4 n Year 2022, Vol. 69, No. 4 ActaChimicaSlovenica ActaChimicaSlovenica ActaChimicaSlovenica ActaChimicaSlovenica SlovenicaActaChim A cta C him ica Slovenica 69/2022 Pages 733–947 Pages 733–947 n Year 2022, Vol. 69, No. 4 http://acta.chem-soc.si 4 69/2022 4 ISSN 1580-3155 The significance of nitrogen- and oxygen-heterocycles in many areas of life is well-known, however, the preparation of new derivatives remains important. The multicomponent synthesis of potentially biologically active heterocycles containing a phosphonate or a phosphine oxide moiety was performed (page 735). 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 Ručigaj, 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 Eva Mihalinec, Slovenian Chemical society, 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. 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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. 4 4 n Year 2022, Vol. 69, No. 4 ActaChimicaSlovenica ActaChimicaSlovenica ActaChimicaSlovenica ActaChimicaSlovenica SlovenicaActaChim A cta C him ica Slovenica 69/2022 Pages 733–947 Pages 733–947 n Year 2022, Vol. 69, No. 4 http://acta.chem-soc.si 4 69/2022 4 ISSN 1580-3155 The significance of nitrogen- and oxygen-heterocycles in many areas of life is well-known, however, the preparation of new derivatives remains important. The multicomponent synthesis of potentially biologically active heterocycles containing a phosphonate or a phosphine oxide moiety was performed (page 735). 735–755 Feature Article Multicomponent Synthesis of Potentially Biologically Active Heterocycles Containing a Phosphonate or a Phosphine oxide Moiety Nóra Popovics-Tóth and Erika Bálint 756–771 Feature Article Adaptation of the Crystal Structure to the Confined Size of Mixed-oxide nanoparticles Darko Makovec ScientiFic pAper FeAture Article 772–778 Organic chemistry Synthesis, Characterization, Anti-Glycation, and Anti- oxidant Activities of Sulfanilamide Schiff Base Metal Chelates Muhammad Yaqoob, Waqas Jamil, Muhammad Taha and Sorath Solangi Graphical Contents 796–802 Biomedical applications Evaluation of the Stability of Hydrocortisone Sodium Succinate in Solutions for Parenteral Use by a Validated HPLC-UV Method Katja Mihovec, Žane Temova Rakuša, Enikő Éva Gaál and Robert Roškar 787–795 inorganic chemistry Tetranuclear Copper(II) Complexes Derived from 5-Bromo-2-((2-(2-hydroxyethylamino)ethylimino) methyl)phenol: Synthesis, Characterization, Crystal Structures and Catalytic oxidation of olefins Xiao-Jun Zhao, Su-Zhen Bai and Ling-Wei Xue 779–786 Organic chemistry Synthesis, Crystal Structure and Biological Activity of Two Triketone-Containing Quinoxalines as HPPD Inhibitors Xinyu Leng, Chengguo Liu and Fei Ye 803–810 Applied chemistry Use of Total organic Carbon Analyzer in Isotherm Measurements of Co-Adsorption of VoCs and Water Vapor from the Air Dragana Kešelj, Dragica Lazić and Zoran Petrović 811–825 chemical, biochemical and environmental engineering Enhanced Adsorption of Methylene Blue by Chemically Modified Materials Derived from Phragmites australis Stems Bui Thi Minh Nguyet, Nguyen Huu Nghi, Nguyen Anh Tien, Dinh Quang Khieu, Ha Danh Duc and Nguyen Van Hung Graphical Contents 848–862 chemical, biochemical and environmental engineering Hybrid Polymer Composite of Prussian Red Doped Polythiophene forAdsorptive Wastewater Treatment Application Mohd Mustafa, Shabnum Bashir, Syed Kazim Moosvi, Mohd. Hanief Najar, Mubashir Hussain Masoodi and Masood Ahmad Rizvi1 837–847 Organic chemistry Ionic Liquid Supported on Magnetic Graphene oxide as a Highly Efficient and Stable Catalyst for the Synthesis of Triazolopyrimidines Azar Jahanbakhshi, Mahnaz Farahi and Yeganeh Aghajani 826–836 chemical, biochemical and environmental engineering Assessment of the Capability of Magnetic nanoparticles to Recover neodymium Ions from Aqueous Solution Ana Ambrož, Irena Ban and Thomas Luxbacher 863–875 Biochemistry and molecular biology Synthesis and Biological Evaluation of Some Hydrazide-Hydrazone Derivatives as Anticancer Agents Kadriye Akdağ, Fatih Tok, Sevgi Karakuş, Ömer Erdoğan, Özge Çevik and Bedia Kaymakçıoğlu 876–883 Organic chemistry Preparation and Characterization of niCoFe2o4 nanoparticles as an Effective Catalyst for the Synthesis of Trisubstituted Imidazole Derivatives Under Solvent- free Conditions Leila Hemmesi and Hossein Naeimi Graphical Contents 905–912 inorganic chemistry Zinc(II) Complex Containing oxazole Ring: Synthesis, Crystal Structure, Characterization, DFT Calculations, and Hirshfeld Surface Analysis Karwan Omer Ali, Hikmat Ali Mohamad, Thomas Gerber and Eric Hosten 896–904 inorganic chemistry The Paramagnetic or Spin Crossover Iron(III) Complexes Based-on Pentadentate Schiff Base Ligand: Crystal Structure, and Magnetic Property Investigation Zhijie Xu, Shuo Meng, Tong Cao, Yu Xin, Mingjian Zhang, Xiaoyi Duan, Zhen Zhou and Daopeng Zhang 884–895 chemical, biochemical and environmental engineering Cost-Effective Control of Molecular Weight in Ultrasound-Assisted Emulsion Polymerization of Styrene Ibrahim Korkut, Fuat Erden and Salih Ozbay 913–919 inorganic chemistry Synthesis, Crystal Structures and Antibacterial Activities of N,N’-Ethylene-bis(3-bromosalicylaldimine) and Its Copper(II) and Cobalt(III) Complexes Xue-Song Lin, Yong-Gang Huang, Rui-Fa Jin and Ya-Li Sang 920–927 Biochemistry and molecular biology Synthesis and In Vitro Cytotoxicity of novel Halogenated Dihydropyrano[3,2-b]Chromene Derivatives Salehe Sabouri, Ehsan Faghih-Mirzaei and Mehdi Abaszadeh Graphical Contents 944–947 AUTHoR InDEX 837–943 Organic chemistry In vitro Assessment of Antiprotozoal and Antimicrobial Activities of Fractions and Isolated Compounds from Pallenis hierochuntica Vincent O. Imieje, Abiodun Falodun and Ahmed A. Zaki 928–936 Organic chemistry Syntheses, Crystal Structures and Xanthine oxidase Inhibitory Activity of Aroylhydrazones Yong-Jun Han, Xue-Yao Guo and Ling-Wei Xue S95–S97 Trideset let delovanja Šole eksperimentalne kemije na Institutu »Jožef Stefan«: trideset let motiviranja mladih generacij in utrjevanja poti naravoslovnega izobraževanja Melita Tramšek, Evelin Gruden in Marko Jeran S98–S106 Slavnostna akademija ob 70-letnici Slovenskega kemijskega društva DruŠtVene VeSti 735Acta Chim. Slov. 2022, 69, 735–755 Popovics-Tóth and Bálint: Multicomponent Synthesis of Potentially Biologically ... DOI: 10.17344/acsi.2022.7648 Feature article Multicomponent Synthesis of Potentially Biologically Active Heterocycles Containing a Phosphonate or a Phosphine Oxide Moiety Nóra Popovics-Tóth and Erika Bálint* Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, Műegyetem rkp. 3., H-1111 Budapest, Hungary * Corresponding author: E-mail: balint.erika@vbk.bme.hu Tel.: +36-1-463-1111/5886 Received: 07-05-2022 Abstract Several multicomponent synthetic approaches were elaborated for plenty of novel nitrogen or oxygen heterocycles con- taining a phosphonate or a phosphine oxide moiety. All multicomponent reactions were optimized through a model reaction in respect of the heating mode, molar ratio of the starting materials, atmosphere, catalyst, temperature, reac- tion time and solvent applied, and then, the extended preparation of small libraries of structurally-related compounds was performed. Most of the reactions could be considered as “green syntheses”, as they were carried out in the absence of any catalyst and/or solvent using microwave (MW) irradiation or even at ambient temperature. The scaling-up of a MW-assisted synthesis was also elaborated in a continuous flow MW system. Altogether more than 150 heterocyclic or- ganophosphorus compounds were synthesized, among them several derivatives showed moderate or promising activity against the HL-60 cell line and Bacillus subtilis bacteria. Keywords: Multicomponent reactions, Heterocycles, Organophosphorus compounds, Microwave chemistry, Biological activity 1. Introduction In modern synthetic chemistry, the application of efficient and simple reaction routes for the preparation of organic compounds has become more and more impor- tant. Therefore, multicomponent reactions (MCRs) attract growing interest. In MCRs, the components react with each other in a “one-pot” manner without isolation of any intermediates, which may save time and energy.1,2 They can be considered as ideal synthetic methods due to their features, such as the quick and simple procedure, as well as energy saving and high atom efficiency.3–5 In general, complex structures can be easily formed from inexpensive and simple starting materials by these transformations.6–9 In addition, these properties make them suitable to cre- ate large libraries of structurally-related compounds.10–12 MCRs especially show their importance in the synthesis of heterocycles. Heterocycles are present in human and animal or- ganisms, as well as in plants as components of nucleic ac- ids, sugars, enzymes, hormones, vitamins, pigments and hemoglobin.13–17 In addition, their importance is further enhanced by many synthetic members, such as drugs, pes- ticides, fine chemicals and cosmetics.18–22 In the last few decades, the multicomponent synthesis of heterocycles containing phosphonate moieties has become more and more important, due to their promising biological prop- erties.23,24 The aim of our research work was to synthesize po- tentially biologically active nitrogen heterocycles bear- ing a phosphonate or a phosphine oxide moiety, such as oxoisoindolinyl)phosphonates and -phosphine oxides 1, (1,2-dihydroisoquinolinyl)phosphonates and -phos- phine oxides 2, (dihydropyrimidinone)phosphonates 3, (1,2,3-triazol-5-yl)phosphonates 4, as well as ((1,2,3-tri- azol-4-yl)methyl)phosphinates and -phosphates 5 (Figure 1). In addition, oxygen heterocycles containing a phos- phonate or a phosphine oxide moiety, such as (amino- chromenyl)phosphonates and -phosphine oxides 6, as well as 1-alkyl- and 1-alkoxy-1H-phoshindole-1-oxides 7 were also aimed to be investigated. 736 Acta Chim. Slov. 2022, 69, 735–755 Popovics-Tóth and Bálint: Multicomponent Synthesis of Potentially Biologically ... Figure 1. Target molecules Several derivatives containing the above-mentioned target backbones have biological effects in a wide variety of indications (Figure 2). Some oxoisoindoline carboxylic acid 8 or carboxylic amide derivatives 9 can be found in the literature, which are anticancer or analgesic agents.25,26 Dihydroisoquinolines are effective in a variety of indica- tions, such as antidepressants, sedatives, antitumor (e.g., Crispine A (10)) or as antibacterial drugs.27–30 Several 3,4-dihydropyrimidin-2(1H)-one carboxylates are applied as antitumor (e.g., Monastrol (11) or Piperastrol (12)), an- tihypertensive, anti-inflammatory, antibacterial, antiviral or antifungal agents.31–33 The 1,2,3-triazole derivatives 13 may have antibacterial, antiviral, antifungal, anticancer or anti-inflammatory effect.34–36 From among O-heterocy- cles, 4H-chromenes have various utilizations, especially in pharmaceutical industry, such as the antiallergic and anti- asthmatic sodium chromoglycate (14), or in the cosmetic and dye industry, as well as in the agriculture.37–39 Figure 2. Biologically active N- and O-heterocyclic derivatives The biological activity of the phosphorylated het- erocyclic compounds is less investigated; however, a few important examples can also be found (Figure 3). For ex- ample, 3,4-dihydropyrimidin-2(1H)-one phosphonates 15 have anti-inflammatory effect.40 The 1,2,3-triazolyl phosphonate derivative 16 showed anti-HIV effect.41 Some 2-amino-4H-chromenylphosphonate derivatives 17 have antioxidant and anticancer activity,42,43 and a few (chromonylaminomethyl)phosphonates 18 also showed antitumor effect.44 While benzo[b]phospholoxide 19 is used in the optoelectronic industry, e.g. in OLEDs.45 Figure 3. Biologically active phosphorylated heterocyclic com- pounds Our main aim was to develop effective and simple methods for the preparation of phosphorylated N- and O-heterocyclic derivatives via multicomponent reactions, as far as possible, in the absence of any solvent and/or cat- alyst. We aimed at providing comprehensive study on the reactions, and the formation of diverse molecular libraries. We also investigated the in vitro cytotoxicity and antibac- terial activity of the compounds synthesized. Furthermore, a phosphine oxide derivative was aimed to be utilized as a precursor of a phosphine ligand in the synthesis of a tran- sition metal complex. 2. Multicomponent Synthesis of N-Heterocycles 2. 1. Synthesis of (Oxoisoindolin-1-yl) phosphonates A solvent- and catalyst-free MW-assisted meth- od was developed for the synthesis of (oxoisoindolinyl) phosphonates by the Kabachnik–Fields reaction followed by intramolecular cyclization of 2-formylbenzoic acid, aliphatic primary amines and dialkyl phosphites. In the literature, only a few examples can be found for the con- densation of 2-formylbenzoic acid, aromatic amines, ami- no alcohols or phenylethylamine derivatives and dialkyl phosphites. The reactions were carried out under thermal heating or under MW conditions usually for long reac- tion times (1–5 h) and in a solvent (methanol, ethyl ac- etate).46–49 In a few cases, the transformations were per- formed in the presence of a catalyst or an additive, such as NaH,50 T3P®51 or OSU-6.52 737Acta Chim. Slov. 2022, 69, 735–755 Popovics-Tóth and Bálint: Multicomponent Synthesis of Potentially Biologically ... In the first step, the reaction of 2-formylbenzo- ic acid, butyl-, cyclohexyl- or benzylamine and diethyl phosphite was studied and optimized in respect of the heating mode, the molar ratio of starting materials, the temperature and the reaction time.53 After the optimiza- tion, the model reaction was extended for the preparation of further (oxoisoindolinyl)phosphonate derivatives 20– 22 (Scheme 1). Carrying out the catalyst- and solvent-free MW-assisted condensation of 2-formylbenzoic acid, bu- tylamine and dimethyl-, diethyl-, diisopropyl-, dibutyl- or dibenzyl phosphite at 60 °C for 10 min, the correspond- ing dialkyl (2-butyl-3-oxo-2,3-dihydro-2H-isoindol-1-yl) phosphonates 20a–e were synthesized in high yields (81– 94%). Starting from cyclohexylamine and various dialkyl phosphites (dimethyl-, diethyl-, diisopropyl-, dibutyl- or dibenzyl phosphite), under the optimized conditions (60 °C, 30 min) five new (oxoisoindolinyl)phosphonates 21a–e were formed in yields of 70–84%. After that, the re- action was also performed applying benzylamine as the amine component, and five (oxoisoindolin-1-yl)phospho- nates 22a–e were synthesized with high yields (80–90%) at 60 °C for 20 min. Finally, the three-component reaction of 2-formyl- benzoic acid, butylamine and ethyl phenyl-H-phosphinate as the P-reagent was also performed at 60 °C, for 10 min. The desired (oxoisoindolin-1-yl)phosphinate 20f was ob- tained in a yield of 78%, as a mixture of diastereomers in a ratio of almost 1:1. The mechanism of the condensation was also inves- tigated by in situ Fourier transform infrared (FT-IR) spec- troscopy by the model reaction of 2-formylbenzoic acid (FBA), butylamine (BA) and diethyl phosphite (DEP) in ethanol. At first, the signal of the solvent (ethanol) was re- corded, then the starting materials were added in ten-min- ute intervals. In the next step, the mixture was heated to 60 °C with an oil bath, and the IR spectrum of the mix- ture was measured continuously. In the time-dependent IR spectrum, the characteristic absorptions of the reaction components (FBA, DEP, BA and 20b) can be seen (Figure 4). The lactone form of FBA had a strong absorption band at νC=O = 1756 cm-1. In the case of DEP, signals at 964 cm-1 (νP–O–C) and 1254 cm–1 (νP=O) could be seen. BA was identified by the δC-H (1381 cm–1) and δN-H (1605 cm–1) absorptions. Diethyl (2-butyl-3-oxo-2,3-dihydro-2H- Scheme 1. The reaction of 2-formylbenzoic acid, alkyl amines and dialkyl phosphites or ethyl phenyl-H-phosphinate Figure 4. The time-dependent IR spectrum of the reaction of 2-formylbenzoic acid (FBA), butylamine (BA) and diethyl phosphite (DEP) in ethanol 738 Acta Chim. Slov. 2022, 69, 735–755 Popovics-Tóth and Bálint: Multicomponent Synthesis of Potentially Biologically ... isoindol-1-yl)phosphonate (20b) had a νC=O characteristic absorption at 1690 cm–1. During the measurement, the signals of the starting materials decreased, while the signal of product 20b in- creased, as it was expected. The signal of FBA decreased after the addition of BA, however, no signal of an interme- diate, for instance an imine, appeared. The reason for the decrease of the signal of FBA is the change of IR properties of FBA in the reaction mixture. Furthermore, to increase the productivity, the syn- thesis of some (oxoisoindolinyl)phosphonates (20b, 21b, 22b) was elaborated in a continuous flow MW system. The equipment contained a dual HPLC pump and CEM® MW reactor with a commercially available CEM® continuous flow cell. The FBA in ethanol (pump A) and the mixture of amines and DEP in ethanol (pump B) were fed separately. The temperature was monitored and controlled by the IR sensor of the MW reactor. The leaving mixture was cooled down to 25 °C and was passed through a back-pressure regulator operating at 250 psi (17 bar). At first, the continuous flow reaction of FBA, BA and DEP was carried out, and it was complete with 1.5 equiv- alents of both amine and dialkyl phospite, at 60 °C under a residence time of 30 min (at a flow rate of 0.25 mL/min) (Table 1, Entry 1). Starting from cyclohexylamine, a longer residence time of 45 min (at a flow rate of 0.15 mL/min) was needed to obtain a complete conversion (Table 1, En- try 2). While in the case of benzylamine, a residence time of 40 min (a flow rate of 0.18 mL/min) was applied, and the ratio of the (oxoisoindolinyl)phosphonate derivative 22b was 100% (Table 1, Entry 3). The productivity of the flow method was 2.3 g/h, 1.4 g/h and 1.8 g/h in the case of compounds 20b, 21b, 22b, which were 1.5–2 times higher as compared to the batch method (1.8 g/h, 0.6 g/h and 1.0 g/h, respectively). The productivity of the batch process was calculated for one h, based on the net reaction time of several consecutive reactions. In all, 16 (oxoisoindolin-1-yl)phosphonate deriva- tives 20–22 were synthesized, among them, 14 were new compounds. By the catalyst- and solvent-free MW-assisted method, good results were obtained at a lower temperature for shorter reaction times compared with the literature procedures. The mechanism of the condensation was stu- died by in situ FT-IR spectroscopy, and experiments were successfully performed in a continuous flow MW system to increase the productivity. 2. 2. Synthesis of (Oxoisoindolin-1-yl) phosphine Oxides Our aim was to carry out the special Kabachnik– Fields reaction of FBA, primary amines and secondary phosphine oxides, which is a new method for the synthesis of (oxoisoindolinyl)phosphine oxides. In the literature ex- amples, the desired compounds were formed by multistep reactions, applying special reagents and conditions and in most cases, low yields were obtained.54–61 In our research work, the three-component conden- sation of FBA, butyl-, cyclohexyl-, benzylamine or aniline and diphenylphosphine oxide was studied.62 An efficient method was elaborated by us, where complete conversion was obtained in the absence of any catalyst, at room tem- perature, after short reaction times (10–20 min) in ace- tonitrile. The condensation was extended to various sec- ondary phosphine oxides, such as bis(p-tolyl)-, bis(3,5-di- methylphenyl)-, bis(2-naphthyl)- or dibenzylphosphine oxides (Scheme 2). In the case of dibenzylphosphine ox- ide, a longer reaction time of 25 min was necessary to ob- tain full conversion. Altogether, 18 new (oxoisoindolinyl) Table 1. Condensation of FBA, primary amines and diethyl phosphite (DEP) under continuous flow MW conditions Entry R Flow rate τ Conversiona Yieldb Productivity [g/h] [mL/min] [min] [%] [%] Batch method Flow method 1 nBu 0.25 30 100 95 (20b) 1.8 2.3 2 cHex 0.15 45 100 86 (21b) 0.6 1.4 3 Bn 0.18 40 100 91 (22b) 1.0 1.8 aBased on GC. bIsolated yield. 739Acta Chim. Slov. 2022, 69, 735–755 Popovics-Tóth and Bálint: Multicomponent Synthesis of Potentially Biologically ... phosphine oxides 23–26 were isolated in excellent yields (94–99%). After that, the Kabachnik–Fields reaction of FBA, butylamine and various P-stereogenic phosphine ox- ides (tert-butyl(phenyl)phosphine oxide, 2-methylphe- nyl(phenyl)phosphine oxide, 2-methoxyphenyl(phenyl) phosphine oxide, 2-, 3- or 4-trifluoromethylphenyl(phe- nyl)phosphine oxide, biphenyl(phenyl)phosphine oxide or 1-naphthyl(phenyl)phosphine oxide) was carried out (Scheme 3). Applying the optimized conditions (no cata- lyst, at 25 °C, for 10–20 min in acetonitrile), eight (3-oxoi- soindolin-1-yl)phosphine oxides 27–34 were synthesized in high yields (94–98%). Due to the P-stereogenic centre on the phosphorus atom, the products 27–34 were formed as a mixture of two diastereomers. The diastereomeric ratio (dr) was close to 50:50 for most of the compounds 27–34 synthesized. 2-Trifluormethylphenyl(phenyl)phos- phine oxide as the P-reagent was an exception, in that case, the diastereomeric ratio was 35:65. It should be noted that the diastereomers of 1-naphthyl(phenyl) (2-butyl-3-oxo- 2,3-dihydro-2H-isoindol-1-yl)phosphine oxide (34) could be separated by column chromatography because of the bigger difference of the size of the functional groups on the phosphorus atom (phenyl and naphtyl groups). One of the (oxoisoindolinyl)phosphine oxide (23a) was reduced to an (oxoisoindolinyl)phosphine 35, which was utilized as a phosphine ligand in the synthesis of a plat- inum(II) complex 36 (Scheme 4). In the first step, the de- oxygenation of diphenyl (2-butyl-3-oxo-2,3-dihydro-2H- isoindol-1-yl)phosphine oxide (23a) was performed with phenyl silane (PhSiH3) as the reducing agent. The reaction was carried out under inert atmosphere, applying MW ir- radiation at 140 °C for 6 h. The phosphine derivative 35 was not isolated, but it was further reacted with 0.5 equiv. of dichlorodibenzonitrile platinum(II) (Pt(PhCN)2Cl2) precursor at 25 °C in dichloromethane. The monodentate platinum(II) complex 36 was isolated by column chroma- tography in a yield of 80%. The complex 36 was formed in a relative configu- ration of trans, based on platinum-phosphorus coupling constant (1JPt-P) in the 31P NMR spectra. It is known in the literature that the 1JPt-P between 3400 to 3600 Hz sug- gests cis complexes, while the 1JPt-P coupling constant is 2500–3000 Hz in the case of trans arrangements.63 In our case, the 1JPt-P coupling constant was 2519 Hz. The relative orientation of the trans-36 platinum(II) complex was also confirmed by X-ray diffraction measurements. In addition, it was observed in the 31P NMR spec- trum that the central signal consisted of two very close peaks in a ratio of nearly 1:1. This can be explained by the chirality centre on the oxoisoindoline ring, which caused the formation of the complex trans-36 as a mixture of homo- and heterochiral diastereomers. To conclude, an efficient, simple, one-pot method was developed for the synthesis of (oxoisoindolinyl)phos- phine oxides by the Kabachnik–Fields reaction followed Scheme 2. The reaction of FBA, primary amines and secondary phosphine oxides Scheme 3. The reaction of 2-formylbenzoic acid, butylamine and P-stereogenic secondary phosphine oxides 740 Acta Chim. Slov. 2022, 69, 735–755 Popovics-Tóth and Bálint: Multicomponent Synthesis of Potentially Biologically ... by intramolecular cyclization of FBA, primary amines and secondary phosphine oxides. The condensation was extended to P-stereogenic secondary phosphine oxides as well. In all, 26 (3-oxoisoindolin-1-yl)phosphine oxide de- rivatives 23–34 could be synthesized in excellent yields at room temperature after short reaction times (10–60 min). After deoxygenation, diphenyl (2-butyl-3-oxo-2,3-dihy- dro-2H-isoindol-1-yl)phosphine oxide (23a) was utilized in the synthesis of a platinum(II) complex trans-36. 2.3. Synthesis of (Dihydroisoquinolin-1- yl)phosphonates and α-Amino-(2- alkynylphenyl)-methylphosphonates In the literature, the Kabachnik–Fields reaction of 2-alkynylbenzaldehydes, primary amines and dialkyl phosphites was carried out in the presence of various ca- talysts. α-Amino-(2-alkynylphenyl)-methylphosphonates were prepared at room temperature or at 60 °C after 4 h in 1,2-dichloroethane, using magnesium perchlorate (Mg(OCl4)2) or Lewis acids (FeCl3, In(OTf)3, Bi(OTf)3, Yb(OTf)3) as the catalysts.64,65 However, (1,2-dihydroi- soquinolin-1-yl)phosphonates were obtained in the pres- ence of a silver or a copper salt (AgOTf or CuI) in etha- nol or in 1,2-dichloroethane at 60 °C for 4–6 h.64,65 Under ultraso nic conditions, a surfactant-type copper catalyst (C12H25SO3Na and CuSO4) was used in water.66 In anoth- er example, the condensation was performed applying a chiral spirocyclic phosphonic acid as a chiral additive, and the optically active (dihydroisoquinolinyl)phosphonates were obtained at ‑10 °C after 3 days.67 (Dihydroisoquinoli- nyl)phosphonates were also synthesized by a ring-closure method, starting from α-amino-(2-alkynylphenyl)-meth- ylphosphonates in the presence of silver triflate (AgOTf).68 In our research work, the Kabachnik–Fields reaction of 2-alkynylbenzaldehydes, aniline and dialkyl phosphites was studied and optimized in respect of the molar ratio of the starting materials, the temperature, the reaction time, the additive or catalyst and the solvent.69 Based on our results, depending on the conditions, α-amino-(2-alk- ynylphenyl)-methylphosphonates 37–43 or (1,2-dihy- droisoquinolin-1-yl)phosphonates 44–50 could be syn- Scheme 4. Deoxygenation of (oxoisoindolinyl)phosphine oxide (23a) and formation of platinum(II) complex trans-36 Scheme 5. T3P®-mediated Kabachnik–Fields reaction of 2-alkynylbenzaldehydes, aniline and dialkyl phosphites 741Acta Chim. Slov. 2022, 69, 735–755 Popovics-Tóth and Bálint: Multicomponent Synthesis of Potentially Biologically ... thesized selectively. An efficient procedure was developed for the preparation of α-amino-(2-alkynylphenyl)-meth- ylphosphonates 37–43 at room temperature for 1 h in the presence of T3P® (propylphosphonic anhydride) as an additive (Scheme 5). Then, the model reaction was ex- tended to different alkinylbenzaldehydes (2-(phenyleth- ynyl)-, (2-(p-tolylethynyl)-, 4-fluoro-2-(p-tolylethynyl)-, 2-((4-methoxyphenyl)ethynyl)- and 2-((4-chlorophenyl) ethynyl)-benzaldehyde), as well as dialkyl phosphites (di- ethyl-, dibutyl- and dibenzyl phosphite), and seven new derivatives 37–43 were prepared in yields of 87–98%. The condensation may take place through an imine intermediate, which may form by the reaction of 2-alki- nylbenzaldehyde and aniline. The role of the T3P® is pro- moting dehydration. After the addition of the phospho- rus reagent to the double bond of the intermediate, the α-amino-(2-alkynylphenyl)-methylphosphonates 37–43 are formed. Performing the three-component reaction at 60 °C for 1 h, in the presence of 5 mol% of copper chloride (CuCl) as the catalyst, and using 2-alkynylbenzaldehyde and aniline in a small excess (1.2 equiv.), (1,2-dihydroi- soquinolin-1-yl)phosphonates 44–50 were synthesized se- lectively (Scheme 6). After the optimization, by changing the 2-alkynylbenzaldehydes and dialkyl phosphites, seven new (dihydroisoquinolin-1-yl)phosphonates 44–50 were prepared in good to high yields (79–86%). In contrast to the literature, in our method, we applied a cheaper catalyst and shorter reaction time. The first step of the formation of (1,2-dihydroiso- quinoline)phosphonates 44–50 is the CuCl-catalyzed Kabachnik–Fields reaction of 2-alkynylbenzaldehydes, aniline and dialkyl phosphites. After that, the catalyst in- teracts with the triple bond of the α-amino-(2-alkynylphe- nyl)-methylphosphonates 37–43, which makes the intra- molecular nucleophile attack possible by the amino group, causing the ring closure step. Altogether seven new α-amino-(2-alkynylphe- nyl)-methylphosphonates 37–43 were prepared in a short- er reaction time (1 h) under milder conditions (25 °C) by the T3P®-mediated process developed by us as compared to the literature methods, which were carried out in the pres- ence of Mg(OCl4)2 or Lewis acids for long reaction times. Furthermore, seven new (1,2-dihydroisoquinolin-1-yl) phosphonates 44–50 were also synthesized using a small excess (1.2 equiv.) of alkynylbenzaldehyde and amine, in acetonitrile instead of a halogenated solvent (1,2-dichlo- roethane) in a shorter reaction time (1 h instead of 4–6 h) using a cheaper catalyst (CuCl), than in the literature. 2. 4. Synthesis of (Dihydroisoquinolin-1-yl) phosphine Oxides The Reissert-type reaction of isoquinoline, different acetylenes and secondary phosphine oxides or phosphine sulfides for the synthesis of (1,2-dihydroisoquinolin-1-yl) phosphine oxide derivatives was studied in the literature, however, only in two cases.70,71 The condensations were performed at high temperature (70–72 °C) for long re- action times (1.5–12 h), applying 1.1–1.5 equiv. excess of isoquinoline and acetylenes. However, starting from acyl- phenylacetylenes, longer reaction times (45–72 h) were used.71 In two other examples, the Reissert-type reaction was performed with dialkyl phosphites in the absence of any catalyst and solvent at room temperature for 2–4 h.72,73 The (1,2-dihydroisoquinolin-1-yl)phosphonates were ob- tained in yields of 52–90%. The Reissert-type reaction of isoquinoline, diethyl acetylenedicarboxylate and diphenylphosphine oxide was investigated, and the effect of the solvent, catalyst, tem- perature and reaction time was investigated.74 A complete conversion was obtained using equivalent amount of the starting materials in acetonitrile, at room temperature af- ter 10 min. Under the optimized conditions, the conden- sation of isoquinoline, dimethyl or diethyl acetylenedicar- boxylate and diphenyl-, bis(p-tolyl)-, bis(3,5-dimethyl- phenyl)phosphine oxide or ethyl phenyl-H-phosphinate Scheme 6. CuCl-catalyzed Kabachnik–Fields reaction of 2-alkynylbenzaldehydes, aniline and dialkyl phosphites 742 Acta Chim. Slov. 2022, 69, 735–755 Popovics-Tóth and Bálint: Multicomponent Synthesis of Potentially Biologically ... was performed, and eight (1,2-dihydroisoquinolin-1-yl) phosphine oxide derivatives (51 and 52) were formed in yields of 65–85% (Scheme 7). In the case of ethyl phe- nyl-H-phosphinate, the desired dialkyl (E)-2-[1-(ethox- y(phenyl)phosphoryl)isoquinolin-2(1H)-yl]maleate de- rivatives (51f and 52f) were obtained as a mixture of dias- tereomers in a ratio of 60:40. Starting from dibenzyl-, or di(2-naphthyl)phosphine oxides as the P-reagent, a small excess (1.2 equiv.) of iso- quinoline and dialkyl acetylenedicarboxylate, as well as somewhat longer reaction time (1 h) were applied to ob- tain complete conversion. Thus, further four new (1,2-di- hydroisoquinolin-1-yl)phosphine oxides (51d,e and 52d,e) were synthesized in yields of 70–73%. The mechanism of the formation of (1,2-dihydroi- soquinolin-1-yl)phosphine oxides 51 and 52 can be ex- plained by the nucleophile addition of isoquinoline to di- akyl acetylenedicarboxylates, forming a zwitterion inter- mediate. Then the products 51 and 52 are formed after the reaction of the intermediate with the P-reagent. In summary, an efficient, rapid process was devel- oped for the synthesis of (dihydroisoquinoline)phosphine oxides 51 and 52 by the Reissert-type reaction of isoquino- line, dialkyl acetylenedicarboxylates and secondary phos- phine oxides or ethyl phenyl-H-phosphinate. As compared to the literature, a complete conversion was obtained in shorter reaction time (10 min instead of 1.5–72 h) and in most cases, without the excess (1.1–1.5 equivalents) of isoquinoline and acetylene. In all, 12 dialkyl (E)-2-[1- (phosphoryl)isoquinolin-2(1H)-yl]maleate derivatives 51 and 52 were synthesized, among them 11 compounds were new. 2. 5. Synthesis of (Dihydropyrimidinone) phosphonates Only a few examples can be found in the literature for the Biginelli reaction of β-ketophosphonates, alde- hydes and urea. In one example, the condensation was performed in the presence of 15 mol% of zinc triflate (Zn(OTf)2) in toluene, at high temperature (110 °C) for 3 h.40 In another case, 50 mol% of p-toluenesulfonic acid (PTSA) was used as a catalyst in boiling acetonitrile for longer reaction time (24 h).75 Finally, the condensation was performed with ytterbium triflate (Yb(OTf)3) in tolu- ene, at reflux temperature for 12 h.76 In all cases, urea was used in excess. Based on the literature data, the Biginelli reaction of β-ketophosphonates does not take place start- ing from aliphatic aldehydes. The Biginelli reaction of diethyl (2-oxopropyl)phos- phonate, benzaldehyde and urea was studied by us.77 The conditions (heating mode, temperature, reaction time, molar ratio of the starting materials, catalyst and sol- vent) were changed to maximize the conversion. Based on our results, a  new solvent-free MW-assisted method was developed for the synthesis of (dihydropyrimidinone) phosphonates 53–57 by the Zn(OTf)2-catalyzed Biginelli reaction. During optimization it was found that besides starting materials and the desired (dihydropyrimidinone) phosphonate 53a, a by-product containing a styryl group at position six was also in the reaction mixture, which could be formed by the aldol condensation of the prod- uct 53a and benzaldehyde. The optimal parameters for the MW-assisted synthesis of (dihydropyrimidinone)phos- phonates were applying 1.5 equiv. of benzaldehyde and 2 equiv. of urea, in the presence of 15 mol% of Zn(OTf)2 at 100 °C for 2 h. After that, the condensation was carried out with different β-ketophosphonates (dimethyl or diethyl (2-oxopropyl)phosphonate), substituted benzaldehydes (benzaldehyde, 2-chlorobenzaldehyde, 3-chlorobenza- ldehyde, 4-chlorobenzaldehyde, 4-fluorobenzaldehyde, 2-fluoro-4-iodobenzaldehyde, 3-methylbenzaldehyde, 4-hydroxybenzaldehyde, 4-nitrobenzaldehyde, 3,4,5-tri- methoxybenzaldehyde) and urea derivatives (urea or N-methylurea) (Scheme 8). In all 20 (dihydropyrimidi- none)phosphonates 53 and 54 were obtained in yields of 53–81% after column chromatography, and among them, 14 were new derivatives, not yet described in the literature. Scheme 7. Reissert-type reaction of isoquinoline, diakyl acetylenedicarboxylates, and secondary phosphine oxides or ethyl phenyl-H-phosphinate 743Acta Chim. Slov. 2022, 69, 735–755 Popovics-Tóth and Bálint: Multicomponent Synthesis of Potentially Biologically ... Starting from N-methylurea, dimethyl or diethyl (2-oxopropyl)phosphonate and benzaldehyde, further two new compounds were prepared in slightly lower yields (Scheme 9). In contrast with the literature procedures, our meth- od was also suitable when using aliphatic aldehydes in the Biginelli reaction of β-ketophosphonates (Scheme 10). The condensation of dimethyl or diethyl (2-oxopropyl) phosphonate, butyraldehyde or isovaleraldehyde and urea was accomplished successfully, and further four new com- pounds 56 and 57 were isolated in yields of 41–43%. In counclusion, a new solvent-free MW-assisted process was elaborated for the preparation of (3,4-dihy- dropyrimidin-2-(1H)-one)phosphonates 53–57 by the Biginelli reaction of β-ketophosphonates, substituted benzaldehydes and urea derivatives. As compared to the literature examples, the desired compounds 53–57 could be obtained in shorter reaction time (2 h instead of 3–24 h) without solvent. The condensation was also success- fully performed starting from aliphatic aldehydes. In our research work, a molecular library of 26 (dihydropyrimid- inone)phosphonate derivatives 53–57 was created, of which 20 compounds were new. 2. 