies1,10,45 that metals, pesticides and nanoparticles disrupt the internal balance in fish, cause serious toxic effects at the molecular, biochemical, and cellular levels, and even death. Similarly, in the present research significant biochemical and oxidative stress re- sponses were observed in the O. niloticus following expo- sures of CuSO4, CuO-NPs, and CuSO4+CuO-NPs. Table 1 shows the alterations in plasma enzyme ac- tivities of O. niloticus in response to the separate or com- bined effects of CuSO4 and CuO-NPs. Changes in the plas- ma/serum biochemical parameters in response to envi- ronmental pollutants occur rapidly and therefore these parameters are attributed as biomarkers of the toxic effects of chemicals. Among these biochemical parameters, ALT, AST, ALP, and LDH are liver-originated enzymes. These enzymes are intracellular enzymes. Because ALT, AST, ALP, and LDH are sensitive to contaminants, they are rec- ommended as key enzymes in the evaluation of hepatic cell damage and most liver diseases. These enzyme levels in blood plasma are low. However, due to the damage of hepatocyte cell membranes in the presence of toxicants that can cause cellular damage in the liver, their levels may increase by passing into the intercellular fluid and then into the blood. In the current work, all tested plasma en- zyme activities of O. niloticus increased, especially at 21-d, under the effect of CuSO4 and CuO-NPs and their combi- nation compared that in the control, observing a statisti- cally significant difference (F = 60.289, p = 0.000 for ALT; F = 22,458, p = 0.000 for AST; F = 19.035, p = 0.001 for ALP; F = 13,233, p = 0.002 for LDH). It is estimated that these increases in the plasma enzyme activities occur due to cellular damage caused by both copper forms in the fish liver. Similar elevation trends in the enzyme activities of fish blood serum were also found by Fırat et al. (2011)46 for Nile tilapia O. niloticus after metals (copper and lead) and pesticide (cypermethrin) treatments. The researchers con- cluded that all tested pollutants induced significant in- creases in the serum ALT, AST, ALP, and LDH activities as a result of chemical toxicity on the liver. Also, it was re- ported that iron oxide nanoparticles and zinc nanoparti- cles increase serum ALT, AST, ALP, and LDH activities in O. niloticus. 21,47 In another investigation, it was observed that there was a significant elevation in serum ALT, AST, and ALP activities in CuONPs-exposed fish groups com- pared to the control group.26 Table 1. Effects of individual and co-exposure of CuSO4 and CuO- NPs on plasma enzyme activity of O. niloticus Group 4 days 21 days ALT activity (U/L) Control 18.21 ± 0.48 a 18.44 ± 0.77 a 0.05 mg/L CuSO4 20.49 ± 0.93 a 27.07 ± 0.68 b 0.05 mg/L CuO-NPs 31.15 ± 0.74 b 34.66 ± 0.56 c 0.05 mg/L Cu-Mix 34.28 ± 0.53 b 44.72 ± 0.39 d AST activity (U/L) Control 136 ± 4.5 a 128 ± 5.6 a 0.05 mg/L CuSO4 127 ± 6.1 a 169 ± 3.9 b 0.05 mg/L CuO-NPs 141 ± 5.4 a 197 ± 6.1 c 0.05 mg/L Cu-Mix 173 ± 3.3 b 213 ± 5.2 c ALP activity (U/L) Control 25.34 ± 0.51 a 24.79 ± 0.63 a 0.05 mg/L CuSO4 24.89 ± 0.47 a 33.21 ± 0.70 b 0.05 mg/L CuO-NPs 24.60 ± 0.39 a 34.59 ± 0.66 b 0.05 mg/L Cu-Mix 31.93 ± 0.41 b 36.05 ± 0.39 b LDH activity (U/L) Control 422 ± 12 a 429 ± 18 a 0.05 mg/L CuSO4 431 ± 22 a 558 ± 11 b 0.05 mg/L CuO-NPs 417 ± 27 a 573 ± 23 b 0.05 mg/L Cu-Mix 552 ± 19 b 581 ± 17 b Data are expressed as mean ± standard error (n = 6). Small letters (a, b, c and d) are used to determine the differences between treatment groups at the same time. There is a statistical difference between data denoted by different letters (p < 0.05, Student-Newman-Keuls test). Cu-Mix: CuSO4 + CuO-NPs Energy may be urgently needed to cope with stress- ful situations that occur under the influence of toxic sub- stances in the fish. Cortisol and glucose, important stress metabolites, play an active role in energy requirement pro- cesses in such cases. Under stress, the fish brain releases excessive amounts of catecholamines and corticosteroid hormones, which in turn increase the breakdown of liver glycogen, causing elevated blood glucose levels.48 In our work, plasma cortisol and glucose levels of O. niloticus sig- 85Acta Chim. Slov. 2022, 69, 81–90 Fırat et al.: Effects of Individual and Co-exposure ... nificantly elevated in response to both alone- and co-expo- sure of CuSO4 and CuO-NPs at 4 and 21 days (Table 2). Increases in the plasma metabolite levels of fish treated with 0.05 mg/L of CuSO4, CuO-NPs, and CuSO4+CuO-NPs at 4 days were found to be 47%, 51%, and 56% for cortisol (F = 26.100, p = 0.000), and 59%, 64%, and 86% for glucose (F = 20.916, p = 0.000), respectively. We concluded that the plas- ma cortisol and glucose levels increased depending on meet the increasing energy needs in stress situations caused by these chemicals. Similar to our study findings, it was ob- served that exposures to various toxicants such as metals (Zn, Cd, and Zn+Cd) and metal oxide nanoparticles (CuO- NPs) in O. niloticus caused significant elevations in serum glucose and cortisol levels.47,49 The researchers emphasized in these studies that increases in glucose and cortisol levels might be important processes in dealing with stress caused by toxicants. In the study conducted by Soliman et al. (2021)5 15 mg/L CuSO4 or CuO-NPs significantly increased blood glucose levels of O. niloticus. The plasma/serum BUN and creatinine levels are measured frequently to assess the kidney dysfunction and damage caused by chemicals. In toxicological researches, these parameters have been used as biochemical indicators to provide valuable information about renal functions. In our investigation, the creatinine and BUN were signifi- cantly elevated by all tested chemicals at 21 days (Table 2). Significant increases in levels of the creatinine (F = 12.576, p = 0.002) and BUN (F = 19.109, p = 0.001) were found with the treatments of CuSO4 (64% and 52%) and CuO- NPs (65% and 93%), while marginally significant eleva- tions in these parameters were noted in fish exposed to CuSO4+CuO-NPs (148% and 171%). The increased plas- ma creatinine and BUN levels may demonstrate the signif- icant pathological alterations of fish kidneys associated with toxicity of all tested copper compounds. In agreement with our results, Canli et al. (2018)9 reported that O. niloti- cus after exposure to 1, 5, 25 mg/L of metal oxide nanopar- ticles (Al2O3, CuO, and TiO2) for 14-d showed striking el- evations in the serum creatinine and BUN levels, as their levels elevated nearly 10 folds. The researchers noted in- creased creatinine and blood urea nitrogen may reflect kidney failure as a result of nanoparticle toxicities. Also, a significant dose-dependent increase in BUN and creati- nine levels was reported in O. niloticus exposed to 10, 20 and 50 mg/L CuO-NPs for 25 days.26 The levels of plasma proteins are closely related to liv- er function as most of these proteins are synthesized in this tissue.50 Various chemicals can cause significant changes in plasma total protein levels, which may indicate their effects on protein metabolism in the liver. Cholesterol, another biochemical parameter, is an important component of cell membranes. Compared with the control, the individual and combined effects of CuSO4 and CuO-NPs declined total protein levels (F = 14.261, p = 0.000) after 21 days whereas they did not cause a significant change in cholesterol levels during both exposure periods (F = 0.426, p = 0.742) (Table 2). Declined total protein levels may be the result of in- creased protein degradation or reduced protein synthesis in the fish liver caused by these chemicals. These findings are in agreement with the results of Fırat et al. (2011)46 who noted O. niloticus exposed to lead and cypermethrin for 21 days showed significant decreases in the serum total protein levels. The exposures of CuO-NPs and CuO-bulks declined serum total protein levels of O. niloticus.47 Also, 21-d expo- sure of 0.5 and 1.0 mg/L silver-NP (Ag-NP) declined serum total protein levels of Cyprinus carpio (common carp).51 In another study, significant changes in the serum cholesterol levels of O. niloticus were not observed following exposures of Al2O3-, CuO-, and TiO2-NPs.9 Pollutants such as metals, pesticides, and met- al-based nanoparticles that enter aquatic ecosystems from Table 2. Effects of individual and co-exposure of CuSO4 and CuO-NPs on plasma metabolite level of O. niloticus Group 4 days 21 days Cortisol level (ng/dL) Control 4.67 ± 0.17 a 4.78 ± 0.11 a 0.05 mg/L CuSO4 6.86 ± 0.13 b 6.16 ± 0.22 b 0.05 mg/L CuO-NPs 7.04 ± 0.21 b 6.20 ± 0.19 b 0.05 mg/L Cu-Mix 7.29 ± 0.16 b 6.77 ± 0.34 b Glucose level (mg/dL) Control 51.44 ± 0.63 a 53.61 ± 0.71 a 0.05 mg/L CuSO4 81.88 ± 0.74 b 75.18 ± 0.46 b 0.05 mg/L CuO-NPs 84.25 ± 0.52 b 76.09 ± 0.84 b 0.05 mg/L Cu-Mix 95.73 ± 0.81 b 98.57 ± 0.84 c Cholesterol level (mg/dL) Control 211 ± 3.51 a 205 ± 4.63 a 0.05 mg/L CuSO4 217 ± 2.12 a 221 ± 5.27 a 0.05 mg/L CuO-NPs 208 ± 3.05 a 214 ± 2.71 a 0.05 mg/L Cu-Mix 223 ± 2.42 a 230 ± 4.30 a Total Protein level (g/dL) Control 4.30 ± 0.11 a 4.33 ± 0.08 a 0.05 mg/L CuSO4 4.28 ± 0.13 a 3.40 ± 0.06 b 0.05 mg/L CuO-NPs 4.31 ± 0.07 a 3.28 ± 0.15 b 0.05 mg/L Cu-Mix 4.34 ± 0.08 a 3.17 ± 0.10 b BUN level (mg/dL) Control 0.015 ± 0.002 a 0.014 ± 0.002 a 0.05 mg/L CuSO4 0.015 ± 0.001 a 0.023 ± 0.003 b 0.05 mg/L CuO-NPs 0.016 ± 0.002 a 0.027 ± 0.003 b 0.05 mg/L Cu-Mix 0.017 ± 0.003 a 0.038 ± 0.004 c Creatinine level (mg/dL) Control 0.022 ± 0.003 a 0.023 ± 0.002 a 0.05 mg/L CuSO4 0.022 ± 0.002 a 0.035 ± 0.002 b 0.05 mg/L CuO-NPs 0.024 ± 0.002 a 0.038 ± 0.003 b 0.05 mg/L Cu-Mix 0.025 ± 0.003 a 0.057 ± 0.002 c Data are expressed as mean ± standard error (n = 6). Small letters (a, b and c) are used to determine the differences between treatment groups at the same time. There is a statistical difference between data denoted by different letters (p < 0.05, Student-Newman-Keuls test). Cu-Mix: CuSO4 + CuO-NPs 86 Acta Chim. Slov. 2022, 69, 81–90 Fırat et al.: Effects of Individual and Co-exposure ... natural or anthropogenic sources can cause oxidative stress in fish by producing ROS. It is well known ROS con- taining highly dangerous radicals such as hydroxyl and superoxide anion cause serious damage to cells. To cope with oxidative stress, there are mechanisms in cells that prevent ROS formation and/or repair cellular damage caused by them. One of the most important of these mech- anisms is antioxidant defence systems. This system con- sists of enzymatic antioxidants such as CAT, SOD, GPX, GR and GST, or non-enzymatic antioxidants such as GSH. It has been emphasized by many researchers that cellular antioxidant defence systems can be used as biomarkers of oxidative damage caused by metal-based nanoparticles and metals.1,44,52 CAT and SOD constitute the cell’s first line of de- fence against ROS and play important biological roles in protecting cells from oxidative stress.53 In the current study, CAT and SOD activities indicated a significant de- crease at the end of 21 days in both liver (F = 15.707, p = 0.001; F = 38.458, p = 0.000, respectively) and gill (F = 17.750, p = 0.001; F = 14.149, p = 0.001, respectively) of fish exposed to individually or in a mixture of CuSO4 and CuO-NPs (Table 3). When compared to the control group, these declines in the fish liver in the treatment groups of CuSO4, CuO-NPs, and CuSO4+CuO-NPs were found to be 38%, 46%, and 48% for CAT, and 41%, 42%, and 51% for SOD, respectively. Considering the biological roles of these enzymes in antioxidant defence, the decreases in SOD and CAT activities under the effect of both copper forms may cause a decrease in the defence abilities of cells against the toxic effects of superoxide and hydroxyl radi- cals. Similar results to our study were also observed in the research conducted by Tunçsoy et al. (2017)44. They re- ported that the SOD and CAT activities reduced in the liv- er and gill tissues of O. niloticus exposed to 20 µg/L CuO- NPs. Also, it was found that the gill tissue SOD and CAT activities of O. niloticus, which was exposed to 1.0 and 5.0 mg / L TiO2-NP for 4 and 14 days, decreased significantly at the end of the first exposure period.1 These researchers noted that depending on reduced SOD and CAT activities the cells may remain vulnerable to the toxicity of radicals and suffer from oxidative stress. Ag NP and bulk Ag parti- cle exposure caused consistent decreases in both SOD and CAT activities in estuarine ragworm (Nereis diversicol- or).54 GPX protects the cell against damage induced by hy- drogen peroxide. Therefore, this enzyme, like CAT, plays significant roles in cellular defence against ROS. Changes in GPX activity affect the defence abilities of cells against toxicants. In our study, liver GPX activity of O. niloticus decreased after 4 days in CuSO4 (29%), CuO-NPs (39%), and CuSO4+CuO-NPs (43%) (F = 10.937, p = 0.003) (Ta- ble 3). Declined GPX activity may cause the accumulation of H2O2 in the cell. Due to the decreasing activities of both CAT and GPX enzymes under the effect of both copper forms, the insufficient removal of H2O2 may induce this ROS to turn into hydroxyl radical and thus cause damage to cell components. Consistent with our results, in C. carpio exposed to different concentrations of ZnO-NPs for 14 days, 50 mg/L nanoparticle concentration declined the liver, gill, intestine and brain GPX activities.55 GR, like CAT and SOD, protects cells against oxida- tive stress as an antioxidant that forms the primary line of defence against oxidative damage. It also plays an import- ant role in GSH metabolism. GST, another antioxidant enzyme, has very effective and important roles in detoxi- fication processes in cells. This enzyme catalyses the GSH conjugation to xenobiotics, protecting cells and their components from the harmful effects of these chemicals. Our research showed that in response to the tested all copper forms, GR and GST activities increased in both tissues at 4 days and decreased in the liver at 21 days (F = 8.382, p = 0.008; F = 20.878, p = 0.000, respectively) (Table 3). The induction of GR and GST activities may be an ad- aptation response to the toxic effects of CuSO4 and CuO- NPs. Similarly, it was reported that the gill GR and GST activities of O. niloticus increased after TiO2-NPs expo- sure as a rapid adaptation response to neutralize the tox- icity of this nanoparticle.1 The inhibition of GST activity may be related to decreased intracellular GSH levels in the effect of these chemicals, as determined in our study. In parallel with the results in our study, a similar decrease in GST activity was found in the tissues of freshwater fish, Labeo rohita (Indian major carp), treated with Ag-NP for 28 days.22 GSH, a cysteine-rich and low molecular weight tripeptide, acts in the cell as a protective agent against many toxic compounds.56 Therefore, maintaining intracel- lular levels of GSH is crucial in both normal cell function and neutralization of toxic stress. Under the single and combined effect of CuSO4 and CuO-NPs, the liver and gill GSH levels of O. niloticus increased at 4 days whereas they decreased at 21 days (F = 31.336, p = 0.000; F = 12.103, p = 0.002, respectively) (Table 3). Increases in GSH levels are may be important in neutralizing the toxic effects of both copper forms on the cells. However, the decrease in its lev- els with increasing time of exposure may be the result of the toxic effect of the chemicals on the synthesis of GSH or the increased cellular utilization of this tripeptide under oxidative stress. Similar to our study results, it was noted that the GSH level of the gill and liver tissues of C. carpio significantly increased in the treatment group of 0.5 mg/L ZnO-NP at 14 days.55 GSH levels increased in the initial periods of defence responses against aquatic pollutants.57 In another investigation, the effect of ZnO and ZnO-NP caused a decrease in the liver GSH levels of Danio rerio (zebrafish).58 Lipid peroxidation disrupts the selective permeabil- ity of cell membranes and can initiate processes that cause serious damage to cells. Lipid peroxidation has been at- tributed as one of the most important markers of oxida- tive damage caused by toxicants such as metals, pesticides, 87Acta Chim. Slov. 2022, 69, 81–90 Fırat et al.: Effects of Individual and Co-exposure ... and nano-metals in aquatic organisms. MDA is one of the lipid peroxidation products and increases in its levels pro- vide critical information about the oxidative stress of tox- icants and the severity of this stress. In our research, CuSO4 and CuO-NPs exposures, either separately or in combination, after 21 days caused significant increases in MDA levels of liver (F = 10.855, p = 0.003) and gill (F = 6.747, p = 0.014) (Table 3). The levels of MDA elevate as a result of lipid peroxidation that occurs due to copper-in- duced ROS. These increases in MDA levels most likely demonstrate that these chemicals induce oxidative stress in fish tissues. In agreement with the current investiga- tion, it was reported a similar elevation in the levels of tissue MDA, clearly indicating the lipid peroxidation in 5 and 50 mg/L ZnO-NP treated the fish, C. carpio, for 10 and 14 days.55 Also, CuSO4 and Cu-NPs increased lipid peroxidation in the gill tissue of Oncorhynchus mykiss (rainbow trout).59 In another study, an elevation in MDA levels was observed in rat liver following aluminium chlo- ride administration.60 In a study investigating the com- parative toxicity of copper oxide bulk and nanoparticles on fish, it was found that CuO-NPs have a more toxic ef- Table 3. Effects of individual and co-exposure of CuSO4 and CuO-NPs on tissue oxidative stress parameters of O. niloticus Liver Gill Group 4 days 21 days 4 days 21 days CAT activity (U/mg) Control 470 ± 13 a 461 ± 15 a 165 ± 6.8 a 172 ± 3.8 a 0.05 mg/L CuSO4 481 ± 16 a 285 ± 20 b 171 ± 5.5 a 129 ± 4.4 b 0.05 mg/L CuO-NPs 493 ± 21 a 247 ± 16 b 166 ± 4.7 a 122 ± 2.9 b 0.05 mg/L Cu-Mix 497 ± 18 a 241 ± 21 b 164 ± 2.3 a 98 ± 1.7 c SOD activity (U/mg) Control 27.40 ± 0.62 a 27.98 ± 0.43 a 21.70 ± 0.51 a 21.95 ± 0.44 a 0.05 mg/L CuSO4 27.89 ± 0.54 a 16.65 ± 0.34 b 20.97 ± 0.34 a 14.13 ± 0.26 b 0.05 mg/L CuO-NPs 26.71 ± 0.78 a 16.24 ± 0.59 b 22.06 ± 0.65 a 13.60 ± 0.51 b 0.05 mg/L Cu-Mix 28.22 ± 0.83 a 13.83 ± 0.27 c 21.14 ± 0.49 a 13.19 ± 0.74 b GPX activity (U/mg) Control 0.51 ± 0.02 a 0.52 ± 0.04 a 0.31 ± 0.03 a 0.30 ± 0.02 a 0.05 mg/L CuSO4 0.36 ± 0.04 b 0.50 ± 0.04 a 0.30 ± 0.03 a 0.34 ± 0.04 a 0.05 mg/L CuO-NPs 0.31 ± 0.03 b 0.48 ± 0.05 a 0.33 ± 0.02 a 0.31 ± 0.02 a 0.05 mg/L Cu-Mix 0.29 ± 0.04 b 0.47 ± 0.03 a 0.31 ± 0.03 a 0.35 ± 0.04 a GR activity (U/mg) Control 0.081 ± 0.003 a 0.085 ± 0.004 a 0.035 ± 0.002 a 0.034 ± 0.003 a 0.05 mg/L CuSO4 0.104 ± 0.004 b 0.064 ± 0.005 b 0.045 ± 0.003 b 0.033 ± 0.002 a 0.05 mg/L CuO-NPs 0.108 ± 0.003 b 0.063 ± 0.003 b 0.047 ± 0.002 b 0.030 ± 0.005 a 0.05 mg/L Cu-Mix 0.133 ± 0.002 c 0.058 ± 0.004 b 0.051 ± 0.004 b 0.029 ± 0.003 a GST activity (U/mg) Control 29.18 ± 0.84 a 31.41 ± 0.64 a 14.76 ± 0.57 a 15.28 ± 0.63 a 0.05 mg/L CuSO4 37.14 ± 0.69 b 24.49 ± 0.33 b 18.61 ± 0.73 b 14.91 ± 0.49 a 0.05 mg/L CuO-NPs 44.85 ± 0.51 c 23.55 ± 0.48 b 18.89 ± 0.89 b 15.13 ± 0.54 a 0.05 mg/L Cu-Mix 47.29 ± 0.77 c 17.91 ± 0.21 c 23.04 ± 0.61 c 14.77 ± 0.42 a GSH level (µmol/mg) Control 2.61 ± 0.14 a 2.72 ± 0.18 a 1.49 ± 0.05 a 1.54 ± 0.04 a 0.05 mg/L CuSO4 3.40 ± 0.23 b 2.08 ± 0.15 b 1.85 ± 0.04 b 1.23 ± 0.03 b 0.05 mg/L CuO-NPs 3.52 ± 0.19 b 1.65 ± 0.22 c 1.96 ± 0.05 b 1.22 ± 0.03 b 0.05 mg/L Cu-Mix 4.16 ± 0.17 c 1.51 ± 0.13 c 1.99 ± 0.06 b 1.17 ± 0.02 c MDA level (nmol/mg) Control 2.11 ± 0.03 a 2.04 ± 0.03 a 1.73 ± 0.02 a 1.74 ± 0.03 a 0.05 mg/L CuSO4 2.06 ± 0.02 a 2.89 ± 0.04 b 1.75 ± 0.03 a 2.13 ± 0.02 b 0.05 mg/L CuO-NPs 2.07 ± 0.04 a 2.97 ± 0.03 b 1.72 ± 0.02 a 2.22 ± 0.04 b 0.05 mg/L Cu-Mix 2.05 ± 0.03 a 3.58 ± 0.02 c 1.71 ± 0.03 a 2.32 ± 0.03 b Data are expressed as mean±standard error (n = 6). Small letters (a, b, c and d) are used to determine the differences between treatment groups at the same time. There is a statistical difference between data denoted by different letters (p<0.05, Student-Newman-Keuls test). Cu-Mix: CuSO4 + CuO-NPs 88 Acta Chim. Slov. 2022, 69, 81–90 Fırat et al.: Effects of Individual and Co-exposure ... fect than CuO-bulks in liver and gill tissues of O. niloticus in most oxidative stress parameters.47 Similar to our study results, it was determined in other studies that the combined effect of chemicals had more toxic effects. The combined toxic effects of silica nanoparticles (SiNPs) and methylmercury (MeHg) on ze- brafish D. rerio, a good model organism for toxicological researches, had more severe toxicity than the single expo- sure alone.61 Concomitant (iron oxide nanoparticles+mer- cury) exposure displayed a synergistic response to that of individual responses of either iron oxide nanoparticles or mercury which was evident by significant increases in GST and lipid peroxidation of the gills of Anguilla Anguilla (Eu- ropean eel).62 In an investigation determining impact of co-exposure of aldrin, a pesticide, and titanium dioxide nanoparticles at biochemical and molecular levels in Ze- brafish (D. rerio), it was observed that the combined effect of chemicals on oxidative stress parameters was generally higher than the effect alone.63 Similarly, the combined ef- fect of carbon nanotubes as nanomaterial and carbofuran as pesticide on A. ribeirae (fish) was found to be higher than the effect of these chemicals alone.16 4. Conclusions The current investigation demonstrated that almost all biochemical and oxidative stress parameters examined were negatively affected by CuSO4 and CuO-NPs, alone or in combination and that these chemicals caused cytotoxic and oxidative damage in O. niloticus. Also, our results il- lustrate that CuSO4 and CuO-NPs have similar toxic ef- fects in the fish; however, the combined effects of these two chemicals were higher than on the individual exposure regarding the biochemical changes and the oxidative stress observed in O. niloticus. Acknowledgment This study was partially funded by the Scientific Re- search Projects Unit of Adiyaman University (Grant of FE- FYL/2018-0002). Ethical approval All applicable international, national, and/or institu- tional guidelines for the care and use of animals were fol- lowed. 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Nanodelci bakrovega oksida (CuO-NP) se npr. v svetu pogosto uporabljajo kot nanoma- terial. Bakrov sulfat (CuSO4) se po vsem svetu uporablja kot fungicid v kmetijstvu in kot algicid v ribogojništvu. Danes vse večja uporaba teh kemikalij vzbuja zaskrbljenost zaradi njihovih možnih učinkov na zdravje vodnih organizmov in ekoloških tveganj. Zato so bili v pričujoči raziskavi ovrednoteni toksični učinki CuSO4 in CuO-NP, samostojno in v kom- binaciji, z uporabo biokemijskih markerjev (plazemsko-biokemijski ter škržni in jetrni oksidativni stres) pri sladkovod- nih ribah Oreochromis niloticus. Ribe so bile izpostavljene 0,05 mg/L CuSO4, CuO-NP in CuSO4 + CuO-NP 4 in 21 dni. Predvsem po 21 dneh sta CuSO4 in CuO-NP, samostojno in v kombinaciji, na splošno povečala nivo plazemske alkalne fosfataze, aspartat aminotransferaze, alanin aminotransferaze, laktatne dehidrogenaze, kortizola, glukoze, kreatinina, dušika iz sečnine v krvi in tkivnih proteinov, medtem ko sta zmanjšala nivo skupnega malondialdehida v tkivih, tkivne superoksidne dismutaze, katalaze, glutation-S-transferaze, glutation reduktaze in glutationa. Posledično naši rezultati kažejo, da imata CuSO4 in CuO-NP podobne toksične učinke pri ribah, vendar je sočasna izpostavljenost CuO-NP in CuSO4 bolj strupena kot učinki posameznih kemikalij. Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License 91Acta Chim. Slov. 2022, 69, 91–97 Mostaghni et al.: A Facile Synthesis of Bioactive Five- ... DOI: 10.17344/acsi.2021.7021 Scientific paper A Facile Synthesis of Bioactive Five- and Six-membered N-heterocyclic Aromatic Compounds Using AlCoFe2O4 as a Green Catalyst Fatemeh Mostaghni, Homa Shafiekhani and Nosrat Madadi Mahani Department of Chemistry, Payame Noor University, P.O. Box 19395-4697 Tehran, Iran * Corresponding author: E-mail: mostaghnif@yahoo.com Received: 06-19-2021 Abstract Nitrogen-containing heterocycles have been extensively studied due to their broad biological and pharmaceutical ap- plications. In this study, we synthesized five- and six-membered nitrogen-containing rings through one-pot multicom- ponent reaction using an aluminium-doped cobalt ferrite nano-catalyst. The nano-catalyst was prepared by the co-pre- cipitation method from the corresponding metal salts. The obtained results show that the proposed catalyst has a high efficiency and has enabled the formation of the desired products with high efficiency and purity. In addition, simplicity of operation, facile purification of products, shorter reaction times, mild reaction conditions, easy separation and recy- clability of the catalyst, are the main advantages of this catalyst. Keywords: Green catalyst; magnetic nano-catalyst; N-heterocyclic aromatic compounds; one-pot multicomponents re- actions 1. Introduction Heterocyclic compounds have significant impor- tance in medicinal chemistry.1–3 Among them, nitro- gen-containing heterocycles, are the most important het- erocyclic moieties of choice for the many bio-active compounds.4–6 In recent years, imidazoles, benzimidazoles and pyr- idines are subject of intense investigations because of their wide biological and pharmaceutical applications. Numer- ous biological activities have been described for imidazole derivatives such as anti-viral, antimicrobial, antifungal, anti-tumor, anti-tubercular, and anti-inflammatory activi- ties.7–18 These compounds contain two nitrogen atoms placed at 1 and 3 position in their ring structure, which possesses both acidic and basic characteristics. Since both nitrogen atoms can carry hydrogen atom, these com- pounds appear in two tautomeric forms. In addition, imid- azole moieties are very polar compounds, and possess af- finity towards enzymes and protein receptors.19 Also, medicinal properties of pyridin ring, particu- larly 2,4,6-triarylpyridines known as Krohnke pyridines, include antimalarial, anaesthetic, antiparasitic, and antitu- mor activity, as well as agents that are used for photody- namic cell specific cancer therapy.20–23 In the past few decades, various methods included synthesis performed by various metal oxide catalysts in the presence or absence of a solvent as well as microwave-as- sisted synthesis, have been reported for the synthesis of imidazoles,24–29 and Krohnke pyridines.30–37 In recent years, there has been increasing interest to develop a simple, efficient and eco-friendly, benign organic transformations catalyzed by cheap and recyclable metals or their salts, avoiding the harmful and pollutant conditions. In this study, heterogeneous AlCoFe2O4 nanocata- lyst was used as a suitable catalyst in one-pot multi-com- ponent reactions for preparation of bioactive five- and six-membered N-heterocyclic aromatic compounds. This catalyst was considered suitable for this purpose due to mild reaction conditions, short reaction times, easy sepa- rability and recyclability of the catalyst. In addition, AlCoFe2O4 nano-particles have high saturation magnetization, strong anisotropy, high coerciv- ity and excellent physiochemical stability.38–41 Many preparation methods for AlCoFe2O4 na- no-particles have been reported, such as the ball milling, co-precipitation, hydrothermal synthesis, sol-gel, and re- action in a micro-emulsion.42–46 Here, we synthesized the magnetic nano-catalyst (AlCoFe2O4) using co-precipita- tion method from metalic salts. 92 Acta Chim. Slov. 2022, 69, 91–97 Mostaghni et al.: A Facile Synthesis of Bioactive Five- ... 2. Experimental 2. 1. Material and Methods All materials and reagents were purchased from Merck, and Aldrich and used without further purification. XRD patterns prepared using powder X-ray diffraction (Bruker diffractometer, Cu-Kα X-rays of wavelength λ = 1.5406 Å). Melting points of the synthesized compounds were determined on Electrothermal-9200 melting point apparatus. IR spectra was recorded on KBr Pellets by Shi- madzu 8400S FT-IR spectrophotometer. 1H and 13C NMR spectra were recorded on a Bruker Advanced DPX spec- trometer (400 MHz for 1H NMR and 100 MHz for 13C NMR). SEM image provided by the scanning electron mi- croscope (FE-SEM, Mira3 Tescan). The absorption spectra was recorded on UV-VIS spectrophotometer (Perkin El- mer, Lambda 35). 2. 2. Synthesis of Nanocatalyst CoFe2–xAlxO4 nanocatalyst was prepared by the co-precipitation method from aqueous solutions of CoCl2, FeCl3 and aluminum nitrate. First, stoichiometric amounts of the three metallic salts were individually dis- solved in 10 mL of deionized water. Then, the three me- tallic solutions were poured in the reaction vessel and stirred vigorous for 30 minutes at room temperature. Polyethylene glycol (average molecular weight: 4000, Qualigen) was then added to the above solution as a sur- factant. Finally, the mixed solution was neutralized to pH 8 by drop-by-drop addition of 25% ammonia solution. The resulting solution was kept under stirring at 60 °C for 1 hour. The obtained particles were thoroughly washed several times with distilled water and filtered. Finally, the precipate was dried at 80 °C and calcined at 500 °C for 3 hours. The synthesized nanocatalyst was characterized by powder X-ray diffraction (XRD), scanning electron mi- croscopy (SEM-EDS), vibrating sample magnetometry (VSM), difuse reflectance and UV-vis spectroscopy. 2. 3. General Procedure for the Preparation of 2,4,6-Triphenylpyridines Various amounts of nano-particle nanocatalyst were added to a stirred solution of acetophenones (2 mmol), ar- omatic aldehydes (1 mmol), and ammonium acetate (4 mmol). Then, the mixture was stirred at various tempera- tures under solvent-free condition. At the end of the reac- tion (monitored by TLC), the catalyst was separated using an external magnet and the reaction mixture was treated with ethanol to form crystals. The crude product was ob- tained by filtration and washed with ethanol. Purification of the crude product was performed by re-crystallization in ethanol. Characterization data of selected compounds: 2,4,6-Triphenylpyridine (Table 3, entry 1) IR (KBr): ν 3071, 1590, 1493, 1471, 1033, 871, 665 cm–1; 1H NMR (400 MHz, DMSO-d6): δ 7.45–7.62 (m, 9H), 7.95 (d, J = 7.6 Hz, 2H), 8.19 (s, 2H), 8.24 (d, J = 7.5 Hz, 4H); 13C NMR (100 MHz, DMSO-d6): δ 117.17, 127.3, 127.5, 128.6, 128.9, 129.5, 130.1, 138.8, 139.4, 150.3, 157.4. 2,6-Bis(4-fluorophenyl)-4-phenylpyridine (Table 3, en- try 2) IR (KBr): ν 1601, 1595, 1506, 1421, 1330, 1223, 1160, 1116, 1075, 987, 825 cm–1; 1H NMR (400 MHz, DM- SO-d6): δ 7.37 (m, 5H), 7.51 (m, 2H), 8.13 (q, 2H), 8.23 (s, 2H), 8.32–8.41 (m, 4H); 13C NMR (100 MHz, DMSO-d6): δ 164.21, 161.67, 155.78, 155.53, 148.59, 148.52, 138.71, 134.96, 135.08, 134.12, 134.00, 129.68, 129.56, 129.27, 129.14, 128.86, 128.65, 126.87, 116.42, 116.26, 116.13, 115.89, 115.71, 115.56, 115.35. 2,6-Bis(4-fluorophenyl)-4-(4-chlorophenyl)pyridine (Table 3, entry 3) IR (KBr): ν 1612, 1592, 1510, 1416, 1328, 1210, 1162, 1105, 1073, 994, 823 cm–1; 1H NMR (400 MHz, DM- SO-d6): δ 7.36 (m, 3H), 7.61 (m, 3H), 8.09 (m, 2H), 8.21 (s, 2H), 8.38 (m, 4H); 13C NMR (100 MHz, DMSO-d6): δ 164.21, 161.72, 155.49, 155.24, 154.98, 148.55, 137.43, 137.42, 135.12, 134.19, 133.94, 129.68, 129.50, 129.32, 129.13, 128.94, 128.59, 116.68, 116.45, 116.27, 116.12, 115.94, 115.72, 115.52, 115.31. 2,6-Bis(4-bromophenyl)-4-(4-fluorophenyl)pyridine (Table 3, entry 4) IR (KBr): ν 1658, 1592, 1505, 1411, 1330, 1210, 1159, 1107, 1070, 997, 819 cm–1; 1H NMR (400 MHz, DM- SO-d6): δ 7.30 (t, J = 8.8 Hz, 2H), 7.72–7.82 (m, 5H), 7.88 (s, 1H), 7.92–8.02 (m, 4H), 8.10 (d, J = 8.4 Hz, 2H); 13C NMR (100 MHz, DMSO-d6): δ 116.3, 116.5, 122.0, 127.8, 131.0, 131.7, 131.72, 131.8, 131.9, 132.3, 136.9, 143.7, 162.7, 165.2, 188.6. 2,6-Bis(4-bromophenyl)-4-(4-methoxyphenyl)pyridine (Table 3, entry 5) IR (KBr): ν 3008, 2937, 1665, 1596, 1511, 1463, 1329, 1309, 1261, 1219, 1175, 1037, 984, 819 cm–1; 1H NMR (400 MHz, DMSO-d6): δ 3.81 (s, 3H), 7.05 (d, J = 8.2 Hz, 2H), 7.72–7.88 (m, 8H), 8.11 (d, J = 8.2 Hz, 4H); 13C NMR (100 MHz, DMSO-d6): δ 56.1, 115.2, 120.3, 128.1, 128.7, 131.3, 131.8, 132.4, 138.3, 145.5, 162.3, 189.1. 2. 4. General Procedure for the Preparation of 2,4,5-Triphenyl-1H-imidazoles Various amounts of AlCoFe2O4 nanocatalyst were added to a stirred solution of aromatic aldehydes (1 mmol), benzoin (1 mmol), and ammonium acetate (4 mmol) in ethanol (10 mL). Then, the mixture was stirred at various temperature. At the end of the reaction (monitored by 93Acta Chim. Slov. 2022, 69, 91–97 Mostaghni et al.: A Facile Synthesis of Bioactive Five- ... TLC) the magnetic nanocatalyst was separated using an external magnet. Then, the reaction mixture was diluted with 50 mL of cold water. Finally, the crude product was collected by filtration and washed with EtOH. Purification of the crude product was performed by re-crystallization from acetone: water (9:1). Characterization data of select- ed compounds were as follow: 2,4,5-Triphenyl-1H-imidazole (Table 4, entry 1) IR (KBr): ν 3445, 3082, 2853, 1641, 1504, 1462, 1398, 1128, 1070, 966, 766, 698 cm–1; 1H NMR (400 MHz, DMSO-d6): δ 7.22 (dd, 1H, J = 7.2 Hz), 7.34 (dd, 2H, J = 7.2 Hz), 7.39 (dd, 2H, J = 7.2 Hz), 7.44–7.51 (m, 6H), 7.59 (d, 2H, J = 7.6 Hz), 8.10 (d, 2H, J = 7.2 Hz), 12.71 (br, 1H); 13C NMR (100 MHz, DMSO-d6): δ 125.2, 126.5, 127.0, 127.7, 128.1, 128.2, 128.3, 128.4, 130.3, 131. 0, 135.1, 137.1, 145.3. 2-(4–Chlorophenyl)-4,5–diphenyl–1H-imidazole (Ta- ble 4, entry 2) IR (KBr): ν 3411, 3060, 2854, 1608, 1485, 1428, 1433, 1325, 1158, 833 cm–1; 1H NMR (CDCl3): δ 12.76 (s, 1H), 7.84 (d, 2H, J = 8.6 Hz), 7.54 (d, J = 6.8 Hz 4H), 7.37–7.28 (m, 8H); 13C NMR (CDCl3): δ 144.82, 137.52, 135.19, 132.24, 130.83, 130.80, 129.24, 128.96, 128.54, 128.48, 128.49, 128.14, 127.15, 126.75, 126.34. 2-(4-Hydroxyphenyl)-4,5-diphenyl-1H-imidazole (Ta- ble 4, entry 3) IR (KBr): ν 3438, 2964, 2828,1602, 1482, 1445, 1374, 1317, 1130, 1034, 968, 764, 692 cm–1; 1H NMR (400 MHz, CDCl3): δ 12.51 (s, 1H), 7.20–7.52 (m, 10H), 6.99 (d, 2H, J = 7.8 Hz), 6.89 (d, 2H, J = 7.5 Hz); 13C NMR (CDCl3): δ 158.3, 156.55, 145.46, 129.18, 128.37, 127.93, 126.92, 124.74, 119.08, 116.54, 112.82. 2-(4-Methoxyphenyl)-4,5-diphenylimidazole (Table 4, entry 4) IR (KBr): ν 1256, 1616, 2465, 2988, 3428 cm–1; 1H NMR (400 MHz, DMSO-d6): δ 12.52 (s, br, 1H), 8.05 (d, J = 8.6 Hz, 2H), 7.50 (d, J = 6.3 Hz, 4H), 7.35 (m, 6H), 7.08 (d, J = 8.7 Hz, 2H), 3.83 (s, 3H); 13C NMR (100 MHz, DM- SO-d6): δ 159.37, 145.81, 132.81, 127.84, 127.47, 126.56, 126.24, 123.13, 113.28, 54.45. 2-(4-Nitrophenyl)-4,5-diphenylimidazole (Table 4, en- try 5) IR (KBr): ν 3438, 3293, 2856, 1605, 1541, 1484, 1335, 1323, 1225, 1026, 838 cm–1; 1H NMR (400 MHz, DM- SO-d6) δ 7.45–7.52 (m, 2H), 7.74 (d, J = 4 Hz, 1H), 7.81– 7.86 (m, 3H), 8.22–8.26 (m, 4H), 8.36–8.39 (m, 5H), 13.58 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ 159.2, 137.1, 133.2, 131.9, 131.2, 130.8, 129.2, 128.8, 128.6, 128.4, 128.1, 127.5, 127.1, 126.4, 126.1, 125.8, 124.3, 122.6. 3. Results and Discussion 3. 1. Characterization of the Nanocatalyst Figure 1 shows the single-phase spinel nature of the synthesized nano catalyst was confirmed by the sharp peaks at 2θ (23.74, 29.89, 35.52, 43.14, 54.16, 57.10, 62.99), which are accredited to (111), (220), (311), (400), (422), (511) and (440). All the peaks were indexed within a single Figure 1. XRD patterns of AlCoFe2O4 Figure 2. EDS of AlCoFe2O4 94 Acta Chim. Slov. 2022, 69, 91–97 Mostaghni et al.: A Facile Synthesis of Bioactive Five- ... phase cubic spinel structure with Fd3m space group (JCPDS Card No. 86-2267). The crystallite size was calculated from intensity of the highly difracted peak (311) according to the Debye– Scherrer formula giving the crystal sizes of 6.63. To deter- mine the elemental compositions of synthesized nanocat- alyst, quantitative analysis was performed using EDS (Fig. 2). The obtained results approved the characteristic peaks of Al, Co, Fe, and O with 7.46, 9.73, 24.70 and 58.12 atom- ic percentage, respectively. The above results indicated that the AlCoFe2O4 nanocatalyst has been successfully synthesized without any impurities. Figure 3 shows the microstructure of syn- thesized AlCoFe2O4 nanocrystals. As can be seen in this photograph, it was understood that the nanoparticles were cubic shape with an average diameter size less than 45 nm with some agglomeration. The cubic structure of spinel ferrites is mainly due to the lack of Co2+ ions at octagonal sites, which leads to the absence of co-operative active Jahn–Teller distortion. Couplings at the atomic level, including the coupling between electron spins and between the electron spin and the angular momentum of the electron orbital are two fac- tors that create magnetic properties in materials. Figure 4 showed the hysteresis loops of AlCoFe2O4 nanoparticles measured using a vibrating sample magnetometer (VSM). As can be seen, the saturation magnetization (MS), the remanent magnetization (Mr) and coercivity (Hc) val- ues obtained for the sample were 18.05 emu/gr, 394 Oe, and 4.44 emu/gr, respectively. The low values of remanent mag- netization and coercivity of the nanoparticles are consistent with the properties of the soft magnetic material. Diffuse reflectance (DRS) and UV-vis spectra of the sample were obtained using a V-670, JASCO spectrophotometer. DRS spectra of synthesized nanocatalyst is shown in Figure 5. Figure 3. FESEM image of AlCoFe2O4 nanocrystals Figure 4. Hysteresis loop of AlCoFe2O4 nanoparticles Figure 5. Diffuse reflectance spectra of AlCoFe2O4 nanocatalyst 95Acta Chim. Slov. 2022, 69, 91–97 Mostaghni et al.: A Facile Synthesis of Bioactive Five- ... As can be seen in the Figure 5, the synthesized nano- catalyst exhibited high value of diffuse reflectance percent- age. The energy band gap of photocatalyst is determined using the absorption spectra according to the Tauc equa- tion as follow: (1) Where α is the absorption coefficient, β is the ab- sorption constant and Eg is the energy gap. The value of n for the direct optical gap is 1. According to Figure 6, the direct optical gap value is 1.43 ev. short reaction time and in high yield. It was also found that further increase in temperature led to a decrease in effi- ciency. This can be related to the possibility of side reac- tions. According to the results presented in Table 1, further increases in the amount of the catalyst did not have any significant effect on the reaction time and efficiency. However, reaction 2 was also completed immediately and the pure product was obtained simply by recrystalliz- Figure 6. Plot (αhv)2 vs. photon energy (inset) of AlCoFe2O4 nano- catalyst 3. 2. Evaluation of Catalytic Properties To evaluate the catalytic properties of the synthe- sized nanoparticles, the synthesis of pyridine and imidaz- ole derivatives through one-pot three-component conden- sation was selected as the model reaction. Catalytic efficiency and optimal reaction conditions were evaluated by performing reactions at different temperatures and ap- plying different amounts of nanocatalysts. The first deriva- tive in each reaction was selected as the representative to optimize this reaction. The results are presented in Tables 1 and 2. The results presented in Table 1 clearly show that at 120 °C and in the presence of 15 mol% of the catalyst, the reaction 1 led to the formation of triphenylpyridine at Table 1. Optimization of reaction conditions for preparing 1,3,5-triphenylimidazole Entry Catalyst (mol%) T (°C) Time Yield (%) 1 None r.t 10 h – 2 None 50 10 h trace 3 None 80 10 h 19 4 2 r.t 5 h 35.5 5 2 50 10 min 53 6 2 80 10 min 49 7 5 r.t 5 h 83.5 8 5 50 10 min 98 9 5 80 10 min 78.5 10 7 r.t 5 h 84 11 7 50 10 min 99 12 7 80 10 min 73 Table 2. Optimization of reaction conditions for preparing 2,4,6-triphenylpyridine Entry Catalyst (mol%) T (°C) Time Yield (%) 1 7 80 3 h – 2 7 100 3 h 23 3 7 120 3 h 67 4 10 80 3 h 34 5 10 100 3 h 64 6 10 120 3 h 91 7 15 80 3 h 52 8 15 100 3 h 75 9 15 120 3 h 97 10 20 80 3 h 54 11 20 100 3 h 79 12 20 120 3 h 98 Table 3. Preparation of 2,4,6-triarylpyridine derivatives using AlCoFe2O4 nanocatalyst Entry Ar Ar‘ Time (min) yield (%) m.p. (°C) 1 Ph Ph 60 97 135–137 2 4-F-C6H4- Ph 60 95 174–176 3 4-F-C6H4- 4-Cl-C6H4- 90 94 224–226 4 4-Br-C6H4- 4-F-C6H4- 90 96 148–150 5 4-Br-C6H4- 4-OMe-C6H4- 60 98 177–189 96 Acta Chim. Slov. 2022, 69, 91–97 Mostaghni et al.: A Facile Synthesis of Bioactive Five- ... ing from ethanol without the need for any chromatograph- ic technique. Table 2 shows that the optimal conditions were obtained with 5 mol% of catalyst at 50 °C. The reac- tion yield did not increase significantly with increasing the amount of catalyst. However, the reactions with the other benzaldehyde derivatives and aromatic ketones was inves- tigated to develop the method. The results are presented in Tables 3 and 4. The results indicate that the performance of the cat- alyst was efficient without the formation of by-products with the wide range of aromatic aldehydes containing elec- tron acceptor and electron donor substituents. In general, this catalyst has produced pure products with high effi- ciency at short reaction times and low reaction tempera- tures which makes this catalyst economically and environ- mentally useful. Furthermore, the catalyst was also successfully recycled and used for at least 5 times without significant reduction in catalytic activity. We believe that the proposed catalyst is a green and efficient catalyst com- pared to the previously reported different catalysts. 4. Conclusion In this research, we synthesized aluminum-doped cobalt ferrite nanocatalyst using co-precipitation method. The characterization results showed that the nanoparticles were cubic shape with an average diameter size less than 45 nm with some agglomeration. The low remanent magneti- zation and coercivity of the nanoparticles are consistent with the properties of the soft magnetic material. Further- more, the prepared nanoparticles were used as an effective green catalyst for one-pot multicomponent reactions. The results showed that the synthesized nanoparticles were able to create the desired heterocycle products with high effi- ciency and purity. In both types of reactions, a wide range of acetophenones and aromatic aldehydes containing elec- tron acceptor groups as well as electron donor groups on the aromatic ring provided the desired products at good isolated yields. In addition, simplicity of operation, facile purification of products, shorter reaction times, mild reac- tion conditions, easy separation and recyclability of the cat- alyst, were the main advantage of this catalyst. Acknowledgments We are grateful to Payam Noor University for encor- agements. 5. References 1. E. El-Sayed, A. Fadda, A. El-Saadaney, Acta Chim. Slov. 2020, 67, 1024–1034. DOI:10.17344/acsi.2019.5007 2. G. H. Elgemeie, R. A. Mohamed, Heterocycl. Commun. 2014, 20, 257–269. DOI:10.1515/hc-2014-0156 3. J. R. Vishnu, A. Sethi, R. Pratap, The Chemistry of Heterocycles, 1st Ed. Elsevier, Amsterdam, 2019 4. H. Beyzaei, S. Sargazi, G. Bagherzade, A. Moradi, E. Yarmo- hammadi, Acta Chim. Slov. 2021, 68, 109–117. DOI:10.17344/acsi.2020.6208 5. T. Chaban, J. 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DOI:10.1016/j.ceramint.2013.09.087 Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License Povzetek Dušikove heterociklične spojine so predmet številnih raziskav, saj izkazujejo široko biološko in farmacevtsko uporab- nost. V tej študiji smo sintetizirali pet- in šestčlenske dušikove obročne sisteme s pomočjo “one-pot” multikomponentne reakcije z uporabo kobaltovega feritnega nanokatalizatorja, dopiranega z aluminijem. Nanokatalizator smo pripravili z metodo so-obarjanja iz ustreznih kovinskih soli. Rezultati kažejo, da je katalizator visoko učinkovit in da omogoča tvorbo željenih produktov z visokimi izkoristki in čistotami. Enostavnost izvedbe ter preprostost čiščenja produktov, krajši reakcijski časi, milejši reakcijski pogoji, enostavnost ločbe in ponovna uporabnost katalizatorja so glavne odlike tega katalizatorja. 98 Acta Chim. Slov. 2022, 69, 98–107 Sadat-Mansouri et al.: Synthesis and Application of Silica Supported ... DOI: 10.17344/acsi.2021.7050 Scientific paper Synthesis and Application of Silica Supported Calix[4]arene Derivative as a New Processing Aid Agent for Reducing Hysteresis of Tread Rubber Compounds Used in Low Rolling Resistance Tires Seyedeh Nazanin Sadat-Mansouri,1 Nasrin Hamrahjou,1 Saeed Taghvaei-Ganjali1,* and Reza Zadmard2 1 Chemistry Department, IA-University, North Tehran Branch, Tehran 1651153311, Iran 2 Chemistry and Chemical Engineering Research Center of Iran, Tehran, Iran * Corresponding author: E-mail: s_taghvaei@iau-tnb.ac.ir Received: 07-13-2021 Abstract Rolling resistance is one of the most important properties of a tire which is highly dependent on the viscoelastic prop- erties of its rubber compounds. There are a lot of ways to reduce this parameter both in construction improvement of the tire and changing in rubber compound formulation especially in tire tread formulation. Rubber scientists have been trying to introduce new processing aid agents beyond the traditional tire components for reducing the rolling resistance. In this study, a unique structure of silica-supported calix[4]arene (SS-CSC[4]A) has been synthesized and applied as a processing aid agent in tire tread formulation. Fourier-transform infrared spectroscopy (FTIR), Nuclear Magnetic Resonance (1HNMR and 13CNMR), 29Si CP/MAS spectroscopy, thermal gravimetric analysis (TGA), elemental analysis, and acid-base titration were used to characterize its structure. Scanning Electron Microscopy (SEM) use to investigate the effect of prepared material on qualification of filler dispersion in the rubber matrix. The viscoelastic properties of the prepared rubber compound were measured by Dynamic Mechanical Thermal Analysis (DMTA) which showed the great decrease in rolling resistance of rubber compound based on SS-CSC[4]A as a processing aid agent. The mechanical and rheological properties of obtained tread rubber compound measured by tensometer and MDR rheometer showed no sensible changes in these properties. Keywords: Rolling resistance; tire tread compound; silica-supported calix[4]arene; dynamic mechanical thermal analy- sis; processing aid agent; wet grip. 1. Introduction Styrene-butadiene rubbers (SBRs) are a group of synthetic elastomers which is a random copolymer of sty- rene and butadiene.1 These rubbers have a huge contribu- tion to the production of car tires2 because of their unique properties especially excellent abrasion resistance and good aging stability.3,4 It is notable that the ratio of styrene and butadiene has an important role in the properties of the final product in order to enhance the ecumenical prop- erties of tread compound in a passenger car tire, SBR is normally blended with BR. SBR and BR are used as elastomers because of their amorphous structures. The absence of crystalline struc- tures in these polymers gives rise to the low mechanical properties which hinder their uses.5 One of the most effec- tive ways to improve the mechanical properties of these polymers is to prepare their compounds with the addition of fillers such as carbon black or silica.6 Although, the addi- tion of such a rigid additive to the SBR and BR blends can increase their mechanical strength, it can complicate the process ability, decrease the adhesion and rises the friction between the tires and the surface which results in the high rolling resistance, heat build-up and fuel consumption.7,8 As a best of our knowledge, high performance rub- ber tread is in high demand, for example, high wet skid resistance (WSR) and low rolling resistance, which is in line with the concept of “green tires”.9 Thus, it is necessary 99Acta Chim. Slov. 2022, 69, 98–107 Sadat-Mansouri et al.: Synthesis and Application of Silica Supported ... to find alternative processing aid agents for using in rubber compounds to reduce heat build-up, hysteresis and rolling resistance. Generally, the processing aid agents used in rubber industry are polymers with low molecular weight, resins, fatty acids, and other hydrocarbon compounds.10 The processing aid agent is a material that will be improved process ability and will be reduced plasticity. C5 (aliphatic hydrocarbon resin) and C9 (aromatic hydrocarbon resin) can do as processing and reinforcing agents. SP-1068 (phe- nolic resin) acts as a tackifying resin that makes strong hy- drocarbon bonds with the rubber compound. G90 (cou- marone indene resin) also acts as a tackifier and processing aid agent and belongs to the highly polar groups of pro- cessing aid agents. Recently, Song11 has reported a silica-based process- ing aid agent, utilizing terpene phenol resin (TPR) which enhanced the mechanical and fatigue properties, Kukreja et al.12 found that the addition of palm oil in an NBR rub- ber matrix improved the aging resistance and plasticizing efficiency, Asharf et al.13 reported that poly (methyl meth- acrylate) (PMMA) compounds with palm oil exhibited enhanced mechanical properties. Veiga et al.14 investigated the replacement of carbon black by silica-organosilane coupling agent system and the number of processing steps on the mechanical properties, rolling resistance, and wet grip of truck tire treads. Hua et al.15 studied the effect of vinyl and phenyl group content on the physical, dynamic and mechanical properties of HVBR and SSBR. Mensah et al.16 explored the physico-mechanical properties of vari- able rubber blends including epoxide natural rubber (ENR), polybutadiene rubber (BR), and solution polymer- ized styrene-butadiene rubber (SSBR) filled by silanized silica and carbon black mixtures. Calix[n]arenes are basket-shaped (vase-shaped) macrocyclic or cyclic oligomers composed of repeating pa- ra-alkyl phenolic monomers linked by methylene bridges to form a hydrophobic cavity and are simply functional- ized both at the upper rim and lower rim. These com- pounds are based on the hydroxyl alkylation products of aldehydes and phenols with a defined upper rim, lower rim and a central annulus.17 Taghvaei-Ganjali and colleagues employed calix[4] arenes and their derivatives in sensors,18 construction of polyurethane foams,19 membrane electrode,20 improve- ment of mechanical properties and thermal stability of polyurethane composite21 and removal agents for ions.22 As other researchers have reported, calix[n]arene deriva- tives can be used as heterogeneous catalytic system,23 binders24 and for detection of tryptophan.25 Our research group studied the use of calix[4]arene derivatives as sorbent,26 filler,27 tackifier resin,28 anti-rever- sion agent29 in rubber industry. Li et al. investigated the ef- fect of phenolic antioxidants based on calixarene on the antioxidative properties of natural rubber.30 Malekzadeh et al.31 studied the influence of a silane coupling agent based on calix[4]arene on the properties of nano-silica filled rub- ber compound. As could be seen, the possible influence of calix[n]arenes as a processing aid agent on tire tread per- formance has not been examined in present literatures. In the present study, a novel calixarene silica based 5, 11, 13, 17-tetrahydroxy 25, 26, 27, 28-tetrakis[chlorosulfo- nyl]calix[4]arene-bonded silica gel (SS-CSC[4]A), has been synthesized and applied as a processing aid agent in BR/SBR based rubber compounds in order to decrease of heat build-up and hysteresis which leads to reduction of rolling resistance. 2. Experimental 2. 1. Materials Emulsion polymerized styrene butadiene rubber (E-SBR1502) was provided by Takhte Jamshid Petrochem- ical Company (Iran) and high-cis polybutadiene rubber 96% (BR) was purchased from Arak Petrochemical Com- pany (Iran). N-330 carbon black was supplied by Pars Company (Iran). Aromatic oil (290, dark brown) was pur- chased from Iranol Company (Iran). The Hydrocarbon Resin (C5) was provided by Lesco Chemical (China). p-tert-Butylphenol was purchased from Merck (Germany) and applied without any purification and another three types of resins (C9, G90 and SP1068) were supplied by Taizhou Huangyan Donghai Chemical Company (China). N-cyclohexyl-2-benzothiazole sulfonamide (CBS), 1, 3-Diphenylguanidine (DPG) and 2, 2, 4-trimethyl-1, 2-di- hydroquinoline (TMQ) and sulfur were supplied by Taizhou Huangyan Donghai Chemical Company (China). N-isopropyl-N’-phenyl-p-phenylene (IPPD) was provided by Nocil (India). The silica (Ultrasil VN3) was purchased from Evonik Company (Germany). Bis[3-(triethoxysilyl) propyl]tetrasulfide (TESPT) was purchased from Shin-Et- su company (Japan). Stearic acid (PALMAC 1600) was supplied by Acidchem Company (Malaysia) and Zinc ox- ide (ZnO) was manufactured by Sepid Oxide Shokuhie Company (Iran). Paraffin wax was supplied by Behran Company (Iran). N-(cyclohexylthio) phethalimide (PVI) was purchased from Changde Dingyuan (China). Sili- ca-supported calix[4]arene derivative was synthesized in our research group.32 All analytical grades of the reagents and solvents used in this study were provided by Merck Company (Germany) and were analytical pure grades. 2. 2. Synthesis Synthetic strategy for preparation of silica-supported calix[4]arene derivative (SS-CSC[4]A) according to our published papar32 has been illustrated in Figure 1. 2. 2. 1. Synthesis of p-tert-Butyl calix[4]arene (1) p-tert-Butyl calix[4]arene was synthesized according to previously described method by Gutsche and Iqbal.33 100 Acta Chim. Slov. 2022, 69, 98–107 Sadat-Mansouri et al.: Synthesis and Application of Silica Supported ... Yield: 62%; mp: 342–344 °C; ATR-FTR: υmax (cm–1) = 3169, 2955, 1200, 1401; 1H NMR (400 MHz, CDCl3), δ(ppm): 1.21 (s, 36H, CH(CH3)3), 3.51 (d, 4H, ArCH2Ar, J 12.8 Hz), 4.26 (d, 4H, ArCH2Ar, J 12.8 Hz), 7.06 (s, 8H, Ar–H), 10.34 (s, 4H, OH); 13C NMR: (100 MHz, CDCl3), δ(ppm): 31.3, 32.4, 34.0, 126.2, 128.4, 144.5, 146.6. 2. 2. 2. Synthesis of Chlorosulfonyl-Calix[4]arene (2) Chlorosulfonyl-Calix[4]arene (CSC[4]A) was pre- pared in accordance with a previous method described by Coquiere et al.34 with some modification. A mixture of p-tert-butylcalix[4]arene (2 mmol) and dichloromethane (25 mL) was placed in a three necked 100 mL round-bot- tom flask equipped with a magnetic stirrer, reflux con- denser and septum. The mixture was stirred for 15 min at room temperature in an inert atmosphere of nitrogen gas. Chlorosulfonic acid (5 mL) was slowly added by syringe at a rate to keep the temperature between 0 and 5 °C. When the addition of chlorosulfonic acid was finished, the solu- tion mixture was refluxed for 2 h under vigorous stirring. After cooling, dry ether (30 mL) was added and the result- ing oil after separating was triturated several times with methanol. CSC[4]A as a tan powder was given. Yield 50%; mp > 230 °C; ATR-FTIR: υmax (cm–1) = 2881, 2829, 1455, 1936, 650, 455–1000. 1H NMR: (500 MHz, DMSO-d6, TMS), δ(ppm): 3.94 (8H, s, ArCH2Ar), 7.39 (8H, s, Ar–H) and 11.39 (4H, s, 8OH); 13C NMR: (125 MHz, DMSO-d6), δ(ppm): 138.3 (ArC–SO2), 30.4 (ArCH2Ar); MS-FAB: m/z 817.0 (M+, calcd 817.5) 2. 2. 3. Synthesis of Silica-Supported Calix[4] arene Derivative (SS-CSC[4]A) (3) 5 g of mesoporous silica gel was activated by reflux- ing with concentrated sulfuric acid and nitric acid [4:1] at 140 °C for 4 h to remove any adsorbed metal ions. The solution was filtered and obtained white powder was washed with distilled water until the neutral pH was gained. The residual solid was washed with acetone, meth- anol and dichloromethane, respectively and dried in an oven at 300 °C for 2 h to remove adsorbed surface water and maximize the number of silanol groups on the surface. Activated silica gel was put in a stream of dry nitrogen for 1 h and was used immediately. 100 mL round-bottom flask, equipped with a reflux condenser, a gas inlet tube for conducting of HCl gas over silver nitrate solution was used. It was charged with 50 mL anhydrous xylene, 1.5 g of activated silica gel and 1 g of CSC[4]A. The mixture was allowed to reflux under contin- uous stirring and a dry nitrogen atmosphere at 140 °C for 72 h. It was mentioned in order to prevent the crashing of silica gel particles and as a result changing the special sur- face area of silica particles during the reaction, the me- chanical stirring was not used in this reaction. Instead, the stirring was done by bubbling of nitrogen gas over the re- action mixture. After carrying out the reaction, the sus- pension was vacuum filtered using a sintered glass funnel (porosity 3) and the residue was washed in sequence with dichloromethane (5 mL), diethyl ether (5 mL), methanol (5 mL) and hexane (5 mL). The unreacted CSC[4]A inside the pores of silica gel was extracted with acetone at reflux temperature for 12 h in a soxhlet system. The acetone solu- tion was checked by thin layer chromatography and there was no evidence of unreacted CSC[4]A. The final product was dried in an oven at 150 °C for 12 h and kept in the desiccator. SS-CSC[4]A was characterized by various physical techniques such as elemental analysis for C and Si, ATR- FTIR spectra for functional group confirmation, TGA for confirmation of covalently anchored organic groups and Solid-State 29Si CP/MAS (cross-polarization/magic-angle spinning) NMR for the conformation of chemically bond- ing between silica gel and CSC[4]A. Figure 1. The synthetic strategy for silica-supported calix[4]arene. 101Acta Chim. Slov. 2022, 69, 98–107 Sadat-Mansouri et al.: Synthesis and Application of Silica Supported ... The percentage of carbon, sulfur, hydrogen which was obtained from elemental analysis and resulting acidic properties of SS-CSC[4]A are given in Table 1. The carbon and sulfur contents were assigned to the loading of CSC[4] A over silica gel. The bonded amount was found to be 92.82 µmol/g (0.219 µmol/m2) according to the carbon content shown in Table 1. Further, the sulfur content of SS-CSC[4]A was 0.36 mmol/g. The number of H+ deter- mined by acid-base titration was 0.17 mmol/g in the hy- drolyzed sample. This value is half of the sulfur content, indicating that only two ester units took place onto SS- CSC[4]A and two acidic sites exist on the surface. The TGA curve of SS-CSC[4]A shows two distinct stages of weight loss. The first weight loss is between 30 °C and 130 °C is attributed to the physically adsorbed water.35 The second weight loss is observed between 160 °C and 900 °C which can be related to the decomposition of calix- arene, corresponding to 92.82 µmol of CSC[4]A content per gram of silica. FT-IR spectra were taken for a bare silica gel, CSC[4]A and SS-CSC[4]A. The major peaks of bare silica gel spectrum are: (a) a large broad band between 3200 and 3400 cm−1 attributed to the presence of the OH stretching frequency of the surface silanol group and ad- sorbed water (b) an intense peak at 1000–1250 cm−1 re- lated to the antisymmetric Si–O–Si (siloxane) stretching in the amorphous silica (c) a band near 800 cm−1 is asso- ciated with the symmetric Si–O–Si stretching (d) a peak in the region 1600–1650 cm−1 is due to the bending mode of associated water molecules (e) the appearance of a peak at 900 cm−1 is related to Si–OH bending frequency. The infrared spectrum of the SS-CSC[4]A presented the same set of bands related to the silica gel, however some additional bands at 2883, 2827, 1457, 1936 and 650 which are assigned to the methylene asymmetric stretching, methylene symmetric stretching, C–H bending mode, benzene ring and C–S stretching mode respectively. Also, the appearance of the several peaks in the region between 1000 and 1500 cm−1 is a characteristic adsorption band of calixarenes. So, It is possible that the characteristic peaks of S=O group in compound SS-CSC[4]A which can be seen at 950–1040 cm−1, overlapped with the strong broad band of siloxane in the compound SS-CSC[4]A. It is rea- sonable to mention here, because of the intense and broad band of siloxane in the region 1100 cm−1, the intensities of the other bands in the spectrum are relatively small. To clarify the spectrum between 1200 and 4000 cm−1, it was scaled up and magnified to show the differences more clearly. Considering the possible heterogeneity in the thickness of samples and the changes in the infrared beam positions, the band area of Si–OH bending fre- quency at 900 cm−1 after and before immobilization were calculated using the silica band at 1100 cm−1 as a refer- ence band. The decrease of absorbance in the 900 cm−1 band region of SS-CSC[4]A in comparison with the spec- trum of bare silica gel indicates that the Si–O–H vibra- tion was affected due to the immobilization. These results which are obtained from IR spectrum are closely in agreement with published data.36–38 Direct evidence for chemical attachment of macro- cyclic functionalized CSC[4]A to silica surface was done by 29Si CP/MAS solid state NMR. Normally, the spectra of the bare silica gel shows three resonance peaks at −90, −100 and −110 ppm correspond to germinal silanol (Si(OH)2, Q2), free silanol (SiOH, Q3) and siloxane (SiOSi, Q4) respectively.39 Because the resolution of the spectra is not sufficient to distinguish these signals, only one broad band was seen in the spectra. As a result of the introduc- tion of functionalized calixarenes, the cross polarized 29Si- MAS NMR spectra displayed unsymmetrical pattern when compared to the bare silica material.40, 41 These results in- dicated the covalent-attachment of organic groups on the silica surface. 2. 3. Preparation of Rubber Compounds In this study, seven tire tread compounds were pre- pared. The formulations of compounds are shown in Table 2. All compounds were mixed on a two-roll mill (Hiva Ma- chinery Company, Iran) according to ASTM D3182 and vulcanization were done in 160 °C. 2. 4. Characterization 2. 4. 1. Cure Characteristics Cure properties of tire tread compounds including scorch time (ts2), cure time (tc90), minimum torque (ML), maximum torque (MH) and cure rate index (CRI) were measured at 160 °C by a Moving Die Rheometer (MDR 2000) made by HIWA company according to ASTM D5289 standard. The curing characteristics of compounds have been shown in Table 3. 2. 4. 2. Mechanical Properties The mechanical properties of prepared compounds including tensile strength, stress at 100% elongation Table 1. The results of elemental analysis and titration of SS-CSC[4]A. Compound Elemental analysis Titration %C %H %S Bonded amount (µmol/g) Acid capacity (mmol H+/g) SS-CSC[4]A 3.11 0.45 1.15 92.82 0.17 102 Acta Chim. Slov. 2022, 69, 98–107 Sadat-Mansouri et al.: Synthesis and Application of Silica Supported ... Table 2. Tire Tread Compound Formulations. Ingredient Component content (phr*) A B C D E F G BR 30 30 30 30 30 30 30 SBR 70 70 70 70 70 70 70 Carbon Black N330 45 45 45 45 45 45 45 Silica 20 20 20 20 20 20 20 Silane (TESPT) 1 1 1 1 1 1 1 ZnO 3 3 3 3 3 3 3 Stearic acid 2 2 2 2 2 2 2 Sulfur 1.6 1.6 1.6 1.6 1.6 1.6 1.6 P.Wax 2 2 2 2 2 2 2 IPPD 1.5 1.5 1.5 1.5 1.5 1.5 1.5 TMQ 1 1 1 1 1 1 1 Aromatic oil 37 37 37 37 37 37 37 CBS 1.4 1.4 1.4 1.4 1.4 1.4 1.4 DPG 2 2 2 2 2 2 2 PVI 0.3 0.3 0.3 0.3 0.3 0.3 0.3 Aliphatic hydrocarbon Resins (C5) – 2 – – – – – Aromatic hydrocarbon Resins (C9) – – 2 – – – – Coumarone Indene Resin (G90) – – – 2 – – – Phenolic Tackifying Resin (SP1068) – – – – 2 – – Para tert butyl phenol – – – – – 2 – SS-CSC[4]A – – – – – – 2 *phr represented the mass parts per 100 mass parts of BR/SBR blend. (M100), 300% elongation (M300), and elongation at break were measured by a Universal Testing Machine (model; M350-5kN, Testometric Company, UK) according to the ASTM D412 C test method. Dumbbell-shaped specimens Table 3. The results of curing behavior of rubber compounds. Sample ML (dN.m) MH (dN.m) MH-ML (dN.m) Tc90 (Min) TS2 (Min) CRI (Min–1) A 1.343 ± 0.213 7.570 ± 0.121 6.227 ± 0.234 13.672 ± 0.223 7.06 ± 0.211 15.125 ± 0.444 B 1.655 ± 0.111 8.277 ± 0.154 6.621 ± 0.054 14.721 ± 0.358 7.729 ± 0.276 14.301 ± 0.591 C 1.655 ± 0.132 8.139 ± 0.148 6.483 ± 0.229 13.823 ± 0.311 7.203 ± 0.257 15.106 ± 0.213 D 1.655 ± 0.112 8.691 ± 0.076 7.035 ± 0.211 14.125 ± 0.298 6.967 ± 0.122 13.972 ± 0.274 E 1.793 ± 0.163 8.415 ± 0.181 6.621 ± 0.017 13.832 ± 0.301 6.767 ± 0.130 14.156 ± 0.479 F 1.793 ± 0.224 8.691 ± 0.159 6.897 ± 0.326 15.46 ± 0.388 7.667 ± 0.199 12.832 ± 0.395 G 1.793 ± 0.194 8.691 ± 0.271 6.897 ± 0.077 14.682 ± 0.390 7.428 ± 0.295 13.784 ±0.565 Table 4. The results of mechanical properties of rubber compounds. Sample Processing Tensile Elongation Modulus Modulus aid agent strength (MPa) at break (%) @ 100% (MPa) @ 300% (MPa) A Blank 13.179 ± 0.876 647.693 ± 28.251 1.742 ± 0.027 5.042 ± 0.038 B C5 13.227 ± 0.726 725.75 ± 24.344 1.493 ± 0.056 4.273 ± 0.096 C C9 14.251 ± 0.44 746.179 ± 4.855 1.529 ± 0.085 4.436 ± 0.158 D G90 14.958 ± 0.252 736.452 ± 4.583 1.729 ± 0.045 4.897 ± 0.105 E SP-1068 14.549 ± 0.13 754.237 ± 6.797 1.820 ± 0.053 4.824 ± 0.086 F P-tert 13.897 ± 0.319 745.163 ± 38.728 1.564 ± 0.131 4.443 ± 0.300 G SS-CSC[4]A 14.175 ± 0.306 712.71 ± 23.45 1.762 ± 0.109 4-927 ± 0.230 (2 mm thickness, 25 mm width and 100 mm length) were cut from molded sheets. The average values of the mea- sured quantities and their standard errors were reported in Table 4. 103Acta Chim. Slov. 2022, 69, 98–107 Sadat-Mansouri et al.: Synthesis and Application of Silica Supported ... 2. 4. 3. Dynamic Mechanical Thermal Analysis (DMTA) Dynamic mechanical thermal properties of tire tread compounds were examined by using a dynamic mechani- cal thermal analyzer (DMTA; Tritec-2000; England) at temperature range from –140 °C to 90 °C in tension mode deformation and a frequency of 1.0 Hz according to ASTM E1640. The results have been shown in Tables 5 and 6. 2. 4. 4. Scanning Electron Microscopy (SEM) The degree of filler dispersion in compounds G and A (blank) was tested by scanning electron microscopy (SEM, Philips-XL 30, Netherlands) with an accelerating voltage of 25 kV. The samples were supper-coated with gold to increase their electric conductivity before the ex- amination. 3. Results and Discussions 3. 1. Curing Characteristics The cure properties that include scorch time (ts2), optimum cure time (tc90), maximum and minimum torque (MH and ML), the difference in torque (ΔM = MH-ML), and the cure rate index (CRI) of different tire tread com- pounds (A-G) are shown in Table 3. These data show that there are no significant changes in cure properties of rubber compounds by changing in processing aid agents. 3. 2. Mechanical Properties In order to investigate the mechanical properties, the results of tensile strength, elongation at break, modulus at 100% and modulus at 300% after optimum vulcanization can be seen in Table 4. The results of mechanical properties of rubber com- pounds A-G show that SS-CSC[4]A acts as C9 resins ac- cording to aromatic behavior and there are slightly in- creasing in tensile strength and elongation at break in comparison with blank compound A without any signifi- cant changes in modulus %100 and %300. 3. 3. DMTA Dynamic mechanical thermal analysis (DMTA) of tire tread compounds due to prediction tire tread perfor- mance, mainly heat build-up, hysteresis and rolling resis- tance as an indicator of fuel consumption efficiency, is im- portant in tire industry. For this purpose, the dependence of the loss factor (tan δ) on temperature at a constant fre- quency could be characterized. Therefore, the tan δ values at about 90 °C, 60 °C, 25 °C, 0 °C and –10 °C are used to predict heat build-up, rolling resistance, dry grip, wet grip and ice grip, respectively.15, 42–50 The loss factor that is the ratio between the loss modulus to storage modulus (tan δ = E”/E’), is related to the macromolecule’s movements and phase transition in the polymers.50–52 The lower value of tan δ at 60 °C, 90 °C causes lower hysteresis (lower rolling resistance) and lower heat build-up, and therefore lower fuel consumption efficiency (The main mechanism of en- Table 5. The results of dynamic mechanical thermal properties of rubber compounds A-G. Sample Processing Tg (°C) Tan δ E’G E’R E’ 30 °C CLD aid agent (max) (MPa) (MPa) (MPa) (mol/m2) A Blank –44.2 2.64E + 03 0.5328 3.134 10.31 0.3466 B C5 –44.5 2.68E + 03 0.5369 4.622 10.53 0.5112 C C9 –44.0 2.69E + 03 0.5505 5.841 10.71 0.6460 D G90 –40.8 2.29E + 03 0.5116 4.876 11.27 0.5393 E SP-1068 –43.9 2.61E + 03 0.4891 7.498 15.89 0.8293 F P-tert –44.0 2.44E + 03 0.5188 5.030 10.87 0.5563 G SS-CSC[4]A –44.3 2.73E + 03 0.5324 5.421 11.31 0.5990 Table 6. tan δ at various temperature for compounds A-G derived by DMTA. Sample Processing tan δ @ aid agent 90 °C 60 °C 25 °C 0 °C –10 °C A Blank 0.1627 0.1517 0.1668 0.1735 0.1773 B C5 0.1378 0.1359 0.1564 0.1681 0.1746 C C9 0.0922 0.1079 0.1413 0.1648 0.1739 D G90 0.1321 0.1460 0.1615 0.1741 0.1797 E SP-1068 0.1357 0.1512 0.1590 0.1672 0.1694 F P-tert 0.1243 0.1326 0.1571 0.1717 0.1760 G SS-CSC[4]A 0.1068 0.1176 0.1441 0.1549 0.1646 104 Acta Chim. Slov. 2022, 69, 98–107 Sadat-Mansouri et al.: Synthesis and Application of Silica Supported ... ergy loss in tread compound in a rolling tire is the Payne effect which has not been studied here). On the other hand, the higher value of tan δ at low temperatures (–10 °C, 0 °C, 25 °C) indicates better grip properties of tire on the roads surface.53,54 Figures 2–4 and Tables 5–6 represent the influence of different processing aid agents on tire tread performance and dynamic proper- ties for tire tread compounds A-G. As shown in Figure 3 and Table 5, C and E com- pounds had the highest and the lowest values of tan δmax (peak of the tan δ). The almost same values of tan δmax for the G and the blank compounds demonstrate that the amount of rubber chains participating in the glass transi- tion for both compounds is in the same order.55 Regarding this fact the value of E’ at 30 °C directly relates to the dry handling property of a tire56, the highest value of E’ at 30 °C for the E compound indicates that the compound pos- sessed the best dry handling property. The E’ value at 30 °C for the G compound increased about 9.7% compared to the obtained parameter for the blank compound. The val- ue of cross-linking density (CLD) parameter in Equation (1) was obtained by the following equation55: CLD = E’/3RT (1) where E’, R, and T are corresponded to the minimum stor- age modules, the universal gas constant, and the absolute temperature at the rubbery plateau zone, respectively. Based on Equation (1), the higher value of E’ at the rubbery region results in a higher degree of cross linking density. Therefore, from Table 5 it can be clearly seen that the E compound possessed the highest modulus at rub- bery and so the highest degree of cross linking density. The higher E’ value at the rubbery zone for the G com- pound compared to the blank compound led to the incre- ment of the cross linking density (about 72.8%) for the SS-CSC[4]A containing compound. The value of E’ at the glassy region for the G compound is the highest in com- parison with other compounds and was about 3.4% more than the blank compound indicating the stronger struc- tural interactions of SS-CSC[4]A in G compound with the matrix. Tg is another parameter that can be obtained from the DMTA analysis. As can be seen, the lowest and highest values of Tg respectively belong to the B and E compounds. The Tg of tire tread compound decreases from –44.2 °C to –44.3 °C when there is SS-CSC[4]A. To understand the reason, the SS-CSC[4]A structure and its effect on silica particle dispersion have been studied. SS-CSC[4]A is a ca- lixarene, that its cavity can relieve the force on itself by moving its flexible bonds, then it is accepted that when SS- CSC[4]A is added as mobile macromolecule to compound with cured chain, Tg must be decreased. On other hand, the addition of SS-CSC[4]A to compound provides better silica dispersion and more interaction between rubber chain and silica particle, which means we have to provide more energy for chain movement and this energy must be provided by higher temperature. According to the results, there is a trade-off between increasing and decreasing Tg when SS-CSC[4]A is added to compound, DMTA results show just 0.1 °C decrease in Tg, so the SS-CSC[4]A flexible bonds is the Tg controller. In general, it is expected that with a gradual decrease in Tg, the values of tan δ will be increased. But as can be seen in results, tan δ values are lower than the blank com- pound at all temperatures in the presence of SS-CSC[4]A. At high temperature when it is well above Tg, the move- ment of the chains is very fast and long range. Regardless of the flexible structure, with the presence of SS-CSC[4]A as an external factor makes it difficult to move the polymer chain, so tan δ values were decreased. According to Figure 4 and Table 6, the values of tan δ at 60 °C and 90 °C of compound G are decreased com- pared to blank compound (A), where the rolling resis- tance and heat build-up decreased by 22.5% and 34.4%, respectively. Which means the fuel consumption efficien- cy and heat build-up performance of compound G con- taining SS-CSC[4]A are improved. The fuel consumption of a passenger car will be reduced by 1–2% if the rolling resistance of tire is reduced by 10%, according to the lit- eratures.57–59 But at lower temperature due to better dispersion of the silica particle and more interaction between matrix and filler, increasing of tan delta for silica filled in compar- ison to CB based compounds are mainly due to the higher polymer volume fraction in them. In fact, at a lower tem- perature, due to increase in stiffness of filler agglomerate and cluster, the release of occluded rubber is more difficult than the higher temperature. As in the case of silica com- pound, we have lower values of occluded rubber and thus polymer volume fraction is higher than CB compounds. As a result, tan δ values and grip decrease in G compound compared to blank compound. Consequently, the use of SS-CSC[4]A as a processing aid agent in tire tread compounds, is beneficial for reduc- Figure 2. Storage modulus (E’) versus temperature curves of rubber samples containing various processing aid agent. 105Acta Chim. Slov. 2022, 69, 98–107 Sadat-Mansouri et al.: Synthesis and Application of Silica Supported ... ing heat build-up, hysteresis and rolling resistance. This is because of better filler dispersion, stronger rubber-filler Figure 3. Loss factor (tan δ) versus temperature curves of rubber samples containing various processing aid agent. Figure 4. Loss factor (tan δ) values at different temperature of rubber samples containing various processing aid agent. Figure 5. SS-CSC[4]A molecular structure. Figure 6. SEM images of compounds A and G. interaction, higher crosslink density and lower filler-filler interaction. 106 Acta Chim. Slov. 2022, 69, 98–107 Sadat-Mansouri et al.: Synthesis and Application of Silica Supported ... 9. R. Huang, Q. Pan, Z. Chen, K. Feng, Appl. Sci. 2020, 10, 13, 4478. DOI:10.3390/app10134478 10. N. C. Kim, S. H. Song, Int. J. Polym. 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According to the structure of SS-CSC[4]A, there is dual structural behavior. A fair dispersity to the silica as filler via lower rim silica-supported moiety and good physical connection to rubber matrix via upper rim moi- ety of aromatic based calixarene. As a result, better silica disperses is observed in the rubber matrix in the presence of SS-CSC[4]A as dispersing agents. 4. Conclusion In this study in order for improvement of rolling re- sistance and heat build-up of tire tread compound a unique processing aid agent, SS-CSC[4]A was synthesized. FTIR and NMR proved the structure of SS-CSC[4]A. DMTA re- sults showed a reduction in rolling resistance (22.5%) and heat build-up (34.4%), due to the effect of SS-CSC[4]A on silica dispersion and interaction between matrix and filler. SEM results showed a great silica dispersion when there is SS-CSC[4]A in the compound, and the tensile test did not illustrate any tangible changes for the compound contain- ing SS-CSC[4]A. 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Sae-oui, K. Suchiva, U. Thepsuwan, W. Intiya, P. Yodjun, C. Sirisinha, Rubber Chem. Technol. 2016, 89, 2, 240–250. DOI:10.5254/rct.15.84859 Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License Povzetek Kotalni upor (ang. rolling resistance) je ena najpomembnejših lastnosti pnevmatik, ki je zelo odvisna od viskoelastičnih lastnosti njenih komponent. Obstaja več načinov za zmanjšanje tega parametra, tako s samo konstrukcijo pnevmatike kot tudi s spreminjanjem njene sestave, še posebej pri načrtovanju profilnega dela. Raziskovalci na tem področju zato poskušajo z uvajanjem novih aditivov, ki bi zmanjšali kotalni upor. V tej študiji smo na silikatno osnovo sintetizirali derivat kaliks[4]arena (SS-CSC[4]A) edinstvene strukture in ga uporabili kot polnilo profilnega dela pnevmatik. FTIR, magnetno resonanco (1HNMR and 13CNMR), 29Si CP/MAS spektroskopijo, termično gravimetrično analizo (TGA), elementno analizo in titracijo smo uporabili za karakterizacijo. Učinkovitosti dispergiranja polnila v gumi smo določili z vrstično elektronsko mikroskopijo (SEM). Viskoelastične lastnosti tako pripravljene gume smo izmerili z dinamično mehanično termično analizo (DMTA), ki je pokazala znatno zmanjšanje kotalnega upora v primerjave z gumo brez SS-CSC[4]A polnila. Meritve opravljene s tenzometrom in MDR reometrom pa niso pokazale drugih bistvenih razlik mehanskih in reoloških lastnosti. 108 Acta Chim. Slov. 2022, 69, 108–115 Dere et al.: A Novel Solid-State PVC-Membrane ... DOI: 10.17344/acsi.2021.7053 Scientific paper A Novel Solid-State PVC-Membrane Potentiometric Dopamine-Selective Sensor Based on Molecular Imprinted Polymer Nurşen Dere,1 Zuhal Yolcu2 and Murat Yolcu2,* 1 Giresun University, Center Research Laboratory Application and Research Center, Giresun /Turkey 2 Giresun University, Science and Arts Faculty, Chemistry Department, Giresun /Turkey * Corresponding author: E-mail: murat.yolcu@giresun.edu.tr Received: 09-21-2021 Abstract A novel solid-state polyvinylchloride (PVC) membrane potentiometric dopamine-selective microsensor was construct- ed based upon dopamine-imprinted polymer (DOP-IP) used as the ionophore in the membrane structure. The optimum membrane composition was determined as 4% (w/w) DOP-IP, 69% (w/w) bis(2-ethylhexyl) sebacate (DOS), 26% (w/w) PVC, and 1% (w/w) potassiumtetrakis(4-chlorophenyl) borate (KTpClPB). The detection limit of the microsensor was determined to be 3.71 × 10–7 mol L–1. The microsensor exhibited a super-Nernstian response for dopamine over the concentration range of 10–6−10–1 mol L–1, with a short response time (<15 s) and a slope of 60.3 ± 1.3 mV per decade (R2: 0.9998) over seven weeks. The microsensor was effectively performed in a pH range of 4.0−8.0 and a temperature range of 5−30 °C. The microsensor has been successfully demonstrated for the rapid, accurate, selective and reproducible determination of dopamine in pharmaceutical formulations with the recovery of 104.3–104.8%. The obtained results were in good harmony with the UV-Vis results at a confidence level of 95%. Keywords: Dopamine; solid-state microsensor; molecular imprinted polymer; potentiometry 1. Introduction Dopamine is one of the most important neurotrans- mitters that play specific roles in various physiological and pathological processes in the central nervous, cardiovas- cular, hormonal and renal systems of the human body, modulated by their levels in various brain tissues.1–3 De- termination of dopamine is important in the diagnosis, monitoring and prevention of certain diseases, such as Parkinson’s, schizophrenia, HIV infections, hyperurice- mia, and a type of arthritis.4 There are many instrumental methods for dopamine determination, such as chromatog- raphy,5 fluorimetry,6 colorimetry,7 spectrophotometry,8 and electrochemistry.9 These methods require both expen- sive equipment and complex sample preparation, and time. Electrochemical methods have several advantages compared to expensive instrumental methods. Especially, when evaluated in terms of ion-selective electrodes; elec- trochemical methods provide superiority such as short re- sponse time, low detection limit, simple design, low cost, wide operating range, high selectivity, minimum sample pretreatment, accuracy and precision, easy measurement process.10 The molecular imprinting method involves the po- lymerization of a functional monomer and crosslinker around a template which is removed using different sol- vents after the synthesis process.11 This technique is a very suitable method for polymeric material formation with molecular recognition cavities created by the addition of template molecules during the process.12 As a result, mo- lecular imprinted polymers (MIPs) provide a wide range of binding sites with various affinities and selectivity that are interrelated to the template molecule in size, function- ality, and shape.13 The imprinted polymers have several advantages such as good physical and chemical stability, high selectivity and low cost.14–16 MIPs are widely used in drug release,17 solid-phase extraction,18 enzyme mim- ics,19 chromatographic separation,20 cancer biomarkers,21 and sensors.22 Different potentiometric sensors based on MIP have been reported.23–25 Several electrochemical sensors have also been reported for dopamine determina- tion.26–30 109Acta Chim. Slov. 2022, 69, 108–115 Dere et al.: A Novel Solid-State PVC-Membrane ... In this work, a novel potentiometric dopamine-selec- tive microsensor, that is solid-state PVC-membrane type, was designed using dopamine-imprinted polymer (DOP- IP) as an ionophore. The performance characteristics (limit of detection, linearity, slope with standard deviation, re- sponse time, selectivity, repeatability, reproducibility, pH, and temperature ranges, etc.) of the microsensor were in- vestigated in detail. The microsensor was successfully used for dopamine determination in the content of the pharma- ceutical formulations. The potentiometric results were compared with the UV-Vis spectroscopic results. 2. Experimental 2. 1. Reagents Dopamine (DOP), methacrylic acid (MA), azobi- sisobutyronitrile (AIBN), ethylene glycol dimethacrylate (EGDMA), ethanol (EtOH), methanol, acetic acid, tetra- hydrofuran (THF), high molecular weight polyvinylchlo- ride (PVC), o-nitrophenyl octyl ether (NPOE), bis(2-eth- ylhexyl) sebacate (DOS), dibutyl sebacate (DBS), potassium tetrakis (4-chlorophenyl) borate (KTpClPB), graphite, solvents, and all other salts were purchased from Sigma-Aldrich. Epoxy resin (Ultrapure SU 2227) and hardener (Desmodur RFE) were supplied from Victor and Bayer AG, respectively. 2. 2. Apparatus A multi-channel potentiometer supported by a com- puter program device and designed in our laboratory was used for the potentiometric measurements. Ag/AgCl elec- trode (Basi-MF-2079-RE-5B) was operated as a reference electrode. A Jenway 3040 model ion analyser was used for pH measurements. A Shimadzu AUX220 model analytical balance was used for measuring weight. A Kubota 4200 model centrifuge was used for centrifugation. Deionized water was supplied from a Sartorius Stedim Ariium 611UV model ultra-deionized water device. A Memmer (GmbH & Co. KG D.91126 Typ: WNB 14) shaker was used for the removal of dopamine molecules from the polymer. The solutions were homogenized using an Ultrasonic LC30 (Germany) stirrer. A Jeol JSM-6610 model instrument was used for scanning electron microscopy (SEM) analysis. A Thermo Scientific Evaluation Array UV-Vis spectropho- tometer was used for the spectroscopic determination of dopamine. 2. 3. Synthesis of Dopamine-Imprinted Polymer The dopamine-imprinted polymer (DOP-IP) was synthesized according to the method described in the lit- erature.31 The preparation process of the DOP-IP is sche- Figure 1. Schematic representation of the DOP-IP preparation process 110 Acta Chim. Slov. 2022, 69, 108–115 Dere et al.: A Novel Solid-State PVC-Membrane ... matized in Figure 1. A 59 mg dopamine, 0.4 mL MA and 1.24 mL EGDMA were dissolved in 6.2 mL EtOH in a glass bottle. The mixture, pure nitrogen gas passed through for 20 min, was sonicated in a water bath for 30 min. Then 0.02 mg AIBN was added to the mixture. The mixture was heated to 60 °C in a thermostatically adjust- ed oil bath on a magnetic stirrer for 21 hours. A colorless translucent bulk of solid polymer was obtained. Polymer particles containing dopamine molecules (DOP-P) were washed with EtOH and filtered. Methanol/acetic acid (90/10; v/v) solution was repeatedly used for removal of the dopamine molecules until not detecting any dopa- mine in the filtered solution by UV-Vis method. Final polymer particles (DOP-IP) were then vacuum dried at 50 °C. The non-imprinted polymer (NIP) was synthe- sized by following the same procedure without dopa- mine. 2. 4. Fabrication of Solid-State Dopamine- Selective Microsensor The solid-state dopamine selective microsensor used was manufactured according to the method described in our previous study.32 The first stage, named as the solid contact production, of sensor fabrication, which occurs in two steps; involves the preparation of an amount of 300 mg of graphite, 210 mg of epoxy, and 90 mg of hardener in 3 mL of THF. A copper wire of about 10 mm length and 2 mm radius is dipped into this mixture several times until a thickness of about 0.5 mm is obtained, and left to dry for a day under laboratory conditions. The second stage con- tains the preparation of a selective membrane mixture. An amount of 10–15 mg of DOP-IP, 167.5–172.5 mg of NPOE, DOS or DBS, 65–67.5 mg of PVC, and 2.5 mg of KTpClPB were thoroughly mixed in 2.5 mL THF. Finally, the solid contact formed in the first stage is dipped 4–5 times in the membrane mixture and the prepared sensor is left to dry under laboratory conditions for 1 day. After these proce- dures, the performances of the microsensor are investigat- ed in detail. 2. 5. Analysis Procedure of the Pharmaceutical Samples The dopamine contents of the pharmaceutical sam- ples were determined using both DOP-selective microsen- sor and UV-Vis spectrophotometric method in commer- cially available drug: Dopasel® (200 mg/5 mL). The drug sample was diluted with deionized water before the poten- tiometric and UV-Vis (at 280 nm) measurements. 3. Results And Discussion 3. 1. SEM Analysis Scanning Electron Microscopy (SEM) was used for the investigation of surface morphologies of the polymers (NIP, DOP-P and DOP-IP). Figure 2a–f shows the relevant SEM images with the structural differences of the particles. When the general surface morphology is examined; it is seen that the polymers have different particle sizes, however, have spherical shapes as similarities. The NIP particles (Fig- ure 2e–f) are substantially larger in size than the MIP parti- cles (Figure 2a–d). Moreover, it is seen that an enhanced surface area and pores were observed on the DOP-IP sur- face (Figure 2c–d) than the DOP-P surface (Figure 2a–b). This situation can be considered as a result of the imprinting process. Consequently, the relatively porous surfaces of DOP-IP possess the specific cavities and suitableinteraction sites for the sorption of dopamine molecules. 3. 2. Optimum Membrane Composition It is known that PVC-membrane sensors are signifi- cantly dependent not only on the structure of the iono- phores but also on the ratio of membrane components, polymers, plasticizers and other additives. These effects on sensors; in addition to lowering the detection limit of the sensors, also increases the sensitivity and selectivity. The effects of PVC membrane components on the potentio- metric response of the DOP-selective microsensor were Table 1. Potentiometric performance characteristics of DOP–selective microsensors No Membrane Composition (mg/250 mg) Potentiometric Behavior PVC NPOE DOS DBS KTpClPB MIP Slope, Linear Detection mV/decade* range, mol L–1 limit, mol L–1 I 65 172.5 – – 2.5 10 49.6 ± 2.6 10–5−10–1 5.30 × 10–7 II 65 – 172.5 – 2.5 10 60.3 ± 1.3 10–6−10–1 3.71 × 10–7 III 65 – – 172.5 2.5 10 45.3 ± 2.5 10–5−10–1 5.72 × 10–6 VI 65 167.5 – – 2.5 15 40.7 ± 2.8 10–4−10–1 2.84 × 10–5 V 65 – 167.5 – 2.5 15 50.6 ± 2.2 10–4−10–1 6.22 × 10–5 VI 65 – – 167.5 2.5 15 42.1 ± 3.0 10–4−10–1 4.87 × 10–5 VII 67.5 172.5 – – – 10 45.1 ± 1.8 10–5−10–1 2.62 × 10–6 VIII 67.5 – 172.5 – – 10 53.8 ± 1.6 10–6−10–1 4.95 × 10–7 IX 67.5 – – 172.5 – 10 44.3 ± 2.1 10–5−10–1 1.36 × 10–6 *The average value of three determinations ± standard deviation 111Acta Chim. Slov. 2022, 69, 108–115 Dere et al.: A Novel Solid-State PVC-Membrane ... investigated using different plasticizers (NPOE, DOS and DBS) and the results are summarized in Table 1. It can be seen that the best potentiometric performances (slope, de- tection limit, linear range) are for sensor number-II com- pared to the others. The potentiometric performance of the DOP-selective microsensor, which was prepared ac- cording to the optimum membrane composition, was in- vestigated in more detail. The potentiometric response of the DOP-selective microsensor was investigated in the standard dopamine solutions prepared in the concentration range of 10–8−10–1 mol L–1 (Figure 3). It was determined that the sensor ex- hibited a linear response to dopamine as a super Nernst behaviour (60.3 ± 1.3 mV) in the concentration range of 10–6–10–1 mol L–1 with a lower detection limit of 3.71 × 10–7 mol L–1 and a short response time (t95) of <15 s ac- cording to the IUPAC recommendations.33 The calibration graphs of microsensors prepared with DOP-IP and NIP are shown in Figure 4. The performance of the DOP-IP- based sensor is better than the NIP-based sensor, and it can be said that this situation in the NIP sensor is due to the non-specific interaction on the NIP surface. Figure 2. SEM images of the DOP-P (a, b), DOP-IP (c, d), and NIP (e, f) 112 Acta Chim. Slov. 2022, 69, 108–115 Dere et al.: A Novel Solid-State PVC-Membrane ... 3. 3. Repeatability and Reproducibility The repeatability (within-day) of the DOP-selective microsensor was investigated. For this purpose; the mea- surements were repeatedly taken in the concentration range of 10–6−10–1 mol L–1 dopamine. The obtained poten- tial-time graph is shown in Figure 5. It can be seen from Figure 5, the behaviour of the developed sensor is highly reproducible. In order to determine the reproducibility (be- tween-days) of the developed DOP-selective microsensor, the changes in the detection limit and slope values of the sensor have been monitored for two months. For this pur- pose, measurements were taken in standard dopamine solutions in the linear operating range of the DOP-selec- tive microsensor on certain days and the obtained slope values against time are shown in Figure 6. As can be seen from Figure 6, especially after 42 days, a significant drift in the slopes indicates that the stability of the sensor has de- teriorated (the initial slope value of 60.3 mV/decade de- creased to 53.1 mV/decade). Therefore, the lifetime of the sensor was estimated to be about 6 weeks. Repeatability and reproducibility of the microsensor showed a differ- ence in potential within 3–5 mV. Figure 3. Potentiometric responses and calibration plot of the DOP-selective microsensor Figure 4. Calibration curves of the DOP-selective sensors based on MIP (•) and NIP () Figure 5. Repeatability measurements of the DOP-selective mi- crosensor Figure 6. Reproducibility of the DOP-selective microsensor (slope values against time) 3. 4. Selectivity The selectivity coefficients of the DOP-selective mi- crosensor were calculated by using the separate solution method (SSM).33 The obtained logarithmic selectivity co- efficients (Log KpotDOP, Xn+) for dopamine molecules over other ions and molecules (Xn+) are summarized in Table 2. The prepared sensor exhibited high selectivity for dopa- mine over the commonly encountered and tested different species. 113Acta Chim. Slov. 2022, 69, 108–115 Dere et al.: A Novel Solid-State PVC-Membrane ... 3. 5. pH Effect In order to examine the effect of pH on sensor re- sponses, 1.0 × 10−3 mol L–1 dopamine solutions were ex- amined in the pH range of 3.0−11.0 (Figure 7). It can be seen from Figure 7; the sensor potential remained signifi- cantly unchanged in the pH range of 4.0−8.0. However, the increase in potential values at low pH values (< 4.0) can be explained by the interaction of hydronium ions on the sen- sor membrane, as interference, and the decrease in poten- tial values at high pH values (> 8) can be explained by the interference of hydroxyl ions. Therefore, the pH: 4.0–8.0 range can be considered the optimum operating range for the proposed sensor. 3. 6. Temperature Effect Temperature is another important property for elec- trochemical sensors. To determine the optimum tempera- ture range of the developed microsensor, the temperatures of the DOP solution were changed from 5 °C to 70 °C. The potential measurements for 10–2 mol L–1 DOP solution are shown in Figure 8. The DOP-selective microsensor can be able to operate in the temperature range of 5−30 °C (± 2 mV) approximately without significant changes on the performance of the microsensor. The performance of the sensor is affected above 30 °C by the temperatures. In ad- dition, it was determined that the sensor was deformed above 30 °C.34 Table 2. Selectivity coefficients of the DOP–selective microsensor Types Log KpotDOP, Xn+ Types Log KpotDOP, Xn+ K+ –2.08 Zn2+ –2.79 Li+ –1.74 Ba2+ –2.67 Na+ –2.52 Ni2+ –2.15 NH4+ –2.11 Cd2+ –3.03 Ca2+ –2.93 Co2+ –2.28 Mg2+ –2.33 Cr3+ –2.06 Cu2+ –2.49 Fe3+ –1.91 Ag+ –3.02 Pb2+ –3.05 Fructose –3.25 Glucose –3.18 Urea –3.17 Lactose –2.01 Triethanolamine –2.05 Thiourea –3.49 Ascorbic acid –1.88 Thioacetamide –3.18 Figure 7. Effect of pH on the DOP-selective microsensor response Figure 8. Effect of temperature on the DOP-selective microsensor performance 3. 7. Sample Analysis The electroanalytical applicability of the prepared DOP-selective microsensor, the dopamine contents in the pharmaceutical samples were determined by the proposed microsensor. The obtained potentiometric results were compared with the results obtained with UV-Vis spectro- Table 3. Determination of DOP in the drug sample Pharmaceutical Label value Amounts of DOP (ppm) * Product Potentiometry UV-Vis Recovery (%) Era (%) t-test f-test Dopasel® 400.0 417.2 ± 4.6 412.5 ± 2.5 104.3 4.25 1.55 3.39 200.0 209.1 ± 5.5 205.3 ± 2.6 104.6 4.55 1.08 4.47 100.0 104.8 ± 5.8 102.2 ± 2.8 104.8 4.80 0.70 4.29 * The average values (ppm) of three determinations ± standard deviation. Era is the relative error for the potentiometry versus label value. t-student’s and f-test level (critical) values are 4.30 and 19.00 at 95% confidence, respectively. 114 Acta Chim. Slov. 2022, 69, 108–115 Dere et al.: A Novel Solid-State PVC-Membrane ... photometric method. The recovery, relative error, t-test and f-test values were calculated and presented in Table 3. As can be seen from Table 3, the student’s t-test and f-test values calculated at the 95% confidence level are lower than the tcritical (4.3) and fcritical (19.0) values, respectively. As a result, it can be concluded that there are no significant differences between the potentiometry and UV-Vis meth- ods. It can be seen that the average values (with the recov- ery of 104.3–104.8% and the relative error of 4.25–4.80%) obtained by the proposed sensor were in satisfactory agreement with the labeled values. 3. 8. Comparison of the proposed sensor with the other DOP-selective sensors The comparison of the developed sensor with both MIP-based and traditional ionophore-based dopa- mine-selective sensors available in the literature is sum- marized in Table 4. The developed sensor is considered to be comparable to the previously reported sensors in most cases as slope, linear range, response time, detection limit, and pH range. The developed microsensor is suitable for miniaturization due to its solid-state structure. The flow- cells with low dead volume can be easily prepared for this type of sensor. Therefore, they have the possibility to be used as detectors for the flow systems, which is another important advantage over conventional sensors. 4. Conclusions In the current study, a novel solid-state type PVC membrane DOP-selective potentiometric microsensor was developed based on DOP-imprinted polymer. The DOP-selective microsensor was successfully applied for the rapid, accurate, selective, and reproducible determi- nation of dopamine in pharmaceutical formulations. The obtained potentiometric results were found to be compat- ible with the results obtained by UV-Vis. The developed sensor has the advantages of fast response time, low de- tection limit, wide linear range, ease of preparation, and low cost. Therefore, the microsensor can be considered to be a notable addition to the list of dopamine selective sen- sors. Acknowledgments The authors acknowledge support by the Giresun University Scientific Research Projects Commission Presi- dency (project no: FEN-BAP-C-281119-81). 5. 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Ryasenskii and I. P. Gorelov, Pharm. Chem. J. 2006, 40, 334–336. DOI:10.1007/s11094-006-0122-7 42. M. Othman, N. M. H. Rizka and M. S. El-Shahawi, Anal. Sci. 2004, 20, 651–655. DOI:10.2116/analsci.20.651 Povzetek Na osnovi polimera, vtisnjenega z dopaminom, ki se je uporabil kot ionofor v membranski strukturi, je bil izdelan nov polivinilkloridni (PVC) membranski potenciometrični mikrosenzor, selektiven za dopamin. Optimalna sestava mem- brane je bila določena kot 4 % (m/m) MIP, 69 % (m/m) bis(2-etilheksil) sebakata (DOS), 26 % (m/m) PVC in 1 % (m/m) kalijevega tetrakis(4-klorofenil) borata (KTpClPB). Meja zaznavanja mikrosenzorja je bila 3,71 × 10–7 mol L–1. Mikrosenzor je pokazal super-nernzijski odziv (angl. super-Nernstian response) na dopamin v razponu koncentracij 10–6–10–1 mol L–1, s kratkim odzivnim časom (<15 s) in naklonom 60,3 ± 1,3 mV na dekado (R2 : 0,9998) znotraj sedmih tednov. Mikrosenzor je bil učinkovit v območju pH 4,0−8,0 in temperaturnem območju 5−30 °C. Uspešna demonstracija mikrosenzorja je pokazala hitro, natančno, selektivno in ponovljivo določanje dopamina v farmacevtskih formulacijah z izkoristkom 104,3–104,8 %. Dobljeni rezultati so dobro kolerilali z rezultati UV-Vis pri stopnji zaupanja 95 %. Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License 116 Acta Chim. Slov. 2022, 69, 116–124 Mahadevaiah et al.: Combustion Synthesis of Nano Fe2O3 ... DOI: 10.17344/acsi.2021.7055 Scientific paper Combustion Synthesis of Nano Fe2O3 and its Utilization as a Catalyst for the Synthesis of Nα-Protected Acyl Thioureas and Study of Anti-bacterial Activities Raghavendra Mahadevaiah,1,3 Lalithamba Haraluru Shankraiah1,* and Latha Haraluru Kamalamma Eshwaraiah2 1 Department of Chemistry, Siddaganga Institute of Technology, B. H. Road, Tumakuru - 572 103, Karnataka, India 2 Department of Electronics & Instrumentation Engineering, Siddaganga Institute of Technology, 10 B. H. Road, Tumakuru - 572 103, Karnataka, India 3 Department of Chemistry, Channabasaveshwara Institute of Technology, B. H. Road, Gubbi, Tumkur - 572 216, Karnataka, India * Corresponding author: E-mail: lalithambasit@yahoo.co.in; hslalithamba@gmail.com Received: 07-15-2021 Dedicated to Dr. Sree Sree Sree Shivakumara Mahaswamiji, Siddaganga Matt, Tumakuru, Karnataka, India Abstract A simple and eco-friendly nano Fe2O3 heterogeneous catalytic system is described for the synthesis of acyl thiourea derivatives from corresponding in situ generated acyl isothiocyanates and amino acid esters in acetone obtained in good yields. The structures of synthesized acyl thioureas were confimed by 1H NMR, 13C NMR, mass, and FTIR analysis. Fe2O3 NPs has been prepared via a solution combustion route using ascorbic acid as the reducing agent and ferric nitrate as the source of iron. The prepared nano material has been characterized by XRD, SEM, UV-Visible, and FTIR analysis. More prominently, the Fe2O3 and other impurities are removed though a simple work-up and the material prepared shows to be effective in catalyzing the conversion of reactants to products in good yields. Further, some of the synthesized acyl thioureas were evaluated for in vitro antibacterial activity against Staphylococcus aureus and Escherichia coli. Keywords: N-protected acyl thioureas; in vitro antibacterial activity; Fe2O3 nanoparticles; solution combustion. 1. Introduction Many acyl thiourea derivatives are well known to pos- sess a diverse range of biological activities such as antican- cer,1 antiviral,2 fungicidal,3 anti-microbial,4 antimycobacte- rial,5 antitumor,6 anti-inflammatory,7 herbicidal,8 anti-aggregating,9 analgesic, and are often employed as local anesthetic, and antihyperlipidemic.10 Therefore, the acyl thiourea linkage has received greater attention because of its potent biological as well as structural aspects. The ability to provide a hydrogen bond donor and acceptor point makes it an efficient anion receptor and enables it to play a key role in some epoxy resin curing agents containing amino function- al groups and to act as chelating agents in catalysis.11–13 Aro- ylthiourea ligands are an important class of compounds in the field of coordination chemistry and catalysis.14–16 Thiourea and its derivatives have long been studied for their use against the corrosion of a wide range of metals in vari- ous corrosion environments.17–18 Also the thiourea and its derivatives have found wide range of applications in agricul- ture, medicine, and analytical chemistry.19–20 In the past, Benjawan and his group synthesized and evaluated the acyl thiourea derivatives for the in vitro evalu- ation of activity against Mycobacterium tuberculosis showing promising results.21 Substituted thioureas are an important 117Acta Chim. Slov. 2022, 69, 116–124 Mahadevaiah et al.: Combustion Synthesis of Nano Fe2O3 ... class of sulphur-containing organic compounds or interme- diates in the synthesis of a variety of heterocyclic compounds such as 2-imino-1,3-thiazolines, imidazole-2-thiones, (benzothiazolyl)-4-quinazolinones. Pyrimidine-2-thiones N-(substituted phenyl)-N-phenylthioureas have been devel- oped as calixarenes containing thioureas as neutral recep- tors towards α,α-dicarboxylate anions, anion-binding sites in a hydrogen-bonding receptor, and N-4-substituted-ben- zyl-N-tert-butylbenzyl thioureas as antagonists in rate DRG neurons and vanilloid receptor ligands.22–28 There are several works demonstrating the synthesis of acyl thiourea derivatives including: (a) reaction of func- tionalized diisothiocyanate with various benzenamines in the presence of PEG-400,29 (b) difluoromethyl pyrazole acyl thiourea derivatives were successfully synthesized us- ing PEG-600 as a phase transfer catalyst,30 (c) The applica- tion of benzoyl / carbethoxy isothiocyanate towards the synthesis of substituted-3-benzoyl/carbethoxy thioureas in the presence of nucleophile such as amine and NH4SCN was investigated,31 (d) reaction of acid chloride with po- tassium thiocyanate to obtain acyl isothiocyanate interme- diate, this is coupled with the amines employing TBAB as an organic catalyst to afford N-(o-fluorophenoxyacetyl) thioureas derivatives,32 (e) Zhong et al. synthesized three different acyl thiourea derivatives of chitosan and their an- timicrobial behavior against four species of bacteria were investigated.33 Therefore, the application of nano Fe2O3 for the synthesis of Nα-protected acyl thioureas is, according to the literature survey, not established yet. Hence, herein we report a simple and efficient route for the synthesis of biologically active acyl thioureas employing nano Fe2O3. Furthermore, the synthesized compounds were screened for in vitro activity in anti-bacterial studies. Nowadays, nano metaloxide semiconductors have attracted a lot of attention, as their properties can be con- trolled by changing the crystallite size, shape, sur- face-to-volume ratio, temperature and also by synthetic routes.34–35 From the view point of the basic research pur- pose Fe2O3 is an important semiconductor for the study of magnetic properties, and polymorphism and structural phase transitions of nanoparticles.36,37 In the last decades, nanostructured iron oxides such as α-Fe2O3, γ-Fe2O3, β-Fe2O3 and Fe2O3, have received remarkable interest from both theoretical and experimental viewpoints be- cause of their potential applications in sensing devices and biomedical applications, such as magnetic resonance im- aging, biosensors, hyperthermia, and drug delivery in can- cer therapy, and detoxification and also in industrial appli- cations.38–41 At the same time there is an increased interest in using iron oxide NPs for the removal of various pollut- ants (As5+, Cr6+, dyes) from wastewaters.42–45 Several synthetic methods have been developed for the preparation of Fe2O3 nanoparticles due to its inherent biocompatible nature, magnetic properties as well its sta- bility towards oxidation.46–49 There are many methods available to prepare nano Fe2O3 including: co-precipita- tion, plasma chemical synthesis, micro emulsions, thermal decomposition, sol-gel, and flame spray pyrolysis etc.50–57 Some of these are expensive and time consuming. Here, we have prepared Fe2O3 nanopowder by eco-friendly sim- ple low-cost solution combustion method using ascorbic acid as the reducing agent.58 Although the synthesis of Fe2O3 nanoparticles has seen substantial progress, the preparation of pure, large surface area nano Fe2O3 pow- ders is still one of the top goals in the field. 2. Experimental Section 2. 1. General All the chemicals were purchased from Sigma-Al- drich and Merck and used without purification. The pathogenic bacterial strains were purchased from National Chemical Laboratory Pune, India. IR spectra were record- ed on Bruker Alpha-II FTIR spectrometer. 1H NMR and 13C NMR spectra were recorded using Bruker AMX 400 MHz spectrometer using TMS as the internal standard and DMSO as the solvent. Mass spectra were recorded on a Micromass Q-ToF Micro Mass Spectrometer. Powder XRD data were recorded on Shimadzu X-ray diffractome- ter (PXRD-7000) using Cu-Kα radiation of wavelength λ = 1.541 Ǻ. IR spectra were recorded on Bruker Alpha-T FTIR spectrometer (KBr windows, 2 cm–1 resolution). The absorption spectrum and band gap were measured using Lambda-35 (Perkin Elmer) spectrophotometer in the wavelength range 200–800 nm diffused reflectance mode. Morphological features were studied by using Hita- chi-7000 Scanning Electron Microscopy. 2. 2. Solution Combustion Synthesis of Nano Fe2O3 1 g of Fe(NO3)3 ∙ 9H2O (acting as oxidizing agent) and 0.6 g of ascorbic acid (acting as reducing agent) were mixed and stirred in 10 mL distilled H2O to get homoge- neous solution. This solution was poured into silica cruci- ble and kept in a preheated muffle furnace at 500 °C. The homogeneous solution first undergoes dehydration, then decomposition and large amounts of gases were released during the process. Fe2O3 NPs were formed within 5 min- utes. In continuation of our interest a facile protocol for the nano Fe2O3 catalyzed synthesis of acyl thioureidopep- tides was developed in peptide chemistry. 2. 3. Reusability and Recyclability of Nano Fe2O3 Catalyst was reused and recycled without any loss of activity and product yield. The nano Fe2O3 can be recycled by a simple protocol after the completion of the reaction. The catalyst was removed by filtration, washed with meth- anol and dried. The recovered catalyst was reused for the 118 Acta Chim. Slov. 2022, 69, 116–124 Mahadevaiah et al.: Combustion Synthesis of Nano Fe2O3 ... second, third and fourth consecutive cycles without any significant loss in catalytic activity. 2. 4. General Procedure for the Synthesis of Nα-Protected Acyl Thioureas To the protected amino acid (1 mmol) dissolved in CH2Cl2 (5 mL), SOCl2 (1.5 mmol) was added and the mix- ture was sonicated at rt for about 40–50 min and moni- tored through TLC. The excess of CH2Cl2 and SOCl2 were carefully eliminated by rotary distillation and the residue was precipitated after the addition of hexane (5 mL) and then filtered and dried to get pure acid chlorides. To the formed acid chloride were added ammonium thiocyanate and acetone, this reaction mixture was refluxed for 1 h and the solvent was removed on a rotary evaporator to obtain a crude product of acyl isothiocyanates. This intermediate was dissolved in CH2Cl2 (30 mL), then subjected to H2O and brine wash and dried over anhydrous Na2SO4. The or- ganic phase containing acyl isothiocyanate intermediate was coupled with neutralized amino acid esters followed by the addition of nano Fe2O3 (0.5 mmol). The reaction mix- ture was stirred for 4–6 h at rt and monitored by TLC. After the completion of the reaction, Fe2O3 was removed by fil- tration and the organic phase was washed with 5% HCl (20 mL), 5% Na2CO3 (20 mL), H2O, and brine and dried over anhydrous Na2SO4 to get crude product of acyl thioureas; then, it was purified by the trituration with hexane–diethyl ether system to afford analytically pure products. 3. Results and Discussion 3. 1. Characterization of Fe2O3 NPs 3. 1. 1. Powder X-Ray Diffraction Technique Figure 1 shows the XRD pattern of Fe2O3 NPs cal- cined at 500 °C. The diffraction peaks observed at 24.18, 33.34, 35.68, 40.85, 49.63, 54.28, 62.55 and 64.22 could be indexed to the (012), (104), (022) (202), (103), and (123) planes respectively, consistent with the standard XRD data of Fe2O3 (JCPDS no. 72-1191). The average crystallite size of the prepared sample was calculated by Debye–Scherrer equation (Equation 1) and it was observed to be 30 nm. (1) 3. 1. 2. Fourier Transform Infrared Spectroscopy From Figure 2 the two peaks at 442 and 530 cm−1 are observed in the FTIR spectrum of the Fe2O3 NPs. In com- parison with the literature, we conclude that these peaks were assigned to the stretching and bending modes of the Fe−O bond. The absorption peaks around 1587 and 1383 cm−1 are due to the asymmetric and symmetric bending vibration of C=O. Figure 1. XRD patterns of the Fe2O3 NPs. Figure 2. FTIR spectrum of Fe2O3 NPs. 3. 1. 3. Scanning Electron Microscopy Analysis Figure 3 shows the SEM image of the prepared nano Fe2O3 which clearly shows that the particles have roughly irregular spongy cave like structure. The size and the shape of the Fe2O3 strongly depend on the preparation technique. Figure 3. SEM images of Fe2O3 NPs. 119Acta Chim. Slov. 2022, 69, 116–124 Mahadevaiah et al.: Combustion Synthesis of Nano Fe2O3 ... 3. 1. 4. UV-Visible Analysis The optical properties of nano Fe2O3 were studied by UV-visible DRS spectroscopy. The spectrum was recorded in the wavelength region between 200 to 1200 nm at rt. The band gap energy of the samples can be evaluated from the Eg measurements using Kubelka–Munk model and the F(R) was estimated using the equation 2 (2) where F(R) is the remission or Kubelka–Munk fuction, and R is the reflectance. A graph was plotted between [F(R)hν]2 and hν, the intercept value is the band gap ener- gy of the Fe2O3 NPs. From the Figure 4a it was observed that Fe2O3 NPs show strong reflectance peak at 510 nm wavelength. From Figure 4b the estimated band gap of nano Fe2O3 was found to be 2.5 eV which was higher when compared with the reported value for the bulk Fe2O3 (2.1 eV) owing to the quantum confinement effect exerted by the nanostructured materials. Thus there is a blue shift of the band edge of Fe2O3 NPs with respect to the bulk Fe2O3. 3. 1. 5. Application of Nano Fe2O3 as the Catalyst for the Synthesis of Nα-Protected Acyl Thioureas of N-Protected Amino Acids To avoid the restrictions, such as cost of synthesis, prolonged reaction conditions, and low yields, the studies were made to develop a well-organized method with high- er yields for the synthesis of acyl thioureas in the presence of nano Fe2O3. Therefore, we described the synthesis of acyl thiourea derivatives of protected amino acids bearing different side chains employing Fe2O3 nano powder under mild reaction conditions. The protocol is based on a three- step strategy, a direct chlorination of the carbonyl group of the protected amino acid with a thionyl chloride followed by the nucleophilic substitution reaction with ammonium thiocyanate in acetone under reflux condition to form acyl isothiocyanates as key intermediates, further being cou- pled with amino acid esters employing nano Fe2O3 as the catalyst leading to the desired acyl thioureas 5a–m in good yields (Scheme 1, Table 1). Basically, acyl thiourea in its basic structure has one sulfur atom, which has six valence electrons. It is believed that the present protocol provides a greater flexibility of amino acids at our convenience and is Figure 4. (a) DRS spectrum; (b) Energy band gap plot of Fe2O3 NPs. R1, R2: H, alkyl, aryl groups Pg (Protecting group): Fmoc (fluorenylmethyloxycarbonyl), Cbz (benzyloxycarbonyl). Scheme 1. Synthesis of acyl thioureas employing nano Fe2O3 a) b) 120 Acta Chim. Slov. 2022, 69, 116–124 Mahadevaiah et al.: Combustion Synthesis of Nano Fe2O3 ... superior to the other methods. Nα-Protected amino acid was dissolved in CH2Cl2 (dichloromethane), thionyl chlo- ride was added and the mixture was sonicated at rt for about 40–50 minutes yielding acyl chlorides 2. Then, the carbonyl group in 2 (in the form of acyl chloride) was modified by the nucleophilic substitution reaction. To the acyl chlorides 2 ammonium isothiocyanate was added, yielding acyl isothiocyanates 3 under reflux condition. The formed acyl isothiocyanate intermediates 3 were trapped with amino acid esters 4 employing nano Fe2O3 as an effi- cient, eco-friendly catalyst to form the final product acyl thioureas 5a–m as monitored by TLC. The reaction was complete in about 6 h and all the compounds were isolat- ed, after a simple work-up, and purified by hexane-diethyl ether system and their structures were confirmed by mass, 1H NMR, 13C NMR, and FTIR spectroscopy techniques. 3. 1. 6. Spectral Data of the Synthesized Compounds 5a–m (S)-Methyl 2-(3-((S)-2-(((9H-Fluoren-9-yl)methoxy) carbonyl)-3-methylbutanoyl)thioureido)propanoate (Fmoc-Val-ψ[CONHCSNH]-Ala-OMe) (5a). Yield 80%, m.p. 184 °C. 1H NMR (400 MHz, DMSO-d6): δ 1.01 (d, J = 12.0 Hz, 6H), 1.30 (d, J = 8.0 Hz, 3H), 2.0 (br, 1H), 2.70 (m, 1H), 3.60 (m, 1H), 3.70 (s, 3H), 4.45–4.70 (m, 4H), 6.0 (br, 2H), 7.20–7.90 (m, 8H). 13C NMR (100 MHz, DMSO-d6): δ 17.0, 17.80, 32.0, 47.0, 52.30, 54.0, 61.0, 68.0, 126.0, 128.0, 128.40, 129.0, 140.0, 143.80, 156.0, 170.0, 175.0, 187.0. MS: Calcd. for C25H29N3O5S: m/z 506.1726 (M + Na+), found: 506.1060. (S)-Methyl 2-(3-((S)-2-(((9H-Fluoren-9-yl)methoxy) carbonyl)-3-phenylpropanoyl)thioureido)-3-hydroxy- propanoate (Fmoc-Phe-ψ[CONHCSNH]-Ser-OMe) (5b). Yield 81%, m.p. 170 °C. 1H NMR (400 MHz, DMSO-d6): δ 2.10 (s, 1H), 2.90 (d, J = 6.0 Hz, 2H), 3.50 (t, J = 10.0 Hz, 1H), 3.70 (s, 3H), 4.40–4.90 (m, 6H), 6.0 (br, 2H), 7.10– 7.90 (m, 13H), 8.0 (br, 1H). 13C NMR (100 MHz, DMSO-d6): δ 38.0, 46.50, 51.20, 53.0, 62.0, 68.70, 126.0, 127.0, 127.80, 128.0, 128.40, 128.70, 129.0, 140.0, 141.0, 143.0, 156.0, 176.0, 187.10. MS: Calcd. for C29H29N3O6S: m/z 570.1675 (M + Na+), found: 570.2275. (S)-Methyl 2-(3-(2-(Benzyloxycarbonyl)-3-methylbuta- noyl)thioureido)acetate (Cbz-Val-ψ[CONHCSNH]-Gly- OMe) (5c). Yield 85%, m.p. 165 °C. 1H NMR (400 MHz, DMSO-d6): δ 1.0 (d, J = 8.0 Hz, 6H), 2.70 (m, 1H), 3.70 (s, 3H), 4.40–4.50 (m, 3H), 5.30 (s, 2H), 6.10 (br, 2H), 7.10– 7.30 (m, 5H), 8.0 (br, 1H). 13C NMR (100 MHz, DM- SO-d6): δ 20.89, 21.67, 46.67, 56.18, 64.93, 65.26, 126.98, 127.51, 127.54, 129.08, 129.14, 129.28, 129.38, 129.44. MS: Calcd. for C17H23N3O5S: m/z 404.1256 (M + Na+), found: 404.2250. (S)-Methyl 2-(3-((S)-2-(Benzyloxycarbonyl)-4-(meth- ylthio)butanoyl)thioureido)-3-phenylpropanoate (Cbz- Met-ψ[NHCONH]-Phe-OMe)(5d). Yield 86%, m.p. 180 °C. 1H NMR (400 MHz, DMSO-d6): δ 2.0 (s, 3H), 2.30– 2.50 (m, 4H), 3.30–3.50 (m, 4H), 3.70 (s, 3H), 5.30 (s, 2H), 6.10 (br, 2H), 7.10–7.30 (m, 10H), 8.01 (br, 1H). 13C NMR (100 MHz, DMSO-d6): δ 17.10, 29.0, 32.0, 38.0, 52.0, 54.50, 60.0, 66.0, 126.0, 127.40, 127.60, 127.90, 128.50, 129.0, 139.0, 141.0, 156.0, 171.0, 175.50, 187.0. MS: Calcd. for C24H29N3O5S2: m/z 526.1446 (M + Na+), found: 526.2130. (S)-Methyl 2-(3-((S)-2-(tert-Butoxycarbonyl)propanoyl) thioureido)-3-hydroxypropanoate (Boc-Ala-ψ[CONHC SNH]-Ser-OMe) (5e). Yield 81%, Gum. 1H NMR (400 MHz, DMSO-d6): δ 1.29 (s, 9H), 1.50–1.59 (d, J = 6.0 Hz, 3H), 2.0 (br, 1H), 3.40–3.55 (m, 1H), 3.78 (s, 3H), 4.10– 4.25 (t, J = 10.0 Hz, 2H), 4.60–4.75 (m, 1H), 6.45 (br, 1H), 8.01 (br, 2H). 13C NMR (100 MHz, DMSO-d6): δ 19.0, 28.0, 50.0, 51.80, 61.0, 80.0, 155.0, 172.0, 176.20, 188.0. MS: Calcd. for C13H23N3O6S: m/z 372.1205 (M + Na+), found: 372.3041. Table 1. List of Nα-protected acyl thioureas synthesized via scheme 1 Entry Acyl thioureas Yield (%) M.p. (°C) [α]D25 in degrees 5a Fmoc-Val-ψ[CONHCSNH]-Ala-OMe 80 184 –55.45 5b Fmoc-Phe-ψ[CONHCSNH]-Ser-OMe 81 170 –50.91 5c Cbz-Val-ψ[CONHCSNH]-Gly-OMe 85 165 –9.09 5d Cbz-Met-ψ[NHCONH]-Phe-OMe 86 180 –16.82 5e Boc-Ala-ψ[CONHCSNH]-Ser-OMe 81 Gum –22.39 5f Boc-Leu-ψ[CONHCSNH]-Ala-OMe 79 Gum –18.43 5g Fmoc-Phe-ψ[CONHCSNH]-Ala-OMe 83 185 –24.56 5h Fmoc-Leu-ψ[CONHCSNH]-Ile-OMe 84 91 –20.62 5i Fmoc-Ala-ψ[CONHCSNH]-Val-OMe: 90 180 –17.27 5j Fmoc-Try-ψ[CONHCSNH]-Ala-OMe 87 205 –12.73 5k Boc-Val-ψ[CONHCSNH]-Ala-OMe 75 Gum –23.18 5l Fmoc-Ile-ψ[CONHCSNH]-Val-OMe 86 159 –9.09 5m Cbz-Thr-ψ[CONHCSNH]-Val-OMe 90 165 –11.10 121Acta Chim. Slov. 2022, 69, 116–124 Mahadevaiah et al.: Combustion Synthesis of Nano Fe2O3 ... (S)-Methyl 2-(3-((S)-2-(tert-Butoxycarbonyl)-4-methyl- pentanoyl)thioureido)propanoate (Boc-Leu-ψ[CONHC SNH]-Ala-OMe) (5f). Yield 79%, Gum. 1H NMR (400 MHz, DMSO-d6): δ 1.0 (d, J = 10.0 Hz, 6H), 1.25 (d, J = 8.0 Hz, 3H), 1.50 (s, 9H), 1.70–1.85 (m, 3H), 3.60 (m, 1H), 3.70 (s, 3H), 4.50 (t, J = 4.0 Hz, 1H), 6.20 (br, 2H), 8.0 br, 1H). 13C NMR (100 MHz, DMSO-d6): δ 17.0, 22.0, 22.7, 28.40, 41.20, 51.0, 52.10, 55.20, 80.0, 155.0, 171.40, 175.50, 188.0. MS: Calcd. for C16H29N3O5S: m/z 398.1726 (M + Na+), found: 398.2230. (S)-Methyl 2-(3-((S)-2-(((9H-Fluoren-9-yl)methoxy) carbonyl)-3-phenylpropanoyl)thioureido)propanoate (Fmoc-Phe-ψ[CONHCSNH]-Ala-OMe) (5g). Yield 83%, m.p. 185 °C. 1H NMR (400 MHz, DMSO-d6): δ 1.30 (d, J = 8.0 Hz, 3H), 2.90 (d, J = 10.0 Hz, 2H), 3.60 (s, 3H), 3.70 (m, 1H), 4.40–4.90 (m, 4H), 6.02 (br, 2H), 7.10–7.90 (m, 13H), 8.0 (br, 1H). 13C NMR (100 MHz, DMSO-d6): δ 18.0, 37.60, 47.10, 52.0, 54.60, 55.0, 67.0, 126.0, 126.60, 128.0, 128.40, 128.70, 129.0, 140.0, 141.0, 143.0, 156.20, 176.60, 171.0, 187.0. MS: Calcd. for C29H29N3O5S: m/z 554.1726 (M + Na+), found: 554.5204. (2S,3S)-Methyl 2-(3-((S)-2-(((9H-Fluoren-9-yl)me- thoxy)c arbonyl)-4-methylpentanoyl)thiourei- do)-3-methylpentanoate (Fmoc-Leu-ψ[CONHCSNH]- Ile-OMe) (5h). Yield 84%, m.p. 91 °C. 1H NMR (400 MHz, DMSO-d6): δ 1.0–1.30 (m, 14H), 1.70–1.85 (m, 3H), 2.50 (m, 1H), 3.40 (d, J = 6.0 Hz, 1H), 3.68 (s, 3H), 4.40–4.70 (m, 4H), 6.20 (br, 2H), 7.22–7.84 (m, 8H), 8.0 (br, 1H). 13C NMR (100 MHz, DMSO-d6): δ 14.69. 22.30, 22.92, 28.17, 28.25, 37.69, 38.68, 47.85, 50.02, 55.81, 59.63, 78.09, 126.45, 126.61, 128.06, 128.18, 128.25, 138.05, 144.30, 145.25, 155.59, 170.79. MS: Calcd. for C29H37N3O5S: m/z 562.2352 [M+Na]+, found: 562.3050. (S)-Methyl 2-(3-((S)-2-(((9H-Fluoren-9-yl)methoxy) carbonyl)propanoyl)thioureido)-3-methylbutanoate (Fmoc-Ala-ψ[CONHCSNH]-Val-OMe) (5i). Yield 90%, m.p. 180 °C. IR (KBr): 3455, 2062, 1640, 1434, 1275, 706 cm–1. 1H NMR (400 MHz, DMSO-d6): δ 1.10–1.20 (d, J = 10.0 Hz, 6H), 1.50–1.57 (d, J = 8.0 Hz, 3H), 2.70–2.94 (m, 1H), 3.40–3.50 (t, J = 6.0 Hz, 1H), 3.75 (s, 3H), 4.48–4.75 (m, 4H), 6.38–6.47 (br, 2H), 7.16–7.50 (m, 8H), 7.98 (br, 1H). 13C NMR (100 MHz, DMSO-d6): δ 20.92, 21.32, 21.69, 46.67, 54.81, 56.16, 64.93, 65.29, 126.03, 126.98, 127.51, 127.55, 128.47, 129.08, 140.66, 143.76, 156.57, 157.26. MS: Calcd. for C25H29N3O5S: m/z 506.1726 [M+Na]+, found: 506.2440. (S)-Methyl 2-(3-((S)-2-(((9H-Fluoren-9-yl)methoxy) carbonyl)-3-(1H-indol-3-yl)propanoyl)thioureido)pro- panoate (Fmoc-Try-ψ[CONHCSNH]-Ala-OMe) (5j). Yield 87%, m.p. 205 °C. 1H NMR (400 MHz, DMSO-d6): δ 1.27–1.34 (d, J = 8.0 Hz, 3H), 2.0 (br, 1H), 3.05–3.12 (d, J = 6.0 Hz, 2H), 3.30–3.59 (m, 1H), 3.80 (s, 3H), 4.40–4.80 (m, 4H), 6.0 (br, 1H), 6.30 (s, 1H), 7.12–7.98 (m, 12H), 8.12 (br, 1H), 8.85 (br, 1H). 13C NMR (100 MHz, DMSO-d6): δ 14.62, 37.61, 47.75, 49.93, 55.73, 59.53, 77.97, 125.97, 126.15, 126.36, 126.53, 127.97, 128.10, 128.17, 128.53, 129.25, 137.94, 138.01, 144.25, 144.46, 145.19, 155.17, 155.48, 156.23, 170.69. MS: Calcd. for C31H30N4O5S: m/z 593.1835 [M+Na]+, found: 593.3030. (S)-Methyl 2-(3-((S)-2-(tert-Butoxycarbonyl)-3-methyl- butanoyl)thioureido)propanoate (Boc-Val-ψ[CONHCS NH]-Ala-OMe) (5k). Yield 75%, Gum. 1H NMR (400 MHz, DMSO-d6): δ 1.0–1.28 (m, 9H), 1.50 (s, 9H), 2.60 (m, 1H), 3.60 (m, 1H), 3.70 (s, 3H), 6.0 (br, 2H), 8.0 (br, 1H). 13C NMR (100 MHz, DMSO-d6): δ 17.0, 17.40, 28.40, 32.0, 52.0, 55.0, 60.0, 80.0, 156.0, 172.0, 177.0, 187.0. MS: Calcd. for C15H27N3O5S: m/z 384.1569 [M+Na]+, found: 384.2060. (S)-Methyl 2-(3-((2S,3S)-2-(((9H-Fluoren-9-yl)methoxy) carbonyl)-3-methylpentanoyl)thioureido)-3-methylbu- tanoate (Fmoc-Ile-ψ[CONHCSNH]-Val-OMe) (5l). Yield 86%, m.p. 159 °C. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 1.20–1.46 (m, 12H), 1.65–1.90 (m, 2H), 2.70–2.92 (m, 2H), 3.33–3.46 (d, J = 6.0 Hz, 1H), 3.60 (s, 3H), 4.60– 4.79 (m, 4H), 6.10–6.21 (br, 2H), 7.17–7.65 (m, 8H), 8.06– 8.18 (br, 1H). 13C NMR (100 MHz, DMSO-d6): δ 10.0, 15.0, 18.20, 24.80, 30.0, 37.10, 47.0, 52.0, 57.80, 62.70, 67.0, 126.60, 128.40, 128.60, 129.0, 141.0, 143.40, 156.0, 171.40, 175.50, 187.0. MS: Calcd. for C28H35N3O5S: m/z 548.2195 [M+Na]+, found: 548.3090. (S)-Methyl 2-(3-((2S,3S)-2-(Benzyloxycarbonyl)-3-hy- droxybutanoyl)thioureido)-3-methylbutanoate (Cbz- Thr-ψ[CONHCSNH]-Val-OMe) (5m). Yield 74%, Gum. 1H NMR (400 MHz, DMSO-d6): δ 1.0 (d, J = 6.0 Hz, 6H), 1.20 (d, J = 8.0 Hz, 3H), 2.0 (s, 2H), 2.75 (m, 1H), 3.40 (d, J = 4.0 Hz, 1H), 3.70 (s, 3H), 4.20–4.60 (m, 2H), 5.40 (s, 2H), 6.0 (br, 2H), 7.20–7.40 (m, 5H). 13C NMR (100 MHz, DMSO-d6): δ 18.0, 19.0, 30.0, 51.4, 60.0, 62.40, 65.0, 67.60, 127.2, 127.9, 129.0, 140.0, 156.10, 172.0, 175.7, 188.0. MS: Calcd. for C19H27N3O6S: m/z 448.1518 [M+Na]+, found: 448.2024. 3. 2. In vitro Anti-bacterial Activity 3. 2. 1. Determination of Zone of Inhibition by Agar Well Diffusion Method Finally, the synthesized acyl thioureas were tested for the antibacterial activity by agar well diffusion meth- od. For this, the synthesized acyl thiourea samples 5a–j were subjected for the antibacterial activities against two Gram positive bacteria: Escherichia coli (MTCC1692) and Staphylococcus aureus (MTCC 3160) employing agar well diffusion method.59,60 The bacterial pathogens were cultured on Mueller–Hinton broth agar for 24 h at 37 °C.61 The inoculums were prepared by allowing bacteria 122 Acta Chim. Slov. 2022, 69, 116–124 Mahadevaiah et al.: Combustion Synthesis of Nano Fe2O3 ... to grow on media containing nutrient broth at 37 °C with permanent stirring at 250 rpm for overnight. After ob- taining 24 h fresh culture, Mueller–Hinton agar plates were prepared, strains of S. aureus, and E. coli were swabbed uniformly on individual plates using sterile cot- ton swabs. Wells of approximately 6 mm diameter were made on MH agar plates using gel puncture. Synthesized samples, approximately 200 mg were dissolved in 2 mL of DMSO. With a concentration of 500 µg/µL were used to measure the activity of the synthesized molecules and streptomycin sulphate (50 mg/2 mL) was used as the standard antibiotic. To each well, a volume of 50 μL of streptomycin sulphate standard (S) and 100 μL respective samples were added to individual plates. The plates were incubated at 37 °C for 24 h and inhibition zones obtained were measured. Antibacterial activity was evaluated in terms of the diameters of growth inhibition zones (mm). If the growth inhibition zones were less than 10 mm in diameter, confluent growth over the plates were scored as the absence of antibacterial activity, zones of 10–15 mm as weak activity, zones of 15–20 mm as moderate-marked activity, and greater than 20 mm as marked activity. Re- sults are summarized and analyzed in Table 2 and Figures 5 and 6. 4. Conclusions We have developed a highly convenient and effective protocol for the synthesis of substituted acyl thiourea de- rivatives from the carboxyl group of Nα-amino acids and organic acids by nano Fe2O3-catalysed coupling of acyl isothiocyanates and amino acid esters at room tempera- ture in very good yields. Some of the synthesized acyl thiourea derivatives showed promising anti-bacterial ac- tivity against two bacterial pathogens, possibly due to the presence of pharmacologically active keto-thiourea group. The presented protocol underlines the potential applica- bility of nano Fe2O3 as inexpensive, user friendly and effi- cient catalyst prepared via solution combustion method using ascorbic acid as a novel reducing agent. This work can be considered as a very good step towards the emerg- ing trend of heterogeneous and environmentally friendly synthesis of acyl thiourea derivatives from Nα-amino ac- ids. Acknowledgements We thank the Principal, Director, CEO of Siddagan- ga Institute of Technology, Tumakuru, Karnataka, for the research facilities. One of the authors (HSL) is thankful to the Vision Group of Science and Technology, Dept. of In- formation Technology, Biotechnology and Science & Technology, Government of Karnataka for providing funds under CISEE programme (VGST-GRD No. 472) to carry out the present research work by means of a spon- sored project. And also thankful to the Dept. of Science and Technology, Govt. of India for the Instrumental facili- ties under Nano mission project. Table 2. Antibacterial activity of acyl thioureas 5a–j against E. coli and S. aureus Treatment Compound (concentration E. coli S. aureus in µg/µL) S 50 14.55 ± 0.88 12.67 ± 0.33 5a 100 14.67 ± 0.67 13.67 ± 0.88 5b 100 16.00 ± 0.58 10.33 ± 0.67 5c 100 15.67 ± 0.88 10.00 ± 1.0 5d 100 16.67 ± 0.88 12.67 ± 0.33 5e 100 12.67 ± 0.33 13.67 ± 0.88 5f 100 13.66 ± 0.88 11.33 ± 0.88 5g 100 10.67 ± 0.88 10.67 ± 1.2 5h 100 14.67 ± 0.67 12.33 ± 0.33 5i 100 13.67 ± 0.88 13.33 ± 1.2 5j 100 17.33 ± 0.33 31.00 ± 1.2 Figure 6. Antibacterial activity of acyl thioureas against S. aureus. Figure 5. Antibacterial activity of acyl thioureas against E. coli 123Acta Chim. Slov. 2022, 69, 116–124 Mahadevaiah et al.: Combustion Synthesis of Nano Fe2O3 ... Conflict of interest The authors declare that they have no conflict of in- terest. 5. References 1. I. Koca, A. Ozgür, E. Muhammet, M. Gümüs, K. A. Cos, B. Kun, Y. Tutar, Eur. J. Med. Chem. 2016, 122, 280–290. DOI:10.1016/j.ejmech.2016.06.032 2. Z. M. Zhong, R. Xing, S. 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Pharmaceutical Sci. 2013, 49, 343–351. DOI:10.1016/j.ejps.2013.04.006 57. K. Uma, H. S. Lalithamba, M. Raghavendra, V. Chandramo- han, C. Anupama, Arkivoc 2016, (iv), 339–351. DOI:10.3998/ark.5550190.p009.605 58. B. L. Shinde, L. A. Dhale, V. S. Suryavanshi, K. S. Lohar, Acta Chim. Slov. 2017, 64, 931–937. 59. B. Secerov, Z. Andri, N. Abazovi, R. Krsmanovi, M. Mitri, A. Montonea, M. D. Dramianin, Acta Chim. Slov. 2008, 55, 486–491. 60. H. S. Lalithamba, M. Raghavendra, K. Uma, K. V. Yatish, D. Mousumi, G. Nagendra, Acta Chim. Slov. 2018, 65, 354–364. DOI:10.17344/acsi.2017.4034 61. N. T. M. Tho, T. N. M. An, M. D. Tri, T. V. M. Sreekanth, J. S. Lee, P. C. Nagajyothi, K. D. Lee, Acta Chim. Slov. 2013, 60, 673–678. Povzetek Opisujemo enostaven in okolju prijazen heterogeni katalitski sistem, temelječ na Fe2O3 nanodelcih, uporaben za sintezo acil tiosečninskih derivatov iz ustreznih in situ pripravljenih acil izotiocianatov in estrov aminokislin. Sinteza poteka v acetonu in daje produkte z dobrimi izkoristki. Strukture pripravljenih acil tiosečnin smo potrdili z 1H NMR, 13C NMR, masno in FTIR analizo. Nanodelce Fe2O3 smo pripravili s pomočjo sežiga raztopine, z uporabo askorbinske kisline kot reducenta ter železovega(III) nitrata kot vira železovih ionov. Pripravljen nanomaterial smo okarakterizirali z XRD, SEM, UV-vidno in FTIR analizami. Pomembno je, da lahko Fe2O3 in ostale nečistoče po sintezi enostavno odstrani- mo. Katalitski sistem je učinkovit in katalizira pretvorbe reaktantov v produkte z dobrimi izkoristki. Za nekatere izmed pripravljenih acil tiosečnin smo določili in vitro antibakterijske aktivnosti proti Staphylococcus aureus in Escherichia coli. Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License 125Acta Chim. Slov. 2022, 69, 125–132 Lobotka et al.: Acetyl Cellulose Film with 18-crown-6 Ether ... DOI: 10.17344/acsi.2021.7092 Scientific paper Acetyl Cellulose Film with 18-crown-6 Ether for Colorimetric Phosgene Detection Martin Lobotka,1,* Vladimír Pitschmann2,3 and Zbyněk Kobliha1 1 University of Defence, NBC defence institute, Víta Nejedlého 691, 682 01 Vyškov 2 Oritest spol. s r.o., Čerčanská 640/30, 150 00 Prague, Czech Republic 3 Czech Technical University in Prague, Faculty of Biomedical Engineering, nám. Sítná 3105, 272 01 Kladno, Czech Republic * Corresponding author: E-mail: lobotka@oritest.cz Received: 12-08-2021 Abstract The use of a cellulose detection film as a carrier for a colorimetric sensor to detect phosgene and allied compounds to be evaluated primarily visually is studied. For the case study, a benzimidazole-rhodamine dye and an acetyl cellulose film were selected. The detection complex was modified using cyclic ether 18-crown-6 to achieve more desirable analytic properties. The chromatic properties of detection film was verified using reflectance colorimetry in the visible light spec- trum. The employed detection agent demonstrated high sensibility to phosgene vapours, but acid gases, acyl chlorides, base organic solvents, and in higher concentrations, even some organophosphorus substances interfered. The detection film application was adjusted to the in-situ preparation of simple detection devices (a spray or a marker) as well as to manufacture detection strips with beforehand excluded polymer film. Keywords: Crown ether, phosgene, polymer film, acetyl cellulose, chromogenic chemosensor 1. Introduction The highly toxic colourless gas phosgene, mostly in mixtures with other toxic gases, was firstly utilised for military purposes during World War I. From the chem- ical point of view, it is a derivate of carbonic acid with two allied compounds: diphosgene (trichloromethyl carbonochloridate) and triphosgene (bis(trichlorome- thyl) carbonate). This highly reactive gas demonstrates distinct toxic properties due to a significant hydrolytic reaction on tissues and is classified as a pulmonary agent. The allied compound diphosgene has risen to military significance. Concerning diphosgene, it is a colourless liquid ensuring easier manipulation.1 The toxic proper- ties of diphosgene correspond with phosgene, the LC50 dose represents 2,000–3,200 mg m-3 (500–800 ppm) at 1 min exposure.1,2 The permissible value of work exposure due to the NIOSH regulations is 0.4 mg m-3 (0,1 ppm) at 8 h shift.1,3 Phosgene is currently utilised when produc- ing agrochemicals, polycarbonates, and pharmaceutical substances; in 2015, the world production amounted to 8.526 million tonnes.4 The colorimetric detection of phosgene/diphosgene is currently conducted using the well-known reaction with benzylpyridine derivates, for example 4-(4-Nitrobenzyl) pyridine: distinct red colouring. The method has been pre- dominantly applied in the form of detection tubes or paper strips also known as continuous detectors in the form of cards.5–10 Furthermore, the reaction of phosgene with Har- rison’s reagent (4-(dimethylamine) benzaldehyde/diphe- nylamine) produces a yellow condensation product.11–13 Newer detection methods use more complex chromoge- nic agents with organic chemosensor structures, or other heterocyclic compounds, which, after the reaction with an analyte, change colours distinctly. To illustrate the case, the reaction between phosgene and substituted BODIPY ox- ime forming orange colouring or with phenylenediamine/ BODIPY unit as fluorophore can be utilised.14,15 Out of other detection agents, the compounds with coumarin, benzothiazole, imidazole, benzimidazole-rhodamine, o-phenylenediamine/pyronine, o-hydroxyaniline, carbox- yimide, and quinazolinone skeletons were employed for colorimetric and fluorometric detection.16–30 126 Acta Chim. Slov. 2022, 69, 125–132 Lobotka et al.: Acetyl Cellulose Film with 18-crown-6 Ether ... The authors, purposefully and on a long-term basis, concentrate on the issues of phosgene and its allied com- pounds detection. The aim of the work was to propose a different carrier of detection agents in the form of a thin polymer film. It is going to be carry several advantages of the solution, such as the abilities to prepare a detector in the place of detection (in situ), to minimise the usage of chemicals and supporting materials, and to allow the con- tinuous monitoring of the environment to check the pres- ence of toxic substances. 2. Experimental 2. 1. Chemicals and Materials To prepare polymer films, acetyl cellulose (Carl Roth GmBH, Germany), cyclic ether 18-crown-6 (Sig- ma-Aldrich, USA), and acetone as a dissolving agent (Merck, Darmstadt, Germany) were employed. Benzim- idazole/rhodamine B dye was employed as a detection agent (The University of Chemistry and Technology, Prague, CZE; the spectral data correspond with the liter- ature). To verify the method, trichloromethyl chlorofor- mate (diphosgene), ammonia, benzoyl chloride, diethyl ether, hydrochloric acid, nitromethane, pyridine, carbon disulphide, (all Sigma-Aldrich, USA), isopropyl meth- ylphosphonofluoridate (sarin, The University of Defence, CZE), and petrol (Cepro, CZE) were employed. To apply polymer films, a refillable marker with a dosing valve and 4 mm tip 211EM (Molotow, Lahr, Ger- many) was utilised. The objective colorimetric measure- ments were conducted using the reflectance spectropho- tometer Ultrascan XE (HunterLab, Reston, USA) with 9.5 mm entrance slit. The micrographs were taken using a scanning electron microscope (SEM). 2. 2.  Preparation of Polymer Solution with Detection Agent The base solution supply of the polymer was pre- pared by dissolving 4 g of acetyl cellulose in 100 ml of anhydrous acetone in a heated ultrasonic bath. 8 mg of rhodamine-benzimidazole dye was dissolved in 5 ml of the base solution to form a pink solution. The transition of the colouring dye to the colourless form was conducted by adding 750 mg of ether 18-crown-6. The solution turned light pink, colourless after the application on the carrier. 2. 3. Phosgene Detection The concentration of diphosgene in the range from 0.1 to 5 mg/m3 in a toluene solution was prepared in the test chamber with the volume of 0.712 m3 using the forced air circulation. The colouring of the detection film depend- ing on the diphosgene vapour concentration and the expo- sure time ranging from 1 to 10 min were observed. Apart from the intended, primary visual evaluation, the instru- mental reflectance measurement was conducted to objec- tively evaluate the changes in film colouring. After each exposure in the test chamber, the new concentration of the analyte was prepared for the following measurement. The chemosensors were prepared by excluding the thin detection film from 50 µl detection polymer solution. The film was subsequently distributed on a PE pad with the diameter of 16 mm. After the distribution, the film was left for drying at the laboratory temperature for the peri- od of 3 min. To evaluate the changes in the colouring of the detection film, the method of reflectance colorimetry in the colour space CIELAB 10°/D65 was employed. The records were interpreted as the dependence of the reflec- tance on the wavelength of the visible spectrum in the in- terval of λ = 380–750 nm. 3. Results and Discussion 3. 1. Method Characteristics To detect phosgene and its allied compounds, the benzimidazole-rhodamine dye was selected. Historically, the phosgene detection was based on its reaction with the nitrogen of the benzimidazole unit releasing the pink form of the dye (Figure 1) that can be evaluated in the UV spec- trum of electromagnetic radiation.19 Figure 1: Benzimidazole-rhodamine dye and the end product after the reaction with phosgene are presented.19 The previous research already applied the agent into the polymer matrix formed by polyethylene oxide, formed using the technology of electrostatic fibre formation, whose fibrous structure was the functional base for the chemosensor to detect phosgene using colorimetric and fluorescent evaluation.19 The simple release of the polymer film from the solution by evaporating the dissolving agent was employed. This simple method can be easily utilised for the in-situ preparation of simple detection means to monitor toxic substances in the atmosphere, to simplify the whole testing procedure, and at the same time, to min- imise financing costs. Three ways were utilised to employ the detection film. Firstly, it was utilised in the form of the solution ap- plicated by the refillable marker. Secondly, the preparation of simple detection strips made from supporting materials 127Acta Chim. Slov. 2022, 69, 125–132 Lobotka et al.: Acetyl Cellulose Film with 18-crown-6 Ether ... (PE strip) and the released detection film on its surface was utilised. Thirdly, the application of the polymer solu- tion using a mechanical spray for larger areas was utilised. 3. 2. Detection Film Characteristics The naturally released films from acetone were unstable and mostly unable to preserve the transparent Figure 2: The non-modified (the upper row) and the modified 15% ether 18-crown-6 acetyl cellulose film (the lower row) are compared. The natural appearance of the polymer film after the evaporation of the dissolving agent [A], the excluded polymer film with the benzimidazole-rhodamine agent [B], and after being exposed to diphosgene vapours [C] are presented. Figure 3: The micrographs of the acetyl cellulose film (the upper row) and the film with the addition of 15% ether 18-crown-6 (the lower row) are compared at various magnifications. 128 Acta Chim. Slov. 2022, 69, 125–132 Lobotka et al.: Acetyl Cellulose Film with 18-crown-6 Ether ... appearance. Besides, the colouring agent was also una- ble to preserve the transparent appearance and after the evaporation of the dissolving agent, it coloured heavily (Figure 2). The further study of the issue aimed at removing these drawbacks by modifying the film using ether 18-crown-6. The cyclic ether improved the structure of the released film and preserved the detection agent in the transparent form (Figure 2). The homogeneity of the distribution of the detection agent inside the polymer film was significantly improved. The growing percentage of ether 18-crown-6 in the polymer decreases its rigidity; with over 20% percent- age of ether 18-crown-6, it formed a gel-like mixture. The 15% value of ether 18-crown-6 mass in the acetone solu- tion of the polymer was selected to reach the compromise between the sufficient film rigidity and the positive impact on the detection. The stabilisation of the colouring agent can be prob- ably assigned to the ability of the cyclic ether to bind a hydrogen cation inside the cyclic molecule in the form of hydronium cation.31–33. After evaluating the impact of the addition of ether 18-crown-6 to the structure of the released acetyl cel- lulose films, the scanning electron microscopy of the samples without the detection agent was conducted. The micrographs demonstrated much higher heterogeneity of the modified films (Figure 3) in comparison with the naturally released ones and this probably contributed to the better accessibility of the analytic agent on their sur- faces. 