6. Synthesis of (1,2,3-Triazol-5-yl) phosphonates In the literature, the 1,3-dipolar cycloaddition of azides and alkynyl phosphonates is the most common way for the synthesis of (1,2,3-triazol-5-yl)phosphonates, how- ever, in most cases, the reaction was not selective, since (1,2,3-triazol-4-yl)phosphonates were also obtained be- sides (1,2,3-triazol-5-yl)phosphonates. In three cases, the reactions were carried out in the absence of any catalyst, in different solvents, such as tolu- ene,78 diethyl ether79 or water.80 In refluxing toluene, the cy- Scheme 8. Biginelli reaction of β-ketophosphonates, substituted benzaldehydes and urea Scheme 9. Biginelli reaction of β-ketophosphonates, benzaldehyde and N-methylurea Scheme 10. Biginelli reaction of β-ketophosphonates, aliphatic aldehydes and urea 744 Acta Chim. Slov. 2022, 69, 735–755 Popovics-Tóth and Bálint: Multicomponent Synthesis of Potentially Biologically ... cloaddition of ethyl (diethoxyphosphinyl)propynoate and methyl azidoacetate was performed.78 Trifluoromethylated triazolylphosphonates were synthesized in diethyl ether by the reaction of tert-butyl azidoacetate to diisopropyl (3,3,3-trifluoroprop-1-ynyl)phosphonate at room tempera- ture for long reaction time (20 h).79 When the click reaction of a phosphorylalkyl azide and tetramethoxy acetylenedi- phosphonate was performed at room temperature for 36 h in water, the desired product was obtained in a high yield.80 The reaction time could be reduced to 2 h at a higher tem- perature of 60 °C with the same yield. Next, the cycloaddi- tion of benzyl azide and diethyl ethynylphosphonate deriv- atives was performed applying copper(II) sulfate pentahyd- rate (CuSO4 ∙ 5H2O) and sodium ascorbate as a catalyst in DMF at 170 °C for 12 h.81 The desired (1,2,3-triazol-5-yl) phosphonates were formed selectively, and were obtained in good to high yields (83–92%). Finally, a MW-assisted sol- vent- and catalyst-free method was also published, where the ratio of the (1,2,3-triazol-4-yl)phosphonate and the (1,2,3-triazol-5-yl)phosphonate derivative was 34:66.82 In the literature, there is only one example regard- ing the domino synthesis of (1,2,3-triazol-5-yl)phospho- nates.83 The condensation of azides, terminal alkynes, and various dialkyl phosphites was performed using CuCl as a catalyst in acetonitrile at room temperature for 20 h under air atmosphere. In our work, the synthesis of (1,2,3-triazol-5-yl)phos- phonates 58–65 was optimized through the three-compo- nent reaction of phenylacetylene, benzyl azide and dibutyl phosphite in respect of the catalyst, base, solvent, molar ratio of the starting materials, atmosphere, temperature, as well as the reaction time.84 The best result was obtained using 1.1 equiv. of the azide derivative, 2 equiv. of dialkyl phosphite in the presence of 10 mol% of CuCl and 2 equiv. of triethylamine (TEA) in acetonitrile at room temperature after 8 h, using continuous air bubbling. During the opti- mization, the reaction mixtures contained two triazole de- rivatives. One of them was the desired (1,2,3-triazol-5-yl) phosphonate and the other compound was the product of the click reaction of phenylacetylene and benzyl azide. After the optimization, the CuCl-catalyzed domi- no reaction of phenylacetylene, benzyl azide and dibutyl phosphite was extended to various benzyl azides (ben- zyl-, 4-methylbenzyl-, 2-fluorobenzyl-, 3-fluorobenzyl-, 4-fluorobenzyl- or 4-(trifluoromethyl)benzyl azide) and dialkyl phosphites (dimethyl-, diethyl-, dipropyl-, di- isopropyl-, dibutyl- or dipentyl phosphite) (Scheme 11). After column chromatography, 13 (1,2,3-triazol-5-yl) phosphonate derivatives 58–63 were obtained in yields of 30–62%, of which 11 were new compounds. Next, the domino reaction was also carried out starting from aliphatic azides (octyl or isooctyl azide), phenylacetylene and different dialkyl phosphites (dime- thyl-, diethyl- or dibutyl phosphite) under the optimized conditions (with 10 mol% of CuCl and 2  equiv. of TEA at room temperature for 8 h, in acetonitrile). Further four new (1,2,3-triazol-5-yl)phosphonates 64 and 65 were syn- thesized in yields of 58% and 28%, respectively. The synthesis of (1,2,3-triazol-5-yl)phosphonates 58–65 was efficiently performed by the three-component domino reaction of phenylacetylene, various azides and dialkyl phosphites in the presence of CuCl and TEA. In all, 17 (1,2,3-triazol-5-yl)phosphonate 58–65 derivatives were synthesized in good yields, among them 15 were new compounds. 2.7. Synthesis of [(1,2,3-Triazol-4-yl)methyl] phosphinates and [(1,2,3-Triazol-4-yl) methyl]phosphates The synthesis of (1,2,3-triazol-4-yl)phosphonates can be performed by the Cu(I)-catalyzed 1,3-dipolar (Hu- isgen) cycloaddition—also known as the click reaction— of azides and phosphorylated alkynes.85,86 By the click reaction of benzyl azide and ethyl eth- ynylphosphonate, 1,2,3-triazolyl-4-phosphonate and 1,2,3-triazolyl-5-phosphonate were synthesized without catalyst in toluene at reflux temperature.87 In two cases, triazoles containing bisphosphonate unit were obtained by the 1,3-dipolar cycloaddition of organic azides and propargyl-substituted bisphosphonates at room tempera- ture after long reaction times (24–68 h).88,89 In one case, the reaction was carried out in the presence of copper io- dide (CuI) as a catalyst and N,N-diisopropylethylamine Scheme 11. Synthesis of (1,2,3-triazolyl)phosphonates 58–65 by CuCl-catalyzed domino reaction 745Acta Chim. Slov. 2022, 69, 735–755 Popovics-Tóth and Bálint: Multicomponent Synthesis of Potentially Biologically ... (DIPEA) as a base, in THF.88 In another example, CuSO4∙5 H2O and sodium ascorbate was used as a catalyst, and the solvent was the mixture of tert-butyl alcohol and water.89 The click reaction of azides and ethynyl- or propargyl-sub- stituted phosphonates was carried out with CuSO4∙5 H2O and sodium ascorbate and α-CF3-α-aminophospho- nates containing triazole unit were formed in yields of 38–92%.90 A triazole-functionalized phosphate flame-re- tardant monomer was synthesized by the cycloaddition of 2-azidoethanol and triprop-2-ynyl phosphate at 85 °C for 12 h in toluene.91 In our research work, we aimed at the study of the Cu(I)-catalyzed click reaction of propynyl phosphinates, propynyl phosphates—which were prepared by esterifica- tion of the  corresponding phosphinic acid—and organic azides.92 At first, the parameters (heating mode, temper- ature, reaction time and load of the catalyst) of the click reaction of benzyl azide and prop-2-ynyl diphenylphosph- inate were investigated in the presence of CuSO4∙5 H2O and sodium ascorbate in the mixture of tert-butyl alco- hol and water (4:1). The optimal conditions were 3 mol% of CuSO4∙5H2O, 5 mol% of sodium ascorbate and 60 °C for 10 min. In the next step, the cycloaddition of ben- zyl-, 4-methylbenzyl-, 2-fluorobenzyl-, 3-fluorobenzyl-, 4-fluorobenzyl- or 4-(trifluoromethyl)benzyl-, octyl-, isooctyl-, cyclohexyl- or phenyl azide and prop-2-ynyl di- phenylphosphinate were performed, and 10 new (1,2,3-tri- azol-4-yl)methyl diphenylphosphinate derivatives 66a–j were isolated in yields of 63–91% (Scheme 12). Carrying out the click reaction of azides mentioned above with diethyl prop-2-ynyl phosphate, the conversion was not complete under the optimized conditions found earlier (60 °C, after 10 min) (Scheme 13). In this case, a slightly longer reaction time (30 min) had to be used. In all, 10 new (1H-1,2,3-triazol-4-yl)methyl diethyl phos- phates 67a–j were synthesized in yields of 51–75%. To sum up, a simple, fast and efficient method was developed for the synthesis of (1H-1,2,3-triazol-4-yl) methyl phosphinates 66a–j and (1H-1,2,3-triazol-4-yl) methyl diethyl phosphates 67a–j by the cycloaddition of azides and prop-2-ynyl phosphinate or diethyl prop-2- ynyl phosphate. The target compounds were prepared in the presence of CuSO4∙5H2O and sodium ascorbate under mild conditions (60 °C) after short reaction times (10–30 min). In all, 20 novel derivatives 66 and 67 were synthe- sized. 3. Synthesis of O-Heterocycles 3. 1. Synthesis of (2-Amino-3-cyano-4H- chromen-4-yl)phosphonates and -phosphine Oxides A few publications can be found for the three-com- ponent synthesis of (2-amino-3-cyano-4H-chromen-4-yl) phosphonates starting from salicylaldehydes, malononi- trile and dialkyl phosphites or trialkyl phosphites. In most Scheme 12. Synthesis of (1,2,3-triazol-4-yl)methyl diphenylphosphinates 66a–j by click reaction Scheme 13. Synthesis of (1,2,3-triazol-4-yl)methyl diethyl phosphates 67a–j by click reaction 746 Acta Chim. Slov. 2022, 69, 735–755 Popovics-Tóth and Bálint: Multicomponent Synthesis of Potentially Biologically ... cases, the reactions were performed in the presence of a basic catalyst and solvent. The condensations were carried out with diethylamine,93 dibutylamine,43 triethylamine,94 dimethylaminopyridine,95 imidazole,96 ethylenediamine diacetate,97 lithium hydroxide,96 potassium phosphate,98 magnesium oxide99 or indium chloride100 in ethanol, or with iron oxide,101 iodine42 or β-cyclodextrin102 in water. A few examples can be found for the use of special sol- vents, such as polyethylene glycol,103 ionic liquids104 or the mixture of urea and choline chloride.105 In four cases, the reactions were carried out without solvents, however, spe- cial catalysts (silica-bonded 2-HEAA-3 catalyst,106 ZnO nano-rods,107 iodine108) or a simple catalyst in a large ex- cess (3.5 equiv. of tetramethylguanidine)109 were needed. In the literature, there is no example for the condensation of salicylaldehydes, malononitrile and secondary phos- phine oxides. The condensation of salicylaldehydes, malononitrile and dialkyl phosphites was studied through a model reac- tion.110 The effect of various basic catalysts, solvent, tem- perature and reaction time was investigated. Based on our results, pentamethyldiethylenetriamine (PMDTA) was the most effective among the bases. A complete conversion was achieved with 10 mol% PMDTA in the absence of any solvent at 60–80 °C after 15–30 min (Scheme 14). A total of 18 (2-amino-3-cyano-4H-chromen-4-yl)phosphonate derivatives 68–73 were synthesized in yields of 70–96%, of which 13 were new compounds. The products were isolat- ed from the reaction mixture by a simple filtration. Start- ing from ethyl phenyl-H-phosphinate as the phosphorus Scheme 14. PMDTA-catalyzed reaction of salicylaldehydes, malononitrile and dialkyl phosphites Figure 5. The crystal structure of 68d and 72e (2-amino-3-cyano-4H-chromen-4-yl)phosphonates 747Acta Chim. Slov. 2022, 69, 735–755 Popovics-Tóth and Bálint: Multicomponent Synthesis of Potentially Biologically ... reagent, the desired (aminochromenyl)phosphinate 68f was obtained in a yield of 86%, as a mixture of diastere- omers in a ratio of 1:1. The crystal structures of dibutyl (2-amino-3-cy- ano-4H-chromen-4-yl)phosphonate (68d) and dibenzyl (2-amino-3-cyano-8-ethoxy-4H-chromen-4-yl)phospho- nate (72e) were determined by X-ray diffraction (XRD), as well (Figure 5). In both derivatives (68d and 72e), an intermolecular N–H···O=P hydrogen bonding between the amino group and the phosphonate oxygen atom was Scheme 15. PMDTA-catalyzed reaction of salicylaldehydes, malononitrile and secondary phosphine oxides Figure 6. The crystal structure of 74a–c (2-amino-3-cyano-4H-chromen-4-yl)phosphine oxides 748 Acta Chim. Slov. 2022, 69, 735–755 Popovics-Tóth and Bálint: Multicomponent Synthesis of Potentially Biologically ... found. However, the amino group as a hydrogen bond donor was observed to be involved in the formation of two interactions in the case of the butyl ester (68d). The other interaction was a centrosymmetric N–H···N hydro- gen bond with the cyano group. Due to these interactions, hydrogen-bonded layers were formed. In the benzyl ester (72e), besides the centrosymmetric N–H···O=P interac- tions, centrosymmetric C–H···N interactions between the chromenyl ring and the cyano group of two adjacent molecules are present, resulting in the hydrogen-bonded chain. According to the proposed mechanism of the for- mation of (2-amino-3-cyano-4H-chromen-4-yl)phospho- nates 68–73, at first, the Knoevenagel condensation of the salicylaldehyde and malononitrile takes place. Next, imi- nocoumarine is formed by the intramolecular Pinner-like reaction from the 2-(2-hydroxybenzylidene)malononitrile intermediate. Finally, the phospha-Michael addition of di- alkyl phosphites leads to (2-amino-3-cyano-4H-chromen- 4-yl)phosphonates 68–73. The PMDTA-catalyzed condensation of salic- ylaldehydes (salicylaldehyde or 5-fluoro-, 2-chloro-, 3-bromo-, or 3-ethoxysalicylaldehyde), malononitrile and P-reagents was extended to secondary phosphine oxides (such as diphenyl-, bis(p-tolyl)-, bis(3,5-dimeth- ylphenyl)- or bis(2-naphthyl)phosphine oxides), as well. A new family of compounds, (2-amino-3-cyano-4H- chromen-4-yl)phosphine oxides 74–78 were formed with 5 mol% PMDTA at 60 °C after 15 min, in acetoni- trile (Scheme 15). In our work, 20 new (aminochrome- nyl)phosphine oxides 74–78 were synthesized in excel- lent (86–95%) yields. Single crystals were also grown from three deriva- tives 74a–c in acetonitrile and their structures were in- vestigated by XRD (Figure 6). Based on our results, an intermolecular N–H···O=P hydrogen bond is formed between the amino group and the phosphine oxide side chain. Furthermore, the amino group is involved in a centrosymmetric N–H···N interaction with the cyano group of the adjacent molecule. In the case of (diphenyl) (2-amino-3-cyano-4H-chromen-4-yl)phosphine oxide (74a) and [bis(3,5-dimethylphenyl)](2-amino-3-cyano- 4H-chromen-4-yl)phosphine oxide (74c), N–H···O=P interactions lead to the formation of layers. A difference can be observed in the crystal structure of [bis(p-tolyl)] (2-amino-3-cyano-4H-chromen-4-yl)phosphine oxide (74b), where a centrosymmetric N–H···N interactions (between the amino and the cyano groups) and cen- trosymmetric N–H···O=P hydrogen bonds led to the for- mation of hydrogen-bonded chains. Summarizing our results, the model reaction of sa- licylaldehyde, malononitrile and dialkyl phosphites was studied and optimized. By our solvent-free PMDTA-cata- lyzed method, 18  (2-amino-3-cyano-4H-chromen-4-yl) phosphonate derivatives 68–73 were prepared in good to high yields (70–96%). Our method was also suitable for the domino Knoevenagel-phospha-Michael reaction of secondary phosphine oxides, and 20 new (2-amino-3- cyano-4H-chromen-4-yl)phosphine oxides 74–78 were synthesized, which are members of a new family of com- pounds in the literature. 4. Synthesis of P-Heterocycles 4. 1. Synthesis of 1-Alkyl-1H-phoshindole-1- oxides and 1-Alkoxy-1H-phoshindole-1- oxides In the literature, phoshindole-1-oxide derivatives were prepared by the intermolecular radical cycloaddition of secondary phosphine oxides or phosphinates and inter- nal alkynes.111 In the examples, several oxidizing agents were used, and in general, a long reaction time (8–24 h) was applied to obtain complete conversion, for example: Ag2O (8–10 h),112,113 AgOAc (4–18 h),114–116 Mn(OAc)2/MnO2 (4 h),117 K2S2O8 (24 h)118 or N-etoxy-2-methylpyridinium tetrafluoroborate (48 h).119 In one case, a shorter reaction time of 30 min was enough, however, beside the oxidant (tert-butyl hydroperoxide), a catalyst (CuSO4) and a base (NH3) were necessary.120 Our aim was to find a fast and simple method for the synthesis of phoshindole-1-oxides. Scheme 16. Cycloaddition of secondary phosphine oxides and ethyl phenylpropiolate or diphenylacetylene 749Acta Chim. Slov. 2022, 69, 735–755 Popovics-Tóth and Bálint: Multicomponent Synthesis of Potentially Biologically ... The first step of our work was the study and optimi- zation of the MW-assisted cycloaddition of diphenylphos- phine oxide and ethyl phenylpropiolate in respect of the oxidant, temperature and reaction time in acetonitrile as the solvent.121 It was found that complete conversion could be obtained under MW conditions, applying 1.5 equiva- lents of diphenylphosphine oxide, 2 equivalents of Ag2O as the oxidizing agent, at 100  °C for 2 h in acetonitrile (Scheme 16). Using the optimal conditions, the  reaction of diphenyl acetylene and diphenylphosphine oxide or tert-butyl(phenyl)phosphine oxide was performed. The three benzophosphole oxide derivatives 79a, 79b and 80b were obtained after column chromatography, in yields of 80–93%. In the next series of experiments, the cycloaddition was extended to alkyl phenyl-H-phosphinates and dif- ferent acetylenes in the presence of Ag2O (Scheme 17). The MW-assisted reaction of ethyl phenyl-H-phosph- inate and ethyl phenylpropiolate was optimized in re- spect of temperature and reaction time. Based on our results, the reaction was complete after a slightly longer (3 h) reaction time as compared to the reactions car- ried out with secondary phosphine oxides. Then, the cycloaddition was performed starting from further alkyl phenyl-H-phosphinates (n-propyl-, isopropyl-, n-butyl-, isobutyl-, n-pentyl-, n-octyl- and adamantyl phenyl-H-phosphinate) and ethyl phenylpropiolate or diphenyl acetylene. In all, 13 1-alkoxy-1H-phoshindole- 1-oxides were synthesized in yields of 56–98%. Slight- ly lower yields (56–68%) were obtained starting from n-pentyl-, n-octyl- and adamantyl phenyl-H-phosphi- nate, due to the steric hindrance. A single crystal was grown of 1-isopropoxy-2,3-di- phenylphosphindole 1-oxide (83b) and the structure was analyzed by XRD (Figure 8). The analysis showed the for- mation of hydrogen-bonded wavy layer through two inter- molecular C–H···O=P hydrogen bonds between two phe- nyl rings and the O=P group. The layers formed a 3D net- work via C–H···π interactions between the phenyl groups of adjacent molecules. In order to investigate the efficiency of our MW-as- sisted method, two scaled-up reactions were also per- formed at a “gram-scale”. The condensation of diphe- nylphosphine oxide or ethyl phenyl-H-phosphinate and diphenyl acetylene was carried out on a 25-times-bigger scale. The  desired 1,2,3-triphenylphosphindole 1-oxide (79b) and 1-ethoxy-2,3-diphenylphosphindole 1-oxide (81b) were obtained in yields of 94% and 70%. To sum up, a MW-assisted, fast (2–3 h instead of 8–24 h) and efficient approach for the synthesis of ben- zo[b]phosphole oxides 79–88 by the oxidative cycload- dition of secondary phosphine oxides or alkyl phe- nyl-H-phosphinates with acetylenes (diphenylacetylene or ethyl phenylpropiolate) was developed. Altogether 16 derivatives 79–88 were prepared, among them 12 were new. Scheme 17. Condensation of alkyl phenyl-H-phosphinate and ethyl phenylpropiolate or diphenylacetylene Figure 8. The crystal structure of 1-isopropoxy-2,3-diphenylphosphindole 1-oxide (83b) 750 Acta Chim. Slov. 2022, 69, 735–755 Popovics-Tóth and Bálint: Multicomponent Synthesis of Potentially Biologically ... 5. Biological Activity Investigations The in vitro cytotoxicity and antibacterial activity of all compounds synthesized was also investigated. In Table 2 only the most active derivatives are shown. The cytotoxicity evaluations were performed on three different cell lines, such as human lung adenocar- cinoma (A549), mouse fibroblast (NIH/3T3) as healthy cell line and human promyelocytic leukemia (HL-60) using the fluorescent Resazurin assay as described pre- viously.122 Positive controls were doxorubicin for A549 and NIH/3T3 (IC50 = 0.31 ± 0.24 µM and 5.65 ± 0.81 µM, respectively) and bortezomib for HL60 (IC50 = 7.42 ± 2.60 nM). The antibacterial activity of the compounds was tested on green fluorescent protein (GFP) produc- ing Bacillus subtilis (Gram‐ositive) and Escherichia coli (Gram-negative) bacterial cells. The GFP producing bac- teria are effective tools for screening for the antibacterial activity, since the GFP signal measured by fluorimetry is proportional to the number of the bacterial cells. Ac- tive compounds kill bacterial cells, which results in the decrease in the GFP fluorescence signal, therefore it is suitable for evaluating the antimicrobial effect of differ- ent agents. Positive controls were doxycycline and gen- tamicin for Bacillus subtilis (IC50 = 0.04 ± 0.01 µM and 0.49 ± 0.14 µM) and for Escherichia coli (IC50 = 0.10 ± 0.02 µM and 4.23 ± 0.99 µM) bacterial cells. The IC50 values (50% inhibiting concentration) determined are shown in Table 2. Among (3-oxo-2,3-dihydro-2H-isoindol-1-yl)phos- phine oxides 23–34, derivatives containing 3,5-dimeth- ylphenyl- or naphthyl groups on the phosphorus atom showed activity.62 The N-butyl and N-benzyl bis(3,5-di- methylphenyl) (3-oxo-2,3-dihydro-2H-isoindol-1-yl) phosphine oxides 23c and 25c were slightly active in HL- 60 cell line. However, against Grampositive bacteria (B. subtilis), the same derivatives (23c and 25c) showed prom- ising activity, as their IC50 values (4.60 ± 1.13 µM and 3.61 ± 1.25 µM) were close to the reference value. In the case of bis(2-naphthyl) (2-butyl-3-oxo-2,3-dihydro-2H-isoin- dol-1-yl)-phosphine oxide (23d), no antibacterial activity was shown, but against all the three investigated cell lines (A549, NIH/3T3 and HL-60) modest cytotoxicity was ob- served. The IC50 value was the smallest against HL-60 cells (12.26 ± 1.02 µM). The biological activity of the α-amino-(2-alkynyl- phenyl)methylphosphonates 37–43 was investigated, as well. According to our results, some butyl esters showed modest activity against HL-60 cells. The IC50 value of the chloro (41) or the unsubstituted derivatives (42) were in the range of 13–15 µM. The results of the bioactivity tests of the (1,2-dihy- droisoquinoline)phosphonates 44–50 also showed that the butyl esters were more active as compared to the other derivatives. Compounds containing methyl (47), meth- oxy (48), or chloro group (49) on the para position of the phenyl group, showed in vitro cytotoxicity. The (1,2-dihy- droisoquinoline)phosphonate 47 was effective in A549, NIH/3T3 and HL-60 cell lines. In addition, the IC50 value was close to the reference against human promyelocytic leukemia cells (4.36 ± 1.31 µM). The (1,2-dihydroisoquinoline)phosphine oxides containing 3,5-dimethylphenyl (51c and 52c) or naph- thyl groups (51e) on the phosphorus atom showed in vitro cytotoxicity and antibacterial activity.74 Dimethyl and diethyl (E)-2-{1-[bis(3,5-dimethylphenyl)phos- phoryl]isoquinolin-2(1H)-yl}maleates (51c and 52c) were slightly active in HL-60 cell line, however the IC50 value was closer to the reference in the case of the methyl ester 51c (IC50 = 4.58 ± 1.08 µM vs. 12.59 ± 1.18 µM). Compound 52c also showed modest activity against B. subtilis (IC50 = 9.06 ± 1.01 µM). Among the (1,2-dihydroisoquinoline)phosphine oxides, dimethyl (E)-2-{1-[di(naphthalen-2-yl)phosphoryl]isoquino- lin-2(1H)-yl}maleate (51e) had the most significant in vitro cytotoxicity against HL-60 cells (IC50 = 3.58 ± 1.16 mM). Based on the IC50 values of (1,2,3-triazolyl)phos- phonates, some derivatives were active against HL-60 cells.84 The dimethyl [1-(4-methylbenzyl)-4-phenyl- 1,2,3-triazol-5-yl]phosphonate (59a), dipropyl (1-benzyl- 4-phenyl-1,2,3-triazol-5-yl)phosphonate (58c), dibutyl [1-(2-fluorobenzyl)-4-phenyl-1,2,3-triazol-5-yl]phospho- nate (60e) and dibutyl [1-(4-trifluoromethyl)-4-phenyl- 1,2,3-triazol-5-yl]phosphonate (62e) showed activity in the range of 9–13 µM. Among chromenylphosphonates, the dibenzyl (2-amino-3-cyano-4H-chromen-4-yl)phosphonates 68e, 69e, 70e, 71e and 72e were the best candidates.110 The an- ti-cancer activity in NIH/3T3 cell line was close to the ref- erence in the case of the unsubstituted (68e) or the 8-bro- mo derivatives (71e) (IC50 = 8.73 ± 1.17 µM or 9.33 ± 1.18 µM, respectively). In addition, all benzyl esters synthesized (68e, 69e, 70e, 71e and 72e) showed good or moderate ac- tivities against HL-60 cells. The IC50 value obtained was the smallest in the case of the 6-fluoro (69e) or 8-bromo (71e) substituted (2-amino-3-cyano-4H-chromen-4-yl) phosphonates (IC50 = 3.62 ± 1.38 µM or 4.79 ± 1.08 µM, respectively). The biological activity investigations showed that the [bis(3,5-dimethylphenyl)](2-amino-3-cyano-4H- chromen-4-yl)phosphine oxides were effective against human promyelocytic leukemia cells and Gram-pos- itive bacteria.110 The IC50 values of the 6-fluoro (69e) and 8-bromo derivatives (71e) were in the rage of 10 µM in HL-60 cell line. The growth of B. subtilis was reduced the most by the unsubstituted, 6-fluoro and 5-chloro [bis(3,5-dimethylphenyl)](2-amino-3-cyano- 4H-chromen-4-yl)phosphine oxides (74c, 75c and 76c) (IC50 = 8.92 ± 1.21 µM, 5.03 ± 1.28 µM and 5.29 ± 1.38 µM, respectively). 751Acta Chim. Slov. 2022, 69, 735–755 Popovics-Tóth and Bálint: Multicomponent Synthesis of Potentially Biologically ... Summarizing the results of the biological activity investigations, heterocyclic phosphonates (butyl, benzyl) or phosphine oxides (3,5-dimethylphenyl, naphthyl) con- taining larger groups on the phosphorus atom showed promising activity against human promyelocytic leukemia (HL-60) cells and B. subtilis Gram-positive bacteria. 6. Conclusions In conclusion, the multicomponent synthesis of the target N- and O-heterocycles containing a phospho- nate or a phosphine oxide moiety and the synthesis of P-heterocyclic derivatives was elaborated successfully. The procedures developed are more effective and accept- Table 2. In vitro cytotoxicity and antibacterial activity of the compounds synthesized.a Compound R1 R2 In vitro Cytotoxicity] Antibacterial Activity [IC50, µM IC50, μM] A549 NIH/3T3 HL-60 B. subtilis E. coli 3,5-diMeC6H3 nBu (23c) >30 >30 17.55±1.70 4.60±1.13 >10 3,5-diMeC6H3 Bn (25c) >30 >30 18.31±1.33 3.61±1.25 >10 2-naphthyl nBu (23d) 28.2±1.05 25.94±1.06 12.26±1.02 >10 >10 Bu Cl (41) >30 >30 13.66±1.08 >10 >10 Bu H (42) >30 >30 15.09±1.17 >10 >10 F Me (47) 11.64±1.11 14.17±1.38 4.36±1.31 >10 >10 H OMe (48) >30 >30 13.16±1.22 >10 >10 H Cl (49) >30 13.58±1.09 13.33±1.14 >10 >10 3,5-diMeC6H3 Me (51c) >30 >30 4.58±1.08 >10 >10 3,5-diMeC6H3 Et (52c) >30 >30 12.59±1.18 9.06±1.01 >10 2-naphthyl Me (51e) >30 >30 3.58±1.16 >10 >10 Me 4-MeC6H4CH2 (59a) >30 19.8±1.2 11.0±1.2 >10 >10 nPr Bn (58c) >30 >30 12.6±1.7 >10 >10 nBu 2-FC6H4CH2 (60e) >30 27.5±1.1 11.7±1.2 >10 >10 nBu 4-CF3C6H4CH2 (62e) >30 23.1±1.2 9.7±1.1 >10 >10 OBn H (68e) 26.46±1.02 8.73±1.17 6.25±1.06 >10 >10 OBn 6-F (69e) >30 21.2±1.71 3.62±1.38 >10 >10 OBn 5-Cl (70e) >30 23.49±1.09 7.51±1.02 >10 >10 OBn 8-Br (71e) 28.65±1.22 9.33±1.18 4.79±1.08 >10 >10 OBn 8-OEt (72e) >30 27.99±1.06 14.37±1.24 >10 >10 3,5-diMeC6H3 H (74c) >30 >30 >30 8.92±1.21 >10 3,5-diMeC6H3 6-F (75c) >30 >30 10.06±1.25 5.03±1.28 >10 3,5-diMeC6H3 5-Cl (76c) >30 >30 >30 5.29±1.38 >10 3,5-diMeC6H3 8-Br (77c) >30 >30 9.80±1.33 >10 >10 Doxorubicin 0.31±0.24 5.65±0.81 – – – Bortezomib – – 7.42×10-3± – – 2.60×10-3 Doxycycline – – – 0.126±0.029 0.10±0.02 Gentamicin – – – 0.115±0.001 4.23±0.99 a Data were expressed as mean ± standard deviation. 752 Acta Chim. Slov. 2022, 69, 735–755 Popovics-Tóth and Bálint: Multicomponent Synthesis of Potentially Biologically ... able according to the principles of “green chemistry” as compared to the literature data. Altogether more than 150 derivatives were synthesized and fully character- ized, and most of them were new compounds. Accord- ing to the biological activity investigations, it was found that in the case of phosphonates butyl- and benzyl es- ters of α-amino-(2-alkynylphenyl)-methylphosphonates, (1,2-dihydroisoquinolinyl)phosphonates, (1,2,3-triazol- 5-yl)phosphonates and (aminochromenyl)phosphonates were effective, however, from among phosphine oxides, those derivatives showed promising antibacterial and/or anticancer effects, which contained large groups (3,5-di- methylphenyl or 2-naphtyl) on the phosphorus atom, es- pecially (oxoisoindolyl)phosphine oxides, (1,2-dihydroi- soquinolinyl)phosphine oxides, and (aminochromenyl) phosphine oxides. 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Slov. 2022, 69, 735–755 Popovics-Tóth and Bálint: Multicomponent Synthesis of Potentially Biologically ... Povzetek Razvili smo več multikomponentnih sinteznih pristopov do množice novih dušikovih in kisikovih heterociklov, ki vse- bujejo fosfonatne ali fosfin oksidne skupine. Za vse multikomponentne reakcije smo na primerih modelnih reakcij opti- mizirali reakcijske parametre: način segrevanja, množinsko razmerje izhodnih spojin, atmosfero, katalizatorje, tempera- ture, reakcijske čase in topila. Na tak način dobljeni reakcijski parametri so bili uporabljeni za priprave majhnih knjižnic strukturno sorodnih spojin. Večino reakcij lahko smatramo kot skladnih s principi »zelene kemije«, saj so potekale brez prisotnosti katalizatorjev in/ali topil ter pod pogoji obsevanja z mikrovalovi (MW) ali celo že pri sobni temperaturi. S po- močjo pretočnega MW sistema smo nekatere mikrovalovne sinteze izvedli tudi na večji skali. Skupno smo pripravili več kot 150 heterocikličnih organofosforjevih spojin; nekateri izmed pripravljenih derivatov so izkazali zmerne do obetavne aktivnosti na HL-60 celično linijo ter na bakterije Bacillus subtilis. 