3. 3. Verifying Methods of Using Detection Films Detection marker Filling the cartridge of the refillable marker by the solution of the detection film allows to produce simple detection devices in the place of detection (in situ) (Fig- ure 4). The advantage of the solution is the sufficient ca- pacity of the cartridge that ensures conducting the tens of tests using a single cartridge; this means an extremely low cost per use. The application is possible on all ma- terials with the good potential of the easy evaluation of the colour change, for example white cotton fibre, paper, plastic material, or ceramics. The 5 ml filling of the de- tection polymer solution suffices for approximately 500 tests. Strip detector The PE strips with the 12 × 80 mm dimensions were marked with the glued label with 5 mm circular openings. 10 µl of the solution of the detection film was applied to the openings using a micropipette. After the evaporation of the dissolving agent, the simple detection device with 5 mm active surface that can be packed into the hermetic packaging (Figure 4) was produced. Detection spray The simple hand spray was the last laboratory-veri- fied application of the detection film. The volatile dissolv- ing agent helps to the quick drying of the film on the base which lasted only a few tens of seconds. The commercially easily accessible hand spray suitable for the application on larger surfaces was utilised (Figure 4). 3. 4. Sensibility, Stability, and Interference Measuring the dependence of diphosgene concen- tration on the colouring intensity of the detection film is depicted in the Figure 5. The absorption maximum of the detection film is in 570 nm wavelength range. The detec- tion limit when visually evaluated is in the case of 10 min exposure on the border of 0.125 mg/m3. LOD was deter- mined on the set of 5 samples tested at the given concen- tration and the exposure time which produced colour perception in an observer. The sensibility of the solution when exposed to phosgene satisfies the requirements of workplace hygiene according to NIOSH. The work verified the stability of the detection films during long-term diphosgene measurement in the atmos- phere. The long-term measurements manifested them- selves in the measurable decrease of the film colouring; nevertheless, the effect was infinitesimal when evaluated visually (Figure 6). The solution is feasible when consid- ering the visual evaluation and/or continuous atmosphere monitoring. Figure 4: The proposed application utilises either the polymer solu- tion for the in-situ release of the detection films (1 – the detection marker, 3 – the detection spray) or the beforehand released film on the suitable carrier (2 – the test strip). The pink colouring was reached by the exposure of the detection devices to diphosgene va- pours. 129Acta Chim. Slov. 2022, 69, 125–132 Lobotka et al.: Acetyl Cellulose Film with 18-crown-6 Ether ... 3.4 Interference Substance Influence on Detection Film The detection agent is sensitive to acidic gases, or the substances which produce acidic products when in the presence of atmospheric humidity; the acidic products protonate the imidazole nitrogen in the molecule.34 The reaction of colouring films on the selected most significant interfering substances was instrumentally measured (Fig- ure 7). Based on the measured data, it is evident that the most significant interfering substance is hydrogen chlo- ride which leads to the highly sensitive reaction with the detection film, as in the case of diphosgene. Benzoyl chlo- ride is a less significant interferent; in the concentration of 2.8 mg/m3 and 2 min exposure time, it does not produce any colour change of the film. Due to the possible need of toxic substance analysis in the military, the nerve agent sarin (GB) was tested as an analyte; however, at the selected 0.5 mg/m3 sarin con- centration and 10 min exposure time, no colouring of the detection film was observed. The colour reaction was ob- Figure 5: The colouring of the detection film at the diphosgene concentration of 2.2 mg/m3 (A) and 4.4 mg/m3 (B) depending on the exposure time is measured. The low concentrations (C) were measured at 10 min exposure time. The colouring examples of the detection film (D) (diphosgene concentration/exposure time) were given at 1) 0.25 mg/m3/5 min; 2) 0.5 mg/m3/5 min; 3) 2.2 mg/m3/2 min; 4) 4.4 mg/m3/2 min. Figure 6: The colouring of the detection film after the diphosgene vapour exposure at the concentration of 1 mg/m3 and 5, 10 min ex- posure is presented. The sample set was placed in uncontaminated environment for 6 h and subsequently exposed to the correspond- ing analyte concentration. 130 Acta Chim. Slov. 2022, 69, 125–132 Lobotka et al.: Acetyl Cellulose Film with 18-crown-6 Ether ... servable at the high concentrations of sarin, over 10 mg/ m3. Contrarily, the colour reaction of the detection system was not observed in aliphatic and aromatic hydrocarbons, chlorinated dissolving agents, base substances, and oth- er dissolving agents (the Table 1). The colour reaction of the diphosgene after the exposure to common dissolving agents and chemicals was examined. The detection film demonstrated high affinity to pyridine when its vapours significantly inhibited the colour reaction even after longer period after the interrupting the exposure. Nitromethane achieved the same, even though a bit weaker result. Am- monia, on the other hand, inhibited the reaction only slightly as it was quickly released from the film. 4. Conclusions The tested solution provides a cheap and simple car- rier of the detection chemicals which represents a possible alternative to the currently commonly used carriers (paper or silica gel) The proposed cellulose carrier, modified with ether 18-crown-6, represents the possibility to prepare simple detection devices in the place of detection (in situ) owing to the thin polymer film with evenly distributed de- tection chemicals on its surface. To verify the abilities of the carrier, the rhodamine-imidazole dye integrated into the polymer matrix was utilised. This simple detection de- vice in the form of a PE card with the applied detection film was exposed to diphosgene vapours. The ascertained LOD when evaluated visually and 10 min exposure time amounted to 0.125 mg/m3 (0.03 ppm). All the considered detection solutions (a marker, a strip sensor, and/or a spray) were functional. The proposed polymer carrier is suitable also for the integration of analytical agents sensitive to other toxic va- Figure 7: The colour reaction of the detection film depending on the exposure time concerning the most significant interfering sub- stances, for example (A) benzoyl chloride at the concentration of 2.8 mg/m3 and (B) hydrogen chloride at the concentration of 5.8 mg/ m3, were measured. Table 1: The overview of the interferences of the proposed detection film, the substances were tested in the form of concentrated gases. Chemicals The reaction of the film to chemicals The reaction of the film to diphosgene after the exposure to chemicals ammonia No reaction Slight decrease in sensibility benzoyl chloride Pink-purple colouring, weaker than – gaseous hydrogen chloride diethyl ether No reaction Detection potential preserved hydrogen chloride Pink-purple colouring, significant – interference car petrol No reaction Detection potential preserved nitromethane No reaction Decrease in sensibility pyridine No reaction Significant decrease in sensibility sarin (GB) 0.5 mg/m3, 10-min exposure – no reaction, Detection potential preserved the reaction noticed over concentrated vapours carbon disulphide No reaction Detection potential preserved 131Acta Chim. Slov. 2022, 69, 125–132 Lobotka et al.: Acetyl Cellulose Film with 18-crown-6 Ether ... pours and gases which will be the topic of the subsequent development of the research. 5. References 1. E. Halámek, Z. Kobliha, V. 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ISBN: 978-0-470-51234-0. 32. J. L. Atwood, S. G. Bott, A. W. Coleman, K. D. Robinson, S. B. Whetstone, C. M. Means, J. Am. Chem. Soc. 1987, 109 (26), 8100–8101. DOI:10.1021/ja00260a033 33. J. L. Atwood, S. G. Bott, C. M. Means, A. W. Coleman, H. Zhang, M. T. May, Inorganic Chemistry, 1990, 29 (3), 467–470. DOI:10.1021/ic00328a025 34. X. Zhongwei, Ch. Mingliang, Ch. Jianming, H. Jiahuai, H. Shoufa, RSC Advances, 2014, 4, 374–378. 132 Acta Chim. Slov. 2022, 69, 125–132 Lobotka et al.: Acetyl Cellulose Film with 18-crown-6 Ether ... Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License Povzetek Proučevali smo uporabo celuloznega detekcijskega filma kot nosilca za kolorimetrični senzor za detekcijo fosgena in sorodnih spojin na podlagi vizualne evalvacije. Za demonstracijo koncepta smo izbrali benzimidazol-rodaminsko bar- vilo in acetilcelulozni film. Detekcijski kompleks smo modificirali z uporabo cikličnega etra 18-krona-6, da smo dosegli bolj ugodne analitične lastnosti. Kromatske lastnosti detekcijskega filma smo preverili z odbojno kolorimetrijo v spektru vidne svetlobe. Uporabljen detekcijski agent je pokazal visoko občutljivost na hlape fosgena, vendar pa so interferirali plini kislin, acil kloridi, bazična organska topila in v višjih koncentracijah celo nekatere organofosforne snovi. Aplikacija detekcijskega filma je bila prilagojena in situ pripravi enostavnih detekcijskih naprav (razpršilo ali marker) ter izdelavi detekcijskih trakov s predhodno izločeno polimerno folijo. 133Acta Chim. Slov. 2022, 69, 133–146 Sun et al.: Modulation of Cerium Carbonate Crystal Growth ... DOI: 10.17344/acsi.2021.7097 Scientific paper Modulation of Cerium Carbonate Crystal Growth by Polyvinylpyrrolidone using Density Functional Theory Deyun Sun,1,2,3 Yanhong Hu,1,2,3,* Mao Tang,1,2,3 Ze Hu,1,2,3 Peng Liu 1,2,3 Zhaogang Liu1,2,3 and Jinxiu Wu1,2,3 1 College of Materials and Metallurgy, Inner Mongolia University of Science and Technology, Baotou 014010, China; 2 Key Laboratory of Rare Earth Hydrometallurgy and Light Rare Earth Application in Inner Mongolia Autonomous Region, Baotou 014010, China; 3 Key Laboratory of Green Extraction and Efficient Utilization of Light Rare Earth Resources, Ministry of Education, Baotou 014010, China * Corresponding author: E-mail: Bthyh@163.com Received: 08-12-2021 Abstract Cerium carbonate crystal morphology is predicted using density functional theory (DFT) simulations in this paper. In the nucleation phase, the ketone group in polyvinylpyrrolidone (PVP) will preferentially bind to Ce3+ to form complexes and provide heterogeneous nucleation sites for the system, prompting the nucleation of cerium carbonate crystals. In the growth stage, due to the adsorption of PVP, the probability of (120) crystal plane appearing in the equilibrium state is the greatest, resulting in the formation of hexagonal flake cerium carbonate crystals with (120) crystal plane as the oblique edge. Experimentally, hexagonal sheet cerium carbonate crystals were successfully prepared using PVP as a template agent. Therefore, DFT can be used to predict the morphology of cerium carbonate crystals, which not only elucidates the growth mechanism of cerium carbonate crystals, but also greatly reduces the experimental cost. Keywords: Cerium carbonate hydrate; Polyvinylpyrrolidone (PVP); Self-assembly template method; Density functional theory; Morphology control 1. Introduction Rare earth ore resources in Bayan Obo, Baotou rank first in the world, with light rare earths accounting for more than 98%, while 50% of them are cerium resources. Due to its special electronic structure, cerium oxide has supe- rior oxygen storage and redox ability. It is widely used in many fields such as electronic ceramics, polishing powder, catalyst, sensors, fuel cells, UV absorption, etc.1–11 It has become an indispensable material for high-tech industries and cutting-edge innovations. The cerium oxide with differ- ent morphologies has obvious functional differences, which will have a huge impact on the performance of products. Therefore, the development of industrial preparation tech- nology of rare earth compounds with special morphology is very important. So far, cerium oxide with special mor- phology has been prepared by physical, chemical and many other ways at home and abroad.12–14 Using polymers as self-assembly templates to regulate material properties pro- vides a new idea for the artificial synthesis of crystal materi- als and bio-intelligent materials with special functions.15–18 The self-assembly-template method is favored by more and more experts and scholars because of its simplicity, low cost and strong controllability. Sodium polystyrene sulfonate (PSS), nonylphenol polyoxyethylene ether (NPEO), polyal- lylammonium chloride (PAH), polyvinylpyrrolidone (PVP) and ethylenediaminetetraacetic acid disodium (EDTA-2Na) were used as templates in our research group. Spherical ceri- um oxide for catalytic materials, shuttle-shaped cerium ox- ide for improving glass properties, hexagonal flake cerium oxide for solid oxide fuel cells, and flower-like cerium oxide for UV absorption were prepared by self-assembly tem- plate method.19–22 Lin Wang et al. synthesized polyaniline (PANI) with nanoscale spherical or string-like morpholo- gy using PS-b-P2VP as a templating agent for modulation by template-self-assembly method, and the average diam- eter of each sample was found to be less than 200 nm and showed a tendency to decrease with increasing pH.23 Using chitosan as a new carbon and nitrogen source precursor and triblock amphiphilic copolymer (F127) as a soft template, nitrogen-doped mesoporous carbon nanoparticles (NMCs) with pore size distribution between 3.05 and 6.09 nm were 134 Acta Chim. Slov. 2022, 69, 133–146 Sun et al.: Modulation of Cerium Carbonate Crystal Growth ... successfully prepared by Xianshu Wang et al. The analysis revealed that the nitrogen-doped mesoporous carbon ma- terials have well-developed pores, and the nanoparticles have a spherical shape with an average diameter of about 300–400 nm and a worm-like mesoporous structure.24 With the rapid progress of science and technology and the development of interdisciplinary, the method of com- puter theoretical calculation to synthesize new materials has attracted more and more attention of scientists. Ab initio quantum chemistry method can be used to study the nucle- ation mechanism and growth mode of materials from the atomic or molecular scale, and disclose the mechanism that cannot be explained in experiments. The morphology con- trol mechanism of cerium carbonate crystals, the precursor of cerium oxide, was studied in our previous research, by molecular dynamics method, and the interaction between the template agent and the crystal surface was simulated. The relationship between the template agent and the crys- talline surface of cerium carbonate crystals was revealed from the energy perspective, and the growth mechanism of cerium carbonate crystals was further disclosed.25–26 Ning Liu et al. successfully predicted the crystal habit of FOX-7 to be spindle-shaped under vacuum conditions with the help of molecular dynamics method. The crystalline habit of FOX-7 in H2O/DMF solution conditions varied signifi- cantly with temperature conditions, and the crystalline hab- it in different ratios of solvents was blocky when the tem- perature was 298 K.27 Balbuena Cristian et al. studied the synthesis of silver nanoparticles with polyvinylpyrrolidone as capping agent, the nucleation of atomic clusters and the subsequent growth of nanoparticles by molecular dynam- ics, finding that the formation of crystals follows Ostwald ‘ s law of phase transition. As the process progresses, a series of ordered structures appear inside the particle: icosahedral, body-centered cubic and face-centered cubic, and finally a block-silver equilibrium configuration.28 This paper uses a combination method of computa- tional simulation and experimental research. The interac- tion mechanism of PVP with Ce3+ and H2O in aqueous solution before precipitation and the adsorption of PVP on the main crystal plane after precipitation were calcu- lated and simulated. The process of morphological change of cerium carbonate crystals under the regulation of PVP is explained from the atomic point of view by analyzing the electronic structure and energy change in different cas- es.29–33 And the morphology of cerium carbonate crystals was predicted. Then, the cerium carbonate crystals were successfully prepared by the self-assembly-template meth- od using CeCl3 as raw material, NH4HCO3 as precipitant and PVP as template agent. The phase structure, morphol- ogy and dimensions of cerium carbonate crystals were characterized by Scanning electron microscope (SEM), Transmission electron microscope (TEM) and Diffraction of X-rays (XRD). The experimental results are compared and analyzed with the simulated prediction results to veri- fy the correctness of the prediction results. 2. Material and Methods 2. 1. Experimental Materials The cerium chloride (CeCl3) used in this experiment was prepared by dissolving and de-hybridizing industrial ce- rium carbonate provided by Baogang Rare Earth High-Tech Company, Inner Mongolia, China, and hydrochloric acid (analytically pure) provided by Tianjin Damao Chemical Reagent Factory, Tianjin, China. NH4HCO3 and anhydrous ethanol were produced by Tianjin Beilian Fine Chemicals Development Co. Ltd., Tianjin, China, and polyvinylpyrro- lidone was produced by Shanghai Maclean Biochemical Technology Co. The selected chemicals were analytically pure, which could be used without further purification. The water used in all experiments was deionized water. 2. 2. Preparation Method of Ce2(CO3)3 Crystals At room temperature, deionized water was added to a certain concentration of CeCl3 solution to dilute to 0.05 mol/L, and then PVP was added to prepare the mixed solu- tion of PVP and CeCl3. After stirring at a constant speed for 15 min, 0.05 mol/L NH4HCO3 solution was dropped into the mixed solution of CeCl3 and PVP at a certain speed by a peristaltic pump for 1 h. After dripping, the solution was stirred with a stirring paddle at constant speed for 15 minutes before aging. The precipitate obtained by aging at room temperature was filtered, washed and dried to obtain Ce2(CO3)3 crystals. The prepared Ce2(CO3)3 crystals were characterized by SEM, TEM and XRD. 2. 3. Characterization XRD was performed using Bruker D8 Advance X-ray diffractometer with CuKa radiation(graphite mon- ochromator). The crystal structure was determined using CuKa radiation(40kV, 40mA), 4° step and the geometric scanning Bragg–Brentano(θ–θ) and the angle range from 5–60°(2θ)were performed. SEM images were taken with Quanta 400 produced by Holland JEI Company, fitted with a field emission source, and working at 15 kV. All samples were mounted on copper stubs and sputter-coated with gold prior to examination. TEM images were taken with a JEM-2100 transmission electron microscope produced by JEI Company, Netherlands, with electron diffraction selected to characterize the samples, observe the morphol- ogy and analyze the crystal structure. 2. 4. Computing Method In this paper, the Vienna Ab Initio simulation soft- ware package34–37 was adopted to perform DFT calculation using GGA (Generalized Gradient Approximation) meth- od.38–41 The nucleation and growth of cerium carbonate crystals before and after precipitation were studied using 135Acta Chim. Slov. 2022, 69, 133–146 Sun et al.: Modulation of Cerium Carbonate Crystal Growth ... the LOBSTER software package involving chemical bond- ing analysis.42–43 Since Ce is in the +3 valence ionic state in cerium carbonate crystals, making only one electron in the 4f orbital. And the 4f orbitals are usually significantly low- er in energy than the 5d orbitals and are accompanied by an extremely contracted radial distribution of the orbitals, exhibiting a hemi-nucleation-like property and therefore not involved in bonding.44 For the sake of brevity, the den- sity of states (DOS) of the PBEsol+U pseudopotential con- sidering the f-orbital electrons, DOS that treats the f-orbit- ing electrons as a simplified pseudopotential of the inner layer electrons, and fractional density of states (PDOS) of each element were calculated using the PBEsol function, as shown in Figure 1. Without considering the peak inten- sity, it is found that in the electronic structures calculated by these two methods, there is not much deviation in the positions of the peaks produced by the total and partial density of states of C, H and O, except Ce. The PBEsol+U pseudopotential is compared with the simplified pseudo- potential and it is found that the spin-up α and spin-down β orbitals are not equal in energy in the 6s and 5p orbitals of Ce. The spin-up α orbitals near the Fermi energy level have DOS for f and d orbitals but not on the β orbitals and exhibit 100% spin polarization for the f orbitals. This is be- cause Ce is not pure +3 valence, and the electrons in its 4f orbitals are partially out of domain, so there is a mixed 4f and 5d feature on the PDOS, and the f orbitals have a cor- responding effect on the 6s and 5p orbitals making their α and β orbitals unequal in energy. In this way, although the mixed f-d orbitals are near the Fermi energy level, they do not interact with other elements in the cell because the f orbitals are occupied by only one electron and have low energy. Therefore, the density of states with little differ- ence is reflected in the total DOS diagram. In view of the above situation, considering the calculation efficiency, the f-orbit electrons of Ce are selected as the simplified pseu- dopotential of the inner electrons in the calculation meth- od, while PBEsol functional is selected as the functional file.44–45 Other relevant calculation parameters are as fol- lows: the plane wave truncation energy ENCUT is 350 eV, and the point K in Brillouin zone is divided by Gamma point with grid size of 4 × 3 × 2. The pseudopotential file Ce selects the simplified pseudopotential of + 3 valence, and the pseudopotential of other elements such as H, O, Cl, N, C, selects the PAW pseudopotential of + 1, –2, –1, + 5, + 4 valence. Until the energy difference between the two iteration steps is stabilized below 10–7 eV and the force is stabilized less than –0.05 eV/Å. Considering the spin polarization of the whole system, the Ce outer electron in the simplified pseudopotential is 6s25p6, and there is no solitary electron. Therefore, the system can be represented by a single molecular orbital Ψi, which can be regarded as a closed-shell system. The whole calculation process is op- timized with interatomic static force at 0 K. The optimized structure is a relatively stable ground state structure with high similarity to the experimental value. Figure 1. DOS and PDOS plots calculated by PBEsol functional (a) DOS and PDOS plots calculated by PBEsol+U pseudopotential (b) DOS and PDOS plots calculated by PBEsol simplified pseudopoten- tial (c) PDOS plots calculated by PBEsol+U for Ce For the analysis of chemical bonds, we used the LOBSTER package to calculate the COHP (Crystal Orbital Hamiltonian) as well as the ICOHP (Integral of COHP) 136 Acta Chim. Slov. 2022, 69, 133–146 Sun et al.: Modulation of Cerium Carbonate Crystal Growth ... mechanism of polymer PVP monomer with other elements in the system. The mechanism of PVP modulation of ceri- um carbonate crystals after the addition of precipitant was simulated, and the cerium carbonate crystal structure was derived from the ICSD (Inorganic Crystal Database) crys- tal library as shown in Figure 2. Three crystal planes (010), (001) and (120), which are easy to be exposed were select- ed. According to the atomic ratios of the cerium carbonate crystal, the crystal plane layers with thickness of 9.3546 Å, 9.037 Å and 7.989 Å were cut respectively. These atomic layers were put at the bottom to construct a slab configu- ration with a top vacuum layer thickness of 13 Å. Accord- ing to different thickness, the atoms at the bottom of each crystal plane are fixed at a ratio of 60%, and the rest are released. (001) crystal plane is fixed from the bottom with 0–9.4 Å thickness atoms; (010) crystal plane is fixed from the bottom with 0–5.4 Å thickness atoms; (120) crystal plane is fixed from the bottom with 0–4.8 Å thickness at- oms. The entire slab configuration is optimized according to the above convergence criteria and meets the relevant convergence requirements. Figure 2. Ce2(CO3)3 crystal structure diagram, in which the green ball represents the Ce element, and each Ce element is surrounded by 4 carbonate ions and 4 water molecules In the AIMD (Ab Initio Molecular Dynamics) simu- lation,49–51 using “Calculation” function in “Material Stu- dio” software, scaling based on actual experimental density in a certain proportion, the slab model of mixed aqueous solution system of Ce3+, Ce32– nd polymer PVP monomer was constructed. According to the density of 1 g/cm3, 8 Ce3+, 12 Ce32–, 20 H2O and 2 PVP polymer monomers were added to make them randomly distributed in the slab model. The nucleation and growth of Ce2(CO3)3 crystals in solution were analyzed by molecular dynamics simula- tions, and the set of uniformly distributed Ce3+ Ce32– and H20 in the slab model was used as the initial configuration. The NVT ensemble at 300 K is selected, and these values method. COHP can obtain bonding information from the calculation results of energy band structure; the bond- ing, non-bonding and anti-bonding interactions between paired atoms in the material can be determined. COHP, which is the Hamiltonian matrix multiplied by the corre- sponding density of states matrix, is calculated as shown in equation (1), (1) where fj represents the occupation number, εj represents the energy band energy, R represents the atom, L repre- sents the atomic orbital, j represents the energy band (mo- lecular orbital), HRL,RʹLʹ represents the Hamiltonian matrix element, and NRL,RʹLʹ (ε) represents the DOS (density of states) matrix. Bonding contribution reduces the energy of the system, and COHP is negative; the inverse bond con- tribution increases the system energy, and COHP is pos- itive; the non-key contribution is represented by the zero value of COHP. In practical applications, positive, negative and zero values of-COHP are commonly used to represent bond, anti-bond and non-bond interactions. The integral over the entire occupied orbital COHP is usually denot- ed as ICOHP, which allows a quantitative analysis of the bonding strength between pairs of atoms. It is defined by equation (2): (2) Analysis of the absolute value of ICOHP alone has no meaning, only comparative analysis of its relative value can reflect the significance of ICOHP. In comparison, the smaller the value of ICOHP, the stronger the stability of bonding and vice versa.43,46–47 2. 5. Modeling During the static calculation, the system before pre- cipitation was an aqueous solution system of CeCl3 and polymer PVP. Scaling to a scale based on actual experi- mental density with “Calculation” in “Material Studio” software,48 the slab model of the aqueous solution system mixed with CeCl3 and polymer PVP was constructed; ac- cording to the density of 1 g/cm3, 2 Ce, 6 Cl, 20 H2O, and 3 PVP molecular monomers were added to make them ran- domly distributed in the slab model. Following the above convergence criteria, we optimized the whole slab model, calculated its COHP, ICOHP, and studied the interaction 137Acta Chim. Slov. 2022, 69, 133–146 Sun et al.: Modulation of Cerium Carbonate Crystal Growth ... represent the synthesis conditions in the experiment. The total simulation time for the equilibrium motion was 1500 ps with a time step of 1 fs, during which data were collect- ed every 500 ps for subsequent analysis. In the optimization of PVP molecules, considering that the use of DFT calculations for macromolecular poly- mers will occupy too many resources, while the electronic structure analysis of the crystal surface only needs to con- sider the interaction between a small part of the crystal and the polymer monomer atoms, and the PVP polymer monomer has similar properties to the PVP polymer, only the PVP polymer monomer (Figure 3) was taken for struc- tural optimization, and then its adsorption relationship with the crystal surface and subsequent electronic struc- ture analysis were investigated. Figure 3. Schematic diagram of PVP monomer molecule 3. Results and Discussion 3. 1. Mechanism Study 3. 1. 1. The Interaction Mechanism of PVP in the System Before Precipitation In the constructed slab model, the results before and after optimization are shown in Figure 4. The two Ce3+ in the constructed slab model are #7_Ce and #8_Ce, respec- tively, and it is found that the PVP, H2O and Cl– in the sys- tem are gradually aggregated around Ce3+, in which C=O in PVP, O in H2O, and Cl– will interact around the center Ce3+ with a tendency to form bonds, while H atoms in H2O will form hydrogen bonds with C=O in PVP. As the atoms move to the position where the force is the least, the atoms in the entire system will aggregate toward the Ce3+ posi- tion to form the corresponding complex. The ICOHP and bond lengths between the major bond-forming atoms in the system are shown in Table 1. The analysis revealed that although the bond length of the chemical bond formed be- tween PVP-Ce3+was the longest, its ICOHP value was the smallest. It is because of the large atomic radius and atomic mass of Ce3+, and its unique electronic structure as well as chemical properties. Thus, although the bond length be- tween PVP-Ce3+ is the longest, the stability is stronger than that between PVP-H2O and H2O-H2O to form chemical bonds. This shows that the bonding effect between Ce3+ and PVP is the greatest, so when no precipitation agent is added, the PVP molecules in the system will preferentially complex with Ce3+ to form a more stable complex. Table 1. The bond length and ICOHP value between each atom af- ter the optimization before precipitation is completed atomNU atomNU bond length (Å) -ICOHP(eV) O in PVP Ce3+ 2.38 –3.57 O in PVP H2O 1.65 –1.17 H2O H2O 1.86 –0.90 3. 1. 2. Analysis of Crystal Planes Easily Exposed Without Polymer PVP According to the research results of Li Erxiao and others,26 crystal planes easy to display in PVP adsorp- tion cerium carbonate crystal were selected, and the (001), (010), (120) crystal planes before and after the Figure 4. Optimization diagram of each atom in the system before precipitation 138 Acta Chim. Slov. 2022, 69, 133–146 Sun et al.: Modulation of Cerium Carbonate Crystal Growth ... Table 2. Relative crystal plane parameters of crystal planes easily exposed when PVP is not added planes Erel Natoms Esurf Ebulk A σ (meV) (eV) (eV) (Å2) (J/m2) 001 2.19 152 –766.41 –6.11 77.23 17.1 010 3.94 152 –748.27 –6.11 133.29 11.1 120 1.36 304 –1514.18 –6.11 301.35 9.48 3. 1. 3. Analysis of Crystal Planes Easily Exposed after Adding Polymer PVP Figure 6 shows the adsorption structure diagram of the PVP monomer on the (001), (010), (120) crystal planes of cerium carbonate crystals after calculation by DFT. The length of the Ce-O bond formed by the O contained in the ketone group in the PVP monomer and the Ce3+ of the (010) crystal plane is the smallest, 2.37 Å, while the Ce-O bond length formed by O and Ce3+ of (120) crystal plane is the longest, 2.42Å. The Ce-O bonds after adsorption is shorter than the Ce-O bonds in the cerium carbonate crys- tal. From the analysis of the C-O bonds length inside the PVP monomer, it is found that, except for the (001) crystal plane adsorption which causes the C-O bonds bond length to remain unchanged, the (010) and (120) crystal plane adsorption will cause the C-O bonds bond length to be elongated. From the analysis of the Ce-O-C bonds angle Figure 5. (a) cerium carbonate crystal (001) crystal plane before and after optimization, (b) cerium carbonate crystal (010) crystal plane before and after optimization, (c) cerium carbonate crystal (120) crystal plane before and after optimization optimization of each crystal plane cut out are shown in Figure 5. It is found that after the optimization of these three crystal planes, the reconstruction of the crystal planes has occurred, and the bonding distance between the surface layer atoms and the bottom layer atoms has slightly increased. This is because the top of the con- structed surface is in a vacuum layer. The surface atoms are only subject to the interaction between themselves and the bottom atoms, resulting in different forces from the internal atoms. The formula for calculating the surface energy of crystal plane is shown in Formula 1,52 where σ is the surface energy of the crystal; Erel is the relaxation energy, indicates the energy released when the crystal plane is optimized to a stable state; Esurf is the energy of the slab configuration after optimization; Natoms is the number of atoms in the slab configuration; Ebulk is the energy of a single atom in the bluck structure and represents the ratio of the ener- gy of the unit cell to the number of atoms in the unit cell after optimization. The calculated results of (001), (010) and (120) crystal planes are shown in Table 2, indicating that the surface energy of the (120) crystal plane was the lowest, 9.48 J/m2, and its growth rate was the slowest; the (001) crystal plane had the highest energy, 17.1 J/m2, and its growth rate was the fastest. The order of growth speed between crystal planes is (001) > (010) > (120). (3) 139Acta Chim. Slov. 2022, 69, 133–146 Sun et al.: Modulation of Cerium Carbonate Crystal Growth ... formed by the adsorption of Ce3+ from the PVP monomer ketone group, it is found that the bond angle formed by the adsorption of the (010) crystal plane is the smallest, 155.25°, and the bond angle formed by the adsorption of the (120) crystal plane is the largest, 169.76°. From the analysis of the bond angles of O-C-N and O-C-C bonds formed inside PVP monomer after adsorption, it is found that the bond angles of O-C-N bonds and O-C-C bonds after adsorption are smaller than the bond angles before adsorption. It is because the Ce-O bond formed by the ke- tone group and Ce3+ in PVP monomer after adsorption has a certain interaction with C and N attached to O in- side PVP monomer, which makes the bond angle smaller in general. The adsorption energy of the molecules adsorbed on the crystal surface calculation is shown in formu- la 2,52 where Ebind is the adsorption energy of PVP ad- sorbs the cerium carbonate crystal face, Etot is the total energy of the system after PVP adsorbs the cerium car- bonate crystal face, and Esolv is the single point energy of the PVP monomer. Esurf is the total energy of the ceri- um carbonate crystal face system without adsorption of Figure 6. (a) The best adsorption structure of PVP monomer on the surface of stoichiometric cerium carbonate (001), showing two side views in the plane model, (b) the best PVP monomer on the surface of stoichiometric cerium carbonate (010) Adsorption structure, showing two side views in the plane model, (c) the best adsorption structure of PVP monomer on the surface of stoichiometric cerium carbonate (120), showing two side views in the plane model 140 Acta Chim. Slov. 2022, 69, 133–146 Sun et al.: Modulation of Cerium Carbonate Crystal Growth ... ing the ICOHP value after PVP adsorption on each crys- tal plane, the ICOHP value of (120) crystal plane is the smallest, which is –3.49eV, indicating that the stability of PVP monomer and (120) crystal plane adsorption is the strongest. Figure 7. (a) 10 ligand structure diagram; (b) 8 ligand structure di- agram Figure 8. (a) PVP monomer adsorption (001) crystal plane COHP map, (b) PVP monomer adsorption (010) crystal plane COHP map, (c) PVP monomer adsorption (120) crystal plane COHP map 3. 1. 5. AIMD Simulation of Growth Process of Cerium Carbonate Crystals Regulated by PVP In this section, an ab initio molecular dynamics (AIMD) method is used to simulate the process of PVP regulating the growth of cerium carbonate crystals at the atomic scale. Figure 9 shows sequential snapshots of the complex formed by Ce32– and PVP and Ce3+ from the ini- tial state to 1500ps. In the initial state, the atoms are uni- formly distributed in the system. With the passage of sim- ulation time, Ce32– gradually shifts to the heterogeneous nucleation site formed between PVP and Ce3+. When the simulation time reaches 1500ps, it can be seen that growth of cerium carbonate crystals along the PVP adsorption di- rection is inhibited, leading to the growth of cerium car- bonate crystals in other directions. PVP monomer. The crystal planes calculated according to Formula 2 are shown in Table 3. The adsorption en- ergy of each crystal plane is analyzed. When the adsorp- tion energy is positive, additional absorption energy is required for the occurrence of adsorption, and when the adsorption energy is negative, the energy is released. The definition of adsorption indicates that the adsorption energy of whichever crystal plane and PVP monomer is negative. It shows that the PVP monomer needs to re- lease energy after adsorbing (001), (010), (120) crystal planes. Comparing the size of adsorption energy, it is found that the adsorption energy of (120) crystal plane is the largest. The adsorption energy difference between (001) and (010) crystal planes is only about 2eV, indicat- ing that PVP molecules are more likely to adsorb (120) crystal planes during the growth of cerium carbonate crystal, and inhibit the growth of crystal planes, so that they can finally be exposed. (4) Table 3. After adding PVP, PVP monomer and easy to show surface adsorption related energy parameter table planes Etot(eV) Esolv(eV) Esurf(eV) Ebind(eV) 001 –1023.43 –110.31 –768.60 –144.51 010 –1008.87 –110.31 –752.21 –146.34 120 –1930.42 –110.31 –1527.74 –292.37 3. 1. 4. Electronic Structure Analysis of the Growth Mechanism of Cerium Carbonate Crystals Regulated by PVP In order to further study the interaction mechanism between PVP and cerium carbonate crystal surface, the COHP value of PVP monomer adsorption on different crystal faces was calculated, and its electronic structure was analyzed. When cerium ions are exposed on the crystal surface, the central cerium ion loses two water molecular ligands along the +b axis, which reduces the original 10-li- gand structure (Figure 7a) to an 8-ligand structure (Figure 7b), and coordination unsaturation occurs, therefore the cerium ions have a greater tendency to be adsorbed by the polar element O in the system. The COHP of each crystal plane adsorbed by PVP is shown in Figure 8, and the COHP value of each crystal plane adsorbed by PVP monomer is analyzed. It is found that the three crystal planes (001), (010), and (120) have basically the same bonding conditions after adsorption of the PVP monomer, only when the (100) crystal surface is adsorbed, a negative peak of -COHP value appears near the fermi level, which indicates that there is an unstable component at this position. Because the peak is small, it almost has no effect on the bonding stability. Compar- 141Acta Chim. Slov. 2022, 69, 133–146 Sun et al.: Modulation of Cerium Carbonate Crystal Growth ... 3. 2. Simulation of PVP Controlling Cerium Carbonate Crystal Growth Process Figure 10 is a simulation process diagram of using polymer PVP liquid phase precipitation method to con- trol the morphology of cerium carbonate crystals, which is mainly divided into the following three stages: 1. In the first stage, CeCl3, PVP and H2O are added to the slab configuration and optimized to a stable state. The ketone groups in the system and PVP will preferentially form complexes, providing heterogeneous nucleation sites for subsequent crystal crystallization. 2. In the second stage, with the addition of precip- itant, the complex formed by PVP and Ce3+ is combined to produce heterogeneous nucleation. With the continu- ous addition of the precipitating agent, Ce32– combines at the heterogeneous nucleation point where PVP molecules Figure 9. Snapshot of AIMD simulation of PVP-controlled cerium carbonate crystal growth process at 300K Figure 10. Process diagram of PVP regulating cerium carbonate crystal growth 142 Acta Chim. Slov. 2022, 69, 133–146 Sun et al.: Modulation of Cerium Carbonate Crystal Growth ... are adsorbed and gradually grows into cerium carbonate crystals. In the process of crystal growth, according to the symmetry of cerium carbonate crystal PBNB, the growth of (010) crystal plane along the C axis and + B axis is inhib- ited, (001) crystal plane along the C axis is inhibited, (120) crystal plane along the axis (210) and C axis is inhibited. Moreover, the (120) crystal plane has the strongest PVP adsorption ability and the slowest growth rate, making the (120) crystal plane most likely to appear in the system. 3. In the third stage, after crystal gradually fills the whole solution system, each crystal face fragment will combine with each other. According to the classical crystal growth theory,53 the crystal face with a faster growth rate will disappear, while the crystal face with a slower growth rate will eventually re- main. For the PVP molecules are adsorbed on the (120) crys- tal plane and hinder the (120) crystal plane, the growth rate of (120) crystal plane is the slowest compared with other crys- tal planes such as (001) and (010) crystal planes, so the (120) crystal plane has a greater probability of being retained, and each crystal face finally grows around the (120) crystal face into a hexagonal plate-shaped cerium carbonate crystal. 3. 3. Experimental Verification Analysis 3. 3. 1. SEM Analysis of Products in Different Growth Stages of Cerium Carbonate Crystals Figure 11 shows the SEM images of the cerium car- bonate crystal morphology prepared using the liquid phase precipitation method, with CeCl3 as the Ce source, PVP as the template, NH4HCO3 as the precipitant, when the Ce3+ concentration is 0.03M, the pH value of the initial solution is 2, and the R value (The ratio of Ce32– to Ce3+) is about 2:1. Figure 11 (a) is the SEM image obtained when the pre- cipitating agent (NH4HCO3) is added dropwise for 10 min- utes. At this time, the cerium carbonate crystals are slightly rounded at both ends, slender and fusiform, without obvi- ous edges and corners. This is because at early reaction stage cerium carbonate crystal grows into an amorphous state, and the crystal lattice is not perfect yet. Figure 11 (b) is the SEM image obtained when the precipitating agent (NH4H- CO3) is dropped for 30 minutes. The cerium carbonate crys- tals have been transformed from the original fusiform shape to the angular, narrow and long hexagonal flake. With the extension of the precipitant dropping time, the morphology of the cerium carbonate crystals remained in the shape of hexagonal flake, and the length of each side changed signif- icantly. The length of the sides on both sides of the tip part gradually became longer, and the length of the length direc- tion gradually became shorter. It indicates that the growth process of cerium carbonate crystals is regulated by PVP. At the beginning of the reaction, because the reaction speed is too fast, PVP has not yet played a regulatory role. Ceri- um carbonate crystals rapidly nucleate and grow into a long and narrow spindle shape. As the reaction progresses, PVP is selectively adsorbed on the crystal surface of cerium car- bonate, which makes Cerium carbonate crystals eventually grow into hexagonal flake. 3. 3. 2. Analysis of Crystal Morphology Figure 12 shows the transmission electron microsco- py (TEM) of the hexagonal flake cerium carbonate crystal. Figure 11. SEM images of different reaction stages of cerium car- bonate crystals: (a) 10 min, (b) 30 min, (c) 50 min 143Acta Chim. Slov. 2022, 69, 133–146 Sun et al.: Modulation of Cerium Carbonate Crystal Growth ... From the figure, it can be measured that the angles formed by each plane of the hexagonal flake cerium carbonate crystal are about 90° and 135°. Since the cerium carbonate crystal belongs to the orthorhombic system, formula 3 can be used to calculate the angle of each crystal plane, where cos φ is the cosine of the angle between the two crystal planes, (h1 k1 l1), (h2 k2 l2), is the crystal plane index of two crystal planes. The unit cell parameters obtained from PDF card (#38-0377) are: a = 9.482 Å, b = 16.938 Å, c = 8.965 Å. The cosine values of the angles between the crystal planes are listed in Table 4 (Supplementary materials). (5) Figure 12. Transmission electron microscope image of hexagonal flake cerium carbonate crystal The diffraction pattern of hexagonal flake cerium carbonate crystal is shown in Figure 13. According to the measurement of the ruler in the diffraction pattern from near to far, the distance R between the surrounding spots and the central spot is calculated. According to d=1/R, the crystal plane spacing d of each spot around the center dif- fraction spot is calculated, and the PDF card (#38-0377) of the cerium carbonate crystal is found to preliminarily determine the crystal plane represented by each spot. Ac- cording to the angles of crystal planes measured in Figure 12, it is determined that the sides of the hexagonal flake ce- rium carbonate crystal are (002), (040), and (240) respec- tively by looking up Table 4 (Supplementary materials). Figure 14 shows XRD images of cerium carbonate crystals regulated by PVP. The four strongest peaks are (020), (040), (060), and (200) crystal planes. These four crystal planes belong to the {100} crystal plane family. Because (240) crystal plane is inclined plane of hexagonal flake crystal, the intensity of (240) crystal plane peak is rel- atively weak in XRD. Therefore, the hexagonal flake ceri- um carbonate crystal with (240) crystal plane as inclined plane and {100} crystal plane group as top and side can be seen in the SEM. 4. Conclusions The morphology of cerium carbonate crystal con- trolled by PVP was simulated by computer. When adding PVP without precipitating agent, the ketone group in PVP would preferentially complex with Ce3+. After adding pre- cipitant, heterogeneous nucleation points of cerium car- bonate crystal would be formed around the complex, and the cerium carbonate crystal would grow gradually around the nucleation points. By calculating the interaction rela- tionship between exposed Ce3+ and polymer PVP on three crystal faces of cerium carbonate (120), (010) and (001), it is found that the absolute value of adsorption energy of (120) crystal surface is the largest, and the gap of adsorp- tion energy of (010) and (001) is only about 2eV, which indicates that due to the adsorption of PVP in the growth process of cerium carbonate crystal, the growth resistance of the (120) plane is much greater than that of the (010) and (001) plane. In the equilibrium state, the probabili- ty of forming (120) crystal plane is the greatest, thus the hexagonal plate-like cerium carbonate crystal structure Figure 13. Diffraction pattern of hexagonal flake cerium carbonate crystal Figure 14. XRD pattern of hexagonal flake cerium carbonate crystal 144 Acta Chim. Slov. 2022, 69, 133–146 Sun et al.: Modulation of Cerium Carbonate Crystal Growth ... that grows around the (120) crystal plane will eventual- ly appear. The experimentally prepared cerium carbonate crystals were analyzed by SEM, TEM, and XRD. The mor- phology of the prepared cerium carbonate crystals is a hexagonal sheet-like cerium carbonate crystal with (240) crystallographic planes as bevels and {100} crystallograph- ic families as top and sides. It is found that the morpholo- gy of cerium carbonate crystal prepared in the experiment was similar to that of cerium carbonate crystal simulated by computer. Therefore, density functional theory can be used to predict the morphology of cerium carbonate crys- tals, which not only elucidates the growth mechanism of cerium carbonate crystals, but also greatly reduces the ex- perimental cost. Acknowledgments The financial support from National Science Foun- dation of China (21666029); Ministry of education inno- vation team project (IRT1065); Inner Mongolia Natural Science Foundation (2016MS0223); grassland talents indi- vidual training project; Inner Mongolia Autonomous Re- gion Science and Technology Major Project (2019ZD023) is gratefully acknowledged. 5. References 1. T Z Gao, X L Yu, Y X Zhang, S Q Zhang, F Zhou, L Nie. Preparation and UV-Shielding Properties of Ceria with Differ- ent Morphologies. Wet Metallurgy, 2018,37(06):497–500. DOI:10.13355/j.cnki.sfyj.2018.06.014. 2. Z M Wang, X L Zhu, Y M Li, Z Y Shen, J L Zuo. Controlla- ble preparation of flake and spherical CeO2 nanoparticles and their photocatalytic properties. Journal of Artificial Crystals, 2017,46(08):1559–1563+1586. DOI:10.1088/1475-7516/2017/08/022 3. YN Feng, JZ Gan, XH Chen, J Zhang, HJ Duan, ZX Cui, YQ Xue. 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Eksperimentalno smo uspešno pripravili heksagonalne plasti kristalov cerijevega karbonata z uporabo PVP kot templata. Ugotovili smo, da metodo DFT lahko uporabimo za napoved morfologije kristalov cerijevega karbonata, s čemer pripomoremo k boljšemu razumevanju mehanizma rasti kristalov cerijevega karbonata in občutno zmanjšamo stroške eksperimenta. Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License 147Acta Chim. Slov. 2022, 69, 147–156 Tuncer et al.: Synthesis, Structure, Thermal Decomposition ... DOI: 10.17344/acsi.2021.7126 Scientific paper Synthesis, Structure, Thermal Decomposition and Computational Calculation of Heterodinuclear NiII – ZnII Complexes Yaprak Gürsoy Tuncer,1 Hasan Nazır,1 Kübra Gürpınar,1 Ingrid Svoboda,2 Nurdane Yılmaz,3 Orhan Atakol1 and Emine Kübra İnal1,* 1 Ankara University, Faculty of Science, Department of Chemistry, 06100, Ankara, Turkey 2 TU-Darmstadt, Materialwissenschaft, Strukturforschung, Alarich-Weiss Strasse 2, 64287, Darmstadt, Germany 3 Kastamonu University, Faculty of Education, Department of Mathematics and Science Education, 37200, Kastamonu, Turkey * Corresponding author: E-mail: inal@science.ankara.edu.tr Received: 09-07-2021 Abstract Mononuclear NiL complex was prepared by the use of bis-N,N’-salicylidene-1,3-propanediamine and Ni(II) salts. NiL was treated with ZnBr2 and pyrazole and 3,5-lutidine coligands in a dioxane medium to prepare the following diheter- onuclear complexes: [NiL · ZnBr2 · (pyrazole)2] and [NiL · ZnBr2 · (3,5-lutidine)2]. The complexes were characterized by elemental analysis, TG, IR and mass spectrometry. The effects of heterocyclic one- and two- nitrogen atoms containing co-ligands were also examined. Theoretical formation enthalpies, dipole moments and the relative levels of HOMO and LUMO energies were determined by the use of Gaussian09 program. The occupancy levels of the atomic orbitals were determined by the NBO analysis of Gaussian09. The effect of pyrazole and lutidine upon the complex formation was evaluated by the use of X-ray diffraction, TG and theoretical calculations. NiL complex with lutidine forms a square pyramidal conformation since lutidine is a much stronger coligand than pyrazole. Keywords: Salpn type ligand; Ni(II)-Zn(II) dinuclear complex; square-pyramidal coordination; thermal decomposition; heterocyclic coligand 1. Introduction Bis-N,N’-salicylidene-1,3-propanediamine (LH2) has been known to give homo- and heteropolynuclear complexes with Fe(II), Co(II), Ni(II), Cu(II), Zn(II) and Cd(II) ions since 1990.1–3 This compound that is classified as a Schiff base and a tetradentate ONNO type ligand gives heterodinuclear complexes with Lewis acids such as ZnCl2 and ZnBr2, and polynuclear complexes with a µ-bridge forming co-ligands such as acetate,4–9 formate,10,11 ni- trate,7,12,13 nitrite,14 benzoate,15,16 pseudohalogen or azides.17–20 In complexes prepared with Lewis acids, it is very common that one or two solvent molecules enter the coordination sphere. The complex maintains its existence with the coordination of solvent molecules. If these solvent molecules are thermally removed from the structure, the dinuclear structure decomposes.21–23 In addition to LH2 ligand giving mononuclear NiL and CuL complexes with Ni(II) and Cu(II), the resulting mononuclear complexes may be utilized to obtain polynu- clear complexes. The molecular models of NiL and CuL mononuclear complexes were first reported in 1985.24 In this study, it was determined that Cu(II) complex had a squashed tetrahedral and Ni(II) complex a square planar coordination sphere. If there are Lewis acids present in the medium, these Lewis acids are coordinated especially to NiL mononuclear complex through phenolic oxygens. As a result, Lewis acids withdraw electrons from NiL unit which decreases electron density upon Ni(II) and enables it to coordinate the solvent molecules or coligands present in the medium (Figure 1). Mononuclear NiL complex can form a square pyra- midal coordination sphere by coordinating H2O molecule 148 Acta Chim. Slov. 2022, 69, 147–156 Tuncer et al.: Synthesis, Structure, Thermal Decomposition ... if there is no Lewis acid in the medium.25 Generally, trinu- clear complexes are formed if there are coligands capable of establishing µ-bridges (Y) such as HCOO–, C6H5COO–, AcO–, NO2–, NO3–.4–16 In these trinuclear complexes, NiY2 is located in the center. Terminal groups are the solvent molecules such as DMF or dioxane coordinated by NiL.4– 9,26 If coligands containing more than one nitrogen donor, such as pyrazole or dicyandiamide, are added to the medi- um, polynuclear complexes are formed.27 This study is devoted to determine the type of coor- dination sphere formed by NiL and ZnBr2 together with one or two nitrogen-containing coligands. In a previous study, it was reported that NiL mononuclear complex forms a square pyramidal or octahedral coordination sphere with ZnCl2, ZnBr2, and 4-methylpyridine (4-pico- line).28 Based upon the picoline concentration there formed a square pyramidal complex with a [NiL·ZnCl2 · (4-picoline)] or an octahedral coordination sphere with [NiL·ZnBr2·(4-picoline)2] stoichiometries. The major tar- get of this study is to investigate the complexes formed if the reaction medium contains more than one heteroatom such as pyrazole and triazole. In this context, the coligands chosen were multi heteroatom containing pyrazole and a single heteroatom containing 3,5-lutidine, complexes were prepared in DMF and dioxane media. The complexes obtained were characterized by IR spectroscopy, elemental analysis, mass spectrometry and thermogravimetric analysis. The goal of the study was to prepare two complexes and elucidate the differences be- tween their thermal behavior. Two complexes designed for the study were obtained as single crystals, their molecular models and unit cell structures were determined by X-ray diffraction methods. The determination of the number of pyrazoles coordinated by NiL unit and their locations was one of the major targets of the study since pyrazole coordi- nation has various isomerization possibilities. The main purpose of the study is to investigate the difference be- tween pyrazole complexes containing multiple nitrogen donors and lutidine complexes containing a single nitro- gen donor. In previous studies, it has been reported that NiL and pyrazole give a polynuclear complex. As a result, both ligands give mononuclear complexes, the interesting thing is that the difference between them is obtained by thermogravimetric analysis, not by XRD study. The re- moval temperatures of the coligands from the structure enabled us to evaluate the strengths of the ligands upon the molecular structure. The variation in the thermal behavior of complexes [NiL · ZnBr2 · (pyrazole)2] (1) and [NiL · ZnBr2 · (3,5-luti- dine)2] (2) was elucidated by thermal analysis and theoret- Figure 1. Dinuclear complex formation with the effect of Lewis acid in the medium. 149Acta Chim. Slov. 2022, 69, 147–156 Tuncer et al.: Synthesis, Structure, Thermal Decomposition ... ical calculations were carried out upon the molecular structures, by using Gaussian 09 software.29 With natural bond analysis (NBO) in Gaussian 09 program, the occu- pancy levels of d orbitals of the central atoms, the molecu- lar dipole moment of the complexes, the electron distribu- tions, the relative energy levels of the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO), the energy difference between HOMO and LUMO were calculated. The values of the the- oretical and experimental bond lengths and bond angles were compared. All the theoretical results were interpreted according to the strength of the ligands. 2. Experimental All the reagents used in the study were supplied from Sigma-Aldrich and used without further purification. In this study, Shimadzu IRAffinity-1 FTIR spectrometer equipped with three reflectional ATR units was used for IR spectra with 4 cm–1 accuracy. C, H, and N analyses were performed on Eurovector 3018 CHNS analyzer. Metal analyses were carried out on GBC Avanta PM Model atomic absorption spectrometer using FAAS mode. Com- plex (2–3 mg) was dissolved in 1 mL HNO3 (63%) with heating, diluted to 100 mL and given to nebulizer of atom- ic absorption spectrometer for metal analysis. The mass spectra of the ligands were obtained by Shimadzu QP2010 Plus GCMS apparatus equipped with a direct inlet (DI) unit with an electron impact ionizer (EI). DI temperature was varied between 40–300 °C and ionization was carried out with electrons with 70 eV energy. The NMR spectra were recorded on the Bruker Ultrashield 300 MHz NMR spectrometer using d6-DMSO solution as a solvent. The thermogravimetric analyses were carried out by Shimadzu DTG 60H. In thermogravimetric analyses, the tempera- ture was varied between 30–600°C. These analyses were performed at 5, 10, 15, 20 and 25 °C min–1 heating rates and under N2 atmosphere in Pt pans. The calibration of the instrument was done with metallic In and Zn. 2. 1. Preparation of bis-N,N’-salicylidene-1,3- propanediamine (LH2) The Schiff base was prepared via condensation reac- tion in EtOH using 2-hydroxybenzaldehyde and 1,3-di- aminopropane. 2-hydroxybenzaldehyde (0.1 mol, 12.20 g) was dissolved in 120.0 mL of warm EtOH, then 0.05 mol (3.70 g) of 1,3-diaminopropane was added to and heated up to the boiling point. The mixture was left aside for 4–5 h and yellow crystals were precipitated, then the crystals were filtered and dried in air (25.90 g), yield: 91%, mp: 58 °C (determined by TG). Anal. Calcd for C17H- 18N2O2: C, 72.32; H, 6.43; N, 9.92. Found: C, 71.95; H, 6.33; N, 10.09. IR ν, cm–1: 2627 (OH), 3021–3019 (CH), 2929- 2862 (CH), 1629 (C=N), 1608 (C=C), 1274-1151 (C–O), 762 (CH). λmax: 243nm, ε: 7045 dm3 mol–1 cm–1 in DMSO, λmax: 242 nm, ε: 7865 dm3 mol–1 cm–1 in MeOH. 1H NMR (CH3COCH3-d6) δ 13.51 (s, 1H) (O−H), 8.60 (s, 1H) (−CH=), 7.43 (d, J = 1.8 Hz) (HAr), 7.32 (t, J = 1.8 Hz) (HAr), 6.88 (t, 1.8 Hz) (HAr), 3.68 (t, J = 7.2 Hz) (N−CH2−), 2.01 (p, J = 7.2 Hz) (−CH2−). 13C NMR (CH3COCH3-d6) δ 166.6, 161.1, 132.7, 132.1, 119.1, 118.9 (CAr), 116.9 (−C=N), 58.5 (N−CH2−), 31.9 (−CH2−). MS m/z: 282 [M]+, 161 [HO−C6H4−CH=N−CH2−CH2−CH2]+, 148 [HO−C6H4−CH=N−CH2−CH2]+ (base peak), 134 [HO− C6H4−CH=N−CH2]+, 120 [HO−C6H4−CH=N]+, 107 [HO−C6H4−CH2]+, 77 [C6H5]+. 2. 2. Preparation of the Complexes The complexes were prepared in two steps. The mononuclear NiL complex synthesized in the first step was converted into the dinuclear complex in DMF or dioxane medium in the second step. 2. 2. 1. Preparation of Mononuclear Complex (NiL) NiL was prepared by ammonia in an ethanol solu- tion of LH2 and NiCl2 · 6H2O outlined in the literature.33 0.01 mol of LH2 (2.82 g) was dissolved in 100.0 mL of hot EtOH under stirring. 10.0 mL of concentrated ammonia (20%) solution was added and the mixture was heated up to boiling temperature. A solution of 0.01 mol NiCl2 · 6H2O (2.36 g) in 30.0 mL hot water was added to this mix- ture. After the mixture was left on the bench for an hour, the light green precipitate of NiL·NH3 was filtered and dried at 150 °C for 4–5 h (3.45 g), yield: 95%, mp: 311 °C. The light green crystals are coordinatively ammonia bond- ed and leaves ammonia at 150 °C, the color of the complex changes to brown (NiL). The brown complex was recrys- tallized in EtOH:dioxane mixture (1:1, v/v). Anal. Calcd for C17H16N2O2Ni: C, 60.28; H, 4.76; N, 8.27; Ni, 17.33. Found: C, 60.55; H, 3.17; N, 7.93; Ni, 17.19. IR ν, cm–1: 3061–3030 (CH), 2922–2866 (CH), 1607 (C=N), 1589- 1541 (C=C), 1475 (CH), 1228–1124 (C–O), 725–744 (CH). MS m/z: 340 (isotope peak, because of 60Ni isotope), 338 [M]+ (base peak), 219 [Ni–O–C6H4–CH=NH–CH2– CH2–CH2]+, 205 [Ni–O–C6H4–CH=NH–CH2–CH2]+, 179 [Ni–O–C6H4–CH=NH]+, 134 [O–C6H4–CH=NH– CH2]+, 107 [HO–C6H4–CH2]+, 58 [Ni]+. 2. 2. 2. Preparation of Complex 1, [NiL · ZnBr2 · (pyrazole)2] 0.001 mol of NiL (0.340 g) was dissolved in 50.0 mL hot DMF under stirring and heated up to 100–110 °C. A solution of 0.001 mol ZnBr2 (0.226 g) and 0.002 mol pyra- zole (0.140 g) in 30.0 mL hot MeOH was added to this solution. The mixture was left on the bench for 2–4 days at room temperature. The light purple crystals were filtered 150 Acta Chim. Slov. 2022, 69, 147–156 Tuncer et al.: Synthesis, Structure, Thermal Decomposition ... and dried in air (0.41 g), yield: 58%. mp: 190 °C (decompo- sition). Anal. Calcd for C23H24Br2N6NiO2Zn: C, 39.56; H, 3.18; N, 12.03; Ni, 8.40; Zn, 9.36; Br, 22.88. Found: C, 40.17; H, 3.27; N, 11.93; Ni, 8.01; Zn, 9.47; Br; 21.83. IR ν, cm–1: 3335 (NH), 3120 (CH), 3034–3017 (CH), 2929–2861 (CH), 1631–1618 (C=N), 1593-1552 (C=C), 1475 (CH), 1298–1118 (C–O), 759 (CH). MS m/z: 338 (molecular peak of NiL and base peak), 179, 132, 107, 77, 58, 44. 2. 2. 3. Preparation of Complex 2, [NiL · ZnBr2 · (3,5-lutidine)2] This complex was prepared as given above using 0.001 mol of NiL (0.340 g), 0.001 mol of ZnBr2 (0.226 g) and 0.002 mol of 3,5-lutidine (0.220 g). The mixture was left on the bench for 2–4 days at room temperature. The light purple crystals were filtered and dried in air (0.59 g), yield: 76%. mp: 157 °C (decomposition). Anal. Calcd for C31H34Br2N4NiO2Zn: C, 44.52; H, 4.42; N, 7.68; Ni, 8.06; Zn, 8.97; Br, 21.93. Found: C, 40.08; H, 3.93; N, 7.35; Ni, 7.73; Zn, 8.59; Br, 21.81. IR ν, cm–1: 3031–3009 (CH), 2921–2865 (CH), 1618 (C=N), 1595-1550 (C=C), 1475 (CH), 1301–1107 (C–O), 752 (CH). MS m/z: 338 (molec- ular peak of NiL), 107 (coligand and base peak), 92, 79, 71, 58, 43. 2. 3. X-Ray Crystallography A single crystals of [NiL · ZnBr2 · (pyrazole)2] (1) and [NiL · ZnBr2 · (3,5-lutidine)2] (2) were analyzed on Oxford Diffraction Xcalibur Single Crystal X-ray Diffractometer with a sapphire CCD detector using MoKα radiation (λ = 0.71073 Å) operating in ω/2θ scan mode. The unit-cell di- mensions were determined and refined by using the angu- lar settings of 25 automatically centered reflections in 2.588° ≤ θ ≤ 26.369° for 1 and 2.556°–27.894° for 2. The data was collected at 293(2) K. The empirical absorption corrections were applied by the semi-empirical method via the CrysAlis CCD software.30 The model was obtained from the results of the cell refinement and the data reduc- tions were carried out using the solution software SHELXL 2014-6.31 The structure of the complexes was solved by di- rect methods using in WinGX package.32 The treatment of hydrogen atoms was made geometrically. Supplementary material for structure has been deposited to the Cam- bridge Crystallographic Data Center as CCDC no: 1949380, 1949381 (deposit@ccdc.cam.ac.uk or http:// www.ccdc.cam.ac.uk). 3. Results 3. 1. X-Ray Studies The Ortep drawing obtained from X-ray diffraction studies of complexes 1 and 2 were depicted in Figures 2 and 3. The crystal data and data collection conditions of these complexes were tabulated in Table 1, the bond lengths and the bond angles are shown in Table 2. As seen in Figures 2 and 3, Ni(II) ion in both com- plexes is in an octahedral coordination sphere between the O2N2 donors of the Schiff base, pyrazole and the two nitro- gens of lutidine. On the other hand, Zn(II) ions are located in a distorted tetrahedral coordination sphere between two phenolic oxygen and two bromine atoms. However, based on the angle values given in Table 2, it can be concluded that the distortion value of the coordination sphere is high- ly extensive. The bond lengths in the equatorial plane of the octahedral coordination sphere of 1, Ni(II) and donor at- oms, are around 2 Å while the axial bond lengths change between 2.138 and 2.178 Å. The corresponding values are 2.154–2.338 Å for 2. The equatorial bond lengths of Ni(II) donor atom are approximately 2 Å while axial bond lengths Figure 2. The Ortep drawing of 1. Figure 3. The Ortep drawing of 2. 151Acta Chim. Slov. 2022, 69, 147–156 Tuncer et al.: Synthesis, Structure, Thermal Decomposition ... differ from each other. The lengths of the bond between pyrazole and lutidine nitrogen atoms indicate that the coli- gands are located axially in the octahedral coordination sphere. In fact, the largest angle among these three atoms is formed between these two atoms. The angle of N4NiN6 in 1 was measured as 176.4° and the angle of N3NiN4 in 2 was measured as 175.4°. In addition, in the coordination of Zn(II) the bond angles for 1 were found to be between 121.44°–82.52° and 79.07°– 117.95° for 2, respectively, showing a high tetrahedron dis- tortion for these compounds. Pyrazole coligand participates in the coordination through a double bond nitrogen atom. The N-H nitrogen of the pyrazole ring does not participate in the coordina- tion. Since the electron pair present on this atom is donat- ed to the π system of the ring, there is no electron pair left to donate to Ni(II) ion. 3. 2. Thermal Analysis The TG and DTA curves of 1 and 2 are given in Fig- ure 4. The thermoanalytical data of these complexes are tabulated in Table 3. As can be seen from Figure 4 and Table 3, pyrazole coligands are separated from the structure in a single step. On the other hand, the removal of lutidine from the struc- ture is a two-step process. In this process which is de- scribed as the first thermal reaction in Table 3, the coli- gands are removed from the structure leaving a NiL mononuclear complex and ZnBr2 behind. The thermo- gravimetric curve of 1 depicted in Figure 4 displays a sin- Table 1. Crystal data and data collection conditions. 1 2 Molecular Formula C23H24Br2N6NiO2Zn C31H34Br2N4NiO2Zn Molar mass/ g mol–1 700.38 778.52 T/ K 293(2) 293(2) Crystal System Monoclinic Monoclinic Space Group P21/n P21/c a /Å 9.0086(3) 9.1210(5) b /Å 15.7423(6) 18.9500(10) c /Å 18.2777(7) 18.9770(10) Alpha 90 90 Beta 98.856(4) 101.916(6) Gamma 90 90 V /Å3 2561.17(16) 3209.4(3) Z 4 4 Calc. Density/ g cm–3 1.816 1.611 µ /mm–1 4.825 3.858 F (000) 1392 1568 Reflections Collected 11155 24685 Reflections Unique 5229 7144 R1, wR2 (2σ) 0.0591, 0.1678 0.0773, 0.1992 R1, wR2 (all) 0.0835, 0.1868 0.1699, 0.2489 Data / Parameters 5229/ 320 7144/ 370 GOOF of F2 1.058 1.022 Largest Difference Peak Hole /e Å–3 1.031, –1.866 1.072, –1.514 CCDC No 1949380 1949381 Table 2. The selected bond lengths and angles around the coordina- tion sphere of the complexes. Bond Lengths / Å Bond Angles / ° 1 N1–Ni1 2.027(5) N7–N6–Ni1 125.8(5) N2–Ni1 2.019(5) Zn1–O1–Ni1 98.9(19) N3–N4 1.336(8) Zn1–O2–Ni1 99.0(19) N4–Ni1 2.138(6) N2–Ni1–N1 99.2(2) N6–N7 1.324(8) N2–Ni1–O2 170.0(2) N4–Ni1 2.138(6) N1–Ni1–O2 90.8(2) N6–Ni1 2.178(6) N2–Ni1–O1 90.6(2) O1–Zn1 1.971(5) N1–Ni1–O1 169.7(2) O1–Ni1 2.042(5) O2–Ni1–O1 79.5(18) O2–Zn1 1.978(4) N2–Ni1–N4 91.1(2) O2–Ni1 2.031(4) N1–Ni1–N4 87.4(2) Ni1–Zn1 3.049(10) O2–Ni1–N4 90.6(2) Zn1–Br1 2.325(12) O1–Ni1–N4 89.3(2) Zn1–Br2 2.328(11) N2–Ni1–N6 92.1(2) N1–Ni1–N6 90.6(2) O2–Ni1–N6 86.4(2) O1–Ni1–N6 92.1(2) N4–Ni1–N6 176.4(2) O1–Zn1–O2 82.5(18) O1–Zn1–Br1 119.6(15) O2–Zn1–Br1 109.2(15) O1–Zn1–Br2 113.1(15) O2–Zn1–Br2 121.4(15) Br1–Zn1–Br2 109.4(4) 2 N1–Ni1 2.024(7) N2–Ni1–O1 170.6(3) N2–Ni1 2.004(8) N2–Ni1–N1 98.9(3) N3–Ni1 2.154(7) O1–Ni1–N1 90.1(3) N4–Ni1 2.338(8) N2–Ni1–O2 92.0(3) O1–Zn1 2.017(5) O1–Ni1–O2 79.1(2) O1–Ni1 2.020(5) N1–Ni1–O2 169.1(3) O2–Zn1 1.967(5) N2–Ni1–N3 92.8(3) O2–Ni1 2.030(5) O1–Ni1–N3 89.6(2) Ni1–Zn1 3.072(14) N1–Ni1–N3 92.7(3) Zn1–Br2 2.326(16) O2–Ni1–N3 87.1(2) Zn1–Br1 2.332(15) N2–Ni1–N4 86.0(3) O1–Ni1–N4 90.9(3) N1–Ni1–N4 91.8(3) O2–Ni1–N4 88.4(2) N3–Ni1–N4 175.4(3) N2–Ni1–Zn1 130.9(2) O1–Ni1–Zn1 40.4(14) N1–Ni1–Zn1 130.2(2) O2–Ni1–Zn1 39.0(15) N3–Ni1–Zn1 83.5(19) N4–Ni1–Zn1 93.9(2) O2–Zn1–O1 80.6(2) O2–Zn1–Br2 112.2(16) O1–Zn1–Br2 110.7(16) 152 Acta Chim. Slov. 2022, 69, 147–156 Tuncer et al.: Synthesis, Structure, Thermal Decomposition ... gle step endothermic mass loss between 197–232 °C corre- sponding to two pyrazole molecules (Table 3). The theoretically calculated mass of two pyrazole molecules in 1 was 19.42% while the experimentally determined value was 20.10%. Subsequently, a mass loss of about 10% was observed at around 380 °C which is the dissociation tem- perature of NiL mononuclear complex.23 The situation in 2 is entirely different. Two lutidine coligands in a complex molecule detach from the structure in two identical stages with two equal mass losses. The first mass loss of approximately 13.49% occurs in a temperature range of 157–202 °C. Subsequently, a second mass loss of 12.98% was observed between 202–264 ° C. Since the mass of lutidine is 13.76% of the mass of the complex, lutidines leave the structure one by one by two consecutive endo- thermic reactions. The residual NiL and ZnBr2 mixture gives a mass loss of 10% at 380 °C corresponding to the dissociation of NiL complex. 3. 3. Computational Results The relative energy levels of HOMO and LUMO, di- pole moments and formation energies obtained by using the sets in the Gaussian 09 program are given in Table 4. The orbital occupation values of the donor atoms are tabu- lated in Table S1 and the types of orbitals are given in Table S2. Figure 5 shows ESP maps and HOMO-LUMO images of the complexes. Figure 4. a. TG curves, b. DTA curves of 1 and 2 (black: 1, [NiL·ZnBr2·(pyrazole)2], red: 2, [NiL·ZnBr2·(3,5-lutidine)2]. Table 3. Thermoanalytical data of the complexes prepared. 1st Thermal Reaction 2nd Thermal Reaction Complex Removal of coligands Decomposition of NiL residue Temperature Calcd mass Final mass Temperature Final mass range / °C loss / % loss/ % range / °C loss/ % 1 197–232 1st pyrazole loss: 9.71 Total loss: 20.10 ± 0.58 380–420 11.42 ± 1.27 Total loss: 19.42 2 157–202–264 1st lutidine loss: 13.76 1st mass loss: 13.49 ± 0.35 Total loss: 27.52 2nd mass loss: 12.98 ± 0.77 Total loss: 26.47 ± 0.52 380–402 10.11 ± 2.17 Table 4. The relative energy levels of HOMO and LUMO, dipole moments and formation energies of the complexes prepared in the study, calculated with Gaussian 09. Complex EHOMO / eV ELUMO / eV ∆E / eV µ / D IP / eV EA / eV ∆H°f / kJ mol–1 1 –6.167 –2.569 3.598 12.947 6.167 2.569 2173.76 2 –6.070 –2.497 3.573 12.501 6.070 2.497 2113.91 Optimization – b3lyp/6-31G(d), NImag: 0, IP: ionization potential, EA: electron affinity 153Acta Chim. Slov. 2022, 69, 147–156 Tuncer et al.: Synthesis, Structure, Thermal Decomposition ... The dipole moments, formation enthalpies and rela- tive energy levels of HOMO and LUMO of the complexes came out to be highly similar. This is a highly expected outcome since the two complexes are very similar to each other. In both complex Ni(II)ion is in O2N4 octahedral co- ordination sphere while Zn(II) is located in a neighboring tetrahedral O2Br2 coordination sphere. Pyrazole and luti- dine donate electrons to Ni(II) ion while Zn(II) ion at- tracts the electrons towards bromine atoms via phenolic oxygens. That is why chelate rings assume partially posi- tive and bromine atoms partially negative charges as clear- ly seen in ESP maps given in Figure 5. Since the diameter of the molecule is large, it is quite normal for the dipole moment to be high. Among the data obtained from NBO studies, the electron occupation val- ues indicate that Ni(II) ion is in octahedral coordination. When focusing on Ni(II) ions, it can easily be seen that three d orbitals are occupied and the remaining two con- tain empty sites. This is expected for the octahedral crystal field splitting theory. On the other hand, Zn(II) possesses 10 d electrons, all d orbitals were found to be filled. There are two nitrogen atoms in the pyrazole ring. One of the nitrogen atoms has a hydrogen atom and an electron pair, the other one attached to the ring with a double bond. However, both nitrogens donate electrons to the π system of the aromatic ring and there are unfilled p orbitals in ni- trogen atoms in the ring. This distribution is more homo- geneous in iminic nitrogens. 4. Discussion The difference between the two complexes is not clear from IR data. The most important result from IR data is the difference between the C=N vibrations of the ligand and the complexes. While C=N vibration was observed at 1608 cm–1 in ligand, it was observed at 1598 and 1595 cm–1 in complexes. This data proves that iminic nitrogen is co- Figure 5. HOMO-LUMO images and ESP maps of the complexes. 154 Acta Chim. Slov. 2022, 69, 147–156 Tuncer et al.: Synthesis, Structure, Thermal Decomposition ... ordinated to the structure. It is already known that when the imine nitrogen is coordinated to a metal, the stretch vibration shifts to a low energy by 10–30 cm–1. Apart from this, O–H stretches observed around 2600 cm–1 due to the strong hydrogen bonds are not observed in the complex spectra. In complexes, vibrations between nitrogen and metal ion in coligands cannot be determined from the spectra because IR spectra were recorded with ATR equip- ment and it is not possible to observe vibrations less than 600 cm–1 with ATR. However, in the theoretical calcula- tions, the Ni–N(pyrazole) stretches can be observed at 334 cm–1 for 1, at 450 cm–1 for 2; the Ni–N(imine) stretches can be observed at 423 cm–1 for 1, at 472 cm–1 for 2; the Ni–O(phenol) stretches can be observed at 623 cm–1 for 1, at 602 cm–1 for 2 (Figure S1). The most important difference between the com- plexes is the variation of coordinative bond lengths ob- served in X-ray diffraction patterns. In 1, the pyrazole molecule is attached to Ni(II) with electron pair of the non-hydrogen bonded nitrogen atom of the pyrazole ring. The distance between two pyrazoles with Ni(II) ion is very close to each other, as seen in Table 2, these distances are 2.138 and 2.178 Å. However, the situation is different in 2. The two lutidines have different distances to Ni(II) ion. These distances are found to be 2.154 Å and 2.338 Å. This is also seen in the TG and DTA curves. As can be seen in Figure 4, both pyrazole coligands in 1 leave the structure in a single-stage process. The same situation is not valid for 2, the removal of lutidine from the complex structure takes place in two distinctive stages. This shows that the coordi- native effects of pyrazole and lutidine are different, lutidine is a stronger ligand than pyrazole. This is an expected re- sult because the pyrazole ring is a more acidic and elec- tron-withdrawing group,34 lutidine is a better electron-do- nating ligand. If the phenolic oxygens of NiL unit coordinate a Lewis acid, the electrons of phenolic oxygen are attracted by Lewis acid resulting in the decrease of the electron density around the Ni(II) ion provided by the phenolic oxygens of the ligand to Ni(II) ion. Under this condition, Ni(II) ion compensates for the decreasing elec- tron density by the coordination of solvent molecules or coligands present in the medium. If the electrons provided by a single coligand are sufficient, a square pyramidal co- ordination sphere is formed. If the electrons provided are not sufficient, then an octahedral coordination sphere oc- curs by the coordination of two coligands. In fact, in simi- lar studies carried out by picoline, there were square-pyra- midal or octahedral coordination spheres formed depending upon the picoline concentration.28 If the coli- gand concentration in the medium is sufficiently high then an octahedral coordination sphere is formed by the addi- tion of a new coligand to the square pyramidal structure of Ni(II) ion (Scheme 1). This situation is clearly illustrated in Figure 4. While the pyrazole molecules are thermally discarded from the structure with a single-stage process, this takes in two dis- tinctive processes in the case of lutidine. Therefore, the distance of the lutidine molecules to Ni(II) ion is different. DTA curves verify the fact that lutidine molecules are re- moved from the structure by two distinctive endothermic reactions. The total mass loss observed in these endother- mic reactions is approximately equal to the mass of two lutidine molecules. Similarly, in the case of using pyrazole as a coligand, the mass loss in a single endothermic reac- tion is equal to the mass of two pyrazole molecules. The visual observation of the chelate rings that oc- curred in both complexes showed that they have semi- chair conformation. Both complexes give a six-membered chelate ring with two nitrogen molecules of the Schiff base, a trimethylene bridge connecting to these two nitrogen at- oms, and a central Ni(II) ion. The interplanar angles were calculated by the use of Parst program.35 For 1, the angle between the atomic planes of C8–C9–C10 and C8–N1– N2–C10 was 62.85°, the angle between C8–N1–N2–C10 and N1–Ni1–N2 was 7.35°. The ideal value of these angles in chair conformation is 62°. Under these conditions, one side of the chelate ring is in a stressed position and it ap- pears to be a semi-chair structure. Scheme 1. Schematic preparation reactions of the complexes. 155Acta Chim. Slov. 2022, 69, 147–156 Tuncer et al.: Synthesis, Structure, Thermal Decomposition ... On the other hand, for 2 these angles are 50.74 and 10.77°. In both complexes, the aromatic rings of the coli- gands are not in the same plane. The angle between the two pyrazole planes in 1 is 20.74° and the angle between two lutidine planes in 2 is 57.39° (Figure 1). These values are similar to the data in the literature, the angles between the N1-Ni-N2 plane and C8-N1-N2-C9 plane have been reported between 5.0 and 8.9°.23,24,36 The theoretical study results, unfortunately, do not clearly show the difference between the complexes. Almost all the values of the two complexes are quite close to each other. The energy differences of HOMO-LUMO orbitals and dipole moments are approximately the same in these two complexes. The electron occupation values in the d or- bitals of Ni(II) ion obtained from NBO analysis are close. In these two complexes, the occupation values of the dxy, dz2, dx2-y2 orbitals are the same, only there is a slight differ- ence in the dyz and dzx orbitals. As can be seen from Table 5, the occupancy value of the dyz orbital for 1 is 1.14 elec- trons, the dzx orbital is 1.60 electrons, the same orbitals have occupancy levels of 1.02 and 1.71 electrons in 2. This result shows that the energy of the dyz orbital in 2 is higher and according to the crystal field theory, the lutidine coli- gand offers more electrons to Ni(II) central ion. However, the difference is not significant and the second-order per- turbation results in the NBO analysis reveal that there is no difference between the numerical values obtained from the two complexes and it is not possible to determine the electron donation effects of the coligands from the theo- retical calculations, but at this point, thermal analysis brings an advantage. It is possible to interpret the differ- ence between the strengths of the two coligands using thermogravimetric results. The stronger electron-donat- ing coligand lutidine can form an intermediate stable com- pound of [NiL·ZnBr2·(3,5-lutidine)] in the dinuclear com- plex, although a pyrazole molecule cannot offer enough electrons, [NiL·ZnBr2·(pyrazole)] molecule does not form, instead [NiL·ZnBr2·(pyrazole)2] complex is formed with two pyrazole molecules. In this study, the complex was prepared at different 3,5-lutidine concentrations, but all the complex stoichiometries obtained were [NiL·Zn- Br2·(3,5-lutidine)2] and [NiL·ZnBr2·(3,5-lutidine)] could not be prepared. However, thermogravimetry shows that this complex can be prepared. This work also proves the importance of thermogravimetry in the study of com- plexes. 5. Conclusion Lewis acids can attract electrons from the oxygens of the coordination sphere of bis-N,N’-salicylidene-1,3-pro- panediamine-Ni(II) complex forming polynuclear µ-com- plexes. This results in a decrease of the electron density upon Ni(II) ion. Therefore, Ni(II) ion coordinates the sol- vent molecules or the coligands present in the medium by withdrawing electrons. If the coligand possesses a suffi- ciently high electron density, it forms a square pyramidal coordination sphere. If the electron density of the coligand is not sufficiently high, then Ni(II) ion attaches two coli- gands forming an octahedral coordination sphere. Acknowledgments This research did not receive any specific grant from funding agencies. The authors (Y. Gürsoy Tuncer and K. Gürpınar) thank to The Scientific and Technological Re- search Council of Turkey (TUBITAK) for financial sup- port (Project number: 118F128). The authors declare that there is no conflict of interest. 6. References 1. C. Fukuhara, K. Tsuneyoshi, N. Matsumoto, S. Kida, M. Mi- kuriya, M. Mori, Dalton Trans. 1990, 11, 3473–3479; DOI:10.1039/DT9900003473 2. A. Gerli, K. S. Hagen, L. G. Marzilli, Inorg. Chem. 1991, 30, 4673–4676; DOI:10.1021/ic00024a043 3. S. Mirdya, M.G.B. Drew, A.K. Chandra, A. Banerjee, A. Fron- tera, S. Chattopadhyay, Polyhedron 2020, 179, 114374; DOI:10.1016/j.poly.2020.114374 4. S. Uhlenbrock, R. Wegner, B. Krebs, Dalton Trans. 1996, 18, 3731–3736; DOI:10.1039/dt9960003731 5. F. Ercan, O. 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B 1982, 38, 1149–1152; DOI:10.1107/S0567740882005184 Povzetek Enojedrni kompleks NiL smo pripravili z uporabo bis-N,N‘-saliciliden-1,3-propandiamina in Ni(II) soli. NiL smo reagi- rali s ZnBr2, pirazolom in 3,5-lutidinom kot soligandoma v dioksanu in izolirali diheterojedrna kompleksa: [NiL · ZnBr2 · (pyrazole)2] in [NiL · ZnBr2 · (3,5-lutidine)2]. Kompleksa smo okarakterizirali z elementno analizo, TG, IR in masno spektrometrijo. Proučili smo učinek heterocikličnih ligandov. Z uporabo programa Gaussian09 smo izračunali tvorbene entalpije, dipolne momente ter energije HOMO in LUMO orbital. Zasedenost atomskih orbital smo določili z NBO ana- lizo. Vpliv pirazola in lutidina na tvorbo kompleksa smo ovrednotili z uporabo rentgenske difrakcije, TG in teoretičnih izračunov. Kompleks NiL z lutidinom tvori kvadratno piramidalno konformacijo, saj je lutidin veliko močnejši koligand kot pirazol. Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License 157Acta Chim. Slov. 2022, 69, 157–166 Liu: Synthesis, Characterization, X-Ray Crystal Structures ... DOI: 10.17344/acsi.2021.7167 Scientific paper Synthesis, Characterization, X-Ray Crystal Structures and Antibacterial Activity of Zinc(II) and Vanadium(V) Complexes Derived from 5-Bromo-2-((2-(methylamino) ethylimino)methyl)phenol Cheng Liu School of Medicine, Huaqiao University, Quanzhou 362021, P. R. China * Corresponding author: E-mail: liucheng_hqu@163.com Received: 09-24-2021 Abstract Three new zinc(II) and one vanadium(V) complexes, [Zn2Cl2L2] (1), [Zn2I2L2] (2), [ZnCl2(HL)] (3), and [V2O2(μ-O)2L2] (4), where L is 5-bromo-2-((2-(methylamino)ethylimino)methyl)phenolate, have been synthesized and characterized by elemental analyses, IR and UV-Vis spectra, as well as molar conductivity. Structures of the complexes were confirmed by single crystal X-ray diffraction. Complexes 1 and 2 are isostructural dinuclear zinc compounds, with the Zn atoms in square pyramidal coordination. The Zn atoms in the mononuclear complex 3 are in tetrahedral coordination. Complex 4 is a dinuclear vanadium(V) compound, with the V atoms in octahedral coordination. The complexes were assayed for antibacterial activities by MTT method. Keywords: Schiff base; Zinc complex; Vanadium complex; X-ray diffraction; Antibacterial activity 1. Introduction Schiff bases are important compounds due to their easy preparation, good coordination, and excellent biolog- ical activities.1 For the past few decades, the coordination chemistry of Schiff base ligands has been the subject of great interest. Schiff bases are capable of forming coor- dinate bonds with various inorganic metal salts through azomethine group.2 Recently, metal complexes with bio- logically active ligands have received considerable atten- tion. The biological activities of the organic ligands can be enhanced during coordination with metal salts.3 It has been reported that chelation is a good way to cure many diseas- es like cancer.4 Zinc is the second most abundant metal in the human body, and zinc homeostasis alterations have been linked to many diseases like neuropsychiatric disor- ders, bone diseases, and skin disorders.5 Zinc homeostasis causes a variety of health problems that include growth retardation, immunodeficiency, hypogonadism, and neu- ronal and sensory dysfunctions.6 Recently, zinc complexes have attracted much attention in the field of cancer ther- apy based on the facts that zinc is significantly non-toxic even at higher doses compared with other metals, which is beneficial to biocompatibility.7 Zinc complexes also show biological activities like anticancer, DNA binding, antiox- idant, antibacterial, and antitumor.8 Moreover, vanadium chemistry has attracted great attention due to its interest- ing structural features and biological relevance.9 Many va- nadium complexes were synthesized and found to show medicinal properties like insulin mimetic activity.10 They also show anticancer, antitumor, antifungal and antibac- terial activities.11 Some complexes with tridentate Schiff base ligands have been reported.12 Herein, we report the synthesis, characterization, and single crystal structures of three new zinc(II) and one vanadium(V) complexes, [Zn2Cl2L2] (1), [Zn2I2L2] (2), [ZnCl2(HL)] (3), and [V2O2(μ-O)2L2] (4), where L is 5-bromo-2-((2-(methyl- amino)ethylimino)methyl)phenolate. The antibacterial activity of the compounds against Gram-positive bac- terial strains (B. subtilis, S. aureus and St. faecalis) and Gram-negative bacterial strains (E. coli, P. aeruginosa and E. cloacae) by MTT method was studied. 2. Experimental 2. 1. Materials and Physical Methods 4-Bromosalicylaldehyde, N-methylethane-1,2-di- amine, zinc chloride, zinc iodide, vanadium(IV)oxy acetylacetonate and sodium azide were purchased from 158 Acta Chim. Slov. 2022, 69, 157–166 Liu: Synthesis, Characterization, X-Ray Crystal Structures ... Aldrich. The solvents used in the synthesis and biological assay were obtained from Xiya Chemical Co. Ltd. and used as received. Elemental analyses for C, H and N were per- formed on a Perkin-Elmer 2400 II analyzer. FT-IR spectra were recorded as KBr pellets on Bruker Tensor-27. UV- Vis spectra were recorded on Lambda 35 spectrophotom- eter. Single crystal X-ray diffraction was carried out with a Bruker Apex II CCD diffractometer. Molar conductivity was measured in methanol with a DDS-11A molar con- ductivity meter. 2. 2. Synthesis of 5-Bromo-2- ((2-(methylamino)ethylimino)methyl) phenol (HL) 4-Bromosalicylaldehyde (2.0 g, 0.010 mol) and N-methylethane-1,2-diamine (0.74 g, 0.010 mol) were stirred at reflux for 30 min in methanol (50 mL). The sol- vent was removed by distillation under reduced pressure to give yellow product. The solid was re-crystallized from ethanol to give the Schiff base. The yield was 0.22 g (85%). Anal. Calc. (%) for C10H13BrN2O: C, 46.71; H, 5.10; N, 10.89. Found (%): C, 46.55; H, 5.21; N, 10.72. IR data (KBr, cm–1): 3412, 3277, 1638. UV–Vis data [methanol, λ/nm (ε/L mol–1 cm–1)]: 230, 265, 310, 405. 1H NMR (500 MHz, DMSO-d6) δ 10.53 (s, 1H, OH), 10.16 (s, 1H, NH), 8.51 (s, 1H, CH=N), 7.53 (d, 1H, ArH), 7.45 (s, 1H, ArH), 7.11 (d, 1H, ArH), 3.71 (t, 2H, CH2), 3.23 (d, 3H, CH3), 2.88 (m, 2H, CH2). 2. 3. Synthesis of [Zn2Cl2L2] (1) 4-Bromosalicylaldehyde (0.20 g, 1.0 mmol) and N-methylethane-1,2-diamine (0.074 g, 1.0 mmol) were stirred at reflux for 30 min in methanol (30 mL). Then, zinc chloride (0.14 g, 1.0 mmol) and sodium azide (0.065 g, 1.0 mmol) dissolved in 20 mL methanol were added. The final mixture was further stirred at reflux for 1 h. Diffrac- tion quality single crystals were obtained after a few days by slow evaporation of colorless solution of the complex in open atmosphere. The yield was 0.19 g (53%). Anal. Calc. (%) for C20H24Br2Cl2N4O2Zn2: C, 33.65; H, 3.39; N, 7.85. Found (%): C, 33.46; H, 3.51; N, 7.73. IR data (KBr, cm–1): 3310, 1649. UV–Vis data [methanol, λ/nm (ε/L mol–1 cm– 1)]: 226 (17,530), 268 (15,540), 333 (5,780). ΛM (10–3 mol L–1 in methanol): 35 Ω–1 cm2 mol–1. 2. 4. Synthesis of [Zn2I2L2] (2) This complex was synthesized by the similar meth- od as described for complex 1, with zinc chloride replaced by zinc iodide (0.32 g, 1.0 mmol). The diffraction quality block like colorless single crystals that deposited over a period of 5 days were collected by filtration and washed Table 1. Crystallographic data and refinement details for the complexes 1 2 3 4 Chemical Formula C20H24Br2Cl2N4O2Zn2 C20H24Br2I2N4O2Zn2 C10H13BrCl2N2OZn C22H32Br2N4O8V2 Molecular weight 713.89 896.79 393.40 742.22 Crystal color, habit Colorless, block Colorless, block Colorless, block brown, block Crystal size, mm 0.25×0.23×0.23 0.22×0.20×0.17 0.20×0.20×0.15 0.19×0.18×0.16 Crystal system Orthorhombic Orthorhombic Triclinic Monoclinic Space group Aba2 Aba2 P-1 C2/c Unit cell dimensions: a, Ǻ 14.6638(18) 15.064(2) 7.3196(18) 25.308(2) b, Ǻ 21.2929(19) 21.180(2) 14.086(2) 6.8247(17) c, Ǻ 8.2244(17) 8.739(2) 14.539(2) 16.7172(16) α, ° 90 90 81.282(1) 90 β, ° 90 90 76.416(1) 105.867(2) γ, ° 90 90 75.596(1) 90 V, Ǻ3 2567.9(7) 2788.3(8) 1404.4(5) 2777.4(8) Z 4 4 4 4 ρcalcd, g cm–3 1.847 2.136 1.861 1.775 μ, mm–1 5.213 6.824 4.959 3.602 θ Range collected, deg 2.36–25.50 2.35–25.50 1.45–25.50 1.67–25.49 Tmin and Tmax 0.3557 and 0.3802 0.3152 and 0.3900 0.4371 and 0.5233 0.5477 and 0.5964 Reflections collected/unique 7270/2356 7321/2399 7483/5172 7107/2573 Observed reflections (I ≥ 2s(I)) 2134 1834 3957 2049 Data/restraints/parameters 2356/1/146 2399/1/146 5172/0/309 2573/0/175 GOOF on F2 1.056 1.087 1.051 1.073 R1, wR2 (I ≥ 2s(I)) 0.0375, 0.0980 0.0551, 0.1357 0.0406, 0.0954 0.0348, 0.0866 R1, wR2 (all data) 0.0443, 0.1065 0.0791, 0.1497 0.0599, 0.1088 0.0494, 0.0928 159Acta Chim. Slov. 2022, 69, 157–166 Liu: Synthesis, Characterization, X-Ray Crystal Structures ... with methanol. The yield was 0.18 g (40%). Anal. Calc. (%) for C20H24Br2I2N4O2Zn2: C, 26.78; H, 2.70; N, 6.25. Found (%): C, 26.59; H, 2.63; N, 6.34. IR data (KBr, cm–1): 3310, 1646. UV–Vis data [methanol, λ/nm (ε/L·mol–1·cm–1)]: 212 (18,360), 241 (17,460), 268 (15,655), 336 (5,460). ΛM (10–3 mol L–1 in methanol): 31 Ω–1 cm2 mol–1. 2. 5. Synthesis of [ZnCl2(HL)] (3) This complex was synthesized by the similar meth- od as described for complex 1, but without sodium azide. The diffraction quality block like colorless single crystals that deposited over a period of 5 days were collected by filtration and washed with methanol. The yield was 0.20 g (51%). Anal. Calc. (%) for C10H13BrCl2N2OZn: C, 30.53; H, 3.33; N, 7.12. Found (%): C, 30.40; H, 3.41; N, 7.27. IR data (KBr, cm–1): 3122, 1632. UV–Vis data [methanol, λ/ nm (ε/L·mol–1·cm–1)]: 225 (19,210), 245 (17,830), 268 (13,380), 365 (5,110). ΛM (10–3 mol L–1 in methanol): 40 Ω–1 cm2 mol–1. 2. 6. Synthesis of [V2O2(μ-O)2L2] (4) 4-Bromosalicylaldehyde (0.20 g, 1.0 mmol) and N-methylethane-1,2-diamine (0.074 g, 1.0 mmol) were stirred at reflux for 30 min in methanol (30 mL). Then, vanadium(IV)oxy acetylacetonate (0.26 g, 1.0 mmol) dis- solved in 20 mL methanol was added. The final mixture was further stirred at reflux for 1 h. Diffraction quali- ty single crystals were obtained after a few days by slow evaporation of brown solution of the complex in open at- mosphere. The yield was 0.26 g (70%). Anal. Calc. (%) for C22H32Br2N4O8V2: C, 35.60; H, 4.35; N, 7.55. Found (%): C, 35.75; H, 4.23; N, 7.46. IR data (KBr, cm–1): 3251, 1650, 934, 848. UV–Vis data [methanol, λ/nm (ε/L·mol–1 cm–1)]: 223 (16,630), 252 (15,120), 363 (4,330). ΛM (10–3 mol L–1 in methanol): 27 Ω–1 cm2 mol–1. 2. 7. X-Ray Structure Determination Intensity data of the complexes were collected at 298(2) K on a Bruker Apex II CCD diffractometer using graphite-monochromated MoKa radiation (λ = 0.71073 Å). For data processing and absorption correction the packages SAINT and SADABS were used.13 Structures of the complexes were solved by direct and Fourier meth- ods and refined by full-matrix least-squares based on F2 using SHELXL.14 The non-hydrogen atoms were refined anisotropically. The hydrogen atoms have been placed at geometrical positions with fixed thermal parameters. Crystallographic data of the complexes are summarized in Table 1. Selected bond lengths and angles are listed in Table 2. 2. 8. Antibacterial Activity Antibacterial activity of the complexes was tested against B. subtilis, S. aureus, S. faecalis, P. aeruginosa, E. coli, and E. cloacae using MTT medium. The minimum inhibitory concentrations (MICs) of the compounds were determined by a colorimetric method using MTT dye.15 Penicillin and Kanamycin were tested as reference drugs. A stock solution of the Schiff base ligand and the complex- es (50 μg mL–1) in DMSO was prepared and quantities of the compounds were incorporated in specified quantity of sterilized liquid medium. A specified quantity of the medium containing the compounds was poured into mi- cro-titration plates. Suspension of the microorganism was prepared to contain approximately 105 cfu mL–1 and ap- plied to micro-titration plates with serially diluted com- pounds in DMSO to be tested, and incubated at 37 °C for 24 h for bacteria. After the MICs were visually determined on each micro-titration plate, 50 μL of phosphate buffered Table 2. Selected bond distances (Å) and angles (°) for the complexes 1 Zn1‒O1 2.030(4) Zn1‒N1A 2.080(5) Zn1‒O1A 2.103(4) Zn1‒N2A 2.215(6) Zn1‒Cl1 2.268(2) O1‒Zn1‒N1A 140.3(2) O1‒Zn1‒O1A 75.84(16) O1‒Zn1‒N2A 96.1(2) O1‒Zn1‒Cl1 108.37(17) N1‒Zn1‒Cl1A 110.86(18) O1‒Zn1‒Cl1A 113.16(17) N2‒Zn1‒Cl1A 103.84(17) 2 Zn1‒O1 2.000(7) Zn1‒N1A 2.037(12) Zn1‒O1A 2.117(7) Zn1‒N2A 2.193(10) Zn1‒I1 2.5873(19) O1‒Zn1‒N1A 135.6(5) O1‒Zn1‒O1A 76.1(3) O1‒Zn1‒N2A 94.8(4) O1‒Zn1‒I1 108.8(3) N1‒Zn1‒I1A 114.9(4) O1‒Zn1‒I1A 109.9(3) N2‒Zn1‒I1A 105.1(3) 3 Zn1‒O1 1.950(3) Zn1‒N1 2.007(4) Zn1‒Cl1 2.2447(16) Zn1‒Cl2 2.2158(15) Zn2‒O2 1.940(4) Zn2‒N3 2.009(4) Zn2‒Cl3 2.2377(17) Zn2‒Cl4 2.2095(17) O1‒Zn1‒N1 96.33(15) O1‒Zn1‒Cl2 111.63(12) N1‒Zn1‒Cl2 113.18(13) O1‒Zn1‒Cl1 108.65(13) N1‒Zn1‒Cl1 111.15(13) Cl2‒Zn1‒Cl1 114.42(6) O2‒Zn2‒N3 96.77(17) O2‒Zn2‒Cl4 109.71(13) N3‒Zn2‒Cl4 113.77(14) O2‒Zn2‒Cl3 110.72(13) N3‒Zn2‒Cl3 109.38(13) Cl4‒Zn2‒Cl3 115.02(7) 4 V1‒O1 1.875(2) V1‒O2 1.6529(19) V1‒O3 1.574(2) V1‒N1 2.140(3) V1‒N2 2.113(2) V1‒O2A 2.2764(19) O3‒V1‒O2 106.43(11) O3‒V1‒O1 101.21(11) O2‒V1‒O1 99.09(9) O3‒V1‒N2 92.27(11) O2‒V1‒N2 94.48(10) O1‒V1‒N2 157.25(10) O3‒V1‒N1 96.48(11) O2‒V1‒N1 155.66(9) O1‒V1‒N1 84.11(10) N2‒V1‒N1 76.15(10) O3‒V1‒O2A 171.10(10) O2‒V1‒O2A 79.35(9) O1‒V1‒O2A 84.26(8) N2‒V1‒O2A 80.39(8) N1‒V1‒O2A 76.96(8) 160 Acta Chim. Slov. 2022, 69, 157–166 Liu: Synthesis, Characterization, X-Ray Crystal Structures ... saline (PBS 0.01 mol L–1, pH 7.4: Na2HPO4·12H2O 2.9 g, KH2PO4 0.2 g, NaCl 8.0 g, KCl 0.2 g, distilled water 1000 mL) containing 2 mg mL–1 of MTT was added to each well. Incubation was continued at room temperature for 4–5 h. The content of each well was removed, and 100 μL of isopropanol containing 5% 1 mol L–1 HCl was added to extract the dye. After 12 h of incubation at room tem- perature, the optical density (OD) was measured with a microplate reader at 570 nm. 3. Results and Discussion 3. 1. Chemistry The Schiff base HL was facile synthesized by the reaction of 4-bromosalicylaldehyde with N-methy- lethane-1,2-diamine in methanol (Scheme 1). The com- plexes were prepared in a similar method, by the reaction of HL with various inorganic salts (Scheme 2). The dinu- clear zinc complexes 1 and 2 were prepared in the presence of sodium azide, even though it is not a component of the compounds. Interestingly, the mononuclear complex 3 was obtained with no sodium azide presented during the synthesis. The solubility values of complexes 1, 2, 3 and 4 in methanol are 63, 54, 47 and 61 mg/mL, respectively. The conductivity values of the complexes (27–40 Ω–1 cm2 mol–1) indicated that they are non-electrolytes in metha- nol solution.16 3. 2. IR and UV-Vis Spectra Study The weak absorptions at 3310-3122 cm–1 for complex- es 1-3, and 3251 cm–1 for complex 4 are assigned to νN-H. The characteristic imine stretching for the complexes 1-4 is observed at 1632-1650 cm–1.17 The Schiff base ligand coor- dination through the phenolate oxygen is indicated by the absorption bands of the Ar–O bonds at 1178-1205 cm–1 in the complexes 1-4.18 In general, the infrared spectra of com- plexes 1 and 2 are similar to each other, due to the isostruc- tural nature. The V=O bonds of complex 4 are indicated by the absorption at 848 and 934 cm–1, which may be assigned to symmetric and asymmetric ν(O=V=O) vibration.19 Scheme 2. The synthetic procedure for the complexes. Scheme 1. The synthetic procedure for HL. 161Acta Chim. Slov. 2022, 69, 157–166 Liu: Synthesis, Characterization, X-Ray Crystal Structures ... The absorption spectral data of the complexes were measured in methanol. In the complexes, peaks between 212–245 nm, 252–268 nm and 333–363 nm are assigned to π→π*, n→π* and ligand to metal charge transfer transi- tions, respectively.20 3. 3. Structure Description of Complexes 1 and 2 Molecular structures of complexes 1 and 2 are shown in Figs. 1 and 2, respectively. The complexes are phenolate O bridged dinuclear zinc compounds. The molecules of the complexes possess crystallographic two fold rotation symmetry. The Zn atom is in square pyramidal coordina- tion, with the basal plane defined by the phenolate O, im- ino N and amino N atoms of one Schiff base ligand, and one phenolate O atom of the other Schiff base ligand, and with the apical position occupied by one chloride ligand, viz. Cl for 1 and I for 2. In general, the coordination ge- ometry around the Zn atoms in both complexes displays distortion, as evidenced by the coordinate bond lengths and angles. The Zn‒O and Zn‒N bonds in both complexes are similar and range from 2.030(4) to 2.215(6) Å for com- plex 1, and range from 2.000(7) to 2.193(10) Å for complex 2. The cis and trans bond angles in the basal planes are 75.84(16)-96.1(2)° and 140.3(2)-142.8(2)° for complex 1, and 76.1(3)-94.8(4)° and 135.6(5)-144.9(4)° for complex 2. The bond angles among the apical and basal donor atoms are 103.84(17)-113.16(17)° for complex 1, and 105.1(3)- 114.9(4)° for complex 2. All the bond angles indicate that the square pyramidal coordination is distorted from ideal Fig. 1. Molecular structure of complex 1. Atoms labeled with the suffix A are related to the symmetry operation 1 – x, 1 – y, z. Fig. 3. Molecular packing diagram of complex 1, viewed along the b axis. Hydrogen bonds are drawn as dashed lines. Fig. 2. Molecular structure of complex 2. Atoms labeled with the suffix A are related to the symmetry operation 1 – x, 1 – y, z. Fig. 4. Molecular packing diagram of complex 2, viewed along the b axis. Hydrogen bonds are drawn as dashed lines. 162 Acta Chim. Slov. 2022, 69, 157–166 Liu: Synthesis, Characterization, X-Ray Crystal Structures ... model. Even though, the coordinate bond values are com- parable to those observed in similar Schiff base zinc com- plexes with square pyramidal coordination.21 The average deviation (0.001(5) Å for complex 1, 0.051(5) Å for com- plex 2) of the four basal donor atoms and the displacement (0.681(3) Å for complex 1 and 0.695(3) Å for complex 2) of the Zn atoms from the least-squares planes defined by the four donor atoms indicate that the O2N2 cavities afford almost perfect planes to the Zn centers. The two benzene rings of the Schiff base ligands form a dihedral angle of 56.4(5)° for complex 1, and 65.2(5)° for complex 2. In the crystal structures of complexes 1 and 2 (Figs. 3 and 4), the complex molecules are linked through C‒H···- Cl and C‒H···I hydrogen bonds (Table 3), respectively, to form two dimensional network. 3. 4. Structure Description of Complex 3 Molecular structure of complex 3 is shown in Fig. 5. The complex is a mononuclear zinc compound. The asymmetric unit of the compound contains two independ- ent molecules. The Schiff base ligand adopts zwitterionic form, with the H atom of the phenol group transfer to the amino group. The Zn atom is coordinated by the phenolate O and imino N atoms of the Schiff base ligand, and two Cl ligands, forming tetrahedral geometry. The coordination geometry around the Zn atoms displays distortion, as ev- idenced by the coordinate bond lengths and angles. The Zn‒O and Zn‒N bonds in the molecules are similar and range from 1.940(4) to 2.009(4) Å. The bond angles are 96.33(15)-114.42(6)° for Zn1, and 96.77(17)-115.02(7)° for Zn2. The coordinate bond values are comparable to those observed in similar Schiff base zinc complexes with tetrahedral coordination.22 In the crystal structure of the complex (Fig. 6), the complex molecules are linked through C‒H···Cl, N‒H···Cl and N‒H···O hydrogen bonds (Table 3), to form two di- mensional network. 3. 5. Structure Description of Complex 4 Molecular structure of complex 4 is shown in Fig. 7. The complex is an oxo O bridged dinuclear vanadium compound, with V···V separation of 3.050(1) Å. There are two methanol molecules of crystallization, which con- nect to the complex molecule through O‒H···O hydrogen bonds (Table 3). The molecule of the complex possesses crystallographic inversion center symmetry. The V atom is in octahedral coordination, with the equatorial plane defined by the phenolate O, imino N and amino N atoms of one Schiff base ligand, and one bridging O atom (O2), and with the axial positions occupied by terminal O atom (O3) and the other bridging O atom (O2A). The coordina- tion geometry around the V atoms displays distortion, as evidenced by the coordinate bond lengths and angles. The V‒O and V‒N bonds in the equatorial plane are range from 1.653(2) to 2.140(3) Å, and the axial bonds are 1.574(2) and 2.276(2) Å. The obvious difference of the axial bonds from the equatorial bonds is caused by the Jahn-Teller ef- fects. The cis and trans bond angles in the equatorial plane are 76.15(10)-99.09(9)° and 155.66(9)-157.25(10)°. The Fig. 5. Molecular structure of complex 3. Atoms labeled with the suffix A are related to the symmetry operation 1 – x, 1 – y, z. Fig. 6. Molecular packing diagram of complex 3, viewed along the a axis. Hydrogen bonds are drawn as dashed lines. 163Acta Chim. Slov. 2022, 69, 157–166 Liu: Synthesis, Characterization, X-Ray Crystal Structures ... bond angles among the axial and equatorial donor atoms are 79.96(8)-106.43(11)°. All the bond angles indicate that the octahedral coordination is distorted from ideal model. Even though, the coordinate bond values are comparable to those observed in similar Schiff base vanadium com- plexes.23 The average deviation (0.063(5) Å) of the four equatorial donor atoms and the displacement (0.315(2) Å) of the V atom from the least-squares plane defined by the four donor atoms indicate that the O2N2 cavity afford somewhat distortion to the V center. In the crystal structure of the complex (Fig. 8), the complex molecules are linked through C‒H···O and N‒H···O hydrogen bonds (Table 3), to form two dimen- sional network. 3. 6. Antibacterial Activity of the Compounds The Schiff base ligand and the complexes were screened for antibacterial activities against three Gram-positive bac- terial strains (B. subtilis, S. aureus, and St. faecalis) and three Gram-negative bacterial strains (E. coli, P. aeruginosa, and E. cloacae) by MTT method. The MICs of the compounds against the bacteria are presented in Table 4. The Schiff base ligand has weak activity against B. subtilis, S. aureus and E. coli, while no activity against the remaining bacteria. In general, the complexes have higher activity than the Schiff base ligand. The zinc complexes 1 and 2 have equal activi- ties against all the bacterial strains. Both complexes showed strong activity against B. subtilis, S. aureus and E. coli (MICs Table 3. Hydrogen bond distances (Å) and bond angles (°) for the complexes D–H∙∙∙A d(D–H) d(H∙∙∙A) d(D∙∙∙A) Angle (D–H∙∙∙A) 1 C7–H7∙∙∙Cl1i 0.93 2.74 3.583(6) 151 2 C7–H7∙∙∙I1ii 0.93 3.00 3.719(13) 136 3 N2–H2A∙∙∙Cl1iii 0.90 2.53 3.274(4) 140(6) N2–H2A∙∙∙Cl1 0.90 2.92 3.551(4) 128(6) N2–H2B∙∙∙O1iv 0.90 1.91 2.793(5) 166(6) N4–H4A∙∙∙Cl2 0.90 2.51 3.277(5) 144(6) N4–H4A∙∙∙Cl3 0.90 2.87 3.444(5) 123(6) N4–H4B∙∙∙O2iv 0.90 1.97 2.856(6) 168(6) C6–H6∙∙∙Cl4v 0.93 2.83 3.647(6) 148(6) C19–H19B∙∙∙Cl4iv 0.97 2.73 3.628(6) 154(6) 4 O4–H4∙∙∙O2 0.82 1.93 2.738(3) 170(5) N2–H2∙∙∙O4vi 0.91 2.60 3.169(4) 121(5) N2–H2∙∙∙O1vii 0.91 2.34 3.007(3) 130(5) C3–H3∙∙∙O4viii 0.93 2.52 3.400(5) 157(5) C7–H7∙∙∙O3ix 0.93 2.58 3.020(4) 109(5) C8–H8B∙∙∙O3ix 0.97 2.57 3.240(4) 126(5) C9–H9B∙∙∙O4vi 0.97 2.54 3.217(5) 127(5) Symmetry codes for i): 1/2 + x, 1 – y, –1/2 + z; ii): –1/2 + x, 1 – y, –1/2 + z; iii): 2 – x, 1 – y, – z; iv): 1 + x, y, z; v): 1 – x, 1 – y, 1 – z; vi): x, –1 + y, z; vii) 1/2 – x, 1/2 – y, 1 – z; viii) 1/2 – x, 3/2 – y, 1 – z; ix) 1/2 – x, –1/2 + y, 1/2 – z. Fig. 7. Molecular structure of complex 4. Atoms labeled with the suffix A are related to the symmetry operation 1/2 – x, 1/2 – y, 1 – z. Fig. 8. Molecular packing diagram of complex 4, viewed along the b axis. Hydrogen bonds are drawn as dashed lines. Table 4. MICs (μg mL–1) of the compounds and related materials Tested Gram positive Gram negative material B. subtilis S. aureus St. faecalis P. aeruginosa E. coli E. cloacae HL 25 12.5 > 50 > 50 12.5 > 50 1 3.12 1.56 25 > 50 6.25 > 50 2 3.12 1.56 25 > 50 6.25 > 50 3 1.56 0.39 6.25 > 50 12.5 > 50 4 12.5 6.25 12.5 > 50 12.5 > 50 Penicillin 1.56 1.56 1.56 6.25 6.25 3.12 Kanamycin 0.39 1.56 3.12 3.12 3.12 1.56 164 Acta Chim. Slov. 2022, 69, 157–166 Liu: Synthesis, Characterization, X-Ray Crystal Structures ... = 1.56-6.25 μg mL–1), weak activity against St. faecalis (MIC = 25 μg mL–1), and no activity against P. aeruginosa and E. cloacae. Complexes 1 and 2 have similar activity against S. aureus and E. coli when compared with the reference drugs Penicillin and Kanamycin. In general, complex 3 has better activity against the Gram positive bacteria than complexes 1 and 2. However, complex 3 has worse activity against the Gram negative bacteria than 1 and 2. Complex 3 has high- er activity against S. aureus and similar activity against B. subtilis when compared with the reference drugs. Complex 4 has effective activity against S. aureus, but weak or no ac- tivity against the remaining bacteria. As a comparison with the zinc complexes derived from 4-fluoro-2-((pyridin-2-ylmethylimino)methyl)phe- nol and 4-fluoro-2-((2-(hydroxymethyl)phenylimino) methyl)phenol, the current zinc complexes have weaker activity against B. subtilis, St. faecalis, P. aeruginosa and E. coli, but higher activity against S. aureus.24 The complexes have similar activities against S. aureus and E. coli when compared with the zinc complexes derived from 5-bro- mo-2-((2-(diethylammonio)ethylimino)methyl)phenol.12f 4. Conclusion Two new dinuclear zinc(II) complexes, one new mononuclear zinc complex, and one new dinuclear vana- dium(V) complex have been synthesized and character- ized. Crystal structures of the complexes are determined and described. The Zn atoms in the dinuclear zinc com- plexes are in square pyramidal coordination. The Zn atom in the mononuclear zinc complex is in tetrahedral coordi- nation. The V atom in the dinuclear vanadium complex is in octahedral coordination. 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Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License Povzetek Sintetizirali smo tri nove koordinacijske spojine cinka(II) in eno vanadija(V), [Zn2Cl2L2] (1), [Zn2I2L2] (2), [ZnCl2(HL)] (3), and [V2O2(μ-O)2L2] (4), kjer je L = 5-bromo-2-((2-(metilamino)etilimino)metil)fenolat, ter produkte karakterizirali z elementno analizo, IR in UV-Vis spektroskopijo ter meritvami molarne prevodnosti. Strukture produktov smo določili z monokristalno rentgensko analizo. Produkta 1 in 2 sta izostrukturni dvojedrni cinkovi spojini s cinkovimi atomi v kvadratno piramidalni koordinaciji. Cinkovi atomi v enojedrni spojini 3 so tetraedrično koordinirani. Spojina 4 je dvo- jedrna z vanadijevimi atomi v oktaedrični koordinaciji. Antibakterijsko učinkovitost produktov smo preverili z metodo MTT. 167Acta Chim. Slov. 2022, 69, 167–186 Dolničar et al.: The Students’ Perceptions Using 3DChemMol ... DOI: 10.17344/acsi.2021.7181 Scientific paper The Students’ Perceptions Using 3DChemMol Molecular Editor for Construction and Editing of Molecular Models Danica Dolničar,1,* Bojana Boh Podgornik1 and Vesna Ferk Savec2 1 Faculty of Natural Sciences and Engineering, University of Ljubljana, Snežniška 5, 1000 Ljubljana, Slovenia 2 Faculty of Education, University of Ljubljana, Kardeljeva ploščad 16, 1000 Ljubljana, Slovenia * Corresponding author: E-mail: danica.dolnicar@ntf.uni-lj.si Received: 09-30-2021 Abstract The paper presents a study in which 54 university students were introduced to a newly developed, free, web-based 3DChemMol molecular editor with a toolbar, which they then evaluated. The tool aims to increase representational competence related to submicroscopic representations. Students who used the software for the first time, were instructed to create molecular models using the model building/editing tools in three activities with varying levels of difficulty: 1) building a simple model (butanoic acid), 2) converting one model (hexane) into two models, 3) converting from a non-cyclic to a cyclic structure (glucose). It took students from two up to 15 minutes to accomplish each of the activities. Several types of help were available in the 3DChemMol molecular editor toolbar to assist students during their activi- ties, including a video tutorial, button hovering, action status display, and a help menu. Undo/redo and restart options were also available. Students’ completion level, difficulties, and use of the help features were investigated using student self-evaluation questionnaires. The 3DChemMol molecular editor proved to be a useful support for students in complet- ing simple chemistry activities. Students were successful in model building, although they encountered some specific difficulties, especially in steps that involved spatial operations, such as rotating the selected part of molecule around the bond. In students’ perception, the video tutorials were the preferred and most frequently used type of help, and the undo function was considered essential. The results suggest that the 3DChemMol molecular editor can be used effectively in introductory chemistry courses at the tertiary level, whether for direct instruction, self-study, or other forms of support in the pedagogical process. The results and new findings of this study will be used to further optimize the interface in future versions of the evaluated tool. Keywords: Representational competence; submicroscopic representations; learning chemistry; 3D model building; model editing tool 1. Introduction 1. 1. Visualization and Molecular Models in Chemistry Education The concept of visualization can be understood in three ways:1 visualization of objects (physical or graphic representations, static or dynamic, analog or digital, can be accompanied by sensory data), introspective visualization (mental models), and interpretive visualization (making meaning from the previous two forms). Vekiri2 states that graphical representations allow for more efficient process- ing of information compared to verbal representations, which reduces working memory load. The adoption of vis- ualization is not automatic but a function of prior knowl- edge.3 Understanding the core ideas introduced in chemis- try education involves engagement with their representa- tions and the associated phenomena.4 Johnstone5,6 was the first to propose three levels of representation of scientific concepts and processes: (1) macroscopic (e.g., chemistry experiments), (2) submicroscopic (e.g., molecular mod- els) and (3) symbolic (e.g., chemical formulae). The three types of representations relate to phenomena perceived through our senses and support explanations at qualita- tive and quantitative levels.4 Students often struggle with understanding and using the triplet concept. 3D models of molecules represent the submicroscopic representation, 168 Acta Chim. Slov. 2022, 69, 167–186 Dolničar et al.: The Students’ Perceptions Using 3DChemMol ... the use of which is important to bridge the gap between the macroscopic and symbolic levels.7 Kozma and Russell8 defined representational com- petence in science education as a set of five distinct abili- ties of students: to analyze the features of representations, transform between representations, create new representa- tions, explain the usefulness of representations, and ex- plain the advantages of representations. Activities aimed at improving representational competence support spatial thinking9 which is critical for understanding 3D spatial concepts in STEM (science, technology, engineering and mathematics) disciplines.10 The use of physical and virtual molecular models promotes representational competence11–13 and fosters spatial understanding,14 although the impact of spatial ability on success is influenced by learning strategy and task demands.15 Students who used models were more likely to implement new concepts, transform from 2D to 3D representations, and answer visual-spatial tasks. In the past, physical modeling kits with balls and sticks or mag- nets were used to construct 3D analog models of chem- ical compounds.16–18 Later, molecular modeling software brought chemical visualizations into the digital virtual realm.19–21 Since then, numerous stand-alone and web- based applications for viewing and manipulating chemi- cal structures have become available, such as, ArgusLab, Avogadro, BALLView, Biovia discovery studio visualizer, Chime, Chimera, JME molecular editor, Jmol/JSmol, Os- cail X, Pymol, RasMol, Spartan, SwissPDB Viewer, Tinker, Chemis 3D Molecular Viewer Applet, VMD, Yasara, and others.20,22–26 Some reported course activities and research in- volved the construction or use of physical models by stu- dents.27–32 Thayban et al.33 found that virtual models were more effective than physical model in learning symmetry. On the other hand, the use of physical or virtual molecular models was found to assist students in solving chemistry problems that require spatial thinking.34 Studies at all levels of chemistry education indicate that in order to construct correct mental models of chem- ical compounds, students should be engaged in construct- ing and manipulating three-dimensional (3D) visualiza- tions.35,36 The construction of submicroscopic models is part of representational competencies. Kelly and Akaygun37 suggested that visualizations are too often used only as a method of direct instruction. Instead of being passive observers students should become interactive participants and critical thinkers. In a survey38 that was part of the workshop for molecular visualization in science education researchers, educators, and software developers discussed the role of molecular modeling in college chemistry and were asked about the features of molecular representation and the types of interactions with molecular visualization that most help students. The responses suggested that students should be able to create their own visualizations and interact with existing ones. In some reported course activities, students were us- ing molecular modeling software. Some of the advantages over physical modeling are flexibility in model building, switching between different representations, and accura- cy of structural representations.39 According to Kozma,8 the construction, calculations and manipulation of mo- lecular models support the laboratory practice of synthe- sis by looking at reaction sites and speculating on reac- tion mechanisms. Clauss and Nelsen40 used WebMo and Gaussian to teach students the fundamentals of computa- tional chemistry by performing ab initio and DFT (density functional theory) calculations in an undergraduate lab- oratory course with the goal of gaining a deeper under- standing of their experimental work. Linenberger et al.41 conducted a guided experiment using the student version of Spartan to discover the relationship between structure and molecular properties, e.g., through measurements, calculating dipole moments, and studying electron density potential maps and molecular shapes. Raiyn and Rayan42 reported on the impact of a workshop using ChemDraw in a college chemistry course that significantly improved students’ understanding of 3D structure and polarity, boiling point, and isomerism. Rothe & Zygmunt43 used Gauss View 5 and Gaussian in an undergraduate chemical reaction engineering course to promote understanding of the relationship between molecular properties and mac- roscopic concepts such as internal energy, enthalpy, rate constants, and activation energies. In a web-based chem- istry course, Dori et al.44 gave first-year students the task of using Weblab and IsisDraw to create molecular models, calculate molecular weight, and construct the hybridiza- tion and electric charge distribution of carbon atoms. On the posttest, which required higher-order thinking skills, the experimental students showed better reasoning skills and a better ability to transfer between levels of representa- tion than the control group. Ealy45 introduced molecular modeling using Spartan Pro to a general chemistry labora- tory. Students performed measurements and investigated properties such as symmetry, electrostatic potential, and dipole. The experimental group performed significantly better than the control group, and the test results at the end of the semester also showed that a transfer of knowl- edge had occurred. In an ethnographic study by Kozma,46 students who first conducted experiments in the laborato- ry and then constructed molecular models using Spartan. When using the computer modeling software, students referred to chemical concepts (e.g., atoms, bonds, elec- tronegativity, dipole moment) more frequently than in the laboratory session. Yet, they did not relate the models to the materials they synthesized. Molecular modeling was used by pre-service teachers in combination with class- room materials and mind map tools to learn hydrogen bond.47 Kolar et al.48 suggested the didactic use of com- putational chemistry to create models of amides to illus- trate acid-base properties. Winfield et al.49 have developed activities that incorporate model building in the iSpartan 169Acta Chim. Slov. 2022, 69, 167–186 Dolničar et al.: The Students’ Perceptions Using 3DChemMol ... tool to teach conformations of alkanes. Similarly, Johnson et al.50 reported integrating of iSpartan into the classical organic chemistry laboratory experiment to help students learn about the stability of alkenes. Conformational analy- sis of small molecules using Vega ZZ software was used by Soulère51 in an undergraduate chemistry course. User-friendliness of graphical interfaces to opti- mize small and medium sized molecules has enabled the possibility to introduce computational chemistry tools to the undergraduate level.52 Rodriguez-Becerra et al.53 described the use of educational computational tools on pre-service chemistry teachers, with Avogadro used for model building. Due to the identified deficiency in educational use of molecular modeling in chemistry classes by teachers and/or students,54 molecular modeling was introduced into chemistry education by Aksela et al.,55 developing pedagogical solutions, training mentors, creating teaching materials and investigating their effectiveness. The mode- ling approach was adopted by schools and the experiences were shared in a book.56 The Edumol.fi web application was used.57 1. 2. Tools for Building Molecular Models in Teaching Organic Chemistry At the beginning of this study, we analyzed existing molecular modeling tools for teaching organic chemistry at the university undergraduate level in order to select the most appropriate tool to serve as the basis for the devel- opment of a new tool, 3DChemMol molecular editor.58 Its editing functionality and help tools are described and eval- uated in this article. Some of the external factors influencing the poten- tial for wider adoption of molecular visualization tools for teaching and active learning could be their suitability for a particular level of education (primary/secondary and college), their focus (small molecules, macromolecules, crystal structures), the presence of editing feature (mo- lecular modeling), functionalities (display of properties), and their cost and convenience. The degree of complexi- ty and the usability of the user interface could also play a role. With the advent of web-based technologies (HTML5, CSS, WebGL, canvases, and the use of JavaScript), there has been a shift from standalone applications and web applications requiring plug-ins to readily available web- based tools.59 In terms of availability, molecular modeling tools have been developed that are open source.60 In this study we focus on the software that is suitable for educa- tion, focuses on small molecules, allows molecular mode- ling and is freely available. Some of the tools are compared in Table 1. Due to immediate availability, we limited our choice to web-based applications that do not require in- stallation. These criteria exclude tools such as Spartan20 (proprietary, standalone), Web Doodle Web Components61 (proprietary, web-based), Avogadro62 and Jmol63 (free, standalone), leaving us with mainly web-based tools. We also excluded web tools that are viewers only (e.g. 3dmol. js64) or those that involve creating a 3D model by drawing a 2D structure (e.g. MolView65). The remaining web-based interfaces were based on JSmol,66 a web version of Jmol. They included interfaces for the creation of 3D models: CheMagic,67 MolCalc68 and 3DChemMol.58 The latter was developed by the first author of this study. The original JSmol editing module is menu-based, cumbersome to use, and lacks a functional undo and help function. CheMagic has implemented both, but the functionalities of the tool (as in JSmol) are all visible at once, which can be distract- ing if you are only focused on editing. MolCalc’s editing feature creates the input for the computational software. It is simple and efficient but uses only basic editing func- tions. 3DChemMol was designed to structure the JSmol functionalities into multiple toolbars accessible from the main menu, including editing, with additional interactive functions with toolbars for model exploration (e.g., elec- tronegativity, measurement, symmetry, creating confor- mations and isomers, model comparison, and exercises). It was chosen for our study because the new editing interface is intended to resemble that of familiar 2D editing tools. 1. 3. Motivation and Aims of the Study The aim of this study was to evaluate the newly de- veloped 3DChemMol molecular editor tool and to investi- gate university students’ first encounter with a 3D struc- ture editing tool while performing three specific activities Table 1: Characteristics of selected freely available user interfaces for 3D model building Tool name Type Technology GUI elements Characteristics Avogadro S C++, Qt Menus, toolbars, dialogs Editing dialog, mode switching for rotation Jmol S Java Menus, toolbar, dialogs Editing menu on right click JSmol (original) W JavaScript, JQuery Menus (right click) Editing menu on right click CheMagic (JSmol) W JavaScript, JQuery Dashboard buttons All tool functionalities at once, editing buttons, undo, help MolCalc (JSmol) W JavaScript, JQuery Buttons Basic editing (adding, deleting), input for computational software 3DChemMol (JSmol) W JavaScript, JQuery Menus, toolbars Editing toolbar, undo, help Types: S = standalone, W = web-based 170 Acta Chim. Slov. 2022, 69, 167–186 Dolničar et al.: The Students’ Perceptions Using 3DChemMol ... for creating and editing molecular models. The research questions were as follows: – RQ1: How successful were students in performing sim- ple chemistry activities using the 3DChemMol molecular editor, and how was their success related to the time re- quired and the perceived difficulty of the activity? – RQ2: What types of difficulties did students encounter when performing activities with 3DChemMol molecular editor? What was the cause and a possible remedy? – RQ3: How did students use the different types of help available in 3DChemMol molecular editor and addition- al support when they encountered problems? 2. Methods 2. 1. Participants A total of 54 students of the University of Ljubljana participated in the study. They were enrolled in the second year of study (aged 20 to 21) at the Faculty of Education (17 students, 31.5%) or the Faculty of Health (37 students, 68.5%) in the study year 2020/21. They had already taken basic chemistry courses in general and inorganic chem- istry; therefore, basic knowledge and understanding of chemistry principles and basic ICT skills were assumed. Introduction to building 3D models of chemical com- pounds was designed as a foundation for organic chem- istry and other higher level chemistry courses that follow in their program of study. Apart from the field of study, there were no additional differences between the groups, important for the purpose of this research. 2. 2. Materials 2. 2. 1. Model Building Tool The editing module of the web-based tool 3DChem- Mol molecular editor (http://www2.arnes.si/~supddol- n/3dchemmol), previously created by the author of this study,58 was used to construct the molecular models. The tool is based on JSmol software for visualization and edit- ing of 3D molecular models. Model creation is performed in 3D using a graphical user interface consisting of the model window and toolbar (Figure 1). The tool contains basic model building functionalities, but also some ad- vanced features that allow the creation of different confor- mations and isomers. The available model interactions (e.g., clicking or drag- ging on atoms/bonds) depend on the current action mode. There are four atom action modes (add/edit, delete, move, invert-substitute switch) and three bond action modes (add/ edit, delete, rotate around bond). Switching between action modes is done by selecting a mode from the list. One of the additional elements implemented in the tool is the Undo/Redo function, which did not work in the original JSmol application. Four types of help are integrated and available at all times: a) status indicator of the currently available action mode, displayed at the bottom of the model window (op- tional), b) explanations of button actions when hovering the mouse over them, c) help menu with image and text explanations of the toolbar, d) video tutorial with examples of structure building, also available from the help menu. One of the standard functions of model building is geometry optimization. The tool also allows to quickly cre- ate an image from the model window. 2. 2. 2. Problem Set Three simple activities were designed to guide stu- dents in building and editing models using our tool. Each activity required students to create or edit a specific mole- cule with a limited number of actions. – Activity 1: Build a simple model of the molecule – buta- noic acid (new model, add/change atoms, change bond type). This activity did not require any change in action mode – all the functions needed to build a model were already present. – Activity 2: Convert from one to two models of the mol- ecules – hexane to ethene and butane (delete bonds, de- lete atoms, manually add hydrogen atoms, change bond type). – Activity 3: Convert from a non-cyclic to a cyclic model of the molecule – glucose (add bonds, rotate branches around a bond, change bond type). The full list of steps for each activity can be found in Table 2. All activities included common features such as changing the bond type (with some differences) and auto- matic geometry optimization. At the end of each activity, students had to create an image of the final model of the molecule. Time for each activity was not limited. Some steps required a simple click on a toolbar but- ton, while others required direct interaction with the mod- el or a combination of both (Table 2). The model interac- tions available depended on the current action modes.Figure 1: User interface of the 3DChemMol molecular editor 171Acta Chim. Slov. 2022, 69, 167–186 Dolničar et al.: The Students’ Perceptions Using 3DChemMol ... For each activity, students were provided with the editing tool interface, which contained the initial model on the left half of the screen and activity instructions in Google Forms on the right half (Figure 2). The activity in- structions consisted of a) general information about the availability of free model rotation, undo/redo functions, and various types of help; b) an image of the 3D output model (which was also displayed in the interface); c) a Table 2: Steps for each activity with the required interaction with the toolbar and the 3D model Interaction with the toolbar Interaction with the 3D model Step # Step content Button Type Mode Atom Atom Bond click change change click drag click Activity 1: Building a simple model of the molecule 1 New model x 2 Adding C atoms x 3 Adding heteroatoms x x 4 Changing the bond type x 5 Model centering x 6 Geometry optimization x 7 Creating an image x Activity 2: Converting one model into two models of the molecules 1 Deleting bonds x x 2 Changing the bond type x x 3 Deleting atoms x x 4 Adding hydrogen (manually) x x x 5 Geometry optimization x 6 Creating an image x Activity 3: Converting from a non-cyclic to a cyclic form of the molecule 1 Adding a bond x 2 Changing the bond type x 3 Geometry optimization x 4 Rotating a branch around the bond x x 5 Geometry optimization x 6 Creating an image x Figure 2: Activity display for the first activity (left: interface for model building, right: activity instructions) 172 Acta Chim. Slov. 2022, 69, 167–186 Dolničar et al.: The Students’ Perceptions Using 3DChemMol ... short, annotated video tutorial explaining relevant actions on another example model; d) step-by-step instructions on how to build the target model, which referred to indi- vidual actions rather than elements of the interface; e) an image of the 3D target model. Students could scroll up and down through the instructions. 2. 2. 3. Students’ Self-evaluation Questionnaires For each activity, a self-evaluation questionnaire was included at the end of the activity instructions (Google Forms) with the following items/questions: – degree of activity completion – completion level (Likert scale 1–5: 1 = started, 5 = fully completed); – time spent on the activity (in minutes, as reported by students); – perception of activity difficulty – perceived difficulty level (Likert scale 1–5: 1 = easy, 5 = difficult); – type(s) of help used (multiple choice: a) video tutorial (single view), b) video tutorial (multiple views), c) hov- er on toolbar, d) current action status, e) help menu); – other actions used (multiple choice: a) free view rota- tion, b) undo, c) redo, d) restart activity); – severity of difficulties encountered for each step of the activity – step difficulty level (Likert scale 1–5: 1 = no difficulties, 5 = severe difficulties); – difficulty description (text). Prior to the study, two researchers (the co-authors of the study) optimized the instrument by performing a face validity69 check. They completed the suggested activ- ities and reviewed the questionnaires and then suggested changes and adjustments. 2. 3. Data Collection The testing was conducted in May 2021 and was su- pervised by the authors in an online format. The Zoom videoconferencing tool and a web browser were used to display the tool and instructions with the questionnaires. Students consented to data analysis. Prior to testing, a standardized introductory pro- tocol was used that included clarification of purpose, in- structions, voluntary participation, and acknowledgement of participation. The research was approved by the com- petent authorities of University of Ljubljana. None of the students had any prior experience with the tool. The teach- er first gave a general introduction/demonstration of the entire 3DChemMol molecular editor. Students had access to the interface. Students were then given links to the ac- tivities. After completing each activity, they completed the questionnaire and moved on to the next activity. 2. 4. Data Analysis Data from the students’ self-evaluation questionnaires were collected in Google Spreadsheets and transferred to Excel and Statistical Package for the Social Sciences (SPSS), version 26 for analysis, which was performed for each of the three activities. – Mean scores were calculated for continuous and or- dinal questionnaire items, including completion lev- el, time spent, perceived activity difficulty level, and step difficulty levels. Step difficulty mean was also calculated for each activity. The two multiple-choice questions (type of help used, other items used) were transformed into multiple dichotomous variables, one for each response (1 if the response was selected and 0 if it was not). Means were calculated for each response. – The distributions of the variables were examined using the frequency of the results expressed as a percentage of students. This was done for ordinal items and multi- ple-choice responses, and also for time spent on activity, where scores were first divided into five groups. – Correlations between parameters were calculated using Spearman correlation coefficient (rs). – The open-ended questions from the student self-eval- uation questionnaires were also recorded in Google Spreadsheets and transferred to Excel. The students’ responses were coded using a coding table. The coding table was derived from a qualitative analysis of 20% of the questionnaires (n = 11 participants); the reliability of the coding was ensured by independent coding by two researchers (the authors of this article). Finally, both evaluations were contrasted at the points where differ- ences occurred and, after consideration, the more appro- priate one was selected. Altogether, 99% reliability was achieved. 3. Results and Discussion 3. 1. Completion Level of the Activities Completion level of the activities was measured by the self-evaluation questionnaire. For each activity, the time spent on the activity and the perceived level of diffi- culty were also reported. 3. 1. 1. Means and Distributions Students were relatively successful in completing the simple chemistry activities, as measured on a Likert scale of 1 to 5. The average score was above 4 for all 3 activities (Figure 3). For the first two activities, the completion level was very high with 91 and 96% of students reporting that they completed the activity, compared to only 53% for the third activity (Figure 4). The completion time, measured in minutes, showed that the majority of students took between 3 and 5 minutes for each of the first two activities, while most students took 6–10 minutes for the last activity (Figure 5), with a signifi- cantly higher mean (Figure 3). 173Acta Chim. Slov. 2022, 69, 167–186 Dolničar et al.: The Students’ Perceptions Using 3DChemMol ... Figure 3: Mean scores with SD (whiskers) by activity for activity completion level (1–5), time spent on activity (min), and perceived activity difficulty (1–5) Figure 4: Distribution of activity completion levels by activity (5 = fully completed) Figure 6: Distribution of perceived activity difficulty by activity (5 = difficult) Figure 5: Distribution of time spent on activity by activity Perceived difficulty, expressed on a Likert scale of 1–5 (5 being difficult), showed that the second activity was considered the easiest with a mean of 1.81, and the third activity was considered the most difficult, with a mean of 3.18 (Figure 3). The most common response for activity 1 was difficulty level 2, for activity 2 was difficulty level 1, and for activity 3 was difficulty level 3 (Figure 6). 3. 1. 2. Correlations No significant correlation was found between time spent and activity completion (Table 3). Some students took more time, but still completed the activity. An exam- ple is a comment on activity 1: “I had trouble adding atoms at first but figured it out after a few minutes.” As expected, time spent correlated positively with perceived difficulty (most strongly for the second – overall easiest activity). Students who spent more time on the activity perceived it to be more difficult. The negative correlation between completion and perceived difficulty was significant for the third – the hardest overall activity – suggesting that stu- dents who did not complete the activity perceived it to be more difficult. For example, a student’s comment was: “It is difficult to have spatial orientation.” The lower correlation between perceived difficulty and completion level for the first two activities was due to the high completion levels for these activities. Similar correlations between perceived difficulty as a determinant of Web search performance and time have been found in a study by Kim.70 Table 3: Spearman correlations between completion level (Compl.), time spent (Time) and perceived difficulty of activities (Perc. diff.) Param. Compl. Time Perc. diff. Activity 1: Building a simple model of the molecule Compl. 1.000 Time –0.069 1.000 Perc. diff. –0.239 0.404b 1.000 Activity 2: Converting one model into two models of the molecules Compl. 1.000 Time –0.081 1.000 Perc. diff. –0.266 0.584b 1.000 Activity 3: Converting from a non-cyclic to a cyclic form of the molecule Compl. 1.000 Time –0.158 1.000 Perc. diff. –0.469b 0.435b 1.000 bp < 0.01 174 Acta Chim. Slov. 2022, 69, 167–186 Dolničar et al.: The Students’ Perceptions Using 3DChemMol ... 3. 2. Difficulties During the Activities 3. 2. 