121. Á. Tajti, B. Kovács, N. Popovics-Tóth, F. Perdih, E. Bálint, Tetrahedron 2021, 102, 132527–132533. DOI:10.1016/j.tet.2021.132527 122. G. J. Szebeni, Á. Balázs, I. Madarász, G. Pócz, F. Ayaydin, I. Kanizsai, R. Fajka-Boja, R. Alföldi, L. Hackler Jr., L. G. Puskás, Int. J. Mol. Sci. 2017, 18, 2105–2121. DOI:10.3390/ijms18102105 Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License 756 Acta Chim. Slov. 2022, 69, 756–771 Makovec: Adaptation of the Crystal Structure to the Confined Size ... DOI: 10.17344/acsi.2022.7775 Feature article Adaptation of the Crystal Structure to the Confined Size of Mixed-oxide Nanoparticles Darko Makovec Department for Materials Synthesis, Jožef Stefan Institute, Jamova ulica 39, SI-1000 Ljubljana, Slovenia. * Corresponding author: E-mail: darko.makovec@ijs.si Received: 08-29-2022 Abstract Chemical composition and crystal structure are central to defining the functional properties of materials. But when a material is prepared in the form of nanoparticles, the structure and, as a consequence, the composition will also fre- quently change. Understanding these changes in the crystal structure at the nanoscale is therefore essential not only for expanding fundamental knowledge, but also for designing novel nanostructures for diverse technological and medical applications. The changes can originate from two thermodynamically driven phenomena: (i) a crystal structure will adapt to the restricted size of the nanoparticles, and (ii) metastable structural polymorphs that form during the synthesis due to a lower nucleation barrier (compared to the equilibrium phase) can be stabilized at the nanoscale. The changes to the crystal structure at the nanoscale are especially pronounced for inorganic materials with a complex structure and composition, such as mixed oxides with a structure built from alternating layers of several structural blocks. In this arti- cle the complex structure of nanoparticles will be presented based on two examples of well-known and technologically important materials with layered structures: magnetic hexaferrites (BaFe12O19 and SrFe12O19) and ferroelectric Aurivil- lius layered-perovskite bismuth titanate (Bi4Ti3O12). Keywords: Nanoparticles; crystal structure; hexaferrites; Aurivillius structure; layered perovskites; polymorphs 1. Introduction The functional properties of materials change signif- icantly when they are prepared in the form of nanoparti- cles. In the scientific literature these changes are usually associated with one of two fundamental reasons. The size effect can simply originate from the large surface-to-vol- ume ratio of nanoparticles. The properties, which are defined by processes occurring on the surfaces (e.g., ad- sorption capacity, catalytic activity, the rate of solid-state chemical reactions, etc.) will generally improve as the par- ticle size becomes smaller. With the explosion in research devoted to nanoscience at the beginning of this millenni- um, an especially large amount of attention was given to the direct effects of the confined size on some functional properties. Well-known examples are the quantum-con- finement effect observed in semiconducting nanoparticles (i.e., quantum dots) and the superparamagnetism of mag- netic nanoparticles. Much less attention has been given to the changes in properties that are an indirect consequence of the changes in crystal structure that are due to the re- stricted size of nanomaterials. This is because when the size is restricted to such an extent, the crystal structure of nanoparticles can also change significantly. The influence of the confined size on the structure of nanoparticles can range from minor changes in atomic positions to the stabilization of diverse structural varia- tions and metastable structural polymorphs. At the very large surface-to-volume ratios of nanoparticles, the struc- tural changes can simply be related to the relaxation and reorganization of the atoms at the surfaces. However, the adaptation of the structure to the small size frequently cannot be explained merely by the increased surface area as it clearly affects the whole particle, including its interior. The adaptation of the crystal structure generally in- volves systematic displacements of the atoms from their ordered positions and deviations in the occupation of dif- ferent lattice sites. The extent of the changes in the crystal- line structure that can be accommodated with the small size depends on the complexity of the composition and the crystal structure. In simple oxides the changes in the occupation of different lattice sites can be restricted to an increased content of vacancies. Classical thermodynamic calculations indicate that the size-dependent vacancy-for- mation energy and entropy result in an increase in the va- cancy concentration at a reduced crystallite size.1 In mixed oxides comprising different cationic lattice sites (e.g., in 757Acta Chim. Slov. 2022, 69, 756–771 Makovec: Adaptation of the Crystal Structure to the Confined Size ... cubic ferrites with a spinel structure) an additional adap- tation mechanism is possible based on the changes in the distribution of constituting cations over different lattice sites.2–6 Finally, in mixed oxides with a complex structure built from alternating layers of several structural blocks, e.g., a structure of hexagonal ferrites (hexaferrites) and the Aurivillius structures of layered perovskites, the adaptation of the crystal structure to the constricted size of the nano- particles is dominated by the termination of the particle at its surfaces with a specific, low-energy atomic layer.7 That can enable the synthesis of nanoparticles with a specific structure, which can be referred to as a specific structural variation of the bulk structure, stabilized at the nanoscale.8 In addition, the changes in the crystal structure due to the confined size of the nanoparticles can lead to a deviation from the bulk composition,5,7 or at least to an increased flexibility of the composition, i.e., the composition of the nanoparticles can change to a much larger extent without any precipitation of secondary phases when compared to the corresponding bulk.5 Also, the solid solubility of the foreign atoms in the host crystal usually increases with a decreasing size of the nanoparticles.1 In addition to the above-mentioned changes in the crystal structure due to the restricted size of the nanopar- ticles, various metastable structural polymorphs can be stabilized at the nanoscale. Polymorphs are defined as sub- stances that are chemically identical but exist in more than one crystal form. For example, iron(III) oxide appears in five different polymorphs: α (hematite), β, γ (maghemite), ε, and ξ.9 The metastable polymorphs are formed during the initial stages of crystallization, because they have a lower nucleation barrier than the stable phase.10,11 With the particle growth, a metastable polymorph usually trans- forms in an energetically cascading series of polymorphic stages to the equilibrium polymorph. This phenomenon is known as the Ostwald step rule.11 However, if the metasta- ble polymorph has a lower surface energy than the equi- librium polymorph it can remain stable while in the form of small particles with a large surface area.10 For example, with the confined growth of iron(III) oxide nanoparticles dispersed in a silica matrix, the γ, ε, and sometimes β pol- ymorphs appear in sequence before the transformation to the thermodynamically stable α polymorph in the larger particles resembling the bulk.12,13 Even though the poly- morphs stabilized on the nanoscale are usually referred to as “metastable”, their stability can be thermodynamically explained by taking into consideration their large surface area related to their small size.12,13 Strictly speaking, the metastable polymorphs are not a consequence of the adap- tation of the crystal structure to the restricted size; howev- er, as they only appear at the nanoscale they can be consid- ered in the context of the size effect. Metastable polymorphs stabilized at the nanoscale are abundant among simple oxides. Well-known examples of polymorphs for which the stability changes with the na- noparticle size include titania (anatase→brookite→rutile), zirconia (monoclinic→tetragonal), alumina (γ→α), silica (tridymite→cristobalite→quartz), and many others. The polymorphism is technologically very important, as differ- ent polymorphs of the same stoichiometry can have vastly different functional properties, and many metastable poly- morphs represent very important functional nanomateri- als. Iron(III) oxide can be used as a good illustration of the diversity of magnetic properties for different polymorphs.9 The thermodynamically stable phase hematite (α-Fe2O3) is only weakly magnetic. The metastable maghemite (γ-Fe2O3) is soft magnetic with a relatively high saturation magnetization, and maghemite nanoparticles are actually the most frequently used magnetic nanoparticles, espe- cially in medicine.14 In contrast, ε-Fe2O3 is hard magnetic as it exhibits the largest coercive field among all the oxides. The β-Fe2O3 and ξ-Fe2O3 phases are antiferromagnetic.9 Even though the Ostwald step rule should not be re- stricted to simple oxides, reports of polymorphs stabilized at the nanoscale are very scarce for inorganic materials with a complex composition and crystal structure, such as mixed oxides with several constituting ions distributed over many non-equivalent lattice sites within a large unit cell. Such complex materials include mixed oxides with a layered structure, which are the topic of this article. Finally, in contrast to the thermodynamically driven adaptations of the structure to the small size mentioned so far, nanomaterials can exhibit a specific crystal struc- ture because of the reaction kinetics during their synthesis. Usually, mild synthesis conditions, e.g., a low temperature, are used during the synthesis of the nanoparticles to limit the particle growth. Such specific, non-equilibrium syn- thesis conditions can also contribute to deviations from the regular, bulk structure when the material is synthe- sized in the form of nanoparticles. In this feature, article the thermodynamically driv- en adaptations of the crystal structure to the small size of the nanoparticles will be presented using two examples of mixed-oxide nanoparticles with a structure built from alternating layers of two structural blocks: a hexaferrite (BaFe12O19 and SrFe12O19) and an Aurivillius layered-per- ovskite bismuth titanate (Bi4Ti3O12). The mixed oxides with a layered structure represent technologically impor- tant materials. The hexaferrites exhibit extraordinary mag- netic properties dominated by a very large magnetocrys- talline anisotropy constant. As ceramics they represent the most abundant materials (by volume) used today for permanent magnets and are also used in microwave de- vices and absorbers.15 In the form of nanoparticles, hex- aferrites enabled the development of some entirely new types of materials, including the first ferromagnetic fluids (i.e., liquid magnets),16,17 and novel magneto-responsive suspensions18,19 and polymer composites.20 Hexafer- rite nanoparticles were also tested in novel applications, e.g., in novel spin-memory devices21,22 and in medical applications.23,24 Layered-perovskite phases of the Au- rivillius family ((Bi2O2)(An−1BnO3n+1), where A is a large 758 Acta Chim. Slov. 2022, 69, 756–771 Makovec: Adaptation of the Crystal Structure to the Confined Size ... 12-coordinated cation, and B is a small 6-coordinated cation), the Ruddlesden-Popper family (A’n−1A’’2BnO3n+1, where  A’  and  A’’  are alkali, alkaline earth or rare-earth ions) and the Dion-Jacobson family (A’’(A’n−1BnO3n+1)) possess interesting properties such as ferroelectricity, co- lossal magnetoresistance, catalytic activity and supercon- ductivity. The Aurivillius Bi4Ti3O12 ceramics are prom- ising materials for ferroelectric random-access-memory devices and lead-free, high-temperature piezoelectric and pyroelectric devices.25 Bi4Ti3O12 nanoparticles were test- ed for biomechanical energy harvesting,26 sensing,27,28 visible-light photocatalysis,29,30 electrocatalysis,28 and pi- ezocatalysis.31,32 Technologically important families of layered mixed oxides further include high-temperature superconducting cuprates,33 and lithium and sodium transition-metal layered oxides (LiMO2 and NaxMO2, M = transition metal), which are used as the cathode mate- rials in batteries.34,35 In all these materials we can expect that similar mechanisms will govern the adaptation of the crystal structure to the nanoscale. Attempts to study the adaptation of such complex crystal structures to the restricted sizes of nanoparticles have seldom been reported in the scientific literature. To the best of our knowledge also, the metastable polymorphs of layered mixed oxides stabilized at the nanoscale were not reported prior to our work. The reason is primarily related to the difficult synthesis of such complex materi- als in the form of small nanoparticles, which usually in- volves high temperatures that lead to the rapid growth of particles and favour thermodynamically stable phases. The hydrothermal method is one of the few methods enabling the direct synthesis (without a calcination stage) of layered mixed oxides, such as hexaferrites and bismuth titanate. The method involves the precipitation of the constituting cations from the aqueous solutions with a strong hydrox- ide, usually NaOH, followed by a hydrothermal treatment, i.e., the alkaline aqueous suspension of the precipitated hydroxides is heated in a closed autoclave at an elevated temperature (typically around 200 °C) and an equilibrium water pressure.36 On the other hand, the characterization of small nanoparticles with a complex structure is very challeng- ing. Conventional methods based on x-ray diffraction (XRD) are not efficient for the characterization of small nanoparticles. In our research we combined direct atom- ic-resolution high-angle annular dark-field (HAADF) imaging with a probe spherical-aberration corrected scanning-transmission electron microscope (STEM) with other microscopy techniques (energy-dispersive X-ray spectroscopy (EDXS), electron-energy-loss spectroscopy (EELS)) and XRD to examine the nanoparticle structures. HAADF imaging enables “Z-contrast”, as the intensity of the spots representing individual atomic columns in the atomic resolution images of the crystal depends on the col- umn’s average atomic number Z (~Zα with α slightly lower than 2).37 Thus, the columns occupied by the heavy cations (Ba2+, Sr2+, Bi3+) can be clearly resolved from the columns of the lighter cations (Fe3+, Ti4+) (the O2− columns are too light to be visible in HAADF images). 2. Structure of Barium and Strontium Hexaferrite Nanoplatelets 2. 1. Magnetoplumbite Structure Barium hexaferrite (BaFe12O19, or BHF) and stron- tium hexaferrite (SrFe12O19, or SHF) are the simplest members of a large family of hexagonal ferrites (i.e., hexa- ferrites) that can be formed by repeatedly stacking layers of three structural building blocks: the “S” block (MeFe4O8, where Me denotes either a divalent (e.g., Zn2+, Co2+) or trivalent (Fe3+) ion), the “R” block (AFe6O11, where A denotes a large divalent ion Ba2+, Sr2+, or Pb2+), and the “T” block (A2Fe8O14), along the c-axis of the hexagonal structure. The BHF and SHF are also known as M-type hexaferrites. They represent the simplest members of the hexaferrite family with a magnetoplumbite structure composed of only Ba2+/Sr2+ ions and Fe3+ ions arranged in the hexagonal “R” block ((BaFe6O11)2–) and the cubic “S” block ((Fe6O8)2+) (Note that the magnetoplumbite structure is frequently simply referred to as the “hexa- ferrite” structure). The unit cell (S.G.: P63/mmc, a = 5.88 Å, c = 23.18 Å) can be illustrated by the RSR*S* stacking sequence, where the asterisk denotes the rotation of the block by 180° around the hexagonal c-axis. Within the structure, the Fe3+ ions occupy five different lattice sites, i.e., one tetrahedral (4f1), three octahedral (12k, 2a, 4f2), and one trigonal (2b) (see Figure 2(a)).15 Due to the an- isotropic, layered structure, the growth of the hexaferrite crystals is limited in the c-direction, resulting in nanopar- ticles growing in the form of thin hexagonal platelets., i.e., nanoplatelets (NPL).16 2. 2. Hydrothermal Synthesis of Hexaferrite Nanoplatelets Hexaferrite nanoplatelets can be efficiently synthe- sized using a simple and scalable hydrothermal meth- od.7,18,39–45 This method is based on the hydrothermal treatment of an aqueous suspension of the corresponding hydroxides in the presence of a high concentration of hy- droxyl ions. An excess of Ba2+/Sr2+ ions is used to avoid the parallel formation of hematite. After the hydrothermal treatment the product is washed with a dilute acid to dis- solve any Ba/Sr-rich compounds that formed due to the excess Ba/Sr. Ba- and Sr-rich compounds (e.g., BaCO3(s), SrCO3(s)) are all very soluble, whereas the hexaferrite is completely insoluble.7,39–42,45 The evolution of the morphology of the formed NPLs during hydrothermal synthesis is very similar for the two hexaferrites: BHF and SHF. The primary, ultraf- 759Acta Chim. Slov. 2022, 69, 756–771 Makovec: Adaptation of the Crystal Structure to the Confined Size ... ine discoid NPLs, around 10 nm wide and less than 3 nm thick, already form at temperatures below 100 °C (given a sufficient time for the hydrothermal treatment) (Figure 1). The ultrafine NPLs exhibit weak magnetic properties. With an increased temperature of the hydrothermal treat- ment, the size of the NPLs remains almost constant up to approximately 150 °C, when individual NPLs start to grow with the mechanism of Ostwald ripening (Figure 1). With Ostwald ripening the NPLs obtain their charac- teristic shape of hexagonal platelets, which reflects their hexagonal structure. And only after the Ostwald ripening do the NPLs obtain their hard-magnetic properties with a sizable saturation magnetization, characteristic for hexa- ferrites. The Ostwald ripening (sometimes also referred to as a secondary recrystallization) is a special mechanism, where individual particles grow very rapidly at the ex- pense of other particles, which dissolve. Because of this very rapid growth, the phenomenon is also referred to as anomalous or exaggerated growth.The size of particles that grow exaggeratedly is very difficult to control. Hexaferrite NPLs with a size below 100 nm can only be obtained if the hydrothermal treatment is stopped immediately after the start of the exaggerated growth. If the exaggerated growth is allowed to proceed, the widths of the platelets rapidly exceed 1 µm, while their thickness increases much more gradually (Figure 1).7,39 The exaggerated growth can be regulated to some extent with chemical substitutions. For example, if the Fe3+ in the composition of the BHF is par- tially substituted with Sc3+, the exaggerated growth is sup- pressed to some extent, enabling the controlled synthesis of NPLs with a relatively narrow distribution of widths centred around 50 nm.42,44 Alternatively, the growth of NPLs can be mediated with appropriate surfactants, such as oleic acid.39 Figure 1. TEM micrographs of the BHF NPLs hydrothermally synthesized for 24 hours at 80 °C (a), and by heating the autoclave to 150 °C (b), 160 °C (c), and 200 °C (d). 760 Acta Chim. Slov. 2022, 69, 756–771 Makovec: Adaptation of the Crystal Structure to the Confined Size ... 2. 3. Structure of Hexaferrite Nanoparticles Obtained by Exaggerated Growth For the hydrothermal synthesis of hexaferrite NPLs with applicable magnetic properties the exaggerated growth is necessary; however, it must be stopped before the NPLs grow too large. Usually, the NPLs with widths below 100 nm are required for various applications.42,44 For widths around 100 nm, the hexaferrite NPLs remain very thin, i.e., their thickness remains less than two unit- cell parameters of their hexagonal structure in the corre- sponding c-direction (c = 23.18 Å). Already after analyses of the first successfully synthesized NPLs with a high-res- olution transmission electron microscope (HRTEM) they appear to exhibit a specific structure. For the small thickness it seemed logical that their layered structure will be terminated at the basal surfaces always at the same, low-energy crystal plane. In the layered structure, such a specific structure should also result in a deviation from the bulk composition. For example, if the hexaferrite NPLs were to terminate at the basal surfaces with the Ba-con- taining planes, their composition would be Ba-rich, and vice versa. However, our analyses of the BHF NPLs’ struc- ture with a combination of XRD, X-ray absorption fine structure (XAFS), Mössbauer spectroscopy, HRTEM, and EDXS could not unambiguously confirm this hypothesis.46 The structure of the BHF NPLs was revealed later, when we applied HAADF imaging with a STEM.8 Figure 2 (b) shows a HAADF STEM image of an exaggeratedly grown BHF NPL oriented edge-on, i.e., with the basal surfaces parallel to the electron beam. The Ba-containing columns can be clearly resolved from the lighter Fe columns. The positions of the cations in the projected magnetoplumbite structural model are super- imposed over the experimental image of Figure 2 (b) to reveal the NPL structure. The NPL in Figure 2 (b) contains only two Ba-containing R structural blocks and its cationic sublattice terminates at the basal surfaces with a layer of Fe ions at the octahedral Fe(12k) sites, i.e., with the complete S structural block. It should be noted that the oxygen col- umns are not visible on the HAADF images. However, it is expected that the NPLs’ structure is terminated with an oxygen-terminating layer in the air. An analysis of a large number of NPLs showed that the NPLs with widths below 100 nm usually contained only two R blocks. Only seldom did the BHF nanoplate- lets contained three or four R blocks. However, the cati- onic sublattice of the NPLs’ structure always terminated at the basal surfaces with the same, Fe(12k) lattice plane. The content of the R blocks and the specific termination of the NPLs’ structure determine their thickness: the NPL containing two R blocks is ~3.1 nm thick. Moreover, all the NPLs of equal thickness have basically the same struc- ture across the NPL. The structure of a NPL containing two R structural blocks can be represented by the SRS*R*S stacking sequence across the NPL. With growth, the thick- ness of the NPLs cannot increase continuously, but in a discrete, stepwise manner, by gradually adding the SR seg- ments.7,8 The structure of exaggeratedly grown SHF NPLs was similar to that of the BHF NPLs.45 Due to the specific structure the hexaferrite NPLs display a different composition than the BaFe12O19 bulk. The composition of a NPL containing the two R blocks (SRS*R*S stacking) corresponds to the theoretical compo- sition BaFe15O23. This theoretical composition was con- firmed by a quantitative EDXS analysis. With increasing thickness, the nanoplatelet composition gradually ap- proaches that of the bulk.7,8,45 Note that although the the- Figure 2. A schematic representation of the magnetoplumbite structure (a) and HAADF STEM image of a hexaferrite nanoplatelet oriented along the <10-1 0> direction of its magnetoplumbite structure (b). In image (b) the projected magnetoplumbite structure is superimposed to illustrate the positions of the Ba2+ and Fe3+ columns. Different Fe lattice sites (trigonal 2b, tetrahedral 4f1, octahedral 12k, 2a, and 4f2) are marked. (Reproduced from the publication44 by D. Makovec, M. Komelj, G. Dražić, B. Belec, T. Goršak, S. Gyergyek, D. Lisjak, Acta Mater. 2019, 172, 84–91 under the terms of CC BY 4.0 license). 761Acta Chim. Slov. 2022, 69, 756–771 Makovec: Adaptation of the Crystal Structure to the Confined Size ... oretical composition of the nanoplatelets containing two R blocks is equal to the composition of the Fe2+-contain- ing X-type hexaferrite Ba2Fe2+2Fe3+28O46, the stacking of the structural blocks in the X-type hexaferrite is different (RSRSSR*S*R*S*S*) and its unit cell is much larger (c = 84.1 Å) than the thickness of the nanoplatelet.47 Moreover, the specific composition of the thin NPLs is not related to the change in the oxidation state of Fe ions. The experimen- tal determination of Fe valence state of the NPLs using an analysis of the EELS spectra showed the Fe valence to be close to 3+.7 The termination of the NPLs’ structure at the basal surfaces was found to depend on the conditions applied during their preparation. The S-block-terminated struc- ture was actually only found in the NPLs washed with diluted acid after the hydrothermal synthesis (Figure 3 (b)).7,8 If the nanoplatelets were extracted from the reac- tion mixture before the washing step, they were terminat- ed at the basal surfaces with the Ba/O/Fe(2b) mixed planes (Figures 3 (a) and (c)). Interestingly, during the washing not only was the top Ba-containing plane dissolved, but also the first Fe plane beneath (Fe(4f2)) was dissolved from the surface to reveal the S-terminated structure (Figure 3 (a)).7 Experimental observations of the NPLs’ structure termination were supported with ab initio calculations of the relative stability of different terminations of the BHF performed using the density functional theory (DFT). The first calculations of a periodic structure resembling the bulk were performed by Matej Komelj from the Jožef Stefan Institute, Ljubljana, Slovenia. These calculations suggested that in energy terms the most stable surface lay- Figure 3. Two structural models schematically representing the structure across the hexaferrite NPL before (a) and after (b) washing with dilute nitric acid. Figures (c) and (d) show HAADF STEM images of BHF NPLs after washing with diluted acid (c) and after annealing for 2 hours at 700 °C. The projected magnetoplumbite structure is superimposed over the images (c) and (d) to illustrate the positions of the Ba2+ and Fe3+ columns. The termination layer at the basal surfaces of the NPLs is marked with a yellow rectangle. (Adapted from the publication by D. Makovec, B. Belec T. Goršak, D. Lisjak, M. Komelj, G. Dražić, S. Gyergyek, Nanoscale 2018, 10, 14480–14491 with permission from The Royal Society of Chemistry.7) 762 Acta Chim. Slov. 2022, 69, 756–771 Makovec: Adaptation of the Crystal Structure to the Confined Size ... er is obtained with a termination of mixed Ba/O/Fe(2b) planes.8 Such a structure was observed experimentally when the NPLs were annealed at high temperatures. Fig- ure 3(d) shows an atomic-resolution HAADF STEM im- age of the platelet crystal obtained by annealing the BHF NPLs at 700 °C. At that high temperature the NPLs grew to larger, plate-like crystals resembling the bulk. At their flat, basal surfaces they were clearly terminated with the Ba-containing planes.7 The difference in the termination of the structure between the NPLs and the larger crystals after annealing highlights the crucial role of the chemical environment, which was not taken into the consideration during the calculations. We must bear in mind that the NPLs after the hydrothermal synthesis are suspended in an aqueous solution containing high concentrations of dif- ferent ions, mainly originating from an excess amount of Ba2+ ions and a high concentration of NaOH, hydrating the surfaces. Later, the ab initio calculations were extended to thin structures, which resembled the structure of the primary nanoplatelets.7 The calculations of the relative stability of thin structures symmetrically terminating at both basal surfaces at different lattice planes showed the highest sta- bility for a structure terminated with the O layers above the Fe(4f1) of the S block; however, its stability was just slightly higher than the termination at the O-only layers above the Fe(12k) layers, experimentally determined as the termination layers for the washed BHF nanoplatelets. Recently, detailed DFT calculations were performed by the group from Scuola Internazionale Superiore di Studi Avanzati (SISSA), Trieste, Italy, led by Layla Mar- tin-Samos. Their calculations clearly demonstrated the influence of the chemical environment on the equilibrium structure of BHF NPLs. In the absence of water (and other species), the calculations showed that the structure ter- minating with the mixed Ba/O/Fe(2b) planes is the most stable. The same surface is also the most stable in oxygen/ iron-poor (Ba-rich) conditions, whereas the fully hydrox- ylated 12k-O surface is the most stable in oxygen/iron-rich conditions, in line with the experimental observations.48 In conclusion, the BHF and SHF NPLs synthesized via exaggerated growth (i.e., Ostwald ripening) during the hydrothermal treatment show specific structures and com- positions, defined by the termination of the particle at its surfaces with a specific, low-energy atomic layer. The ter- mination layer depends on the chemical environment. As the structure and composition of hexaferrite NPLs are sig- nificantly different from the bulk, they can be considered as a novel structural variation of the hexaferrites stabilized on the nanoscale. An equivalent adaptation mechanism can also be expected for other mixed oxides with a layered struc- ture when they are synthesized in the form of thin NPLs. 2. 4. Structure of Primary Hexaferrite Nanoplatelets The NPLs that grow exaggeratedly during the hy- drothermal synthesis exhibit the same crystal structure for both the hexaferrite analogues: BHF and SHF. In contrast, the primary NPLs, i.e., the nanoparticles that appeared as the first product in the initial stage of the hydrothermal synthesis, exhibit a completely different structure for the two hexaferrite analogues, even though they had a similar discoid morphology. Atomic-resolution HAADF STEM imaging showed that the washed primary BHF NPLs ex- hibit a specific variation of the magnetoplumbite structure – the smallest possible structural segment still maintaining the hexaferrite motif. Figure 4 shows a HAADF STEM im- Figure 4. HAADF STEM images of a primary BHF NPL with corresponding EELS elemental maps for Ba and Fe (the area of the analysis is marked on the image with a red rectangle). An SRS* segment of the hexaferrite structure projected along the <10-1 0> direction is superimposed over the image to illustrate the positions of the Ba2+ and Fe3+ ions. (Adapted from the publication by D. Makovec, B. Belec T. Goršak, D. Lisjak, M. Komelj, G. Dražić, S. Gyergyek, Nanoscale 2018, 10, 14480–14491 with permission from The Royal Society of Chemistry.7) 763Acta Chim. Slov. 2022, 69, 756–771 Makovec: Adaptation of the Crystal Structure to the Confined Size ... age of a primary BHF NPL oriented edge-on. Even though the NPL is not perfectly oriented along the zone axis and an additional layer of adsorbed atoms is visible on its sur- faces, its structure is composed of only one Ba-containing R structural block sandwiched between the two S blocks. The thickness of the SRS*-structured NPLs is therefore less than the dimension of one magnetoplumbite SRS*R* unit cell. Their composition is significantly enriched in Fe com- pared with the theoretical composition corresponding to the chemical formula BaFe18O28.7 Note, that the theoreti- cal composition of the SRS*-structured primary BHF nan- oplatelets is equal to the composition of the Fe2+-contain- ing W-type hexaferrite (BaFe2+2Fe3+16O27) with different stacking of the structural blocks (SSRS*S*R*).47 Even though the structure and composition of the primary BHF NPLs strongly deviates from the bulk, their structure remains a specific variation of the hexaferrites. Their structure can therefore be understood in the context of the adaptation of the magnetoplumbite structure to the restricted size. In contrast to the primary BHF NPLs, the primary SHF NPLs display an entirely different structure. Already during our first study devoted to the hydrothermal synthe- sis of SHF NPLs we noticed that the structure of the prima- ry SHF NPLs deviates from the magnetoplumbite structure. HRTEM images showed a dominant periodicity across the primary SHF NPLs that was considerably smaller (~ 9 Å) than that for the magnetoplumbite (11.5 Å corresponding Figure 5. BF and HAADF STEM images of primary SHF NPLs with the corresponding calculated FFT pat- terns. The NPLs are oriented along the <0001> direc- tion (a) and along the <11-2 0> direction of its complex hexagonal structure (b). A unit cell of the structure is marked with a red rectangle on (b). (Adapted from the publication by D. Makovec, G. Dražić, S. Gyergyek, D. Lisjak, CrystEngComm 2020, 22, 7113−7122 with per- mission from The Royal Society of Chemistry.45) 764 Acta Chim. Slov. 2022, 69, 756–771 Makovec: Adaptation of the Crystal Structure to the Confined Size ... to (0002)).41 However, the NPLs were too small for any re- liable structural analysis with conventional methods (e.g. XRD). Later, direct atomic-resolution HAADF STEM im- aging showed that the primary SHF NPLs exhibit an exotic layered hexagonal structure, not reported before (Figure 5).45 Due to the severe complexity of the new structure and the small thickness of the NPLs, we were not able to propose a specific structural model. However, it was evident that the new layered structure is not a variation of the hexaferrite structure. The structure was described with HAADF STEM images having an unusually large unit cell. A basic perio- dicity unit cell with a* ≈ 28.3 Å, c* ≈ 18.0 Å is marked on the atomic-resolution HAADF image taken along the <11-2 0> direction (Figure 5(b)), however the atomic-resolution images along the <10-1 0> direction suggested doubling of the unit cell in the a-direction.45 Along the c-direction, the structure is composed of five layers of cations: three neigh- bouring layers containing Fe3+ and Sr2+ ions are separated by two Fe3+-only layers. The structure of the discoid NPLs always terminates at the basal surfaces with an Fe3+-only layer. The EDXS analysis showed an Fe-rich composition when compared to the SrFe12O19 bulk. When the prima- ry SHF NPLs were annealed above 500 °C they grew and transformed into the magnetoplumbite structure.45 The complex structure of primary SHF NPLs can- not be formed because of the adaptation of the magne- toplumbite structure to the restricted size. It is clearly a metastable polymorph of the SHF stabilized at the nano- scale. It can, therefore, be explained in the context of the Ostwald step rule, where the phases with a lower thermo- dynamic stability form in the initial stages of the synthesis because of the lower nucleation barrier. With the growth of the nanoparticles at high temperatures the metastable polymorph transforms into the thermodynamically stable magnetoplumbite structure. In conclusion, the primary NPLs, which appear as the first crystalline products of the hydrothermal synthe- sis, exhibit different structures for the two hexaferrites. The structure of the primary BHF NPLs is an SRS* var- iation of the SRS*R*magnetoplumbite structure, whereas the primary SHF NPLs exhibit a different, complex layered structure, which is a metastable polymorph of the SHF. The striking difference in the structure of the primary NPLs can be related to the thermodynamic stability of the two hexaferrites. The stability of the AFe12O19 hexaferrites decreases with the decreasing size of the A ion in the se- ries: Ba2+ > Sr2+ > Ca2+.15 3. Structure of Bismuth Titanate Nanoplatelets and Nanowires 3. 1. Aurivillius Structure Bismuth titanate (Bi4Ti3O12, or BIT) belongs to an Aurivillius ((Bi2O2)(An−1BnO3n+1) family of layered per- ovskites. Its layered structure derives from the high-tem- perature paraelectric phase of tetragonal I4/mmm sym- metry, composed of two alternating layers: a (Bi2O2)2+ layer and a perovskite (Bi2Ti3O10)2− layer stacked along the pseudo-tetragonal c-axis (see Figure 7(a)). With the onset of ferroelectricity below the Curie temperature (TC ~ 675 °C the BIT structure is slightly distorted to monoclinic symmetry P1a1 with parameters a = 5.411 Å, b = 5.448 Å, c = 32.83 Å.49 3. 2. Hydrothermal Synthesis of Bismuth- Titanate Nanoplatelets and Nanowires BIT can be successfully synthesized with the hydro- thermal treatment of an aqueous suspension of precipi- tated Bi3+ and Ti4+ ions in mineralizer hydroxide (NaOH or KOH) with a moderate concentration.27,28,30–32,50–53 The synthesized BIT nanoparticles appear in a wide va- riety of different nano-morphologies, including 2-D platelet crystals, (i.e., rectangular nanoplatelets and nano- sheets),27,28,32,50,52,53 1-D crystals (e.g., nanowires, nano- belts, nanobundles, nanorods),27,28,51–53 and 3-D nanos- tructures assembled from 1-D or 2-D nanoparticles.30,31,51 Our research showed that the morphologies of the prod- uct particles crucially depend on the concentration of the mineralizer hydroxide.52 The nanowires (NWs) formed when the precipitated ions were hydrothermally treated in aqueous solutions of NaOH with lower concentrations, whereas the nanoplatelets (NPLs) were obtained at high- er NaOH concentrations. For example, the hydrothermal treatment for 38 hours at 200 °C produced NWs (from 15 nm to 35 nm wide and from several hundreds of nm to several µm long) in 0.5 mol/L NaOH, whereas the rectan- gular NPLs (approximately 10 nm thick and from 50 nm to 200 nm wide) were synthesized in 2-mol/L NaOH (Figure 6 (a) and (b)). The NWs formed as the first crystalline BIT phase in the initial stages of the hydrothermal treatment independently of the NaOH concentration. Initially, the BIT NWs appeared together with globular aggregates of nanocrystallites with a highly defected perovskite struc- ture. The subsequent morphology evolution depended on the concentration of the hydroxide, which influences the stability of the different phases and the kinetics of the hy- drothermal reactions. At lower NaOH concentrations the NWs grew with the treatment temperature/time, while the perovskite aggregates dissolved. At the higher NaOH con- centrations, the nanowires dissolved, while the NPLs grew epitaxially on the surfaces of the aggregated perovskite na- nocrystallites (Figure 6 (c)).52 3. 