1. Mean Scores by Activity The step difficulty mean for each activity reflects the average amount of difficulties students encountered during steps of an activity. The scores (Figure 7) show the same trend as the time spent and perceived difficulty of the activities (Figure 3). Students reported the greatest step difficulty mean on the third activity and the smallest on the second activity. Means ranged from 1.62 to 1.91, which is relatively low given the Likert scale of 1 and 5. For all activities, some students specifically stated: “No problems,” and several others made no comment. Mean scores are low due to the proportion of steps that are not problematic and those that are less problematic. Examples of repeated com- ments in all activities related to some technical difficulties were: “I can’t save the image.” Figure 7: Step difficulty mean with SD (whiskers) by activity (1 = no difficulties, 5 = severe difficulties) 3. 2. 2. Mean Scores by Interaction Type In the previous section the steps were grouped by activities. Here we grouped steps in multiple ways and calculated step difficulty mean for each group. The grouping in Table 4 by type of interaction shows that bond interactions caused more difficulties than atom in- teractions. Toolbar interactions with button click were the least problematic. Table 4: Step difficulty mean by interaction type Interaction type Step diff. mean Toolbar button click 1.48 Atom interaction 1.76 Bond interaction 1.86 Steps with atom and bond interactions were also classified into four groups (Table 5). Actions that re- quired selection of the atom or bond type on a tool- bar button prior to direct interaction with the model caused fewer difficulties than those that did not require a preceding action on the toolbar. On average, the most difficult actions were those that required a change of ac- tion mode (selection on the toolbar from a list of modes, e.g., add/change, delete). The action requiring a combi- nation of type and mode change was also deemed more difficult. Table 5: Step difficulty means for direct interaction with the model, depending on the preceding action Preceding action Step diff. mean Button type change 1.54 No action 1.80 Button mode change 1.95 Button type + mode change 1.98 Another classification of steps was applied to direct interactions with atoms and bonds: clicking, dragging and repeated actions (Table 6). Repeated mouse clicking caused the most difficulties, followed by mouse dragging. A single mouse click on a bond or on atom was the least problematic. Repeated clicking was related to geometry changes in our case. Table 6: Step difficulty means in direct interaction with the model, depending on the type of mouse interaction and repetition Direct interaction type Step diff. mean Mouse click 1.58 Mouse drag 2.07 Mouse click + repetition 2.90 The last grouping of atom and bond interactions concerned geometry change (Table 7). The fewest diffi- culties arose from automatic geometry optimization. No direct interaction with the model was required. Actions where no significant geometry change occurred (nothing added, no automatic hydrogen adjustment) were consid- ered less problematic. The most difficulties occurred when the geometry was changed, highlighting the importance of spatial abilities. Table 7: Step difficulty means when interacting directly with the model, depending on the type of geometry change Type of geometry change Step diff. mean Geometry optimization 1.35 Small geometry change 1.59 Significant geometry change 1.96 175Acta Chim. Slov. 2022, 69, 167–186 Dolničar et al.: The Students’ Perceptions Using 3DChemMol ... 3. 2. 3. Scores by Step Step difficulty levels for each step of the three activ- ities, presented in Table 8, were ranked from 1 (easiest) to 18 (most problematic). Scores for individual steps ranged between 1.21 and 2.90. Total step difficulty mean was 1.66. Activity 1: Building a model of butanoic acid. The eas- iest steps involved two actions available through a simple button click: creating a new structure (ranked 4 out of 18) and geometry optimization (ranked 5). Moderate difficul- ties were encountered in adding C atoms (rank 9) to build the main skeleton of the structure. This step is crucial. Some of the students reported difficulties, such as: “When click- ing with the mouse, an atom was deleted instead of added.” This was because the mouse was moved when clicking on a hydrogen atom. Instead, the “drag” event was registered, which in Jmol is associated with deleting an atom when ap- plied to a hydrogen atom. Comments also related to add- ing heteroatoms (rank 13): “I can’t position the chain as it is shown in the result.” and “Sometimes atoms are added in strange ways.” Another comment: “In the beginning, I had a lot of problems with adding atoms unevenly.” Students were paying attention to structure but not configuration. Adding and replacing atoms only required clicking on existing atoms. There was not much chance for error, so “strange ways” and “unevenly” likely refers to configura- tions that result in isomers of the target structure. In this first activity, students have not yet learned how to make configuration changes. Adding atoms correctly required good spatial orientation. There were some difficulties with centering the model (ranked 12). Comment: “I had trou- ble centering the model until I found the centering button. It would be beneficial if centering was automatic because centering has to be applied repeatedly when building larger structures.” This difficulty could have to do with fa- miliarity with the center button, but students also forgot that they could not only rotate the model during model construction but also zoom it out. The zoom button was not part of the editing toolbar, but was an available mouse shortcut (mouse wheel). Surprisingly, most of the difficul- ties with this activity occurred when it came to changing the bond type (ranked 15), which should be quite simple by just clicking on a bond to increase its order. Increasing the bond order was not included as a toolbar button but was part of the default add/delete action mode. There was no need to change the action mode. The comment “The number of hydrogens doesn’t automatically adjust.” sug- gests that students tried to use a different method where they selected the bond type and clicked on a bond. This process does not currently adjust the hydrogens. Students did not know the shortcut even though it was shown in the introductory video. The two methods should be made compatible. Creating an image (ranked 14) also caused dif- ficulties for some students, as expressed in a comment: “I can’t convert to an image. Numbers appear instead.” The reason here was that some system configurations automat- ically generated a text file with the structure in mol format Table 8: Steps for each activity with interaction types, step difficulty levels and ranks Step # Step content Button, Type, Mouse click, Geom. Inter. Step Step diff. atom, bond mode chg . drag, rep. chg. type* diff. level rank Activity 1: Building a simple model of the molecule 1 New model c c 1.44 4 2 Adding C atoms a – k y a 1.52 9 3 Adding heteroatoms a t k y t+a 1.67 13 4 Changing the bond type b – k y b 1.93 15 5 Model centering c c 1.63 12 6 Geometry optimization c g c 1.46 5 7 Creating an image c c 1.69 14 Activity 2: Converting one model into two models of the molecules 1 Deleting bonds b m k n m+b 1.49 8 2 Changing the bond type b t k n t+b 1.40 3 3 Deleting atoms a m k n m+a 1.47 6 4 Adding hydrogen a tm d n tm+ad 1.98 16 5 Geometry optimization c g c 1.21 1 6 Creating an image c c 1.47 7 Activity 3: Converting from a non-cyclic to a cyclic form of the molecule 1 Adding a bond a – d y ad 2.16 17 2 Changing the bond type b – k y b 1.57 11 4 Rotating a branch b m kr y m+br 2.90 18 3&5 Geometry optimization c g c 1.37 2 6 Creating an image c c 1.53 10 * Key to interaction types: c – toolbar button click, a – atom interaction, b – bond interaction, t – button type change, m – button mode change, k – mouse click, d – mouse drag, r – repetition, g – geometry optimization, n – small geometry change, y – significant geometry change 176 Acta Chim. Slov. 2022, 69, 167–186 Dolničar et al.: The Students’ Perceptions Using 3DChemMol ... instead of the image file. This technical issue needs to be addressed and fixed in the future. The issue mentioned in a comment: “I had no particular problems constructing the model, but the angles between atoms aren’t the same.” was either related to configuration or the student did not optimize the model geometry correctly. A comment from a student who reported no individual difficulties was: “The correct tool is not visible.” In this case, the comment could refer to shortcuts built into the editor that are not explicitly visible in the toolbar (e.g., changing the bond type in gen- eral mode). This activity did not require any action mode changes but some students had expected them. Activity 2: Splitting the model of hexane into models of butane and ethene (cracking). In this assignment, sev- eral students reported, “I had no problems.” Geometry optimization and bond change were considered the easi- est steps by students (ranked 1 and 3, respectively). Here, bond change was performed by first selecting the bond type from the toolbar (no shortcut used). This method did not automatically adjust the number of hydrogen atoms, but unlike the first activity, the subsequent steps were de- signed to solve this problem. Deleting atoms and bonds did not cause too many difficulties (rank 6 and 8), how- ever, a student commented: “Problems switching between adding and deleting atoms.” The reason is that the delete function is not immediately visible but is in a list of action modes in the toolbar. The most problematic part of the ac- tivity was the manual hydrogen addition (ranked 16). It consists of selecting the hydrogen atom type in the toolbar and then dragging out an existing atom with the mouse. A typical comment was: “Problems with adding the sin- gle H atom due to the fact that addition and modification appear together.” As with the first activity, more than one action is available in Add/Change mode, depending on the type of interaction (click, drag), the object of interaction (atom, bond), and sometimes the type of atom (hydrogen, non-hydrogen). There is no separate button or selection on the toolbar for this action. As with the first activity, students may have been looking for a separate mode and could not find the button. Adding the H atoms by drag- ging was otherwise covered in the tutorial video and also shown in the action mode text help at the bottom of the screen. Interestingly, some of the difficulties were relat- ed to a functionality not being available. A student com- mented: “The button to move one of the models did not work, so I could only rotate the left model.” The reason is that moving and rotating individual models is not possi- ble in edit mode. Only the entire view can be rotated. This functionality could be incorporated in the future, as it is already present in other toolbars of this software. Image creation difficulties were not rated as severe (rank 7) for this activity, although the same technical obstacles were encountered. Comment: “I could not save the image, so I took a screenshot instead.” Perhaps the severity changed or there were other novice difficulties saving the file in the first activity. Activity 3: Converting the noncyclic form to a cyclic form of glucose. The only unproblematic action in this activity was geometry optimization (rank 2). Changing the bond type from double to single bond was perceived moderately difficult (rank 11). Some students remem- bered the shortcut from the first activity, others did not. A typical comment was: “I had a problem changing the bond.” Creating an image was also still an issue (ranked Table 9: Summary of the most frequent difficulties with example student comments Act. # Step # Theme / Step Category* Possible issue Step diff. rank** Example student comment 1 2 Ading C atoms a Interface 9 “When clicking with the mouse, an atom was deleted instead of added.” 3 Adding heteroatoms a Spatial ability 13 “I can’t position the chain as it is shown in the result.” 4 Changing the bond b Interface 15 “The number of hydrogens doesn’t automatically adjust.” 5 Model centering c Interface 12 “I had trouble centering the model until I found the centering button.” 7 Creating an image c Technical 14 “I can’t convert to an image. Numbers appear instead.” 2 4 Adding hydrogen a Interface 16 „Problems with adding the single H atom due to the fact that addition and modifi- cation appear together.“ 3 1 Adding a bond b Interface 17 „I didn‘t know how to connect the O atom to the other side...“ 4 Rotating a branch b Spatial ability 18 “One of the groups was always oriented in the wrong direction.“ „It is difficult to have spatial orientation.“ * Key to categories – interaction types: a – atom interaction, b – bond interaction, c – toolbar interaction ** Key to step difficulty rank: 1 = easiest, 18 = most difficult among all steps 177Acta Chim. Slov. 2022, 69, 167–186 Dolničar et al.: The Students’ Perceptions Using 3DChemMol ... 10) to some, with a comment: “I couldn’t save the image.” Adding a bond between two existing atoms and especially rotating a branch around a bond were the two most prob- lematic steps overall (ranked 17 and 18). The latter step had a difficulty of 2.9, which is one grade above the former step at 2.16. Some students did not know how to connect two atoms, as evident in a comment: “I didn’t know how to connect the O atom to the other side and what the correct rotation was.” or a comment: “Having trouble connecting the structure properly.” Dragging was required in Add/ Modify mode, so no mode change was required in this step and no toolbar button was available. The appropriate ac- tion was demonstrated in the tutorial video and shown in the action status help at the bottom of the screen. Perhaps the model itself was part of the problem. It needed to be properly oriented so that the atoms could be reached with the mouse. Good spatial orientation could be related to this action. This was even more evident when the branch was rotated, as a student wrote in a comment: “I couldn’t get the model aligned the way it was in the picture. One of the groups was always oriented in the wrong direction.” or another student “I couldn’t place the atoms in the posi- tion shown in the resulting image.” The branch rotations around the bond were done in 60-degree increments. Stu- dents had to determine the correct degree of rotation by applying (repeating) the action the appropriate number of times. Another comment “It is difficult to have spatial ori- entation.” suggested that this activity required more spatial orientation than the first two activities. Comment, “It was difficult to begin the activity. Watching the tutorial video was crucial. Still, I had trouble rotating the bonds.” The first sentence (beginning of the activity) refers to the bond addition. Although this activity proved to be the most dif- ficult overall, four students indicated, “No problems.” This is consistent with the research of Harle and Towns who noted that rotational transformations were among the tasks that students had particular difficulty with.71 The most typical themes and categories of students’ difficulties that emerged from the above analysis are list- ed in Table 9. Of the eight themes, three each related to atom and bond manipulations and the remaining two to toolbar interaction. Two of the issues are probably relat- ed to the students’ lack of spatial orientation, which could be improved through training. Another requires solving a technical issue. The rest could be possibly avoided/fixed by redesigning parts of the user interface (e.g. even more visi- ble action status, separation of actions that are too similar, separate buttons instead of mode selection). 3. 2. 4. Correlations There are significant correlations between most steps within an activity in terms of difficulties (Tables 10–12). Mean of step difficulties is included as step mean. In the Table 10: Spearman correlations between step difficulty levels within Activity 1 Step Step Step Description 1 2 3 4 5 6 7 mean 1 New model 1.000 2 Adding C atoms 0.620b 1.000 3 Adding heteroatoms 0.479b 0.646b 1.000 4 Changing the bond type 0.441b 0.508b 0.482b 1.000 5 Model centering 0.408b 0.473b 0.414b 0.505b 1.000 6 Geometry optimization 0.426b 0.587b 0.392b 0.343a 0.718b 1.000 7 Creating an image 0.272a 0.408b 0.255 0.238 0.281a 0.506b 1.000 Step mean 0.629b 0.690b 0.625b 0.752b 0.690b 0.631b 0.568b 1.000 a p < 0.05, bp < 0.01 Table 11: Spearman correlations between step difficulty levels within Activity 2 Step Step Step Description 1 2 3 4 5 6 mean 1 Deleting bonds 1.000 2 Changing the bond type 0.562b 1.000 3 Deleting atoms 0.687b 0.740b 1.000 4 Adding hydrogen 0.401b 0.283a 0.332a 1.000 5 Geometry optimization 0.423b 0.672b 0.439b 0.351a 1.000 6 Creating an image 0.185 0.313a 0.254 0.119 0.357b 1.000 Step mean 0.675b 0.657b 0.698b 0.765b 0.537b 0.474b 1.000 a p < 0.05, bp < 0.01 178 Acta Chim. Slov. 2022, 69, 167–186 Dolničar et al.: The Students’ Perceptions Using 3DChemMol ... final step – saving the image of the result – the correlations are not as strong, as the difficulties with image creation were largely a technical issue. Difficulties with rotating a branch around a bound (third activity) also do not cor- relate with all other steps of the activity, as many students had difficulties in this step. 3. 2. 5. Correlations with Completion Level of the Activities The step difficulty mean for each activity correlated positively with time spent and perceived activity difficul- ty and negatively with activity completion (Table 13). The completion level for the second activity was very high, so the correlation with step difficulty mean was not signifi- cant. 3. 3. Help Tools Used During Activities The forms of help available included the tutorial vid- eo, the help menu, the description of the toolbar button when the user hovers over it, and the description of the ac- tions currently available on the structure (atom and bond actions). If students made mistakes, they could undo and Table 13: Spearman correlations of step difficulty mean with completion level (Comp.), time spent (Time) and perceived activity difficulty (Perc. diff.) Step Description Comp. Time Perc. diff. Activity 1: Building a simple model of the molecule 1 New model –0.419b 0.421b 0.522b 2 Adding C atoms –0.421b 0.422b 0.503b 3 Adding heteroatoms –0.235 0.512b 0.553b 4 Changing the bond type –0.135 0.503b 0.290a 5 Model centering –0.231 0.585b 0.353b 6 Geometry optimization –0.290a 0.458b 0.356b 7 Creating an image –0.121 0.224 0.292a Mean –0.334a 0.620b 0.539b Activity 2: Converting one model into two models of the molecules 1 Deleting bonds –0.305a 0.410b 0.307a 2 Changing the bond type –0.341a 0.488b 0.518b 3 Deleting atoms –0.296a 0.494b 0.492b 4 Adding hydrogen (manually) –0.041 0.467b 0.496b 5 Geometry optimization 0.083 0.506b 0.428b 6 Creating an image –0.152 0.205 0.276a Mean –0.215 0.558b 0.599b Activity 3: Converting from a non-cyclic to a cyclic form of the molecule 1 Adding a bond –0.104 0.305a 0.280a 2 Changing the bond type –0.333a 0.298a 0.344a 4 Rotating a branch –0.445b 0.409b 0.468b 3 and 5 Geometry optimization –0.115 0.203 0.236 6 Creating an image –0.211 0.063 0.179 Mean –0.424b 0.493b 0.508b a p < 0.05, b p < 0.01 Table 12: Spearman correlations between step difficulty levels within Activity 3 Step Step Step Description 1 2 4 3 and 5 6 mean 1 Adding a bond 1.000 2 Changing the bond type 0.350a 1.000 4 Rotating a branch 0.276a 0.270 1.000 3 and 5 Geometry optimization 0.285a 0.326a 0.086 1.000 6 Creating an image 0.365b 0.169 0.037 0.331a 1.000 Step mean 0.777b 0.579b 0.630b 0.501b 0.516b 1.000 ap < 0.05, bp < 0.01 179Acta Chim. Slov. 2022, 69, 167–186 Dolničar et al.: The Students’ Perceptions Using 3DChemMol ... redo previous actions. They were free to rotate the models during the construction process. If none of the previous actions helped, they could restart the activity. 3. 3. 1. Distributions Of the above actions with help tools, free rotation and the undo button were used by most students (70–94%) (Figure 8). The frequency of free rotation was lowest in the second activity because fewer configuration changes (de- leting atoms and bonds as opposed to adding them) were made than in the other two activities. Nevertheless, 14% of students reported not rotating the model in the third activity, which involved a larger configuration change when adding a bond to form a ring, as well as rotating a branch around a bond. The number of students who used the undo feature increased by 20% in the third activity, as only 6% of students did without it. This indicates the importance of the undo function, which did not work in the original JSmol interface. Redo function was not used as frequently, although its use increased with each activity and one in four students used it by the third activity. The most commonly used type of help was watching the tutorial video once, followed by the mouse-over button action. About 30% of students reported not watching the video in the first two activities, but in the third activity, the number of multiple video viewings increased significantly: One in three students watched the video more than once, compared to 4–9% in the previous activities. An example of a student comment on this activity is: “Watching the tu- torial video was crucial.” The use of the mouse-over action was comparable in all three activities and was used by less than half of the students. The last two help options (action status and help menu) were used less frequently, increasing from less than 10% in the first two activities to about 15% in the last activity. This could mean that students were not confused about the current action status (work mode) or that they missed the textual status display at the bottom of the screen. Interestingly, they also made little use of the help menu, which could indicate that they found the video tutorials largely sufficient. This is consistent with the con- clusion of a study by Van Der Meij,72 in which video tuto- rials that previewed the training activities were the most effective for learning software. The help menu provided similar information to hovering over the buttons. Finally, the level of activity restarting was low (9%) for the first activity, indicating that building a new structure by adding atoms and changing bonds was not a problem, especially because the undo function was available. This value in- creased slightly in the second activity and significantly in the third activity. Nearly two out of five students estimated that they were too far off course compared to the target model or did not get close enough, so they started over. They were not discouraged and there was no time limit on the activity. In this activity, the importance of good spatial ability was probably most pronounced. Starting over was the chosen strategy. 3. 3. 2. Correlations Interestingly, for all three activities, there was a significant negative correlation between using the video (once) and hovering buttons, suggesting that students who did not watch the video relied on hovering buttons in the toolbar (Table 14). No significant correlation with the four types of help was found for free rotation or the use of the undo button in any of the activities. This could mean that these two functionalities were used by all. In the first activ- ity, the negative correlation with button hovering was also observed for multiple video views. There, the use of redo was positively associated with the help menu and negative- ly associated with watching the video once. In the second activity, use of the help menu was negatively correlated with viewing the video once, indicating that students for whom viewing the video once was sufficient did not use it. With the fewest geometry changes in this activity, students who used free rotation were less likely to use the undo but- ton. In this way, the rotation helped. It is surprising that this was not the case in the third activity, where students could benefit from free rotation even more. There, use of the help menu correlated significantly with other types of help, aside from watching the video once. Students who Figure 8: Distribution of actions with help tools by activity 180 Acta Chim. Slov. 2022, 69, 167–186 Dolničar et al.: The Students’ Perceptions Using 3DChemMol ... needed help used all available types of help. Students who restarted the activity were also more likely to consult the help menu and watch the video multiple times. 3. 3. 3. Correlations with Completion Level of the Activities For the first two activities, activity completion cor- related negatively, and perceived difficulty correlated positively with help menu use (Table 15). Students who did not need to consult the help menu were more likely to complete the activity. Those who did consult the help menu perceived the activity to be more difficult. On the third activity, students who did not have to watch the in- structional video multiple times were more likely to com- plete the activity. Multiple video viewings also correlated positively with perceived activity difficulty. It seems that consulting the static help menu did not help solve the easi- er activities and that the tutorial videos were not sufficient to solve the more difficult activities. One of the possible remedies would be to create help tutorials/videos for in- dividual actions that students found particularly difficult, covering multiple examples. The use of undo correlated with time spent on the first two activities and redo did on the last two activities. Both also correlated positively with perceived difficulty – students who used them found the activities more difficult. With the third activity, the amount of time spent restarting was significantly higher, and these students were less likely to complete the activity they also perceived as more difficult. Starting over did not help enough. 3. 3. 4. Correlations with Difficulties by Activity The difficulty level referenced is the average step dif- ficulty for each activity (step difficulty mean). In the first activity, one video view seemed sufficient for students who reported fewer difficulties overall (Table 16). In the second Table 14: Spearman correlations between actions with help tools Video Video Button Action Help Free Undo Redo Restart once multi hover status menu rotat. button button activity Activity 1: Building a simple model of the molecule Video once 1.000 Video multi –0.416b 1.000 Button hover –0.270a –0.275a 1.000 Action status 0.071 –0.090 0.185 1.000 Help menu –0.152 –0.102 –0.017 –0.090 1.000 Free rotat. –0.168 0.152 0.218 0.135 –0.177 1.000 Undo button 0.006 0.067 0.067 0.184 0.067 –0.205 1.000 Redo button –0.369b –0.090 0.042 0.190 0.398b 0.135 0.029 1.000 Restart activity –0.020 0.118 0.242 0.154 0.118 0.152 0.207 –0.090 1.000 Activity 2: Transformation of one into two models of the molecules Video once 1.000 Video multi –0.058 1.000 Button hover –0.554b –0.154 1.000 Action status –0.084 –0.057 0.072 1.000 Help menu –0.297a –0.064 0.015 0.152 1.000 Free rotat. –0.149 0.130 0.258 0.188 –0.069 1.000 Undo button –0.002 –0.106 0.113 0.009 0.193 –0.301a 1.000 Redo button –0.057 –0.077 0.271a 0.100 0.255 0.135 0.234 1.000 Restart activity –0.057 –0.077 0.156 –0.111 0.065 0.014 0.107 0.012 1.000 Activity 3: Transformation from noncyclical to cyclical form of the molecule Video once 1.000 Video multi –0.610b 1.000 Button hover –0.337a –0.139 1.000 Action status –0.099 0.038 0.259 1.000 Help menu –0.163 0.322a 0.326a 0.298a 1.000 Free rotat. –0.179 0.161 0.132 0.015 –0.006 1.000 Undo button 0.078 0.177 –0.108 0.108 0.100 0.142 1.000 Redo button –0.174 0.098 0.333a 0.269 0.047 0.087 0.139 1.000 Restart activity –0.107 0.315a 0.198 0.002 0.282a –0.164 0.020 0.146 1.000 ap < 0.05, bp < 0.01 181Acta Chim. Slov. 2022, 69, 167–186 Dolničar et al.: The Students’ Perceptions Using 3DChemMol ... activity, more difficulties likely resulted in multiple video views. On the third activity, no correlation was found be- tween difficulty and video views. Difficulty level correlated positively with help menu use on the first two activities. This means that students who had difficulties were more likely to consult the help menu. In both activities where the action mode was changed (activities two and three), the difficulty level correlated with the use of the action sta- tus help. Students who had difficulties consulted this help. Hovering over buttons, free rotation, and restarting the ac- tivity did not significantly correlate with difficulty levels. For all activities, using the undo button, as well as the redo button, were positively correlated with problems. 3. 3. 5. Correlations with Difficulties by Step Activity 1. Consultation of the help menu correlated with step difficulty levels in almost all individual steps (Ta- ble 17). In general, students who had difficulties consulted the help menu. The exception was changing the bond type, where difficulties were inversely correlated with watching the video multiple times. Students who watched the vid- eo multiple times had fewer difficulties with this step. The shortcut for this step was not available in the toolbar but was visible in the action mode description. Those who had difficulties changing the bond type also used the undo and redo buttons. Difficulties with centering the model corre- lated with the use of button hover, indicating difficulty in visually identifying the correct button. Students who used free rotation were less likely to have difficulties with geom- etry optimization. Activity 2. The use of the help menu, as well as the use of the redo button, correlated with difficulty levels in this activity. The exception was manually adding hydrogen, the step that was perceived as the most difficult and, like the shortcut for changing the bond, was not explicitly shown in the toolbar. Undo was used most frequently with the manual hydrogen addition. In this activity, multiple video views correlated with difficulties changing bond type and deleting atoms. Students used multiple videos when they encountered these difficulties. Activity 3. In contrast to the previous two activities, correlations between difficulty and help menu use were absent or low (not significant). For the two most diffi- cult steps, bond addition and branch rotation, there was a low correlation with the use of action status and undo. Two problems were possibly associated with these steps: Table 15: Spearman correlations between actions with help tools and completion level (Comp.), time spent (Time) and perceived difficulty (Perc. diff.) Video Video Button Action Help Free Undo Redo Restart once multi hover status menu rotat. button button activity Activity 1: Building a simple model of the molecule Comp. 0.286a –0.114 0.022 0.090 –0.351b 0.024 –0.070 –0.149 –0.330a Time –0.114 0.079 0.062 –0.207 0.252 0.034 0.286a 0.078 0.033 Perc. diff. –0.108 0.028 0.088 –0.161 0.293a –0.008 0.205 0.033 0.101 Activity 2: Transformation of one into two models of the molecules Comp. 0.0584 0.039 –0.050 0.057 –0.275a 0.085 –0.119 –0.508b –0.215 Time –0.164 0.235 0.059 0.010 0.420b –0.068 0.371b 0.336a 0.185 Perc. diff. –0.106 0.266 0.122 0.129 0.351b 0.090 0.353b 0.502b 0.219 Activity 3: Transformation from noncyclical to cyclical form of the molecule Comp. 0.138 –0.419b 0.050 –0.084 –0.187 –0.187 –0.224 –0.228 –0.315a Time –0.185 0.165 0.308a –0.006 0.371b 0.130 0.147 0.435b 0.349a Perc. diff. –0.195 0.391b 0.106 0.086 0.177 0.181 0.330a 0.379b 0.297a ap < 0.05, bp < 0.01 Table 16: Spearman correlations between actions with help tools and step difficulty mean Activity no. Video Video Button Action Help Free Undo Redo Restart once multi hover status menu rotat. button button activity 1 –0.278a –0.012 0.238 0.117 0.313a –0.068 0.336a 0.243 0.187 2 –0.186 0.287a 0.110 0.305a 0.357b –0.191 0.331a 0.325a 0.151 3 –0.0724 0.169 0.026 0.299a 0.169 0.021 0.296a 0.313a 0.176 ap < 0.05, bp < 0.01 182 Acta Chim. Slov. 2022, 69, 167–186 Dolničar et al.: The Students’ Perceptions Using 3DChemMol ... recognizing the correct action and performing the action correctly. Action status could help with the first part. The tutorial video could help with the second part. Only for branch rotation difficulties was there a low correlation with multiple video views. 4. Conclusions and Implications for Teaching The experience of undergraduate students in con- struction and editing of molecular models of small organic compounds aimed to equip them with the knowledge and ability to create their own presentations and to proceed with further exploration and analysis of model properties in chemistry courses beyond introductory chemistry. The success of the course also depends on the design of the course and the teacher, which would be worth of further study. Manipulation of 3D molecular models has been as- sociated with the development of representational skills, particularly when used to support learning.12,13 Students of all ages encounter problems and misunderstandings when asked to explain chemical phenomena at the submi- croscopic level.73 Molecular modeling has long been used to support experimental work, and to teach fundamental concepts.39 Previous studies have also shown that software usability, expressed as perceived meaningfulness and ease of use, has an impact on learning.74 Spatial ability is an- other factor involved in learning science.75 Its active pro- motion in college-level chemistry and biochemistry has increased, but not to the same extent as other cognitive skills.76 The 3DChemMol molecular editor for building/edit- ing 3D molecular models was used in the study. Features implemented in the user interface allowed for ease of use: a toolbar; separation of the editing function from other functions; the ability to undo and redo changes for mul- tiple steps; various types of help, including video tutorials, button hovering, action status display, and help menu. The 3DChemMol molecular editor incorporating an editing toolbar was tested in a group of 54 university students using three model building/editing activities of varying difficulty: 1) building a simple model, 2) splitting a model into two, 3) creating a cyclic from a non-cyclic structure. Table 17: Spearman correlations between use of help tools and step difficulty level Step Video Video Button Action Help Free Undo Redo Restart once multi hover status menu rotat. button button activity Activity 1: Building a simple model of the molecule 1 New model –0.005 –0.178 0.125 0.015 0.416b 0.014 0.090 0.294a 0.290a 2 Adding C atoms –0.219 –0.047 0.040 –0.043 0.440b –0.149 0.215 0.308a 0.122 3 Adding heteroat. –0.298a 0.176 0.119 –0.065 0.303a 0.088 0.269 0.145 0.090 4 Chg. bond type –0.196 –0.272a 0.237 0.158 0.247 –0.014 0.314a 0.334a 0.050 5 Model centering –0.385b –0.023 0.300a –0.173 0.351b –0.017 0.129 0.227 0.135 6 Geometry optim. –0.192 –0.022 0.117 –0.158 0.520b –0.292a 0.280a 0.158 0.129 7 Creating image –0.048 –0.075 0.170 0.168 0.080 –0.310a 0.139 –0.018 0.115 Mean –0.278a –0.012 0.238 0.117 0.313a –0.068 0.336a 0.243 0.187 Activity 2: Converting one model into two models of the molecules 1 Deleting bonds –0.132 0.120 0.181 0.140 0.272a –0.203 0.158 0.433b 0.119 2 Chg. bond type –0.151 0.390b 0.124 0.204 0.442b –0.039 0.214 0.453b 0.127 3 Deleting atoms –0.078 0.408b 0.120 0.121 0.381b –0.203 0.192 0.329a 0.162 4 Adding hydrogen –0.238 0.137 0.096 0.218 0.2548 –0.097 0.450b 0.118 0.046 5 Geometry optim. –0.356b 0.218 0.098 0.098 0.427b 0.043 0.121 0.299a 0.129 6 Creating image –0.2206 0.185 –0.104 0.243 0.328a –0.122 –0.144 0.214 –0.187 Mean –0.186 0.287a 0.110 0.305a 0.357b –0.191 0.331a 0.325a 0.151 Activity 3: Converting from a non-cyclic to a cyclic form of the molecule 1 Adding a bond 0.032 0.015 0.079 0.203 0.241 –0.096 0.259 0.106 0.059 2 Chg. bond type –0.034 0.069 0.115 0.261 0.162 0.046 0.031 0.408b 0.130 4 Rotating a branch 0.015 0.198 –0.029 0.267 –0.022 0.152 0.228 0.293a 0.250 3,5 Geom. optim. –0.394b 0.061 0.180 0.198 –0.034 0.070 0.123 0.315a –0.185 6 Creating image 0.012 0.071 –0.350a 0.066 –0.196 –0.084 0.123 –0.050 –0.058 Mean –0.0724 0.169 0.026 0.299a 0.169 0.021 0.296a 0.313a 0.176 ap < 0.05, bp < 0.01 183Acta Chim. Slov. 2022, 69, 167–186 Dolničar et al.: The Students’ Perceptions Using 3DChemMol ... In relation with first research question (RQ1), it was found that students were successful overall in using the tool and graphical interface and in completing the ac- tivities. They were excellent on the first two activities and good on the third activity. As expected, the more time they spent on an activity, the more difficult it appeared to them. When they were unable to complete the activity, they per- ceived it to be more difficult. No relationship was found between time spent and success rate. As expected, the average step difficulty of the activity correlated inversely with activity completion and directly with perceived activity difficulty. The more difficulties stu- dents encountered, the more difficult the activity seemed to them; more difficulties also meant more time spent on the activity. When it comes to the second research question (RQ2), it was found that actions for direct model manip- ulation (atoms, bonds) caused more difficulties than using the toolbar buttons. There were more difficulties interact- ing with the model by dragging than by clicking. Steps that involved changing the model configuration or required changing the working mode of the interface were more problematic. It was also found that actions were perceived as easier if they were preceded by a clear mode change. This means that a lot of emphasis needs to be placed on displaying the state of the system so that the user is imme- diately aware of the actions available. The most difficult individual actions reported were 1) rotating a branch around a bond, 2) adding a bond be- tween two existing atoms, and 3) manually adding a hy- drogen atom, but also 4) changing a bond type, 5) creating an image, and 6) adding heteroatoms. Issue #5 was techni- cal in nature. Actions 2 and 3 involved dragging the mouse on or between model atoms. Issues 2–4 had a common denominator: the actions were not implicitly given in the toolbar but were available as part of the default add/change action mode, so students could not discover them without either watching the video tutorial or reading the available actions displayed at the bottom of the screen. Correct ad- dition of bonds and heteroatoms probably requires good spatial orientation, which could be especially true for branch rotation. Action 1 required repeated clicking on a bond until a satisfactory configuration was achieved. The latter was done in 60-degree increments. Difficulties related to the user interface will be ad- dressed in future improvements of the tool, such as high- lighting the action state or even separating actions. Diffi- culties related to spatial abilities could be mitigated by sim- ple video tutorials and exercises focusing on a single issue. Related to the third research question (RQ3), the study indicated that among the four types of help provid- ed, and regardless of reported difficulties, students most frequently watched video tutorials once or used hovering over buttons to indicate button meanings. Use of other forms of help increased only on the third activity, which was perceived as most difficult. Use of the multiple undo feature was high, indicating that it was absolutely neces- sary, and increased with activity difficulty. Similarly, free rotation compensated for the use of the undo function on the second activity. The most difficult and complex activity was found to have a relatively high rate of restarting the activity and re-watching the learning video. When difficulties occurred, students most often used the help menu and the undo/redo actions. Use of the undo function increased for the most difficult steps. For activ- ities/steps that required a mode change, more students consulted the action state that contained the correct an- swer. Individual activities were associated with multiple video views, with video views generally increasing on the most difficult activity. Mouse hovering over the toolbar was used more often when students could not visually identify the correct button. Sometimes the wrong type of help was consulted, such as button hovering (looking for an appropriate action) when no toolbar interaction was re- quired. Reading the action status would have helped there. In other cases, consulting the action status did not con- tain the answer and the tutorial video should be watched. Negative correlations between difficulties and single video views may indicate that the video was a sufficient aid in activity completion for many students. Despite using all the help available (multiple tutori- al video views and restarting the activity), some students were not able to complete the most difficult activity. This could be related to the difficulty of the activity and the need for good spatial orientation and/or mean that the help menus and system status were not fully utilized. Some of the lessons learned in this work, particular- ly the shortcomings of the user interface for editing, have already been implemented and further improvements are planned. Video tutorials became an important part of the help menu. Bond change methods will be unified so that they always include hydrogen adjustment. The toolbar will be upgraded with additional buttons, e.g., for actions that were part of the working modes but were not explicitly present. The action status display will be improved, and video tutorials for individual actions that proved most dif- ficult will be added and immediately available. Alternative help display could be considered, e.g., when you hover over the model parts. The implications for teaching of this study are mul- tifaceted. Using the new tool, students successfully creat- ed 3D models with the help of video tutorials and various types of help. In general, the availability of tools is not yet sufficient for students to use them for learning. Their use must to be encouraged through pedagogical approaches. We suggest that the tool is suitable for direct instruction or self-study. Students can easily use this tool to visualize the structure of chemical compounds during their studies and create images of 3D models to include in their own prod- ucts, such as seminar works, reports, and theses. 3DChemMol could also be used to improve students’ development of chemistry knowledge and representation- 184 Acta Chim. Slov. 2022, 69, 167–186 Dolničar et al.: The Students’ Perceptions Using 3DChemMol ... al skills. Some students may be afraid of special chemistry visualization software because they think it requires spe- cial skills. Because of its simplicity, even students who were not previously familiar with molecular modeling tools and may not have had experience drawing 3D representations or molecules can use it after studying short tutorials. Using 3DChemMol allows students to construct molecular mod- els to visualize the structure of compounds and under- stand their properties, rather than memorizing facts and writing about them. The accessibility of the 3DChemMol tool makes it easy to incorporate into various educational settings. The models created form the basis for further investigation and study of chemistry concepts through display of chem- ical properties. Teachers can use the tool directly in the classroom during lectures or prepare study materials for students in electronic or printed form. For example, vis- ualizations created in 3DchemMol can be part of lectures on various topics. Moreover, it can be used in students’ in- dividual work when they can check their understanding on new examples. Different levels of task difficulty can be accommodated in the tool by the teacher. We are aware that our observational study has some limitations. One of them is the self-reporting nature of the questionnaires. Further insight into students’ behavior and efficiency in building molecular models could be gained by using additional recording and analysis methods, such as eye-tracking, video recording during activity perfor- mance, and structured interviews afterwards. Anoth- er limitation was that the study was focused only on the editing feature of the tool. Future research could include experimental studies such as comparing the usability and effectiveness of other features of the tool (e.g. molecular property display and exploration), comparing it with other 2D and 3D model editing tools, and with building physical models, investigating correlations with other internal or external factors such as students’ spatial skills, representa- tional competence, chemistry knowledge and teaching methods. However, this is already beyond the scope of this study. In further development of 3DChemMol more inter- active online tutorials and exercises tailored to specific chemistry courses could be prepared. Acknowledgements This study was co-financed by University of Ljublja- na (grant no. 704-8/2016-229). The authors would like to thank all of the students who participated in the survey. 5. References 1. K. L. Vavra, V. Janjic-Watrich, K. Loerke, L. M. Phillips, S. P. Norris, J. Macnab, Alta. Sci. Educ. J. 2011, 41, 22–30. 2. I. Vekiri, Educ. Psychol. 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Bergwerf, ACS CHED CCCE Newsl. 2015, https://conf- chem.ccce.divched.org/2015FallCCCENLP9, (accessed: De- cember 20, 2021) 66. R. M. Hanson, J. Prilusky, Z. Renjian, T. Nakane, J. L. Suss- man, Isr. J. Chem. 2013, 53, 207–216. DOI:10.1002/ijch.201300024 67. O. Rothenberger, Virtual Molecular Model Kit, https://che- magic.org/molecules/amini.html, (accessed: December 20, 2021) 68. J. H. Jensen, J. C. Kromann, J. Chem. Educ. 2013, 90, 1093– 1095. DOI:10.1021/ed400164n 69. B. Nevo, J. Educ. Meas. 1985, 22, 287–293. DOI:10.1111/j.1745-3984.1985.tb01065.x 186 Acta Chim. Slov. 2022, 69, 167–186 Dolničar et al.: The Students’ Perceptions Using 3DChemMol ... 70. J. Kim, Inf. Res. 2008, 13, 13–14. DOI:10.1111/j.1469-8749.1971.tb03071.x 71. M. Harle, M. Towns, J. Chem. Educ. 2011, 88, 351–360. DOI:10.1021/ed900003n 72. H. Van der Meij, J. Van Der Meij, Comput. Educ. 2014, 78, 150–159. DOI:10.1016/j.compedu.2014.06.003 73. M. Slapničar, V. Tompa, S. A. Glažar, I. Devetak, J. Pavlin, Acta Chim. Slov. 2020, 67, 904–915. DOI:10.17344/acsi.2020.5908 74. Z. Merchant, E. T. Goetz, W. Keeney-Kennicutt, O. Kwok, L. Cifuentes, T. J. Davis, Comput. Educ. 2012, 59, 551–568. DOI:10.1016/j.compedu.2012.02.004 75. M. Tanweer, Eur. J. Phys. Educ. Sport Sci. 2019, 4, 145–151. DOI:10.5281/zenodo.3407001 76. M. Oliver-Hoyo, M. A. Babilonia-Rosa, J. Chem. Educ. 2017, 94, 996–1006. DOI:10.1021/acs.jchemed.7b00094 Povzetek V članku je predstavljena študija, v kateri je 54 univerzitetnih študentov preizkusilo in ovrednotilo 3DChemMol - no- vo razviti, brezplačni spletni urejevalnik modelov molekul z orodno vrstico. Namen orodja je povečanje reprezentaci- jske kompetence v povezavi s submikroskopskimi predstavitvami. Študenti so programsko opremo uporabili prvič. Z orodjem za gradnjo/urejanje modelov so izdelali modele molekul v naslednjih treh aktivnostih z različnimi stopnjami težavnosti: 1) gradnja preprostega modela (butanojska kislina), 2) pretvorba enega modela (heksan) v dva modela, 3) pretvorba iz neciklične v ciklično obliko (glukoza). Študenti so za izvedbo vsake od aktivnosti potrebovali od dveh do 15 minut. V orodni vrstici urejevalnika 3DChemMol je bilo na voljo več vrst pomoči, ki so študentom olajšale izvajanje aktivnosti, vključno z video vodnikom, prikazom pomoči ob preletu gumbov orodne vrstice z miško, prikazom statusa/ načina dela in menijem pomoči. Na voljo so bile tudi možnosti razveljavitve in ponovne uveljavitve posameznih korakov ter ponovnega začetka celotne aktivnosti. Stopnjo dokončanja aktivnosti, težave in uporabo pomoči smo preučevali s pomočjo vprašalnikov za samoocenjevanje študentov. Urejevalnik molekul 3DChemMol se je izkazal kot koristna pod- pora študentom pri preprostih kemijskih aktivnostih. Študenti so bili pri gradnji modelov uspešni, čeprav so naleteli na nekatere specifične težave, zlasti pri korakih, ki so vključevali prostorske operacije, kot je vrtenje izbranega dela mod- ela molekule okoli vezi. Po mnenju študentov so bila video navodila najprimernejša in najpogosteje uporabljena vrsta pomoči, funkcija razveljavitve pa je bila pri delu bistvenega pomena. Rezultati kažejo, da lahko urejevalnik modelov molekul 3DChemMol učinkovito uporabljamo pri osnovnih predmetih kemije na terciarni ravni izobraževanja, bodisi za poučevanje, samostojno učenje študentov ali druge oblike podpore v pedagoškem procesu. Rezultati in ugotovitve študije bodo uporabljeni tudi za nadaljnjo optimizacijo uporabniškega vmesnika v prihodnjih različicah ovrednotenega orodja. Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License 187Acta Chim. Slov. 2022, 69, 187–199 Abdassalam et al.: Synthesis of New Regioisomers of 5-Nitro-1,4-Naphthoquinone, ... DOI: 10.17344/acsi.2021.7123 Scientific paper Synthesis of New Regioisomers of 5-Nitro-1,4-Naphthoquinone, Evaluation of Antioxidant and Catalase Inhibition Activities Aesha F. SH. Abdassalam,1 Nahide Gulsah Deniz,1 Cigdem Sayil,1,* Mustafa Ozyurek,2 Emin Ahmet Yesil3 and Huseyin Salihoglu1 1 Istanbul University-Cerrahpasa, Engineering Faculty, Department of Chemistry, Division of Organic Chemistry, Avcilar, 34320, Istanbul, Turkey 2 Istanbul University-Cerrahpasa, Engineering Faculty, Department of Chemistry, Division of Analytical Chemistry, Avcilar, 34320, Istanbul, Turkey 3 Istanbul Gedik University, Vocational School, Polymer Technology Programme, Pendik, Istanbul, Turkey * Corresponding author: E-mail: sayil@istanbul.edu.tr Received: 09-04-2021 Abstract The studies on nitronaphthoquinone derivatives are rare in the literature, and the nitro group associated with the aro- matic ring in the quinone system is known to increase the biological activity of naphthoquinone due to its electron-with- drawing properties. In the course of quinone derivatives, the new N(H)-substituted-5-nitro-1,4-naphthoquinones (NQ) as regioisomers were synthesized by reactions of 2,3-dichloro-5-nitro-1,4-naphthoquinone with some heterocyclic ring substituted nucleophiles such as anilines, piperazines, or morpholines, according to a Michael 1,4-addition mechanism. Five NQ regioisomer couples having different functional group (2-chloro-isomers 3, 5, 7, 9 and 13; 3-chloro-isomers 2, 4, 6, 8 and 12) are reported here. All new synthesized compounds were characterized by spectroscopic methods and two-dimensional NMR techniques 1H–1H correlated spectroscopy (COSY). The synthesized NQ regioisomers were evaluated for catalase enzyme inhibitory activities and antioxidant effi- ciency. The synthesized regioisomers were screened for their antioxidant capacity using the cupric-reducing antioxidant capacity (CUPRAC) method. 2-Chloro-3-((2,4-dimethoxyphenyl)amino)-5-nitronaphthalene-1,4-dione (5) showed the highest antioxidant capacity with a 1.80±0.06 CUPRAC-trolox equivalent antioxidant capacity (TEAC) coefficient. Compound 5 also showed strongest catalase enzyme inhibitory activity. The antioxidant capacity results of all 2-chloro regioisomers are higher than the 3-chloro regioisomers. Likewise, also catalase enzyme inhibitory activities results were determined in the same way, except for one regioisomer pair. The catalase was effectively inhibited by the newly syn- thesized compounds, with % inhibition values in the range of 0.71–0.86%. Some of these NQ compounds also showed remarkable antioxidant capacities. Keywords: 5-Nitro-1,4-naphthoquinone; heterocyclic ring; CUPRAC method; Catalase inhibition activity 1. Introduction Naphthoquinone derivatives have been used as anti- bacterial agents for several years already, there are many reports in the 1960s of chemical compounds synthesized with 1,4-naphthoquinone structure and having antibacte- rial properties.1 Later, in the 1980s, there were studies on inhibition of bacteria, along with vitamin K and 1,4-naph- thoquinone. It has been suggested that pharmaceuticals compete in the electronic transport system. Another sug- gestion was the production of ROS and radical semiqui- none and cytotoxicity of naphthoquinone.2,3 It has been shown that amino derivatives such as anilines, piperazines, or morpholines of naphthoquinone improve the biological properties of these derivatives.4 2,2ʹ-[1-(2-Aminoethyl) piperazin-1-yl]-3,3ʹ-dichloro-bis(1,4-naphthoquinone) has showed remarkable antioxidant capacity by using the cupric-reducing antioxidant capacity (CUPRAC) methods and cytotoxic activity against A549 (lung), MCF-7 (breast), DU145 (prostate), and HT-29 (colon) cancer cell lines.4 188 Acta Chim. Slov. 2022, 69, 187–199 Abdassalam et al.: Synthesis of New Regioisomers of 5-Nitro-1,4-Naphthoquinone, ... Furthermore, the amino-1,4-naphthoquinone derivative has been used as synthetic key intermediate or as starting material for synthesis of many compounds.5–10 Some liter- ature reports point to the pro-oxidative effect of 5-hy- droxy-1,4-naphthoquinone (juglone).11,12 Also it has been reported that pyridine, 4H-pyran and thiazolopyrimidine derivatives of 3-hydroxynaphthoquinone have high anti- oxidant activity.13 There are two main ways describing how to prepare the alkyl or arylamino naphthoquinone derivatives. In the first type, the reaction requires a Michael 1,4-addition re- action between the amino compound and 1,4-naphtho- quinone ring to produce 2-amino-1,4-naphthoquinone. The second type involves a nucleophilic substitution reac- tion between the nucleophile with a mono or dihalogenat- ed derivative of 1,4-naphthoquinone to generate the corre- sponding amino derivative.14 The studies on nitronaphthoquinone derivatives are rare in the literature, and the nitro group associated with the aromatic ring in the quinone system is known to in- crease the biological activity of naphthoquinone due to its electron-withdrawing properties and it has been reported that the 2,3-dichloro-5-nitro-1,4-naphthoquinone deriva- tive is more active towards amines and the reaction pro- vides a mixture of two regioisomers.15 Blackburn (2005) has treated 2,3-dichloro-5-nitro-1,4-naphthoquinone with linked resin amine to give very colorful products in high yields.11 The resin under some reducing process and react- ed with 2,3-dichloro-5-nitro-1,4-naphthoquinone in the presence of 2,6-di(tert-butyl)pyridine to give the red res- in-quinone. Treatment with trifluoroacetic acid led to the rapid formation of regioisomeric mixtures of nitroqui- nones. As a comparison between the regioisomers the re- tention factor (Rf) of the 5-nitro isomer was found to be higher than for the 8-nitro isomer. Also, in the 1H NMR, the proton signals of naphthoquinone ring for the first iso- mer are shifted more downfield than the naphthoquinone peaks of the second isomer.15 In a later study, some deriv- atives of 5-nitro-2/3-aminonaphthalene-1,4-dione have been synthesized, studied and tested for biological activi- ties by Samant et al. (2013).16 They found that two regioi- somers of nitronaphthoquinone derivatives, 3-chloro-5-ni- tro-2-((2-(trifluoromethyl)phenyl)amino)naphtha- lene-1,4-dione and 2-chloro-5-nitro-3-((2-(trifluorome- thyl)phenyl)amino)naphthalene-1,4-dione demonstrated strong activity against the sleeping sickness disease (Afri- can human trypanosomiasis) with low cytotoxicity in vit- ro.16 In addition to this, we have previously reported that some regioisomers of 5-nitro-1,4-naphthoquinone con- taining N-substituted group have been synthesized from 2,3-dichloro-5-nitro-1,4-naphthoquinone.10 Catalase (EC 1.11.1.6) as an antioxidant metalloen- zyme capable of degradation of H2O2 is present in many cell types. Lack or malfunction of this class of enzyme may lead to severe disorders such as apoptotic cell death, ane- mia, some dermatological disorders, cardiovascular dis- eases, Wilson disease, hypertension and Alzheimer’s dis- eases.17 Some drugs bind to catalase and elicit enzyme in- hibition; led to H2O2  accumulation and cytotoxicity in cancer cells.18 So, it is important to measure catalase inhi- bition activity in the presence of new catalase inhibitor. In this study, new regioisomeric compounds of 5-ni- tro-1,4-naphthoquinone were synthesized by the reactions of 2,3-dichloro-5-nitro-1,4-naphthoquinone with some heterocyclic ring substituted nucleophiles such as amines, piperazines, or morpholines, according to a Michael 1,4-addition mechanism. Their structures were character- ized by using Fourier transform infrared spectroscopy (FTIR), 1H nuclear magnetic resonance (1H NMR) and two-dimensional techniques (1H–1H correlated spectros- copy (COSY)), attached proton test nuclear magnetic res- onance (APT-NMR), mass spectrometry (MS) and ele- mental analyses. Secondly, these compounds were also tested for their antioxidant capacity in vitro by CUPRAC method and catalase inhibition activities. 2. Experimental 2. 1. Materials and Methods Melting points were measured on a Büchi B-540 melting point apparatus. FTIR spectra (cm–1) were record- ed as KBr pellets in nujol mulls on a Shimadzu IR Prestige 21 model Diamond spectrometer (ATR method). 1H NMR and APT-NMR spectra were obtained using a Varian Uni- ty Inova (500 MHz) spectrometer by using TMS as the in- ternal standard and deuterated chloroform as the solvent. Mass spectra were obtained on a Thermo Finnigan LCQ Advantage MAX LC/MS/MS spectrometer according to ESI probe. Elemental analyses were performed with a Thermo Finnigan Flash EA 1112 elemental analyzer. Prod- ucts were isolated by column chromatography on silica gel (Fluka Silica gel 60, particle size 63–200 μm). Kieselgel 60 F-254 plates (Merck) were used for thin layer chromatog- raphy (TLC). All chemicals were of reagent grade and were used without further purification. Moisture was excluded from the glass apparatus with CaCl2 drying tubes. Sol- vents, unless otherwise specified, were of reagent grade and distilled once prior to use. 2. 2. CUPRAC Assay of Total Antioxidant Capacity The CUPRAC total antioxidant capacity measure- ment method19 depends on the oxidation of an antioxi- dant by cupric neocuproine complex (Cu(II)-Nc) generat- ing yellow-orange colored product (cuprous chelate: Cu(I)-Nc). To a test tube 1 mL CuCl2∙2H2O (10 mM), 1 mL Nc (7.5 mM), 1 mL NH4Ac buffer solution (1.0 M, pH 7), x mL newly synthesized compound, and H2O (1.1 – x mL) (total volume: 4.1 mL) were added in this order and mixed well. The absorbance at 450 nm was recorded 189Acta Chim. Slov. 2022, 69, 187–199 Abdassalam et al.: Synthesis of New Regioisomers of 5-Nitro-1,4-Naphthoquinone, ... against a reagent blank using a Perkin–Elmer Lambda 35 UV–Vis spectrophotometer with a pair of matched quartz cuvettes of 1 cm thickness after 30 min incubation period at room temperature. The calibration graph was then con- structed by plotting the final concentration of each com- pound against the absorbance values which were meas- ured. The result of antioxidant efficiency was expressed as trolox equivalent antioxidant capacity (TEAC) coefficient, mean ±SD of three determinations. 2. 3. Catalase Enzyme Inhibition Activity The catalase enzyme inhibition activity was evaluat- ed by using a modified CUPRAC method described by Bekdeser et al.20 To a test tube 0.5 mL H2O2 (1.0 mM), 1.8 mL H2O,  0.1 mL catalase solution (3.691 U mL–1), and 0.2  mL synthesized compound (1.0  mM, total volume 2.6 mL) were added in this order, mixed and incubated at room temperature for 30 min. After this period, the optical CUPRAC sensor (Cu(II)-Nc-impregnated nafion mem- brane) was taken out and immersed in a test tube consist- ing of 2 mL incubation mixture + 6.2 mL EtOH. After 30 min agitation period, the yellow-orange colored nafion membrane was taken out and its absorbance was meas- ured at 450 nm against that of a blank membrane exclud- ing analyte. 2. 4. Synthesis 2. 4. 1. General Synthesis Procedure 1 for 2,3-Dichloro-5-nitro-1,4-naphthoquinone (1) 2,3-Dichloro-5-nitro-1,4-naphthoquinone (1) was prepared via the following method.21 First, a mixture of the isomer, in which nitro group is substituted at five and six positions, was obtained. These isomers were separated by using column chromatography. The physical properties and characterization methods have been described be- fore.21–23 A complete and unambiguous assignment of 1H shifts was based on a combination of one- and two-dimen- sional techniques (1H and 1H–1H correlated spectroscopy (COSY)), see Figures 2–4. 2. 4. 2. General Synthesis Procedure 2 for Regioisomeric Compounds 2–13 Regioisomeric compounds 2–13 were synthesized by a known previous method.9 2,3-Dichloro-5-ni- tro-1,4-naphthoquinone (1) and nucleophiles (anilines, piperazines, etc.) 1a–f were stirred in 25 mL of absolute ethanol for 3–4 h in the presence of Na2CO3 at room tem- perature. The reaction mixture was monitoring by TLC to establish the end of the reaction. 30 mL of chloroform was added to the reaction mixture. The organic layer was washed with water (3 × 30 mL), and dried with Na2SO4. Evaporator system was used to remove the extra amount of solvent, the residue was then purified by column chroma- tography (Table 1). In the 1H NMR spectra of compounds 1–13, the signals for protons represented H1–3 belong to the naphthoquinone ring (Figure 1). Figure 1. Characterization of quinonoid protons H1–3 of N(H)-sub- stituted 5-nitro-1,4-naphthoquinones 1–13 Synthesis of 2,3-Dichloro-5-nitro-1,4-naphthoquinone (1) Dark yellow crystals, yield: 12 g (42%); Rf 0.3 (EtOAc/ Hexane) (1:6 v/v). M.p. 151–152 °C (lit.19 156–157 °C); 1H NMR (499.74 MHz, CDCl3) δ 7.81 (dd, H1, J = 9.2, 0.98 Hz, 1H, Hnaphth), 7.98 (t, H2, J = 7.8 Hz, 1H, Hnaphth), 8.42 (dd, H3, J = 8.6, 0.98 Hz, 1H, Hnaphth). 13C(APT) NMR (125.66 MHz, CDCl3) δ 121.95, 128.26, 130.04, 131.75, 135.53 (CHarom, Carom), 143.35 (=C-Cl), 143.82 (C-NO2), 172.63, 174.29 (C=O). C10H3Cl2NO4 (Mw 272.04 g/mol). MS [+ESI]: m/z = 271.2 [M]+. Synthesis of 2-(4-(Benzo[d][1,3]dioxol-5-ylmethyl)pip- erazin-1-yl)-3-chloro-5-nitronaphthalene-1,4-dione (2)9 and 3-(4-(Benzo[d][1,3]dioxol-5-ylmethyl)piperaz- in-1-yl)-2-chloro-5-nitronaphthalene-1,4-dione (3)10 Isomer compounds of 2 and 3 were obtained by reac- tion between 2,3-dichloro-5-nitro-1,4-naphthoquinone (1) and 1-piperonylpiperazine (1a) according to the gener- al procedure 2. The mixture was purified by using column chromatography and mixture of ethyl acetate with hexane (1:3 ratio) was used as the mobile phase. Isomer 2: Red solid, Rf 0.80 (EtOAc /Hexane) (1:3 v/v). M.p. 131–132 °C. Isomer 3: Pink solid, Rf 0.73 (EtOAc /Hexane) (1:3 v/v). M.p. 89–91 °C. Isomer 2: Red solid, yield: 0.110 g (20%); Rf 0.80 (EtOAc/Hexane) (1:3 v/v). M.p. 131–132 °C (lit.10 131–132 °C); FTIR (cm−1) ν 3094 (C-Harom), 2912, 2809, 2772, 2659 (C-Haliph), 1679, 1640 (C=O), 1590, 1555 (C=C), 1494, 1438 (C-NO2). 1H NMR (499.74 MHz, CDCl3) δ 2.56 (br s, 4H, Hpiper), 3.52 (s, 2H, CH2), 3.64 (br s, 4H, Hpiper), 5.96 (s, 2H, O-CH2-O), 6.79–6.91 (m, 3H, CHarom), 7.76–7.82 (m, 2H (H1,H2), Hnaphth), 8.34 (dd, J = 9.27, 1.46 Hz, 1H (H3), Hnaphth). 13C(APT) NMR (125.66 MHz, CDCl3) δ 51.15, 53.31 (N-CH2)piper, 62.43 (CH2), 101.12 (O-CH2-O), 108.01, 109.56, 122.60, 127.07, 129.42, 130.49, 132.64, 134.17 (CHarom, Carom), 148.40 (C-NO2), 150.40 (=C-N), 175.74, 179.07 (C=O). Anal. Calcd. for C22H18N3O6Cl (Mw 190 Acta Chim. Slov. 2022, 69, 187–199 Abdassalam et al.: Synthesis of New Regioisomers of 5-Nitro-1,4-Naphthoquinone, ... 455.85 g/mol): C, 57.97; H, 3.98; N, 9.22. Found: C, 58.31; H, 3.59; N, 9.12. MS [+ESI]: m/z = 456.0 [M]+. Isomer 3: Pink solid, yield: 0.223 g (44 %); Rf 0.73 (EtOAc/Hexane) (1:3 v/v). M.p. 89–91°C (lit.10 89–91°C); FTIR (cm−1) ν 3079 (C-Harom), 2905, 2811, 2772 (C-Haliph), 1676, 1644 (C=O), 1590, 1537 (C=C), 1492, 1439 (Carom-NO2). 1H NMR (499.74 MHz, CDCl3) δ 2.52 (br s, 4H, Hpiper), 3.62 (s, 2H, CH2), 3.64 (br,s, 4H, Hpiper), 5.97 (s, 2H, O-CH2-O), 6.81–6.92 (m, 3H, CHarom), 7.67 (dd, J = 9.2, 1.4 Hz, 1H (H1), Hnaphth), 7.81 (t, J = 7.8 Hz, 1H (H2), Hnaphth), 8.21 (dd, J = 9.2, 1.4 Hz, 1H (H3), Hnaphth). 13C(APT) NMR (125.66 MHz, CDCl3) δ 53.26 (N-CH2), 62.39 (CH2), 101.08 (O-CH2-O), 108.07, 109.73, 122.67, 127.63, 129.16, 132.32, 133.70 (CHarom, Carom), 148.35 (C-NO2), 149.33 (=C-N), 173.97, 179.84 (C=O). Anal. Calcd. for C22H18N3O6Cl (Mw 455.85 g/mol): C, 57.97; H, 3.98; N, 9.22. Found: C, 58.21; H, 3.54; N, 9.15. MS [+ESI]: m/z = 456.0 [M]+. Synthesis of 3-Chloro-2-((2,4-dimethoxyphenyl)amino) -5-nitronaphthalene-1,4-dione (4) and 2-Chloro-3- ((2,4-dimethoxyphenyl)amino)-5-nitronaphthalene-1,4 -dione (5) According to the general procedure 2, 0.50 g (1.8 mmol) of 2,3-dichloro-5-nitro-1,4- naphthoquinone (1) was reacted with 0.30 g (2 mmol) of 2,4-dimethoxyaniline (1b) in 25 mL of ethanol at room temperature for 4 hours. The mixture was purified by column chromatography and mixture of ethyl acetate with hexane (1:4 ratio) was used as the mobile phase. Compounds 4 and 5 were obtained as a new regioisomer compounds. Isomer 4: Red solid, Rf 0.52 (EtOAc/Hexane) (1:4 v/v). M.p. 205–206 °C. Isomer 5: Red solid, Rf 0.35 (EtOAc/Hexane) (1:4 v/v). M.p. 198–199 °C. Isomer 4: Red solid, yield: 0.251 g (32%); Rf 0.52 (EtOAc/Hexane) (1:4 v/v). M.p. 205–206 °C; FTIR (cm−1) ν 3316 (N-H), 3083 (C-Harom), 2967, 2917 (C-H), 1675 (C=O), 1585, 1554 (C=C), 1534, 1370 (C-NO2). 1H NMR (499.74 MHz, CDCl3) δ 3.84 (s, 6H, O-CH3), 6.42–6.52 (m, 2H, Harom), 6.93–7.02 (m, 2H, Harom), 7.40 (s, 1H, N-H), 7.73 (d, J = 7.8 Hz, 1H, (H1)Hnaphth), 8.02 (t, J = 7.3 Hz, 1H, (H2), Hnaphth), 8.40 (d, J = 7.8 Hz, 1H, (H3) Hnaphth). 13C(APT) NMR (125.66 MHz, CDCl3) δ 55.58 (O-CH3), 98.65, 103.54 (CHarom), 113.79 (=C-Cl), 119.17, 126.85, 128.30, 128.87, 130.88, 131.73, 135.60 (CHarom, Carom), 143.34 (C-NO2), 148.93 (=C-N), 154.57, 159.64 (=C-OCH3), 172.65, 174.28 (C=O). Anal. Calcd. for C18H13ClN2O6 (Mw 388.73 g/mol): C, 55.61; H, 3.37; N, 7.21. Found: C, 55.31; H, 3.49; N, 7.12. MS [+ESI]: m/z = 387.2 [M–H]+. Isomer 5: Red solid, yield: 0.357 g (46%); Rf 0.35 (EtOAc/Hexane) (1:4 v/v). M.p. 198–199 °C; FTIR (cm−1) ν 3308 (N-H), 3090 (C-Harom), 2970 (C-H), 1679, 1640 (C=O), 1590, 1563 (C=C), 1455, 1339 (C-NO2). 1H NMR (499.74 MHz, CDCl3) δ 3.82 (s, 6H, O-CH3), 6.48 (dd, J = 6.9, 2.4 Hz, 2H, Harom), 6.98–7.20 (m, 1H, Harom), 7.54 (s, 1H, N-H), 7.69 (dd, J = 9.2, 1.4 Hz, 1H, (H1), Hnaphth), 7.87 (t, 7.8 Hz, 1H, (H2), Hnaphth), 8.29 (dd, J = 8.8, 1.2 Hz 1H, (H3), Hnaphth). 13C(APT) NMR (125.66 MHz, CDCl3) δ 55.68 (O-CH3), 98.67, 103.60 (CHarom), 113.34 (=C-Cl), 119.17, 123.71, 126.84, 128.95, 131.05, 133.37, 135.24 (CHarom, Carom), 142.86 (C-NO2), 148.72 (=C-N), 154.11, 159.49 (=C-O-CH3), 173.29, 178.67 (C=O). Anal. Calcd. for C18H13ClN2O6 (Mw 388.73 g/mol): C, 55.61; H, 3.37; N, 7.21. Found: C, 55.83; H, 3.71; N, 7.41. MS [+ESI]: m/z = 387.5 [M–H]+. Synthesis of 3-Chloro-2-((4-methoxyphenyl)amino)-5-ni- tronaphthalene-1,4-dione (6) and 2-Chloro-3-((4-meth- oxyphenyl)amino)-5-nitronaphthalene-1,4-dione (7) According to the general procedure 2, 0.50 g (1.8 mmol) of 2,3-dichloro-5-nitro-1,4- naphthoquinone (1) was reacted with 0.22 g (1.8 mmol) of 4-methoxyaniline (1c) in 25 mL of ethanol at room temperature for 4 hours. The mixture was purified by column chromatography and mixture of ethyl acetate with hexane (1:4 ratio) was used as the mobile phase. Compounds 6 and 7 were obtained as a new regioisomer compounds. Isomer 6: Red solid, Rf 0.51 (EtOAc/Hexane) (1:4 v/v). M.p. 167–168 °C. Isomer 7: Red solid, Rf 0.33 (EtOAc/Hexane) (1:4 v/v). M.p. 129–131 °C. Isomer 6: Red solid, yield: 0.157 g (24%); Rf 0.51 (EtOAc/Hexane) (1:4 v/v). M.p. 167–168 °C; FTIR (cm−1) ν 3226 (N-H), 3084 (C-Harom), 2919, 2850 (C-H), 1684, 1636 (C=O), 1590, 1563 (C=C), 1532, 1376 (C-NO2). 1H NMR (499.74 MHz, CDCl3) δ 3.85 (s, 3H, O-CH3), 5.38 (br s, 1H, NH), 6.85–6.94 (m, 2H, Harom), 7.04–7.10 (m, 2H, Harom), 7.91 (dd, J = 7.9, 1.2 Hz, 1H, (H1), Hnaphth), 7. 97 (t, J = 7.8 Hz, 1H, (H2), Hnaphth), 8.33–8.42 (m, 1H, (H3), Hnaphth). 13C(APT) NMR (125.66 MHz, CDCl3) δ 55.53 (O-CH3), 111.70 (CHarom), 113.36 (=C-Cl), 119.17, 123.17, 126.84, 127.14, 128.95, 131.37, 135.24 (CHarom, Carom), 144.86 (C-NO2), 148.93 (=C-N), 158.64 (=C-OCH3), 173.29, 178.91 (C=O). Anal. Calcd. for C17H11ClN2O3 (Mw 358.73 g/mol): C, 56.92; H, 3.09; N, 7.81. Found: C, 56.79; H, 2.93; N, 7.63. MS [+ESI]: m/z = 357.1 [M–H]+. Isomer 7: Red solid, yield: 0.355 g (54%); Rf 0.33 (EtOAc/ Hexane) (1:4 v/v). M.p. 129–131 °C; FTIR (cm−1) ν 3289 (N- H), 3102, 3002 (C-Harom), 2918, 2843 (C-H), 1680, 1644 (C=O), 1590, 1542 (C=C), 1500, 1329 (C -NO2). 1H NMR (499.74 MHz, CDCl3) δ 3.86 (s, 3H, O-CH3), 6.15 (s, 1H, N-H), 6.74–6.98 (m, 2H, Harom), 7.06– 7.17 (m, 2H, Harom), 7.63–7.74 (m, 2H, (H1,H2), Hnaphth), 8.26- 8.41 (m, 1H, (H3), Hnaphth). 13C(APT) NMR (125.66 MHz, CDCl3) δ 55.49 (O-CH3), 112.04 (=C-Cl), 113.66 (CHarom), 123.12, 126.60, 128.46, 129.17, 131.90, 132.01, 134.60 (CHarom, Carom), 144.25 (C-NO2), 148.23 (=C-N), 157.36 (=C-O-CH3), 173.66, 178.92 (C=O). Anal. Calcd. for C17H11ClN2O3 (Mw 358.73 g/mol): C, 56.92; H, 3.09; N, 7.81. Found: C, 57.13; H, 3.25; N, 7.52. MS [+ESI]: m/z = 357.2 [M–H]+. 191Acta Chim. Slov. 2022, 69, 187–199 Abdassalam et al.: Synthesis of New Regioisomers of 5-Nitro-1,4-Naphthoquinone, ... Synthesis of 3-Chloro-2-((2-morpholinoethyl)amino)-5- nitronaphthalene-1,4-dione (8) and 2-Chloro-3-((2-mor- pholinoethyl)amino)-5-nitronaphthalene-1,4 -dione (9) According to the general procedure 2, 0.50 g (1.8 mmol) of 2,3-dichloro-5-nitro-1,4- naphthoquinone (1) was reacted with 0.24 g (1.8 mmol) of 4-(2-aminoethyl) morpholine (1d) in 25 mL of ethanol at room temperature for 4 hours. The mixture was purified by column chroma- tography and mixture of ethyl acetate with hexane (1:2 ra- tio) was used as the mobile phase. Compounds 8 and 9 were obtained as a new regioisomer compounds. Isomer 8: Red solid, Rf 0.50 (EtOAc/Hexane) (1:2 v/v). M.p. 178–179 °C. Isomer 9: Red solid, Rf 0.33 (EtOAc/Hexane) (1:2 v/v). M.p. 173–174 °C. Isomer 8: Red solid, yield: 0.231 g (34%); Rf 0.50 (EtOAc/Hexane) (1:2 v/v). M.p. 178–179 °C; FTIR (cm−1) ν 3292 (N-H), 3096 (C-Harom), 2918, 2852, 2813 (C-H), 1690 (C=O), 1591, 1561 (C=C), 1519, 1338 (C-NO2). 1H NMR (499.74 MHz, CDCl3) δ 2.50 (t, J = 6.1 Hz, 4H, (CH2-N- CH2) morph), 2.58 (t, J = 6.0 Hz, 2H, N-CH2CH2), 3.75 (t, J = 6.1 Hz, 4H, CH2-O-CH2), 3.85 (q, J = 6.1 Hz, 2H, HN- CH2), 6. 94 (br s, 1H, N-H), 7.65 (dd, J = 7.9, 0.98 Hz, 1H, (H1) Hnaphth), 7.86 (t, J = 7.8 Hz, 1H, (H2) Hnaphth), 8.36 (dd, J = 7.8, 1.5 Hz, 1H, (H3) Hnaphth). 13C(APT) NMR (125.66 MHz, CDCl3) δ 40.87 (NH-CH2-CH2), 52.93 (CH2-N-CH2)morph, 56.50 (NH-CH2-CH2), 66.94 (CH2-O-CH2)morph, 110.36 (=C-Cl), 123.22, 126.09, 129.02, 133.54, 135.24 (CHarom, Carom), 152.16 (C-NO2), 158.93 (=C-N), 178.69, 181.51 (C=O). Anal. Calcd. for C16H16N3O- 5Cl (Mw 365.77 g/mol): C, 52.54; H, 4.41; N, 11.49. Found: C, 52.67; H, 4.70; N, 11.50. MS [+ESI]: m/z = 366.2 [M]+. Isomer 9: Red solid, yield: 0.221 g (32%); Rf 0.33 (EtOAc/Hexane) (1:2 v/v). M.p. 173–174 °C; FTIR (cm−1) ν 3275 (N-H), 3080 (C-Harom), 2957, 2894, 2822 (C-H), 1679, 1640 (C=O), 1609, 1572 (C=C), 1535, 1330 (C-NO2). 1H NMR (499.74 MHz, CDCl3) δ 2.62 (br s, 4H, (CH2-N- CH2) morph), 2.73 (br s, 2H, N-CH2-CH2), 3.70 (br s, 4H, CH2-O-CH2), 3.80–3.94 (m, 2H, HN-CH2), 7. 06 (br s, 1H, N-H), 7.66 (dd, J = 7.9, 0.98 Hz, 1H, (H1), Hnaphth), 7.81 (t, J = 7.8 Hz, 1H, (H2), Hnaphth), 8.23 (dd, J = 7.8, 1.5 Hz, 1H, (H3), Hnaphth). 13C NMR (125.66 MHz, DMSO-d6) δ 40.49 (NH-CH2-CH2), 53.28 (CH2-N-CH2)morph, 57.13 (NH- CH2-CH2), 66.45 (CH2-O-CH2)morph, 98.63 (=C-Cl), 122.98, 128.71, 129.50, 133.30, 134.24 (CHarom, Carom), 148.83 (C-NO2), 160.45 (=C-N), 175.26, 176.76 (C=O). Anal. Calcd. for C16H16N3O5Cl (Mw 365.77 g/mol): C, 52.54; H, 4.41; N, 11.49. Found: C, 52.83; H, 4.13; N, 11.08. MS [+ESI]: m/z = 366.5 [M]+. Synthesis of 3-Chloro-5-nitro-2-((2-(pyrrolidin-1-yl) ethyl)amino)naphthalene-1,4-dione (10) and 2-Chloro- 5-nitro-3-((2-(pyrrolidin-1-yl)ethyl)amino)naphtha- lene-1,4-dione (11) According to the procedure 2, 0.50 g (1.8 mmol) of 2,3-dichloro-5-nitro-1,4- naphthoquinone (1) was reacted with 0.24 g (1.8 mmol) of 2-(pyrrodin-1-yl)ethane-1- amine (1e) in 25 mL of ethanol at room temperature for 4 hours. The mixture was purified by column chromatogra- phy and mixture of ethyl acetate with hexane (1:2 ratio) was used as the mobile phase. Compounds 10 and 11 were obtained as a new regioisomer compounds. Isomer 10: Red solid, Rf 0.71 (EtOAc/Hexane) (1:2 v/v). M.p. 146–147 °C Isomer 11: Red solid, Rf 0.33 (EtOAc/Hexane) (1:2 v/v). M.p. 132–133 °C Isomer 10: Red solid, yield: 0.280 g (46%); Rf 0.71 (EtOAc/Hexane) (1:2 v/v). M.p. 146–147 °C; FTIR (cm−1) ν 3281 (N-H), 3112 (C-Harom), 2958, 2851 (C-H), 1681 (C=O), 1591, 1559 (C=C), 1525, 1335 (C-NO2). 1H NMR (499.74 MHz, CDCl3) δ 1.72 (br s, 4H, (CH2-CH2)pyrro), 2.51 (br s, 4H, (CH2-N-CH2)pyrro), 2.61–2.70 (m, 2H, CH2-N), 3.82 (br s, 2H, HN-CH2), 6.96 (br s, 1H, N-H), 7.61 (d, J = 7.8 Hz, (H1), 1H, Hnaphth), 7.79 (t, J = 7.8 Hz, 1H, (H2), Hnaphth), 8.32 (d, J = 7.8 Hz, 1H, (H3), Hnaphth). 13C(APT) NMR (125.66 MHz, CDCl3) δ 23.34 (CH2-CH2) pyrro, 43.24 (N-CH2), 54.11 (CH2-N-CH2)pyrro, 55.32 (HN- CH2), 109.98 (=C-Cl), 126.02, 128.88, 130.91, 133.43, 135.37 (Carom, CHarom), 148.23 (=C-N) 173.95, 177.32 (C=O). Anal. Calcd. for C16H16ClN3O4 (Mw 363.80 g/mol): C, 54.94; H, 4.61; N, 12.01. Found: C, 54.64; H, 4.39; N, 11.77. MS [+ESI]: m/z = 349.5 [M]+. Isomer 11: Red solid, yield: 0.220 g (35%); Rf 0.33 (EtOAc/Hexane) (1:2 v/v). M.p. 132–133 °C. FTIR (cm−1) ν 3241 (N-H), 3072 (C-Harom), 2968 (C-H), 1681 (C=O), 1590 (C=C), 1531, 1372 (C-NO2). 1H NMR (499.74 MHz, CDCl3) δ 1.50 (br s, 4H, CH2-CH2)pyrro, 2.48 (br s, 4H, CH2-N-CH2)pyrro, 2.58–2.69 (m, 2H, (CH2-N), 3.60 (br s, 2H, HN-CH2), 6.73 (br s, 1H, N-H), 7.47 (d, J = 7.8 Hz, 1H, (H1), Hnaphth), 7.76 (t, J = 7.8 Hz, 1H, (H2), Hnaphth), 8.29 (d, J = 7.8 Hz, 1H, (H3), Hnaphth). 13C NMR (125.66 MHz, DMSO-d6) δ 23.83 (CH2-CH2)pyrro, 45.17 (N-CH2), 54.05 (CH2-N-CH2)pyrro, 60.90 (HN-CH2), 103.93 (=C- Cl), 122.02, 126.05, 128.16, 131.35, 136.42 (Carom, CHarom), 148.03 (=C-N), 166.72 (C-NO2), 172.46, 181.90 (C=O). Anal. Calcd. for C16H16ClN3O4 (Mw 349.77 g/mol): C, 54.94; H, 4.61; N, 12.01. Found: C, 55.03; H, 4.81; N, 12.29. MS [+ESI]: m/z = 348.5 [M]+. Synthesis of 2-(4-Benzylpiperidin-1-yl)-3-chloro-5-ni- tronaphthalene-1,4-dione (12) and 3-(4-Benzylpiperi- din-1-yl)-2-chloro-5-nitronaphthalene-1,4-dione (13) According to the general procedure 2, 0.50 g (1.8 mmol) of 2,3-dichloro-5-nitro-1,4- naphthoquinone (1) was reacted with 0.32 g (1.8 mmol) of 4- benzylpiperidine (1f) in 25 mL of ethanol at room temperature for 4 hours. The mixture was purified by column chromatography and mixture of chloroform with petroleum ether (1:1 ratio) was used as the mobile phase. Compounds 12 and 13 were obtained as a new regioisomer compounds. Isomer 12: Red solid, Rf 0.46 (CHCl3/PE) (1:1 v/v). M.p. 153–154 °C. 192 Acta Chim. Slov. 2022, 69, 187–199 Abdassalam et al.: Synthesis of New Regioisomers of 5-Nitro-1,4-Naphthoquinone, ... Isomer 13: Red oil, Rf 0.37 (CHCl3/PE) (1:1 v/v). Isomer 12: Red solid, yield: 0.332 g (44 %); Rf 0.46 (CHCl3/PE) (1:1 v/v). M.p. 153–154 °C; FTIR (cm−1) ν 2952, 2920, 2853 (C-H), 1718 (C=O), 1606, 1594 (C=C), 1453, 1375 (C-NO2). 1H NMR (499.74 MHz, CDCl3) δ 1.46–1.59 (m, 2H, (CH2)piperi), 1.66–1.93 (m, 3H, (CH2- CH)piperi), 2.53–2.71 (m, 2H, (CH2), 3.12–3.19 (m, 2H, (N- CH2)piperi), 3.69–3.81 (m, 2H, (N-CH2)piperi, 7.07–7.25 (m 5H, Harom), 7.70–7.74 (m, 2H, (H1,H2), Hnaphth), 8.26–8.37 (dd, J = 2.4, 6.8 Hz, 1H, Hnaphth). 13C(APT) NMR (125.66 MHz, CDCl3) δ 33.06 (CH2-CH-CH2)piperi, 37.65 (CH2- CH-CH2)piperi, 42.95 (CH2), 52.20 (CH2-N-CH2)piperi, 119.98 (=C-Cl), 125.79, 126.95, 128.27, 129.08, 130.02, 132.80, 135.51, 140.00 (Carom, CHarom), 151.10 (=C-N), 175.69, 179.36 (C=O). Anal. Calcd. for C22H19ClN2O4 (Mw 410.85 g/mol): C, 64.32; H, 4.66; N, 6.82. Found: C, 64.64; H, 5.02; N, 6.72. MS [+ESI]: m/z = 411.1 [M]+, 433.2 [M+Na]+. Isomer 13: Red oil, yield: 0.226 g (30 %); Rf 0.37 (CHCl3/PE) (1:1 v/v). FTIR (cm−1) ν 3060 (C-Harom), 2970, 2919 (C-H), 1717, 1664 (C=O), 1592, 1561 (C=C), 1452, 1329 (C-NO2). 1H NMR (499.74 MHz, CDCl3) δ 1.38–1.43 (m, 2H, (CH2)piper.), 1.69–1.81 (m, 3H, (CH2-CH)piper.), 2.50–2.55 (m, 2H, (CH2), 3.12–3.18 (m, 2H, (N-CH2)pip- er.), 3.77–3.80 (m, 2H, (N-CH2)piper., 7.05–7.25 (m 5H, Har- om), 7.62–7.70 (m, 1H, (H1), Hnaphth), 7.75–7.85 (m, 1H, (H2)Hnaphth) 8.22 (d, J = 7.8 Hz, 1H, (H3), Hnaphth). 13C(APT) NMR (125.66 MHz, CDCl3) δ 33.05 (CH2-CH- CH2)piperi, 37.60 (CH2-CH-CH2)piper., 42.92 (CH2), 52.20 (CH2-N-CH2)piperi, 119.90 (=C-Cl), 122.70, 126.08, 127.01, 128.33, 129.63, 133.61, 134.14, 139.87 (Carom, CHarom), 148.23 (C-NO2), 151.11 (=C-N), 173.89, 179.35 (C=O). Table 1. The reaction pathway and obtained regioisomeric products 2–13 Reactions and conditions Colour, Rf, yield (%), m.p. (°C) (2): Red solid, Rf: 0.80, yield 20%, m.p. 131–132 (3): Pink solid, Rf: 0.37, yield 44%, m.p. 89–91 (4): Red solid, Rf: 0.52, yield 32%, m.p. 205–206 (5): Red solid, Rf: 0.35, yield 46%, m.p. 198–199 (6): Red solid, Rf: 0.51, yield 24%, m.p. 167–168 (7): Red solid, Rf: 0.33, yield 54%, m.p. 129–131 (8): Red solid, Rf: 0.50, yield 34%, m.p. 178–179 (9): Red solid, Rf: 0.33, yield 32%, m.p. 173–174 (10): Red solid, Rf: 0.71, yield 46%, m.p.146–147 (11): Red solid, Rf: 0.33, yield 35%, m.p.132–133 (12): Red solid, Rf: 0.46, yield 44%, m.p.153–154 (13): Red oil Rf: 0.37, yield 30% 193Acta Chim. Slov. 2022, 69, 187–199 Abdassalam et al.: Synthesis of New Regioisomers of 5-Nitro-1,4-Naphthoquinone, ... Anal. Calcd. for C22H19ClN2O4 (Mw 410.85 g/mol): C, 64.32; H, 4.66; N, 6.82. Found: C, 64.60; H, 4.66; N, 7.16. MS [+ESI]: m/z = 411.2 [M]+, 433.1 [M+Na]+. 3. Results and Discussion 3. 1. Chemistry and Spectral Study The new regioisomers 2–13 were synthesized by the reactions of 2,3-dichloro-5-nitro-1,4-naphthoquinone (1) with some nucleophiles such as amines, piperazines, or morpholines (1a–f) according to a Michael 1,4-addition mechanism and reaction pathways of synthesizes are illus- trated in Table 1. The regioisomers were separated by col- umn chromatography by using a different ratio of solvents. The obtained regioisomers have different color, melting point, retention factor (Rf) and chemical shifts of naphtho- quinone ring protons in 1H NMR spectra. The 1H NMR spectra of the synthesized new regioi- somers indicate that the peaks of the 2-N-substitut- ed-3-chloro-5-nitro-naphthalene-1,4-dione isomer of aro- matic protons (H1–3) are shifted more downfield than the aromatic protons of the 3-N-substituted-2-chloro-5-ni- tro-naphthalene-1,4-dione isomer. Also, it has been found that in the case of mixture of regioisomers, the higher Rf component was shown to be the 2-N-substitut- ed-3-chloro-5-nitro-naphthalene-1,4-dione isomer and the lower Rf component the 3-N-substituted-2-chloro-5-ni- tro-naphthalene-1,4-dione isomer. The comparison of Rf values is compatible with similar values published in the literature.15–16, 24 As a comparison between the 1H NMR spectra of the synthesized new regioisomers 4 and 5; we found that the protons of the naphthoquinone (H1–3) peaks (δ 7.73–8.40 ppm) for the first isomer 3-chloro-2-((2,4-dimethoxyphenyl)amino)-5-nitronaph- thalene-1,4-dione (4) are shifted more downfield than the naphthoquinone peaks (δ 7.69–8.29 ppm) of the second isomer 2-chloro-3-((2,4-dimethoxyphenyl)amino)-5-ni- tronaphthalene-1,4-dione (5). Also, it was found that the isomer 4 has a higher Rf component than the isomer 5. The comparison of Rf values is consistent with the related liter- ature.15 In APT-NMR spectra signals of methoxy group (CH3-O) and carbonyl group for isomer 4 were detected at δ 55.58 and δ 172.65, 174.28 ppm, while in isomer 5 at δ 55.68 and δ 173.29, 178.67 ppm, respectively. 3. 2. 1H–1H Correlated Spectroscopy (COSY) The structures of these 5-nitro-1,4-naphthoqui- none (1) and regioisomers of 5-nitro-1,4-naphthoqui- none (2 and 3) were elucidated by using one- and two-di- mensional NMR techniques in which the differences of positions of nitro group on the naphthalene ring were detected. The three hydrogen signals at the quinone ring of 2,3-dichloro-5-nitro-1,4-naphthoquinone (1) were as- signed in the 1H NMR spectrum (Figure 2) and con- firmed by the 1H–1H COSY (Figure 3). In the 1H NMR spectrum of compound 1, a doublet of doublet (dd) at 7.81 ppm corresponding to H1 that are coupled to H2 (t, 7.98, 1H, 3JH,H = 9.27 Hz), and to H3 (dd, 8.42, 1H, 3JH,H = 9.27 Hz) (Figure 2). All these hydrogens were assigned on the basis of the 1H–1H COSY spectrum, where can be observed that H1 is coupled to H2 and H3, H2 to H1 and H3, and H3 to H2 and H1. From 1H–1H COSY contour map these hydrogens are coupled to each other (Figure 3). The hydrogen signals at the quinone and piperon- ylpiperazine ring of compounds 2 and 3 were assigned in the 1H NMR spectrum (Figures 4 and 7) and also con- firmed by the 1H–1H COSY spectrum (Figures 5, 6 and 8, Figure 2. 1H NMR spectrum of compound 1 194 Acta Chim. Slov. 2022, 69, 187–199 Abdassalam et al.: Synthesis of New Regioisomers of 5-Nitro-1,4-Naphthoquinone, ... 9). For compound 2 a multiplet at 7.76–7.82 correspond- ing to H1 and H2 that are coupled to H3 (8.34, dd, 3JH,H = 9.27 Hz). As expected, H4 and H5 at piperizine ring appear as broad singlets at 3.64 and 2.56 ppm, respectively. A mul- tiplet at 6.79–6.91 ppm corresponds to H7 and H8, respec- tively. In the 1H NMR spectrum of compound 3, a doublet of doublet (dd) at 7.67 ppm corresponding to H1 that is coupled to H2 (t, 7.81, 1H, 3JH,H = 7.8 Hz), and to H3 (dd, Figure 3. 1H–1H COSY contour map of compound 1 Figure 4. The hydrogen signals at the quinone and piperonylpiperazine ring of compound 2 in 1H NMR spectrum 195Acta Chim. Slov. 2022, 69, 187–199 Abdassalam et al.: Synthesis of New Regioisomers of 5-Nitro-1,4-Naphthoquinone, ... 8.21, 1H, 3JH,H = 9.20 Hz) was observed. H4 and H5 appear as broad singlets at 3.64 and 2.52 ppm, respectively. A mul- tiplet at 6.81–6.92 ppm corresponds to H7 and H8, respec- tively (Figures 4 and 7). The interaction of hydrogens of 1, 2, 3, 7, 8 and 4, 5 in compound 2 and 3 were observed on the basis of the 1H–1H COSY spectrums, as can be seen from Figures 5, 8 and Figures 6, 9. H3 is coupled to H2, H2 to H3, and H7 is coupled to H8, H8 to H7 and H4 is coupled to H5, H5 to H4 for compound 2 (Figures 5, 6). As can be observed from Figure 8, H3 is coupled to H1 and H2, H2 to H3 and H1, and H1 is coupled to H2 and H3 also H7 coupled to H8, H8 to H7. As can be seen in Figure 9, H4 is coupled to H5 and H5 to H4 for compound 3. Chemical shifts in ppm of the above mentioned hydrogens are indicated in the figures. 3. 3. CUPRAC Antioxidant Capacities NQ compounds were assayed using the normal CU- PRAC assay (at 25 °C) against trolox (TR) as the reference standard.18 TEACCUPRAC coefficients are defined as the ra- tio of the slope of the curve of the tested compounds to that of TR (Table 2). LODs for synthesized compounds with respect to CUPRAC method were established be- tween 0.74–7.37 µM (n = 10) and RSD% were found to be Figure 5. 1H–1H COSY contour map of aromatic protons of com- pound 2 Figure 6. 1H–1H COSY contour map of piperazine ring protons of compound 2 Figure 7. The hydrogen signals at the quinone and piperonylpiperazine ring of compound 3 in 1H NMR spectrum 196 Acta Chim. Slov. 2022, 69, 187–199 Abdassalam et al.: Synthesis of New Regioisomers of 5-Nitro-1,4-Naphthoquinone, ... less than 4%. Among all the compounds synthesized, com- pound 5 showed the highest antioxidant potency (TEAC- CUPRAC = 1.80±0.06). The TEACCUPRAC coefficient of com- pound 5 was also higher than unity (TEACTR = 1.0). Since TEACCUPRAC coefficient of ascorbic acid (for compari- son)19 and compound 7 is close to 1.0, their antioxidant power are approximately equal to that of TR. Five NQ regioisomer couples having different func- tional group (2-chloro-isomers 3, 5, 7, 9 and 13, as well as 3-chloro-isomers 2, 4, 6, 8 and 12) are reported here (Table 2). Although all isomer couples are just regioisomers, very interesting and dramatic differences in biological activities have been observed. Antioxidant capacity result of isomers showed that it is directly related to the bonding of N-nu- cleophiles at the 2 or 3 position on the naphthoquinone ring. As can be seen from the Table 2, the capacity results of all 2-chloro regioisomers are higher than the 3-chloro regioisomers. Surprisingly, two isomer pairs having – OCH3 functional group also attracted attention in these results. Interestingly, despite having the same functional group, isomer 6 and 7, if we compare the antioxidant result of the two isomers, we see that the difference is more than two times. Likewise, there is a remarkable difference in ca- pacity result of isomers 4 and 5. As shown in Table 2, 4 and 5 had stronger antioxidant capacity than 6 and 7. These results may be related to that the larger the number of the –OCH3 groups in the same structure, the higher is the an- tioxidant capacity of a molecule.25 Figure 8. 1H–1H COSY contour map of aromatic protons of compound 3 Figure 9. 1H–1H COSY contour map of piperazine ring protons of compound 3 Figure 10. The comparison of TEAC coefficients of derivatives with and without NO2 substituents (2, 3, compound A, 8, 9 and com- pound B) 197Acta Chim. Slov. 2022, 69, 187–199 Abdassalam et al.: Synthesis of New Regioisomers of 5-Nitro-1,4-Naphthoquinone, ... A few years ago, our research group synthesized two derivatives not substituted with nitro groups on the aro- matic skeleton (compounds A and B) and reported their antioxidant results.4 Comparing these results with those for the regioisomers 2, 3 and 8, 9 (Table 3 and Figure 10), it is evident that the effect of the electron-withdrawing nitro group in the system is directly reflected on the antioxidant results and higher antioxidant capacities were obtained for nitro derivatives. Regiosomers 8 and 9 showed approxi- mately twice as high antioxidant activity, while regioiso- mers 2 and 3 showed approximately half times higher anti- oxidant activity than compounds B and A, respectively. 3. 4 Catalase Activity The screening of NQ compounds against the cata- lase revealed that most of the compounds have moderate inhibition activity of this enzyme (> 0.7 U/mL, Figure 11). Enzyme activity results were determined in accord- ance with the antioxidant activity results. As can be seen in  Figure 11,  generally the enzyme inhibitory activities results of 2-chloro regioisomers are higher than for the 3-chloro regioisomers except for one regioisomer pair and compounds 5 and 7 revealed significant inhibition activity. Table 2. The linear calibration equations, correlation coefficients, linear concentration ranges, and TEAC coefficients of the NQ compounds using CUPRAC method. Compounds Linear range Calibration equation r TEACCUPRACa (mol L–1) 2 3.28×10–5 – 1.05×10–4 A = 7.60×103 c + 0.12 0.992 0.46±0.02 3 1.23×10–6 – 1.42×10–4 A = 8.10×103 c + 0.05 0.988 0.49±0.02 4 4.09×10–6 – 4.75×10–5 A = 2.44×104 c + 0.04 0.989 1.46±0.07 5 1.26×10–6 – 3.51×10–5 A = 3.16×104 c + 0.09 0.992 1.80±0.06 6 2.89×10–6 – 1.63×10–4 A = 6.90×103 c + 0.07 0.990 0.41±0.01 7 4.45×10–6 – 6.88×10–5 A = 1.57×104 c + 0.06 0.991 0.94±0.04 8 2.13×10–6 – 2.47×10–4 A = 4.68×103 c + 0.04 0.989 0.28±0.01 9 2.05×10–5 – 1.61×10–4 A = 6.32×103 c + 0.18 0.991 0.38±0.02 10 3.77×10–6 – 1.40×10–4 A = 7.95×103 c + 0.08 0.990 0.48±0.03 12 1.39×10–5 – 2.54×10–4 A = 4.29×103 c + 0.11 0.988 0.26±0.01 13 1.76×10–6 – 2.03×10–4 A = 5.69×103 c + 0.04 0.991 0.34±0.02 a TEACcompound = εcompound / εTR εTR = 1.67×104 Lmol–1cm–1 (DMSO). Table 3. Comparison of antioxidant results of substituted NO2 and unsubstituted NO2 derivatives of NQ according to CUPRAC method Compounds TEACCUPRACa Compounds TEACCUPRACa 0.46±0.02 0.28±0.01 2 8 0.49±0.02 0.38±0.02 3 9 0.349±0.02[22] 0.164±0.02[22] Compound A[22] Compound B[22] a TEACcompound = εcompound / εTR εTR = 1.67×104 Lmol–1cm–1 (DMSO). 198 Acta Chim. Slov. 2022, 69, 187–199 Abdassalam et al.: Synthesis of New Regioisomers of 5-Nitro-1,4-Naphthoquinone, ... 4. Conclusions The studies on nitronaphthoquinone derivatives are rare in the literature and the nitro group associated with the aromatic ring in the quinone system is known to in- crease the biological activity of naphthoquinone due to its electron-withdrawing properties. For this reason, 2,3-di- chloro-5-nitro-1,4-naphthoquinone (1) was used as the starting material in this study. The new regioisomeric compounds of 5-nitro-1,4-naphthoquinone 2–13 were synthesized by the reactions of 2,3-dichloro-5-ni- tro-1,4-naphthoquinone with some heterocyclic ring sub- stituted nucleophiles according to a Michael 1,4-addition mechanism. All newly synthesized compounds were char- acterized by electrospray ionisation mass spectrometry (ESI-MS), Fourier transform infrared spectroscopy (FTIR), 1H nuclear magnetic resonance (1H NMR), at- tached proton test  nuclear magnetic resonance (APT- NMR). Two-dimensional technique 1H–1H correlated spectroscopy (COSY) was used for characterization of compound 1 and regioisomers 2 and 3. Their in vitro anti- oxidant capacity and catalase enzyme inhibition activity were investigated. The effect of the electron-withdrawing nitro group in the system was directly reflected on the an- tioxidant results and higher antioxidant capacities were obtained. The compounds 4 and 5 showed comparable an- tioxidant potency to ascorbic acid. The antioxidant capac- ity results of all 2-chloro regioisomers are higher than for the 3-chloro regioisomers. Likewise, also catalase enzyme inhibitory activities results were determined in the same way, except for one regioisomer pair. Supplementary Information (SI) Supplementary information for this article is availa- ble at the journal web site. Acknowledgements We gratefully thank the Research Fund of Istanbul University-Cerrahpasa for financial support of this work (Project Numbers: FDK-2017-24871, FYL-2018-30488, FBA-2019-30472, FBA-2019-32783). Conflict of Interest No potential conflict of interest was reported by the authors. 5. References 1. R. F. Silver, H. Holmes, Can. J. Chem. 1968, 46, 1859–1864. DOI:10.1139/v68-309 2. H. Tomozane, Y. Takeuchi, T. Choshi, S. Kishida, M. Yamato, Chem. Pharm. Bull. 1990, 38, 925–929. DOI:10.1248/cpb.38.925 3. K. Oogose, Y. Hafuri, E. Takemori, E. Nakata, Y. Inouye, S. Nakamura, A. Kubo, J. Antibiot. 1987, 40, 1771–1778. DOI:10.7164/antibiotics.41.1471 4. N. G. Deniz, C. Ibis, Z. Gokmen, M. Stasevych, V. Novikov, O. Komarovska-Porokhnyavets, M. Ozyurek, K. Guclu, D. Kar- akas, E. Ulukaya, Chem. Pharm. Bull. 2015, 63, 1029–1039. DOI:10.1248/cpb.c15-00607 5. S. Kurban, N. G, Deniz, C. Sayil, Bulg. Chem. Comm. 2016, 48, 43–47. 6. C. Sayil, C. Ibis, Russ. J. Org. Chem. 2010, 46, 216–221. DOI:10.1134/S1070428010020119 7. C. Sayil, S. Kurban, C. Ibis, Phosphorus Sulfur Silicon Relat. Elem. 2013, 188, 1855–1867. DOI:10.1080/10426507.2013.796475 8. A. Kacmaz, N. G. Deniz, S. G. Aydinli, C. Sayil, E. Onay-Ucar, E. Mertoglu, N. Arda, Open Chem. 2019, 17, 337–345. Figure 11. Catalase enzyme activities (U mL–1) of the novel NQ compounds. 199Acta Chim. Slov. 2022, 69, 187–199 Abdassalam et al.: Synthesis of New Regioisomers of 5-Nitro-1,4-Naphthoquinone, ... DOI:10.1515/chem-2019-0030 9. S. Kurban, N. G. Deniz, C. Sayil, M. Ozyurek, K. Guclu, M. Stasevych, V. Zvarych, O. Komarovska-Porokhnyavet, V. No- vikov, Heteroat. Chem. 2019, 2019, 1–12. DOI:10.1155/2019/1658417 10. A. Abdassalam, S. Kurban, N. G. Deniz, C. Sayil, J. Chem. Soc. Pak. 2019, 41, 834–840. 11. V. Chobot, F. Hadacek, J. Chem. Ecol. 2009, 35, 383–390. DOI:10.1007/s10886-009-9609-5 12. C. Soto-Maldonado, M. Vergara-Castro, J. Jara-Quezada, E. Caballero-Valdés, A. Müller-Pavez, M. E. Zúñiga-Hansen, C. Altamirano, Electron. J. Biotechnol. 2019, 39, 1–7. DOI:10.1016/j.ejbt.2019.02.001 13. M. A. Berghot, E. M. Kandeel, A. H. Abdel-Rahman, M. Ab- del-Motaal, Med. Chem. 2014, 4, 381–388. DOI:10.4172/2161-0444.1000169 14. A. A. Kutyrev, V. V. Moskva, Russ. Chem. Rev. 1991, 47, 72– 88. DOI:10.1070/RC1991v060n01ABEH001032 15. C. Blackburn, Tetrahedron Lett. 2005, 46, 1405–1409. DOI:10.1016/j.tetlet.2005.01.049 16. B. S. Samant, C. Chakaingesu, Bioorganic  Med. Chem. Lett. 2013, 23, 1420–1423. DOI:10.1016/j.bmcl.2012.12.075 17. A. Nandi, L. J. Yan, C. K. Jana, N. 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M. Wynne, A. Ryan, E. Sim, A. J. Russell, Bioorg. Med. Chem. 2014, 22, 3030–3054. DOI:10.1016/j.bmc.2014.03.015 25. J. Chen, J. Yang, L. Ma, J. Li, N. Shahzad, C. K. Kim, Sci. Rep. 2020, 10, 2611–2620. DOI:10.1038/s41598-020-59451-z Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License Povzetek Raziskave nitronaftakinonskih derivatov so v literaturi redke, čeprav je znano, da prisotnost nitro skupine na aromatskem obroču zaradi svojih elektron-privlačnih lastnosti skupaj s kinonskim sistemom poveča biološko aktivnost tovrstnih naf- tokinonskih sistemov. Z reakcijo med 2,3-dikloro-5-nitro-1,4-naftokinonom in različnimi nukleofili, substituiranimi s heterocikličnimi fragmenti, kot so anilini, piperazini in morfolini, smo s pomočjo Michaelove 1,4-adicije sintetizirali nove regioizomere N(H)-substituiranih-5-nitro-1,4-naftokinonov (NQ). Poročamo o petih regioizomernih parih NQ z različnimi funkcionalnimi skupinami, ki se ločijo po položajih klorovega substituenta (2-kloro izomeri 3, 5, 7, 9 in 13 ter 3-kloro izomeri 2, 4, 6, 8 in 12). Vse nove spojine smo karakterizirali s spektroskopskimi metodami in dvodimenzional- no NMR tehniko 1H–1H korelacijske spektroskopije (COSY). Pripravljenim NQ regioizomerom smo določili inhibitorne aktivnosti na encimu katalaza. S pomočjo metode bakrove redoks antioksidativne kapacitete (CUPRAC) smo določili njihovo antioksidativno delovanje. 2-Kloro-3-((2,4- dimetoksifenil)amino)-5-nitronaftalen-1,4-dion (5) se je izkazal z najvišjo antioksidativno kapaciteto in sicer je koe- ficient CUPRAC-troloks antioksidativne kapacitete (TEAC) znašal 1.80±0.06. Spojina 5 je izkazala tudi najmočnejšo inhibitorno aktivnost na encim katalaza. Ugotovili smo, da je antioksidativna kapaciteta za vse 2-kloro regioizomere večja kot za 3-kloro regioizomere. Za vse spojine, razen za en regioizomerni par, so bili analogni tudi rezultati za inhib- itorno aktivnost na encim katalaza. Nove spojine so učinkovito inhibirale katalazo, odstotek inhibicije je bil v območju 0.71–0.86 %. Nekatere izmed teh NQ spojin so pokazale precejšnje antioksidativne kapacitete. 200 Acta Chim. Slov. 2022, 69, 200–216 Sumrra et al.: Metal Based Bioactive Nitrogen ... DOI: 10.17344/acsi.2021.7182 Scientific paper Metal Based Bioactive Nitrogen and Oxygen Donor Mono and Bis Schiff Bases: Design, Synthesis, Spectral Characterization, Computational Analysis and Antibacterial Screening Sajjad Hussain Sumrra,1,* Wardha Zafar,1 Sabaahatul Ain Malik,1 Khalid Mahmood,2 Syed Salman Shafqat3 and Saira Arif1 1 Department of Chemistry, University of Gujrat, Gujrat 50700, Pakistan; 2 Institute of Chemical Sciences, Bahauddin Zakariya University, Multan, Pakistan; 3 Department of Chemistry, Division of Science and Technology, University of Education, Lahore, Pakistan * Corresponding author: E-mail: sajjadchemist@gmail.com/sajjadchemist@uog.edu.pk Received: 09-30-2021 Abstract The scientific interest in developing the advanced metal based compounds to inhibit and control bacterial infections is continuously rising. Keeping in view their pharmacological significance, two new bioactive symmetrical phenylenedi- amine mono- and bis-Schiff bases, 2-{[(4-aminophenyl)imino]methyl}-6-methoxyphenol (L1) and 2,2’-{benzene-1,2-diylb- is[nitrilomethylylidene]}bis(6-methoxyphenol) (L2) have been synthesized and characterized by using physical techniques, spectral methods, elemental and DFT based computational analysis with B3LYP/6-311++G(d, p) basis set. Furthermore, the synthesized ligands were complexed with VO, Mn, Co, Ni, Cu and Zn ions in ratio [M:L,1:2 and 1:1], respectively. All the complexes exhibited significant antibacterial action against all tested bacterial strains. But overall, the zinc complexes possessed higher antibacterial activity. These results concluded that metal complexes might be promising induction in the upcoming time for medical purposes. Keywords: Symmetrical phenylenediamine mono and bis-Schiff bases; computational study; chelation; bidentate and tetradentate; antibacterial activity. 1. Introduction Schiff base ligands occupy significant importance in coordination chemistry as they have potential to form me- dicinally important metal complexes.1 They have attained greater attention in biological and coordination chemistry because of their facile preparation, structural variability and diversity.2 They are significantly involved in the devel- opment of chelates. Particularly, the Schiff bases having – OH, –SH and –NH2 nucleophilic substituents at ortho po- sition to the azomethine group have appropriate structures for coordination with metal ions forming more stable met- al chelates.3 Schiff base ligands with electron donors such as nitrogen and oxygen atoms have been extensively inves- tigated because of their competent therapeutic potentials.4 They are also significant for their biological action against fungi, bacteria5 and certain types of tumors.6 Nowadays, the design and development of new com- pounds for dealing with resistant microbes have become an important area of antifungal and antibacterial research, as resistance of pathogenic microbes towards already ex- isting antimicrobial drugs is rapidly becoming a world- wide problem. And the discovery of novel metal based antimicrobial agents is one of the most challenging de- mands for pharmacologists and chemists today.7 The work highlights the bioactivity of those compounds of first-row divalent and tetravalent transition metals which are non-toxic, economical as well as readily available for phar- macology. Most of the first-row transition metals are es- sential for biological processes8 such as respiration, cell division, photosynthesis and nitrogen fixation.9 Apart from choice of transition metals, it is also important to properly design the ligand framework as it has the ability to modify the systematic/oral bioavailability of metals, se- 201Acta Chim. Slov. 2022, 69, 200–216 Sumrra et al.: Metal Based Bioactive Nitrogen ... cure some selective target enzymes or DNA, protect as well as supply metal ions at targeted sites.10 Slight modifications in the structure of ligand could significantly increase the pharmacological properties of metal complexes through the endorsement of bioactive metal ions.11 The metal complexes of Schiff bases have been known to possess different biological activities like antifungal,12 enzyme inhibition,13 anticancer,14 anticon- vulsant,15 antitumor,16 anti-proliferative,17 antibacterial,18 antioxidant,19 anti-inflammatory,20 cytotoxic,21 DNA binding22 and antiviral.23 Literature reveals that extensive work have been done on the preparation of various sym- metrical tetradentate Schiff bases from 1,2-diamines with ortho-hydroxyketones/aldehydes.24 The metal complexes of 1,2-diamine have a vital role in both structural and syn- thetic research,25 related to analytical, pharmaceutical, bi- ological and clinical fields.26 With the expansion of our research work describing the synthesis, structure elucidation and pharmacological properties of chemical scaffolds prepared by the condensa- tion of phenylenediamine with aromatic carbonyls,27 here we report the facile synthesis of two new phenylenedi- amine derived mono- and bis-Schiff bases, 2-{[(4-amino- phenyl)imino]methyl}-6-methoxyphenol (L1) and 2,2’-{ben- zene-1,2-diylbis[nitrilomethylylidene]}bis(6-methoxyphe- nol) (L2) and their complexes with VO(IV), Mn(II), Co(II), Ni(II), Cu(II) and Zn(II) metals. All the synthesized com- pounds have been studied in detail for their structure, physicochemical and in vitro antibacterial properties against one Gram positive bacteria (GPB) and one Gram negative bacteria (GNB). Density functional parameters have been utilized for analysing the molecular designs, ac- tive sites, molecular interactions along with biochemical activity. The experimental findings have been compared to their computed results. 2. Experimental 2. 1. Materials Chemicals of analytical grade were used during the research work. o-Phenylenediamine, p-phenylenediamine and 2-hydroxy-3-methoxybenzaldehyde were purchased from Sigma Aldrich which were extremely pure so they were used without further purification. Ethanol and diox- ane were distilled for the additional refinement because they have been used as solvents in the reaction media for the synthetic reactions of ligands and their corresponding metal chelates, respectively. 2. 2. Instrumentation The melting temperatures of ligands and decomposi- tion temperatures of metal complexes were determined using Stuart apparatus. FT-IR spectra were documented using Nicolet FT-IR Impact 400D infrared Spectropho- tometer in working range (4000–400 cm–1). Proton NMR spectra of the mono- and bis-Schiff bases were obtained through Bruker Advance 300 MHz using DMSO (dimeth- yl sulfoxide) solvent. The mass spectra of ligands were tak- en on JEOL MS Route instrument. UV-Vis spectra were recorded on Hitachi UV-3200 spectrophotometer using DMF (dimethyl formamide) solvent. Magnetic moment values for the metal complexes were recorded with the Magnetic Susceptibility Balance (MSB Mk-1) using MnCl2 as a reference. Molar conductivity measurements of metal complexes (0.001M solutions in DMF) were carried out using Inolab Cond 720 Conductivity Bridge at room tem- perature. The antibacterial activity was performed using DMSO solvent on ESCO Laminar Flow Cabinet and Mem- mert incubator at the Department of Biochemistry, Uni- versity of Gujrat, Gujrat, Pakistan. 2. 3. Synthesis of Ligands The phenylenediamine based ligands, 2-{[(4-amino- phenyl)imino]methyl}-6-methoxyphenol (L1) and 2,2’-{ben- zene-1,2-diylbis[nitrilomethylylidene]}bis(6-methoxyphe- nol) (L2) have been synthesized by adopting already published path28 as shown in Scheme 1. The p-phenylene- diamine ligand (L1) was synthesized by adding the equim- olar amount of ethanolic solution of 2-hydroxy-3-me- thoxybenzaldehyde (10 mmol, 1.52 g) over a stirred ethanolic solution of p-phenylenediamine (10 mmol, 1.08 g). Change in colour and precipitate formation in the reac- tion mixture was the first indication of the successful reac- tion. The reaction mixture was continuously stirred and the advancement in the reaction mixture was checked by tak- ing comparative thin layer chromatography (TLC). The fin- ishing point of the reaction was evidenced through a single spot of ligand on the TLC. The precipitates of ligand were then filtered out, rinsed with warm ethanol, dried off and then recrystallized by 1:2 ratio of ethanol and ether to ob- tain the pure product. The o-phenylenediamine ligand (L2) was also synthesized by the same pathway through reflux- ing instead of stirring the ethanolic solution of o-phenyl- Scheme 1. Synthesis of phenylenediamine Schiff base ligands (L1) & (L2) 202 Acta Chim. Slov. 2022, 69, 200–216 Sumrra et al.: Metal Based Bioactive Nitrogen ... enediamine (5 mmol, 0.54 g) with same 2-hydroxy-3-me- thoxybenzaldehyde (10 mmol, 1.52 g) in 1:2 molar ratio. 2. 3. 1. 2-{[(4-Aminophenyl)imino]methyl}-6- methoxyphenol (L1) Yield (%): 82; m.p. (°C): 256; colour: light yellow; IR (KBr, cm–1): 3427 (OH), 3020 (NH2), 2930 (OCH3), 1640 (HC=N), 1389 (C-O); 1HNMR (DMSO-d6, 300 MHz) δ (ppm): 3.82 (s, 3H, OCH3), 6.90-7.26 (m, 7H, Ar-H), 7.53 (s, 2H, NH2), 9.01 (s, 1H, HC=N), 13.17 (s, 1H, OH); MS (ESI) m/z (%): 241.1 ([M]+, 100), 227.1 (24), 209.1 (09), 197.0 (08), 183.0 (05), 154.0 (10), 135.0 (04); Anal. calcd. for C14H14N2O2 (%) C, 69.4; N, 11.56; H, 5.82; found: C, 69.35; N, 11.49; H, 5.78. 2. 3. 2. 2,2’-{Benzene-1,2-diylbis[nitrilomethyly- lidene]}bis(6-methoxyphenol) (L2) Yield (%): 73; m.p. (°C) 173; colour: dark orange; IR (KBr, cm–1): 3431 (OH), 3100 (C-H), 2918 (OCH3), 1638 (HC=N), 1396 (C-O); 1HNMR (DMSO-d6, 300 MHz,) δ (ppm): 3.92 (s, 3H, OCH3), 6.88-6.93 (t, 1H, C8-H, C18-H), 6.95-6.97 (d, 1H, C9-H, C19-H), 7.07-7.13 (dd, 1H, C7-H, C23-H), 7.23-7.28 (d, 1H, C3-H), 7.38-7.46 (m, 1H, C1-H, C2-H), 7.60-7.64 (d, 1H, C6-H), 8.91 (s, 1H, HC=N), 12.97 (s, 1H, OH); MS (ESI) m/z (%): 376.4 ([M]+, 100), 361.2 (36), 253.1 (25), 240.1 (62), 222.1 (28), 211.1 (22), 197 (14). Anal. Calcd. for C22H20N2O4 (%): C, 70.20; N, 7.44; H, 5.36; found: C, 69.96; N, 7.38; H, 5.31. 2. 4. Synthesis of Transition Metal Complexes All the transition metal complexes of p-phenylenedi- amine ligand (L1) were synthesized using 1:2 molar ratio of metal to ligand by accomplishing previously reported protocol.28 The ligand (20 mmol) was dissolved in 10 mL dioxane and magnetically refluxed until the ligand was completely dissolved. Then, the respective metallic salt (vanadyl sulphate hydrate, manganese(II) chloride, co- balt(II) chloride hexahydrate, nickel(II) chloride hexahy- drate, copper(II) chloride dihydrate and anhydrous zinc(II) chloride) solution (10 mmol) was gradually added in the ligand solution with continual stirring. After that, the reaction mixture was further refluxed for 6 hours. The reaction was observed by using TLC at regular intervals. Desired products were obtained in the form of the precip- itates, which were filtered, washed with hot dioxane, dried and then recrystallized using dioxane and ether (1:2) to obtain the pure product. For the synthesis of metal com- plexes of o-phenylenediamine ligand (L2), same procedure was fallowed except that ligand (L1) was replaced with li- gand (L2) and equimolar ratio of ligand and metal salts were used. All complexes were synthesized according to above mentioned procedure and their structures are shown in Scheme 2. 2. 5. Computational Study Both the ligands (L1) and (L2) and their selected 3d-metal complexes have been optimized by density func- tional theory (DFT) to insight their geometries in absence of their well resolved SC-XRD data. For this computation- al study, the Gaussian 09 program29 was employed to exe- cute all the theoretical simulations for molecular dynamics with the help of DFT. Based on the B3LYP method with 6-311++G(d, p) basis sets, the molecular geometries of compounds were thoroughly optimized at their ground state energy levels. The HOMO-LUMO energies together with their energy differences for optimized structures were Scheme 2. Synthesis of phenylenediamine metal based compounds (1)–(12) 203Acta Chim. Slov. 2022, 69, 200–216 Sumrra et al.: Metal Based Bioactive Nitrogen ... calculated to analyse their quantum chemical parameters. The structural characteristics, stability and molecular quantities i.e., chemical softness, hardness and electroneg- ativity, electrophilicity were determined satisfactorily.30 The computed FT-IR spectra of the studied molecular sys- tems have also been established from their optimized geo- metrical structures through B3LYP/6-311++G(d, p) func- tions. The natural bond orbital (NBO) analysis, Mulliken atomic charges (MAC) and molecular electrostatic poten- tial (MEP) maps of the studied molecules have been as- sessed at the same functional, whereas UV−Vis spectra have been computed employing the TD-DFT (time-de- pendent density functional theory) accompanied by the afore-mentioned functional. Furthermore, all the input data files were structured using Gaussview 5.0.31 While GaussSum,32 Avogadro,33 Chemcraft34 and Gaussview 5.0 programs have been utilized to interpret and visualize the outcomes of the optimized structures, computed spectra along with the summary of their geometrical parameters like bond lengths and bond angles. 2. 6. Antibacterial Study Anti-bacterial action of the prepared compounds was evaluated against three G- bacteria (GNB) i.e., Salmonella typhi, Klebsiella pneumonia, Escherichia coli and one Gram+ bacteria (GPB) i.e., Staphylococcus aureus through disc dif- fusion method.35 Standard drugs ampicillin and streptomy- cin were used to compare the results. Equivalent quantity of both nutrient broth and agar-agar were mixed up in dis- tilled water. The synthesized media, filter paper strips and petri dishes were autoclaved for about 30 minutes at 121 °C for sterilization. Then, the semi-liquid media was trans- ferred to petri dishes and allowed to solidify. After that, the bacterial inoculum was spread over the media by means of glass spreader. Later on, the filter paper strips were placed at the regular distance on the solidified media. Then, 10 µL of DMSO, sample solutions and standards having same con- centration (2 mM in DMSO) were poured onto the strips via micropipette. In this analysis, DMSO and standards act- ed as negative and positive controls, correspondingly. The plates were properly labelled for each sample and standard, tightly wrapped and placed in incubator at 37 °C tempera- ture for 24 hours. Finally, the inhibition zones of the tested samples and standards were checked and recorded in milli- metre (mm) against each bacterial strain. 3. Results and Discussion The symmetrical Schiff base ligands (L1) and (L2) were prepared in 1:1 and 1:2 molar ratio by condensation reaction of o-vanillin with p-phenylenediamine and o-phenylenediamine, respectively. The synthesized ligands Table 1. Physical, analytical and elemental details of metal complexes (1)–(12) No Compound Formula M.W (g/mol) Calculated (%) (Found) M.P (°C) Colour Yield (%) C H N M (1) VO(L1)2 C28H26N4O5V 547.47 61.20 4.77 10.20 9.27 300+ Pine green 65 (61.08) (4.65) (10.11) (9.14) (2) Mn(L1)2(H2O)2 C28H30N4O6Mn 573.50 58.64 5.27 9.77 9.58 283–285 Dark brown 78 (58.59) (5.21) (9.68) (9.45) (3) Co(L1)2(H2O)2 C28H30N4O6Co 577.49 58.23 5.24 9.70 10.20 277–279 Russet Brown 70 (58.18) (5.19) (9.64) (10.07) (4) Ni(L1)2(H2O)2 C28H30N4O6Ni 577.25 58.26 5.24 9.71 10.17 289–291 Cyan 72 (58.18) (5.16) (9.63) (10.09) (5) Cu(L1)2(H2O)2 C28H30N4O6Cu 582.11 57.77 5.19 9.62 10.92 293–295 Olive green 82 (57.65) (5.08) (9.53) (10.88) (6) Zn(L1)2(H2O)2 C28H30N4O6Zn 583.94 57.59 5.18 9.59 11.20 264–266 Off–white 72 (57.51) (5.07) (9.50) (11.06) (7) VO(L2) C22H18N2O5V 441.33 59.87 4.11 6.35 11.54 265–267 Greenish black 72 (59.73) (4.05) (6.31) (11.47) (8) Mn(L2)(H2O)2 C22H22N2O6Mn 465.37 56.78 4.77 6.02 11.81 276–278 Brownish green 68 (56.64) (4.72) (5.96) (11.76) (9) Co(L2)(H2O)2 C22H22N2O6Co 469.35 56.30 4.72 5.97 12.56 248–250 Dark violet 77 (56.17) (4.67) (5.90) (12.50) (10) Ni(L2)(H2O)2 C22H22N2O6Ni 469.1 56.33 4.73 5.97 12.51 260–262 Dark grey 83 (56.18) (4.68) (5.93) (12.43) (11) Cu(L2)(H2O)2 C22H22N2O6Cu 473.97 55.75 4.68 5.91 13.41 230–232 Greenish black 82 (55.65) (4.64) (5.88) (13.47) (12) Zn(L2)(H2O)2 C22H22N2O6Zn 475.83 55.53 4.66 5.89 13.74 239–241 Off–white 81 (55.41) (4.60) (5.83) (13.66) 204 Acta Chim. Slov. 2022, 69, 200–216 Sumrra et al.: Metal Based Bioactive Nitrogen ... were moisture and air stable. Both the ligands were com- pletely soluble in DMSO, DMF and dioxane at room tem- perature. Both these ligands were then reacted with metal- lic salts VOSO4 ∙ H2O, MnCl2 ∙ 2H2O, NiCl2 ∙ 6H2O, CoCl2 ∙ 6H2O, CuCl2 ∙ 2H2O and ZnCl2 to synthesize 3d-metal complexes in metal to ligand molar ratio of 1:2 and 1:1, respectively (Scheme 1). All the as-synthesized metal che- lates were microcrystalline solids having intense colours except zinc complexes which were colourless. The metal complexes exhibited greater range of decomposition points with regard to their respective ligands as a result of strong bonding in metal chelates. The structures of synthe- sized phenylenediamine Schiff bases and their corre- sponding 3d-metal chelates were explored on the basis of their spectral, physical and micro-analytical results. The non-electrolytic behaviour of the metal chelates was spec- ified by their minor conductance values. The spectral re- sults together with elemental analysis agreed well with the proposed structures of the as-synthesized compounds, verifying their high purity (given in Table 1). 3. 1. FT-IR Spectra The IR spectra of both ligands showed a typical peak of azomethine linkage (HC=N) at 1638-1640 cm–1 that gave a clue about the condensation of amine (-NH2) group of phenylenediamine with carbonyl (C=O) group of 2-hy- droxy-3-methoxybenzaldehyde. Moreover, the ligands also exhibited different bands at 3427–3431, 2918–2930 and 1389–1396 cm–1 because of the existence of v(OH), v(OCH3) and v(C-O) groups, respectively.36 The vibra- tional spectrum of ligand (L1) demonstrated a peak at 3020 cm–1 (Figure S1) signifying the non-participation of one amino (NH2) group of p-phenylenediamine moiety in the condensation process, consequently validating the syn- thesis of mono-Schiff base (L1). While the characteristic peaks of both amino (NH2) groups of o-phenylenediamine moiety were missing in the spectrum of ligand (L2) con- firming the synthesis of bis-Schiff base (Figure S2). IR spectra of the phenylenediamine Schiff bases (L1) and (L2) have been compared with metal complexes and showed that the Schiff bases were bonded with metal ions in bidentate and tetradentate mode, respectively (Table 2). Coordination of both ligands with 3d-metallic ions oc- curred via oxygen atom of benzaldehyde by the deproton- ation of phenolic group and nitrogen atom of azomethine (Figure S3-S4). The coordinating action of the azome- thine-N with the metal atoms was confirmed from the shifting of the IR band of azomethine (CH=N) linkage from 1638-1640 cm–1 to lower frequency at 1620–1630 cm–1.37 The absence of band at 3427–3431 cm–1 because of v(OH) group accompanied by the shifting of v(C–O) band from 1389-1396 cm–1 to 1381–1388 cm–1 indicated the deprotonation of phenolic group of the ligands and its co- ordination with metal ions.38 Existence of new weak bands at 431–440 and 523– 539 cm–1 in the metal complexes, were allocated to v(M–O) and v(M–N) vibrations, correspondingly39 and these vi- brational bands were not observed in the spectra of the uncomplexed ligands. A peak was emerged at 968–972 cm–1 only in the vibrational spectra of VO(IV) complexes (1) and (7) which was owing to v(V=O). New broad peaks appearing in all the complexes except vanadium complex- es at 3423–3479 cm–1 were due to existence of H2O mole- cules.40 There was no change in IR bands of methoxy (OCH3) group indicating that it was not involved in the complexation.41 All these evidences confirmed that the li- gands bonded with the respective metal cations by azome- thine-N along with benzaldehydic-O by the deprotonation of phenolic group. 3. 2. UV-Vis Spectra The experimental UV-Vis spectra of all the as-syn- thesized complexes were recorded in dimethylformamide using 10-1 M concentration. The UV–Vis spectra of ligands have shown a band at 282–297 nm attributed to π–π* elec- tronic structure of aromatic ring system. However, the Table 2. Magnetic, conductivity and IR spectral data of metal complexes (1)–(12) No. µeff ΩM (B.M) (Ω–1cm2mol–1) v (cm–1) (1) 1.72 13.5 3153 (NH2), 1625 (C=N), 972 (V=O), 538 (V-N), 434 (V-O) (2) 5.78 12.9 3479 (H2O), 3128 (NH2), 1622 (C=N), 535 (Mn-N), 432 (Mn-O) (3) 4.34 15.4 3477 (H2O), 3140 (NH2), 1626 (C=N), 547 (Co-N), 445 (Co-O) (4) 3.07 17.6 3467 (H2O), 3152 (NH2), 1620 (C=N), 528 (Ni-N), 440 (Ni-O) (5) 1.81 19.2 3423 (H2O), 3167 (NH2), 1621 (C=N), 540 (Cu-N), 439 (Cu-O) (6) Dia 15.6 3470 (H2O), 3145 (NH2), 1627 (C=N), 549 (Zn-N), 435 (Zn-O) (7) 1.76 12.7 1622 (C=N), 972 (V=O), 528 (V-N), 439 (M-O) (8) 5.85 17.2 3429 (H2O), 1620 (C=N), 535 (Mn-N), 431 (Mn-O) (9) 4.21 14.8 3475 (H2O), 1628 (C=N), 537 (Co-N), 437 (Co-O) (10) 3.11 22.3 3445 (H2O), 1625 (C=N), 528 (Ni-N), 440 (Ni-O) (11) 1.87 15.4 3470 (H2O), 1623 (C=N), 539 (Cu-N), 437 (Cu-O) (12) Dia 18.3 3473 (H2O), 1630 (C=N), 530 (Zn-N), 433 (Zn-O) 205Acta Chim. Slov. 2022, 69, 200–216 Sumrra et al.: Metal Based Bioactive Nitrogen ... other absorption bands were documented as λmax at the 339–364 and 395–407 nm owing to n–π* electronic transi- tions by the azomethine linkage together with charge transfer, correspondingly.42 The vanadium(IV) complexes, (1) and (7) exhibited their characteristic bands in the range of 374–387, 529–536 and 733–747 nm as a result of the electronic transitions that were referred to B2→Eπ, B2→B1 and B2→A1 thus validating their predicted square pyramidal geometry.43 The three bands in the UV-Vis spectra of manganese(II) complexes (2) and (8) were ob- served at 221–233 nm 251–267 nm and 342–356 nm due to intra-ligand electronic transitions and 6A1g→4Eg there- fore verifying their proposed octahedral geometry. The cobalt(II) complexes (3) and (9) had shown a high energy band of 327-348 nm along with the low ener- gy absorption bands ranging from 506-586 and 1135-1157 nm because of 4T1g(F)→4T1g(P) and 4T1g(F)→4T2g(F) elec- tronic transitions evidencing their anticipated octahedral geometrical structure.44 The three electronic bands of nickel(II) complexes (4) and (10) were observed in the range of 389–425, 617–645 and 1024–994 nm owing to 3T2g(F)→3T1g(P), 3A2g(F)→3T1g(F) and 3A2g(F)→3T2g(F) electronic transitions proving their estimated octahedral geometry. The copper(II) complexes, (5) and (11) demon- strated two absorption bands at 518–524 and 631–674 nm due to 2B1g→2Eg and 2B1g→2A1g excitations as well as a highly intense band as a result of metal→ligand charge transfer (MLCT) at 338–345 nm suggesting their octahe- dral geometry.45 Only a strong band owed to MLCT at 322–336 nm was recorded for zinc(II) complexes (6) and (12), signifying the absence of d–d transitions and con- firming their anticipated octahedral geometry.46 3. 3. 1H-NMR Spectra 1HNMR spectra of the phenylenediamine Schiff bas- es (L1) and (L2) were determined in DMSO-d6. All the ar- omatic as well as heteroaromatic protons were observed in their estimated ranges. In the spectra of both ligands, the distinctive singlet peak of imine (HC=N) proton was spot- ted at 8.91–9.01 ppm. While in the spectrum of ligand (L1), the singlet peak of two amino (NH2) protons was ob- served at 7.53 ppm, signifying that only one amino group of p-phenylenediamine was condensed with o-vanillin, indicating the synthesis of mono-Schiff base ligand (Fig- ure S5). While the absence of both amino (NH2) group of o-phenylenediamine (Figure S6) specified the synthesis of bis-Schiff base ligand (L2). The peaks of methoxy (OCH3) group protons of both Schiff bases were observed as singlet at 3.82-3.92 ppm. The aromatic protons were appeared in the range 6.88–7.64 ppm. Furthermore, the phenolic (OH) group protons were found at 12.97–13.17 ppm as singlet. Absence of aldehydic (CH=O) group protons in the spec- tra of both ligands, confirmed that the condensation phe- nomenon was occurred between phenylenediamine and o-vanillin. 3. 4. Mass Spectra The mass spectra of both phenylenediamine Schiff bases showed the molecular weights (m/z) and base peaks (%). The base peak as well as molecular ion peak of the li- gand (L1) was found at m/z 241.1 due to [C14H13N2O2]+ fragment (Figure S7). While the ligand (L2) displayed mo- lecular ion peak of C22H20N2O4 fragment at m/z 376.4 that was equivalent to its molecular weight (Figure S8). This was also the most stable fragment. Similarly, all the daugh- ter fragments were obtained by the cleaving action of exo- cyclic as well as endocyclic (C=N) and (C=C) groups. The results of mass spectra evidently confirmed the synthesis of both phenylenediamine Schiff bases with their pro- posed structures. 3. 5. Molar Conductivity and Magnetic Moment Measurements The molar conductivity measurements of the synthe- sized metal complexes have been carried out at room tem- perature using dimethylformamide solvent and the con- ductivity readings are depicted in Table 2. The complexes were found to be non-electrolytes in nature as their molar conductance values were observed in the range 12.7–22.3 Ώ–1cm2mol–1.47 The findings of molar conductivity analy- sis declared all the metal complexes as neutral having no free anions outside the coordination sphere. Magnetic moments have contributed valuable details regarding the number of unpaired electrons together with stereochemistry of metal cations, leading towards the de- termination of the appropriate geometries of the complex- es. Depending on the magnetic influence of the unpaired electrons, some metal complexes have shown greater val- ues of magnetic moments while others presented lesser magnetic moments (as given in Table 2). The vanadyl com- plexes (1) and (7) had the magnetic moment values of 1.72–1.76 B.M which showed one unpaired electron and thus confirmed a square pyramidal configuration for both VO(IV) complexes.48 The Mn(II) complexes (2) and (8) have exhibited 5.78–5.85 B.M that pointed towards the presence of five unpaired electrons suggesting octahedral arrangement.49 The Co(II) complexes (3) and (9) dis- played the magnetic moments at 4.21–4.34 B.M, signifying complexes have high spins with three unpaired electrons present in an octahedral geometry.50 The measured mag- netic moments of nickel complexes (4) and (10) were found at 3.07–3.11 B.M representing the existence of two unpaired electrons thus signifying an octahedral configu- ration for Ni(II) complexes.51 The magnetic moments for Cu(II) complexes (5) and (11) have been observed as 1.81- 1.87 B.M representing only one unpaired electron for each copper metal ion with d9-system indicating octahedral ge- ometry for copper complexes.52 The Zn(II) complexes (6) and (12) had zero magnetic moment showing no unpaired electron thus both the complexes were found to be dia- magnetic in nature as expected.53 206 Acta Chim. Slov. 2022, 69, 200–216 Sumrra et al.: Metal Based Bioactive Nitrogen ... 3. 6. Molecular Geometric Parameters Complete process of geometry optimization has been accomplished without any symmetry restriction by em- ploying B3LYP level of DFT in combination with basis set 6-311++G(d, p). The vibrational analysis employing DFT/ B3LYP/6-311++G(d, p) level of theory has also been car- ried out to further validate the stability related with opti- mized geometrical structures. No hypothetical frequency was detected from the vibrational scrutiny of investigated compounds, which signified the completion of their geom- etry optimization. Figures 1 & 2 illustrate the molecular structure of all the studied compounds with atom number- ing. The optimized geometrical elements including bond angles together with bond lengths were estimated with DFT study by the B3LYP level, and the representative out- comes are given in Table S1 (Supplementary Information). For complexes (2), (3) and (4), the bond lengths of C4-N7, N7-C14 and C11-O15 were increased, because of the reason that the chelation occurred via N7, O15 with metallic centres (Mn, Co and Ni). Similarly for complexes (7), (11) and (12), increase in C5-N13, N13-C15, C4-N14, N14-O24, O16-C11 and O25-C21 bond lengths was ob- served as a result of the complexation via N13, N14, O16, O25 with metallic centres (V, Cu and Zn). This increase in bond lengths signified that the ligands (L1) and (L2) coor- dinated via N7, O15 and N13, N14, O16, O25 with diva- lent and tetravalent metallic centres, respectively. The for- mations of the M–N and M–O bonds resulted in the weakness of C–N and C–O bonds, correspondingly. All the bond lengths of ligands (L1) and (L2), which were involved in coordination have shown an increase in the lengths signifying the establishment of M–N and M–O bonds in all the studied metal complexes. Moreover, the Figure 1. View of optimized geometrical structures of p-phenylenediamine ligand (L1) and its derived metal complexes (2), (3) and (4) 207Acta Chim. Slov. 2022, 69, 200–216 Sumrra et al.: Metal Based Bioactive Nitrogen ... other bond lengths of the ligands were also significantly influenced by the coordination. Bond lengths of M–N bonds were greater than that of M–O bonds that gave the indication for stronger coordination of metal centers with oxygen atom of benzaldehyde by the deprotonation of phenolic group rather than nitrogen atom of azomethine linkage. 3. 7. Frontier Molecular Orbitals (FMOs) Analysis The highest occupied molecular orbital (HOMO) designates the electron donation while the lowest unoc- cupied molecular orbital (LUMO) designates electron ac- ceptance aptitude. The electronic transitions produced in consequence of the dipole moments arising amongst the ground and excited states of studied chemical entities are responsible for the optical features, electrical attributes and molecular chemical stability together with reactivity. In most of the cases, a transitions occur from HOMO to LUMO. Furthermore, the energy gap (ΔE) between these molecular orbitals is the main parameter to assign and explain reactivity and stability of the studied com- pounds.54 For newly synthesized phenylenediamine ligands (L1)–(L2) and their selective metal complexes, the energies Figure 2. View of optimized geometrical structures of o-phenylenediamine ligand (L2) and its derived metal complexes (7), (11) and (12) 208 Acta Chim. Slov. 2022, 69, 200–216 Sumrra et al.: Metal Based Bioactive Nitrogen ... of molecular orbitals including LUMO and HOMO, along with their energy differences have been computed by em- ploying B3LYP/6-311++G(d, p) basis sets (as shown in Ta- ble 3). While the FMO’s interpreting the distribution of electron charge density are illustrated in Figure 3. The DFT obtained results have shown that ligands (L1) and (L2) ex- hibited 491 and 749 molecular orbitals, respectively. From these orbitals, 64-65 and 99–100 were established as HO- MO-LUMO for both ligands, correspondingly. It is evalu- ated that the FMO energy gap calculated for HOMO→LU- MO in ligand (L1) was more than that in ligand (L2). The FMO energy gap HOMO→LUMO was 3.693 eV for (L1), which decreased to 3.653 eV in (L2). This reduction in the band difference for (L2) may be because of the presence of noncovalent attraction as well as extended conjugation in contrast to (L1). In both studied structures of the ligands, an evident intramolecular transfer of charge occur from the middle part (HOMO) to the ultimate part (LUMO), therefore giv- ing appropriate explanations to use these studied ligands in phenomenon of charge transfer. Overall, the acquired details signified that the ligand (L2) has shown small ener- gy difference contrary to other ligand (L1), which describes greater intra-molecular charge transfer (ICT) communica- tion within the ligand (L2). The trend for ∆EHOMO→LUMO was obtained as; (L1) 3.693 > (L2) 3.654 > (3) 3.085 > (12) 2.967 > (11) 2.926 > (4) 2.912 > (7) 2.847 > (2) 1.728. The molecules having minor frontier orbital band difference are highly chemical reactive as well as more polarizable. The calculated values of ∆EHOMO→LUMO suggested that the studied complexes have small band gap. Therefore, the complexes were found to be more reactive than the li- gands. It could be observed from figure that for both ligands (L1) and (L2), the electron density of HOMO and LUMO is concentrated on the entire structures with the exception of methoxy groups. Likewise, in LUMO of complex (2), the charge density is only accomulated at half part of the com- plex. Whie in the LUMO of all the other stidied complexes, the charge density is distributed over the entire complex structure. But in the HOMO of complex (2) and (7), the charge density is only focused at central part, mainly at the central metal ions and phenylenediamine-azomethine fragments. Whereas in the HOMO of all the other studied complexes, the charge density is dispersed over the com- plete complex structure. Figure 3. Frontier molecular orbitals (FMOs’) illustrating distribution of electronic charge density in ligands and their selected metal complexes 209Acta Chim. Slov. 2022, 69, 200–216 Sumrra et al.: Metal Based Bioactive Nitrogen ... 3. 8. Chemical Reactivity Parameters The FMO band gap (ΔE) is a remarkable factor to investigate and describe the chemical reactivity parame- ters such as the accepting and donating ability of the stud- ied molecules along with their hardness and softness.55 Molecules with a high FMO energy difference are kineti- cally least reactive and more stable, which makes them chemically hard in nature. While the molecules with small FMO band gaps are kinetically more reactive in nature which makes them chemically soft and less stable with more polarizability. Global reactivity parameters56 like electron affinity (EA), ionization potential (IP), global softness (σ), chemical potential (μ), global hardness (η), global electrophilicity (ω) in addition to electronegativity (χ) can be determined using FMO energy gap employing equations S1−S6 and the values are shown in Table 3. Elec- tronegativity is considered as the most important chemical parameter that describes the competency of any chemical system for attracting the electrons. The stability of any molecule is specified by the negative readings of the chem- ical potential (μ). This remarkable study could play an im- perative impact in the domain of experimental investiga- tion and particularly in the biological assay of chemical systems.57 It can be witnessed from the table that the ion- ization potential (5.886 eV) in ligand (L2) is more, having most negative chemical potential (–4.059 eV), greater val- ues of electron affinity (2.232 eV) and electronegativity (4.059 eV) than (L1). In addition, there exist a straight link between the FMO energy gap and the hardness, conse- quently the compound with a more FMO energy differ- ence is the chemically less reactive. As a result, the calcu- lated FMO energy differences and hardness are greater, while softness readings are smaller for (L1) in comparison to (L2), signifying that (L2) is more reactive and less stable. The detailed comparison of global reactivity parameters for all the studied compounds is illustrated by Figure 4. 3. 9. Molecular Electrostatic Potential (MEP) Analysis Molecular electrostatic potential (MEP) map is linked to electronic charge density and with the help of it, the chemical reactivity and noncovalent interactions such as nucleophilic and electrophilic attack sites can be com- prehend. MEP map is the graphical interpretation of the three dimensional electronic charge. With the help of this 3D map, the physical as well as chemical characteristic fea- tures of any chemical structure can also be elucidated.58 In the MEP map, the charge is distributed around the mole- cule in space which helps to understand the hydrogen bonding, reactive positions for the attack by electrophiles and nucleophiles as well and biological recognition proce- dures. It is known as supportive parameter that provides assistance to characterize the zone, size, positive, negative and shape of an investigated chemical structure. The elec- trostatic potential values are assessed with different shades. The negative region of electrostatic potential is denoted by red colour, while the blue and green colours represent the positive and less positive region of MEP respectively. Dif- ferent code colours in the terms of potential fallows fol- lowing order; blue > green > yellow > orange > red. Table 3. FMO energies and their energy gaps (∆E) for phenylenediamine based ligands and their selected metal complexes Descriptor (eV) Compounds (L1) (L2) (2) (3) (4) (7) (11) (12) ELUMO –1.796 –2.232 –1.483 –1.039 –1.484 –2.235 –1.891 –1.860 EHOMO –5.489 –5.886 –3.211 –4.124 –4.396 –5.082 –4.817 –4.827 ∆EHOMO→LUMO 3.693 3.654 1.728 3.085 2.912 2.847 2.926 2.967 Ionization Potential (IP) 5.489 5.886 3.211 4.124 4.396 5.082 4.817 4.827 Electron Affinity (EA) 1.796 2.232 1.483 1.039 1.484 2.235 1.891 1.860 Global Hardness (η) 1.847 1.827 0.864 1.543 1.456 1.424 1.463 1.484 Chemical Potential (µ) –3.643 –4.059 –2.347 –2.582 –2.940 –3.659 –3.354 –3.344 Global Softness (S) 0.271 0.274 0.579 0.324 0.343 0.351 0.342 0.337 Electronegativity (χ) 3.643 4.059 2.347 2.582 2.940 3.659 3.354 3.344 Electrophilicity (ω) 3.593 4.509 3.188 2.160 2.968 4.701 3.845 3.768 Figure 4. Comparison of global reactivity parameters for ligands vs metal complexes 210 Acta Chim. Slov. 2022, 69, 200–216 Sumrra et al.: Metal Based Bioactive Nitrogen ... By using density functional B3LYP/6-311++G(d, p) basis set, MEP is designed over optimized geometrical structures of the studied compounds and the MEP plots are displayed in Figure 5. An analysis of the MEP plots suggested that in the investigated compounds, the red co- lour (negative region) was localized around Ohydroxyl and Omethoxy atoms. Hence, it is an electron rich part and could be potential place for the attack by electrophile. In con- trast, the blue colour (positive region) defining the elec- tron deficient area was localized around Namino and Nazome- thine atoms together with some of the hydrogen alongside carbon atoms could be a favourable site for the attack by nucleophiles. Whereas, the green zone indicated the aver- age potential, i.e., the part in the middle of two margins. From blue, green and red colours, it was obvious that all these different reaction positions were present in all stud- ied compounds. The studied metal complexes possess electron deficient sites with smaller electronegativity which are the preferred positions for an attack by nucleop- hilic species. The computed MEP plots were found to be in agreement with the computed atomic charges contained by studied chemical molecules. Thus, signifying that on complexation, the intra-molecular charge dispersal pro- duced an electropositive zone at the central metal atoms that might influence their various physicochemical prop- erties. 3. 10. Natural Bond Orbital (NBO) Analysis NBO analysis is an effective practice that proficiently interprets the intramolecular interactions and delocaliza- tion of electronic charge density. It provides proper details to analyse and explain the intra-molecular hydrogen bond- ing as well as transfer of electronic charge from the filled orbitals to free orbitals by employing the second-order Fock matrix. By means of equation 1, the stabilization energies of the investigated molecules have been calculated, and some significant NBO interactions are depicted in Table S2. NBO analysis indicated the existence of resonance/π-conjugation attributable to delocalization of π-electrons in addition to strong intra-molecular primary or secondary hyper-conju- gative interactions in the investigated compounds. From the findings attained in valence hybrids of NBO's, the NBO analysis also provides significant perceptions about the po- larity of different bonds within the studied molecular sys- tems. The NBO results also signify the involvement of extra valence orbitals in the composition of natural bond orbitals that has notable contribution regarding the stabilization en- ergy within the examined molecules.59 The NBO analysis is also important as it explains the nature of any definite bond through evaluating the interactions between donating and accepting orbitals. (1) In this equation, E(2) defines the stabilization energy, qi symbolizes the vacancy of the contributor orbital, F(i,j) denotes the diagonal while εi and εj indicate the off diago- nal NBO Fock matrix features. In this NBO study, interac- tions between electron donating and accepting orbitals are exposed by the stabilization energy E(2) value. The larger value of stabilization energy E(2) indicates that greater in- teraction is found between electron acceptors and donors. The overlapping of σ(C-C) with σ*(C-C) bonding orbitals causes molecular interaction, that is producing the intra- molecular charge to stabilize the system. When there is higher electronic charge density in C-C antibonding orbit- al, these interactions are observed which weakens corre- sponding bonds.60 The electron density due to single and double bond of the conjugated ring exhibits strong delo- calization within the chemical systems. The interaction energy inferred from second order perturbation theory analysis of Fock Matrix. The analysis was performed for Figure 5. MEPs and colour pattern for the investigated ligands and their selected metal complexes 211Acta Chim. Slov. 2022, 69, 200–216 Sumrra et al.: Metal Based Bioactive Nitrogen ... studied compounds by investigating all the promising in- teractions regarding occupied Lewis style donors and va- cant non Lewis type acceptor, while their status of energies is estimated by second order perturbation theory. Usually, four major types of electronic transitions in- cluding σ→σ*, LP→σ*, π→π*, along with LP→π* are seen for any studied molecule. The observed π→π* type of tran- sitions have an additional estimation, while transitions such as LP→σ* as well as LP→π* demonstrated quite ap- propriate values for stabilization energy E(2). Moreover, the least E(2) values were shown by σ→σ* electronic transi- tions. The highest readings for π→π* transitions were π(C2−C3)→π*(C1−C6), π(C21−C22)→π*(N14−C24) with stabilization energies of 21.46 and 21.54 kcal/mol in li- gands (L1) and (L2), correspondingly. Although, some oth- er π→π* electronic transitions having significant values of stabilization energy were also observed like π(C8− C13)→π*(C9−C10), π(C4−C5)→π*(C1−C6), π(C11−C12)→ π*(N7−C14), π(C9−C10)→π*(C8−C13) and π(C1−C6)→π* (C2−C3) in (L1) with 18.68, 18.67, 18.38, 17.77 and 16.25 kcal/mol, whereas π(C3−C4)→π*(C1−C2), π(C5−C6)→ π*(C1−C2), π(C7−C8)→π*(C9−C10), π(C1−C2)→π*(C3− C4), π(C18−C23)→π*(C19−C20), π(C19−C20)→π*(C18−C23) and π(C9−C10)→π*(C7−C8) in (L2) with stabilization ener- gy of 20.54, 20.54, 20.54, 20.09, 18.30, 17.61 and 17.34 kcal/mol, respectively. These electronic transitions strong- ly stabilize both the ligands (L1) and (L2). Similar type of interaction linked to resonance in the structure was observed between the oxygen lone pair O15, O12 and the anti-periplanar C11−C12, C7-C8 antibond which gave 27.24 and 65.17 kcal/mol energy of stabiliza- tion in (L1) and (L1), respectively. It is evident from the NBO analysis that strong interactions, charge transfer properties, stabilization energies, coordination tendency and stability exist in the studied ligands (L1) and (L2). Likewise, the foremost π→π* interactions including; π(C29‒C30)→π*(N24‒C31), π(C29‒C30)→π*(N24‒C31), π(C2‒ C3)→π*(C4‒C5), π(C24‒C25)→π*(N16‒C26) and π(C20‒ C21)→π*(C24‒C25) produced 18.39, 38.68, 10.64, 17.72 and 23.42 kcal/mol stabilization energies for complexes (2), (4), (7), (11) and (12) correspondingly. In addition, the LP→π* transitions were observed as; C10→π*(C8-C9), C12→π*(N7-C14), C23→π*(N15-C25), C22→π*(C20-C21) and O14→π*(C9-C10) with higher stabilization energies of 39.85, 125.84, 69.47, 41.41 and 64.55 kcal/mol for com- plexes (2), (4), (7), (11) and (12) respectively. This NBO analysis showed extended hyperconjugation and notable intramolecular interactions of studied compounds. 3. 