3. Structure of Bismuth-Titanate Nanoplatelets The plate-like shape of the hydrothermally synthe- sized BIT rectangular NPLs is consistent with their pseu- do-tetragonal layered Aurivillius-type structure. Figure 7 765Acta Chim. Slov. 2022, 69, 756–771 Makovec: Adaptation of the Crystal Structure to the Confined Size ... (b) shows the HAADF STEM image of a small BIT NPL, oriented edge-on along the [110] direction of its pseu- do-tetragonal structure. The two layers of the Aurivillius structure, i.e., a (Bi2O2)2+ layer and a (Bi2Ti3O10)2– per- ovskite-like layer, can be clearly distinguished. The NPL structure always terminates at the large {001} surfaces with the (Bi2O2)2+ layers. Thus, it is expected that the thin BIT NPLs will be Bi-rich compared to the Bi4Ti3O12 bulk. However, the BIT NPLs were generally much thicker than the hexaferrite NPLs presented above, usually more than 10 nm thick, so that their actual composition approached to the bulk composition. Figure 6. TEM images of bismuth-titanate NPLs (a) and NWs (b) synthesized with hydrothermal treatment of the precipitated Bi and Ti ions for 38 hours at 200 °C in the aqueous solution of NaOH with the concentration of 2 mol/L and 0.5 mol/L, respectively. (c) A schematic presentation of the morphology evolution during hydrothermal synthesis. Independently of the NaOH concentration, a mixture of NWs and globular nanocrystalline particles with a defected perovskite structure forms first. With temperature/time of the treatment in NaOH with a lower concentration, the per- ovskite aggregates dissolve, while the NWs grow. At a higher NaOH concentration, the NWs dissolve, while on the surfaces of the perovskite aggre- gates the NPLs grow epitaxially. ((a) and (b) are reproduced from the publication by D. Makovec, N. Križaj, A. Meden, G. Dražić, H. Uršič, R. Kostan- jšek, M. Šala, S. Gyergyek, Nanoscale 2022, 14, 3537–3544,52 (c) adapted from the publication by D. Makovec, N. Križaj, S. Gyergyek, CrystEngComm 2022, 24, 3972–398, with permission from The Royal Society of Chemistry.52) 766 Acta Chim. Slov. 2022, 69, 756–771 Makovec: Adaptation of the Crystal Structure to the Confined Size ... 3. 4. Structure of Bismuth-Titanate Nanowires The 1-D shape of the NWs, which always form in the initial stages of the hydrothermal synthesis, is not consistent with the layered Aurivillius structure (the pseudo-tetragonal layered structure will tend to form rectangular platelet crystals). Indeed, the XRD of the BIT NWs suggested a new structure, entirely different to the Aurivillius structure characteristic for the BIT.52 (As ex- plained below, the NW structure does not contain any perovskite-like layers). EDXS analyses showed the same composition for the two BIT morphologies within the un- certainty of the method. The chemical ICP-OES analysis further confirmed the EDXS analyses and showed that so- dium from the NaOH used as the mineralizer hydroxide for the synthesis was not incorporated into the NW struc- ture in a significant concentration. Electron diffraction in the TEM suggested an orthorhombic unit cell. Based on the orthorhombic cell and the cell parameters obtained from the electron diffraction it was possible to find a unit cell that gave a satisfactory LeBail fit to the experimental powder XRD pattern with the refined unit-cell parame- ters: a = 3.804(1) Å, b = 11.816(3) Å, and c = 9.704(1) Å.53 Atomic-resolution STEM imaging confirmed that the NWs exhibit a different structure to that of the NPLs. Figure 8 shows HAADF STEM images of NWs orient- ed normal to the electron beam (in the [010] zone axis) and along the beam (in the [100] zone axis). Based on the HAADF STEM imaging and the analysis of the XRD pat- tern, a tentative arrangement of the cations in the structure was proposed (Figure 8 (a)). The structure of the NWs is composed of two layers stacked along the c-direction of the orthorhombic unit cell: a layer composed of two par- allel rows of Bi atoms in a zig-zag arrangement (marked with “B” in Figure 8 (c)) and a layer of two rows of Ti at- oms in a zig-zag arrangement, where every sixth Ti is ex- changed with Bi (marked with “T”). The B layer resembles the (Bi2O2)2+ layer of the Aurivillius structure, while the T layer is much thinner than the perovskite (Bi2Ti3O10)2- layer of the Aurivillius structure. The arrangement of the Ti atoms in the T layer is consistent with two layers of edge-sharing (TiO6)2− octahedra, as opposed to the cor- ner-sharing (TiO6)2− octahedra of the perovskite layers. The proposed cation arrangement in the NW structure predicts a Bi:Ti ratio of 7:5, which deviates from the BIT composition (Bi:Ti = 4:3). The excess Bi, predicted by the model, is most probably compensated by a random partial insertion of the Ti layers parallel to the (010) planes, visi- ble along the [100] direction (Figure 8 (e)).53 If the BIT NWs were annealed at high temperature, they transformed to the Aurivillius structure. In the pow- der obtained by annealing the NWs for 2 hours at 525 °C some nanoparticles were just partially transformed. A HAADF STEM analysis (Figure 9) showed that the trans- formation of the NW structure to the Aurivillius (AU) structure is topotactic ((100)NW || (110)AU, (001)NW || (001)AU), (010)NW || ( -1 10)AU).53 The analysis also strong- ly suggested that the transformation does not involve any changes to the composition (the precipitation of second- ary phases was not detected). This is a strong indication that the NWs are actually a metastable polymorph of BIT stabilized at the nanoscale. With growth at high tempera- tures the metastable NW structure transforms to the ther- modynamically stable Aurivillius structure. 4. Final Remarks and Future Directions The chemical composition and the crystal structure of materials tend to define their functional properties. Nowadays, discoveries of entirely new crystal structures of Figure 7. Schematic representation of the magnetoplumbite structure (a), and BF (b) and HAADF (c) STEM images of BIT NPL oriented along [110] direction of the pseudo-tetragonal Aurivillius structure (b). The projected structural models are superimposed over the image (c) to illustrate the positions of the Bi3+ and Ti4+ ions. The arrow marks a defected perovskite block (Adapted from the publication by D. Makovec, N. Križaj, S. Gyergyek, CrystEngComm 2022, 24, 3972 – 398, with permission from The Royal Society of Chemistry.52) 767Acta Chim. Slov. 2022, 69, 756–771 Makovec: Adaptation of the Crystal Structure to the Confined Size ... inorganic materials are seldom. However, when materials are synthesized in the form of nanoparticles, their crystal structure can change significantly because of its adaptation to the restricted size. The extent of these structural changes increases with the increasing complexity of the structure. In this article, research at the Department for Materials Synthesis, Jožef Stefan Institute, devoted to the adaptation of complex structures built from alternating layers of two structural blocks to the restricted size of nanoparticles was presented based on two examples of well-known and tech- nologically very important materials, the hexaferrites BHF and SHF, and the Aurivillius layered perovskite, BIT. Both examples confirmed that the nanoparticles with layered structures will normally adopt a plate-like shape and a spe- cific structure defined by a termination at surfaces with a specific, low-energy atomic layer. For a small thickness of the platelet crystal, the structure can significantly deviate from the ordered bulk structure. Moreover, the adaptation of the crystal structure to the restricted size will result in a deviation from the bulk composition. With the defined structure and composition, which are different to those of the bulk, the NPLs can even be considered as new com- pounds or at least variations of the bulk structures. The adaptation of the structure to the restricted size will certainly contribute to changed material prop- erties when compared to the bulk properties. This aspect of the size effect has seldom been studied. Since the size has a large effect on the properties per se, it is difficult to estimate the extent of the size effect, which is specifical- ly related to the changed structure. However, there are some indications of positive effects of the structural ad- aptation on the functional properties of nanoparticles. For example, we can speculate that the SRS*R*S-structured hexaferrite NPLs exhibit increased magnetization due to the surface termination with the S block, because the S block has a larger theoretical saturation magnetization Figure 8. Cation arrangement in a tentative model of an orthorhombic structure of BIT NW (a), TEM (b) and HAADF STEM (c) images of NW orient- ed along the [010] direction, and HAADF STEM images ((d) and (e)) of NW oriented along the [100] direction of the orthorhombic structure. The projected model proposed for the NW structure is also superimposed over the images (c) and (e) to illustrate the positions of the Bi3+ and Ti4+ ions. The unit cell of the NW structure is marked with a red rectangle on the HAADF image. (Adapted from the publication by D. Makovec, N. Križaj, A. Meden, G. Dražić, H. Uršič, R. Kostanjšek, M. Šala, S. Gyergyek, Nanoscale 2022, 14, 3537–3544 with permission from The Royal Society of Chemistry.53) 768 Acta Chim. Slov. 2022, 69, 756–771 Makovec: Adaptation of the Crystal Structure to the Confined Size ... than the R block.8 We also found one example where the BHF nanoplatelets behave completely differently than the bulk. A partial substitution of Fe3+ ions in BHF with Sc3+ ions greatly improves the saturation magnetization of the NPLs. The effect was surprising since the substitution of diamagnetic Sc strongly decreases the saturation magnet- ization of bulk BHF. Ab-initio calculations pointed to a specific, two-dimensional magnetic ordering in the NPLs as the most probable reason for the opposite effect of the Sc substitution in the NPLs to that in the bulk.44 The metastable polymorphs, which are frequently obtained because they have a lower nucleation barrier compared to the thermodynamically stable phase, have seldom been considered in the context of size effect, even though they remain stable only while in the form of small particles, i.e., at the nanoscale. Many well-known and technologically very important nanomaterials, e.g., magnetic maghemite nanoparticles and catalytic anatase nanoparticles, are actually metastable polymorphs sta- bilized on the nanoscale. Interestingly, metastable pol- ymorphs were not reported for complex materials such as mixed oxides with a layered structure. To the best of our knowledge the structure of primary SHF NPLs repre- sented the first reported inorganic metastable polymorph with a complex, layered structure, stabilized at the nano- scale. The NPLs with the newly discovered SHF structure exhibit weak magnetic properties, comparable to those of the SRS*-structured BHF NPLs. Other functional prop- erties, e.g., catalytic, electronic, etc., were not compared. To show that the discovery of a new structure is not only a special case specific for the SHF, we looked for new metastable polymorphs in other well-known materials with a layered structure. The discovery of the new BIT polymorph clearly demonstrated that the metastable pol- ymorphs can generally also be expected to form in mixed oxides with a layered structure. Observations of the fer- roelectric domains with TEM and piezo-response force microscopy indicated the ferroelectric nature of the BIT NWs polymorph;53 however, their functional properties remain to be studied. Nevertheless, the discovery of new polymorphs demonstrated the immense potential of the stabilization of new metastable polymorphs of complex functional materials for the discovery of new nanoma- terials. It is possible that a metastable polymorph of a known complex functional material, which will exhib- it improved or even entirely new functional properties, could be discovered. Both aspects of the influence of the size effect on the crystal structure of nanoparticles, i.e., the adaptation of the structure to the restricted size and the stabilization of met- astable polymorphs on the nanoscale, are also important in the context of materials synthesis. For example, the ad- aptation of the hexaferrite structure to the small size of hy- drothermally synthesized nanoplatelets will influence the final composition of hexaferrite ceramics after sintering.54 Moreover, we showed that the metastable polymorphs have an important role in the hydrothermal synthesis of BIT nanoparticles.52 Advances in controlled synthesis enabling the syn- thesis of small nanoparticles of inorganic materials with a complex structure, on the one hand, and the advances in structural characterization based on advanced electron microscopy, on the other, will pave the way to further dis- coveries of nanoparticles with new and interesting crystal Figure 9. HAADF STEM images of a BIT particle from the NW sample annealed for 2 hours at 525 °C. The particle is composed of a NW sandwiched between two lamellas with the Aurivillius (AU) structure ([010]NW||[110]AU). Projected models of the AU structure and the NW structure are super- imposed over (b) to illustrate the positions of the Bi2+ and Ti4+ ions. (Reproduced from the publication by D. Makovec, N. Križaj, A. Meden, G. Dražić, H. Uršič, R. Kostanjšek, M. Šala, S. Gyergyek, Nanoscale 2022, 14, 3537–3544 with permission from The Royal Society of Chemistry.53) 769Acta Chim. Slov. 2022, 69, 756–771 Makovec: Adaptation of the Crystal Structure to the Confined Size ... structures. And with new structures, new properties of the nanoparticles can be expected. Acknowledgements I would like to thank my colleagues at the Depart- ment for Materials Synthesis, Jožef Stefan Institute, Prof. Dr. Darja Lisjak and Asst. Prof. Dr. Sašo Gyergyek for a fruitful collaboration. The research contribution and ex- perimental help of former and current Ph.D. students and coworkers is also acknowledged: Dr. Darinka Primc, Dr. Blaž Belec, Tanja Goršak, Nina Križaj, and Bernarda Anželak. I am particularly grateful to Prof. Dr. Goran Dražić and other colleague microscopists from the Na- tional Institute of Chemistry and Jožef Stefan Institute for their help with the microscopy. I also have to acknowledge Prof. Dr. Matej Komelj from the Department for Nanos- tructured Materials, Jožef Stefan Institute, Prof. Dr. Lay- la Martin-Samos, Dr. Matic Poberžnik, and Dr. Gabriela Herrero-Saboya from SISSA, Trieste, for the theoretical considerations, Prof. Dr. Anton Meden from Faculty of Chemistry and Chemical Technology, University of Lju- bljana for the XRD analyses of the BIT nanoparticles, Prof. Dr. Rok Kostanjšek from the Biotechnical Faculty, Univer- sity of Ljubljana, for the help with TEM specimen prepa- ration using microtome, and Dr. Martin Šala from the National Institute of Chemistry for the ICP chemical anal- yses. The financial support from the Slovenian Research Agency (ARRS) within research core funding No. P2-0089 is greatly appreciated. 5. References 1. G. 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Koch, Determination of Core Structure Periodicity and Point Defect Density along Dislocations. Ph.D. Thesis, Arizona State University, 2002. 92. D. Lisjak, A. Mertelj, Prog. Mater. Sci. 2018, 95. 286–328. DOI:10.1016/j.pmatsci.2018.03.003 Povzetek Uporabne lastnosti materialov so v veliki meri določene s sestavo in kristalno strukturo materialov. Struktura materiala in posledično tudi njegova sestava se lahko znatno spremenita, če material pripravimo v obliki nanodelcev. Poznava- nje sprememb v kristalni strukturi zaradi končne dimenzije nanomaterialov je torej pomembno tako s stališča širjenja osnovnega znanja, kot tudi za načrtovanje novih nanomaterialov za uporabo v tehnologiji in medicini. Spremembe v strukturi so lahko posledica dveh različnih pojavov: (i) kristalna struktura se prilagodi končni velikosti nanodelcev, in (ii) z majhno velikostjo delcev lahko stabiliziramo različne metastabilne strukturne polimorfe, ki nastanejo med sintezo v tekočem zaradi nižje energijske pregrade za nukleacijo v primerjavi z energijsko pregrado potrebno za nukleacijo rav- notežnih faz. Omenjene spremembe v kristalni strukturi so posebej pogoste pri anorganskih materialih s kompleksno strukturo in sestavo, kot so zmesni oksidi s plastovito strukturi sestavljeno iz več strukturnih blokov. Pričujoči članek pojasnjuje kompleksno strukturo nanodelcev na primerih dveh dobro znanih in tehnološko zelo pomembnih materi- alov s plastovito strukturo: magnetnih heksaferitov (BaFe12O19 in SrFe12O19) in feroelektričnega bizmutovega titanata (Bi4Ti3O12) s plastovito perovskitno strukturo Aurivilliusovega tipa. Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License 772 Acta Chim. Slov. 2022, 69, 772–778 Yaqoob et al.: Synthesis, Characterization, Anti-Glycation, and ... Scientific paper Synthesis, Characterization, Anti-Glycation, and Anti-Oxidant Activities of Sulfanilamide Schiff Base Metal Chelates Muhammad Yaqoob,1 Waqas Jamil,1,* Muhammad Taha2 and Sorath Solangi1 1 Institute of Advanced Research Studies in Chemical Sciences, University of Sindh, Jamshoro, Pakistan 2 Institute for Research and Medical Consultations (IRMC), Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia * Corresponding author: E-mail: waqas.jamil@usindh.edu.pk Received: 02-21-2022 Abstract The current study reports synthesis, structure establishment, anti-glycation, and anti-oxidant activities of ligand 4-[(2-hydroxynaphthalene-1-ylmethylene)-amino]-benzenesulfonamide (L) and its coordination compounds with Mn(II), Co(II), Ni(II), Cu(II), and Zn(II) metal ions. The analytical techniques used (UV-Vis, FT-IR, CHN/S) confirmed the bidentate nature of the ligand, coordinating via O and N atoms in 2:1 ligand-to-metal ratio. The TG/DTA anylsis displayed that these compounds are thermally stable. Furthermore, the synthesized compounds were evaluated for their anti-glycation and antioxidant potential and showed significant activities with IC50 values range 184.11–386.34 µM and 37.05–126.27 µM, respectively. The Mn (IC50 = 184.11 ± 2.11 µM), Ni (IC50 = 211.26 ± 1.46 µM), Cu (IC50 = 254.56 ± 1.16 µM), and Zn (IC50 = 276.43 ± 2.14 µM) metal complexes exhibited substantial anti-glycation activity and comparatively better activity than the standard rutin (IC50 = 294.4 ± 1.50 µM), whereas Zn complex (IC50 = 37.05 ± 1.53 μM) also showed better DPPH radical scavenging activity than the standard tert-butyl-4-hydroxyanisole (IC50 = 44.7 ± 1.21 µM). Keywords: 4-[(2-Hydroxynaphthalene-1-ylmethylene)amino]benzenesulfonamide (L), Coordination Compounds, An- ti-oxidant activity, Anti-glycation activity 1. Introduction Coordination chemistry deals with the study of co- ordination compounds or metal complexes. The group of ten elements (i.e. V, Cr, Fe, Mn, Co, Cu, Ni, Mo, Zn, and Cd) form many complexes with various biomolecules to execute different biological functions.1,2 These metal com- plexes are required for our bodies in very small quantities, but their excess or deficiency can cause many serious dis- eases.3,4 The clinical and commercial importance of metal complexes as medicinal drugs is increasing day by day for the treatment of various diseases.5,6 The synthesis of metal complexes as chemotherapeutic agents in clinical applica- tion has shown significant progress in medicinal chemis- try to fight against several human diseases, such as to treat different types of cancers, tumors, diabetes mellitus, an- ti-inflammation, possessing antifungal activity and acting against a wide range of bacterial diseases.7–9 Recently, the metal complexes of transition elements have shown great importance in materials synthesis, catalysis and photo- chemistry.10,11 The platinum metal complexes including cisplatin, carboplatin and nedaplatin are widely used drugs for cancer chemotherapy.12 Copper(II), zinc(II), vanadi- um(V) and oxidovanadium(V) metal complexes have been reported for their excellent antibacterial urease en- zyme inhibition and catalytic properties.13–17 According to the literature, sulfonamides have a va- riety of bioactivities, including antitumor, antimalarial, antimicrobial, antithyroid, antidiabetic, anti-HIV/AIDS, anti-parasitic, antiepileptic, and dihydropteroate syn- thetase inhibitors activities.18–21 Sulfonamide metal che- lates derivatives have also been reported for their anti-in- flammatory, antidiabetic, anti-HIV, anticancer, anti- carbonic anhydrase, diuretic, hypoglycemic, antithyroid, antimalarial, antitumor, anti-angiogenic, anti-tubercular, antibacterial, and antifungal activities.22–25 Moreover, gold sulfonamide chelates were also found to have applications for the treatment of skin disorders and rheumatoid arthri- tis.26 DOI: 10.17344/acsi.2022.7422 773Acta Chim. Slov. 2022, 69, 772–778 Yaqoob et al.: Synthesis, Characterization, Anti-Glycation, and ... Schiff bases are widely studied compounds due to their structural resemblance with the natural bioactive molecules and ease of synthesis of diverse structures.27,28 The importance of Schiff base complexes in supramolecu- lar chemistry, catalysis and material science, separation and encapsulation processes, biomedical applications and formation of compounds with unusual properties and structures has been well recognized and reviewed.29–31 Sulfonamides Schiff base complexes with cobalt(II), cop- per(II), nickel(II) and zinc(II) have shown significant in vitro antibacterial, antifungal, and cytotoxic properties. The N,N-chelating half-sandwich ruthenium(II) para-cy- mene complexes containing sulfonamide moieties also showed a broad range of therapeutic applications, which include the inhibition of various isoforms of carbonic an- hydrases (CAs).32 Glycation is a reaction of blood sugar with the pro- teins like collagen; when this reaction occurs in a great ex- tent advanced glycation products (AGEs) are formed which may further degrade protein and cause oxidative stress that damage cell membranes and produce diabetic complications such as neuropathy and diabetes retinopa- thy which further increase the rate of the aging processes. Oxidative stress is the main aspect of all living sys- tems which occurs due to the excess of free radicals. Due to oxidative stress, biochemical energy is converted into adenosine triphosphate with the help of oxygen, this bio- chemical reaction generates reactive oxygenated species (ROS). These ROS can damage lipids, proteins, and DNA by oxidation and cause many diseases such as cancer, brain disorders, rheumatoid arthritis, atherosclerosis, obesity, aging, diabetes and skin disease.33 Antioxidant compounds are used as health-protecting factors in food playing an important role in preventing many diseases. The antioxi- dant compounds obtained from plants such as carotenes, phenolic acids, vitamin C and E, phytate, and phytoestro- gens were found to be very helpful in decreasing the risks of many diseases. A number of synthetic compounds have also been reported having remarkable anti-oxidant prop- erties.34 The cosmetic and food industries are funded by several companies to promote the research of the synthesis of glycation-inhibiting ingredients and anti-oxidants in order to discover new anti-aging compounds for keeping skin youthful for a prolonged period. The latest research focuses on finding the ways for the inhibition of AGEs for- mation, and on reducing oxidative stress with the objective of promoting health by treating degenerative changes and mitigating the effect of lifestyle-related diseases.35 Earlier, we have reported anti-glycation and anti-ox- idant properties of isatin containing hydrazide Schiff base metal complexes.36 In the continuation of exploring metal complexes for bioactivities, herein we have synthesized 4-[(2-hydroxynaphthalen-1-ylmethylene)amino]ben- zenesulfonamide Schiff base ligand (L) and its metal (Mn, Co, Ni, Cu, Zn) complexes for the evaluation of anti-glyca- tion and anti-oxidant activities. 2. Experimental 2. 1. Physical Parameters Elemental (CHN/S), TG/DTA, and UV-Vis and met- al content analyses were done on Perkin Elmer`s 2400 Se- ries II and Diamond TG/DTA, Lambda 35 UV-Vis spec- trophotometer, and Analyst 800 atomic absorption spectrophotometer, respectively. FT-IR were performed by Thermoscientific iS10 IR spectrophotometer in the region 4000–600cm–1. Bruker 300 MHz spectrometer was used for 1H NMR experiments. EI-MS were measured on Finni- gan MAT-311A (Germany) mass spectrometer. Molar conductance was measured on Thermoscientific Orian 5 Star meter. 2. 2. Materials and Methods Analytical grade chemicals (Sigma-Aldrich) sulfan- ilamide, 2-hydroxynaphthaldehyde, acetic acid, ammoni- um acetate and metal salts viz MnSO4∙H2O, Co(CH- 3COO)2∙4H2O, Ni(CH3COO)2∙4H2O, CuCl2∙2H2O, and Zn(CH3COO)2∙2H2O were used for synthesis. Bovine se- rum albumin (BSA) was obtained from Research Organics (Cleveland, USA). 2. 3. Synthesis of 4-[(2-Hydroxynaphthalen-1- ylmethylene)amino]benzenesulfonamide ligand (L) 0.5 mol of 2-hydroxynaphthaldehyde was added to 50 mL methanol with 2 to 3 drops of acetic acid and then added equimolar amount of sulfanilamide and refluxed for about 8 hours. The solvent was evaporated, precipitate ob- tained, dried and recrystallized from ethanol (Figure 1). Figure 1. Synthesis of 4-[(2-hydroxynaphthalene-1-ylmethylene)amino]benzenesulfonamide ligand (L) 774 Acta Chim. Slov. 2022, 69, 772–778 Yaqoob et al.: Synthesis, Characterization, Anti-Glycation, and ... Yield 80%, m.p. 278 °C. FT-IR νmax 3290 (-OH, NH), 1622 (-C=N-), 1347 (O=S=O), 1586 (-C=C-) cm–1. 1H NMR (DMSO-d6, 300 MHz) δ 6.87 (s, 1H, OH), 9.68 (s, 1H, CH=N), 8.52 (d, 2H, Ar, J = 8.7 Hz), 7.97 (d, 2H, Ar, J = 8.3 Hz, 6.88 (d, 1H, J = 7.4 Hz), 7.02 (d, 1H, J = 7.4 Hz), 7.58 (d, 2H, Ar, J = 7.6 Hz), 7.44 (m, 2H). EI-MS m/z 326 (M+) 310, 246, 170, 157, 153, 77. Anal. Calcd for C17H14N2O3S: C, 62.56; H, 4.32; N, 8.85; S, 9.82. Found: C, 62.51; H, 4.30; N, 8.79; S, 9.80. 2. 4. Synthesis of 4-[(2-Hydroxynaphthalene- 1-ylmethylene)amino] benzenesulfonamide Ligand Metal Chelates MnSO4∙H2O, Co(CH3COO)2∙4H2O, Ni(CH- 3COO)2∙4H2O, CuCl2∙2H2O, and Zn(CH3COO)2∙2H2O metal salts were refluxed with the ethanolic solution of 4-[(2-hydroxynaphthalene-1-ylmethylene)amino]ben- zenesulfonamide ligand (L) and ammonium acetate for 6 h. Then, the solvent was evaporated and the obtained pre- cipitates were isolated and washed with water. The struc- tures of these compounds were confirmed by UV/Vis, FT- IR spectroscopy and CHN/S analysis, while thermal stability was measured by TG/DTA analysis. Mn(L)2 ∙ 2H2O. Yield 83%, m.p. 235 °C. FT-IR νmax 3480 (H2O), 3278 (-OH, NH), 1615 (-C=N-), 1348 (O=S=O), 1587 (-C=C-) cm–1. Anal. Calcd for C34H26N4O6S2Mn: C, 55.06; H, 4.08; N, 7.55; S, 8.65. Found: C, 55.01; H, 4.01; N, 7.51; S, 8.60. Electrical conductance (DMF, µScm–1): 5.22. Co(L)2 ∙ 2H2O. Yield 84%, m.p. 170 °C. FT-IR νmax 3480 (H2O), 3278 (-OH, NH), 1615 (-C=N-), 1348 (O=S=O), 1587 (-C=C-) cm–1. Anal. Calcd for C34H30N4O8S2Co: C, 54.76; H, 4.06; N, 7.51; S, 8.60. Found: C, 54.73; H, 4.01; N, 7.47; S, 8.51. Electrical conductance (DMF, µScm–1): 9.53. Ni(L)2 ∙ 2H2O. Yield 87%, m.p. 250 °C. FT-IR νmax 3480 (H2O), 3278 (-OH, NH), 1615 (-C=N-), 1348 (O=S=O), 1587 (-C=C-) cm–1. Anal. Calcd for C34H30N4O8S2Ni: C, 54.78; H, 4.06; N, 7.52; S, 8.60. Found: C, 54.70; H, 4.02; N, 7.46; S, 8.64. Electrical conductance (DMF, µScm–1): 0.25. Cu(L)2 ∙ 2H2O. Yield 85%, m.p. 210 °C. FT-IR νmax 3480 (H2O), 3278 (-OH, NH), 1615 (-C=N-), 1348 (O=S=O), 1587 (-C=C-) cm–1. Anal. Calcd for C34H30N4O8S2Cu: C, 54.43; H, 4.03; N, 7.47; S, 8.55. Found: C, 54.40; H, 4.01; N, 7.41; S, 8.49. Electrical conductance (DMF, µScm–1): 53.9. Zn(L)2 ∙ 2H2O. Yield 81%, m.p. 250 °C. FT-IR νmax 3480 (H2O), 3278 (-OH, NH), 1615 (-C=N-), 1348 (O=S=O), 1587 (-C=C-) cm–1. Anal. Calcd C34H30N4O8S2Zn: C, 54.29; H, 4.02; N, 7.45; S, 8.53. Found: C, 54.25; H, 4.01; N, 7.38; S, 8.44. Electrical conductance (DMF, µScm–1): 2.02. 2. 5. Anti-Oxidant (DPPH Radical Scavenging) Protocol 1,1-Diphenyl-2-picrylhydrazyl (DPPH) free radical was used to measure the scavenging activity of ligand and metal complexes by using literature protocols. The reac- tion matrix consists of 5 µL test sample (1 mM in DMSO) and 300 µM DPPH (95 µL) and ethanol as the solvent. Af- ter 30 min of incubation at 37 °C, the absorbance of test samples was measured at 515 nm. All the samples were tested in triplicate. The following formula was used to cal- culate percent radical scavenging activity, whereas DMSO was used as a control. 50% of DPPH scavenge radicals represented by IC50 values. tert-Butyl-4-hydroxyanisole was used as the con- trol. The anti-oxidant activities with IC50 values were measured according to the reported procedures.37 2. 6. Anti-Glycation Activity Bovine Serum Albumin (10 mg/mL), anhydrous D-glucose (14 mM), and 0.1 M phosphate buffer (pH 7.4) containing sodium azide (30 mM) and various concentra- tions of the tested compounds in DMSO were incubated at 37 °C for 9 days. After 9 days, fluorescence (excitation, 330 nm; emission, 440 nm) was measured against blank. Rutin was taken as the standard anti-glycation agent. The AGE % inhibition was calculated by given formula: The anti-glycation activities with IC50 values were measured according to the reported procedures.38 3 Result and Discussion 3. 1. Chemistry The literature revealed that metal complexes may be useful candidates in drug development process, therefore these compounds were synthesized and evaluated for their various physical parameters as well as bioactivities. The ligand and its metal chelates were coloured, non-hygroscopic in nature, stable in air, have sharp melt- ing points and were obtained with good yield. All metal chelates including the ligand were insoluble in hexane, chloroform, water, and ethanol although they were found to be soluble in dimethyl sulfoxide (DMSO) and dimethyl formamide (DMF). The electrical conductance values for metal chelates in DMF solvent were found to be 0.25 to 53.9 µS/cm–1. These values indicate the non-electrolytic 775Acta Chim. Slov. 2022, 69, 772–778 Yaqoob et al.: Synthesis, Characterization, Anti-Glycation, and ... nature of metal chelates and the ligand are shown in Table S.1 (see Supp. data). 3. 2. Molecular Formula of the Ligand and Metal Complexes The CHN/S elemental micro-analysis data agree well with the proposed formulae for 4-[(2-hydroxynaphtha- lene-1-ylmethylene)amino]benzenesulfonamide ligand (L) and also confirm the composition of all synthesized metal chelates (Figure 2). The elemental analysis results show that calculated values are in close agreement with the values found. Elemental analysis confirmed the formula of the ligand and its metal complexes with 1:2 metal ligand ratio indicating the bidentate nature of the ligand as shown in Table S.2 (see Supp. data). Figure 2. Proposed reaction for the synthesis of metal chelates 3. 3. Electronic Spectra The UV-Vis spectra (see Supp. data Figures S.3–S.8) of 4-[(2-hydroxynaphthalene-1-ylmethylene)amino]ben- zenesulfonamide (L) and its metal chelates were deter- mined in DMSO solutions and show absorption bands at a longer wavelength with increasing intensity as shown in Table S.3 (see Supp. data). The ligand showed characteris- tic absorption bands at 315 and 364 nm. These bands were assigned to π → π* intra ligand transitions. The UV-Vis spectra of all metal complexes showed bathochromic shifts [Mn(L)2, 467 nm], [Co(L)2, 471 nm], [Ni(L)2, 473 nm], [Cu(L)2, 470 nm], [Zn(L)2, 472 nm] that were taken as an indication for metal complexation. These shifts might be attributed to the d-d-transitions. There were characteristic electronic transitions within the range of 260 nm to ap- proximately 380 nm, that were also observed; these bands being unique for the electronic inter-ligand π → π* transi- tions. Ligand to metal charge transfer (LMCT) peaks were also observed in a distinct region, i.e. within the range of 412 nm onwards, and these are a characteristic feature of nitrogen and oxygen atoms charge transfer to the central metal atoms. 3. 4. IR Spectroscopy The data obtained from FT-IR spectra (see Supp. data Figures S.9–S.14) of some important functional groups of 4-[(2-hydroxynaphthalene-1-ylmethylene)ami- no]benzenesulfonamide (L) and its metal chelates are pre- sented in Table S.4 (see Supp. material). The IR spectrum of the ligand showed strong absorption bands at 1622 and 3290 cm–1, which were attributed to the characteristic band of the ν(-C=N-) and ν(-OH) or -NH groups respec- tively. The sharp bands observed at 1347 cm–1 are due to -S=O stretching vibration. FT-IR spectral calculation re- vealed that for the ligand, which may act as a bidentate according to its structure, is expected that FT-IR measure- ments will be highly indicative with respect to the compl- exation behavior with various metal ions. Peaks in 3400 to 3500 cm–1 region support the observation of water mole- cules participating in the complex formation; this being further confirmed by CHN/S and thermogravimetric data. In the case of metal complexes, the peaks for azomethine group (-C=N-) were shifted from 1622 cm–1 to 1615, 1612, 1610, 1609, 1608 cm–1 and hydroxy group (–OH) peaks were shifted from 3290 cm–1 to 3278, 3260, 3248, 3240, 3237 cm–1 for Mn(L)2, Co(L)2, Ni(L)2, Cu(L)2 and Zn(L)2 complexes, respectively. The changes in the frequency of the peaks indicated that these two groups are involved in coordination. Only spectra of metal complexes showed these new bands, which were thus established as those par- ticipating in these donor groups. The band at 1347 cm–1 for the -SO2 group remains almost unaltered in the che- lates, demonstrating that -SO2 group is not contributing to the coordination. 3. 5. Thermogravimetric Analysis of Ligand and Its Metal Chelates The ligand 4-[(2-hydroxynaphthalene-1-ylmethyl- ene)amino]benzenesulfonamide and its metal chelates were subjected for thermal stability profile. According to the TGA thtrmograms (see Supp. data Figures S.15–S.20) the ligand showed no weight loss upon heating till 250 °C. The further TGA process of the ligand was carried out which showed thermal decomposition in two stages. In the first stage the TGA curve of thermal decomposition was observed between 250–300 °C with weight loss of 1.85%, while the second stage was observed between 300–350 °C with weight loss of 39.5%. DTA thermogram showed one exothermic peak at 328 °C, while two endothermic peaks appeared at 69.42 °C and 270 °C, these may be due to some physical or chemical change phenomenon occurring dur- ing weight loss, such as melting, phase change, chemisorp- tions etc. The TGA of Mn(L)2 complex showed thermal decomposition in three stages. In the first stage the TGA curve of thermal decomposition was observed between 150–200 °C with weight loss of 5.78%, this might be due to the dehydration process. The second and the third stages were observed between 200–250 °C (13.7%), and 250–350 °C (28.1%.) These stages correspond to the decomposition of organic part of metal complex. The DTA thermogram showed three endothermic peaks which were observed at 172 °C, 219 °C, and 326 °C. The TGA of Co(L)2 complex showed thermal decomposition in two stages. In the first stage the TGA curve of thermal decomposition was ob- served between 150–200 °C with weight loss of 3.6%, the second stage was observed within the temperature range 776 Acta Chim. Slov. 2022, 69, 772–778 Yaqoob et al.: Synthesis, Characterization, Anti-Glycation, and ... 200–350 °C with weight loss of 59.04%. These weight loss- es are linked with the loss of water molecules and disinte- gration of the ligand molecule. The two endothermic peaks were spotted at 115 °C and 225 °C in DTA. The TGA of Ni(L)2 complex showed thermal decomposition in three stages, i.e. 150–250 °C, 250–350 °C and 350–410 °C with weight loss of 39.9%, 11.79%, and 16.05%, respectively. These weight losses are due to the dehydration breakdown of ligand. In the DTA thermogram three endothermic peaks appeared at 127 °C, 325 °C, and 388 °C. The TGA of Cu(L)2 complex showed thermal decomposition in two stages. In the first stage the TGA curve of thermal decom- position was observed between 150–200 °C with weight loss of 4.14% (dehydration), while the second thermal pu- trefaction chelating molecule was observed at 200–350 °C with weight loss of 28.07%. Three endothermic peaks were marked in DTA thermogram at 91 °C, 246 °C, and 300 °C. The thermal disintegration of Zn(L)2 complex was ob- served in three stages. In the first stage the thermal decom- position was observed at 150–270 °C with weight loss of 4.23%, possibly due to the dehydration; the second at 270– 370 °C (28.7%), while the third stage was observed at 400– 460 °C with weight loss of 10.89%. The DTA thermogram showed five endothermic peaks which were observed at 108 °C, 148 °C, 251 °C, 346 °C, and 441 °C. These values are closely related to the calculated values. The metal che- lates total weight loss thermal stability was found to be as Cu > Zn > Mn > Co > Ni (Table S.5, see Supp. data). 3. 6. Structural Interpretation The data of spectroscopic, elemental and thermal analyses revealed that metals are coordinated via N and O atoms of ligand molecules in 1:2 M/L ratio (Figure 3). It is reported in the literature that copper can form the octahe- dral coordinated metal complexes such as [Cu(Hmb- m)2(OAc)2], which was reported as an octrahedral com- plex. Hmbm is bonded to the Cu(II) ion in a chelating mode through its nitrogen and oxygen atoms, and two carboxylic oxygen atoms complete the octahedral coordi- nation. The coordination of a water molecule was con- firmed by FT-IR and XRD data.40 For the same cause, the synthesized complexes have the octahedral geometry. 4. Biological Screening 4. 1. Anti-Glycation Activity 4-[(2-Hydroxynaphthalene-1-ylmethylene)amino] benzenesulfonamide ligand (L) and its metal chelates were tested for their anti-glycation activity, using rutin (IC50 = 294.4 ± 1.50 μM) as the standard (Table 1). They showed excellent anti-glycation activity. The ligand (IC50 = 265.11 ± 1.86 μM) and its metal chelates including Mn(L)2 (IC50 = 184.11 ± 2.11 μM), Zn(L)2 (IC50 = 211.26 ± 2.14 μM), Ni(L)2 (IC50 = 254.56 ± 1.73 μM), Cu(L)2 (IC50 = 276.43 ± 1.16 μM) showed outstanding anti-glycation activity, whereas, Co(L)2 (IC50 = 386.34 ± 1.46 μM) was observed as a weak anti-glycating agent. Among them, Mn(L)2 com- plex showed the highest activity and it was found to be many fold more active than the standard rutin. However, Zn(L)2, Ni(L)2, and Cu(L)2 are comparatively less active than Mn(L)2 complex, although they were also found to have better activities than the standard. The activity pat- tern of these complexes can therefore be depicted as: Mn(L)2 > Zn(L)2 > Ni(L)2 > Cu(L)2 > Co(L)2 (see Supp. data S.21–S.24). It was found that these chelates have capa- bility to interact with proteins or glucose in a great extent and can obstruct the advancement of glycation. The active compounds may insert into hydrophobic cavities of BSA followed by the inhibition of advance glycation. Mn(L)2 has elevated level of insertion into the slots of BSA protein. Table 1. Anti-glycation Activity of Ligand and Respective Chelates S# Compounds Anti-Glycation IC50 (μM ± SEMa) 1. Ligand 265.11 ± 1.86 2. Mn(L)2∙2H2O 184.11 ± 2.11 3. Co(L)2∙2H2O 386.34 ± 1.46 4. Ni(L)2∙2H2O 254.56 ± 1.73 5. Cu(L)2∙2H2O 276.43 ± 1.16 6. Zn(L)2∙2H2O 211.26 ± 2.14 7. Rutin 294.4 ± 1.50 a SEM is the standard error of the mean; b rutin is standard inhibitor for anti-glycation activity 4. 2. Antioxidant Assay 4-[(2-Hydroxynaphthalene-1-ylmethylene)amino] benzenesulfonamide ligand (L) and its metal chelates were assessed for DPPH radical scavenging activity (Table 2). Ligand (IC50 = 65.58 ± 1.29 μM) itself was found to be weakly active, while its metal complex Zn(L)2 (IC50 = 37.05 ± 1.53 μM) showed excellent antioxidant activity as com-Figure 3. The proposed structures for metal chelates 777Acta Chim. Slov. 2022, 69, 772–778 Yaqoob et al.: Synthesis, Characterization, Anti-Glycation, and ... pared to the standard tert-butyl-4-hydroxyanisole (IC50 = 44.7 ± 1.21 μM). The metal complexes such as Mn(L)2 (IC50 = 76.1 ± 1.44 μM), Cu(L)2 (IC50 = 86.11 ± 1.12 μM), Co(L)2 (IC50 = 112.14 ± 1.11 μM), and Ni(L)2 (IC50 = 126.27 ± 1.54 μM) were found to be less active than the standard. The order of anti-oxidant potential of these che- lates is Zn(L)2 > Mn(L)2 > Cu(L)2 > Co(L)2 > Ni(L)2. Table 2. Antioxidant Activity of Ligand and Respective Chelates S# Compounds DPPH Radical Scavenging Activity IC50 (μM ± SEMa) 1. Ligand 65.58 ± 1.29 2. Mn(L)2∙2H2O 76.1 ± 1.44 3. Co(L)2∙2H2O 112.14 ± 1.11 4. Ni(L)2∙2H2O 126.27 ± 1.54 5. Cu(L)2∙2H2O 86.11 ± 1.12 6. Zn(L)2∙2H2O 37.05 ± 1.53 7. tert-butyl-4-hydroxyanisoleb 44.7 ± 1.21 a SEM is the standard error of the mean; b tert-butyl-4-hydroxyan- isole is standard inhibitor for antioxidant activity 5. Conclusion 4-[(2-Hydroxynaphthalene-1-ylmethylene)amino] benzenesulfonamide ligand (L) and its Mn(L)2, Co(L)2, Ni(L)2, Cu(L)2, Zn(L)2 chelates were investigated for an- ti-glycation and DPPH radical scavenging activity. Among these chelates Mn(L)2 (IC50 = 184.11 ± 2.11 μM), Zn(L)2 (IC50 = 211.26 ± 2.14 μM), Ni(L)2 (IC50 = 254.56 ± 1.73 μM), and Cu(L)2 (IC50 = 276.43 ± 1.16 μM) showed notable anti-glycation potential while Zn(L)2 (IC50 = 37.05 ± 1.53 μM) showed excellent DPPH radical scavenging activity. The results show that these complexes have excellent po- tential towards anti-glycation activity. So, it is concluded that these complexes may serve as organometallic lead compounds in the drug development process to cure dia- betic complications. However, further studies on the mech- anisms of antioxidation and anti-glycation are required. Acknowledgement The authors are thankful to the University of Sindh, Jamshoro and Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia to support this research work. Conflict of Interest There is no conflict of interest Supplementary Data Tables S.1–S.5 and figures S.1–S.24 (proton NMR and mass spectrum of the ligand; UV-Vis, FT-IR and TGA/DTA spectra of the ligand and complexes; structure of complexes). 5. References 1. T. E. Brown, H. E. LeMay, B. E. Bursten, C. Murphy, P. Wood- ward, Chemistry: The Central Science, 12th edition. Pearson Education, Inc., publishing as Pearson Prentice Hall, 2012. 2. V. U. Rani, G. Jyothi, G. N. Rao, B. B. V. Sailaja, Acta Chim. Slov. 2010, 57, 916–921. 3. B. Nagy, A. Maicaneanu, C. Indolean, S. Burca, L. S. Dumi- trescu, C. Majdik, Acta Chim. Slov. 2013, 60, 263–273. 4. M. Strlič, J. Kolar, V.-S. Šelih, D. Kočaar, B. Pihlar, Acta Chim. Slov. 2003, 50, 619−632. 5. I. Ott, Coord. Chem. Rev. 2009, 253, 1670–1681. 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Za sintetizirane spojine smo določili tudi anti-glikacijske aktivnosti (IC50 vrednosti v območju 184.11–386.34 µM) ter antioksidativne lastnosti (IC50 vrednosti v območju 37.05–126.27 µM). Kovinski kom- pleksi Mn (IC50 = 184.11 ± 2.11 µM), Ni (IC50 = 211.26 ± 1.46 µM), Cu (IC50 = 254.56 ± 1.16 µM) in Zn (IC50 = 276.43 ± 2.14 µM) so izkazali precej boljše anti-glikacijske aktivnosti kot standard rutin (IC50 = 294.4 ± 1.50 µM). Kompleks s Zn (IC50 = 37.05 ± 1.53 μM) pa je pokazal boljšo sposobnost lovljenja radikalov na DPPH testu kot standard terc-butil-4-hi- droksianizol (IC50 = 44.7 ± 1.21 µM). 779Acta Chim. Slov. 2022, 69, 779–786 Lenget al.: Synthesis, Crystal Structure and Biological Activity of Two ... DOI: 10.17344/acsi.2022.7516 Scientific paper Synthesis, Crystal Structure and Biological Activity of Two Triketone-Containing Quinoxalines as HPPD Inhibitors Xinyu Leng,1 Chengguo Liu2 and Fei Ye1,* 1 Department of Chemistry, College of Arts and Sciences, Northeast Agricultural University, Harbin 150030, China 2 Department of State Assets Management, Northeast Agricultural University, Harbin 150030, China * Corresponding author: E-mail: yefei@neau.edu.cn Tel: +86-451-55190070 Received: 04-03-2022 Abstract Two new triketone-containing quinoxaline derivatives were designed by fragment splicing strategy and synthesized us- ing 3,4-diaminobenzoic acid and substituted cyclohexanedione as starting materials. Both compounds were character- ized by IR, 1H and 13C NMR, HRMS and X-ray diffraction. 3-Hydroxy-5-methyl-2-(quinoxaline-6-carbonyl)cyclohex- 2-en-1-one (6a) crystallized in the triclinic system, space group Pī, a = 7.9829(2) Å, b = 8.1462(2) Å, c = 10.7057(3) Å, α = 84.3590(10)°, β = 89.7760(10)°, γ = 87.4190(10)°, Z = 2, V = 692.12(3) Å3, F(000) = 296, Dc = 1.335 Mg/m3, µ(MoKα) = 0.095 mm–1, R = 0.0683 and wR = 0.1983. 3-Hydroxy-5,5-dimethyl-2-(3-ethoxyquinoxaline-6-carbonyl)cyclohex-2-en- 1-one (6b) crystallized in the monoclinic system, space group P21/c, a = 10.1554(6) Å, b = 9.6491(6) Å, c = 17.7645(10) Å, β = 90.784(2)°, Z = 4, V = 1740.59(18) Å3, F(000) = 720, Dc = 1.299 Mg/m3, µ(MoKα) = 0.092 mm–1, R = 0.0462 and wR = 0.1235. Physicochemical property comparison and ADMET prediction showed that compound 6a had similar properties to the commercial herbicide mesotrione. Molecular docking results showed that the interactions between 6a and AtHPPD were similar to mesotrione. Moreover, the extended aromatic ring system and the additional alkyl form more interactions with the surrounding residues. Examination of AtHPPD inhibition and herbicidal activity showed that 6a had similar inhibition values to mesotrione and had a superior inhibitory effect on Echinochloa crus-galli. Keywords: Triketone-containing quinoxaline derivatives; Synthesis; Single-crystal structure; Molecular structure infor- mation; Herbicidal activity 1. Introduction Herbicides that inhibit 4-hydroxyphenylpyruvate di- oxygenase (EC 1.13.11.27, HPPD) have been used in agri- culture for weed control since the 1970s.1 HPPD is one of the α-keto acid-dependent, non-heme, Fe(II)-dependent enzymes belonging to the 2-His-1-carboxylate facial tri- ad family, and is involved in tyrosine catabolism, which is necessary for most aerobic organisms.1–3 L-tyrosine is converted to 4-hydroxyphenylpyruvic acid (HPPA) by a transamination catalyzed by tyrosine aminotransferase (TAT). Subsequently, HPPA is converted to homogentisic acid (HGA) by a complex biochemical reaction catalyzed by HPPD. In plants, HGA is converted to plastoquinone and tocopherol,4–7 and its absence leads to bleaching symptoms, necrosis, and plant death.8,9 Therefore, HPPD is an important enzyme class discovered in recent years that targets herbicides. HPPD inhibitor herbicides are characterized by broad spectrum weed control, flexibili- ty, remarkable plant selectivity, good environmental com- patibility, low toxicity, and high efficiency.9–11 Based on their structure, they can be classified into three categories: Triketones, isoxazoles, and pyrazoles.12,13 Triketone deriv- atives are among the best-studied herbicides and generally contain both triketone and aromatic ring components.14,15 Unfortunately, with the widespread use of HPPD herbi- cides recently, more and more weeds have developed re- sistance to these active ingredients, which is not limited to the target site.16,17 This highlights the importance of devel- oping new HPPD herbicides to effectively manage weed resistance and improve weed control efficiency. Inspired by our previous reports and interest in HPPD herbicides,18–22 two novel triketone-containing quinoxaline derivatives were designed and synthesized (Figure 1). Comparisons of physical and chemical properties, ADMET parameters (absorption, distribution, metabolism, excretion, and tox- icity), and molecular docking were performed. The biolog- ical results showed that compound 6a had similar inhibi- tion values to the commercial herbicide mesotrione. 780 Acta Chim. Slov. 2022, 69, 779–786 Lenget al.: Synthesis, Crystal Structure and Biological Activity of Two ... 2. Experimental 2. 1. Materials and Characterization All reagents were purchased from Shanghai Alad- din Biochemical Technology Co. and were of analytical grade that could be used without any purification. Melt- ing points were measured using a Shanghai INESA melt- ing point instrument (WRS-3) and were uncorrected. The IR spectrum was recorded in KBr pellets using a Bruker ALPHA -T instrument. The NMR spectrum was record- ed with a Bruker AV -400 MHz spectrometer (Bruker Company, DEU) using CDCl3 as solvent and tetramethyl- silane (TMS) as internal standard. High-resolution mass spectrometry (HRMS) data were obtained using a Bruk- er micrOTOF-Q II 10410 spectrometer. X-ray diffraction data were obtained using a RAPID-AUTO area detector diffractometer. 2. 2. Preparation of the Quinoxaline-6- carboxylic acid (2) In a three-neck flask (100 mL), 3,4-diaminobenzoic acid (10 mmol) was stirred in distilled water, 10% sodium dioctyl sulfosuccinate (SDOSS, 10 mmol) and substituted diketones (1, 12 mmol) were added, and the mixture was stirred at room temperature for 4 h.23 After the reaction, the mixture was filtered under vacuum, the filter cake was dried and recrystallized with EtOH/water to give quinox- aline-6-carboxylic acid (2). 2. 3. Preparation of the Acid Chloride (3) Compound 1 (2 mmol) was dissolved in CH2Cl2 (40 mL) in a three-neck flask (100 mL), to which sulfoxide chloride (3 mmol) and DMF (0.1 mL) were added and re- fluxed for 2 hours.24 Compound 3 was isolated by remov- ing the solvent. 2. 4. Preparation of Enol Ester Compounds (5) Compound 3 (2.4 mmol) and substituted 1,3-cy- clohexanedione (2.1 mmol) were dissolved in CH2Cl2 (30 mL), and triethylamine (Et3N, 2.3 mol) was added drop- wise and reacted at 0 °C for 6 h.25 After completion of the reaction, the mixture was washed three times with aque- ous HCl (50 mL, 1 M), followed by washing with saturated sodium chloride solution (50 mL), drying with anhydrous MgSO4, and then removing the solvent by filtration un- der reduced pressure, leaving a solid residue. Compound 5 was obtained by purifying the crude product by silica gel column chromatography (ethyl acetate : petroleum ether = 1:3). 2. 5. Preparation of Triketone-Containing Quinoxalines (6) The synthetic pathway of 6a and 6b is shown in Fig- ure 2. Compound 5 (1 mmol), Et3N (12 mmol), CH3CN (13 mmol), and acetone cyanohydrin (AC, 5 mmol) were mixed in CH2Cl2 (30 mL) and the reaction was carried out at 25 °C for 6 h.26 After completion of the reaction, the solu- tion was washed three times with aqueous HCl (30 mL, 1 M), followed by washing with saturated aqueous NaCl (30 mL), drying with anhydrous MgSO4, and evaporation of the solvent. Compound 6 was obtained by purifying the crude product by silica gel column chromatography (ethyl acetate : petroleum ether = 4:1). Supporting information includes IR, 1H NMR, 13C NMR, and HRMS information for compounds 6 (Figures S1-S8). 3-Hydroxy-5-methyl-2-(quinoxaline-6-carbonyl)cyclohex- 2-en-1-one (6a), Yellow solid; yield: 58%; m.p. 132.5–133.5 °C, IR (KBr, cm–1) 3063–2847 (-CH2-, =CH), 1651–1608 (C=O), 1578–1543 (C=C), 1H NMR (400 MHz, CDCl3, ppm) δ 16.81 (s, 1H, OH), 8.90 (s, 2H, Ar-H), 8.26–7.81 (m, 3H, Ar-H), 2.83 (s, 1H, CH), 2.67–2.29 (m, 4H, 2×CH2), 1.17 (d, J = 6.1 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3, ppm) δ 197.82, 196.18, 193.95, 146.02, 145.53, 144.16, 142.17, 140.08, 129.59, 129.08, 128.79, 112.95, 26.74, 20.82. HRMS (ESI): calculated for C16H14N2O3 [M+H]+ 283.1077, found 283.1080. 3-Hydroxy-5,5-dimethyl-2-(3-ethoxyquinoxaline-6-car- bonyl)cyclohex-2-en-1-one (6b) Yellow solid; yield: 42%; m.p. 162.7–163.5 °C; IR (KBr, cm–1) ν 3039–2904 (-CH2-, =CH), 1670–1661 (C=O), 1550 (C=C), 1H NMR (400 MHz, CDCl3, ppm) δ 17.01 (s, 1H, OH), 8.49–7.58 (m, 4H, Ar-H), 4.51–4.45 (m, 2H, CH2), 2.77–2.60 (m, 2H, Figure 1. Design of the target compounds. 781Acta Chim. Slov. 2022, 69, 779–786 Lenget al.: Synthesis, Crystal Structure and Biological Activity of Two ... CH2CO), 2.42 (s, 2H, CH2), 1.48 (t, J = 7.1 Hz, 3H, CH3), 1.18 (s, 6H, 2×CH3). 13C NMR (100 MHz, CDCl3, ppm) δ 197.14, 196.11, 194.02, 157.78, 148.05, 146.13, 142.02, 139.80, 129.13, 128.64, 128.42, 112.37, 77.37, 76.74, 74.66, 51.96, 45.97, 31.13, 28.33. HRMS (ESI): calculated for C19H20N2O4 [M+H]+ 341.1500, found 341.1496. 2. 6. Crystal Structure Determination Compound 6 was dissolved in EtOAc to form a nearly saturated solution. The crystals grew during the volatilization of the solvent at room temperature in the dark. The crystal was mounted on a RAPID-AUTO area detector diffractometer with MoKα radiation (λ = 0.71073 Å) at 293(2) K. The crystal structures were solved by direct methods and refined using SHELXS-97 and SHELXL-97.27,28 The symmetric equivalent reflectance was used to optimize the shape and size of the crystal. The H atom was then constrained to an ideal geometry with a C-H distance of 0.93–0.98 Å. The Uiso(H) value for the me- thyl H atoms was set to 1.5 Ueq(C) and 1.2 Ueq(C) for the other H atoms. Crystal packing diagrams were prepared using the xp software. Cambridge Crystallographic Data Center under supplemental publication numbers CCDC 2150405 (6a) and 2150406 (6b). Copies of the data are available free of charge on request from CCDC, 12 Un- ion Road, Cambridge CB21EZ, UK [www.ccdc.cam.ac.uk/ data_request/cif]. 2. 7. AtHPPD Inhibitory Experiments in Vitro Homogentisate 1,2-dioxygenase (HGD) and Arabi- dopsis thaliana HPPD (AtHPPD) were prepared and pu- rified according to methods described in the literature.29 Mesotrione, compounds 6, and AtHPPD were preincubat- ed for 25 minutes, then a mixture of appropriate amounts of HGD, HPPA, FeCl2 (1 mM), ascorbic acid (20 mM), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, 20 mM) buffer (pH 7.0) was added sequential- ly.9,30 An initial screening of the inhibitory effect of each compound was performed at a concentration of 10 μM to determine the final concentration range of the IC50. The test compounds were dissolved in DMSO and diluted with buffer to various concentrations before use. The IC50 of residual activity was calculated by fitting the curves for different concentrations of the compounds at specific sub- strate concentrations. 2.8. Herbicidal Activity Assay All test weeds were purchased from the seed mar- ket in Harbin, China. Mesotrione and compounds 6 were tested against the weeds Echinochloa crus-galli (EC), Setar- ia faberi (SF), Digitaria sanguinalis (DS), Amaranthaceae Amaranthus retroflexus (AR), and broadleaf Abutilon jun- cea (AJ) by post-emergence application.10 Mesotrione and compounds 6 were prepared using DMSO as solvent and Tween 80 (0.1 g/mL) as emulsifier. These solutions were diluted with distilled water to the required concentration and then sprayed on the test plants in the greenhouse. The clay soil was Mollisols-Cryolls clay loam with a pH of 7.3. Approximately 15 weed seeds from EC, SF, DS, AR, and AJ were planted in paper cups, covered with 1.5 cm of soil, and grown in a greenhouse at 18–28 °C and 78% humidity. Broadleaf weeds and monocotyledonous weeds were treated at the two-leaf stage and one-leaf stage, re- spectively. Once weeds reached the appropriate stage, they were treated with the inhibitors at doses of 0.045 and 0.090 mmol/m2 (approximately 150 and 300 g ai/ha). Seeds of the positive control group were treated with the commer- cial herbicide mesotrione. After 10 days of treatment with these compounds, herbicide activity was measured visual- ly, with each treatment repeated three times.12 2. 9. Computational Chemistry Physical and chemical property comparison, AD- MET prediction, and molecular docking were performed using Discovery Studio 2019 (DS, Biovia Inc., CA, USA), and electronegativity was calculated using SYBYL-X 2.0. The 3D structure of mesotrione, compounds 6a and 6b was created using Chem3D 15.1, and the molecular struc- tures were further optimized using the MM2 minimization module. The crystal structure of the protein was down- Figure 2. The synthetic route for the compounds 6. 782 Acta Chim. Slov. 2022, 69, 779–786 Lenget al.: Synthesis, Crystal Structure and Biological Activity of Two ... loaded from Protein Data Bank (PDB ID: 5YY6, http:// www.rcsb.org/pdb). 5YY6 was processed before molecular docking, all hydrogen atoms were added, all heteroatoms, ligands and water molecules were removed. Then, the Clean Protein Tool in DS was used to complete incomplete residues, remove excess protein conformations, hydrogen- ate, and assign the associated charges. The active site was defined using a binding sphere of the native ligand. Molec- ular docking was performed using the CDOCKER mod- ule, and parameters were set to default values. The crystal structure of 5YY6 contained the native ligand 94L, and the ligand molecules at the active site of the complex were ab- stracted and redocked into the binding pocket. The root mean square deviation (RMSD) was calculated.3 3. Results and Discussion 3. 1. Description of the Crystal Structures and Hydrogen Bonding The crystallographic data and structural refinement details for 6a and 6b are given in Table S1. 6a crystal- lized in triclinic Pī-space group with a unit cell volume of 692.12(3) Å3, the cell dimensions are: a = 7.9829(2) Å, b = 8.1462(2) Å, c = 10.7057(3) Å, α = 84.3590(10)°, β = 89.7760(10)°, γ = 87.4190(10)°, and Z = 2. 6b crystallizes in monoclinic P21/c space group with a unit cell volume of 1740.59(18) Å3, the cell dimensions are: a = 10.1554(6) Å, b = 9.6491(6) Å, c = 17.7645(10) Å, β = 90.784(2)°, and Z = 4. The molecular structures of compounds 6a and 6b with the numbering of the atoms are shown in Figure 3. Selected bond lengths and bond angles of 6a and 6b are listed in Table S2. The molecule is not coplanar; both crys- tal structures of compounds 6a and 6b consist of two aro- matic moieties: a cyclohexanedione (A) and a quinoxaline (B). In compound 6a, for example, the C-C bond length was entirely within the range of the typical C-C bond length (1.54 Å).30 The bond length of C(15)-O(3) was 1.3162(15), which was shorter than the typical C-O length (1.42 Å); the bond length of C(10)=C(15) (1.3841(16) Å) and C(9)-O(1) (1.2490(15) Å) was also longer than the typical C=C length (1.34 Å) and C=O length (1.21 Å).31–33 These results suggest a conjugative effect between carbon- yl, hydroxyl, and C=C bond. In addition, the C(6)-C(9) bond length (1.4892(16) Å) was shorter than the typical C-C length, which could be due to a π-π conjugation be- tween carbonyl and benzene of quinoxaline. The dihedral angles of part A and part B in compound 6a and 6b were 50.32° and 53.80°, respectively. And the cyclohexanedione in both compounds belongs to the half-chair conforma- tion. The presence of π-π packing interactions, hydrogen bonding, and van der Waals forces resulted in an ordered Figure 3. Molecular structures of compounds 6a and 6b. Figure 4. Molecular packing diagram of 6a and 6b, hydrogen bonds are shown as dashed lines. 783Acta Chim. Slov. 2022, 69, 779–786 Lenget al.: Synthesis, Crystal Structure and Biological Activity of Two ... crystal arrangement with high symmetry and regularity (Figure 4). Compound 6a formed the packing through the hydrogen bonds C(7)-H(7)…O(1), C(2)-H(2)…O(2), and C(12)-H(12B)…N(2). Compound 6b formed the packing through hydrogen bonds C(4)-H(4B)…O(1). The hydro- gen bonding data are shown in Table 1. As shown in Figure 5, the distance between aromatic rings is within the limit- ed range of typical π-π packing interaction (d1 = 3.8537(1) Å, d2 = 3.6850(1) Å, d3 = 3.8537(1) Å, d4 = 3.7647(2) Å). 3. 2. Spectroscopic Studies Compounds 6 were confirmed by IR, 1H and 13C NMR, and HRMS. Let us take compound 6a as an exam- ple. The IR analysis confirmed the presence of methylene and = CH- group at 3063–2847 cm–1, carbonyl at 1651– 1608 cm–1, and C=C bond at 1578–1543 cm–1. The NMR data indicated the possible structure of the compound. The 1H NMR signals at δ 16.81 ppm are associated with the hydroxyl group of enol. The signals at δ 7.81–8.90 ppm are associated with the five Ar-H of the pyrazine and benzene rings. The signal at δ 2.83 ppm is associated with the hy- drogen on the tertiary carbon. The signals at δ 2.37–2.68 ppm and 1.17 ppm are associated with the methylene and methyl groups, respectively. The 13C NMR data at δ 193.95–197.82 ppm show the presence of the carbon atom of enol. The signals at δ 128.79–146.02 ppm relate to the pyrazine and benzene rings and signals at δ 112.95 ppm refer to the carbon between three enols. The signals at δ 20.82–20.74 are characteristic of the remaining saturated carbon atoms. The [M+H]+ ion of 6a was calculated with Chemdraw 15.1 as 283.1007; the actual signal found was 283.1080. 3. 3. AtHPPD Inhibition and Herbicidal Activities The IC50 values against AtHPPD in vitro of mesotri- one and target compounds 6 are shown in Table 2. The IC50 values of mesotrione, 6a, and 6b were 0.23, 0.46, and 6.41 μM, respectively. Compound 6a showed similar inhibition values as mesotrione, possibly because they share the same skeletal structure. The herbicidal effects of mesotrione and compounds 6 against EC, SF, AJ, DS, and AR are listed in Table 2. Weeds treated with compounds 6a and 6b showed similar symptoms to mesotrione, suggesting that these tar- get compounds are potential HPPD inhibitors. All com- pounds tested showed no inhibition in monocotyledonous weeds (AG, DS, and AR). Compound 6a showed similar inhibitory activity against EC and SF as mesotrione. Nota- bly, 6a had the superior EC inhibitory activity, suggesting that the scaffold of compounds 6 could be further modi- fied as herbicides. Table 2. Inhibitory activities and post-emergence herbicidal activi- ties (inhibition rating 0–100) of compounds 6a and 6b (150 g ai/ha). compounds IC50 (μM) Inhibition (%)a EC SF AJ DS AR Mesotrione 0.23 B A G E G 6a 0.46 A B G G G 6b 6.41 F F G G G a Rating scale of inhibition percentage in relation to the untreated control: A, 100%; B, 99–90%; C, 89–70%; D, 69–50%; E, 49–30%; F, 29–20%; G,0–19%. The comparisons of physical and chemical proper- ties were studied and are shown in Table 3. Certain sim- Table 1. Hydrogen bonding parameters in the structures of 6a and 6b. D-H…A d(D-H) d(H…A) d(D…A) Symmetry codes ∠DHA 6a C(7)–H(7)…O(1)a 0.9302(11) 2.514(1) 3.2713(15) 1–x, –y, 1–z 138.754(75) C(2)–H(2)…O(2)b 0.9295(17) 2.4973(13) 3.1763(21) 1–x, –y, –z 130.071(103) C(12)–H(12B)…N(2)c 0.9699(16) 2.7456(13) 3.5689(20) 1–x, 1–y, –z 143.073(99) 6b C(4)–H(4B)…O(1)d 0.9703(14) 2.5100(11) 3.3190(18) 1–x, –0.5+y, 0.5–z 140.789(86) Figure 5. π-π packing interactions between two molecules. 784 Acta Chim. Slov. 2022, 69, 779–786 Lenget al.: Synthesis, Crystal Structure and Biological Activity of Two ... ilarities of these compounds at the molecular level were confirmed by the hydrogen bond acceptors (HBAs), hy- drogen bond donors (HBDs), rotatable bonds (RBs), aro- matic rings (ARs), and electronegativity. When comparing the log p of 6a and 6b, it was found that 6a has a relatively low value that favors herbene transfer and absorption,34 and that compound 6a has a similar surface area (SA) to mesotrione, which is advantageous for compound 6a to enter the active pocket. In addition, experiments on AtH- PPD inhibition and herbicide activity showed that com- pound 6a had a stronger inhibitory effect than compound 6b. This is likely due to the p-π conjugation between pyra- zine and ethoxy, with ethoxy acting as an electron donor and enhancing the inhibitory effect. 3. 4. ADMET Prediction ADMET prediction has received special attention in drug development. The predicted parameters of mesotri- one, 6a and 6b are shown in Table 4. Both compounds 6a and mesotrione were similar in terms of solubility degree, cytochrome P450 2D6 (CYP2D6) prediction, and plasma protein binding ability (PPB). Apparently, compound 6a was better absorbed than mesotrione. CYP2D6 predic- tion showed that these two compounds could successfully pass through the first stage of metabolism. The PPB pre- diction values of the two compounds were not correct, which could lead to low bioavailability because they do not attach to the carrier protein.35,36 On the other hand, in the case of compound 6b, although it has good CYP2D6 prediction and absorption degree, its solubility degree and PPB prediction value are unsatisfactory, leading to a decrease in its activity. In conclusion, compound 6a has similar pharmacokinetic properties to the commercial herbicide mesotrione, confirming that it has some pros- pect of weed control. Table 4. The ADMET prediction of mesotrione, 6a and 6b. Mesotrione 6a 6b Solubility Levela 4 3 2 Absorption Levelb 1 0 0 CYP2D6 Predictionc false false false AlogP98d 0.093 1.698 2.776 PPB# predictione false false True a Solubility Level: Categorical solubility level. 2: Yes, low. b Absorption Level: Absorption Level. 0: Good absorption. c CYP2D6: cytochrome P450 2D6. <0.161: false, non-inhibitor; >0.161: true, inhibitor. d AlogP98: the logarithm of the partition coefficient between n-oc- tanol and water. <4.0: Binding is<90%; >4.0: Binding is>90% and Binding is <95% e PPB: Plasma Protein Binding ability. <−2.209: ≥90%, false; >−2.209: ≤90%, true. 3. 5. Molecular Docking Molecular docking was an essential tool for comput- er-aided drug design (CADD), which correctly predicted Table 3. Comparison of physical and chemical properties of mesotrione, 6a and 6b. Mesotrione 6a 6b Structure MWa 340.33 282.29 340.37 Logpa 0.26 1.68 2.78 HBAsa 7 5 6 HBDsa 2 1 1 RBsa 4 2 4 ARsa 1 2 2 SAa 317.34 280.81 362.96 electronegativityb Figure 6. The ligand compared using the CDOCKER docking method (the newly docked ligand was red and the native ligand was green). 785Acta Chim. Slov. 2022, 69, 779–786 Lenget al.: Synthesis, Crystal Structure and Biological Activity of Two ... the interaction between the inhibitor and herbicide target enzymes.37 To verify the feasibility of molecular docking, the native ligand 94L was redocked to the target protein. The superposition of the conformation of the native ligand with the newly docked conformation is shown in Figure 6. The conformation of the native and redocked ligand 94L was almost completely overlapping with an RMSD value of 0.5549 Å (< 2 Å), confirming the accuracy of the CDOCK- ER docking procedure.3 The mesotrione, 6a and 6b were selected for the mo- lecular docking experiments to predict the binding pattern with 5YY6. Compounds 6a and 6b hardly differed from mesotrione in terms of geometric complementarity of the binding position in the active pocket. Mesotrione and 6a occupied the active pocket almost completely, whereas 6b occupied only part of the pocket (Figure S9 in the Sup- porting Information). The enol structure and carbonyl groups in these compounds all coordinate with Co2+. In compound 6a, the distances between the Co2+ and O at- oms were 2.02 Å and 1.86 Å, respectively, similar to mes- otrione. The benzene moiety of compound 6a formed a π-π-stacked interaction with PHE424, similar to that of mesotrione, and HIS226 formed a new interaction with the O atom, further improving the binding ability of the ligand. The additional interactions of compound 6a with PRO280 and VAL269 resulted in strong binding to the re- ceptor, which may explain the similarity of the AtHPPD inhibitory activity of compound 6a with mesotrione. 4. Conclusion In summary, two new triketone-containing quinox- aline derivatives were developed and synthesized as nov- el HPPD herbicides. Both compounds exhibited certain HPPD inhibitory activities. In particular, compound 6a showed similar AtHPPD inhibition and herbicidal activity to the commercial herbicide mesotrione, as demonstrated by physical and chemical property comparisons, ADMET prediction, and molecular docking study. Acknowledgements This work was supported by the National Nature Sci- ence Foundation of China (grant number 22077014) and the Under-graduate SIPT Program of Northeast Agricul- tural University (grant number 202110224014). Supporting information includes the crystallographic data and structure refinement details for compounds 6 (Table S1), selected bond lengths and bond angles for crystals of compounds 6 (Table S2), the IR, 1H, 13C NMR and HRMS spectra of compounds 6 (Figures S1-S8), the receptor-ligand interaction, and coordination patterns of mesotrione and compounds 6 with AtHPPD (Figure S9). 5. References 1. E. Rocaboy-Faquet, L. Barthelmebs, C. Calas-Blanchard, T. Noguer, Talanta., 2016, 146, 510–516. DOI:10.1016/j.talanta.2015.09.030 2. L. Serre, A. Sailland, D. Sy, P. Boudec, A. Rolland, E. Pebay– Peyroula, C. Cohen–Addad, Structure, 1999, 8, 977–988. DOI:10.1016/S0969-2126(99)80124-5 3. Y. Fu, S. Q. Zhang, Y. X. Liu, J. Y. Wang, S. Gao, L. X. Zhao, F. Ye, Ind. 