11. Mulliken Atomic Charge (MAC) Analysis In chemistry, chemical reactivities, electromagnetic spectra accompanied by NMR chemical shifts, electric po- tentials and dipole moments are considered as recogniz- able parameters of molecules which could have direct asso- ciation with atomic charges in the chemical systems. Various theories regarding the structural features of chem- ical systems entirely depend on the concept of Mulliken atomic charges. The calculation of atomic charges for the preferred chemical systems by computational chemistry play supportive role to evaluate and explain the experimen- tal data. It is particularly important to deeply comprehend the characterization of atomic charges to understand and describe the applications of any chemical system.61 The Mulliken atomic charges of investigated compounds were determined by employing B3LYP level and 6–311++G(d, p) basis set. The Mulliken charges are listed in Tables S3 & S4, while their histograms are presented in Figure 6 & 7. The charge dispersion of the studied compounds displayed that the all oxygen and nitrogen atoms in addition to the carbon atoms linked with oxygen and nitrogen atoms were negatively charged. Whereas, greater positive values were found for the other carbon and hydrogen atoms together Figure 6. Histogram for Mulliken atomic charges of ligands (L1) & (L2) 212 Acta Chim. Slov. 2022, 69, 200–216 Sumrra et al.: Metal Based Bioactive Nitrogen ... with metals (Mn, Co, Ni, V, Cu and Zn) in the studied met- al complex (2), (3), (4), (7), (11) and (12). 3. 12. Computed UV−Vis Analysis UV−Visible spectroscopy gave valuable explanation on the charge transfer potentials of the investigated com- pounds. To get the insights concerning the theoretical UV- Vis spectra of the ligands together with their selected met- al complexes, TD-DFT calculation were executed at B3LYP/6-311++G(d, p) level. The transition energy values, maximum absorption wavelength (λmax), oscillator strength (fos) together with minor and major molecular orbital transitions of the studied compounds are tabulated in Table S5. Whereas, the theoretical UV-Vis spectra are shown in Figure S9. Figure 7. Histogram for Mulliken atomic charges of studied metal complexes 213Acta Chim. Slov. 2022, 69, 200–216 Sumrra et al.: Metal Based Bioactive Nitrogen ... 3. 13. Computed IR Analysis The synthesized ligands (L1) and (L2) having 32 and 48 atoms correspondingly comprising carbon, oxygen, hydro- gen and nitrogen atoms. The harmonic vibrational frequen- cies of both ligands were computed using B3LY- P/6-311++G(d, p). Both the ligands contain 90 and 138 vibrational modes correspondingly with singlet spin and point group symmetry C1. The calculated vibrational modes of atoms were assigned using the animation option of Gauss View software. All the vibrational assignments associated with theoretical infrared spectral values were taken into con- sideration while only the most prominent vibrational fre- quencies with high accuracy are listed in Tables S6 & S7. Figure 8 illustrates the theoretically simulated scaled infra- red spectra of all studied compounds. The theoretical fre- quencies were found to be greater than the experimentally observed values and the obvious reason would be the overes- timation of the computed vibrational modes because of the negligence of anharmonicity in the actual chemical system. With the aim to reduce the frequency values analo- gous to that of experimental values, electron correlation would be included in density functional theory. The over- all practice is to scale down the computed vibrational fre- quencies to compare the frequency values obtained after the experiment. The scaling factor approach is very valu- able for correlating the theoretical vibrational frequencies to the practically investigated frequencies. Thus, the scal- ing factor (0.9742) of B3LYP/6-311++G(d, p) was utilized to precise the systematic defects i.e., to neglect of basis set defects along with some enharmonic effects.62 Experimen- tal infrared spectra of ligands were compared with the the- oretically simulated scaled infrared spectra, followed by comprehensive frequency assignments. The important functional groups are described here in detail: O-H vibrations: The O–H stretching vibration was found at 3744, 3123 cm−1 (theoretical) and 3427, 3230 cm−1 (experimental) for ligand (L1) and (L2), respectively. NH2 vibrations: The vibrational modes for NH2 were observed at 3478 and 3574 cm−1 (theoretical) and 3020 cm−1 (experimental). HC=N vibration: The HC=N vibrations were detect- ed at 1617, 1619 cm−1 (theoretical) and 1640, 1638 cm−1 (experimental) for ligand (L1) and (L2), correspondingly. The DFT-based theoretical vibrational modes have obtained to be consistent with the practically determined findings. 3. 14. Antibacterial Activity The as-synthesized compounds were investigated for in vitro antibacterial activity against three G- bacteria (GNB) i.e., Salmonella typhi, Klebsiella pneumonia, Esche- richia coli along with one G+ bacteria (GPB) i.e., Staphylo- coccus aureus. Two reference drugs i.e., ampicillin (SD1) and streptomycin (SD2) were used to compare the results of antibacterial activity of investigated compounds (Table 4, Figure 9). The results showed that DMSO had no inter- ference on antibacterial activity of the compounds. Both the uncomplexed ligands exhibited antibacterial activity against all bacteria except (L2) that showed no activity against Klebsiella pneumonia. Overall, ligand (L1) exhibit- ed more activity. It showed maximum and minimum ac- tivity against Staphylococcus aureus and Klebsiella pneumo- nia with 17 and 05 mm zones of inhibition, respectively. While (L2) exhibited highest activity against Escherichia coli with 18 mm zone of inhibition. All the complexes exhibited significant antibacterial activity against all bacteria except complexes (2) and (4) that were inactive against Klebsiella pneumonia and Staph- ylococcus aureus, correspondingly. Among all the com- plexes, the zinc complexes were found to be the most ac- tive. The complexes (12) and (6) displayed the maximum activity of 29 and 28 mm against Staphylococcus aureus. While both complexes (12) and (6) exhibited moderate in- hibitory activity of 23 mm against Escherichia coli, corre- spondingly. The complex (3) inhibited 23, 22, 21 and 16 mm zones of Klebsiella pneumonia, Salmonella typhi, Esch- erichia coli and Staphylococcus aureus, respectively. The complexes (7) and (8) demonstrated least antibacterial profile with 6 and 7 mm inhibition zones against Staphylo- coccus aureus and Salmonella typhi, correspondingly. Figure 8. Computed IR spectra for phenylenediamine Schiff base ligands and their selected metal complexes 214 Acta Chim. Slov. 2022, 69, 200–216 Sumrra et al.: Metal Based Bioactive Nitrogen ... Table 4. Antibacterial activity (inhibition zone/mm) of ligands and their metal complexes Compounds Salmonella Klebsiella Escherichia Staphylococcus typhi (G-) pneumonia (G-) coli (G-) aureus (G+) (L1) 15 05 15 17 (L2) 17 0 18 16 (1) 14 22 06 19 (2) 21 23 11 0 (3) 22 23 16 21 (4) 21 0 21 0 (5) 19 15 17 20 (6) 20 23 19 28 (7) 14 20 05 06 (8) 07 16 23 23 (9) 21 09 07 09 (10) 18 21 22 19 (11) 22 18 15 20 (12) 19 22 23 29 (SD1) 29 38 35 30 (SD2) 35 39 37 40 SD1 = Ampicillin, SD2 = Streptomycin 4. Conclusion In the current situation of increasing global drug re- sistance coupled with the scarcity of efficient antibacterial drugs, two new symmetrical ligands (L1)–(L2) and their derived metal chelates were synthesized and experimen- tally characterized through physical, elemental, spectral data along with computational study by DFT/B3LY- P/6-311++G(d,p) approach. The spectral assignments of all the metal based phenylenediamine compounds con- firmed that the deprotonated bidentate and tetradentate Schiff base ligands bonded with 3d-metal cations through phenolic oxygen and azomethine nitrogen along with two water molecules resulting in the formation of a stable six-membered chelate ring. Based on the magnetic mo- ments and electronic spectra, an octahedral geometry was recommended for all the divalent metal complexes except for tetravalent vanadyl complexes that exhibited square pyramidal geometry, thus correlating accurately with the assessed molecular formula. On the basis of molar con- ductivity, all the metal complexes formed were declared as neutral having no free anions outside the coordination sphere. The theoretically obtained structural features ac- corded effectively with experimentally determined struc- tural findings. This reasonable constancy validated that the chosen DFT method might be a realistic approach to com- prehend some other characteristic features of the studied compounds. The charge transfer properties, kinetic stabil- ity and chemical reactivity of the studied compounds were evaluated by FMO analysis. Antibacterial activity of all the Figure 9. Antibacterial activity of phenylenediamine Schiff base derived compounds against Gram-positive and Gram-negative bacteria 215Acta Chim. Slov. 2022, 69, 200–216 Sumrra et al.: Metal Based Bioactive Nitrogen ... phenylenediamine derived compounds was evaluated. The results of bioactivity concluded that both the phenylenedi- amine ligands have shown significant antibacterial poten- tial which was further intensified upon chelation owing to the transference of charge from metal to ligand. Overall, the Zn(II) complex possessed higher antibacterial activity. The results conclude that these metal based compounds have the aptitude to be converted into drug candidates and this study will be valuable to design and develop promis- ing metal-based drugs to treat microbial infections. Acknowledgements The authors are grateful to the Higher Education Commission (HEC) of Pakistan for providing financial as- sistance through the NRPU Project # 7800. Conflict of Interest All authors declared that they have no conflict of in- terest. 5. References 1. W. Zafar, S. H. Sumrra, Z. H. Chohan, Eur. J. Med. Chem. 2021, 222, 113602. DOI:10.1016/j.ejmech.2021.113602 2. H. Kargar, R. Behjatmanesh-Ardakani, V. Torabi, M. Kashani, Z. Chavoshpour-Natanzi, Z. Kazemi, V. 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DOI:10.1016/j.saa.2019.117200 61. A. S. Athmani, F. Madi, I. Laafifi, M. Cheriet, N. Issaoui, L. Nouar, R. Merdes, J. Struct. Chem. 2019, 60, 1906–1916. DOI:10.1134/S0022476619120060 62. V. S. Kumar, Y. S. Mary, K. Pradhan, D. Brahman, Y. S. Mary, R. Thomas, M. S. Roxy, C. V. Alsenoy, J. Mol. Struct. 2020, 1199, 127035. DOI:10.1016/j.molstruc.2019.127035 Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License Povzetek Zanimanje za razvoj naprednih spojin na osnovi kovin, ki zavirajo in kontrolirajo bakterijske okužbe, neprestano narašča. Sintetizirali smo dve novi bioaktivni simetrični mono- in bis- Schiffovi bazi na osnovi fenilendiamina, 2-{[(4-aminofenil) imino]metil}-6-metoksifenol (L1) in 2,2‘-{benzen-1,2-diilbis[nitrilometililiden]}bis(6-metoksifenol) (L2). Spojini smo kar- akterizirali s fizikalnimi metodami, spektroskopijo, elementno analizo in DFT računalniško analizo z metodo B3LY- P/6-311++G(d, p). Sintetizirali smo koordinacijske spojine obeh novih ligandov z VO, Mn, Co, Ni, Cu in Zn v množinskih razmerjih [M:L, 1:2 in 1:1]. Vse tako pripravljene koordinacijske spojine imajo dobro antibakterijsko delovanje, z najboljšim delovanjem v primeru cinkovih kompleksov. Rezultati kažejo, da so tovrstne spojine obetavne za medicinske aplikacije. 217Acta Chim. Slov. 2022, 69, 217–226 Žener, et al.: Metal and Non-Metal Modified Titania: ... DOI: 10.17344/acsi.2021.7200 Scientific paper Metal and Non-Metal Modified Titania: the Effect of Phase Composition and Surface Area on Photocatalytic Activity Boštjan Žener,1 Lev Matoh,1 Martin Reli,2 Andrijana Sever Škapin3,4 and Romana Cerc Korošec1,* 1 Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, 1000 Ljubljana, Slovenia; 2 Institute of Environmental Technology, VŠB-Technical University of Ostrava, 17. listopadu 15/2172, Ostrava-Poruba, Czech Republic; 3 Slovenian National Building and Civil Engineering Institute, Dimičeva 12, 1000 Ljubljana, Slovenia 4 Faculty of Polymer Technology - FTPO, Ozare 19, 2380, Slovenj Gradec, Slovenia * Corresponding author: E-mail: romana.cerc-korosec@fkkt.uni-lj.si Received: 10-11-2021 Abstract The application of TiO2 photocatalysis in various environmental fields has been extensively studied in the last decades due to its ability to induce the degradation of adsorbed organic pollutants. In the present work, TiO2 powders doped and co-doped with sulfur and nitrogen and modified with platinum were prepared by particulate sol-gel synthesis. PXRD measurements revealed that the replacement of HCl with H2SO4 during synthesis reduced the size of the crystallites from ~ 30 nm to ~20 nm, increasing the surface area from ~44 m2/g to ~80 m2/g. This is consistent with the photocatalytic activity of the samples and the measured photocurrent behavior of the photocatalysts. The results showed that the prop- erties of the powders (i.e., surface area, crystallite size, photocurrent behavior) depend strongly not only on the type but also on the amount of acid and dopants used in the synthesis. Doping, co-doping and modification of TiO2 samples with nitrogen, sulfur and platinum increased their photocatalytic activity up to 6 times. Keywords: Titanium dioxide; powders; doping; photocatalysis; photocurrent; SEM 1. Introduction Titanium dioxide is considered to be one of the most contemporary important materials. It occurs in nature in three polymorphic modifications: anatase, rutile and brookite, among which rutile is the most abundant and thermodynamically stable. On the contrary, anatase has the highest photocatalytic activity, which can be attributed to the highest number of hydroxyl groups on the surface.1 Furthermore, three metastable phases can be produced synthetically, one of which is β-TiO2, which crystallizes in a monoclinic crystal system.2,3 Due to its favourable prop- erties, including its high chemical stability, non-toxicity, low price and high refractive index (value of µ is 2.70 for rutile and 2.55 for anatase) TiO2 is used for a wide number of applications in a variety of fields, for example as a white pigment in paints, plastic, paper, toothpastes and chewing gums, replacing the toxic lead oxides.4 It is also used in the fields of photovoltaics, electrochemistry and photocataly- sis.5–9 Due to its ability to mineralize adsorbed organic pol- lutants to CO2 and H2O, photocatalysis has been re- searched extensively with regard to its application in the fields of water remediation and air purification.10–12 Oxi- dation of adsorbed organic pollutants can occur directly on the surface of the photocatalyst.13 In the case that ad- sorption is not favourable due to the same electric charge on both pollutant and catalyst itself, reactive hydroxyl rad- ical, formed via oxidation of water with holes, can start degradation reactions of pollutants in a solution. To en- hance photocatalytic efficiency, it is necessary to prevent recombination between holes and electrons on route to the surface or on the surface sites.14 218 Acta Chim. Slov. 2022, 69, 217–226 Žener, et al.: Metal and Non-Metal Modified Titania: ... Various studies have been aimed at increasing the photocatalytic activity of TiO2, which can be achieved by increasing the surface area of TiO2, or through metal and non-metal doping. The surface area of the photocatalyst can be increased by decreasing its particle size.15,16 Sam- ples with a smaller crystallite size have a larger number of surface active sites, which should also increase its photo- catalytic activity. Wang et al., however, concluded that an optimum particle size of 11 nm exists for the degradation of chloroform in water. This was attributed to an increased recombination rate, which offsets the ultra-high surface area.17,18 Maira et al. found an optimum particle size of 7 nm for gas phase photooxidation of trichloroethylene. The diminished activity for samples with crystallites, smaller than 7 nm, was attributed to changes in electronic and structural properties.19 The surface area of the photocata- lyst can also be increased by adding polymers during the synthesis. The calcination that follows removes the poly- mer chains, leaving behind a mesoporous framework of TiO2, with an increased surface area.20–23 Doping TiO2 with metals also increases its photocat- alytic activity, by facilitating free electron capture and thus extending the lifetime of photogenerated electron-hole pairs.24 The capture results in an efficient separation of electron-hole pairs, thus inhibiting recombination and in- creasing the photocatalytic activity of TiO2 by enhancing the mass transfer of holes and possibly electrons to the sur- face.25 It has to be mentioned, however, that metal centres can also act as recombination centres and thus lower the photocatalytic activity. TiO2 is usually doped with noble metals, such as Ag, Pt and Pd.26–28 Other metals include Cu, V, Cr, Ni, as well as In.29–31 TiO2 can be synthesized using various synthetic pro- cedures, including hydrothermal, microwave-assisted and sonochemical methods, and miniemulsion techniques.32–35 Sol-gel synthesis offers many benefits compared to the synthetic methods mentioned above, including low cost, simplicity and low preparation temperatures. Because of this, it is a well-established procedure for the preparation of metal oxide nanoparticles.36 The method is based on in- itial hydrolysis of a precursor (e.g. TiCl4, titanium alkox- ides), which is followed by reactions of condensation (ox- olation and olation). Reactions result in the formation of sols, which are defined as stable suspensions of colloidal particles that can polymerize to form gels under certain conditions. On the other hand, stable sol can be deposited by dip- or spin coating onto a substrate and is then subject- ed to a drying process. During this process, the free –OH groups begin to link together, resulting in xerogels.37 In this work, we focussed on the preparation and characterization of sulfur and nitrogen doped and co- doped and platinum modified TiO2 powders. Synthesis, structural properties and photocatalytic efficiency of the corresponding thin film was already published.38 Since chemically equivalent thin films and powders often behave in different way, we have also systematically studied pow- dered samples, which is the main focus of the presented paper. Different analytical methods were used for their characterization and determination of the photocatalytic activity. Prepared samples were characterized by X-ray dif- fraction (XRD), specific surface area (BET) and photocur- rent measurements. Morphology of the powders was ex- amined using a field emission scanning electron microsco- py. The photocatalytic activity of the powders was deter- mined by monitoring the rate of oxidation of isopropanol to acetone using FTIR spectroscopy. 2. Experimental 2. 1. Synthesis The method of synthesis, as used to synthesize metal and non-metal doped and co-doped TiO2 powders, has been described previously in detail elsewhere.38 The synthe- sis procedure is described in the supplementary material. Sample names, types and amounts of dopants and acids added during the synthesis are presented in Table 1. Table 1. Sample names, types and amounts of dopants and acids added during the synthesis. Nominal Amount Dopant; amount (mL) and Sample dopant of dopant type of acid source relative to TiO2 added (atom %) REF / / 18; HCl Urea_15 N; urea 15 18; HCl Thiourea_15 S; Thiourea 15 18; HCl S2 S; H2SO4 / 3.3; H2SO4 S3 S; H2SO4 / 4.95; H2SO4 S3_N0.5 S; H2SO4 / N; NH4NO3 0.5 4.95; H2SO4 S3_N2 S; H2SO4 / N; NH4NO3 2 4.95; H2SO4 S3_urea15 S; H2SO4 / N; urea 15 4.95; H2SO4 S3_thiourea15 S; H2SO4 / S; Thiourea 15 4.95; H2SO4 S; H2SO4 / S3_N0.5+1%Pt N; NH4NO3 0.5 4.95; H2SO4 Pt; H2PtCl6 1 S; H2SO4 / S3_N0.5+2%Pt N; NH4NO3 0.5 4.95; H2SO4 Pt; H2PtCl6 2 S; H2SO4 / S3_N0.5+3%Pt N; NH4NO3 0.5 4.95; H2SO4 Pt; H2PtCl6 3 S; H2SO4 / S3_urea15+1%Pt N; urea 15 4.95; H2SO4 Pt; H2PtCl6 1 S; H2SO4 / S3_urea15+2%Pt N; urea 15 4.95; H2SO4 Pt; H2PtCl6 2 219Acta Chim. Slov. 2022, 69, 217–226 Žener, et al.: Metal and Non-Metal Modified Titania: ... The measured amounts of different dopants were deter- mined with XPS measurements and are given in.38 2. 2. Characterization XRD patterns were measured using a PANalytical X’Pert PRO MPD instrument in the 2θ range of 20–80° with a step of 0.034° using CuKα1 radiation. The average diameters of crystallites and phase compositions (amounts of polymophic modifications in % ) of the samples and corresponding error values were calculated with Rietveld analysis using TopasR software.39 Structural model (ICSD codes 92363 for anatase and 171670 for β-TiO2) was used for the calculations. The specific surface area of the powders was deter- mined through the measurement of nitrogen adsorp- tion-desorption isotherms by a Tristar 3000, Micromerit- ics (USA) instrument. The measurements were performed at –196 °C (77 K). The samples were outgassed under vac- uum for 16 h at 110 °C (383 K). The mass of the samples in the analyser was ≈ 0.1 g. The specific surface area was cal- culated from the adsorption measurements in the relative pressure (p/p0) range of 0.05–0.25. Photocurrent responses were recorded using a three electrode system, where Ag/AgCl and a Pt wire were used as reference and counter electrodes, respectively. The working electrode was prepared by depositing the meas- ured sample on the conductive side of an ITO foil. 0.1 M KNO3 was used as an electrolyte. The measurements were carried out in the range of wavelengths from 250 nm to 450 nm at an applied external potential of 1 V. This ensures that the highest number of photogenerated electrons travel to the working electrode. In this way the charge carriers (free electrons and holes) were successfully separated, thus preventing recombination. Before each measurement, the cell was purged with argon in order to ensure an oxygen free environment.40,41 In the measured range from 250 nm to 450 nm, the wavelength is being changed by 10 nm step. The photocurrent signal drops down when the shutter closes to switch the wavelength for another 10 nm. The morphology and the size of the particles of pre- pared powders were examined using a field-emission scanning electron microscope FE-SEM (FEI InspectTM F50 and Ultra Plus Zeiss). Accelerating voltage was set to 2 kV. Images were obtained with detection of secondary electrons. 2. 3. Photocatalytic Activity Tests The photocatalytic activity of the powder samples under UV and visible light exposure was determined by measuring the rate constant of oxidation of isopropanol to acetone and further oxidation leading to CO2 and H2O as the final products using FTIR spectroscopy. Commercially available Hombikat UV 100 (DE) – anatase nanopowder, with a primary crystal size <10 nm and specific surface area >250 m2/g was used for comparison. The reactions are presented in Equation (2):42 (2) Generally, the first step (k0) is considered to be a zero order reaction, whereas the second step (k1) is considered to be first order reaction. The method is presented in detail elsewhere.43 In the first step, approximately 50 mg of the powder was suspended in 3 mL of 1-butanol. This suspen- sion was then evenly distributed in a standard Petri dish and dried for 2 hours at 50 °C. Each dried sample was then put in a sealed gas-solid flow reactor system and then in- jected with 8 µL of isopropanol. Once the adsorption equi- librium was reached, as indicated by flat line in the isopro- panol concentration profile, the sample was illuminated with a 300 W Xe lamp (Newport Oriel Instrument) with an infrared filter. The spectrum of this Xe lamp is similar to sun illumination. The working distance between the Petri dish and the lamp was 6 cm. The temperature and relative humidity were set to 23 ± 2 °C and 25 ± 5 %, re- spectively. The isopropanol degradation and acetone for- mation and degradation processes (see equation 2) were followed by monitoring the calculated area of their charac- teristic peaks at 951 cm−1 and 1207 cm−1, respectively, in the IR spectra obtained by a FT-IR spectrometer (Per- kin-Elmer Spectrum BX II). The examples of characteristic FTIR output at three diferent reaction times with related explanations are presented in Figure S1 (Supplementary material). 3. Results and Discussion 3. 1. X-Ray diffraction (XRD) XRD patterns of the prepared powders are presented in Figure 1. It can be seen from the results that anatase is the only polymorphic modification present in samples synthesized with HCl (REF, Urea_15 and Thiourea_15). On the contrary, patterns of samples synthesized with H2SO4 include peaks of β-TiO2, which crystallizes in a monoclinic crystal system and cannot be found in nature. Table 2 shows the share of polymorphic phases and calcu- lated diameters of crystallites (both were calculated using the Rietveld analysis) in characterized samples. The results show that the amount of β-TiO2 in samples synthesized with H2SO4 varies from 15.6 ± 0.3 % to 45.4 ± 0.3 %. The highest amounts are present in samples S3_urea15 and S3_thiourea15 (38.7 ± 0.3 % and 45.4 ± 0.3 %), whereas samples S3 exhibits the lowest (15.6 ± 0.3 %). Moreover, crystallites found in samples synthesized with H2SO4 (15.1 ± 0.3 – 23.5 ± 0.2 nm) were smaller compared to samples synthesized with HCl (26.9 ± 0.7 – 30.7 ± 0.8 nm). The same trend was observed in our previously published work, which focused on thin films.38 As with thin film sys- 220 Acta Chim. Slov. 2022, 69, 217–226 Žener, et al.: Metal and Non-Metal Modified Titania: ... tems, this can be attributed to the formation of TiOSO4 in samples synthesized with H2SO4, which inhibits the crys- tallization of anatase. The smallest crystallites were found in samples S3_N2 and S3_urea15 (15.1 ± 0.3 to 17.5 ± 0.2 nm). Decreasing the crystallite size of TiO2 usually results in an increase in surface area, which can have a positive effect on the photocatalytic activity of the samples. In most samples, the calculated crystallite sizes of β-TiO2 are smaller to those of anatase. When comparing the size of crystallites in powders to those in thin films (as published in our previous work), we observed an interesting phenomenon. In the case of samples synthesized with H2SO4 we found larger crystal- lites in powder form (sizes of ~ 15 – 23 nm in powders compared to 8–12 nm in thin films). This can be explained by the unlimited growth of crystallites in powders, unlike in thin films, where growth is limited by the thickness of the film and the substrate. In direct contrast, when analys- ing samples synthesized with HCl we found larger crystal- lites in thin film samples compared to powders (sizes of 40 – 60 nm and ~ 27 – 31 nm, respectively). We attribute this to the partial crystallization and subsequent growth of TiO2 nanoparticles during the thermal treatment after each deposition (the final layer was prepared from three successive depositions, after each deposition thermal treatment was performed at 300 °C).38 Samples with added platinum also exhibit peaks at 40, 47.5 and 67.5° 2θ, which correspond to metallic plati- num. 3. 2. Specific Surface Area (BET) Results of surface area measurements are presented in Table 2. Measurements for six of these samples (REF, Urea_15, Thiourea_15, S3, S3_N0.5 and S3_N0.5+1% Pt) Table 2. The share of different polymorphic modifications, sizes of crystallites in powder samples calculated from XRD patterns using Rietveld re- finement and the specific surface areas for examined samples. Amount Calculated diameters Amount of Calculated diameters BET specific Sample of anatase of anatase β-TiO2 of β-TiO2 surface area (wt%) crystallites (nm) (wt%) crystallites (nm) (m2/g) REF 100 30.7 ± 0.8 / / 44.1 ± 0.4 Urea_15 100 29.6 ± 0.7 / / 24.2 ± 0.2 Thiourea_15 100 26.9 ± 0.7 / / 49.5 ± 0.3 S2 75.1 ± 0.3 21.1 ± 0.1 24.9 ± 0.3 19.0 ± 0.5 63.2 ± 0.3 S3 84.4 ± 0.3 23.5 ± 0.2 15.6 ± 0.3 19.0 ± 0.7 80.2 ± 0.2 S3_N0.5 81.1 ± 0.4 22.3 ± 0.2 18.9 ± 0.4 18.8 ± 0.6 84.5 ± 0.3 S3_N2 69.9 ± 0.3 16.8 ± 0.1 30.1 ± 0.3 15.1 ± 0.3 103.4 ± 0.4 S3_urea15 61.3 ± 0.3 17.5 ± 0.2 38.7 ± 0.3 16.5 ± 0.3 101.7 ± 0.4 S3_thiourea15 54.6 ± 0.3 18.7 ± 0.2 45.4 ± 0.3 19.3 ± 0.3 92.1 ± 0.3 S3_N0.5+1%Pt 82 ± 1 21.0 ± 0.4 18 ± 1 18 ± 1 83.7 ± 0.2 S3_N0.5+2%Pt 77 ± 2 17.9 ± 0.6 23 ± 2 20 ± 3 80.5 ± 0.4 S3_N0.5+3%Pt 74 ± 2 17.1 ± 0.8 26 ± 2 20 ± 3 84.1 ± 0.4 S3_urea15+1%Pt 69 ± 1 21.0 ± 0.6 31 ± 1 22 ± 2 82.3 ± 0.3 S3_urea15+2%Pt 73 ± 1 20.7 ± 0.6 27 ± 1 23 ± 2 84.2 ± 0.4 Figure 1. X-Ray diffraction patterns of powders. The designations A, B and Pt indicate the peaks for the corresponding TiO2 polymor- phic modifications and platinum. 221Acta Chim. Slov. 2022, 69, 217–226 Žener, et al.: Metal and Non-Metal Modified Titania: ... have already been published in our previous article. This study expands on those results.38 In general, samples synthesized with H2SO4 have higher surface areas (63.2 ± 0.3 to 103.4 ± 0.4 m2/g) com- pared to samples synthesized with HCl (24.2 ± 0.2 to 49.5 ± 0.3 m2/g). The higher surface area can be attributed to smaller crystallites found in samples synthesized with the addition of H2SO4, which is also observed from the XRD measurements. It can be seen from the results presented in Table 2 that the addition of urea (sample Urea_15 has specific sur- face area of 24.2 ± 0.2 m2/g) significantly decreases the surface area compared to the undoped sample REF (44.1 ± 0.4 m2/g). By increasing the volume of H2SO4 added we also increase the surface area of the sample (63.2 ± 0.3 m2/g for S2, 80.2 ± 0.2 m2/g for S3). The addition of NH4NO3 (samples S3_N0.5 and S3_N2), urea (sample S3_urea15) and thiourea (sample S3_thiourea15) to sam- ple S3 also increase the surface area of the samples (surface areas 84.5 ± 0.3 to 103.4 ± 0.4 m2/g). This can be explained by an additional decrease in the size of crystallites in these samples as compared to those in sample S3. The specific surface area does not change significant- ly when adding 1 % and 3 % of Pt to sample S3_N0.5. On the contrary, a decrease in surface area is observed when adding 2 % of Pt to sample S3_N0.5 (from 84.5 ± 0.3 m2/g for sample S3_N0.5 to 80.5 ± 0.4 m2/g for sample S3_ N0.5+2%Pt) and when adding 1 % or 2 % of Pt to sample S3_urea15 (from 101.7 ± 0.4 m2/g for S3_urea15 to 82.3 ± 0.3 m2/g and 84.2 ± 0.4 m2/g for sample S3_urea15+1%Pt and S3_urea15 + 2%Pt, respectively). 3. 3. Scanning Electron Microscopy (SEM) Figure 2 shows FE-SEM micrographs of samples REF, S3_N0.5 and S3_thiourea15. Substituting HCl (sample REF) with H2SO4 (samples S3_N0.5 and S3_thiourea15) during the synthesis has resulted in the formation of more porous powders, which is in agreement with the results of BET spe- cific surface area measurements. Furthermore, we have ob- served crystallites of sizes 31–35 nm in the SEM image of sample REF, which confirms the results of the XRD meas- urements. SEM images of samples synthesized with H2SO4 show crystallites ranging in size from 20–22 nm (sample S3_N0.5) and 18–23 nm (sample S3_thiourea15), which is also in agreement with the results of XRD measurements. We can also observe pores with sizes ranging from 70-100 nm (sample S3_N0.5) and 70-80 nm (sample S3_thiourea15). 3. 4. Photocurrent Measurements Figure 3 shows the results of photocurrent measure- ments for different samples. The reason why there is a very low photocurrent response in UV region is due to the fact 150 W Xe lamp was used as the light source. Xe lamps have very low intensity in UV region below 300 nm. By adding urea (sample Urea_15) to the undoped sample REF, the photocurrent response has decreased. We can attribute this to the lower surface area of sample Urea_15, which was observed from BET measurements. Contrarily, the addition of thiourea (sample Thiourea_15) has resulted in an increase in photocurrent response, which is attributed to the higher surface area of the sample Thiourea_15. Figure 2. FE-SEM micrographs of powders: REF, S3_N0.5 and S3_ thiourea15 at 200,000x magnification. Accelerating voltage was set to 2 kV. Images were obtained with detection of secondary elec- trons. 222 Acta Chim. Slov. 2022, 69, 217–226 Žener, et al.: Metal and Non-Metal Modified Titania: ... By substituting HCl with H2SO4 during the synthe- sis (samples S2 and S3) the current has increased. As with sample Thiourea_15, this can be explained by the higher surface area of samples S2 and S3 compared to the sam- ples synthesized with HCl. Because sample S3 has a high- er surface area than sample S2, a higher photocurrent is induced when irradiating the former. The results, as pre- sented in Figure 3b, show that the addition of 0.5 % of NH4NO3 (sample S3_N0.5), urea (sample S3_urea15) or thiourea (sample S3_thiourea15) to sample S3 has result- ed in a decrease in photocurrent response, despite the increase in surface area. The response has increased when adding 2 % of NH4NO3 (sample S3_N2), which also has the highest surface area of all the samples. From this we can deduce that the amount of induced photocurrent de- pends not only on the surface area, but also on the amount of added dopants. Band gap energies for selected samples have already been measured and published by Žener et al. 38 Results for samples with added Pt are presented in Figures 3c and 3d. When comparing these samples with those without the added Pt (samples S3_N0.5 and S3_ urea15), we can see that the addition of 1 % and 2 % of Pt had a positive effect on the photocatalytic response, de- spite the lower surface area of these samples. When irradi- ating the sample with 3 % of added Pt (sample S3_N0.5 + 3%Pt) the amount of induced photocurrent has decreased dramatically, which could be due to the increased Pt block- ing light to the photocatalyst. Moreover, platinum particles can also act as recombination centres. 3. 5. Photocatalytic Activity Concentration profiles for isopropanol and acetone for selected samples are presented in Figure 4. The photo- catalytic activity of powders under UV and visible light irradiation was tested by monitoring the oxidation of iso- propanol to acetone. It can be seen that the isopropanol curve is unstable before the UV and visible light is switched on, which can be attributed to the adsorption of isopropanol onto the surface of the sample and reactor system. After the UV and visible light is switched on the acetone concentration increases, while the isopropanol concentration decreases. Reactions are presented in Equa- tion (2) in the experimental section. In the initial steps of the photocatalysis, we can approximate the reaction to be of zero order, because the photocatalytic oxidation of iso- propanol to acetone is faster than the subsequent oxida- tion of acetone. Zero order kinetics can be described with Equation (3): (3) In this equation c and c0 represent concentration of acetone and the initial concentration of acetone, respec- tively in ppm, while t is time in hours and k0 is the zero-or- der rate constant (units ppm/h). Therefore, the initial slope of the acetone concentration curve is equal to k0 (Equation (3)), which was determined from line equations in Figure 4. This presents a good basis to compare photocatalytic ac- tivity of different TiO2 powders.43 Zero order rate con- stants (k0) for different samples are presented in Table 3. Figure 3. Photocurrent responses recorded under applied external potential of 1 V. 223Acta Chim. Slov. 2022, 69, 217–226 Žener, et al.: Metal and Non-Metal Modified Titania: ... The addition of urea (sample Urea_15) to the un- doped sample REF has resulted in an increase in the pho- tocatalytic activity (k0 = 122 ± 4 ppm/h and 68 ± 1 ppm/h for sample Urea_15 and REF, respectively). In our previ- ously published work, we reported a very high band-gap value in sample REF (3.44 eV). The addition of urea nar- rows the band-gap due to nitrogen doping, and conse- quently increases photocatalytic activity even under UV light.38,44 On the contrary, addition of thiourea (sample Thiourea_15) decreases the photocatalytic activity of the sample (k0 = 50 ± 2 ppm/h) when compared to REF, de- spite the former having a higher surface area (49.5 ± 0.3 m2/g for sample Thiourea_15 and 44.1 ± 0.4 m2/g for REF) and higher photocurrent response. This could be explained by higher recombination rates of the charge carriers in sample Thiourea_15. Additional measurements would, however, be needed to confirm this theory. By using H2SO4 instead of HCl in the synthesis (sam- ples S2 and S3) we obtained samples with smaller-sized crystallites, which results in higher porosity (24.2 ± 0.2 to 49.5 ± 0.3 m2/g for samples synthesized with HCl; 63.2 ± 0.3 Figure 4. Concentration profiles of isopropanol and acetone for: (a) REF; (b) Urea_15; (c) S3; (d) S3_N2; (e) S3_N0.5 + 1%Pt and (f) S3_urea15 + 1%Pt; y in the line equation represents concentration of acetone (in ppm) and x represents time of experiment in reactor (in hours) Table 3. Photocatalytic activities of different samples, determined by observing the oxidation of isopropanol to acetone (rate constant k0). Activity under UV and Sample visible light exposure - k0 (ppm/h) HOMBIKAT UV 100 337 ± 2 REF 68 ± 1 Urea_15 122 ± 4 Thiourea_15 50 ± 2 S2 183 ± 2 S3 310 ± 3 S3_N0.5 260 ± 2 S3_N2 328 ± 3 S3_urea15 304 ± 2 S3_thiourea15 225 ± 2 S3_N0.5+1%Pt 343 ± 2 S3_N0.5+2%Pt 251 ± 3 S3_N0.5+3%Pt 260 ± 3 S3_urea15+1%Pt 420 ± 3 S3_urea15+2%Pt 420 ± 2 224 Acta Chim. Slov. 2022, 69, 217–226 Žener, et al.: Metal and Non-Metal Modified Titania: ... and 80.2 ± 0.2 m2/g for samples S2 and S3, respectively) and a far higher photocurrent response. For these reasons, sam- ples S2 and S3 show much higher activity than samples syn- thesized with HCl (k0 = 183 ± 2 ppm/h for sample S2 and 310 ± 3 ppm/h for sample S3). S3 has a higher surface area and higher photocurrent response compared to S2, and consequently much higher photocatalytic activity. The addition of NH4NO3 (samples S3_N0.5 and S3_ N2), urea (sample S3_urea15) and thiourea (sample S3_ thiourea_15) to sample S3 increases the specific surface areas (84.5 ± 0.3 to 103.4 ± 0.4 m2/g). Despite this, only one sample has a higher photocatalytic activity than the sample without additions (sample S3, k0 = 310 ± 3 ppm/h, sample S3_N2, k0 = 328 /h). This can be explained by the results of the photocurrent measurements, which have shown decreased responses (compared to sample S3) in all but sample S3_N2, which also has the highest surface area (103.4 ± 0.4 m2/g). Higher activity can also be attributed to the addition of nitrogen, which increases the amount of oxygen vacancies and reduces the band gap energy, result- ing in higher photocatalytic activity.38 The decreased activ- ity in samples S3_urea15 and S3_thiourea15 can be attrib- uted to higher percentages of β-TiO2, found in these two samples, since the photocatalytic activity of β-TiO2 is gen- erally much lower than that of anatase.45–47 The addition of Pt to sample S3_N0.5 does not in- crease its surface area, in fact in the case of the sample with 2 % Pt added (sample S3_N0.5 + 2% Pt) it actually de- creases (84.5 ± 0.3 m2/g for sample S3_N0.5 and 80.5 ± 0.4 m2/g for sample S3_N0.5 + 2% Pt). Despite this, the sam- ple S3_N0.5 + 1%Pt shows much higher photocatalytic activity (k0 = 343 ± 2 ppm/h), compared to sample S3_N0.5 (k0 = 260 ± 2 ppm/h). We attribute this to Pt acting as an efficient trap for free electrons, thus inhibiting recombina- tion (confirmed with photocurrent measurements), whilst also improving the free electron transfer to adsorbed pol- lutants. The activity of sample S3_N0.5+3%Pt is equal to the activity of sample S3_N0.5, but the activity of sample S3_N0.5 + 2%Pt has reduced slightly. Doping the sample S3_urea15 with Pt (samples S3_ urea15 + 1%Pt and S3_urea15+2%Pt) significantly lowers its surface area (82.3 ± 0.3 m2/g for sample S3_urea15 + 1%Pt and 84.2 ± 0.4 m2/g for sample S3_urea15 + 2%Pt). Furthermore, results of the photocurrent measurements have shown that the addition of Pt significantly increases the photocurrent response, resulting in far higher photo- catalytic activity in samples S3_urea15 + 1%Pt (k0 = 420 ± 3 ppm/h) and S3_urea15 + 2%Pt (k0 = 420 ± 2 ppm/h) compared to sample S3_urea15 (k0 = 304 ± 2 ppm/h). It was found out that metal and non-metal doping, as well as addition of HPC significantly increase the photocatalytic activity of powders under UV and visible light irradiation. In the case of samples S3_urea15 + 1%Pt and S3_urea15 + 2%Pt the photocatalytic activity is even higher than that of selected anatase sample available on the market: HOMBIKAT UV 100 (k0 = 337 ± 2 ppm/h) (see Table 3).48 4. Conclusions In the present work TiO2 powders, doped with sulfur and nitrogen and modified with platinum were prepared by means of particulate sol-gel synthesis in order to in- crease the photocatalytic activity of undoped sample, while the organic polymer hydroxypropyl cellulose (HPC) was added to increase the surface area of the photocatalyst. By substituting HCl with H2SO4 during the synthesis, the resulting samples contained smaller crystallites (26.9 ± 0.7 – 30.7 ± 0.8 nm for samples synthesized with HCl; 15.1 ± 0.3 – 23.5 ± 0.2 nm nm for samples synthesized with H2SO4). We attributed the smaller crystallite size to the formation of TiOSO4, which inhibits the crystallization of TiO2. Additionally, we observed the presence of β-TiO2 in samples synthesized with H2SO4. The highest percentage of β-TiO2 was found in sample S3_thiourea15 (45.4 ± 0.3 %). The afore-mentioned decrease in the size of crystallites led to a higher specific surface area for samples synthe- sized with H2SO4 (63.2 ± 0.3 to 103.4 ± 0.4 m2/g) com- pared to those synthesized with HCl (24.2 ± 0.2 to 49.5 ± 0.3 m2/g). The addition of NH4NO3, urea and thiourea to sample S3 increased its porosity. Sample S3_N2 had the highest surface area (103.4 ± 0.4 m2/g) and the smallest crystallites of anatase (16.8 ± 0.1 nm). The addition of Pt did not increase the sample’s porosity, in some cases it even decreased it. FE-SEM images confirmed the increased po- rosity of samples synthesized with H2SO4. Additionally, smaller crystallites were found in samples synthesized with H2SO4, confirming the results of X-Ray diffraction. The increased porosity of the samples synthesized with H2SO4 also resulted in greater photocurrent respons- es in these samples compared to those synthesized with HCl. Despite increasing the specific surface area, the addi- tion of NH4NO3, urea and thiourea to S3 yielded a lower photocurrent response, with the exception of sample S3_ N2, which also had the highest surface area. In all but one case the addition of platinum resulted in greater photocur- rent responses. Samples synthesized with H2SO4 exhibit higher pho- tocatalytic activity compared to samples synthesized with HCl, which can be explained by larger surface areas and higher photocurrent responses. Out of all the samples with added NH4NO3, urea or thiourea, only sample S3_N2 has a higher photocatalytic activity compared to sample S3. This is also the only sample in this group which exhibit a greater photocurrent response than sample S3. The addi- tion of platinum to sample S3_urea significantly increased its photocatalytic activity, which is in agreement with the results of photocurrent measurements. Out of all the sam- ples, samples S3_urea15 + 1%Pt and S3_urea15 + 2%Pt showed the highest photocatalytic activity (k0 = 420 ± 3 and 420 ± 2 ppm/h), which was even higher than the activ- ity of the well-known pure anatase photocatalyst HOMBIKAT UV 100 (k0 = 337 ± 2 ppm/h). We were able to significantly increase the photocatalytic activity of pow- 225Acta Chim. Slov. 2022, 69, 217–226 Žener, et al.: Metal and Non-Metal Modified Titania: ... ders under UV and visible light irradiation by increasing the surface area and photocurrent response with non-met- al and metal doping. Acknowledgements The authors acknowledge the financial support from the Slovenian Research Agency (research core funding Nos. P1-0134 and P2-0273, while part of the work was conducted under project No. NC-0002). B. Ž. is grateful to Slovenian Research Agency for the position of young re- searcher enabling him the doctoral study. M. R. also ac- knowledges the Operational Programme Research, Devel- opment and Education, project No. CZ.02.1.01./0.0/0.0/17 _049/0008419 „COOPERATION“. The authors thank to Mojca Opresnik from the National Institute of Chemistry for BET measurements and to Edi Kranjc (also from the National Institute of Chemistry) for performing XRD measurements. The authors also acknowledge dr. Amalija Golobič for her help with Rietveld analysis. 5. References 1. R. Fagan, D. E. McCormack, D. D. Dionysiou, S. C. Pillai, Mater. Sci. Semicond. Process. 2016, 42, 2–14. DOI:10.1016/j.mssp.2015.07.052 2. R. Marchant, L. Brohan, M. Tournox, Mater. Res. Bull. 1980, 15, 1129–1133. DOI:10.1016/0025-5408(80)90076-8 3. S. León-Ríos, R. Espinoza González, S. Fuentes, E. Chávez Ángel, A. Echeverría, A. E. 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PXRD meritve kažejo, da zamenjava HCl s H2SO4 med sinteznim postopkom zmanjša velikost kristalitov iz ~30 nm na ~20 nm, pri čemer se pov- eča tudi specifična površina iz ~44 m2/g na ~80 m2/g. Opažanja korelirajo z izmerjeno fotokatalitsko aktivnostjo vzorcev in izmerjenim fototokom. Rezultati kažejo, da so lastnosti prahov (specifična površina, velikost kristalitov, obnašanje fototoka) odvisne ne le od vrste uporabljene kisline, temveč tudi od njene količine in uporabljenega dopanta. Dopiranje z žveplom, kodopiranje z žveplom in dušikom in modifikacija prahov TiO2 s platino povečajo fotokatalitsko aktivnost tudi do šestkrat. Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License 227Acta Chim. Slov. 2022, 69, 227–234 Zhang et al.: Synthesis, Crystal Structure and Separation Performance ... DOI: 10.17344/acsi.2021.7218 Scientific paper Synthesis, Crystal Structure and Separation Performance of p-tert-butyl(tetradecyloxy)calix[6]arene Wei Zhang,1 Zhi-qiang Cai,1,* Xiao-min Shuai,1 Wei Li,1 Qiu-chen Huang,1 Ruo-nan Chen,1 Qi-qi Zang,2 Fei-fei Li2 and Tao Sun2,* 1 Liaoning Province Professional and Technical Innovation Center for Fine Chemical Engineering of Aromatics Downstream, School of Petrochemical Engineering, Shenyang University of Technology, Liaoyang 111003, P. R. China. 2 College of Chemistry and Chemical Engineering, Henan Key Laboratory of Function-Oriented Porous Materials, Luoyang Normal University, Luoyang 471934, P. R. China. * Corresponding author: E-mail: kahongzqc@163.com; E-mail: suntao2226@163.com Received: 10-19-2021 Abstract This work describes the investigation of separation performance of the p-tert-butyl(tetradecyloxy)calix[6]arene (C6A- C10-OH) as stationary phase for gas chromatography (GC) separations. Its structure was characterized by IR, 1H NMR, 13C NMR, MS and single-crystal X-ray diffraction analysis. The C6A-C10-OH column shows good separation capacity for aliphatic, aromatic and cis-/trans- isomers. Especially, it exhibits multiple molecular recognition interactions for the analytes with a wide range of polarity, including dispersion, π-π, H-bonding and dipole-dipole interactions. The present work provides experimental and theoretical basis for the designing of the new calixarene stationary phases in GC anal- yses. Keywords: Calixarene; crystal structure; separation performance 1. Introduction Calixarenes are the third generation of supramo- lecular compound after crown ethers and cyclodextrins.1 These macrocyclic compounds have attracted extensive attention in the field of separation science because of it has unique physicochemical properties such as adjustable cavity size, good solubility, structural stability and so on.2 Their hydrophobic cavities are composed of benzene ring units, and its upper and lower rims are p-tert-butyls and phenolic hydroxyl groups, respectively, which are easy to derivatize.3 In recent years, calixarene derivatives with di- verse structures have been widely used in various fields, such as catalysis, molecular recognition, energy and sep- aration analysis.4 Scheme 1. The C6A-C10-OH capillary column for GC separation. 228 Acta Chim. Slov. 2022, 69, 227–234 Zhang et al.: Synthesis, Crystal Structure and Separation Performance ... GC has widely applied in many fields including envi- ronmental analysis, petrochemical industry, food analysis and pharmaceutical analysis due to its excellent character- istics such as good selectivity, high sensitivity, rapid anal- ysis and low cost.5 In GC, it is the key to choose a suitable stationary phase for the separation of compounds with close nature. In recent years, our group has been engaged in the research of new calixarene chromatographic sta- tionary phases. In 2019, we first reported the amphiphilic calixarene (C4A-NH2) and used it to separate aromatic amine isomers.6 Subsequently, we reported the study of calix[6]arene and calix[8]arene derivatives as stationary phases for GC.7 These results indicated that calixarenes and their derivatives are suitable as GC stationary phases with good chromatographic selectivity. First, we synthesized a new calixarene compound (C6A-C10-OH) in this work. The upper rim is p-tert-bu- tyl, and the lower rim is long alkyl chain and phenolic hy- droxyl. Second, we characterized the molecular structure of C6A-C10-OH by IR, 1H NMR, 13C NMR, MS and sin- gle-crystal X-ray diffraction analysis. Then, it was coated on the inner wall of capillary column by static method, and its chromatographic separation performance was in- vestigated (Scheme 1). 2. Experimental 2. 1. Materials and Methods An Agilent 7890A gas chromatograph was used for GC analyses. Thin layer chromatography (TLC) was per- formed on silica-gel plates (HF254). 1H NMR spectrum and 13C NMR spectrum used TMS (tetramethylsilane) as the internal standard and exported on a Bruker BioSpin 400 MHz instrument. Chemical shifts (δ) were expressed in ppm. IR spectrum was reported on a Bruker Platinum ART Tensor II FTIR spectrometer. MALDI-TOF-MS was reported on a Bruker BIFLEX III mass spectrometer. Sin- gle Crystal data of C6A-C10-OH were gained on a Bruker D8 VENTURE X-ray diffractometer. All reagents and sol- vents were not further treated, and all from commercial way. 2. 2. Synthesis of the C6A-C10-OH Firstly, NaH (1.24 g, 51.67 mmol), p-tert-butylca- lix[6]arene (1.50 g, 1.54 mmol) and DMF (25 mL) were added to a 50 mL round bottom flask. The reactants were reacted at room temperature for 1 h. Afterwards, 1-bro- modecane (4.30 g, 19.19 mmol) was added to mixed solu- tion, raised the temperature to 85 °C and reacted for 10 h. After the reaction, the solvent was concentrated to obtain a yellow solid. Then, dichloromethane was used to dis- solve the obtained yellow solid and rinsed three times with deionized water (15 mL). Then, the anhydrous magnesium sulfate was used to remove water and vacuum drying. Fi- nally, a light yellow oily product was obtained. Using col- umn chromatography [dichloromethane/petroleum ether (v/v = 1:4)] to purify the product of the previous step, and the final white solid product was gained with a yield of 85.34%. 1H NMR (400 MHz, CDCl3, ppm): δ 7.39 (s, 4H, CH), 7.04 (s, 8H, CH), 4.05 (m,8H, CH2), 3.84 (s, 12H, CH2), 1.60 (m, 8H, CH2), 1.42 (m, 8H, CH2), 1.27 (s, 54H, CH3), 1.22 (m, 48H, CH2), 0.86 (t,12H, CH3); 13C NMR (100 MHz, CDCl3, ppm): δ 151.57, 151.15, 146.93, 142.79, 132.78, 126.23, 125.70, 77.48, 77.16, 76.84, 34.29–33.67, 32.19–31.90, 31.90–30.69, 30.16–29.01, 26.50, 23.05– 22.74, 14.29; IR (KBr), cm–1: v(OH) 3373, v(CH3) 2955, v(CH2) 2929, v(CH2) 2858, v(C=C) 1484, v(C=C) 1460, v(C-O-C) 1188, v(CH2) 722; ESI-MS(m/z): [M+K]+calcd for C106H164O6, 1572.253; found, 1572.166. 2. 3. X-Ray Structure Determination Took a small amount of white solid of the C6A-C10- OH and dissolved it in dichloromethane. After two days, dichloromethane volatilizes completely, we gained the white crystal for single crystal diffraction analysis. The dimensions of white crystal (C106H164O6) were 0.17mm × 0.12mm × 0.12mm, and which were measured on a Bruk- er D8 VENTURE diffractometer equipped with graph- ite-monochromatic Mo Kα radiation (λ = 1.54178 Å) us- ing an ω scan mode at 103(2) K. A total of 55912 reflec- tions were gathered in the range of 2.457° < θ < 70.168° (index ranges: –12 ≤ h ≤ 12, –43≤k≤42 and –30 ≤ l ≤ 30) and 17547 were independent (Rint = 0.0745), of which 11531 observed reflections with I > 2σ(I) were applied in the refinements and structure determination. Used the intrinsic phasing methods to confirm the structure with Table 1. Crystal data of the C6A-C10-OH Crystal size 0.170 × 0.120 × 0.120 mm3 Formula C106H164O6 Molecular weight 1534.36 T (K) 103(2) K Crystal system Monoclinic Space group Cc a (Å) 10.5965(5) b (Å) 35.9665(16) c (Å) 25.1721(11) α (°) 90 β (°) 92.663(3) γ (°) 90 V (Å)3 9583.2(7) Z 4 Dc (g/cm3) 1.063 F(000) 3392 Goodness-of-fit on F2 1.075 Reflection collected 55912 R1, wR2 [I > 2σ (I)] 0.0852, 0.2445 R1, wR2 (all data) 0.1135, 0.2743 R1 = ∑(||Fo|–|Fc||)/∑|Fo| ... wR2 = (∑w(Fo2–Fc2)2/∑w(Fo2)2)1/2 229Acta Chim. Slov. 2022, 69, 227–234 Zhang et al.: Synthesis, Crystal Structure and Separation Performance ... the SHELXT 2014 program and reported by the Fourier technique.8 The non-hydrogen atoms were purified aniso- tropically. Through theoretical calculation, we reached the hydrogen atom combined with carbon atom. The structure was purged by the full-matrix least-squares techniques on F2 with SHELXL-2017.8 The final refinement gave R = 0.0852 and wR = 0.2743 (w = 1/[σ2(Fo2)+(P)2+P)], where P = (Fo2+2Fc2)/3), S = 1.075, (∆/σ)max = 0.002, (∆ρ)max = 0.694 and (∆ρ)min = –0.278 e/Å3. Other crystal structure data of C6A-C10-OH are shown in Table 1. 2. 4. Preparation of the Capillary Column The static coating method was used to make the C6A-C10-OH capillary column.9 Firstly, the dichlo- romethane was used to rinse the empty column (0.25 mm × 10 m) to remove impurities. The rinsed capillary column was filled by a NaCl-MeOH saturated solution to rough inner wall of capillary column. Then, it was rised from 40 °C to 200 °C and maintained at 200 °C for 3 h. Next, the C6A-C10-OH stationary phase was dissolved in dichlo- romethane (2 mL) and injected into the treated column. When the column was completely full of stationary phase solution, sealed one end of the chromatographic column and connected the other end to the vacuum environment at 40 °C to evaporate the excess dichloromethane solution. Temperature process started at 40 °C hold for 30 minutes rise to 160 °C at the rate of 1 °C/min and maintained at 160 °C for 7 h. The experimental process was operated in nitrogen atmosphere. 3. Results and Discussion 3. 1. Synthesis and Characterization Scheme 2 exhibits the synthetic process of C6A-C10- OH and it was obtained by one-step reaction. Meanwhile, IR, 1H NMR, 13C NMR, MS and single-crystal X-ray dif- fraction analysis interpreted the molecular structure of C6A-C10-OH. In the IR, the peak value of 2955 cm–1 was C-H antisymmetric stretching vibration on the p-tert-bu- tyl. The peak values of 1484 and 1460 cm–1 was the C=C stretching vibration on benzene ring units. The peak value of 1188 cm–1 was the C-O-C stretching vibration of ether, the peak value of 722 cm–1 was the CH2 plane rocking vi- bration of alkyl chains in the compound. In the 1H NMR, the proton absorption peaks of the aromatic rings were observed at 7.39 and 7.04 ppm in the low field, the proton absorption peaks of the bridged methylene in the benzene rings were discovered at 3.84 ppm, and the proton absorp- tion peaks of methyl on p-tert-butyls and alkyl chains were found at 1.27 and 0.86 ppm, respectively. The integral area of each peak was consistent with the expected number of protons. Moreover, the structure of C6A-C10-OH was also characterized by 13C NMR. 3. 2. Crystal Structure of the C6A-C10-OH Fig. 1 presents the molecular structure of the C6A- C10-OH, the crystal data and selected bond lengths are listed in the Table 1 and the Table 2 respectively. Its mo- lecular structure consisted of six benzene rings, the four alkyl chains and two phenolic hydroxyl groups at the lower of C6A. The torsion angle of C(5)B-C(21)B-C(22)B-C(27) B was –90.0(8)°, which suggested that the two blue ben- zene rings were perpendicular to each other in space. The torsion angle of C(4)B-C(5)B-C(6)B-C(1)B was 0.1(10)°, which further proved that the carbon atoms on the ben- zene ring were coplanar. In the Table 2, the bond lengths of C(23)B-C(24)B (1.387(10) Å) and C(22)B-C(23)B (1.391(11) Å) were almost equal because these two bonds were in the same benzene ring. The bond length of O(1) B-C(11)B (1.416(9) Å) is shorter than C(11)B-C(12)B (1.519(11) Å). This is because the electronegativity of oxy- gen atom was larger than that of carbon atom, so that the bond energy of C=O was stronger than that of C=C. More- over, Fig. 2 depicts the molecular packing in the unit cell and Table 3 gives that the hydrogen bond lengths and bond angles in the structure.10 Scheme 2: Synthesis of the C6A-C10-OH 230 Acta Chim. Slov. 2022, 69, 227–234 Zhang et al.: Synthesis, Crystal Structure and Separation Performance ... Fig. 1. Molecular structure of the C6A-C10-OH Fig. 2. Packing diagram of the C6A-C10-OH with 50% probability level ellipsoids Table 2. Selected Bond Lengths (Å) and Bond Angles (°) for the C6A-C10-OH Bond Dist. Bond Dist. Bond Dist. C(28)B-C(29)B 1.515(13) C(21)B-C(22)B 1.522(9) O(1)B-C(11)B 1.416(9) C(24)B-C(28)B 1.526(11) C(5)B-C(21)B 1.531(11) C(11)B-C(12)B 1.519(11) C(23)B-C(24)B 1.387(10) C(4)B-C(5)B 1.399(9) C(12)B-C(13)B 1.544(11) C(22)B-C(23)B 1.391(11) O(1)B-C(4)B 1.409(9) C(13)B-C(14)B 1.449(13) Angle (°) Angle (°) Angle (°) C(29)B-C(28)B-C(24)B 111.7(7) C(22)B-C(21)B-C(5)B 111.5(6) O(1)B-C(11)B-C(12)B 106.5(6) C(23)B-C(24)B-C(28)B 122.8(7) C(4)B-C(5)B-C(21)B 120.0(6) C(11)B-C(12)B-C(13)B 113.6(7) C(24)B-C(23)B-C(22)B 123.3(7) C(5)B-C(4)B-O(1)B 120.8(7) C(14)B-C(13)B-C(12)B 113.7(8) C(23)B-C(22)B-C(21)B 119.7(6) C(4)B-O(1)B-C(11)B 117.6(6) C(13)B-C(14)B-C(15)B 118.5(9) 231Acta Chim. Slov. 2022, 69, 227–234 Zhang et al.: Synthesis, Crystal Structure and Separation Performance ... 3. 3. Separation Performance of the C6A-C10-OH The column efficiency of C6A-C10-OH column was 2400 plates/m. Afterwards, the aromatic and cis-/trans- isomers were used to study its separation performance. The results shown that the C6A-C10-OH column achieved baseline resolution for above analytes. Fig. 3 shows the separation of aromatic isomers of different polarity on the C6A-C10-OH column, such as substituted benzenes, trimethylbenzene and trichloroben- zene isomers. The C6A-C10-OH column presented excel- lent peak shapes for the benzene analytes and had a good resolution (R > 1.5). The C6A-C10-OH stationary phase had the good separation capacity for aromatic isomers due to its unique 3D cavity and aromatic framework, which can provide π-π interactions between stationary phase and aromatic analytes. In addition, cis-/trans- isomers were used to study the interaction mechanism between them in the C6A-C10-OH column. Fig. 4 displays that the separations of cis-/trans- iso- mers on the C6A-C10-OH column, including 1,3-dichlo- ropropene, 1,2,3-trichloropropene, nerolidol and nerol/ geraniol. The results exhibited that the cis-/trans- isomers were well separated. It is worth to note that the C6A-C10- OH column had outstanding resolution for analytes with close boiling point, such as nerol (b.p. 226 °C)/geraniol (b.p. 229 °C), the boiling point difference was only 3 °C. This proved that the C6A-C10-OH column had excellent separation ability for the analytes of similar structure and physicochemical properties. 3. 4. Relationship Between Molecular Structure and Retention Behavior In order to further study the relationship between mo- lecular structure and retention behavior of the C6A-C10-OH stationary phase, we investigated its polarity and selectivity Table 3. Hydrogen Bond Lengths (Å)and Bond Angles (°) for the C6A-C10-OH D-H...A d(D-H) d(H...A) d(D...A) <(DHA) O(2)A-H(2)A...O(1)A 0.84 2.08 2.891(8) 162.9 O(2)B-H(2)B...O(1)C 0.84 2.04 2.874(7) 170.3 Fig. 3. GC separations of aromatic isomers. Temperature process: 40 °C (keep 1 min) up to 160 °C (keep 5 min) at 10 °C/min and gas flow rate at 0.6 mL/min. Fig. 4. GC separations of cis-/trans- isomers. Temperature process: 40 °C (keep 1 min) up to 160 °C (keep 5 min) at 10 °C/min and gas flow rate at 0.6 mL/min. 232 Acta Chim. Slov. 2022, 69, 227–234 Zhang et al.: Synthesis, Crystal Structure and Separation Performance ... in comparison to C6A-C10 stationary phase (the previous work of our group).7 The McReynolds constants of the C6A- C10-OH and C6A-C10 stationary phases were determined by the five probe compounds at 120 °C. Their sum and aver- age values were used to characterize their general polarity and average polarity. In general, it can be regarded as non-po- lar and moderately polar when the polarities of stationary phases are less than 100 and between 100 and 400 respective- ly.11 As shown in table 4, the average value of C6A-C10 sta- tionary phase was 89, belonging to nonpolar, but the average value of C6A-C10-OH stationary phase was 129, indicating its moderate polarity. This polarity difference may derive Table 4. McReynolds constants of the C6A-C10-OH and C6A-C10 columns Stationary phases Xʹ Yʹ Zʹ Uʹ Sʹ General polarity Average C6A-C10-OH 68 153 99 165 161 645 129 C6A-C10 41 124 73 115 92 445 89 Xʹ, benzene; Yʹ, 1-butanol; Zʹ, 2-pentanone; Uʹ, 1-nitropropane; Sʹ, pyridine. Temperature: 120 °C. Table 5. The boiling point of alcohols and n-alkanes Alcohols n-Alkanes Compound Molecular formula Boiling point Compound Molecular formula Boiling point 1-nonanol C9H20O 215 °C n-dodecane C12H26 216 °C 1-decanol C10H22O 233 °C n-tridecane C13H28 235 °C 1-undecanol C11H24O 241 °C n-tetradecane C14H30 254 °C 1-dodecanol C12H26O 260 °C n-pentadecane C15H32 268 °C Fig. 5. GC separations of the alcohols and n-alkanes on the C6A-C10-OH and C6A-C10 columns. Temperature process: 40 °C (keep 1 min) up to 160 °C (keep 5 min) at 10 °C/min and gas flow rate at 0.6 mL/min. 233Acta Chim. Slov. 2022, 69, 227–234 Zhang et al.: Synthesis, Crystal Structure and Separation Performance ... from the different structures, the C6A-C10-OH stationary phase contained two unsubstituted phenolic hydroxyl groups, so its polarity was higher than C6A-C10 stationary phase and may offer H-bonding and dipole-dipole interac- tions for the separations of polar analytes. Fig. 5 presents the separetions of the alcohols and n-alkanes on the C6A-C10-OH and C6A-C10 columns with the same separation conditions, respectively. The boiling points of analytes are listed in the Table 5. As shown, the C6A-C10-OH column exhibited the excellent resolving ability and good peak shapes for n-alkanes and alcohols. Interestingly, the C6A-C10-OH stationary phase exhibited stronger retention trend for the polar alcohols than the non-polar n-alkanes, such as the analyte pairs of n-dodecane/1-nonanol (b.p. 216 °C/b.p. 215 °C), n-tr- idecane/1-decanol (b.p. 235 °C/b.p. 233 °C), n-tetrade- cane/1-undecanol (b.p. 254 °C/b.p. 241 °C) and n-penta- decane/1-dodecanol (b.p. 268 °C/b.p. 260 °C). However, the alcohols and alkanes were eluted in the order of boiling points on the C6A-C10 column. The above results showed that the retention behaviors of alco- hols and alkanes in the two columns are quite different. This is because they have different molecular structures. C6A-C10-OH has two phenolic hydroxyl groups at the lower rim, so there are strong H-bonding and dipole-di- pole interactions between the stationary phase and the po- lar analytes. The interactions between C6A-C10 and the linear analytes are mainly dispersion interactions, because the lower rim of its aromatic skeleton are all alkyl chain substituents. The above results proved that the C6A-C10- OH stationary phase had multiple molecular recognition interactions for different types of analytes due to its unique molecular structure, including dispersion, H-bonding and dipole-dipole interactions. 4. Conclusion This work presents the investigation of the C6A- C10-OH stationary phase for GC separations. Its mo- lecular structure was characterized by IR, 1H NMR, 13C NMR, MS and single-crystal X-ray diffraction analysis. As demonstrated, the C6A-C10-OH stationary phase pre- sents good separation capacity for aliphatic, aromatic and cis-/trans- isomers. Importantly, it exhibits prolonged re- tention trend for alcohols mainly due to the H-bonding and dipole-dipole interactions with the phenolic hydrox- yl groups. In short, this work illustrates the outstanding separation ability of the C6A-C10-OH stationary phase for diverse analytes owing to its distinct molecular structure and multiple interactions. Supplementary Material CCDC 2093329 contains the supplementary data for this paper. The data can be obtained free of charge from the Cambridge Crystallographic Data Centre via http:// summary.ccdc.cam.ac.uk/structure-summary-form. Acknowledgements The authors thank the financial support of the Natural Science Foundation of Liaoning Province (20180550016) and the Scientific Research Foundation of the Education Department of Liaoning Province (LJGD2020015). 5. References 1. S. Shinkai, Tetrahedron.1993, 49, 8933–8968. DOI:10.1016/S0040-4020(01)91215-3 2. (a) H. J. Kim, M. H. Lee, L. Mutihac, J. Vicens, J. S. Kim, Chem. Soc. Rev. 2012, 41, 1173–1190. DOI:10.1039/C1CS15169J (b) C. Schneider, U. Menyes, T. Jira, J. Sep. Sci. 2010, 33, 2930–2942. DOI:10.1002/jssc.201000281 (c) D. Guillaume, L. Roy, J. 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Delo ponuja eksperimentalno in teoretsko podlago za razvoj novih stacionarnih faz na osnovi calixarenov v GC analizi. 235Acta Chim. Slov. 2022, 69, 235–242 Lei: Two Oxidovanadium(V) Complexes ... DOI: 10.17344/acsi.2021.7296 Scientific paper Two Oxidovanadium(V) Complexes with Hydrazone Ligands: Synthesis, Crystal Structures and Catalytic Oxidation Property Yan Lei School of Materials and Environmental Engineering, Chengdu Technological University, Chengdu 611730, P. R. China * Corresponding author: E-mail: leiyan222@126.com Received: 11-23-2021 Abstract Two new oxidovanadium(V) complexes, [VOL1(aha)]DMF (1) and [VOL2(mat)] (2), where L1 and L2 are the dianionic form of N’-(4-bromo-2-hydroxybenzylidene)-3-methyl-4-nitrobenzohydrazide and N’-(3,5-dibromo-2-hydroxybenzylidene) pivalohydrazide, respectively, and aha and mat are the monoanionic form of acetohydroxamic acid and maltol, respectively, have been synthesized and structurally characterized by physico-chemical methods and single crystal X-ray determination. X-ray analysis indicates that the V atoms in the complexes are in octahedral coordination. Crystal structures of the com- plexes are stabilized by hydrogen bonds. The catalytic property for epoxidation of styrene by the complexes was evaluated. Keywords: Vanadium complex; hydrazone ligand; crystal structure; catalytic property 1. Introduction Hydrazones bearing typical –CH=N–NH–C(O)– group are a kind of Schiff base compounds, which repre- sent one of the most attractive series of ligands in coordina- tion chemistry.1 The hydrazone ligands can adopt both ketone and enol forms during the coordination with vari- ous transition and rare earth metal atoms, to form com- plexes with versatile structures and properties like antibac- terial, enzyme inhibition, magnetism, catalytic and photo- luminescence.2 In the last few years, a number of complex- es with hydrazone ligands have been reported to have fasci- nating catalytic properties, such as oxidation of sulfides, polymerization and asymmetric epoxidation.3 Among the hydrazone complexes, those with V centers are of particu- lar interest for their catalytic applications.4 Maltol and ace- tohydroxamic acid are bidentate ligands in vanadium com- plexes.4b,5 In pursuit of new maltolate and acetohydro- xamate coordinated vanadium complexes with hydrazone ligands, we report herein two new oxidovanadium(V) Scheme 1. The ligands. 236 Acta Chim. Slov. 2022, 69, 235–242 Lei: Two Oxidovanadium(V) Complexes ... complexes, [VOL1(aha)]DMF (1) and [VOL2(mat)] (2), where L1 and L2 are the dianionic form of N’-(4-bro- mo-2-hydroxybenzylidene)-3-methyl-4-nitrobenzohydra- zide (H2L1) and N’-(3,5-dibromo-2-hydroxybenzylidene) pivalohydrazide (H2L2), respectively, and aha and mat are the monoanionic form of acetohydroxamic acid (Haha) and maltol (Hmat), respectively (Scheme 1). 2. Experimental 2. 1. Materials VO(acac)2, 4-bromosalicylaldehyde, 3,5-dibromosa- licylaldehyde, acetohydroxamic acid, and maltol were pur- chased from Aldrich. All other reagents with AR grade were used as received without further purification. 2. 2. Physical Measurements Infrared spectra (4000–400 cm–1) were recorded as KBr discs with a FTS-40 BioRad FT-IR spectrophotome- ter. 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. Solu- tion 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 collect- ed on a Bruker SMART CCD area diffractometer with graphite monochromated Mo-Kα radiation (λ = 0.71073 Å) at 298(2) K. Absorption corrections were applied by us- ing the multi-scan program.6 The structures of the com- plexes were solved by direct methods and successive Fou- rier difference syntheses, and anisotropic thermal parameters for all nonhydrogen atoms were refined by full-matrix least-squares procedure against F2.7 All non-hydrogen atoms were refined anisotropically. The amino H atom of complex 1 was located from a difference Fourier map and refined isotropically, with N‒H distance restrained to 0.90(1) Å. The remaining hydrogen atoms were located at calculated positions, and refined isotropi- cally with Uiso(H) values constrained to 1.2 Uiso(C) and 1.5 Uiso(O and methyl C). The C20 and O8 atoms in complex 1, and the C10 atom in complex 2 are refined as isotropic behavior due to their disorder manner. The crystallo- graphic data and experimental details for the structural analysis are summarized in Table 1. 