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S. Sakkiah, S. Thangapandian, S. John, Y. J. Kwon, K. W. Lee, Eur. J. Med. Chem., 2010, 45, 2132–2140. DOI:10.1016/j.ejmech.2010.01.016 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 V prispevku je opisana priprava dveh novih triketonskih kinoksalinskih derivatov, ki vsebujeta triketone, s strategijo spajanja posameznih fragmentov, sintetiziranih iz 3,4-diaminobenzojske kisline in substituiranega cikloheksandiona kot izhodnih molekul. Oba kinoksalinska derivata so okarakterizirali z IR, 1H in 13C NMR, HRMS in rentgensko di- frakcijo. 3-Hidroksi-5-metil-2-(kinoksalin-6-karbonil)cikloheks-2-en-1-on (6a) kristalizira v triklinskem kristalnem sistemu, v prostorski skupini Pī, a = 7.9829(2) Å, b = 8.1462(2) Å, c = 10.7057(3) Å, α = 84.3590(10)°, β = 89.7760(10)°, γ = 87.4190(10)°, Z = 2, V = 692.12(3) Å3, F(000) = 296, Dc = 1.335 Mg/m3, µ(MoKα) = 0.095 mm–1, R = 0.0683 and wR = 0.1983. 3-Hydroxy-5,5-dimethyl-2-(3-ethoxyquinoxaline-6-carbonyl)cyclohex-2-en-1-one (6b) crystallized in the monoclinic system, space group P21/c, a = 10.1554(6) Å, b = 9.6491(6) Å, c = 17.7645(10) Å, β = 90.784(2)°, Z = 4, V = 1740.59(18) Å3, F(000) = 720, Dc = 1.299 Mg/m3, µ(MoKα) = 0.092 mm–1, R = 0.0462 and wR = 0.1235. Primerjava fizikalno-kemijskih lastnosti in ADMET napovedi so pokazale, da ima spojina 6a podobne lastnosti kot komercialni her- bicid mezotrion. Rezultati molekulskega modeliranja so pokazali, da so interakcije med 6a in AtHPPD podobne tistim pri mezotrionu. Poleg tega razširjeni aromatski obročni sistem in dodatne alkilne skupine povečajo interakcije z okolico. Raziskave inhibicije AtHPPD in herbicidnega delovanja so pokazale, da ima kinoksalin 6a podobne vrednosti inhibicije kot mezotrion in boljši inhibitorni učinek na Echinochloa crus-galli. 787Acta Chim. Slov. 2022, 69, 787–795 Zhao et al.: Tetranuclear Copper(II) Complexes Derived from 5-Bromo-2-((2-(2-hydroxyethylamino) ... DOI: 10.17344/acsi.2022.7530 Scientific paper Tetranuclear Copper(II) Complexes Derived from 5-Bromo- 2-((2-(2-hydroxyethylamino)ethylimino)methyl)phenol: Synthesis, Characterization, Crystal Structures and Catalytic Oxidation of Olefins Xiao-Jun Zhao, Su-Zhen Bai and Ling-Wei Xue* School of Chemical and Environmental Engineering, Pingdingshan University, Pingdingshan Henan 467000, P. R. China * Corresponding author: E-mail: pdsuchemistry@163.com Received: 04-14-2022 Abstract An acetate bridged tetranuclear copper(II) complex, [Cu4L2(μ2-η1:η1-CH3COO)6(CH3OH)2] (1), and a chloride, phe- nolate and azide co-bridged tetranuclear copper(II) complex, [Cu4L2Cl2(μ-Cl)2(μ1,1-N3)2]2CH3OH (2), where L is the deprotonated form of the Schiff base 5-bromo-2-((2-(2-hydroxyethylamino)ethylimino)methyl)phenol (HL), have been synthesized and characterized by elemental analysis, IR and UV spectra, and single crystal X-ray diffraction. Single crystal X-ray analysis revealed that the Cu atoms in both complexes are in square pyramidal geometry. In complex 1, two [CuL] units and [Cu2(μ2-η1:η1-CH3COO)4] core are linked through two acetate ligands. In complex 2, [Cu2LCl(μ-Cl)] units are linked together by two end-on azido ligands. The Schiff base ligand coordinates to the Cu atoms through four N and O donor atoms. The molecules of both complexes are linked through hydrogen bonds to generate three dimensional networks. The catalytic property of the complexes for epoxidation reactions of some alkenes was studied using tert-bu- tylhydroperoxide as the terminal oxidant under mild conditions in acetonitrile. Keywords: Schiff base; copper complex; crystal structure; tetranuclear complex; catalytic property. 1. Introduction Transition metal complexes with Schiff bases as li- gands have received much attention for their structures, biological, pharmaceutical, magnetic and catalytic proper- ties.1 The complexes have been widely studied on the cat- alytic processes in many fundamentally and industrially important reactions.2 Among the catalytic reactions, the epoxidation of olefins is of remarkable interest, because the products are necessary precursors for the production of fine chemicals. Copper complexes with Schiff base lig- ands are of particular interest due to their versatile struc- tures and catalytic properties.3 Some copper complexes have been used as catalysts for the epoxidation reactions. Among them, those with Schiff base ligands have received particular attention.4 A number of reports used hydro- gen peroxide as oxidant in the catalytic reactions. How- ever, due to the explosive nature of hydrogen peroxide, industrial processes prefer to use tert-butylhydroperoxide (TBHP) as the oxidant.5 Although the catalytic properties of Schiff base copper(II) complexes toward oxidation re- actions both in homogeneous and heterogeneous condi- tions are well documented, catalytic oxidation of alkenes involving tetranuclear Schiff base copper(II) complex- es has rarely reported. Notably, TBHP has seldom been used as an oxidant in the catalytic oxidation reactions by copper(II) complexes as homogeneous catalysts.6 In this work, two new tetranuclear copper(II) complexes, namely [Cu4L2(μ2-η1:η1-CH3COO)6(CH3OH)2] (1) and [Cu4L- 2Cl2(μ-Cl)2(μ1,1-N3)2]2CH3OH (2), where L is the depro- tonated Schiff base 5-bromo-2-((2-(2-hydroxyethylami- no)ethylimino)methyl)phenol (HL; Scheme 1), have been synthesized, characterized and studied on their catalytic epoxidation efficacy towards some alkenes. Scheme 1. The Schiff base HL. 788 Acta Chim. Slov. 2022, 69, 787–795 Zhao et al.: Tetranuclear Copper(II) Complexes Derived from 5-Bromo-2-((2-(2-hydroxyethylamino) ... 2. Experimental 2. 1. Materials and Methods All solvents used were of AR grade and used as re- ceived. 4-Bromosalicyaldehyde, 2-(2-aminoethylamino) ethanol, copper acetate monohydrate, copper chloride dihydrate and sodium azide were purchased from Aladin Chemical Co. Ltd. and were used as received. Styrene, cy- clooctene, cyclohexene and TBHP were purchased from Aldrich and were used as received. Infrared spectra (4000– 400 cm–1) were recorded as KBr discs with a FTS-40 Bio- Rad FT-IR spectrophotometer. The electronic spectra were recorded on a Lambdar 35 spectrometer. Microanalyses (C, H, N) of the complex were carried out on a Carlo-Erba 1106 elemental analyzer. Solution electrical conductivity was measured at 298K using a DDS-11 conductivity meter. GC analyses were performed on a Shimadzu GC-2010 gas chromatograph. Crystallographic data of the complexes were collected on a Bruker SMART 1000 CCD area dif- fractometer with graphite monochromated Mo-Kα radia- tion (λ = 0.71073 Å) at 298(2) K. Caution! Transition metal azido complexes are po- tentially explosive especially in the presence of organic ligands. Although we have not encountered any problem during our study, yet a small quantity of materials should be prepared and it should be handled with care. 2. 2. X-Ray Crystallography Absorption corrections were applied by using the SADABS program.7 Structures of the complexes were solved by direct methods and successive Fourier difference syntheses, and anisotropic thermal parameters for all non- hydrogen atoms were refined by full-matrix least-squares procedure against F2 using SHELXTL and SHELXL-97 packages.8 All non-hydrogen atoms were refined aniso- tropically. The amino and hydroxyl H atoms of the Schiff base ligands in both complexes were located from differ- ence Fourier maps and refined isotropically. The N−H and O−H distances were restrained to 0.90(1) and 0.85(1) Å, respectively. The crystallographic data and experimental details for the structural analysis are summarized in Table 1, and selected bond lengths and angles are listed in Table 2. 2. 3. Synthesis of [Cu4L2(μ2-η1:η1- CH3COO)6(CH3OH)2] (1) 4-Bromosalicylaldehyde (1.0 mmol, 0.20 g) and 2-(2-aminoethylamino)ethanol (1.0 mmol, 0.10 g) were mixed and stirred in methanol (30 mL) for 30 min at 25 °C. Then, copper acetate monohydrate (2.0 mmol, 0.40 g) was added. The final mixture was further stirred for 30 min. The deep blue solution was evaporated to remove three quarters of the solvents under reduced pressure, yielding deep blue solid product of the complex. Yield: 0.41 g (69%). Well- shaped single crystals suitable for X-ray diffraction were obtained by re-crystallization of the solid from methanol. Anal. calcd for C34H46Br2Cu4N4O16 (%): C 34.59, H 3.93, N 4.75. Found (%): C 34.37, H 4.02, N 4.83. IR data (KBr, cm– 1): 3285, 3067, 2929, 2873, 1632, 1565, 1512, 1431, 1418, 1349, 1299, 1248, 1207, 1193, 1138, 1097, 1056, 1020, 995, 930, 911, 875, 803, 682, 623, 468, 443. UV-Vis data in ace- tonitrile [λmax (nm), ε (L mol–1 cm–1)]: 227, 6.89 × 103; 249, 6.91 × 103; 270, 4.57 × 103; 365, 1.51 × 103; 640, 83. 2. 4. Synthesis of [Cu4L2Cl2(μ-Cl)2(μ1,1- N3)2]2CH3OH (2) 4-Bromosalicylaldehyde (1.0 mmol, 0.20 g) and 2-(2-aminoethylamino)ethanol(1.0 mmol, 0.10 g) were Table 1. Crystallographic data for the single crystal of the complexes 1 2 Empirical formula C34H46Br2Cu4N4O16 C24H36Br2Cl4Cu4N10O6 Formula weight 1180.73 1116.41 Crystal system Triclinic Monoclinic Space group P1̄ C2/c a, Å 8.1594(11) 21.6461(16) b, Å 10.4407(13) 9.7999(15) c, Å 12.9744(12) 18.9698(17) α, ° 88.6530(10) 90 β, ° 84.1000(10) 116.908(2) γ, ° 77.3890(10) 90 V, Å3 1072.9(2) 3588.4(7) Z 1 4 F(000) 592 2208 Data/restraints/parameters 3960/2/280 3341/2/234 Goodness-of-fit on F2 1.070 1.032 R indices [I > 2σ(I)] R1 = 0.0299, wR2 = 0.0808 R1 = 0.0281, wR2 = 0.0645 R indices (all data) R1 = 0.0360, wR2 = 0.0838 R1 = 0.0376, wR2 = 0.0679 789Acta Chim. Slov. 2022, 69, 787–795 Zhao et al.: Tetranuclear Copper(II) Complexes Derived from 5-Bromo-2-((2-(2-hydroxyethylamino) ... mixed and stirred in methanol (30 mL) for 30 min at 25 °C. Then, copper chloride dihydrate (2.00 mmol, 0.34 g) and sodium azide (2.00 mmol, 0.13 g) were added. The fi- nal mixture was further stirred for 30 min. The deep blue solution was evaporated to remove three quarters of the solvents under reduced pressure, yielding deep blue solid product of the complex. Yield: 0.43 g (77%). Well-shaped single crystals suitable for X-ray diffraction were obtained by re-crystallization of the solid from methanol. Anal. cal- cd for C34H46Br2Cu4N4O16 (%): C 25.82, H 3.25, N 12.55. Found (%): C 43.75, H 4.31, N 15.56. IR data (KBr, cm–1): 3415, 3243, 2962, 2083, 1658, 1585, 1538, 1470, 1445, 1420, 1383, 1293, 1265, 1207, 1138, 1086, 1065, 1021, 989, 930, 910, 846, 805, 725, 686, 626, 608, 591, 556, 463. UV-Vis data in acetonitrile [λmax (nm), ε (L mol–1 cm–1)]: 226, 1.45 × 104; 245, 1.14 × 104; 275, 7.27 × 103; 367, 2.60 × 103; 640, 74. 2. 5. Catalytic Reactions The catalytic reactions were performed according to the procedure described as follows. Substrate (10 mmol), solvent (8 mL) and the complex as catalyst (0.005 mmol) were mixed in a flask. The mixture was equilibrated to 65 °C. Then, TBHP (20 mmol) was added to the mixture. The final mixture was further stirred for 24 h. The products of the oxidation reactions at different time intervals were col- lected and identified by gas chromatograph. 3. Results and Discussion 3. 1. Chemistry The synthetic procedure of the complexes is shown in Scheme 2. The Schiff base 5-bromo-2-((2-(2-hydrox- yethylamino)ethylimino)methyl)phenol was formed by reaction of 4-bromosalicylaldehyde and 2-(2-aminoethyl- amino)ethanol in methanol, which was not isolated and used directly to prepare the complexes. Complex 1 was synthesized by reaction of the Schiff base with copper ace- tate monohydrate, and complex 2 was synthesized by reac- tion of the Schiff base with copper chloride dihydrate and sodium azide. The reaction progresses are accompanied by Table 2. Selected bond distances (Å) and bond angles (º) for the complexes 1 Cu(1)–O(1) 1.894(2) Cu(1)–N(1) 1.950(2) Cu(1)–O(3) 1.9801(19) Cu(1)–N(2) 2.028(2) Cu(1)–O(2) 2.348(2) Cu(2)–O(5) 1.962(2) Cu(2)–O(7) 1.964(2) Cu(2)–O(6A) 1.970(2) Cu(2)–O(8A) 1.977(2) Cu(2)–O(4) 2.143(2) O(1)–Cu(1)–N(1) 92.95(10) O(1)–Cu(1)–O(3) 87.61(9) N(1)–Cu(1)–O(3) 164.84(10) O(1)–Cu(1)–N(2) 176.83(10) N(1)–Cu(1)–N(2) 84.18(10) O(3)–Cu(1)–N(2) 95.51(9) O(1)–Cu(1)–O(2) 101.13(10) N(1)–Cu(1)–O(2) 105.97(10) O(3)–Cu(1)–O(2) 88.75(9) N(2)–Cu(1)–O(2) 78.46(9) O(5)–Cu(2)–O(7) 89.25(10) O(5)–Cu(2)–O(6A) 168.40(9) O(7)–Cu(2)–O(6A) 90.03(9) O(5)–Cu(2)–O(8A) 90.00(10) O(7)–Cu(2)–O(8A) 168.33(9) O(5)–Cu(2)–O(4) 94.32(9) O(7)–Cu(2)–O(4) 101.00(9) O(6)–Cu(2)–O(4A) 97.18(9) O8–Cu(2)–O(4A) 90.67(9) 2 Cu(1)–N(1) 1.882(3) Cu(1)–O(1) 1.891(2) Cu(1)–N(2) 1.936(3) Cu(1)–Cl(1) 2.2248(8) Cu(1)–O(2) 2.280(2) Cu(2)–O(1) 1.9344(18) Cu(2)–N(3) 1.947(3) Cu(2)–N(3B) 1.955(3) Cu(2)–Cl(2) 2.1834(9) Cu(2)–Cl(1) 2.4889(9) N(1)–Cu(1)–O(1) 92.33(10) N(1)–Cu(1)–N(2) 85.05(12) O(1)–Cu(1)–N(2) 172.71(10) N(1)–Cu(1)–Cl(1) 156.24(8) O(1)–Cu(1)–Cl(1) 88.77(6) N(2)–Cu(1)–Cl(1) 96.37(8) N(1)–Cu(1)–O(2) 110.16(10) O(1)–Cu(1)–O(2) 95.45(9) N(2)–Cu(1)–O(2) 79.14(11) Cl(1)–Cu(1)–O(2) 93.35(6) O(1)–Cu(2)–N(3) 169.70(11) O(1)–Cu(2)–N(3B) 94.55(10) N(3)–Cu(2)–N(3B) 76.96(15) O(1)–Cu(2)–Cl(2) 95.16(7) N(3)–Cu(2)–Cl(2) 95.13(9) N(3)–Cu(2)–Cl(2B) 147.00(10) O(1)–Cu(2)–Cl(1) 80.48(6) N(3)–Cu(2)–Cl(1) 96.78(10) N(3)–Cu(2)–Cl(1B) 109.98(9) Cl(2)–Cu(2)–Cl(1) 102.71(3) Symmetry codes: A: 1 – x, 1 – y, 2 – z; B: 1/2 – x, 3/2 – y, 1 – z. 790 Acta Chim. Slov. 2022, 69, 787–795 Zhao et al.: Tetranuclear Copper(II) Complexes Derived from 5-Bromo-2-((2-(2-hydroxyethylamino) ... an immediate color change of the solution from yellow to deep blue. The elemental analyses are in good agreement with the general formulae determined by single crystal X-ray determination. The molar conductivities (ΛM = 27 Ω–1 cm2 mol–1 for 1 and 35 Ω–1 cm2 mol–1 for 2) measured in methanol are consistent with the values expected for non-electrolyte.9 3. 2. Structure Descripiton of Complex 1 The ORTEP plot of complex 1 is shown in Fig. 1. The molecule of the complex possesses crystallographic inversion center symmetry. The two [CuL] units and the central [Cu2(μ2-η1:η1-CH3COO)4] core are linked through two μ2-η1:η1-acetate ligands. In the central [Cu2(μ2-η1:η1- CH3COO)4] core (tetraacetatodicopper(II)), the two Cu atoms has a distance of 2.627(1) Å. The central Cu atom is coordinated by five acetate oxygen atoms, forming a won- derful square pyramidal geometry. The cis and trans angles in the basal plane are in the ranges of 88.36(9)–90.03(9)° and 168.33(9)–168.40(9)°, respectively. The bond angles among the apical and basal donor atoms are in the range of 90.67(9)–101.00(9)°. The Cu-O bond lengths are compara- ble to those observed in acetate bridged copper complex- es.10 The Cu atom in [CuL] unit is coordinated in a square pyramidal geometry, with the phenolate oxygen (O(1)), imino nitrogen (N(1)) and amino nitrogen (N(2)) of the Schiff base ligand, and the acetate oxygen (O(3)) defining the basal plane, and with the hydroxyl oxygen (O(2)) of the Schiff base ligand occupying the apical position. The Cu(1) atom deviates from the basal plane by 0.122(2) Å. The square pyramidal coordination is distorted from ideal model, as evidenced by the bond angles. The cis and trans angles in the basal plane are in the ranges of 84.18(10)– 105.97(10)º and 164.84(10)–176.83(10)°, respectively. The bond angles among the apical and basal donor atoms are in the range of 78.46(9)–105.97(10)°. The distortion is mainly caused by the strain created by the five-membered chelate Scheme 2. The preparation of the complexes. Fig. 1. ORTEP diagram of complex 1 with 30% thermal ellipsoid. Unlabeled atoms are related to the symmetry operation 1 – x, 1 – y, 2 – z. Hydrogen bonds are shown as dashed lines. 791Acta Chim. Slov. 2022, 69, 787–795 Zhao et al.: Tetranuclear Copper(II) Complexes Derived from 5-Bromo-2-((2-(2-hydroxyethylamino) ... rings Cu(1)-N(1)-C(8)-C(9)-N(2) and Cu(1)-N(2)-C(10)- C(11)-O(2). The Cu(1)-O and Cu(1)-N bond lengths are comparable to those observed in Schiff base copper com- plexes.11 The intramolecular hydrogen bond between N(2) and O(4) locks the conformation of the Schiff base ligand. In the crystal structure of the complex, the molecules are linked through O‒H···O and C‒H···Br hydrogen bonds (Table 3), to form three-dimensional network (Fig. 2). Fig. 2. Molecular packing structure of complex 1 linked by hydro- gen bonds. 3. 3. Structure Descripiton of Complex 2 The ORTEP plot of complex 2 is shown in Fig. 3. The molecule of the complex possesses crystallographic inversion center symmetry. The two [Cu2LCl(μ-Cl)] units are linked together by two end-on azido ligands. The Cu atom in the [Cu2LCl(μ-Cl)] unit is coordinated in a square pyramidal geometry, with the phenolate oxygen (O(1)), imino nitrogen (N(1)) and amino nitrogen (N(2)) of the Schiff base ligand, and the bridging chloride atom (Cl(1)) defining the basal plane, and with the hydroxyl oxygen (O(2)) of the Schiff base ligand occupying the apical po- sition. The Cu(1) atom deviates from the basal plane by 0.159(2) Å. The square pyramidal coordination is distort- ed from ideal model, as evidenced by the bond angles. The cis and trans angles in the basal plane are in the ranges of 85.05(12)–96.37(8)° and 156.24(8)–172.71(10)°, re- spectively. The bond angles among the apical and basal donor atoms are in the range of 79.14(11)–110.16(10)°. The distortion is mainly caused by the strain created by the five-membered chelate rings Cu(1)-N(1)-C(8)-C(9)-N(2) and Cu(1)-N(2)-C(10)-C(11)-O(2), and the four-mem- bered chelate ring Cu(1)-O(1)-Cu(2)-Cl(1). The Cu(1)-O and Cu(1)-N bond lengths are comparable to those ob- served in Schiff base copper complexes.10 In the central azido bridged [Cu2(μ1,1-N3)2] core, the two Cu atoms has a distance of 3.054(1) Å. The central Cu atom is coordinated in a square pyramidal geometry, with the phenolate oxy- gen (O(1)) of the Schiff base ligand, the terminal chloride ligand (Cl(2)), and two azido nitrogen (N(3) and N(3A)) defining the basal plane, and with the bridging chloride ligand (Cl(1)) occupying the apical position. The Cu(2) atom deviates from the basal plane by 0.255(2) Å. The square pyramidal coordination is distorted from ideal model, as evidenced by the bond angles. The cis and trans angles in the basal plane are in the ranges of 76.96(15)– 95.16(7)° and 147.00(10)–169.70(11)°, respectively. The bond angles among the apical and basal donor atoms are Table 3. Hydrogen bond distances (Å) and bond angles (º) for the complexes D–H∙∙∙A d(D–H) d(H∙∙∙A) d(D∙∙∙A) Angle (D–H∙∙∙A) 1 O(2)–H(2)∙∙∙O(3)#1 0.84 1.90 2.7318 168 C(9)–H9B∙∙∙Br(1)#2 0.97 2.93 3.6688 134 2 N(2)–H(2)∙∙∙Cl2#3 0.89 2.51 3.2061 135 O(2)–H(2)A∙∙∙O(3)#4 0.84 1.82 2.6347 164 O(3)–H(3)∙∙∙Cl2#5 0.82 2.28 3.0680 162 C(5)–H(5)∙∙∙Cl2#6 0.93 2.76 3.4815 136 C(8)–H8A∙∙∙Cl1#3 0.97 2.68 3.4459 137 C(8)–H8B∙∙∙O(2)#7 0.97 2.50 3.4587 171 C(9)–H(9A)∙∙∙N(6)#5 0.97 2.52 3.2450 132 Symmetry codes: #1: 1 – x, – y, 1 – z; #2: – x, 1 – y, 1 – z; #3: 1/2 – x, 1/2 – y, 1 – z; #4: 1/2 – x, 1/2 + y, 1/2 – z; #5: 2 – x, 1 – y, 1 – z; #6: 1 – x, 1 – y, 1 – z; #7: 1/2 – x, –1/2 + y, 1/2 – z. Fig. 3. ORTEP diagram of complex 2 with 30% thermal ellipsoid. Unlabeled atoms are related to the symmetry operation 1/2 – x, 3/2 – y, 1 – z. Fig. 4. Molecular packing structure of complex 2 linked by hydro- gen bonds. 792 Acta Chim. Slov. 2022, 69, 787–795 Zhao et al.: Tetranuclear Copper(II) Complexes Derived from 5-Bromo-2-((2-(2-hydroxyethylamino) ... in the range of 80.48(6)–109.98(9)°. The Cu-Cl and Cu-N bond lengths are comparable to those observed in chlorido and azido coordinated copper complexes.12 There are two independent methanol molecules of crystallization. In the crystal structure of the complex, the methanol molecules and the coordination moieties are linked through O‒H···O, N‒H···Cl, O‒H···Cl, C‒H···Cl, C‒H···O and C‒H···N hydrogen bonds (Table 3), to form three-dimensional network (Fig. 4). 3. 4. Infrared and Electronic Spectra The infrared spectra of the complexes were record- ed in the region of 4000–400 cm–1 using KBr pellets. The sharp absorptions at 3285 cm–1 for 1 and 3243 cm–1 for 2 are attributed to the N-H bonds of the Schiff base ligands. Peaks at 3400–3500 cm–1 are attributable to O–H stretch- ing vibrations of solvent or hydroxyl group of the Schiff base ligand. Several weak peaks observed in the range 3100–2870 cm–1 likely to be due to C–H stretches. Com- plex 1 displays peak at 1632 cm–1, and complex 2 displays peak at 1658 cm–1, which are assigned to the C=N stretch- es of the Schiff base ligands.13 The different frequencies are in accordance with the bond lengths of C=N, viz. 1.279(4) Å for 1, and 1.230(4) Å for 2. The Ar–O stretching bands are observed at 1248 cm–1 for 1 and 1265 cm–1 for 2. The asymmetric and symmetric stretching vibrations of the ac- etate groups in 1 appear at 1565 and 1418 cm–1, respective- ly. The difference between νasym(COO) and νsym(COO) (Δν = 147 cm–1), which is smaller than 164 cm–1 observed in ionic acetate, reflects the bidentate bridging coordination mode.14 The occurrence of medium and sharp intensity peaks at 2083 cm–1, suggests the presence of N=N stretch- ing frequency of the end-on azide group.15 In addition, new bands observed in the region of 440–600 cm–1 for the complexes due to ν(Cu–O), ν(Cu–N) and ν(Cu–Cl) .16 In the UV-Vis spectra of the complexes, the bands at 245–250 nm and 270–275 nm are attributed to the π-π* and n-π* transitions of the aromatic rings and the C=N groups.17 The bands at about 365 nm can be attributed to the ligand to Cu(II) charge transfer transition (LMCT).15b The visible spectra of complexes display weak single broad d-d bands centered at about 640 nm, which are consistent with the five-coordinate geometry of the Cu(II) complex.15b 3. 5. Catalytic Property Some aromatic and aliphatic alkenes reacted with TBHP to produce the corresponding oxides and epox- ides in good yields and selectivity in acetonitrile when catalyzed by the two complexes (Figs. 5 and 6). The re- sults are summarized in Table 4. The oxidation of styrene with TBHP gave styrene epoxide in 43% yield for 1 and 45% yield for 2 (selectivity 48–49%) under the homoge- neous conditions. Besides, benzaldehyde with yields of 44–48% was produced. The oxidation of cyclohexene and cyclooctene with TBHP catalyzed by the complexes gave good conversion of 95–98% and 82–85%, respectively. The epoxide yields are about 32–38% for both complexes, while the other oxides like cyclohex-2-en-1-ol, cyclohex- 2-en-1-one and cyclooctane-1,2-diol were produced in high yields (49–61%). The solvent effects on the oxidation reactions of cy- clohexene catalyzed by the complexes have been studied (Table 5). It is clear that acetonitrile is a preferred solvent for this reaction. The proposed mechanism for the catalytic reaction is depicted in Scheme 3. Both copper complexes contain unsaturated penta-coordinated Cu spheres, which can be used as Lewis acidic catalysts under homogeneous condi- tions. The peroxo group of TBHP coordinates to the Cu atoms of both complexes to form the pre-catalysts contain- ing LCu–OOH units. The oxo-functionality transferred to the olefins to produce the oxidized products. Table 5. The catalytic resultsa Complex MeCN MeOH CH2Cl2 CHCl3 1 95 63 85 82 2 98 57 81 78 a The reaction time is 24 h, and the temperature is 70 °C. Table 4. The catalytic resultsa Substrate Complex Conversion (wt%) % Yield of products Epoxide Others 1 87 43 44 2 93 45 48 1 95 38 57 2 98 37 61 1 82 32 50 2 85 36 49 a The reaction time is 24h, and the temperature is 70 °C. 793Acta Chim. Slov. 2022, 69, 787–795 Zhao et al.: Tetranuclear Copper(II) Complexes Derived from 5-Bromo-2-((2-(2-hydroxyethylamino) ... 4. Conclusion Two new tetranuclear copper(II) complexes derived from the Schiff base 5-bromo-2-((2-(2-hydroxyethylami- no)ethylimino)methyl)phenol were prepared and charac- terized. All Cu atoms in the complexes are in square py- ramidal coordination. The μ2-η1:η1-acetate, chloride and end-on azide bridging ligands play important role in the formation of these polynuclear complexes. Both complex- es have good catalytic property for the industrially impor- tant epoxidation reactions of styrene, cyclohexene and cyclooctene under homogeneous condition in acetonitrile with TBHP as the oxidant. Scheme 3. The proposed mechanism of the catalytic reactions by the complexes as catalysts. Fig. 5. The conversion plot for the oxidation reactions with complex 1 as the catalyst. Fig. 6. The conversion plot for the oxidation reactions with complex 2 as the catalyst. 794 Acta Chim. Slov. 2022, 69, 787–795 Zhao et al.: Tetranuclear Copper(II) Complexes Derived from 5-Bromo-2-((2-(2-hydroxyethylamino) ... Supplementary Material CCDC 2059783 (1) and 2059784 (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. 6. References 1. (a) S. H. Sumrra, W. Zafar, S.A. Malik, K. Mahmood, S. S. Shafqat, S. Arif, Acta Chim. Slov. 2022, 69, 200–216; DOI:10.17344/acsi.2022.7842 (b) M. Abdi, A.F. Shojaei, M. Ghadermazi, Z. Moradi-Shoeili, Acta Chim. Slov. 2020, 67, 476–486; DOI:10.17344/acsi.2019.5466 (c) S. Dasgupta, S. Paul, D. Samanta, S. Hansda, E. Zangran- do, D. Das, Inorg. Chim. Acta 2020, 501, 119336; ( DOI:10.1016/j.ica.2019.119336 d) C. Spinu, A. Kriza, Acta Chim. Slov. 2000, 47, 179–185; DOI:10.1023/A:1014014631732 (e) S. Rayati, E. Khodaei, P. Nafarieh, M. Jafarian, B. Elmi, A. 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DOI:10.1007/s11243-020-00415-7 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 štirijedrni kompleks bakra(II) z mostovnimi acetatnimi ligandi, [Cu4L2(μ2-η1:η1-CH3COO)6(CH3OH)2] (1), in štirijedrni kompleks bakra(II) s kloridnimi, fenolatnimi in azidnimi mostovnimi ligandi, [Cu4L2Cl2(μ-Cl)2 (μ1,1-N3)2]2CH3OH (2), pri čemer je L deprotonirana oblika Schiffove baze 5-bromo-2-((2-(2-hidroksietilamino)eti- limino)metil)fenol (HL). Produkta smo karakterizirali z elementno analizo, IR in UV spektroskopijo ter rentgensko monokristalno difrakcijo. Strukturna analiza na monokristalu je v obeh spojinah pokazala kvadratno planarno geo- metrijo okoli bakrovih atomov. V kompleksu 1 dva acetatna liganda povezujeta dve enoti [CuL] z jedrom [Cu2(μ2-η1: η1-CH3COO)4]. V kompleksu 2 so enote [Cu2LCl(μ-Cl)] povezane z azido ligandi. Schiffova baza kot ligand je koordi- nirana na bakrov atom preko štirih N in O donorskih atomov. Vodikove vezi povezujejo molekule obeh kompleksov v tridimenzionalno mrežo. Katalitske lastnosti kompleksov smo preučevali v reakcijah epoksidacije alkenov s tert-butilhi- droperoksidom kot terminalnim oksidantom pod blagimi pogoji v acetonitrilu. 796 Acta Chim. Slov. 2022, 69, 796–802 Mihovecet et al.: Evaluation of the Stability of Hydrocortisone Sodium ... DOI: 10.17344/acsi.2022.7539 Scientific paper Evaluation of the Stability of Hydrocortisone Sodium Succinate in Solutions for Parenteral Use by a Validated HPLC-UV Method Katja Mihovec,1 Žane Temova Rakuša,2 Enikő Éva Gaál2 and Robert Roškar2 1 University Medical Centre Ljubljana, Slovenia 2 University of Ljubljana, Faculty of Pharmacy, Ljubljana, Slovenia * Corresponding author: E-mail: robert.roskar@ffa.uni-lj.si Tel: +386 1 4769 655 Received: 06-30-2022 Abstract This study aimed to determine the in-use stability (t95%) of hydrocortisone sodium succinate (HSS) infusion solutions and provide evidence-based guidelines on their usability. HSS infusion solutions were prepared and stored as recommended by the manufacturer and under common conditions in our hospital. The effects of HSS concentration (1 and 4 mg/mL), solvent (isotonic saline and glucose), temperature (ambient and 30 °C), and light on its stability were evaluated using a validated stability-indicating HPLC-UV method. HSS degradation followed first-order kinetics. No significant difference in its stability was observed between the two evaluated concentrations, solvents and light exposure (t95% between 25 and 30 h). Elevated temperature (30 °C) affected HSS stability and significantly reduced the t95% (4.6–6.3 h). HSS infusion solutions are physically and chemically stable (<5% degradation) for at least 6 h if stored below 30 °C. The in-use stability may be extended up to 24 h if stored below 24 °C. Keywords: Forced degradation study; in-use stability; infusion; injection; Solu-Cortef. 1. Introduction Cortisone is a glucocorticoid hormone synthesized endogenously in the adrenal gland cortex, as a response to stress.1 Its synthetic form – hydrocortisone, mostly as hy- drocortisone sodium succinate (HSS), is used in medi- cines for various conditions, requiring rapid and intense corticosteroid effects, such as acute or chronic adrenal in- sufficiency, various autoimmune and allergic diseases, and septic shock, unresponsive to fluid resuscitation and treat- ment with vasopressors.2,3 Thus, the use of HSS as a con- tinuous infusion is associated with more stable cortisol plasma concentrations and reduced fluctuation in blood glucose levels compared to intermittent boluses.4 This is particularly important in patients with diabetes, as hyper- glycaemia is one of the most common glucocorticoid side effects.4,5 The application of continuous HSS infusion is also well-established practice for critically ill patients in hospital intensive care units (ICUs), also including the ICU of our hospital. For such purposes, commercially available medicinal products, in the form of vials contain- ing freeze-dried powder for solution for injection/infu- sion, are used. Thus, an intravenous infusion is prepared as recommended by the manufacturer – by reconstituting the powder with 2 mL of sterile water for injection and addi- tion of this solution to 100–1000 mL of 5% glucose in wa- ter or isotonic saline solution or 5% glucose in isotonic saline solution under aseptic conditions.2 While the man- ufacturer recommends immediate use after reconstitution with sterile water for injection and disposal of any remain- der, no information is provided on the in-use stability of the diluted HSS solution for infusion.2 HSS is an ester, sus- ceptible to hydrolysis and other degradation reactions (ox- idation and transesterification) in aqueous solutions.6–8 In a broader sense, data on HSS stability can be found in the literature, as HSS stability studies in oral solutions and sus- pensions,9,10 solutions for infusion,11,12 or as compatibility studies with other drugs.13–15 Focusing on stability studies of HSS individually in solutions for infusion, which are limited to isotonic saline solutions, we identified a need for 797Acta Chim. Slov. 2022, 69, 796–802 Mihovecet et al.: Evaluation of the Stability of Hydrocortisone Sodium ... a stability study under clinically relevant real-life condi- tions, since the type of media, temperature, and HSS con- centration may affect its stability.8–10 As medical personnel deal with these issues on daily basis, our primary objective within this study was to evaluate the stability of HSS under common real-life conditions and thus provide evi- dence-based guidelines. For such purpose, we investigated the stability of HSS in solutions for infusion, as commonly prepared in our hospital, by using a stability-indicating HPLC-UV method. We thus evaluated the effect of clini- cally relevant HSS concentration (1 mg/mL and 4 mg/mL), type of reconstitution solvent (isotonic saline and glucose solutions), temperature (24 °C and 30 °C), and light (pro- tected and exposed to daylight) on its stability in infusion solutions. 2. Experimental 2. 1. Chemicals and Preparations HPLC grade acetonitrile (ACN) was purchased from Sigma-Aldrich (Steinheim, Germany). Hydrochloric acid (HCl) and sodium hydroxide (NaOH) solutions (Titrisol®) as well as phosphoric acid (H3PO4) (85%) were purchased from Merck (Darmstadt, Germany). H2O2 solution (30%) was purchased from Honeywell FlukaTM (Seelze, Germa- ny). High purity water was obtained using a Milli-Q A10 Advantage water purification system (Millipore Corpo- ration, Bedford, MA, USA). Solu-Cortef 100 mg powder for solution for injection or infusion (Pfizer, Luxembourg, Luxembourg), and solutions for infusion: 0.9% sodium chloride (S) in 50 mL infusion bags (Baxter, Deerfield, Il- linois, USA), and 5% glucose (G) in 100 mL intravenous (IV) containers (B. Braun, Melsungen, Germany) were used. Due to the lack of an HSS reference standard, its calibration and quality control (QC) solutions, as well as samples for the forced degradation study, were prepared by dissolving a portion of the powder of the medicinal product Solu-Cortef in Milli-Q water. The total powder was initially weighted to calculate the share of HSS, ac- cording to the reported HSS content in the product. 2. 2. Instrumentation and Chromatographic Conditions The analysis was performed on an Agilent 1100/1200 series HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a UV–VIS detector and a Chem- Station data acquisition system. A reversed-phase Luna C18 250 × 4.6 mm, 5 μm particle size column (Phenomenex, Torrance, CA, USA) at 40 °C using 1% (v/v) H3PO4 (mo- bile phase A), and ACN (mobile phase B) in isocratic mode (33% A, 67% B), at a flow-rate of 1.5 mL/min was utilized for the analysis. Detection was performed at 254 nm. The injection volume was 2 µL. The retention time (tr) of HSS was 10.8 min and the total runtime was 13 min. 2. 3. Preparation of Samples for Forced Degradation Study The forced degradation study was performed accord- ing to the ICH guidelines Q1A (R2).16 A stock HSS solu- tion (5 mg/mL) was initially prepared and diluted 5-fold, to obtain samples containing 0.1 M HCl, 0.1 M NaOH, 3% H2O2, or Milli-Q water. Samples with Milli-Q water were used as controls (ambient temperature and protected from light) and to assess the effect of temperature (60 °C) and light (exposure to daylight). All samples, except those for thermal degradation, were stored at ambient tempera- ture (24 °C) and protected from light (except those for the photostability testing). The samples were exposed to stress conditions for 24  hours. The samples were neutralized with HCl or NaOH (when required) or cooled to ambient temperature (thermal stress samples) before analysis. 2. 4. Preparation of Calibration Standards and QC Solutions A stock solution containing 5 mg/mL HSS was initial- ly prepared and further diluted with Milli-Q water to ob- tain calibration standards with the following HSS concen- trations: 0.05 mg/mL, 0.5 mg/mL, 2.0 mg/mL, 3.0 mg/mL and 5.0 mg/mL. QC samples containing 0.1 mg/mL, 1.0 mg/mL, and 4.0 mg/mL HSS were prepared from the initially prepared HSS stock solution in triplicate in the same manner. The calibration and QC solutions were pre- pared and analysed on three consecutive days of the vali- dation. 2. 4. Method Validation The utilized HPLC–UV method was validated fol- lowing the ICH guidelines Q2(R1) in terms of specificity, linearity, precision, accuracy, quantitation limit (LOQ), detection limit (LOD), and sample stability.17 Specificity was evaluated in chromatograms of the used solvents (Milli-Q water, 0.9% sodium chloride solu- tion, 5% glucose solution, and the solvent in the vial of the medicinal product Solu-Cortef, which contains ben- zyl alcohol as a preservative), which were compared with the chromatogram of HSS solution. Specificity was also assessed in forced degradation HSS samples, which were evaluated for chromatographic interferences. Linearity was assessed by linear regression analysis of calibration standards, covering expected HSS concen- trations in solutions for infusion (0.05–5.0 mg/mL). The determination coefficient (R2) > 0.999 was considered ac- ceptable. The QC solutions, prepared at three concentration levels on each day of the validation, were used to evaluate the accuracy, precision, and injection repeatability. Intra- and inter-day accuracy was determined as the ratio be- tween the HSS concentration calculated from the regres- 798 Acta Chim. Slov. 2022, 69, 796–802 Mihovecet et al.: Evaluation of the Stability of Hydrocortisone Sodium ... sion line and its actual concentration. Intra- and inter-day precision was determined by calculating the relative stand- ard deviation (RSD) of the QC solutions in triplicate and injection repeatability was determined as the RSD of six consecutive injections of a QC solution. The acceptance criteria were 100 ± 5% for accuracy, ≤ 5% RSD for preci- sion, and ≤ 2% RSD for injection repeatability. The LOD and LOQ were calculated using the equa- tions LOD = (3.3 × σ)/S and LOQ = (10 × σ)/S, where σ is the standard deviation of the intercepts and S is the aver- age slope of the three regression lines. HSS stability was determined by storing the QC solu- tions at all three concentration levels in the autosampler (6 °C) and analysing them within 24 h. HSS stability, ex- pressed as a share of the initial response, was set at 100 ± 5%. 2. 6. Sample Preparation and HSS in-use Stability Study in Solutions for Infusion The stability of HSS was studied in solutions for in- fusion prepared according to the manufacturer’s instruc- tions, and as prepared in our hospital. A 50 mg/mL HSS solution was initially prepared by adding 2 mL of sterile water for injection to the content of a vial of Solu-Cor- tef, followed by manual shaking. Half of this solution (1 mL) was withdrawn and diluted up to 50 mL with S or G in the original IV containers to an HSS concentration of 1 mg/mL. The 4 mg/mL HSS solutions in S or G were pre- pared in the same way, using two vials of Solu-Cortef (2 × 2 mL diluted with 50 mL of S or G). The pH values of the prepared solutions were measured using a pH meter MP 220 (Mettler Toledo, Switzerland). The effects of different HSS concentrations (1 mg/mL and 4 mg/mL), solvent (S and G), temperature (controlled ambient (24 ± 1 °C) and elevated (30 ± 1 °C), and light exposure (protected (UV-prot) and unprotected (UV)) on HSS stability in solutions for infusion were studied (Table 1). All samples were prepared and stored in the original S and G IV containers in triplicate and analyzed at regular time points within 72 hours. All samples were also visually ex- amined for potential physical changes. 2. 7. In-use Stability Determination and Statistical Analysis The results are expressed as the mean of three par- allels of the samples along with the standard error of the mean. Zero (c = c0 – kt), first (lnc = lnc0 – kt), and sec- ond-order (1/c = 1/c0 + kt) kinetics, where t is time, c is the HSS concentration at time t, c0 is the initial HSS concentra- tion, and k is the reaction rate constant, were fitted to the HSS degradation using the least square regression func- tion. Among them, the model with the highest R-square was selected and applied for in-use stability determina- tion, which was defined as HSS content ≥ 95% of the initial content. The determined rate constants and in-use stabili- ty were compared by 95% confidence intervals. Statistical analyses using a two-sample t-test assuming variances, as previously determined by the F-test for two sets of data, which differ in only one parameter (e.g., different storage temperature, light, HSS concentration, or type of solvent) were performed using MS Excel 2019. Differences with p values < 0.05 were considered significant. 3. Results 3. 1. HSS Forced Degradation Study Among the conditions tested within the forced deg- radation study, HSS proved most susceptible to hydrolytic degradation with only 2% remaining in 0.1 M NaOH and 20% in 0.1 M HCl after 24 hours. HSS was also susceptible to oxidation (56% remaining after 24 hours in 3% H2O2), thermal degradation (63% remaining after 24 hours of storage at 60 °C), and photolytic degradation to a lesser extent (96% remaining after 24 hours). The HSS main deg- radation products (tr 4.0, 5.7, and 6.5 min) formed dur- ing the forced degradation study did not interfere with its chromatographic evaluation (tr 10.9 min) (Figures S1, S2, and S3). 3. 2. Method Validation The specificity of the method was confirmed as all solvents recommended by the manufacturer for dissolution and dilution of the medicinal product Solu-Cortef, did not contain interfering peaks at HSS retention time. Also, no interfering degradation product peaks were formed during the HSS forced degradation study (Figure S2). In addition, the linearity, intra- and inter-day accuracy and precision, Table 1. Conditions during hydrocortisone sodium succinate (HSS) stability study in isotonic saline (S) and 5% glucose (G) solutions for infusion. Sample HSS Solvent Storage Light concentration temperature exposure [mg/mL] [°C] 1 S 24 UV-prot 4 S 24 UV-prot 1 G 24 UV-prot 4 G 24 UV-prot 1 S 24 UV 4 S 24 UV 1 G 24 UV 4 G 24 UV 1 S 30 UV-prot 4 S 30 UV-prot 1 G 30 UV-prot 4 G 30 UV-prot S – isotonic saline (0.9% sodium chloride); G – 5% glucose solution; UV – exposed to daylight; UV-prot – light protected. 799Acta Chim. Slov. 2022, 69, 796–802 Mihovecet et al.: Evaluation of the Stability of Hydrocortisone Sodium ... and injection repeatability of the method were confirmed (Table 2), as all results were within the acceptance crite- ria. The method was found sufficiently sensitive for HSS evaluation within the stability study (LOQ ≤ 3.9% of the expected HSS content). HSS also proved proper stability in the QC solutions, with a < 1% change in its response after 24 hours (Table 2). 3. 3. HSS in-use Stability Study in Solutions for Infusion The determined pH values of the simulated solu- tions for infusion with different HSS concentrations were as follows: 7.04 in S containing 1 mg/mL HSS; 7.26 in S containing 4 mg/mL HSS; 7.42 in G containing 1 mg/mL HSS and 7.52 in G containing 4 mg/mL HSS and were within the pH range, specified by the manu- facturer.2 The obtained results on HSS stability in the simulated solutions for infusion are shown in Figure 1. The increase in temperature (from 24 °C to 30 °C) signif- icantly increased HSS degradation, while the other tested conditions – different HSS concentrations (1 mg/mL and 4  mg/mL), media (S or G), and light exposure only slightly affected HSS stability. No changes in colour, odour or precipitations were detected in the samples during the stability study. Table 2. Validation data of the analytical method for hydrocortisone sodium succinate (HSS) quantification. HSS Range [mg/mL] R2 LOD [mg/L] LOQ [mg/L] calibration 0.05-5.00 1.0000 12.6 39.2 samples QC samples Intra-day accuracy and precision Inter-day accuracy and precision HSS conc. Found conc. Accuracy RSD Found conc. Accuracy RSD Inj. Stability [mg/mL] [mg/mL] (%) (%) [mg/mL] (%) (%) rep. (%) 0.1 0.0981 98.1 1.29 0.0997 99.7 3.30 0.52 100.5 1.0 1.0054 100.5 0.02 1.0087 100.9 0.29 0.20 99.2 4.0 3.9901 99.8 0.46 3.9956 99.9 0.55 0.29 99.2 LOD – detection limit; LOQ – quantitation limit; QC – quality control; conc. – concentration; RSD – relative standard deviation; Inj. rep. – injection repeatability. Figure 1. Hydrocortisone sodium succinate (HSS) stability in the simulated solutions for infusion in isotonic saline (0.9% sodium chloride) (S) and 5% glucose solution (G) under different storage conditions. The results are presented as an average ± standard error of the mean, n = 3. 800 Acta Chim. Slov. 2022, 69, 796–802 Mihovecet et al.: Evaluation of the Stability of Hydrocortisone Sodium ... The fitting of zero, first, and second-order kinetic models showed that HSS degradation in aqueous solu- tions follows first-order kinetics, which was applied to its stability evaluation. First-order reaction rate constants were calculated for HSS degradation in each tested solu- tion and used for the determination of its in-use stabili- ty (Section In-use stability determination and statistical analysis) (Table 3). 4. Discussion The main objective of our study was to determine the chemical and physical stability of HSS at two concentra- tions (1 mg/ml and 4 mg/ml) at ambient temperature after reconstitution with the solvent in the vial of the medic- inal product and dilution with 0.9% sodium chloride or 5% glucose solution. Although the HSS concentration of 4 mg/ml exceeds the highest HSS concentration in in- fusions, prepared according to the manufacturer’s in- structions, it is a clinically relevant concentration in the treatment of ICU patients, for whom a reduction in the infusion volume is very desirable. These two HSS concen- trations represent the most common circumstances, when preparing an HSS infusion, in our hospital and were there- fore selected for the study. HSS stability evaluation was performed by utilizing a stability-indicating HPLC-UV method, based on the method proposed in the European pharmacopeia HSS monograph,18 which was further opti- mized and properly validated following the ICH Q2(R1) guidelines.17 The stability-indicating nature of the meth- od was confirmed by forced degradation studies, as the chromatographic peak of HSS was chromatographically separated from its degradation products, as can be seen in Figure S2, yet the peak purity was not assessed due to lim- itations of the analytical equipment (variable wavelength UV detector). Forced degradation studies also revealed the susceptibility of HSS to degradation under hydrolytic, ox- idative, and thermal conditions. However, these stability issues are not addressed by the manufacturer of the medic- inal product Solu-Cortef, who does not specify the in-use stability of the HSS solution for infusion. Specified in-use stability would be valuable information for the medical personnel with implications for the patients, as HSS is commonly applied as a continuous infusion. However, it is also unaccounted for in the accessible literature. There- fore, the in-use stability of HSS in solutions for infusion was determined within this study, together with the effects of real-life conditions and situations (use of different types and volumes of solution for dilution, temperature varia- tions, and exposure to light). The main conclusion from the performed in-use stability study is that HSS stability is mostly affected by temperature, as the increase in temperature (from 24 °C to 30 °C) significantly increased HSS degradation (reaction rate constants in Table 3) and resulted in significantly shorter in-use stability of ≤ 6.3 hours (in-use stability in Table 3) (t-test, p < 0.001). The destabilizing effect of high- er storage temperature on HSS in S was also demonstrat- ed by Gupta and Ling.16 The determined in-use stability, considering the 95% confidence interval was higher in the samples protected from light (Table 3). However, these dif- ferences were typically not statistically significant (t-test, p > 0.05), which is in line with the findings from the per- formed forced degradation study (Section HSS forced deg- radation study). Analogously, the differences between the stability of HSS at different concentrations in S were not significant (t-test, p > 0.05), whereas G containing a higher HSS concentration (4 mg/mL) was significantly less stable than the 1 mg/mL solution (t-test, p < 0.03) under all three evaluated conditions (24 °C, protected from light; 24 °C, exposed to daylight; and 30 °C, protected from light) (Ta- ble 3). Comparing the HSS stability in solution with the same concentration and stored under the same conditions Table 3. First-order rate constants (k1) and in-use stability (t95%) for hydrocortisone sodium succinate (HSS) at two concentrations (1 mg/mL and 4 mg/mL) in the simulated solutions for infusion under different storage conditions. Simulated k1 [h–1] (CI) In-use stability [h] (CI) solutions for 24 °C, 24 °C, 30 °C, 24 °C, 24 °C, 30 °C, infusion UV-prot UV UV-prot UV-prot UV UV-prot 1 mg/mL 1.84×10–3 1.92×10–3 8.79×10–3 28.0 26.7 5.9 HSS in S (1.86×10–3 – 2.01×10–3) (1.90×10–3 – 1.93×10–3) (7.84×10–3 – 9.74×10–3) (26.6 – 29.3) (26.5 – 27.0) (5.2 – 6.5) 4 mg/mL 1.94×10–3 1.99×10–3 1.03×10–2 26.5 25.8 5.0 HSS in S (1.86×10–3 – 2.01×10–3) (1.91×10–3 – 2.07×10–3) (9.46×10–3 – 1.10×10–2) (25.4 – 27.5) (24.7 – 26.8) (4.6 – 5.4) 1 mg/mL 1.64×10–3 1.76×10–3 7.81×10–3 31.2 29.1 6.6 HSS in G (1.55×10–3 – 1.73×10–3) (1.71×10–3 – 1.81×10–3) (7.46×10–3 – 8.17×10–3) (29.6 – 32.9) (28.2 – 29.9) (6.3 – 6.9) 4 mg/mL 1.98×10–3 2.00×10–3 1.10×10–2 25.9 25.4 5.0 HSS in G (1.91×10–3 – 2.05×10–3) (2.00×10–3 – 2.05×10–3) (9.88×10–3 – 1.05×10–2) (25.0 – 26.9) (25.0 – 25.7) (4.9 – 5.2) CI – 95% confidence interval; S – isotonic saline (0.9% sodium chloride); G – 5% glucose solution; UV – exposed to daylight; UV-prot – light pro- tected. 801Acta Chim. Slov. 2022, 69, 796–802 Mihovecet et al.: Evaluation of the Stability of Hydrocortisone Sodium ... (Table 3), we concluded that the use of different dilution solvents (S or G) does not significantly affect the HSS sta- bility (t-test, p > 0.05), which was expected as they are both recommended as dilution solvents by the manufacturer.2 The determined in-use stability of 24 hours under con- trolled room temperature provides the medical personnel reliable evidence on the usability of the infusion solution, during the application of the infusion. During this time period, all evaluated HSS solutions remained physically stable, as no changes in the organoleptic properties were observed. The microbiological stability was not evaluated within this study. The results of this stability study, per- formed in clinically relevant conditions on the medicinal product, represent a step forward in providing high-qual- ity patient care, which is primarily ensured by the quality of the medicinal product itself, guaranteed by its manufac- turer and the competent regulatory bodies. 5. Conclusion The chemical and physical stability of HSS in solu- tions for infusion under different conditions, which sim- ulate the conditions in hospitals, was assessed within this study. The results, obtained using a stability-indicating HPLC-UV method, revealed HSS degradation in these solutions, which followed first-order kinetics. Based on the stability data, in-use stability (t95%) of at least 24 hours was confirmed at ambient temperature and a significantly lower in-use stability (≤ 6 hours) at 30 °C. Other evaluated conditions (HSS concentration, light exposure, and use of different dilution solvents), did not significantly affect the stability of HSS in the examined solutions for infusion. All evaluated solutions were physically stable within the deter- mined in-use stability. The significant temperature effect on the stability of HSS in solutions for infusion should be considered in hospitals with uncontrolled temperatures and especially during summertime. Acknowledgements This research was financially supported by the Slove- nian Research Agency (ARRS) [P1-0189]. Competing interest: None declared. 6. References 1. M. Q. Almeida, B. B. Mendonca, Clinics (Sao Paulo) 2020, 75, e2022–e2022. DOI:10.6061/clinics/2020/e2022 2. ‘Solu-Cortef – Summary of Product Characteristics (SmPC) – https://www.medicines.org.uk/emc/medicine/7833#gref, (assessed: July 21, 2021). 3. R. H. Straub, M. Cutolo, Rheumatology 2016, 55, ii6–ii14. DOI:10.1093/rheumatology/kew348 4. H. Hoang, S. Wang, S. Islam, A. Hanna, A. Axelrad, C. Brath- waite, P T 2017, 42, 252–255. 5. H. E. Tamez-Pérez, D. L. Quintanilla-Flores, R. Rodríguez- Gutiérrez, J. G. González-González, A. L. Tamez-Peña, World J Diabetes 2015, 6, 1073–1081. DOI: 10.4239/wjd.v6.i8.1073 6. E. R. Garrett, J Pharm Sci 1962, 51, 445–450. DOI:10.1002/jps.2600510511 7. L. Solomun, S. Ibric, V. Pejanovic, J. Djuris, J. Jockovic, P. Stankovic, Z. Vujic, Hem Ind 2012, 66, 647–657. DOI:10.2298/HEMIND120207023S 8. V. Das Gupta, J Pharm Sci 1978, 67, 299–302. DOI:10.1002/jps.2600670305 9. J. Chappe, N. Osman, S. Cisternino, J.-E. Fontan, J. Schlatter, J Pediatr Pharmacol Ther 2015, 20, 197–202. DOI:10.5863/1551-6776-20.3.197 10. A. Manchanda, M. Laracy, T. Savji, R. H. Bogner, Int J Pharm Compd 2018, 22, 66–75. 11. V. D. Gupta, J. Ling, Int J Pharm Compd 2000, 4, 396–397. 12. D. C. Rigge, M. F. Jones, J Pharm and Biomed 2005, 38, 332– 336. DOI:10.1016/j.jpba.2004.12.026 13. J. C. Cradock, L. M. Kleinman, A. Rahman, Am J Hosp Pharm 1978, 35, 402–406. DOI:10.1093/ajhp/35.4.402 14. L. A. Trissel, K. M. King, Y. Zhang, A. M. Wood, J Oncol Pharm Pract 2002, 8, 27–32. DOI:10.1191/1078155202jp087oa 15. Y. W. Cheung, B. R. Vishnuvajjala, K. P. Flora, Am J Hosp Pharm 1984, 41, 1802–1806. DOI:10.1093/ajhp/41.9.1802 16. International Council for Harmonisation. ICH Harmonised Tripartite Guideline. Stability Testing of New Drug Substanc- es and Products Q1A(R2). Geneva, Switzerland, 2003. 17. International Council for Harmonisation. ICH Harmonised Tripartite Guideline. Validation of Analytical Procedures: Text and Methodology Q2(R1). Geneva, Switzerland, 2005. 18. Hydrocortisone Hydrogen Succinate. In: European Pharma- copoeia 10ed. Strasbourg: Council of Europe. 2020:2888-9. 802 Acta Chim. Slov. 2022, 69, 796–802 Mihovecet et al.: Evaluation of the Stability of Hydrocortisone Sodium ... 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 Namen te študije je opredelitev stabilnosti in določitev roka uporabnosti med uporabo (t95%) raztopin za infundiranje z natrijevim hidrokortizonsukcinatom (HSS) ter zagotovitev na dokazih podprtih priporočil o njihovi uporabnosti. Infuzijske raztopine HSS smo pripravili in shranjevali v skladu s priporočili proizvajalca in pri običajnih pogojih v naši bolnišnici. Z validirano stabilnostno indikativno HPLC-UV metodo smo ugotavljali vpliv koncentracije HSS (1 in 4 mg/mL), topila (izotonična fiziološka raztopina in raztopina glukoze), temperature (sobna in 30 °C) in svetlobe na njegovo stabilnost. Razgradnja HSS je sledila kinetiki prvega reda. Ugotovili smo, da različni preiskovani koncentraciji HSS, obe topili in izpostavljenost svetlobi niso značilno vplivali na stabilnost HSS (t95% med 25 in 30 urami), medtem ko je povišana tem- peratura (30 °C) značilno skrajšala t95% (4,6–6,3 ur). Infuzijske raztopine HSS so fizikalno in kemično stabilne (<5 % razgradnja) vsaj 6 ur pri temperaturi do 30 °C in najdlje 24 ur pri temperaturi do 24 °C. 803Acta Chim. Slov. 2022, 69, 803–810 Kešelj et al.: Use of Total Organic Carbon Analyzer in Isotherm ... DOI: 10.17344/acsi.2022.7553 Scientific paper Use of Total Organic Carbon Analyzer in Isotherm Measurements of Co-Adsorption of VOCs and Water Vapor from the Air Dragana Kešelj,1,* Dragica Lazić1 and Zoran Petrović1 1 Faculty of Technology, University of East Sarajevo, Karakaj 34A, 75400 Zvornik, BiH * Corresponding author: E-mail: dragana.keselj@tfzv.ues.rs.ba; draganakeselj@yahoo.com; Tel.: +38766253256 Received: 04-05-2022 Abstract The binary adsorption isotherms of volatile organic compounds (VOCs), and water vapor from the air have been the fo- cus of much research in recent years. The content of adsorbed VOCs in the presence of water vapor can be determined by the volumetric or gravimetric method, in a static or dynamic mode. This study focuses on the adsorption technique in a static mode for isotherm measurement of the co-adsorption of VOCs and water vapor from the air using the gravimetric method. The content of VOCs is determined using a total organic carbon analyzer, while the amount of the adsorbed water was calculated from the difference between total adsorption (VOCs and water) and the adsorbed VOCs. This paper presents several adsorption isotherms with different VOCs (toluene, benzene, methanol, ethanol and isopropyl alcohol) and adsorbents (ZSM-5 zeolite, silica gel and Na-Form mordernite) in the presence of water vapor. The well-known adsorption isotherm models (Langmuir, extended Langmuir, Freundlich, extended Freundlich and Hill) were used to treat experimental results. The adjusted R-Squared (adj. R2) values obtained for those non-linear models for isotherms (total adsorpton (qe,tot) as a function of equilibrium concentration of VOC (Ce) and the adsorbed VOC (qe) as a func- tion of equilibrium concentration of VOC (Ce) are used to determine the best-fit isotherm model. The modeling results showed that the 3-parameter models could fit the data better than the 2-parameter model, with relatively higher adj. R2. Experimental results demonstrate that the presented adsorption technique can be used for isotherm measurement of the co-adsorption of VOCs and water vapor from the air. Keywords: Adsorption, adsorption isotherms, VOCs, water vapor 1. Introduction The binary adsorption isotherms of VOCs and water vapor from the air have been the focus of much research in recent years.1–7 Much research is also dedicated to the ad- sorption of VOCs on zeolites or activated charcoal in the absence of moisture. Under real conditions, water vapor is always present, and its content may sometimes vary and even exceed that of VOCs. The influence of relative hu- midity on VOCs diffusion and adsorption is still not well understood. Research in the past couple of years has shown that the presence of water vapor (moisture) affects the ad- sorption of gases and volatile compounds.8–10 For exam- ple, investigations into the adsorption of dichloroethane, ethyl acetate and benzene on metal–organic frameworks (MOFs) show that the adsorption of these volatile organic compounds decreases in the presence of humidity.11 Very limited experimental research has been done on the effect of humidity on the diffusion coefficient.12 The attraction between water molecules and methanol molecules may weaken the interaction forces between the solid surface and the methanol molecule and reduce the total adsorp- tion capacity of methanol.2,11 A piece of equipment that may be quicker and more effective at measuring pure CO2 adsorption, pure H2O adsorption, and co-adsorption is the DVS Vacuum.12 This has previously been used by Su et al. to measure CO2 and H2O isotherms on an amine-func- tionalised Mg-MOF-74.13 The DVS Vacuum uses a micro- balance to measure the weight change of a sample subject- ed to various conditions. The temperature, pressure, and composition in the sorption chamber can be controlled 804 Acta Chim. Slov. 2022, 69, 803–810 Kešelj et al.: Use of Total Organic Carbon Analyzer in Isotherm ... very accurately.12,13 Today there are different devices which can perform adsorption of a variety of vapors (H2O, MeOH, EtOH, C6H6 and other non-corosive vapors). A unique principle of the DVS Vacuum is the ability to con- trol and measure sorbate entry and exit flows simultane- ously while recording changes in sample mass.12 The determination of adsorption capacity can be done by volumetric or gravimetric method, in a stat- ic or dynamic mode. This study sets out to show how co-adsorption isotherms can be performed in a static mode, using a total organic carbon analyzer for gravi- metric determination of the amount (mass) of adsorbed VOCs. The total adsorption of VOCs and water vapor was determined gravimetrically, from the difference in the mass of the adsorbent after and before the adsorp- tion, while the amount of adsorbed water is calculated from the difference between total adsorption and the adsorbed VOCs. 2. Materials and Methods 2. 1. Materials Zeolites, porous polymers, composites, alumino- phosphates (AlPOs) and silica aluminophosphates (SA- POs), silica gels, activated carbons and metal organic frameworks (MOFs) are important classes of materials used in various sorption-based technologies. In order to obtain representative experimental data for the purpose of the analysis, the study included adsorbents of different physical and chemical properties. The following adsorbents were used for the purpose of this study: highly silicate ZSM-5 zeolite – two samples with different molar ratio SiO2/Al2O3 (Adsorbent 1- SiO2/ Al2O3 = 394 and Adsorbent 2- SiO2/Al2O3 = 926), Na- Form mordernite (Adsorbent 3), and silica gel (Adsorbent 4) (Table 1). Molar ratio SiO2/Al2O3 for adsorbents 1-3 is calculated based on their chemical analysis (mass per- centages of SiO2 and Al2O3), and refers to the ratio be- tween the content of SiO2 and Al2O3 expressed in mass per cent multiplied by the ratio between the molar mass- es of Al2O3 and SiO2. For these adsorbents, apart from the content of SiO2 and Al2O3, the analysis also included determining the content of Na in mass per cent. Loss of annealing for adsorbents 1–3 was calculated in the form of the percentage of the shrinkage in mass after heating at 950 °C, whereas Adsorbent 4 was analyzed for water residue, which also represents the loss of mass in per- centages, but after drying at 160 °C. A low-temperature N2 adsorption for the adsorbents used was employed to determine the pore size as well as the specific surface area using the BET method. The adsorbents were analyzed for the mean diameter of 50% of the particles (d(50)) and the mean diameter of 10% of the particles (d(10)) (Table 1) using a laser diffraction particle size analyzer (Master- sizer 3000). The adsorbents were pre-dried to remove moisture before the experiments. The chemicals used as adsorbates were toluene, ben- zene, methanol, ethanol and isopropyl alcohol. The physi- cal properties of the five VOCs used as adsorbates are list- ed in Table 2. Table 2. Physicochemical properties of adsorbates Chemical Chemical Density Molecular Boiling Vapor formula (g/cm³) weight point pressure (g/mol) ( °C) (kPa) Methanol CH3OH 0.792 32.04 64.7 13.02 (20 °C) Ethanol C2H5OH 0.7893 46.07 78.4 5.95 (20 °C) Isopropyl alcohol (CH3)2CHOH 0.7855 60.10 82.4 4.4 (20 °C) Benzene C6H6 0.8765 78.114 80.1 12.7 (25 °C) Toluene C6H5CH3 0.866 92.14 110.6 2.8 (20 °C) Table 1. Physical and chemical characteristics of Adsorbent 1, 2, 3 and 4 Parameter Adsorbent 1 Adsorbent 2 Adsorbent 3 Adsorbent 4 Loss of annealing, w % 2.5 3.1 6.9 – d(10), μm 1.3 – 2.3 – d(50), μm 2.5 < 10 8.8 15–35 Molar ratio SiO2/Al2O3 384 926 13.5 – Na content, w % 1.2 1.32 4.9 – BET, m2/g 355 434 420 450–550 Water residue, w % – – – < 10 pH (5%) – – – 6–8 Pore size, nm – – – 4.7–8 805Acta Chim. Slov. 2022, 69, 803–810 Kešelj et al.: Use of Total Organic Carbon Analyzer in Isotherm ... 2. 2. Experimental Method and Analysis The adsorbents were pre-dried to remove moisture before the experiments. A mass of 0.5 g of adsorbent is weighed into nine small glass jars with lids. The closed glass jars with adsorbents are placed in the adsorption chambers (2.5 L). Adsorption chambers (2.5 L) with closed glass jars were put in a climate chamber, where they were thermostated and filled with humid air at atmospher- ic pressure. Then the adsorption chambers were closed. A known volume of VOCs in the range of 10–250 μL is in- jected into the adsorption chambers. After allowing all the VOCs to evaporate for an hour, a portion of the gas phase was taken and analyzed using a total organic carbon ana- lyzer (Shimadzu TOC high sensitivity). It represents the concentration of the gas-phase VOCs (C0). For the purpose of the analysis, the manual injection kit is installed on the total organic carbon analyzer (Shi- madzu TOC high sensitivity) (Figure 1). Figure 1. The manual injection kit After determining the initial VOC concentration, the lids on the adsorbent jars are opened and the co-adsorp- tion of VOC and water is performed. Part of the gaseous phase is taken again after equilibration and analyzed on a total organic carbon analyzer; this represents the equilib- rium concentration of VOC (Ce). The amount of adsorbed VOC is calculated using the following equation (1): (1) where: qe – the amount of the adsorbed VOC per gram of adsorbent (g/g) C0 – VOC concentration at the beginning of adsorption (g/m3) Ce – equilibrium concentration of VOC (g/m3) V – adsorption chamber volume (m3) m1 – mass of the adsorbent (g) The total adsorption of VOCs and water vapor (me,- tot) was determined gravimetrically, from the difference in the mass of the adsorbent before and after the adsorption. (2) (3) qe,tot – total mass of adsorbed VOC and water per gram of adsor- bent (g/g) m2 – mass of the adsorbent after co-adsorption of VOC and wa- ter (g). The mass of water adsorbed per gram of the adsor- bent (qe,w) was determined from the difference between the total mass of VOC and water adsorbed per gram of the adsorbent (qe,tot) and the mass of VOC adsorbed per gram of the adsorbent (qe): (4) Freundlich and Langmuir isotherm models are used to discuss the equilibrium behavior of single-component adsorption. The Langmuir isotherm can be written as (5) where: qo – is the maximum amount of adsorbed adsorbate (g/g), Ce – is the equilibrium concentration of the adsorbate (g/m3) and KL – is the Langmuir constant (m3/g). The Freundlich isotherm model mathematically is expressed as: (6) where: KF – is adsorption capacity (m3/g) and 1/n – is adsorption intensity. Freundlich and Langmuir models were used to dis- cuss the equilibrium behavior of single-component ad- sorption.15 The models used for a single-component system are not always suitable for a multicomponent system.15–19 Therefore, the single-component isotherm models were modified to suit a multicomponent system. There are 806 Acta Chim. Slov. 2022, 69, 803–810 Kešelj et al.: Use of Total Organic Carbon Analyzer in Isotherm ... – co-adsorption of methanol-H2O on Adsorbent 3 (rH = 55%, t = 25 °C) (Figure 4); – co-adsorption of ethanol - H2O on Adsorbent 3 (rH = 46%, t = 25 °C) (Figure 5); – co-adsorption of toluene - H2O on Adsorbent 4 (rH = 70%, t = 22 °C) (Figure 6) and – co-adsorption of benzene - H2O on Adsorbent 4 (rH = 50%, t = 26 °C) (Figure 7). The obtained results show that co-adsorption of adsorbate (VOC) and water vapor occurred on all adsor- bents. The shape of all isotherms indicates that there is a resemblance to type I or the lowest concentration part of type IV isotherm. Analyzing the adsorption equilibrium data using a curve fitting tool (user-defined non-linear Langmuir and Freundlich, extended Langmuir, extended Freundlich and Hill models) in Origin software, adjusted R-Squared (R2) and parameters of models were obtained and presented in Table 3. The values of corresponding isotherm parameters obtained by fitting of experimental data on the co-adsorp- tion of isopropyl alcohol -H2O on Adsorbent 1 based on the chosen isotherm models, shows a high value of adj. R2. The highest value of adj. R2 (adj.R2 = 0.9688) for the total adsorbed isopropyl and water vapor and water (qe,tot = f (Ce)) was for the extended Freundlich. The model which gives the best description of adsorption of isopropyl alco- hol from the water vapor- isopropyl alcohol binary system (qe = f (Ce)) is the Freundlich model with adj.R2 = 0.9566. Very close adj. R2 was obtained for the extended Freun- dlich model for qe = f (Ce) (adj. R2 = 0.9495). The order of the isotherm models which best fits the experimental data of co-adsorption of isopropyl alcohol -H2O on Adsorbent 1 for co-adsorption of isopropyl alcohol –water vapor on Adsorbent 1 for qe,tot = f (Ce) is the extended Freundlich >Hill> Freundlich> extended Langmuir> Langmuir (adj. R2 values were 0.9688; 0.9479; 0.9191; 0.90259; 0.4420). On the other hand, for qe = f (Ce) the best fitting non-lineare model for adsorption isotherms is Freundlich > Langmuir > extended Freundlich > Hill > extended Langmuir (adj.R2 values were 0.9566; 0.9536; 0.9439; 0.9495; 0.94792). The order of the isotherm models which best fit the adsorption of methanol and water vapor on Adsorbent 2 qe,tot = f (Ce) is extended Langmuir = Hill > extended Fre- undlich > Langmuir > Freundlich (adj.R2 were 0.99656; 0.99656; 0.9965; 0.9956; 0.93556). Whereas the order of the isotherm models that offers the best representation of measured data for methaonol in the methanol-water va- por binary system is extended Langmuir = Hill > extended Freundlich> Langmuir> Freundlich (adj.R2 values were 0.99228; 0.99228; 0.98899; 0.9345; 0.85256). The values of corresponding isotherm parameters obtained by fitting the experimental data of co-adsorption of methanol and water vapor on Adsorbent 3 shows the fol- lowing order of models Langmuir > extended Langmuir many models for analyzing adsorption equilibrium data, and in this study, apart from the non-linear Langmuir and Freundlich, we also used the extended Langmuir, extend- ed Freundlich and Hill defined models in Origin software. Extended Langmuir and extended Freundlich are hybrid, three-parameter isotherm models, with another constant C added as the third parameter, apart from the constant B. A Hill isotherm model is a three-parameter isotherm model which refers to a modified Langmuir model with constants K I n.20–22 introduced. An indicator of isotherm model suitability used in this study is adjusted R-Squared (adj. R2), which is calculated as a function of Origin soft- ware by the following equation 23: (7) here, n– represents the number of data points in our dataset, k– represents the number of independent variables, and R–  represents the R-squared values determined by the model. The coefficient of determination (R2) is defined by the following equation:21 (8) where: qe –is the amount of the adsorbate adsorbed by the adsorbent during the experiment (g/g), qcal – is the amount of the adsorbate obtained by isotherm mod- els (g/g), and qmexp – is the average value of qe (g/g). The advantage of the non-linear method is that the error distribution does not get altered as in the linear re- gression approach. 