2. 4. Synthesis of H2L1 A methanol solution (20 mL) of 3-methyl-4-nitro- benzohydrazide (1.9 g, 0.010 mol) was added to a methanol solution (20 mL) of 4-bromosalicylaldehyde (2.0 g, 0.010 Table 1. Crystallographic data for the single crystal of the complexes 1 2 Empirical formula C20H21BrN5O8V C18H17Br2N2O6V Formula weight 590.27 568.09 Temperature (K) 298(2) 298(2) Crystal system Monoclinic Orthorhombic Space group P21/n Pca21 a (Å) 8.6210(10) 14.6120(11) b (Å) 26.9525(13) 15.6777(12) c (Å) 10.5418(12) 9.4641(11) α (º) 90 90 β (º) 98.528(1) 90 γ (º) 90 90 V (Å3) 2422.4(4) 2168.1(3) Z 4 4 F(000) 1192 1120 μ, mm–1 2.114 4.179 Rint 0.0640 0.0910 Collected data 14312 19236 Unique data 4514 3834 Observed data [I > 2σ(I)] 2931 2731 Restraints 13 19 Parameters 323 267 Goodness-of-fit on F2 0.994 1.031 R1, wR2 indices [I > 2σ(I)] 0.0507, 0.1162 0.0496, 0.0776 R1, wR2 indices (all data) 0.0893, 0.1357 0.0912, 0.0880 Large diff. peak and hole, e Å–3 0.554, –0.425 0.655, –0.446 237Acta Chim. Slov. 2022, 69, 235–242 Lei: Two Oxidovanadium(V) Complexes ... mol). The mixture was refluxed for 1 h, and three quarter of the solvent was evaporated to give yellow precipitate, which was filtered off and washed several times with methanol. The yield is 92%. Analysis calculated for C15H12BrN3O4: C, 47.64; H, 3.20; N, 11.11%; found: C, 47.47; H, 3.31; N, 11.25%. 1H NMR (d6-DMSO, 500 MHz): δ 2.62 (s, 3H, CH3), 7.07 (d, 1H, ArH), 7.51 (s, 1H, ArH), 7.58 (d, 1H, ArH), 7.86 (d, 1H, ArH), 8.03 (s, 1H, ArH), 8.41 (d, 1H, ArH), 8.69 (s, 1H, CH=N), 10.76 (s, 1H, NH), 11.38 (s, 1H, OH). IR data (KBr, cm–1): 3446 (br, w, νOH), 3222 (sh, w, νNH), 1657 (vs, ν-C(O)-NH-), 1602 (vs, νC=N), 1520 (s, νas NO2 ), 1338 (s, νs NO2 ). UV-Vis data (λmax, nm): 285, 345, 420. 2. 5. Synthesis of H2L2 A methanol solution (20 mL) of pivalohydrazide (1.1 g, 0.010 mol) was added to a methanol solution (20 mL) of 3,5-dibromosalicylaldehyde (2.8 g, 0.010 mol). The mix- ture was refluxed for 1 h, and three quarter of the solvent was evaporated to give colorless precipitate, which was fil- tered off and washed several times with methanol. The yield is 88%. Analysis calculated for C12H14Br2N2O2: C, 38.12; H, 3.73; N, 7.41%; found: C, 38.31; H, 3.62; N, 7.32%. 1H NMR (d6-DMSO, 500MHz): δ 1.26 (s, 9H, C(CH3)3), 7.72 (s, 1H, ArH), 7.83 (s, 1H, ArH), 8.71 (s, 1H, CH=N), 11.22 (s, 1H, NH), 11.75 (s, 1H, OH). IR data (KBr, cm–1): 3438 (br, w, νOH), 3121 (sh, w, νNH), 1653 (vs, ν-C(O)-NH-), 1605 (vs, νC=N). UV-Vis data (λmax, nm): 295, 305, 332, 400. 2. 6. Synthesis of [VOL1(aha)]DMF (1) H2L1 (1.0 mmol, 0.38 g) and [VO(acac)2] (1.0 mmol, 0.26 g) were mixed and stirred in methanol (50 mL) for 30 min at 25 ºC. Then, acetohydroxamic acid (1.0 mmol, 0.075 g) was added and the mixture was stirred for another 30 min. The brown solution was evaporated to remove three quarters of the solvents under reduced pressure, yielding deep brown solid of the complex. Yield: 65%. Well-shaped single crystals suitable for X-ray diffraction were obtained by re-crystallization of the solid from meth- anol. Analysis calculated for C20H21BrN5O8V: C, 40.70; H, 3.59; N, 11.86%; found: C, 40.54; H, 3.70; N, 11.95%. IR data (KBr, cm–1): 3129 (sh, w, νNH), 1661 (vs, ν-C(O)-NH-), 1594 (vs, νC=N), 1522 (s, νas NO2 ), 1340 (s, νs NO2 ), 973 (m, V=O). UV-Vis data (λmax, nm): 260, 328, 410, 545. 2. 5. Synthesis of [VOL2(mat)] (2) H2L2 (1.0 mmol, 0.38 g) and [VO(acac)2] (1.0 mmol, 0.26 g) were mixed and stirred in methanol (50 mL) for 30 min at 25 °C. Then, maltol (1.0 mmol, 0.13 g) was added and the mixture was stirred for another 30 min. The brown solution was evaporated to remove three quarters of the solvents under reduced pressure, yielding deep brown sol- id of the complex. Yield: 73%. Well-shaped single crystals suitable for X-ray diffraction were obtained by re-crystalli- zation of the solid from methanol. Analysis calculated for C18H17Br2N2O6V: C, 38.06; H, 3.02; N, 4.93%; found: C, 38.23; H, 2.95; N, 4.81%. IR data (KBr, cm–1): 1611 (vs, νC=N), 978 (m, V=O). UV-Vis data (λmax, nm): 270, 340, 460. 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 complexes (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 com- position of reaction medium was determined by GC with styrene and styrene epoxide quantified by the internal standard method (chlorobenzene). All other products de- tected by GC were mentioned as others. For each complex the reaction time for maximum epoxide yield was deter- mined 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 inde- pendent 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 H2L1 and H2L2 were synthesized by reaction of 3-methyl-4-nitrobenzohydrazide with 4-bro- mosalicylaldehyde, and pivalohydrazide with 3,5-dibro- mosalicylaldehyde, respectively in methanol (Scheme 2). The complexes 1 and 2 were prepared by the reaction of the hydrazone ligands with VO(acac)2 in the presence of acetohydroxamic acid and maltol (Scheme 3). The reaction progresses are accompanied by an immediate color change of the solution from colorless to deep brown. The hydra- zones were deprotonated during the coordination. The ox- idation of V(IV) in VO(acac)2 to V(V) in both complexes during the reaction in air is not uncommon.4c,8 The molar conductivities (ΛM = 35 Ω–1 cm2 mol–1 for 1 and 30 Ω–1 cm2 mol–1 for 2) are consistent with the values expected for non-electrolyte.9 3. 2. Crystal Structure Description of the Complexes Selected bond lengths and angles for the complexes are listed in Table 2. Single crystal X-ray analysis indicates that the complexes are mononuclear oxidovanadium(V) 238 Acta Chim. Slov. 2022, 69, 235–242 Lei: Two Oxidovanadium(V) Complexes ... compounds. The ORTEP plots of the complexes 1 and 2 are shown in Figs. 1 and 2, respectively. The V atoms in the complexes are in octahedral geometry. In complex 1, the V atom is coordinated by the O2N donor atoms of the hydra- zone ligand L1 and the hydroxyl O atom of the acetylhy- droxamate ligand in the equatorial plane, and by the car- bonyl O atom of the acetylhydroxamate ligand and the oxido O atom at the two axial positions. In complex 2, the V atom is coordinated by the O2N donor atoms of the hy- drazone ligand L2 and the hydroxyl O atom of the malto- late ligand in the equatorial plane, and by the carbonyl O atom of the maltolate ligand and the oxido O atom at the two axial positions. The V atoms displaced toward the axi- al oxido O atoms (O3) by 0.269(1) Å for 1, and 0.322(1) Å for 2, from the equatorial planes of both complexes. The distortion of the octahedral coordination of the complexes can be observed from the bond angles related to the V at- oms. The cis- and trans- angles related to the V atoms at the equatorial planes are in the ranges of 75.22(15)– 100.1(2)º and 154.24(16)–172.90(17)° for 1 and 74.6(2)– 101.3(2)º and 153.6(2)–177.3(2)° for 2. The deviations from the ideal octahedral geometry are mainly origin from the strain created by the five-membered chelate rings V1- N1-N2-C8-O2 and V1-O4-C17-N4-O5 for 1 or V1-O4- C13-C14-O5 for 2. The bond lengths of V–O and V–N of both complexes are similar to each other, and comparable to those in other V complexes in literature.4,10 The termi- nal V1–O3 [1.57–1.58 Å] bond distances of both complex- es agree well with the corresponding values reported for related systems.9 Because of the trans influence of the oxi- do groups, the distances to the O4 atoms (2.20–2.31 Å) are considerably elongated, making the O4 atoms weakly co- ordinated to the V atoms. Such elongation has previously been observed in other complexes with similar structures. The hydrazone ligands coordinate to the V atoms through dianionic form, which can be observed from the bond Scheme 2. The synthesis of the hydrazones. Scheme 3. The synthesis of the complexes. 239Acta Chim. Slov. 2022, 69, 235–242 Lei: Two Oxidovanadium(V) Complexes ... lengths of C8–O2 and C8–N2. The bonds C8–O2 are obvi- ously longer than typical double bonds, while the bonds C8–N2 are obviously shorter than typical single bonds. This phenomenon is not uncommon for hydrazone com- plexes.4,5a,10 The crystal structure of complex 1 is stabilized by N‒H···N and C‒H···O hydrogen bonds (Table 3), to gener- Table 2. Selected bond distances (Å) and bond angles (°) for the complexes 1 2 V1‒O1 1.856(3) 1.841(7) V1‒O2 1.944(3) 1.901(7) V1‒O3 1.579(3) 1.575(6) V1‒O4 2.207(3) 2.309(7) V1‒O5 1.848(3) 1.856(6) V1‒N1 2.089(4) 2.098(7) O3‒V1‒O5 96.46(16) 101.2(3) O3‒V1‒O1 100.10(18) 98.1(3) O5‒V1‒O1 98.23(14) 100.7(3) O3‒V1‒O2 97.63(17) 99.5(3) O5‒V1‒O2 98.18(14) 94.9(3) O1‒V1‒O2 154.24(15) 153.7(3) O3‒V1‒N1 98.39(16) 99.6(3) O5‒V1‒N1 164.42(15) 158.0(3) O1‒V1‒N1 83.78(14) 83.1(3) O2‒V1‒N1 75.23(13) 74.8(3) O3‒V1‒O4 172.88(16) 177.2(3) O5‒V1‒O4 76.53(14) 76.9(3) O1‒V1‒O4 82.37(14) 84.2(3) O2‒V1‒O4 82.31(13) 78.7(3) N1‒V1‒O4 88.50(13) 82.0(3) Table 3. Hydrogen bond distances (Å) and bond angles (°) for the complexes D–H∙∙∙A d(D–H) d(H∙∙∙A) d(D∙∙∙A) Angle (D–H∙∙∙A) 1 N4–H4∙∙∙O8#1 0.90 1.83 2.698(4) 164(5) C15–H15B∙∙∙O8#2 0.96 2.44 3.334(4) 156(5) 2 C6–H6∙∙∙O4#3 0.93 2.54 3.404(5) 154(6) C16–H16∙∙∙O3#4 0.93 2.59 3.347(5) 139(6) Symmetry codes: #1: 1½ + x, ½ – y, ½ + z; #2: 1 + x, y, z; #3: 1½ – x, y, ½ + z; #4: x, y, –1 + z. Fig. 1. ORTEP diagram of complex 1 with 30% thermal ellipsoid. Fig. 2. ORTEP diagram of complex 2 with 30% thermal ellipsoid. Fig. 3. Molecular packing structure of complex 1 linked by hydro- gen bonds (dashed lines). 240 Acta Chim. Slov. 2022, 69, 235–242 Lei: Two Oxidovanadium(V) Complexes ... ate chains along the a axis (Fig. 3). The crystal structure of complex 2 is stabilized by C‒H···O hydrogen bonds (Table 3), to generate chains along the c axis (Fig. 4). 3. 4. IR and UV-vis Spectra of the Compounds The weak and broad absorptions in the region 3400– 3500 cm–1 of the free hydrazones are attributed to the O‒H bonds of the phenol groups. The weak absorptions at 3120–3230 cm–1 for the free hydrazones and complex 1 are assigned to the stretching vibrations of the N–H groups. The intense bands at 1657 cm–1 for H2L1, 1653 cm–1 for H2L2, and 1661 cm–1 for complex 1 are assigned to the vi- brations of the C=O groups.11 The typical bands for the azomethine groups, ν(C=N), are observed at 1590–1611 cm–1 for the compounds.12 The characteristic of the spec- tra of both complexes is the exhibition of sharp bands at about 973 cm–1 for 1 and 978 cm–1 for 2, corresponding to the V=O stretching vibration.13 The appearance of a single band in this region indicates the existence of monomeric six-coordinated V=O units instead of the polymeric units.14 This is approved by the single crystal structure de- termination. The weak bands in the range of 400–650 cm–1 are assigned to the vibrations of the V–O and V–N bonds. In the UV-Vis spectra of the compounds, the bands at 320–350 nm are attributed to the azomethine chromo- phore π-π* transitions. The bands at higher energy (260– 300 nm) are associated with the benzene π-π* transitions.15 The weak bands at 545 nm for 1 and 460 nm for 2 are at- tributed to intramolecular charge transfer transitions from the pπ orbital on the nitrogen and oxygen to the empty d orbitals of the V atoms.16 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 giv- en in Table 4. The data reveals that the complexes as cata- lysts convert styrene most efficiently in the presence of both oxidants. Nevertheless, the catalysts are selective to- wards the formation of styrene epoxides despite of the for- mation 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 epox- ide yield. When the reactions are carried out with PhIO and NaOCl, styrene conversions of complexes 1 and 2 were about 85% and 81%, and 78% and 75%, respectively. It is evident that between PhIO and NaOCl, the former acts as a better oxidant with respect to both styrene con- version and styrene epoxide selectivity. The epoxide yields for the complexes 1 and 2 using PhIO and NaOCl as oxi- dants are 76% and 86%, and 74% and 81%, respectively. 4. Conclusion Two new mononuclear oxidovanadium(V) complex- es derived from hydrazone ligands have been synthesized and characterized. Single crystal X-ray analysis indicates that the V atoms in both complexes are in distorted octa- hedral coordination. The complexes have effective catalyt- ic property for the epoxidation of styrene, with conver- sions over 75% and selectivities over 87%. Supplementary Material CCDC 2123401 for 1 and 2123402 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 work was financially supported by the Scientific Research Foundation of Chengdu Technological Universi- ty (Grant No. 2021RC004). 5. References 1. (a) S. Kanchanadevi, F. R. Fronczek, V. Mahalingam, Inorg. Chim. Acta 2021, 526, 120532; (b) P. H. D. O. Santiago, E. Fig. 4. 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Povzetek Sintetizirali smo dva nova oksidovanadijeva(V) kompleksa, [VOL1(aha)]DMF (1) in [VOL2(mat)] (2), kjer sta L1 in L2 dianionski obliki N‘-(4-bromo-2-hidroksibenziliden)-3-metil-4-nitrobenzohidrazida in N‘-(3,5-dibromo-2-hidroksibe- nziliden)pivalohidrazida ter aha in mat monoanionski obliki acetohidroksaminske kisline in maltola ter ju okarakter- izirali s fizikalno-kemijskimi metodami in monokristalno rentgensko difrakcijo. Rentgenska analiza razkriva, da imajo V atomi v kompleksih oktaedrično koordinacijo. Kristalni strukturi kompleksov sta stabilizirani z vodikovimi vezmi. Proučili smo katalitske lastnosti kompleksov za epoksidacijo stirena. Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License 243Acta Chim. Slov. 2022, 69, 243–250 Veljović et al.: Crystallography and DFT Studies ... DOI: 10.17344/acsi.2021.7329 Scientific paper Crystallography and DFT Studies of Synthesized Tetraketones Elma Veljović,1 Krešimir Molčanov,2 Mirsada Salihović,1 Una Glamočlija,1,3,4 Amar Osmanović,1,* Nevzeta Ljubijankić5 and Selma Špirtović-Halilović1 1 University of Sarajevo, Faculty of Pharmacy, Zmaja od Bosne 8, 71 000 Sarajevo, Bosnia and Herzegovina 2 Ruđer Bošković Institute, Bijenička 54, 10000 Zagreb, Croatia 3 University of Mostar, School of Medicine, Bijeli Brijeg bb, 88000 Mostar, Bosnia and Herzegovina 4 Bosnalijek JSC, Scientific-Research Unit, Jukićeva 53, 71 000 Sarajevo, Bosnia and Herzegovina 5 University of Sarajevo, Faculty of Science, Department of Chemistry, Zmaja od Bosne 35, 71000 Sarajevo, Bosnia and Herzegovina * Corresponding author: E-mail: amar.osmanovic@ffsa.unsa.ba Received: 12-15-2021 Abstract Two tetraketone derivatives, one previously reported and one novel, were synthesized, whose structures have been con- firmed by elemental analyses, NMR, HPLC-MS, and IR spectroscopy. The crystal structures of synthesized tetraketones were determined using X-ray single-crystal diffraction. To analyze the molecular geometry and compare with experi- mentally obtained X-ray crystal data of synthesized compounds 1 (2,2’-((4-nitrophenyl)methylene)bis(5,5-dimethyl- cyclohexane-1,3-dione)) and 2 (2,2’-((4-hydroxy-3-methoxy-5-nitrophenyl)methylene)bis(5,5-dimethylcyclohex- ane-1,3-dione)), DFT calculations were performed with the standard 6-31G*(d), 6-31G**, and 6-31+G* basis sets. The calculated HOMO-LUMO energy gap for compound 1 was 4.60 eV and this value indicated that compound 1 is chem- ically more stable compared to compound 2 whose energy gap was 3.73 eV. Both compounds’ calculated bond lengths and bond angles were in very good accordance to experimental values determined by X-ray single-crystal diffraction. Keywords: Tetraketones; X-ray diffraction; DFT; HOMO-LUMO energies 1. Introduction Tetraketones (2,2’-(arylmethylene)bis(5,5-dimeth- yl-2-cyclohexane-1,3-diones)) represent an important class of compounds that have shown beneficial pharmaco- logical effects in vitro. They are widely used as important precursors in the synthesis of various acridindiones as la- ser dyes and some heterocyclic compounds, xanthendi- ones and thioxanthenes.1 Tetraketones exhibit antioxidant, antibacterial and antiviral effects.2 These compounds are well studied as the inhibitors of the enzyme lipooxygen- ase.3 Tetraketones are being evaluated as prospective med- icines in the treatment of inflammatory diseases, bronchi- olitis, carcinoma, and autoimmune illnesses since lipooxygenases represent a potential target for rational drug design and identification of mechanism-based inhib- itors for these conditions.4,5 These compounds were studied by X-ray crystallogra- phy (X-ray), nuclear magnetic resonance (NMR), and mo- lecular modeling, revealing important information about structure and conformation, such as the presence of intra- molecular hydrogen bonds.6–9 One of the most important studies was conducted by Forsen et al. in 1969 when they determined by NMR that 2,2’-arylmethylene-bis(3-hy- droxy-5,5-dimethyl-2-cyclohexene-1-ones) are found as dienol tautomers. As a result, these compounds are referred to as tetraketones in the literature (Figure 1).10 Figure 1. Tetraketones and their keto-enol tautomeric forms. 244 Acta Chim. Slov. 2022, 69, 243–250 Veljović et al.: Crystallography and DFT Studies ... 2. Materials and Methods 2. 1. Synthesis of Tetraketone (2,2’-(arylmethylene)bis(5,5-dimethyl -2-cyclohexane-1,3-dione)) Derivatives Benzaldehyde (1 mmol), 5,5-dimethylcyclohex- ane-1,3-dione (2 mmol), and diazobicyclo[2.2.2]octane (DABCO) (0.05 g) were refluxed in water (20 mL). Thin-layer chromatography was used to monitor the reac- tion’s flow and completion. Tetraketones are obtained ap- proximately after 20 minutes of reflux. If the reflux is con- tinued for longer (30 minutes or more), reaction leads toward formation of the 9-aryl substituted 3,3,6,6-te- tramethyl-3,4,5,6,7,9-hexahydro-1H-xanthene-1,8(2H)- diones.11 The mixture was cooled to room temperature, filtered, and rinsed with water once the reaction was com- pleted. Recrystallization of the resulting compounds was performed from 96% ethanol.12 All chemicals have been obtained from Merck (Germany). Newly synthesized compounds were obtained through Knoevenagel condensation of aromatic aldehyde and Michael addition with 5,5-dimethylcyclohexandi- one-1,3-dione (Figure 2). In this article, we present two tetraketones, one previously reported (1)13 and the other one novel (2), whose structures have been confirmed by elemental analysis, IR, NMR spectroscopy and HPLC-MS spectrometry. 2. 2. Characterization of Synthesized Products Elemental analysis. For the synthesized tetraketone derivatives, elemental analysis was performed at the Insti- tute of Chemistry, Technology and Metallurgy, Center for Chemistry in Belgrade, Serbia, on the Vario EL apparatus III C,H,N,S/O Elemental Analyzer, Elementar Analysen- systeme GmbH, Hanau, Germany. IR spectroscopy. IR spectra of the synthesized com- pounds were recorded at the Bosnalijek Pharmaceutical Company Ltd., Sarajevo, Bosnia and Herzegovina, on the Shimadzu IR Prestige 21 apparatus in the wavelength range from 4500 to 700 cm–1. NMR. 1H NMR and 13C NMR spectra for the syn- thesized compounds were recorded at the Faculty of Sci- ence in Novi Sad, Serbia, using a Bruker AC 250 E appara- tus. Compounds were recorded in deuterated chloroform using TMS (tetramethylsilane) as a reference. HPLC-MS spectra. The mass spectra were recorded on an HPLC-MS triple quadrupole 6420 autosampler (Agi- lent Technologies, Palo Alto, CA, USA). The recordings were made at a temperature of 573 K and a gas flow of 6 L min–1. As the mobile phase, 0.1% formic acid in 50% meth- anol was used, at a flow rate of 0.2 mL min–1. The spectra were processed using Agilent MassHunter software. Melting point. The melting points of the synthesized compounds were determined at the Department of Phar- maceutical Chemistry, Faculty of Pharmacy in Sarajevo, Bosnia and Herzegovina, using the Melting point appara- tus manufactured by Kruss Optronic, Germany. X-ray diffraction. Single crystal measurements were performed on an Oxford Diffraction Xcalibur Nova R (mi- crofocus Cu tube) at room temperature [293(2) K]. Pro- gram package CrysAlis PRO was used for data reduction.14 The structures were solved using SHELXS97 and refined with SHELXL97.15 The models were refined using the full-matrix least-squares refinement; all non-hydrogen at- oms were refined anisotropically. Hydrogen atoms were modeled as riding entities using the AFIX command. Molecular geometry calculations were performed by PLATON,16 and molecular graphics were prepared using ORTEP-3,17 and CCDC-Mercury.18 Crystallographic and refinement data for the structures reported in this paper are shown in Table 1. Supplementary crystallographic data can be ob- tained free of charge via www.ccdc.cam.ac.uk/conts/re- Figure 2. Synthesis of 2,2’-(arylmethylene)bis(5,5-dimethyl-2-cyclohexane-1,3-dione) derivatives. 245Acta Chim. Slov. 2022, 69, 243–250 Veljović et al.: Crystallography and DFT Studies ... trieving.html (or from the Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033; or deposit@ccdc.cam.ac.uk). CCDC 1990310-1990311 contains the supplementary crystallo- graphic data for this paper.19 2. 3. Computational Details Quantum chemical computations were done for compounds 1 and 2 on a single molecule in vacuo, with comprehensive geometry optimizations using standard Spartan 14 software. At the B3LYP/6-31G*, 6-31G**, and 6-31+G* levels of theory, geometry optimization was per- formed.20 The HOMO (highest occupied molecular orbit- al) and LUMO (lowest unoccupied molecular orbital) en- ergy distributions, as well as the HOMO-LUMO energy gap, were calculated using these levels of theory. The re- sults of the DFT analysis were compared to those experi- mentally obtained crystallographic data. 3. Results and Discussion 3. 1. Chemistry According to described Knoevenagel condensation of aromatic aldehyde and Michael addition with 5,5-di- methyl-1,3-cyclohexandione, we synthesized compounds 1 (2,2’-((4-nitrophenyl)methylene)bis(5,5-dimethylcyclo- hexane-1,3-dione)) and 2 (2,2’-((4-hydroxy-3-me- thoxy-5-nitrophenyl)methylene)bis(5,5-dimethylcyclo- hexane-1,3-dione)) (Figure 3). The characterization of synthesized compounds 1 and 2 was achieved by FT-IR, 1H and 13C NMR spectros- copy, and HPLC-MS spectrometry. 1: Yield: 81%. Mp 198–203 °C. Anal. Calcd for C23H- 27N1O6: C, 66.81; H, 6.58. Found: C, 66.74; H, 6.62. IR (KBr) ν 3000 (Ar-H), 1670 (C=O), 1480 (C=C), 1300 (C- O), 1500 (C=O), 1250 (NO2) cm–1. 1H NMR (600 MHz, CDCl3) δ 11.8 (br s, 1H, OH, disappears with D2O), 8.13 (d, 2H, J2’,3’ = 8.9 Hz, H-3’, H-5’), 7.24 (d, 2H, J2’,3’ = 8.9 Hz, H-2’, H-6’), 5.53 (s, 1H, CH), 2.21–2.57 (m, 8H, 4 × CH2), 1.11 and 1.23 (2 × s, 12 H, 4 × CH3). 13C NMR (150 MHz, CDCl3) δ 190.82 (C=O), 189.46 (C-2), 146.49–146.03 (Ar- C), 127.56, 123.40 (Ar-CH), 114.81 (C-1), 46.91, 46.32 (CH2), 33.18 (CH), 31.39 (C from C(CH3)2), 29.38, 27.38 (CH3 from C(CH3)2). MS  m/z  (relative intensity):  412.2 (M+H). 2: Yield: 88%. Mp 230–232 °C. Anal. Calcd for C24H- 27N1O8: C, 62.73; H, 6.36. Found: C, 63.10; H, 6.08. IR (KBr) ν 3300–2500 (Ar-OH), 3042 (Ar-H), 1730 (C=O), 1607, 1588 (C=C), 1448 (O-CH3), 1320 (C-O), 1200 (Ar- Table 1. Crystallographic data collection and structure refinement details. Compound 1 2 Empirical formula C23H25NO6 C24H27NO8 Formula wt. / g mol–1 411.45 457.47 Space group P bc21 P1 - a / Å 23.5533(3) 8.9958(3) b / Å 12.9754(1) 9.3891(4) c / Å 28.1370(3) 13.9171(6) α / ° 90 98.814(4) β / ° 90 99.380(3) γ / ° 90 90.925(3) Z 16 2 V / Å3 8599.05(16) 1145.04(8) Dcalc / g cm–3 1.271 1.327 μ / mm–1 0.759 0.835 T / K 293(2) 293(2) Radiation vawelength 1.54179 (CuKα) 1.54179 (CuKα) Reflections collected 28964 10363 Independent reflections 13399 4692 Observed reflections (I ≥ 2σ) 12660 4108 Rint 0.0212 0.0207 R (F) 0.0463 0.0488 Rw (F2) 0.1313 0.1470 Goodness of fit 1.034 1.049 No. of parameters 1081 299 No. of restraints 1 0 Flack parameter 0.11(7) – Δρmax, Δρmin (eÅ–3) 0.380; –0.199 0.338; –0.168 Figure 3. Structures of synthesized tetraketones 1 and 2. 246 Acta Chim. Slov. 2022, 69, 243–250 Veljović et al.: Crystallography and DFT Studies ... OH), 1595 (NO2) cm–1. 1H NMR (600 MHz, CDCl3) δ 10.60 (br s, 1H, OH, disappears with D2O), 7.44 (s, 1H, H-6ʹ), 6.90 (s, 1H, H-2ʹ), 5.43 (s, 1H, H-13), 3.83 (s, 3H, OCH3), 2.54–2.22 (m, 8H, H-3, H-11, H-5, H-9), 1.25 (s, 6H, H-15, H-17), 1.12 (s, 6H, H-14, H-16). 13C NMR (150 MHz, CDCl3) δ 190.94 (C-6, C-8), 189.45 (C-2, C-12), 149.59 (C-3ʹ), 144.41 (C-5ʹ), 133.60 (C-4ʹ), 114.71 (C-1, C-7), 117.04 (C-2ʹ), 114.01 (C-6ʹ), 33.18 (C-13), 31.39 (C- 4, C-10), 29.82 (C-15, C-17), 26.87 (C-14, C-16). MS m/z (relative intensity): 460 (M+H). 3. 2. Description of the Structures The compound 1 crystallizes in a non-centrosym- metric space group P bc21 with four symmetry-indepen- dent molecules in the asymmetric unit (i.e. Z’ = 4), labeled as a, b, c, and d (Figure 4). There are two conformers, with a different confor- mation of the ring C2→C6: one is comprised of molecules a and d, and the other of b and c. The rest of the molecule differs less than 3 e.s.d.’s (least-squares overlay is shown in Figure 4. ORTEP-3 drawings of four symmetry-independent molecules in 1 with atom numbering schemes. Displacement ellipsoids are drawn for the probability of 50% and hydrogen atoms are shown as spheres of arbitrary radii. Figure 5. Crystal packing of 1 viewed in the direction [010]. Sym- metry-independent molecules are shown in different colors: a are green, b are blue, c are red and d are gray. 247Acta Chim. Slov. 2022, 69, 243–250 Veljović et al.: Crystallography and DFT Studies ... Figure 8). The compound lacks proton donors and no π-stacking is observed, so 3D packing (Figure 5) is achieved mainly through dispersion interactions and weak C-H∙∙∙O hydrogen bonds (Table 2). The asymmetric unit of 2 contains one molecule (Figure 6), whose geometry and conformation are similar to those of 1 (Figure 8). The molecule possesses a single proton donor, the O8-H8 hydroxyl group, which forms an intermolecular hydrogen bond with atom O5 of the nitro group as an acceptor. Crystal data and structure refine- ment summary of compounds 1 and 2 are given in Tables 2 and 3. Dispersion interactions are responsible for the 3D packing (Figure 7). Figure 6. ORTEP-3 drawing of a molecule of 2 with the atom num- bering scheme. Displacement ellipsoids are drawn for the probabil- ity of 50% and hydrogen atoms are shown as spheres of arbitrary radii. Figure 7. Crystal packing of 2 viewed in the direction [010]. Table 2. Geometric parameters of hydrogen bonds and angles. d(D–H) d(H···A) d(D···A) φ(D–H···A) Cpd (D–H···A) (Å) (Å) (Å) (°) Exp. Calc. Exp. Calc. Exp. Calc. Exp. Calc. Symm. op. on A 1 C1A–H1A∙∙∙O2A 0.98 1.01 2.41 2.38 2.855(3) 2.678 107 101 x, y, z C1A–H1A∙∙∙O3A 0.98 1.01 2.43 2.25 2.858(3) 2.680 106 109 x,y, z C1B–H1B∙∙∙O2B 0.98 1.01 2.42 2.53 2.871(3) 2.701 107 109 x, y, z C1B–H1B∙∙∙O3B 0.98 1.01 2.38 2.40 2.842(3) 2.710 108 110 x, y, z C1C–H1C∙∙∙O2C 0.98 1.01 2.41 2.47 2.863(3) 2.715 107 109 x, y, z C1C–H1C∙∙∙O3C 0.98 1.01 2.39 2.42 2.850(3) 2.370 108 110 x, y, z C1D–H1D∙∙∙O2D 0.98 1.01 2.38 2.45 2.842(3) 2.790 108 110 x, y, z C1D–H1D∙∙∙O3D 0.98 1.01 2.44 2.48 2.865(3) 2.800 106 109 x, y, z C20B–H20B∙∙∙O2A 0.93 0.99 2.54 2.60 3.295(4) 3.100 138 141 x, −1+y,z C20D–H20D∙∙∙O6A 0.93 0.99 2.53 2.58 3.434(4) 3.110 165 163 x, 3/2−y, 1/2+z C22B–H22B∙∙∙O6D 0.93 0.99 2.57 2.61 3.458(4) 3.120 160 161 1−x, 1−y, −1/2+z C22D–H22D∙∙∙O2D 0.93 0.99 2.51 2.56 3.199(3) 3.010 131 129 1−x, 1/2+y, z 2 O8–H8∙∙∙O5 0.82 0.99 1.89 1.84 2.578(3) 2.308 141 140 x,y,z O8–H8∙∙∙N1 0.82 0.99 2.50 2.41 2.911(2) 3.04 113 117 x,y,z C1–H1∙∙∙O2 0.98 1.09 2.35 2.35 2.843(18) 2.82 110 115 x,y,z C1–H1∙∙∙O3 0.98 1.09 2.40 2.44 2.8506(1) 2.81 108 106 x,y,z C12–H12B∙∙∙O1 0.97 1.09 2.53 2.84 3.480(2) 3.10 165 170 1+x,y, z Figure 8. Least-squares overlay of four symmetry-independent molecules of 1 (a is green, b is blue, c is red and d is gray) and 2 (black). 248 Acta Chim. Slov. 2022, 69, 243–250 Veljović et al.: Crystallography and DFT Studies ... Compared calculated and experimentally obtained values in both Table 2 and 3 show very good accordance, differing mostly only in the second decimal place. Similar investigation and comparison of theoretical and experi- mental data, with good accordance for synthesized com- pounds, has been reported before.21,22 3. 3. Analysis of Molecular Orbital The energy gap HOMO-LUMO of the molecules has a role in deciding their bioactivity and is an important pa- rameter for quantum chemistry. The molecule becomes harder and more stable or less reactive when the HO- MO-LUMO energy gap increases.23 The HOMO energy distinguishes electron donor capacity, whereas the LUMO energy distinguishes electron acceptor capacity, and the gap defines chemical stability.24 The energy gap HO- MO-LUMO for the compounds 1 and 2 were calculated by 6-31G*, 6-31G**, and 6-31+G* basis sets and these values were –4.60, –4.57, and –4.58 for compound 1 and –3.73, –3.69, and –3.70 for compound 2. The energies and energy gaps of HOMO and LUMO are shown in Table 4. The HO- MO-LUMO orbital schemes for compounds 1 and 2 are shown in Figure 9 (the positive phases are red, and the negative phases are blue). Compound 1 HOMO electron density demonstrates that the HOMO is localized on carbonyl carbons, methyl, and benzene, while compound 2 HOMO is concentrated on hydroxyl and methoxy groups. The HOMO-LUMO en- ergy gap for compound 1 is 4.60 and for compound 2 is 3.73, indicating that electron density passes from carbonyl carbons, methyl, and methoxy groups to hydroxyl and ni- tro groups. Compound 1 has a larger HOMO-LUMO en- ergy gap than compound 2, making it less reactive and hence more stable. The descriptor of electron donor and acceptor is implicitly explained by the HOMO to LUMO transition to comprehend their interaction capabilities with their target molecules. 4. Conclusions The tetraketones (compounds 1 and 2) were success- fully synthesized with excellent yield by condensation of aromatic aldehyde and Michael addition with 5,5-dimeth- ylcyclohexane-1,3-dione. The synthesized compounds 1 Table 3. Bond lengths [Å] and angles [°] of compounds 1 (molecule A, only) and 2. Bond length Compound 1 Compound 2 and angles Exp. Cal. Exp. Cal. C1-C2 1.526(4) 1.548 1.5204(18) 1.548 C1-C10 1.528(3) 1.557 1.5246(19) 1.556 C10-C11 1.402(3 1.530 1.3944(19) 1.530 C11-C12 1.487(4) 1.520 1.499(2) 1.520 C2-C3 1.402(4) 1.546 1.395(2) 1.545 C21-O8 – – 1.345(2) 1.340 C21-N1 1.470(4) 1.469 – – N1-O6 1.230(4) 1.232 1.211(3) 1.255 C7-O2 1.279(4) 1.211 1.285(19) 1.340 C11-O3 1.292(3) 1.215 1.297(19) 1.215 C2-C1-C10 113.6(2) 113.2 115.2(11) 118.0 C10-C11-O3 123.1(3) 124.0 123.0(13) 122.4 C10-C11-C12 122.2(2) 119.4 121.5(14) 121.1 C2-C3-O1 122.9(3) 120.0 123.3(13) 125.2 C2-C3-C4 122.0(2) 122.9 121.2(13) 119.9 C20-C21-C22 122.0(3) 122.7 116.9(14) 116.4 C21-N1-O5 118.4(3) 120.0 118.2(19) 119.0 C21-N1-O6 118.2(3) 119.8 119.8(16) 120.3 Table 4. Calculated HOMO and LUMO energy values for compounds 1 and 2. Compound 1 Compound 2 Parameters B3LYP/6-31G* B3LYP/6-31G** B3LYP/6-31+G* B3LYP/6-31G* B3LYP/6-31G** B3LYP/6-31+G* EHOMO (eV) –6.85 –6.87 –6.84 –6.85 –6.87 –6.84 ELUMO (eV) –2.25 –2.30 –2.26 –2.53 –2.58 –2.54 Energy gap (∆) 4.60 4.57 4.58 3.73 3.69 3.70 249Acta Chim. Slov. 2022, 69, 243–250 Veljović et al.: Crystallography and DFT Studies ... and 2 were characterized using 1H and 13C NMR, FTIR, HPLC-MS methods, and elemental analysis. Using sin- gle-crystal X-ray diffraction data, we presented the struc- tural details of tetraketone compounds 1 (C23H25NO6) and 2 (C24H27NO8). To analyze the molecular geometry and compare it to experimentally available X-ray crystal data of investigated compounds, DFT calculations were done using a standard 6-31G*(d), 6-31G**, and 6-31+G* basis sets. For compound 2, the computed HOMO-LUMO en- ergy gaps for basis sets 6-31G*(d), 6-31G**, and 6-31+G* were 3.73, 3.69, and 3.70, respectively. Compound 2 is chemically more reactive than compound 1 based on these smaller gap values. The theoretically determined HO- MO-LUMO energy gaps can be employed to describe the biological activity of the title compounds. The crystal structure is stabilized by both intramolecular and inter- molecular hydrogen bonds, with the intermolecular N–H…O hydrogen bond in compound 2 generating the N1 and O8 chain motif. The bond lengths and angles cal- culated for compounds 1 and 2 were in very good accor- dance with the experimental values obtained from X-ray crystal diffraction. 5. References 1. K. Khan, G. Maharvi, M. Khan, A. Jabbar Shaikh, S. Perveen, S. Begum, M. I. Choudhary, Bioorg. Med. Chem. 2006, 14, 344–351. DOI:10.1016/j.bmc.2005.08.029 2. R. W. Lambert, J. A. Martin, J. H. Merrett, K. E. B. Parkes, J. G. Thomas, PCT Int. Appl., WO 9706178 Chem. Abstr. 1997, 126, 212377y. 3. S. Ali, G. M. Maharvi, N. Riaz, N. Afza, A. Malik, A. U. Reh- man, M. Lateef, L. Iqbal, West Indian Med. 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S pomočjo DFT računskih metod s standardnimi baznimi seti 6-31G*(d), 6-31G** in 6-31+G* smo izvedli analizo molekulske geometrije in dobljene rezultate primerjali z eksperimentalnimi, ki so bili pridobljeni z rentgensko difrakcijo pripravljenih spojin 1 (2,2’-((4-nitrofenil)metilen)bis(5,5-dimetilcikloheksan-1,3-di- on)) in 2 (2,2’-((4-hidroksi-3-metoksi-5-nitrofenil)metilen)bis(5,5-dimetilcikloheksan-1,3-dion)). Izračunana vrednost HOMO-LUMO energijske špranje za spojino 1 je 4.60 eV, kar kaže, da je spojina 1 kemijsko bolj stabilna kot spojina 2, katere velikost energijske špranje je 3.73 eV. Izračunane dolžine vezi in koti se za obe spojini zelo dobro ujemajo z eks- perimentalnimi vrednostmi, dobljenimi z rentgensko difrakcijo monokristala. Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License 13. J. Safaei-Ghomi, S. Asadian, S. H. Nazemzadeh, H. Shahba- zi-Alavi, J. Chin. 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Marjan Marinšek Somentorica: prof. dr. Violeta Bokan Bosiljkov SAMOOBNOVA BETONA Z DOLOMITNIM AGREGATOM ZARADI PROCESA ALKALNO KARBONATNE REAKCIJE Datum zagovora: 25. 2. 2021 Andreja PETROVIĆ Mentorica: prof. dr. Polona Žnidaršič Plazl BIOTEHNOLOŠKA PROIZVODNJA LAKAZ IN NJIHOVA UPORABA PRI RAZVOJU MIKROBIOSENZORJA Datum zagovora: 3. 3. 2021 Aleksandar BLAGOJEVIĆ Mentor: prof. dr. Robert Dominko OPTIMIZACIJA ELEKTROKEMIJSKIH LASTNOSTI V PHBQS/CNT NANOKOMPOZITIH Datum zagovora: 4. 3. 2021 Lucija POLAK Mentorica: izr. prof. dr. Gabriela Kalčikova UPORABA RAZLIČNIH METOD ZA ČIŠČENJE TEKSTILNIH ODPADNIH VOD Datum zagovora: 31. 3. 2021 Leon OSTANEK JURINA Mentorica: prof. dr. Polona Žnidaršič Plazl RAZVOJ MIKROFLUIDNE PLATFORME ZA PRIPRAVO IN ANALIZO MIKROKAPLJIC Datum zagovora: 12. 5. 2021 Alen ŠTROS Mentor: prof. dr. 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Vera Župunski PRIPRAVA, GENSKI INŽENIRING IN KARAKTARIZACIJA CELIC ZA POVRNITEV IZRAŽANJA DISTROFINA V MIŠJEM MODELU DUCHENNOVE MIŠIČNE DISTROFIJE Datum zagovora: 17. 12. 2021 Bor KLANČNIK Mentor: prof. ddr. Boris Turk KARAKTERIZACIJA BIOLUMINISCENČNE SONDE ZA GRANCIM B Datum zagovora: 21. 12. 2021 MAGISTRSKI ŠTUDIJSKI PROGRAM 2. STOPNJE – TEHNIŠKA VARNOST Anže HEINDLER Mentorica: doc. dr. Barbara Novosel PRIMERJAVA ORODIJ ZA SIMULACIJO NENADZOROVANEGA SPROŠČANJA PLINOV V OKOLICO Datum zagovora: 18. 2. 2021 Tina LESJAK Mentorica: doc. dr. Klementina Zupan ERGONOMSKA ANALIZA DELOVNEGA MESTA V PODJETJU TALUM D.D. KIDRIČEVO Datum zagovora: 23. 2. 2021 Loti BRUS Mentor: prof. dr. Matevž Pompe OCENA TVEGANJA NA DELOVNEM MESTU RAZVIJALCA ANALIZNIH METOD Datum zagovora: 23. 2. 2021 Tajda BULOVEC Mentor: izr. prof. dr. Simon Schnabl UČINKOVITOST HEPTAFLUOROPROPANA PRI GAŠENJU POŽAROV Z ELEKTRO IZVOROM Datum zagovora: 3. 3. 2021 Peter VIRŠEK Mentorica: doc. dr. Barbara Novosel OBVLADOVANJE TVEGANJA PRI OBRATOVANJU TERMINALOV UTEKOČINJENEGA ZEMELJSKEGA PLINA Datum zagovora: 4. 3. 2021 Tina BREZAR Mentor: izr. prof. dr. Luka Tičar POMEN INŽENIRJA TEHNIŠKE VARNOSTI V DELOVNEM PROCESU Datum zagovora: 17. 3. 2021 Anja MORAN Mentor: izr. prof. dr. Simon Schnabl VPLIV USPOSABLJANJA NA ZNANJE O POŽARNI VARNOSTI ZAPOSLENIH Datum zagovora: 20. 4. 2021 Špela KRZNARIČ Mentor: izr. prof. dr. Simon Schnabl ANALIZA ZNANJA IZ VARSTVA PRED POŽAROM ZAPOSLENIH V ZDRAVILIŠČU Datum zagovora: 3. 5. 2021 Matej VESEL Mentorica: doc. dr. Klementina Zupan PROUČEVANJE ERGONOMSKE UREDITVE PROSTOROV NA FAKULTETI UNIVERZE V LJUBLJANI Datum zagovora: 14. 5. 2021 Miha PIRC Mentor: doc. dr. Mitja Robert Kožuh ANALIZA STANJA PREPREČEVANJA VEČJIH INDUSTRIJSKIH NESREČ V PREMAZNI INDUSTRIJI Datum zagovora: 7. 7. 2021 Laura BOROŠ Mentorica: doc. dr. Klementina Zupan ANALIZA TVEGANJ SUŠ IN POPLAV Datum zagovora: 5. 7. 2021 Peter KOČMAN Mentor: izr. prof. dr. Simon Schnabl Somentor: prof. dr. Robert Dominko VARNOST LITIJ-IONSKIH BATERIJ Datum zagovora: 8. 7. 2021 Laura HRKA Mentorica: doc. dr. Klementina Zupan ERGONOMIJA ŠOLAJOČE POPULACIJE Datum zagovora: 11. 10. 2021 David KLANČIŠAR Mentor: izr. prof. dr. Boštjan Genorio RECIKLAŽA Li-Ion AKUMULATORJEV NA OSNOVI NMC KATOD Datum zagovora: 15. 10. 2021 Tina BESAL Mentorica: doc. dr. Barbara Novosel OCENJEVANJE TVEGANJ PRI TRANSPORTU IN SKLADIŠČENJU METANOLA IN METANALA Datum zagovora: 3. 11. 2021 Jan LIPOVŠEK Mentor: izr. prof. dr. Simon Schnabl ANALIZA EVAKUACIJE IN POTEKA POŽARA IZ KOMPLEKSNEGA OBJEKTA S PROGRAMOM PATHFINDER IN PYROSIM Datum zagovora: 24. 11. 2021 S12 Acta Chim. Slov. 2022, 69, (1), Supplement Društvene vesti in druge aktivnosti DIPLOME - UNIVERZITETNI ŠTUDIJ KEMIJA – 1. STOPNJA Katharina Carola PAVLIN Mentor: prof. dr. Franc Požgan SINTEZA HEKSAARILBENZENOV Z DUŠIKOVIMI KOORDINIRAJOČIMI SKUPINAMI Datum zagovora: 15. 6. 2021 Domen GOSTE Mentorica: prof. dr. Marija Bešter Rogač VODA V IONSKIH TEKOČINAH Datum zagovora: 15. 6. 2021 Ana GOLOB Mentor: izr. prof. dr. Uroš Grošelj SINTEZA TETRAMSKE KISLINE IZ Cbz-Gly-OH IN UPORABA V ORGANOKATALIZIRANIH 1,4-ADICIJAH Datum zagovora: 29. 6. 2021 Jakob HÖFFERLE Mentor: prof. dr. Janez Košmrlj SINTEZA IZBRANIH AROMATSKIH IN HETEROAROMATSKIH GEMINALNIH DIBROMOALKENOV Datum zagovora: 30. 6. 2021 Tilen ZORKO Mentor: prof. dr. Bogdan Štefane FOTOKATALITSKE PRETVORBE NEKATERIH 3-(ALKILAMINO)PROPENOATOV Datum zagovora: 30. 6. 2021 David RIBAR Mentorica: prof. dr. Irena Kralj Cigić RAZVOJ MODELA REVERZNO-FAZNIH KROMATOGRAFSKIH SEPARACIJ NA OSNOVI HANSENOVIH PARAMETROV TOPNOSTI Datum zagovora: 1. 7. 2021 Anže HUBMAN Mentor: prof. dr. Tomaž Urbič ŠTUDIJ FOKUSIRANJA DELCEV V MIKROFLUIDNIH SISTEMIH Z UPORABO MREŽNE BOLTZMANNOVE METODE Datum zagovora: 1. 7. 2021 Simon KEBELJ Mentor: doc. dr. Krištof Kranjc REGIOSELEKTIVNOST IN STEREOSELEKTIVNOST CIKLOADICIJ NA PRIMERIH SUBSTITUIRANIH 3-ACILAMINO-2H-PIRAN-2-ONOV Datum zagovora: 1. 7. 2021 Klara KLEMENČIČ Mentor: doc. dr. Krištof Kranjc SINTEZE IN UPORABA 2H-PIRAN-2-ONOV PRI DIELS-ALDERJEVIH REAKCIJAH TER PREGLED ORGANOKATALIZE Datum zagovora: 1. 7. 2021 Špela POK Mentorica: prof. dr. Helena Prosen PREIZKUS EKSTRAKCIJE BENZOTRIAZOLOV IZ TAL Z UPORABO VROČE VODE Datum zagovora: 6. 7. 2021 Veronika GODEC Mentor: prof. dr. Matevž Pompe DOLOČEVANJE N-GLIKANOV Z MASNO SPEKTROMETRIJO Datum zagovora: 7. 7. 2021 Ana ZIDAR Mentorica: izr. prof. dr. Romana Cerc Korošec UPORABA PLASTOVITIH DVOJNIH HIDROKSIDOV ZA ČIŠČENJE VODE Datum zagovora: 8. 7. 2021 Nika ARTNAK Mentor: izr. prof. dr. Mitja Kolar DOLOČANJE pH IN VSEBNOSTI MAKROELEMENTOV V UMETNIH GNOJILIH Datum zagovora: 8. 7. 2021 Amanda KAČAR Mentor: izr. prof. dr. 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Franc Perdih VPLIV CINKA IN VITAMINOV NA COVID-19 Datum zagovora: 26. 8. 2021 Anja SEVER Mentorica: doc. dr. Saša Petriček 3-HIDROKSIPIRIDIN V BAKROVIH KOMPLEKSIH Datum zagovora: 26. 8. 2021 Lan SKOLIBER Mentor: doc. dr. Andrej Pevec KOFEIN KOT LIGAND V KOORDINACIJSKIH SPOJINAH S PLATINO Datum zagovora: 26. 8. 2021 Teja PELKO Mentor: doc. dr. Krištof Kranjc SINTEZA SUBSTITUIRANIH BICIKLO[2.2.2]OKTENOV S CIKLOADICIJAMI MED 2H-PIRAN-2-ONSKIMI DERIVATI IN MALEINANHIDRIDOM TER NADALJNJE PRETVORBE Z DUŠIKOVIMI NUKLEOFILI Datum zagovora: 30. 8. 2021 Blaž UŽMAH Mentor: prof. dr. Marjan Jereb PRETVORBE KARBONILNIH SPOJIN POD ZELENIMI POGOJI Datum zagovora: 30. 8. 2021 Matej JARC RYDZI Mentor: prof. dr. Jurij Svete SINTEZA IMOBILIZIRANE 4-AMINOBENZOJSKE KISLINE IN NJENA UPORABA V PRIPRAVI IMOBILIZIRANIH Cu(II)-ENAMINON KATALIZATORJEV Datum zagovora: 30. 8. 2021 Anja PEČKAJ Mentorica: doc. dr. 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Drago Kočar DOLOČEVANJE KATIONOV IN KATIONSKE KAPACITETE V OGLJU Datum zagovora: 3. 9. 2021 Tilen ŠIMENKO LALIČ Mentorica: prof. dr. Helena Prosen DOLOČITEV FITOESTROGENOV V PIVU S HPLC Datum zagovora: 3. 9. 2021 Žan ZAKOŠEK Mentorica: izr. prof. dr. Amalija Golobič KARAKTERIZACIJA IZBRANE KERAMIKE IZ TERNARNEGA SISTEMA La2O3-Ta2O5-TiO2 Datum zagovora: 3. 9. 2021 Tjaša STOPAR Mentorica: doc. dr. Nataša Gros METODE ZA DOLOČANJE SEČNINE Datum zagovora: 3. 9. 2021 Sara ŠADL Mentorica: doc. dr. Nataša Gros DOLOČANJE HLAPNIH SNOVI V MINIATURIZIRANIH IN PRETOČNIH SISTEMIH Datum zagovora: 3. 9. 2021 Vid KERMELJ Mentorica: prof. dr. Urška Lavrenčič Štangar ADSORPCIJA FENITOINA IN OKSITETRACIKLINA NA NANODELCE ZnO Datum zagovora: 6. 9. 2021 Tine LIKOVIČ Mentor: izr. prof. dr. Drago Kočar DOLOČITEV IZBRANIH VITAMINOV IN MINERALOV PREHRANSKEGA DODATKA HERBALIFE NUTRITION Datum zagovora: 6. 9. 2021 Gregor PUHAR Mentor: prof. dr. 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Irena Kralj Cigić EKSTRAKCIJA OCETNE KISLINE IN DIETIL FTALATA Z AKTIVNEGA OGLJA Datum zagovora: 7. 9. 2021 Tanja TOPIĆ Mentor: prof. dr. Janez Košmrlj SINTEZA IZBRANIH ALIFATSKIH GEMINALNIH DIBROMOALKENOV Datum zagovora: 7. 9. 2021 Klara ŠPARLEK Mentor: prof. dr. Anton Meden ANALIZA TABLET ZA POMIVALNI STROJ Z RENTGENSKO PRAŠKOVNO DIFRAKCIJO Datum zagovora: 7. 9. 2021 Zala KOPČAVAR Mentor: prof. dr. Matija Strlič DOLOČANJE OCETNE KISLINE V MUZEJSKI ATMOSFERI Datum zagovora: 7. 9. 2021 Andraž PEZDIRC Mentor: prof. dr. Matija Strlič DOLOČANJE ALDEHIDNIH ONESNAŽIL V NOTRANJOSTI ZGRADB Datum zagovora: 7. 9. 2021 Katarina VELKOV Mentor: prof. dr. Matija Strlič DOLOČANJE KLORIDA V RAZGRAJENEM PVC Datum zagovora: 7. 9. 2021 Jaka PRELOG Mentor: prof. dr. Tomaž Urbič MONTE CARLO SIMULACIJE MEHKIH DELCEV Datum zagovora: 8. 9. 2021 Martin RIHTARŠIČ Mentor: izr. prof. dr. Jernej Iskra Z NATRIJEVIM NITRITOM KATALIZIRANO AEROBNO OKSIDATIVNO JODIRANJE AROMATSKIH SPOJIN Datum zagovora: 8. 9. 2021 Simon PAVLIČ Mentorica: doc. dr. Nataša Gros METODE ZA IZDELAVO MIKROPRETOČNIH SISTEMOV Datum zagovora: 8. 9. 2021 Andraž STARIHA Mentorica: izr. prof. dr. Barbara Modec PRIPRAVA AMIDINOV PROPIONITRILA IN BENZONITRILA Datum zagovora: 8. 9. 2021 Larisa FILIP Mentor: izr. prof. dr. Jernej Iskra FOTOKEMIJSKE REAKCIJE FLUORIRANJA Datum zagovora: 9. 9. 2021 Nika METELKO Mentor: izr. prof. dr. Uroš Grošelj SINTEZA PIRAZOLONOV IN NJIHOVA UPORABA V ORGANOKATALIZIRANIH PRETVORBAH Datum zagovora: 10. 9. 2021 Ana CIZERL Mentor: doc. dr. Jakob Kljun SINTEZA HETEROCIKLIČNIH SPOJIN IZ TIOSEMIKARBAZIDOV IN ŠTUDIJA VEZAVE NA CINKOVE MODELNE SPOJINE AKTIVNIH MEST METALOPROTEINOV Datum zagovora: 10. 9. 2021 Barbara DERGANC Mentorica: prof. dr. Ksenija Kogej TEMPERATURNA ODVISNOST SAMO-ASOCIACIJE ATAKTIČNE POLI(METAKRILNE KISLINE) Datum zagovora: 10. 9. 2021 Jakob MUHIČ Mentor: prof. dr. Bogdan Štefane SINTEZA IN PRETVORBE 4-BENZILIDEN-2- FENILOKSAZOLONOV Datum zagovora: 10. 9. 2021 Jan ŠILER HUDOKLIN Mentor: prof. dr. Tomaž Urbič IONSKE TEKOČINE Datum zagovora: 10. 9. 2021 Ivana PODLIPNIK Mentor: doc. dr. San Hadži IZVOR SPREMEMBE TOPLOTNE KAPACITETE PRI ZVITJU DNK: VPLIV SPECIFIČNO VEZANIH MOLEKUL VODE Datum zagovora: 10. 9. 2021 Žiga OGRIN Mentor: izr. prof. dr. Janez Cerar NAPAKE PRI MERJENJIH Z IONOSELEKTIVNIMI ELEKTRODAMI Datum zagovora: 30. 9. 2021 S15Acta Chim. Slov. 2022, 69, (1), Supplement Društvene vesti in druge aktivnosti Lara BREMEC KODELJA Mentor: izr. prof. dr. Miha Lukšič UPORABA KOLOIDNIH SISTEMOV PRI POSTOPKIH ČIŠČENJA PREDMETOV KULTURNE DEDIŠČINE S POUDARKOM NA POLI(VINIL ALKOHOLNIH) HIDROGELIH Datum zagovora: 19. 10. 2021 Veronika BRAČIČ Mentor: prof. dr. Franc Požgan KOROZIJSKA ZAŠČITA ALUMINIJEVE ZLITINE S KONVERZIJSKO IN HIBRIDNO SOL-GEL PREVLEKO Datum zagovora: 3. 11. 2021 Urška BERCKO Mentor: prof. dr. Jurij Reščič RAZISKAVE STRUKTURNIH IN TERMODINAMSKIH LASTNOSTI MEŠANIC NABITIH KOLOIDOV Z NEVTRALNIMI POLIMERI Z RAČUNALNIŠKIMI SIMULACIJAMI MONTE CARLO Datum zagovora: 23. 11. 2021 Eva STERLE Mentor: doc. dr. Krištof Kranjc SUBSTITUIRANI CIKLIČNI DIENOFILI V DIELS- ALDERJEVIH REAKCIJAH POD OKOLJU PRIJAZNIMI POGOJI Datum zagovora: 13. 12. 2021 KEMIJSKO INŽENIRSTVO – 1. STOPNJA Nastja STRAŠEK Mentor: prof. dr. Marjan Marinšek BIOLOŠKO SAMOPOPRAVLJIVI BETONSKI SISTEMI Datum zagovora: 15. 1. 2021 Maja KOŠAR Mentorica: doc. dr. Lidija Slemenik Perše FITOREMEDIACIJA ONESNAŽENIH TAL S TEŽKIMI KOVINAMI: GENSKI INŽENIRING ZA VEČJO UČINKOVITOST PROCESA Datum zagovora: 22. 1. 2021 Tjaša UHELJ GRČA, TJAŠA Mentorica: doc. dr. 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Andreja Žgajnar Gotvajn PREPOVEDANE DROGE V KOMUNALNIH ODPADNIH VODAH Datum zagovora: 8. 6. 2021 Neli KUČIČ Mentorica: prof. dr. Urška Šebenik LAPONITNI HIDROGELI ZA ADSORPCIJO SNOVI Datum zagovora: 5. 7. 2021 Teja BUH Mentorica: doc. dr. Lidija Slemenik Perše VPLIV NATRIJEVEGA LAVRIL ETER SULFATA IN SOLI NA VISKOZNOST DETERGENTA ZA ROČNO POMIVANJE POSODE Datum zagovora: 16. 7. 2021 Katja TURK Mentorica: izr. prof. dr. Gabriela Kalčikova KVANTIFIKACIJA BIOFILMA NA MIKROPLASTIKI Datum zagovora: 27. 8. 2021 Anej BLAŽIČ Mentorica: izr. prof. dr. Gabriela Kalčikova INTERAKCIJE MIKROPLASTIKE Z VODNIMI RASTLINAMI IN MOŽNOST UPORABE FITOREMEDIACIJSKIH TEHNOLOGIJ ZA NJENO ODSTRANJEVANJE IZ POVRŠINSKIH VOD Datum zagovora: 27. 8. 2021 Neža VREČER Mentorica: izr. prof. dr. Gabriela Kalčikova UPORABA RASTLINSKIH ČISTILNIH NAPRAV ZA ODSTRANJEVANJE MIKROPLASTIKE IZ ODPADNIH VOD Datum zagovora: 2. 9. 2021 S16 Acta Chim. 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Marko Hočevar UPORABA INERCIJSKEGA SENZORJA V POVEZAVI S SISTEMOM ZA LASERSKO SKENIRANJE V VINOGRADIH Datum zagovora: 7. 9. 2021 Urban JANJIČ Mentor: prof. dr. Miran Gaberšček GORIVNE CELICE Datum zagovora: 7. 9. 2021 Jaka KOMAR Mentor: izr. prof. dr. Blaž Likozar IZBIRA TOPILA ZA EKSTRAKCIJO 5-HIDROKSIMETILFURFURALA IZ VODNE FAZE Datum zagovora: 7. 9. 2021 Jovana ĐURĐEVIĆ Mentor: prof. dr. Igor Plazl RAZVOJ MATEMATIČNIH MODELOV ZA NAPOVED HITROSTNIH PROFILOV DVOFAZNEGA TOKA V MIKROKANALU Datum zagovora: 7. 9. 2021 Blaž JANČIČ Mentorica: prof. dr. Andreja Žgajnar Gotvajn UČINKOVITOST NAPREDNIH OKSIDACIJSKIH PROCESOV PRI ODSTRANJEVANJU VIRUSOV IZ KOMUNALNE ODPADNE VODE Datum zagovora: 7. 9. 2021 Monika NARTNIK Mentor: izr. prof. dr. Blaž Likozar UTEKOČINJANJE BIOMASE MIKROALG S KATALIZATORJEM Datum zagovora: 7. 9. 2021 Jure BELEJ Mentorica: prof. dr. 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Janez Plavec KARAKTERIZACIJA KRATKIH ZAPOREDIJ DNK, KI POVZROČAJO KOLAPS REPLIKACIJSKIH VILIC Datum zagovora: 27. 8. 2021 Neža ŽERJAV Mentor: doc. dr. Gregor Gunčar FUNKCIONALIZACIJA MONODISPERZNIH DELCEV SILICIJEVEGA DIOKSIDA S KORONAVIRUSNIMA PROTEINOMA ORF8 IN N Datum zagovora: 30. 8. 2021 Maks KUMEK Mentor: doc. dr. Miha Pavšič SISTEM ZA DETEKCIJO HOMOGENO GLIKOZILIRANIH PROTEINOV NA OSNOVI GLIKOZILTRANSFERAZE MGAT-1 Datum zagovora: 31. 8. 2021 Tina LOGONDER Mentorica: prof. dr. Brigita Lenarčič PRIPRAVA KONSTRUKTOV ZA IZRAŽANJE REKOMBINANTNEGA ZUNAJCELIČNEGA DELA ČLOVEŠKEGA PROTEINA EpCAM V SESALSKIH CELIČNIH LINIJAH Datum zagovora: 1. 9. 2021 S18 Acta Chim. Slov. 2022, 69, (1), Supplement Društvene vesti in druge aktivnosti Nina VARDA Mentor: izr. prof. dr. Marko Novinec EVOLUCIJSKA ANALIZA DIPEPTIDIL PEPTIDAZE I Datum zagovora: 2. 9. 2021 Nastja FEGUŠ Mentor: izr. prof. dr. 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Sabina Huč SIMULIRANJE POSLEDIC IZPUSTA NEVARNE SNOVI V PROGRAMU ALOHA Datum zagovora: 30. 9. 2021 DIPLOME – VISOKOŠOLSKI STROKOVNI ŠTUDIJ KEMIJSKA TEHNOLOGIJA – 1. STOPNJA Neža DRNOVŠEK Mentor: izr. prof. dr. Janez Cerkovnik NOVE FORMULACIJE VODIKOVEGA PEROKSIDA Datum zagovora: 15. 1. 2021 Diana OZMEC Mentor: prof. dr. Anton Meden RENTGENSKA PRAŠKOVNA DIFRAKCIJA ZDRAVIL PROTI DEPRESIJI Datum zagovora: 22. 1. 2021 Jaka JENKO Mentorica: doc. dr. Klementina Zupan MATERIALI S FAZNO SPREMEMBO V GRADBENIŠTVU Datum zagovora: 22. 1. 2021 Tajda RODE Mentor: doc. dr. Bojan Kozlevčar BAKROVE IN CINKOVE SPOJINE S 4-AMINOBENZOJSKO KISLINO Datum zagovora: 29. 1. 2021 Sabina TRAKO Mentor: prof. dr. Marjan Marinšek DOLOČITEV KARAKTERISTIČNIH TEMPERATUR (LIKVIDUS IN SOLIDUS) NIKELJ-KROM-ŽELEZOVIH ZLITIN Z UPORABO DTA Datum zagovora: 15. 2. 2021 Žiga PODBEVŠEK Mentor: doc. dr. Bojan Šarac POSTOPKI PRIDOBIVANJA ČISTE VODE Datum zagovora: 8. 3. 2021 Eva KANALEC Mentorica: doc. dr. 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Krištof Kranjc 2H-PIRAN-2-ONI IN NJIHOVI DERIVATI KOT POMEMBNE NARAVNE SPOJINE TER GRADNIKI V ORGANSKI SINTEZI Datum zagovora: 6. 9. 2021 Tjaša ČRNILOGAR Mentor: doc. dr. Jakob Kljun SINTEZA NOVIH TRIAZOLIDINOV IN NJIHOVIH SREBROVIH(I) KOORDINACIJSKIH SPOJIN Datum zagovora: 6. 9. 2021 Nina LESJAK Mentor: viš. pred. dr. Branko Alič VPLIV MOLEKULSKIH MAS NA REOLOŠKE LASTNOSTI POLIMERNIH TALIN IN RAZTOPIN Datum zagovora: 6. 9. 2021 Žiga ŠUBIC Mentor: doc. dr. Krištof Kranjc UPORABA PERICIKLIČNIH REAKCIJ V KLJUČNIH STOPNJAH SINTEZE NARAVNIH SPOJIN Datum zagovora: 6. 9. 2021 Tilen MAGAŠ Mentorica: prof. dr. Irena Kralj Cigić DOLOČANJE ALDEHIDOV V NOTRANJIH PROSTORIH S PASIVNIMI VZORČEVALNIKI Datum zagovora: 7. 9. 2021 Assya BELLAADEM Mentor: prof. dr. Jurij Reščič IZRAČUN PARNEGA TLAKA RAZKUŽIL ZA ROKE Datum zagovora: 7. 9. 2021 Sara STANKO Mentor: prof. dr. Matevž Pompe DOLOČEVANJE NIKOTINAMIDA MONONUKLEOTIDA V PREHRANSKIH DOPOLNILIH Datum zagovora: 7. 9. 2021 Mateja JELIĆ Mentor: prof. dr. Matija Strlič VSEBNOST AGRESIVNEGA CO2 V CELJSKEM VODOVODNEM OMREŽJU Datum zagovora: 8. 9. 2021 Špela KLADNIK Mentor: doc. dr. Martin Gazvoda ORGANOKOVINSKI KOMPLEKSI KOVIN PREHODA Z ACETILENSKIMI LIGANDI Datum zagovora: 8. 9. 2021 Tina JERETINA Mentor: prof. dr. Marjan Jereb PRETVORBA KARBONILNIH SPOJIN Z NEKATERIMI NUKLEOFILI Datum zagovora: 9. 9. 2021 Urša SKUBE Mentor: izr. prof. dr. Mitja Kolar IONSKA KROMATOGRAFIJA KOT METODA ZA DOLOČANJE ANIONOV V LEBDEČIH (PM) DELCIH Datum zagovora: 9. 9. 2021 Aljaž ZADRAŽNIK Mentorica: doc. dr. Nives Kitanovski KARAKTERIZACIJA DVEH KOORDINACIJSKIH SPOJIN MOLIBDENA Datum zagovora: 10. 9. 2021 Nika MALEČKAR Mentor: prof. dr. 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Slov. 2022, 69, (1), Supplement Društvene vesti in druge aktivnosti Rebeka KOGELNIK IZZIVI IN POTENCIALI UPORABE PROCESA OSMOZE PRI OBDELAVI TEKSTILNIH ODPADNIH VODA Datum zagovora: 19. 5. 2021 Rolando KRIVEC KINETIKA ESTERIFIKACIJE MIRISTINSKE KISLINE Z UPORABO KOVINSKEGA KATALIZATORJA Datum zagovora: 19. 5. 2021 Marina MALIĆ BIOLOŠKA AKTIVNOST ANTIOKSIDANTOV V KONOPLJINIH EKSTRAKTIH PRI RAKU PROSTATE Datum zagovora: 19. 5. 2021 Anja SIHER REAKCIJE METILAMINOV Z VODIKOVIMI HALOGENIDI IN NJIHOVA UPORABA V SINTEZI KOORDINACIJSKIH SPOJIN Datum zagovora: 21. 4. 2021 Monika DOKL SIMULACIJA IN OPTIMIRANJE ORGANSKEGA RANKINOVEGA CIKLA ZA IZKORIŠČANJE ODPADNE TOPLOTE PRI PROIZVODNJI ALUMINIJA Datum zagovora: 8. 4. 2021 Jerneja BABIČ ANTIMIKROBNO DELOVANJE GRANATNEGA JABOLKA IN ASIMINE Datum zagovora: 4. 5. 2021 DIPLOME – UNIVERZITETNI ŠTUDIJ UNIVERZITETNI ŠTUDIJ – 1. STOPNJA Andraž OŠTIR SINTEZA KOVINSKIH KATALIZATORJEV Z METODO MOKRE IMPREGNACIJE Datum zagovora: 25. 8. 2021 Nina KOPŠE PRIMERJALNA ŠTUDIJA POGOJEV FERMENTACIJE VODNEGA KEFIRJA Datum zagovora: 17. 11. 2021 Nika KRAJNC DINAMIČNO OPTIMIRANJE PROBLEMOV KEMIJSKEGA INŽENIRSTVA Z UPORABO PROGRAMSKEGA OKOLJA APMONITOR Datum zagovora: 5. 7. 2021 Tina DŠUBAN MED KOT INHIBITOR KOROZIJSKIH PROCESOV Datum zagovora: 9. 9. 2021 Anja KOŠAK BAKER IMOBILIZIRAN NA POROZEN POLIPIRIDIN KOT OBNOVLJIV KATALIZATOR Datum zagovora: 7. 9. 2021 Katja GOLE SINTEZA IN KARAKTERIZACIJA KOVINSKIH KISLINSKIH KATALIZATORJEV Datum zagovora: 25. 8. 2021 Anej PEVEC PREGLED IN OCENA POTENCIALA ENERGETSKE INTEGRACIJE MED RAZLIČNIMI INDUSTRIJSKIMI PODSEKTORJI Datum zagovora: 22. 9. 2021 Blaž ŠKOF DOLOČEVANJE IN IZBOLJŠEVANJE INDEKSA OPERABILNOSTI ZA PROCES HIDRODEALKILACIJE TOLUENA Datum zagovora: 22. 9. 2021 Ana PERPAR TANIN KOT ZAVIRALEC KOROZIJE NA JEKLU Datum zagovora: 13. 7. 2021 Nina KUGL DOLOČEVANJE AKTIVNOSTI ENCIMOV V PIRINI MOKI PO IZPOSTAVITVI V SUPERKRITIČNEMU CO2 Datum zagovora: 2. 9. 2021 Marcel ŽAFRAN RAZVOJ IN VALIDACIJA KOLORIMETRIČNEGA SENZORSKEGA RECEPTORJA ZA ZAZNAVANJE KVARJENJA ŽIVIL Datum zagovora: 9. 9. 2021 Iztok MAJCEN VPLIV RAZMERJA FUNKCIONALNIH SKUPIN PRI TIOL/ EN POLIMERIZACIJI NA TERMOMEHANSKE LASTNOSTI PRODUKTOV Datum zagovora: 2. 9. 2021 Tinkara Marija PODNAR ANALIZA VPLIVA SUŠENJA KEFIRNIH ZRN NA ČAS AKTIVACIJE IN KVALITETO PROBIOTIČNEGA NAPITKA Datum zagovora: 9. 9. 2021 Domen SLEMENŠEK IN SITU FUNKCIONALIZACIJA POROZNIH POLITIOLENOV S FUNKCIONALNIMI ALKENI Datum zagovora: 7. 9. 2021 S27Acta Chim. Slov. 2022, 69, (1), Supplement Društvene vesti in druge aktivnosti Zala SERIANZ DOLOČEVANJE AKTIVNOSTI NEKATERIH ENCIMOV V OLUPKIH MANGA Datum zagovora: 9. 9. 2021 Rok KRAMBERGER TERMOKEMIJSKO SHRANJEVANJE ENERGIJE Datum zagovora: 2. 9. 2021 Miha FERMIŠEK TVORBA KOORDINACIJSKIH SPOJIN MED HIDROLIZIRAJOČIMI TANINI IN FE(II) Datum zagovora: 9. 9. 2021 Rok PUČNIK FUNKCIONALIZIRANI MAGNETNI NANODELCI ZA ODSTRANJEVANJE TEŽKIH KOVIN IZ ODPADNIH VOD Datum zagovora: 2. 9. 2021 Lara PLOHL VSEBNOST VITAMINA D V FARMACEVTSKIH PRIPRAVKIH Datum zagovora: 9. 9. 2021 Nika HOČEVAR MODELIRANJE PROCESA REGULACIJE NIVOJA S POVRATNO – ZANČNO KASKADNO REGULACIJO IN RAZVOJ SIMULACIJSKEGA VMESNIKA Datum zagovora: 9. 9. 2021 Tamara GAVRIĆ UPORABA SPLETNE PLATFORME SIMSCALE ZA SIMULACIJE MEHANIKE TEKOČIN IN PRENOSA TOPLOTE Datum zagovora: 9. 9. 2021 Ana GORIČAN EKSTRAKT ŠIPKA KOT ZELENI INHIBITOR KOROZIJSKIH PROCESOV Datum zagovora: 9. 9. 2021 Teja CESAR VPLIV SHRANJEVANJA STARTER KULTURE KOMBUČE NA HITROST AKTIVACIJE IN KAKOVOST FERMENTIRANEGA NAPITKA Datum zagovora: 2. 9. 2021 Patricija POTISK UPORABA MEŠANIH ŠABLON ZA PRIPRAVO HIERARHIČNO POROZNIH POLIMEROV Datum zagovora: 2. 9. 2021 Anja MEŠL IZOLACIJA KERATINA IZ ODPADNE BIOMASE IN SINTEZA KERATINSKIH NANODELCEV Datum zagovora: 9. 9. 2021 Ema ŠUŠTERŠIČ SEPARACIJA VREDNIH SPOJIN IZ LUPIN GRANATNEGA JABOLKA Datum zagovora: 9. 9. 2021 Zala MESEC IZOLACIJA KEFIRANA IZ KEFIRJA Datum zagovora: 2. 9. 2021 Aleksander Saša MARKOVIČ SINTEZA ? – KONJUGIRANIH POLIMERNIH PEN Z REAKCIJAMI PRIPAJANJA Datum zagovora: 2. 9. 2021 Gal BJELOVUČIĆ RAZVOJ KOLORIMETRIČNEGA SENZORSKEGA RECEPTORJA ZA ZAZNAVANJE BIOGENIH AMINOV Datum zagovora: 9. 9. 2021 Jan GIMPELJ IDENTIFIKACIJA PREVLADUJOČIH FUNKCIONALNIH SKUPIN V PROTIBAKTERIJSKIH UČINKOVINAH Datum zagovora: 9. 9. 2021 Sven GRUBER RAČUNALNIŠKA SIMULACIJA SUBKRITIČNIH IN TRANSKRITIČNIH TOPLOTNIH ČRPALK Datum zagovora: 2. 9. 2021 Tinkara OŠLOVNIK SIMULACIJA IN PRIMERJAVA REGULACIJSKIH MEHANIZMOV ZA REGULACIJO PROCESOV Z MRTVIM ČASOM Datum zagovora: 9. 9. 2021 Klemen ROLA PROIZVODNJA BIOETANOLA IZ LIGNOCELULOZNE BIOMASE Datum zagovora: 2. 9. 2021 Maj VIRANT INKORPORACIJA PROGRAMABILNEGA LOGIČNEGA KRMILNIKA V DINAMIČNI PROCES REGULACIJE PRETOKA Datum zagovora: 9. 9. 2021 Dea SIMONIČ SUBCELIČNA FRAKCIONACIJA HUMANIH MONONUKLEARNIH CELIC PERIFERNE KRVI IN IZOLACIJA PROTEINOV IZ RAZLIČNIH CELIČNIH KOMPARTMENTOV Datum zagovora: 9. 9. 2021 Andreja BEŽAN VPLIV TRANSGLUTAMINAZE NA ORGANOLEPTIČNE IN DRUGE LASTNOSTI JOGURTA Datum zagovora: 2. 9. 2021 David HOMŠAK ŠTUDIJA ADSORPCIJE LIPOFILNIH SNOVI NA BENTONIT Datum zagovora: 22. 9. 2021 S28 Acta Chim. Slov. 2022, 69, (1), Supplement Društvene vesti in druge aktivnosti DIPLOME – VISOKOŠOLSKI STROKOVNI ŠTUDIJ VISOKOŠOLSKI STROKOVNI ŠTUDIJ – 1. STOPNJA Lucija DOLINŠEK STABILNOST LIZOCIMA PRI EKSTREMNIH POGOJIH Datum zagovora: 7. 7. 2021 Maja VERDEV ZAMREŽENJE LIZOCIMA V ENCIMSKE SKUPKE Datum zagovora: 9. 9. 2021 Nika ATELŠEK HOZJAN EKSTRAKCIJA AMIGDALINA IZ JEDRC KOŠČIČASTEGA SADJA IN TESTIRANJE ANTIOKSIDATIVNE AKTIVNOSTI EKSTRAKTOV Datum zagovora: 9. 9. 2021 Klemen GRADIŠNIK SEPARACIJA VREDNIH SPOJIN IZ OREHOVIH LUPIN S POD- KRITIČNO VODO Datum zagovora: 2. 9. 2021 Blaž VIDOVIČ ODSTRANJEVANJE TEŽKIH KOVIN IZ ODPADNE VODE Z UPORABO GLINENEGA ADSORBENTA Datum zagovora: 9. 9. 2021 Borut SOLINA MODELIRANJE OGLJIČNEGA ODTISA METILIRANE MELAMINSKE SMOLE Datum zagovora: 9. 9. 2021 Žan TURK PROIZVODNJA GLIKOLNE KISLINE IZ ODPADNIH PLINOV Datum zagovora: 7. 7. 2021 Eva ROZMAN PROIZVODNJA SEČNINE IZ ODPADNIH PLINOV Datum zagovora: 2. 9. 2021 Urška VTIČ PINOCEMBRIN IZ MEDU KOT POTENCIALNI LOVILEC KEMIJSKIH KARCINOGENOV – RAČUNALNIŠKI PRISTOP Datum zagovora: 9. 9. 2021 Nina BELINA HIDROLIZA TRIACETINA Z UPORABO ENCIMA LIPAZE Datum zagovora: 2. 9. 2021 S29Acta Chim. Slov. 2022, 69, (1), Supplement Društvene vesti in druge aktivnosti UNIVERZA V NOVI GORICI FAKULTETA ZA PODIPLOMSKI ŠTUDIJ 1. januar – 31. december 2021 DIPLOME ŠTUDIJSKI PROGRAM OKOLJE – 1. STOPNJA Milica ČVOROVIĆ DIPLOMSKI SEMINAR Datum zagovora: 13. 7. 2021 S30 Acta Chim. Slov. 2022, 69, (1), Supplement Društvene vesti in druge aktivnosti S31Acta Chim. Slov. 2022, 69, (1), Supplement Društvene vesti in druge aktivnosti V okviru Slovenskega kemijskega društva (http:// www.chem-soc.si) se vzpostavlja Sekcija za okolje, katere osnovno poslanstvo bo spodbujanje sodelovanja, povezo- vanja in izmenjave izkušenj med strokovnjaki z različnih področij kemije, ki se ukvarjajo z okoljskimi tematikami, z namenom ozaveščanja javnosti pri razumevanju aktualnih okoljskih vprašanj in dogodkov. Namen Sekcije za okolje bo tudi spodbujanje pravilne uporabe kemije za ocenjeva- nje in reševanje okoljskih vprašanj ter obravnava po- membnih vidikov okoljske kemije, ki potrebujejo regulaci- jo. Poleg tega bo Sekcija za okolje spodbujala vključitev novih vsebin s področja okoljske kemije v izobraževalni sistem ter sodelovala z mednarodnim okoljskimi organi- zacijami. Mesto predsednika Sekcije za okolje prevzema dr. Marko Štrok (marko.strok@ijs.si), mesto pomočnice in tajnice pa dr. Janja Vidmar (janja.vidmar@ijs.si), oba sode- lavca z Odseka za znanosti o okolju, Institut »Jožef Stefan«. Ob tej priložnosti vabimo vse strokovnjake z različ- nih področij kemije, ki so zainteresirani za okoljsko tema- tiko, da se pridružijo Sekciji za okolje in v njej aktivno so- delujejo. Ob enem bi želeli povabiti študente s področja okolja k udeležbi na letošnjih Slovenskih kemijskih dnevih (https://skd2022.chem-soc.si/), na katerih bo sodelovala tudi novoustanovljena Sekcija za okolje. USTANAVLJANJE SEKCIJE ZA OKOLJE V OKVIRU SLOVENSKEGA KEMIJSKEGA DRUŠTVA S32 Acta Chim. Slov. 2022, 69, (1), Supplement Društvene vesti in druge aktivnosti S33Acta Chim. Slov. 2022, 69, (1), Supplement Društvene vesti in druge aktivnosti 2022 April 2022 4 – 7 NPM-6 (NEW PHOTOCATALYTIC MATERIALS FOR ENVIRONMENT, ENERGY AND SUSTAINABILITY)/PAOT-7 (PHOTOCATALYTIC AND ADVANCED OXIDATION TECHNOLOGIES FOR WATER, AIR, SOIL AND SURFACES Ljubljana, Slovenia Information: https://redoxtech.com/ 26 THE NITROGEN ELEMENT – SUSTAINABLE FOOD PRODUCTION? Online Information: https://www.euchems.eu/events/nitrogen-workshop/ 25 – 30 PETROMASS 2022 - XII INTERNATIONAL MASS SPECTROMETRY CONFERENCE ON PETROCHEMISTRY, ENVIRONMENTAL AND FOOD CHEMISTRY Crete, Greece Information: https://www.petromass2022.com/ May 2022 25 – 27 POLYMERS 2022: NEW TRENDS IN POLYMER SCIENCE: HEALTH OF THE PLANET, HEALTH OF THE PEOPLE Turin, Italy Information: http://polymers2022.sciforum.net June 2022 6 – 10 11TH EUROPEAN CONFERENCE ON SOLAR CHEMISTRY AND PHOTOCATALYSIS: ENVIRONMENTAL APPLICATIONS - SPEA11 Turin, Italy Information: https://www.spea11.unito.it/home 6 – 10 10TH INTERNATIONAL CONFERENCE ON MECHANOCHEMISTRY AND MECHANICAL ALLOYING Cagliari, Italy Information: https://income2022.it/ 9 – 10 58TH SERBIAN CHEMICAL SOCIETY CONFERENCE Belgrade, Serbia Information: https://www.shd.org.rs 12 – 16 XLVI »ATTILIO CORBELLA« INTERNATIONAL SUMMER SCHOOL ON ORGANIC SYNTHESIS Gargnano, Italy Information: https://corbellasummerschool.unimi.it/ KOLEDAR VAŽNEJŠIH ZNANSTVENIH SREČANJ S PODROČJA KEMIJE IN KEMIJSKE TEHNOLOGIJE SCIENTIFIC MEETINGS – CHEMISTRY AND CHEMICAL ENGINEERING S34 Acta Chim. Slov. 2022, 69, (1), Supplement Društvene vesti in druge aktivnosti 13 – 15 SYMPOSIUM ON THE INTERACTIONS BETWEEN SEDIMENTS AND WATER Piran, Slovenia Information: https://www.iasws2022.si/ 13 – 16 4TH TRAINING SCHOOL 'MECHANOCHEMICAL SYNTHESIS AND KINETICS' Cagliari, Italy Information: https://www.mechsustind.eu/ 23 – 24 GREEN AND UNCONVENTIONAL SYNTHESIS, APPROACHES AND FUNCTIONAL ASSESSMENT AIM 2020 (AIM 2020 ADVANCED INORGANIC MATERIALS) Bari, Italy Information: http://www.unconventional-aim2020-bari.it/ž 28 – July 1 26TH INTERNATIONAL SYMPOSIUM ON SEPARATION SCIENCES - ISSS 2022 Ljubljana, Slovenia Information: https://isss2020.si/ 30 – July 2 4TH INTERNATIONAL CONGRESS OF CHEMISTS AND CHEMICAL ENGINEERS OF B&H Sarajevo, Bosnia and Herzegovina Information: icccebih.dktks.ba July 2022 4 – 8 CHEMISTRY FOR CULTURAL HERITAGE - CHEMCH-2020 Ravenna, Italy Information: https://eventi.unibo.it/chemch2022 11 – 13 EUROPEAN CONFERENCE OF RESEARCH IN CHEMISTRY EDUCATION (ECRICE 2020) Rehovot, Israel Information: https://www.weizmann.ac.il/conferences/ECRICE2020/ 13 – 14 TOTAL FOOD 2022: MAXIMISING VALUE FROM THE FOOD CHAIN Nottingham, UK Information: https://www.nottingham.ac.uk/conference/fac-sci/biosciences/total-food/index.aspx 18 – 22 SECOND INTERNATIONAL CONFERENCE ON NONCOVALENT INTERACTIONS 2021- 2022 - ICNI2021 Strasbourg, France Information: http://icni2021.unistra.fr/ August 2022 28 - Sept 1 8TH EUCHEMS CHEMISTRY CONGRESS (ECC8) Lisbon, Portugal Information: https://euchems2022.eu/ September 2022 5 – 9 9TH IUPAC INTERNATIONAL CONFERENCE ON GREEN CHEMISTRY - ICGC-9 Athens, Greece Information: greeniupac2020.org 21 – 23 19. RUŽIČKA DAYS Vukovar, Croatia Information: http://www.ruzickadays.eu 21 – 23 SCS ANNUAL MEETING 2022 Portorož-Portorose, Slovenia Information: https://skd2022.chem-soc.si/ 27 – 30 11TH CENTRAL EUROPEAN CONGRESS ON FOOD AND NUTRITION Čatež ob Savi, Slovenia Information: https://cefood2022.si/ S35Acta Chim. Slov. 2022, 69, (1), Supplement Društvene vesti in druge aktivnosti October 2022 5 – 7 7 MS FOOD DAY Florence, Italy Information: https://www.spettrometriadimassa.it/Congressi/7MS-FoodDay/index.html 23 – 26 31ST INTERNATIONAL SYMPOSIUM ON THE CHEMISTRY OF NATURAL PRODUCTS AND 11TH INTERNATIONAL CONGRESS ON BIODIVERSITY (ISCNP31 & ICOB11) Naples, Italy Information: https://www.iscnp31-icob11.org/index.php November 2022 8 – 11 SOLUTIONS IN CHEMISTRY 2022 Sveti Martin na Muri, Croatia Information: https://solutionsinchemistry.hkd.hr/ S36 Acta Chim. 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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. Whi te, Ac ta Chim. Slov. 2008, 55, 1055–1059. 2. M. F. Kem me re, T. F. Keu rent jes, in: S. P. Nu nes, K. V. Pei ne mann (Ed.): Mem bra ne Tech no logy in the Che mi cal In du stry, Wi ley­VCH, Wein heim, Ger­ many, 2008, pp. 229–255. 3. J. Le vec, Ar ran ge ment and pro cess for oxi di zing an aqu e ous me dium, US Pa tent Num ber 5,928,521, da te of pa tent July 27, 1999. 4. L. A. Bur sill, J. M. Tho mas, in: R. Ser sa le, C. Col le la, R. Aiel lo (Eds.), Re cent Pro gress Re port and Dis cus­ sions: 5th In ter na tional Zeo li te Con fe ren ce, Na ples, Italy, 1980, Gia ni ni, Na ples, 1981, pp. 25–30. 5. J. Sze gez di, F. Csiz ma dia, Pre dic tion of dis so cia tion con stant using mi cro con stants, http://www. che­ ma xon.com/conf/Pre dic tion_of_dis so cia tion _con­ stant_using_mi cro co nstants.pdf, (as ses sed: March 31, 2008) Titles of journals should be abbreviated according to Chemical Abstracts Service Source Index (CASSI). Spe cial No tes • Com ple te cha rac te ri za tion, inc lu ding cry stal struc tu re, should be gi ven when the synthe sis of new com pounds in cry stal form is re por ted. • Nu me ri cal da ta should be re por ted with the num ber of sig ni fi cant di gits cor res pon ding to the mag ni tu de of ex pe ri men tal un cer tainty. • The SI system of units and IUPAC re com men­ da tions for nomenclature, symbols and abbrevia- tions should be followed closely. Additionally, the authors should follow the general guidelines when citing spectral and analytical data, and depositing crystallographic data. • Cha rac ters 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 ru les and re com men da tions of the IUBMB and the In ter na tio nal Union of Pure and Ap plied Che mi stry (IUPAC) should be used for abbreviation of chemical names, nomenclature of chemical com- pounds, enzyme nomenclature, isotopic compounds, optically active isomers, and spectroscopic data. • A conf ict of in te rest occurs when an individual (author, reviewer, editor) or its organization is in- S38 Acta Chim. Slov. 2022, 69, (1), Supplement Društvene vesti in druge aktivnosti volved in multiple interests, one of which could pos- sibly corrupt the motivation for an act in the other. Financial relationships are the most easily identifi- able conflicts of interest, while conflicts can occur also as personal relationships, academic competi- tion, etc. The Edi tors 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 aut hors 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 fund- ing support should be mentioned. The statement of disclosure must be provided as Comments to Editor during the submission process. • Pub lis hed sta te ment of In for med Con sent. Research described in papers submitted to ACSi must adhere to the principles of the Declaration of Helsinki (http://www.wma.net/e/po licy/ b3.htm). 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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. • Con tri bu tions aut ho red by Slo ve nian scien tists are evaluated by non-Slovenian referees. • Pa pers des cri bing mi cro wa ve­as si sted reac­ tions performed in domestic microwave ovens are not considered for publication in Acta Chimica Slovenica. • Ma nus cripts that are not pre pa red and sub mit­ ted in ac cord with the in struc tions for aut hors are not con si de red for pub li ca tion. Ap pen di ces Authors are encouraged to make use of supporting in- formation for publication, which is supplementary ma- terial (appendices) that is submitted at the same time as the manuscript. 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All the listed authors have agreed on the content and the corresponding (submitting) author is re- sponsible 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 sub­ mitted in PDF (for reviewers) as well as in orig- inal MS Word format (as a supplementary file for technical editing); diagrams and graphs are cre- ated 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 ma nus cript has been exa mi ned for spel ling and gram mar (spell chec ked). 5. The tit le (ma xi mum 150 cha rac ters) briefly ex­ plains the con tents of the ma nus cript. 6. Full names (first and last) of all authors together with the afliation address are provided. 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Permission has been obtained for use of copy- righted material from other sources (including the Web). 19. The names, full afliation (department, institution, city and country), e­mail addresses and referenc- es 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 re- viewer must be provided. Authors declare no con- flict 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 manu- script is proposed for graphical abstract. 21. Ap pen di ces (if appropriate) as supplementary material are prepared and will be submitted at the same time as the manuscript. Pri vacy Sta te ment The na mes and email ad dres ses en te red in this journal si te will be used exc lu si vely for the sta ted pur po ses of this jour nal and will not be ma de avai lab le for any ot­ her pur po se or to any ot her party. ISSN: 1580­3155 S40 Acta Chim. Slov. 2022, 69, (1), Supplement Društvene vesti in druge aktivnosti 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 European Federation of Chemical Engineering https://efce.info/ International Union of Pure and Applied Chemistry https://iupac.org/ Brussels News Updates http://www.euchems.eu/newsletters/ Novice europske zveze kemijskih društev EuChemS najdete na: Koristni naslovi Komore za testiranje baterij Vakuumski sušilniki Klimatske komore Donau Lab d.o.o., Ljubljana Tbilisijska 85 SI-1000 Ljubljana www.donaulab.si office-si@donaulab.com BINDER Acta Chimica oglas.indd 1 21. 11. 2021 20:29:54 PRED UPORABO NATANČNO PREBERITE NAVODILO! O TVEGANJU IN NEŽELENIH UČINKIH SE POSVETUJTE Z ZDRAVNIKOM ALI S FARMACEVTOM. www.vitamind3krka.si ZAGOTOVITE SI SONCE. PREPROSTO. VITAMIN D3 Krka ZAGOTOVI priporočeni dnevni odmerek vitamina D. EDINI kot zdravilo brez recepta. BREZ konzervansov, barvil in glutena. V IT A M IN D 3 K rk a vs eb uj e ho le ka lc if er ol . 214803-2021 VITAMIN D3 SIMPLE Ad 205x276 SI.indd 1 7. 12. 2021 13:48:49 Ju ne 1 2 – 15 , 20 22 Pi ra n, S lo ve ni a ht tp s: // w w w .i as w s2 02 2. si Jo že f St ef an In st it ut e N at io na l I ns ti tu te o f Bi ol og y 4 n Year 2022, Vol. 69, No. 1 ActaChimicaSlovenica ActaChimicaSlovenica ActaChimicaSlovenica ActaChimicaSlovenica SlovenicaActaChim A cta C him ica Slovenica 69/2022 Pages 1–250 Pages 1–250 n Year 2022, Vol. 69, No. 1 http://acta.chem-soc.si 1 69/2022 1 ISSN 1580-3155 Glucose-sensitive biosensors are known as glucose-oxidase, protein, and phenyl boronic acid based systems. Sugar-sensitive polymeric particles are produced via crosslinker and monomer in one step and surfactant-free emulsion polymerization technique. Sensitivity of the polymer particles to sugar molecules is monitored in glucose/fructose rich media