3. Results and Discussion The adsorbents used in the study had different char- acteristics. Adsorbents 1 and 2 are hydrophobic, with dif- ferent specific surface areas (355 and 434 m2/g, respective- ly) determined by low-temperature nitrogen adsorption. Adsorbents 3 and 4 are hydrophilic. The adsorbates used in adsorption also had different characteristics: polar (methanol, ethanol), semi-polar (isopropyl alcohol) and non-polar (toluene, benzene). The adsorbates were adsorbed on the adsorbents at atmospheric pressure, defined temperature (t) and relative humidity (rH). Based on the results obtained, we were able to obtain six isotherms – co-adsorption of isopropyl alcohol -H2O on Adsorbent 1 (rH = 65%, t = 25 °C) (Figure 2); – co-adsorption of methanol - H2O on Adsorbent 2 (rH = 60%, t = 25 °C) (Figure 3); 807Acta Chim. Slov. 2022, 69, 803–810 Kešelj et al.: Use of Total Organic Carbon Analyzer in Isotherm ... = Hill > extended Freundlich > Freundlich (adj. R2 were 0.9523; 0.94712; 0.94712; 0.9273; 0.77091). The order of the isotherm models that describe the adsorption of meth- anol from the binary system of methanol-water vapor on Adsorbent 3 (qe = f (Ce)) is extended Freundlich > Fre- undlich > extended Langmuir > Hill > Langmuir (adj. R2 values were 0.94633; 0.94362; 0.93194; 0.9298; 0.4100). The order of the isotherm models which best fit the adsorption of toluene and water vapor on Adsorbent 4 qe,tot = f (Ce) is Hill > extended Freundlich > Freundlich > ex- Figure 2. Adsorption Isotherms a- co-adsorption of isopropyl alco- hol -H2O; b- isopropyl alcohol on Adsorbent 1 (rH = 65%, t = 25 °C) Figure 3. Adsorption Isotherms a-co-adsorption of methanol -H2O; b- methanol on Adsorbent 2 (rH = 60%, t = 25 °C) Figure 5. Adsorption Isotherms a- co-adsorption of ethanol – H2O; b- ethanol on Adsorbent 3 (rH = 46%, t = 25 °C) Figure 4. Adsorption Isotherms a- co-adsorption of methanol-H2O; b- methanol on Adsorbent 3 ( rH = 55%, t = 25 °C) Figure 7. Adsorption Isotherms a-co-adsorption of benzene -H2O; b- benzene on Adsorbent 4 (rH = 50%, t = 26 °C) Figure 6. Adsorption Isotherms a-co-adsorption of toluene -H2O; b-toluene on Adsorbent 4 (rH = 70%, t = 22 °C) 808 Acta Chim. Slov. 2022, 69, 803–810 Kešelj et al.: Use of Total Organic Carbon Analyzer in Isotherm ... tended Langmuir > Langmuir (adj. R2 values were 0.99217; 0.9874; 0.98705; 0.98435; 0.8835), whereas for qe = f (Ce) the order is Langmuir > extended Langmuir > extended Freundlich > Hill > Freundlich (adj. R2: 0.9924; 0.99217; 0.9900; 0.98434; 0.96059). For the data obtained for co-adsorption of benzene- water vapor on Adsorbent 4, the order of the isotherm best fits for qe,tot = f (Ce) is extended Freundlich > Freundlich > Hill > extended Langmuir > Langmuir (adj. R2 values were 0.99296; 0.98337; 0.98041; 0.98037; 0.8800), and for qe = f (Ce) the order is Langmuir > extended Freundlich > Freundlich > extended Langmuir = Hill (adj. R2 0.9804; 0.97957; 0.97764; 0.97732; 0.97732). Figures 2–7 show the best fitting non-linear models for adsorption isotherms of VOC-water vapor co-adsorp- tion. The values of Adjusted R- Squared (R2), which is an indicator of isotherm model suitability, obtained for all six adsorption isotherms for co-adsorption of VOC and water vapor, were lower for the two-parameter models Table 4. Langmuir, Freundlich, extended Langmuir, extended Freundlich and Hill isotherm parameters obtained by non-linear fitting for co-adsorp- tion of VOC (isopropyl alcohol, methanol, ethanol, toluene, benzene) on adsorbents (Adsorbents 1-4) Adsorbate /Adsorbent Isopropyl Methanol/ Methanol/ Ethanol/ Toluene/ Benzene/ Alcohol/ Adsorbent 2 Adsorbent 3 Adsorbent 3 Adsorbent 4 Adsorbent 4 Model Adsorbent 1 Langmuir qe,tot KL(m3/g) 0.3114 0.103 2.608 29.12 0.1554 0.06855 qo(g/g) 0.1448 0.1358 0.1192 0.1018 0.1989 0.1379 Adj. R2 0.4420 0.9956 0.9523 0.4100 0.8835 0.8800 qe KL(m3/g) 0.1044 0.05112 0.592 1.17 0.0707 0.01073 qo(g/g) 0.1786 0.1466 0.0948 0.0859 0.2309 0.2444 Adj. R2 0.9536 0.9345 0.9665 0.9309 0.9924 0.9804 Extended qe,tot A(g/g) 21.2227 0.12882 0.11757 5.91215 17.70209 13.862 Langmuir B(m3/g)1/1–C 0.00335 0.08734 2.81094 0.01368 0.00271 0.00175 C 0.81494 –0.12337 –0.25522 0.89807 0.62488 0.61706 Adj. R2 0.90259 0.99656 0.94712 0.93194 0.98435 0.98037 qe A(g/g) 1.423 0.10571 0.09826 0.1045 0.2083 0.28798 B(m3/g)1/1–C 0.0248 0.00973 0.57812 0.7981 0.06503 0.01038 C 0.5645 –0.9534 0.12835 0.4779 –0.12623 0.05433 Adj. R2 0.9439 0.99228 0.96259 0.9769 0.99217 0.97732 Freundlich qe,tot 1/n 0.18441 0.34577 0.08446 0.10043 0.37272 0.38071 KF (g/g)n 0.07084 0.03052 0.08891 0.07979 0.04787 0.02433 Adj. R2 0.9191 0.93556 0.77091 0.94362 0.98705 0.98337 qe 1/n 0.44875 0.49811 0.21789 0.19472 0.53652 0.73792 KF (g/g)n 0.03162 0.01551 0.0425 0.04533 0.02587 0.0047 Adj. R2 0.9566 0.85256 0.86869 0.92199 0.96059 0.97764 Extended qe,tot A(g/g) 0.0664 0.12972 0.0858 0.07834 0.0507 0.36699 Freundlich B 0.1458 –5.05901 0.2511 0.09095 0.3091 –3.16602 C –0.1341 1.26742 0.2901 –0.05036 –0.04299 0.59346 Adj. R2 0.9688 0.9965 0.9273 0.94633 0.9874 0.99296 qe A(g/g) 0.0334 0.11086 0.0362 0.07912 0.0108 0.32614 B 0.3911 –16.4310 0.5556 0.01 1.415 –6.34167 C –0.03276 1.67984 0.2124 9.65401 0.1731 0.74606 Adj. R2 0.9495 0.98899 0.9636 0.77668 0.9900 0.97957 Hill qe,tot Vmax (g/g) 6.69483 0.12882 0.11757 1.03324 16.18856 16.65986 K 3.49∙1010 8.75983 0.43895 6.341∙109 5.439∙106 2.577∙107 n 0.1869 1.12337 1.25522 0.10983 0.37542 0.38267 Adj. R2 0.9479 0.99656 0.94712 0.9298 0.99217 0.98041 qe Vmax(g/g) 4.26917 0.10571 0.09826 0.1045 0.2083 0.28798 K 44168.7 10.71157 1.87509 1.54257 11.32094 125.2708 n 0.45875 1.9534 0.87165 0.52191 1.12623 0.94567 Adj. R2 0.94792 0.99228 0.96259 0.97687 0.98434 0.97732 809Acta Chim. Slov. 2022, 69, 803–810 Kešelj et al.: Use of Total Organic Carbon Analyzer in Isotherm ... (Langmuir and Freundlich) than for the three-parameter models (extended Langmuir are, extended Freundlich and Hill). This paper confirms that three-parameter models are more suitable for describing the isotherm measure- ments of VOC-water co-adsorption, i.e., multicomponent adsorptions. This can be explained by the fact that another constant is added to a two-parameter model, which allows additional factors characteristic for multicomponent sys- tems to be introduced and quantified (such as the content of particular gaseous components, their molar mass, the surface area occupied by each of these components, etc.). 4. Conclusions The adsorption equilibrium data of co-adsorption of VOC and water vapor from the air on the test adsor- bents included the total adsorbed VOC and water vapor, adsorbed VOC, initial and equilibrium concentrations of VOC. The initial and equilibrium concentrations of VOC were determined using a total organic carbon analyzer. The experimental data for six co-adsorption isotherms of VOC-water vapor were analyzed. The analysis was per- formed using nonlinear models, which are considered to be a better tool for calculating isothermal parameters, and adj. R2 was also used to determinate the best fitting isotherm to the experimental data. The values obtained for adj. R2 indicate a good fit to isotherm models, which clearly demonstrates that the technique used in the present work is suitable for studying the co-adsorption of VOCs and water vapor from the air. Acknowledgments The authors would like to acknowledge the support provided by “Zeochem” doo, Zvornik for funding this work through the project “Adsorption isotherms of volatile organic compounds (VOC) on products of the company “Zeochem” d.o.o”. 5. References 1. C. Gebald, J. A. Wurzbacher, A. Borgschulte, T. Zimmer- mann, Environ. Sci. Technol., 2014, 48, 2497–2504. DOI:10.1021/es404430g 2. S. Xian, Y. Yu, J. Xiao, Z. Zhang, Q. Xia, H. Wang, Z. Li, RSC Adv., 2015, 5, 1827–1834. DOI:10.1039/C4RA10463C 3. R. Veneman, N. Frigka, W. Zhao, Z. 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DOI:10.1016/j.molliq.2016.01.021 810 Acta Chim. Slov. 2022, 69, 803–810 Kešelj et al.: Use of Total Organic Carbon Analyzer in Isotherm ... 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 V zadnjih letih je fokus raziskovanja na binarnih adsorpcijskih izotermah hlapne organske komponente (VOC) in vodne pare iz zraka. Količino adsorbirane VOC v prisotnosti vodne pare lahko določimo volumetrično ali gravimetrično, v statičnem ali dinamičnem načinu. Ta študija je osredotočena na adsorpcijsko tehniko v statičnem načinu za izotermne meritve koadsorpcije VOC in vodne pare iz zraka z uporabo gravimetrične metode. Količino VOC smo določili s po- močjo analizatorja celotnega dušika, medtem ko smo količino vode izračunali iz razlike med celotno adsorpcijo (VOC in voda) in količino absorbirane VOC. V članku predstavljamo nekaj adsorpcijskih izoterm za različne VOC (toluen, benzen, metanol, etanol in izopropilalkohol) in različne adsorbente (zeolite ZSM-5, silikagel in natrijevo obliko mor- dernita) v prisotnosti vodne pare. Za obravnavo eksperimentalnih podatkov smo uporabili dobro znane adsorpcijske modele (Langmuir, razširjeni Langmuir, Freundlich, razširjeni Freundlich in Hill). Za določitev najbolje prilegajočih izotermnih modelov smo uporabili prilagojene vrednosti R2, ki smo jih dobili iz teh nelinearnih modelov (to je krivulj celotne količine adsorbiranih plinov (qe,tot) v odvisnosti od ravnotežne koncentracije VOC (Ce)). Rezultati so pokazali, da dajo modeli s tremi parametri boljše rezultate za prileganje k meritvam kot modeli z dvema parametroma, torej z višjimi vrednostmi R2. Eksperimentalni rezultati pokažejo, da to adsorpcijsko tehniko lahko uporabimo za izotermne meritve ko-adsorpcije VOC in vodnih par iz zraka. 811Acta Chim. Slov. 2022, 69, 811–825 Nguyet et al.: Enhanced Adsorption of Methylene Blue by Chemically ... DOI: 10.17344/acsi.2022.7567 Scientific paper Enhanced Adsorption of Methylene Blue by Chemically Modified Materials Derived from Phragmites australis Stems Bui Thi Minh Nguyet,1 Nguyen Huu Nghi,1 Nguyen Anh Tien,2 Dinh Quang Khieu,3 Ha Danh Duc1 and Nguyen Van Hung1,* 1 Dong Thap University, Cao Lanh City, 81000, Vietnam 2 Ho Chi Minh City University of Education, Ho Chi Minh City, 700000, Vietnam 3 University of Sciences, Hue University, 530000, Vietnam * Corresponding author: E-mail: nguyenvanhung@dthu.edu.vn Received: 05-05-2022 Abstract In this study, the biomass of Phragmites australis was chemically modified using NaOH and subsequently citric acid to produce an effective adsorbent named SA-RPB. The absorbent was characterized using XRD, SEM, BET, and FT-IR methods. The study’s findings indicated that the adsorbent existed mainly as cellulose crystals, contained micropores with an average diameter of 15.97 nm, and had a large number of hydroxyl and carboxyl groups on the surface. The adsorption process of SA-RPB was evaluated through the adsorption of methylene blue (MB) dye in aqueous solution. Adsorption kinetics showed that the pseudo-second-order model well described the adsorption process. The adsorption isotherm process satisfactorily fitted with the Langmuir model with the maximum adsorption capacity of 191.49 mg/g at 303 K. These findings show that MB may be efficiently removed from aqueous solutions using the adsorbent made from the raw biomass of Phragmites australis treated with NaOH and then citric acid. Keywords: Adsorbent; Phragmites australis; Methylene blue; Kinetics; Adsorption mechanism 1. Introduction Dye has been extensively used in various industries such as textile, leather, cosmetics, tanning, paper, food technology, hair coloring, pulp mill, pharmaceuticals, and plastics.1 Wastewater discharged from these industries has reportedly caused severe environmental pollution2 and health problems. Specifically, methylene blue (MB), wide- ly used for coloring cotton, wood, and silk3, can damage humans’ and animals’ eyes and trigger nausea, vomiting, profuse sweating, and mental instability when it passes through the mouth, causing rapid or difficult breathing within short periods of inhaling.4 Therefore, it is practical- ly essential to remove MB from dye wastewater. Many advanced techniques have been developed for removing MB, including the Fenton process and combined electrochemical treatments, electrochemical degradation, reverse osmosis, photodecomposition, coagulation/floc- culation, membrane processing, oxidative degradation, electrocoagulation, and carbonaceous nanomaterials.5–13 In addition, activated carbon has been recognized to ef- fectively remove different dye molecules.14–16 However, these methods are costly, owing to poor regeneration. In recent years, considerable efforts have been made in de- veloping adsorbents derived from plant materials such as mango peels, pistachio shell, cladodes of Opuntia ficus in- dica, peach stone, carbonized watermelon, seed fibers, and potato peels.17–23 Some recent studies have used raw materials or raw materials modified with NaOH to treat MB.26,27 For instance, plant materials modified with citric acid (CA) show good potential for wastewater treatment.20,28,29 Cel- lulose fibrils extracted from P. australis and treated by both NaOH and CA appear to be a better-modified material compared to being treated with only NaOH as described in the previous report.26 P. australis is a type of reed that mainly grows around lakes, rivers, streams, and brackish water worldwide between 10° and 70° northern latitudes.24 This plant type has a high tissue porosity formed by cel- lulose, hemicellulose, and lignin, which are vital constit- uents for developing adsorbents.25 It wildly grows all year round throughout the country of Viet Nam, especially in 812 Acta Chim. Slov. 2022, 69, 811–825 Nguyet et al.: Enhanced Adsorption of Methylene Blue by Chemically ... wetlands. In this study, the biomass of P. australis modified with NaOH and followed by esterification using citric acid (NaOH-then-CA treatment) was investigated in adsorb- ing MB from aqueous solutions. In addition, the effects of environmental parameters on MB adsorption by raw and modified adsorbents were evaluated. 2. Materials and Methods 2. 1. Chemicals and Materials Citric acid (HOC(COOH)(CH2COOH)2, ≥ 99.5%), sodium hypophosphite monohydrate (NaH2PO2.H2O, ≥ 99.5%), sodium hydroxide (NaOH, ≥ 97%), hydrochlo- ric acid (HCl, 37%), sodium chloride (NaCl, ≥ 99.5%), and MB (C16H18N3SCl, 99.5%) were purchased from Sig- ma-Aldrich. Then, MB was diluted with double-distilled water to a range of 125–300 mg/L. The pH was adjusted using NaOH (0.1 M) and HCl (0.1 M). P. australis samples were collected from a wetland in Dong Thap province, Vietnam and cleaned with tap water to remove dirt and other impurities adhered to their sur- faces. The plant stems were dried under the sunlight for four days prior to being finely ground to approximately 1–2 mm sizes. The obtained biomass was rinsed with dis- tilled water and dried in a vacuum oven at 70 °C to a con- stant weight. The product was stored in a desiccator and used as raw P. australis biomass (RPB). 2. 2. Chemical Modifications of P. australis Biomass RPB (5 g) was added to a 250 mL glass beaker con- taining 100 mL NaOH (0.5 M). The solution was stirred at 60 °C and 400 rpm using a magnetic bar for 5 h. After that, the biomass was collected and cleaned with distilled water until the pH of the solution was 7.0. The product was then dried in a vacuum oven at 60 °C for 12 h until it yielded an adsorbent. The biomass modified with NaOH was des- ignated as S-RPB, which was further denatured with a 50 mL (0.1 M) CA solution added to a 2.0 g S-RPB. Then, NaH2PO2.H2O (2.65 g), used as a catalyst, was added to the solution. The biomass was collected after stirring at 60 °C and 400 rpm using a magnetic bar for 5 h. After that, it was soaked in 50 mL distilled water several times until the pH reached 7.0, and then was dried at 60 °C for 12 h, and subsequently arriving at 140 °C for 3 h. The second modi- fied adsorbent was designated as SA-RPB. 2. 3. Characterization of Materials The lignocellulosic composition before and after being modified (as mentioned above) was determined according to the National Renewable Energy Laboratory (NREL) compositional analysis procedure.30 The C, H, N, S, and O contents of the materials were analyzed using a CHNS-O Elemental Analyzer (Thermo, Flash EA1112, USA). Also, the products’ XRD was performed by a Mini- Flex 600 diffractometer (Rigaku, Japan) with a radiation source of Cu Kα, λ = 0.15406 nm. The scanned angle (2θ values) ranged between 5° and 80° with a step size of 0.01°. The adsorbents’ surface morphology was scanned under the scanning electron microscopy (SEM) technique (FEI- SEM NOVA NanoSEM 450-USA). Meanwhile, the sam- ples’ FTIR spectra were recorded by an Infrared Affinity-1S spectrophotometer (Shimadzu) and BET was determined by N2 adsorption–desorption isotherms at liquid nitrogen temperature (77 K) using a Quantachrome TriStar 3000 V6.07A absorption instrument. 2. 4. Determination of Point of Zero Charge (pHPZC) The pHPZC of the adsorbents was determined using the pH drift method described in a previous study.31 For- ty-five milliliters of 0.5 M NaCl with pH values were ad- justed from 2 to 12 by either 0.1 M NaOH or 0.1 M HCl solution. Then, distilled water (50 mL) was added, and the pH values were readjusted, closely noting the initial pH (pHi). Next, an obtained adsorbent was added to each flask at 1.0 g/L, incubated at 180 rpm using a magnetic stir bar for 24 h at room temperature (~30 °C). The differences in the pH (ΔpH) values between the initial pH and final pH (pHf) (ΔpH = pHi – pHf) were plotted against pHi. The points of intersection of the curve with the abscissa at which ΔpH is equal to zero were presented as the pHPZC. 2. 5. Adsorption Tests Adsorption tests were performed by adding an ad- sorbent into a 250 mL glass beaker containing 100 mL MB solution. For the effects of the adsorbent on adsorption, the MB was diluted to 125–300 mg/L, while SA-RPB was used from 0.4 to1.4 g/L. The solution was stirred at 300 rpm, and liquid media were collected from 2 to 105 min. Liquid media samples were centrifuged at 3000 rpm for 5 min to remove solid particles. Also, MB concentrations were measured by an ultraviolet–visual spectrophotome- ter (Spectro UV–2650, Labomed, USA) at a wavelength of 665 nm. The percent removal (R) and adsorption capacity per unit mass (qt) after a specific contact time (t) were cal- culated via Eqs. (1) and (2), respectively, as follows: (1) (2) where C0 (mg/L) and Ct (mg/L) are MB concentrations in liquid media at the initial and time t, respectively, and V is the volume of the solution (L). 813Acta Chim. Slov. 2022, 69, 811–825 Nguyet et al.: Enhanced Adsorption of Methylene Blue by Chemically ... 2. 5. 1. Adsorption Kinetics The adsorption kinetics were fitted to the pseu- do-first-order and pseudo-second-order kinetic models, which are expressed by Eqs. (3) and (4), respectively32,22, below: (3) (4) where k1 (1/min) and k2 (1/min) are rate constants of the pseudo-first-order and pseudo-second-order, respectively; and t (min) is the contact time. 2. 5. 2. Adsorption Mechanisms The Weber–Morris intra-particle diffusion and Boyd models were applied in order to investigate diffu- sion mechanisms. The former model was derived from the Fick’s second law of diffusion as expressed by Eqs. (5):1,4 5) where kpi (mg/gmin1/2) means the diffusion rate constant at stage i, and Ci is the intercept which can be evaluated from the slope of the linear plot of qt versus t1/2. The qt (mg/g) is adsorption capacity per unit weight of adsorbent per time, and t1/2 (min1/2) denotes half adsorption time. The intercept, Ci, relates to the extent of external mass trans- fer during the adsorption, acting as the rate-controlling step. When the linear plot of qt versus t1/2 passes through the origin, intra-particle diffusion is the sole rate-limiting step. However, if the linear plots at each concentration do not pass through the origin, when it indicates that the in- tra-particle diffusion was not only rate controlling step.1 Meanwhile, the Boyd model was implemented to distinguish between film diffusion and intra-particle dif- fusion as expressed by Eqs. (6) and (7):15,16 (6) (7) where Bt is a mathematical function of F representing the fractional attainment of equilibrium at any time t given by Eqs.(8): (8) The plot Bt versus time t (s) is used to anticipate the diffusion limit. If the plot is linear and passes through the origin, it indicates that the pore diffusion occurs. If the lines are linear and pass through the origin, the intra-par- ticle diffusion takes place. However, if the lines are linear but do not pass through the origin or non-linear, the film diffusion controls the adsorption process. 2. 5. 3. Adsorption Isotherms Four isotherm equations, namely the Langmuir, Fre- undlich, Temkin, and Dubinin–Radushkevich, were used to fit the experimental equilibrium isotherm data for MB adsorption on SA-RPB. Adsorption isotherm tests were performed by adding 0.1 g SA-RPB into 100 mL MB at a concentration range of 125–300 mg/L. The initial pH of the MB solution was 6.5, and the controlled temperatures were 30 °C (303 K). The Langmuir model assumes that ad- sorption is localized on a monolayer, and all adsorption sites on the adsorbent homogeneously possess the same adsorption capacity, as expressed by Eqs. (9)35: (9) where Ce (mg/L) is the equilibrium concentration; qe (mg/L) is the amount of adsorbed dye at equilibrium; qmax (mg/g) is the maximum adsorption capacity; and KL (L/ mg) is the Langmuir adsorption equilibrium constant. The equilibrium parameter (RL) is a dimensionless constant of the Langmuir isotherm, expressed by Eqs. (10) 35: (10) where C0 is the highest initial solute concentration. The Freundlich isotherm model assumes that multilayer ad- sorption processes occur on heterogeneous surfaces, ex- pressed by Eqs. (11)36: (11) where KF (mg/g.(L/mg)1/n) and n are Freundlich constants related to the adsorption capacity and adsorption intensity, respectively. The adsorbate–adsorbate interactions can cause a decrease in the heat of adsorption of all the molecules in the layer. The Temkin isotherm reflects the effect of the ad- sorbate interaction on SA-RPB, expressed by Eqs. (12)37: (12) (13) where At (L/g) and b (g.J/mg.mol) are Temkin’s isotherm constants; R (8.314 J/mol.K) is the universal gas constant; T (K) is the absolute temperature. The Dubinin–Radush- kevich isotherm model38 was used to determine the mean free energy of biosorption, expressed by Eqs. (14)–(16): (14) (15) (16) where KDR is a constant related to the adsorption energy (mol2/kJ2); qDR (mg/g) is the Dubinin–Radushkevich iso- 814 Acta Chim. Slov. 2022, 69, 811–825 Nguyet et al.: Enhanced Adsorption of Methylene Blue by Chemically ... therm adsorption capacity; ε (kJ/mol) is the Polanyi po- tential; R is the ideal gas constant; and T (K) is the temper- ature. The free energy of adsorption (E) is considered as either chemical adsorption (E = 8–16 kJ/mol) or physical adsorption (E < 8 kJ/mol). 2. 5. 4. Adsorption Thermodynamics The thermodynamic parameters for MB adsorption onto SA-RPB were evaluated at 303, 313, and 323 K. The Gibb’s free energy change (ΔG°), enthalpy (ΔH°), and en- tropy (ΔS°) were calculated using Eqs. (17)–(19). (17) (18) Adding Eqs. (17) and (18) amounts to Eq. (19): (19) where R (8.314 J/mol K) is the universal gas constant; T (K) the absolute temperature, and KL the Langmuir equi- librium constant. The values of ΔH° and ΔS° can be cal- culated from the slope and intercept, respectively, of the linear plot of lnKL versus 1/T. 2. 6. Reusability After each cycle of batch experiment, the SA-RPB was collected through centrifugation at 5000 rpm for 10 min. The adsorbent was first washed with absolute ethanol and then rinsed with double-distilled water three times. The adsorbent (0.1 mg) was transferred into 100 mL of the rinse media, stirred at 300 rpm for 6 hours, and collected through centrifugation at 5000 rpm for 10 min, followed by being dried for 24 h at 100 °C until its weight stayed constant. 3. Results and Discussion 3. 1. Characterization of Materials The biomass components of P. australis are listed in Table 1. The components of the raw RPB were also deter- mined in previous studies39,40. The cellulose content signif- icantly increased, whereas hemicellulose and lignin con- tents comparatively decreased after the NaOH treatment (S-RPB) and the NaOH-then-CA treatment (SA-RPB) (Table 1). The C content in RBB (46.42%) decreased slight- ly, whereas the O content (45.82%) and the ratio of O to C increased slightly after NaOH-then-CA treatment (Ta- ble 1) was done. Moreover, the percentages of N, S, Si, K, and Mg significantly decreased after the treatment. Acidic and basic solutions are typically used for modifying and/or removing lignin and hemicellulose from plant biomass.41 The treatment with NaOH resulted in the formation of hydroxyl groups on the S-RPB, which then reacted with citric acid, forming an ester linkage to introduce carboxyl groups into SA-RPB.20 Table 1. Chemical compositions of RPB, S-RPB, and SA-RPB Parameter RPB S-RPB SA-RPB Lignocellulosic analysis (dry weight basis), wt% Cellulose (%) 43.31 66.32 71.21 Hemicellulose (%) 30.82 15.17 13.28 Lignin (%) 20.37 12.30 9.19 Elemental analysis (dry weight basis), wt% C (%) 46.42 45.71 45.23 O (%) 45.82 47.72 48.83 H (%) 5.910 5.610 5.720 N (%) 0.232 0.111 0.021 S (%) 0.313 0.222 0.107 O/C (mol/mol) 0.7403 0.7830 0.8097 Si (%) 1.050 0.021 – K (%) 0.454 0.284 0.182 Mg (%) 0.601 0.322 0.110 SEM micrograph images of P. australis biomass be- fore and after the NaOH-then-CA treatment were cap- tured (Fig. 1). The raw material had a surface composed of fibrous rods (Fig. 1(a)). The surface morphology was found to transform, being affected by NaOH. The S-RPB sample retained its tubular structure, but the surface turned out to be more porous and uneven (Fig. 1(b)). The texture was also rough and irregular after being treated with CA (Fig. 1(c)). The treatment with citric acid reduces the cavities on the adsorbent surface. Citric acid clogged the carbon sur- face, which explains for a spotted reduction in the surface area and pore volume of the adsorbent.42 Table 2. Porous textural parameters of RPB, S-RPB, and SA-RPB samples Sample Surface area Pore volume Average pore (m2/g) (dm3/g) diameter (nm) RPB 1.01 2.626 16.64 S-RPB 0.87 2.052 16.86 SA-RPB 0.74 1.935 15.97 The crystallographic structures of the RPB, S-RPB, and SA-RPB were analyzed using the XRD technique (Fig. 1(d)). The results indicated that all samples had two diffraction peaks at angles 2θ of 15.7° and 22.3°, corre- sponding to (101) and (002) planes of cellulose crystals.43 The diffraction intensities were in the order of SA-RPB > S-RPB > RPB (Fig. 1(d)), indicating that NaOH-then- 815Acta Chim. Slov. 2022, 69, 811–825 Nguyet et al.: Enhanced Adsorption of Methylene Blue by Chemically ... CA treatment enhanced the cellulose crystallinity. The increased crystallization attributed to the partial removal of amorphous polymers (hemicellulose and lignin) from plant structures has also been reported.26,44 The specific surface area and porous texture of the obtained samples were evaluated using the nitrogen adsorption–desorption isotherms at 77 K (Table 2). The RRB sample had a specific surface area of 1.01 m2/g, a common property of raw plant biomasses.45 The surface areas decreased by 13.9% after the NaOH treatment and went down to 26.7%, resulting from that of the NaOH-then-CA treatment, while the pore volumes decreased by 21.8% and 26.3%. Besides, the aver- age pore diameter slightly decreased after the treatment. Citric acid can easily penetrate the pore structure because of its small molecular size, causing the pore block of the absorbent.42 These results indicate that the dye adsorption capacity can be boosted due to the formation of hydroxyl and carboxyl groups on the surface of P. australis biomass, and the functional groups might play a more important role than the surface area in MB adsorption. The functional groups on the adsorbent surfaces with differing intensities of the observed peaks during P. aus- tralis biomass modification were analyzed via FTIR (Fig. 2). Adsorption bands corresponding to functional groups were determined according to Reddy.46 A broad peak of approximately 3321 cm−1 corresponded to the stretch- ing vibration of the hydroxyl groups (–OH) for cellulose, hemicellulose, and lignin, whereas the 2918 cm−1 band in- dicated the presence of C–H stretching vibrations of me- thyl and methylene. After the raw material was modified with NaOH and NaOH-then-CA, the stretching vibration bands of OH shifted to 3443 and 3438 cm–1, respective- Fig. 1. Morphology and crystallization of samples; SEM images of (a) RPB, (b) S-RPB, and (c) SA-RPB; (d) XRD patterns of RPB, S-RPB, and SA- RPB samples 816 Acta Chim. Slov. 2022, 69, 811–825 Nguyet et al.: Enhanced Adsorption of Methylene Blue by Chemically ... ly. The band at 1734 cm–1 could be attributed to the C=O bond stretching of acetyl ester groups in hemicellulose, lignin, or both. Fig. 2. FTIR spectra of RPB, S-RPB and SA-RPB samples This adsorption peak was absent from the FTIR spec- trum of the S-RPB sample (Fig. 2) after the alkaline treat- ment because of the removal of hemicellulose and lignin through a process called de-esterification. Moreover, C=O bond stretching was observed after the NaOH-then-CA treatment, owing to the esterification reaction. The band at 1645 cm–1 could be attributed to –COO– stretching of carboxylate groups with the aromatic ring. The band at ap- proximately 1512 cm–1 was associated with C=C stretching vibrations in aromatic rings of lignin, whereas the band at 1427 cm–1 was attributed to the C–H bond deformation of lignin. The peak intensities at 1457 and 1380 cm–1 re- flected C–H symmetric and asymmetric deformations of cellulose, respectively. The appearance of peaks at 1334 and 1327 cm–1 could be attributed to the –OH bending vibration in C–OH and C1–O vibrations in S derivative vi- brations of cellulose, respectively. The signal at 1249 cm–1 corresponded to the –COO vibration of acetyl groups in hemicellulose and lignin.20,47 The adsorption peaks at 1159 and 1111 cm–1 were attributed to C–O–C antisymmetric and anhydroglucose ring vibrations, respectively, whereas the band at 1049 cm–1 corresponded to C–O stretching vi- brations of cellulose, hemicellulose, and lignin.48 A band at 899 cm–1 corresponds to C–H rocking vibrations of cellu- lose.49 The intensities of these parts of S-RPB and SA-RPB decreased, owing to the removal of lignin. The weak ad- sorption peaks of 832–400 cm–1 were probably related to C–H and C=H bending in aromatic rings, C–H bending, and C–O stretching.40,50 The FTIR results indicated abun- dant functional groups of −OH, −COOH, and −COO− on the adsorbent surfaces. 3. 2. pHPZC determination The differences in the pHPZC of RPB, S-RPB, and SA- RPB are shown in Fig. 3(a). The raw P. australis biomass had a pHPZC of 6.72, also obtained in a previous study.27 The pHPZC levels of S-RPB and SA-RPB were 6.17 and 3.10, respectively. The pHPZC level slightly decreased after the NaOH treatment, possibly because of de-esterification and the re- moval of a part of hemicellulose and lignin.51 The pHPZC value of the SA-RPB was significantly lower than those of RPB and S-RPB, which could be attributed to the esteri- fication reaction of hydroxyl on the raw material surface with the carboxyl group of citric acid to increase the car- boxyl group on its surface.20,52 Absorbents with pH values lower than pHPZC absorb compounds with a positive sur- face charge.53 The MB dye with a molecular diameter of 0.8 nm54 was smaller than the pore diameter of SA-RPB (15.97 nm, Table 2); hence, MB could easily penetrate the SA-RPB pore structure. The batch adsorption test results Fig. 3. (a) Plots of point of zero charges of RPB, S-RPB, and SA-RPB (1.0 g/L adsorbent at different pH values); (b) percentage removal efficiency values for MB on RPB, S-RPB, and SA-RPB samples (150 mg/L MB at 6.5 pH value) 817Acta Chim. Slov. 2022, 69, 811–825 Nguyet et al.: Enhanced Adsorption of Methylene Blue by Chemically ... showed that the removal efficiency by SA-RPB adsorbent was 98.11±1.76%, about 68.8 and 35.1% higher than that of RPB and S-RPB, respectively (Fig. 3(b)). The increase in removal efficiency by SA-RPB was due to both NaOH and CA modified to the raw material. Therefore, SA-RPB was used for other experiments. 3. 3. Batch Adsorption 3. 3. 1. Effect of Adsorbent Dosage In this experiment, the effects of the adsorbent dose (SA-RPB) on MB adsorption were examined. As seen in Fig. 4, an increase in the adsorbent mass from 0.4 to 1.0 g/L improved the MB removal rate because of the increased sites available for adsorption. However, the adsorption did not statistically differ at adsorbent doses higher than 1.0 g/L. The adsorption appeared to be in equilibrium when the adsorbent mass reached a particular value, possibly because the number of MB dye molecules available in the solution was insufficient to combine with all effective ad- sorption sites on the adsorbent. Fig. 4. SA-RPB dosage effects on equilibrium adsorption capacity (qe, mg/g) and removal efficiency of MB (R, %) (The tests conducted for 105 min by 150 mg/L MB at 6.5 pH) 3. 3. 2. Effects of Contact Time, Temperature, and Adsorption Kinetics The effect of contact time on MB removal via the SA-RPB adsorbent is depicted in Fig. 5. The adsorption sharply increased within 20 min at the initial stage and then attained equilibrium after 60 min at all temperatures, followed by a maximum removal. About 89.98% of equi- librium adsorption capacity was achieved within 10 min. The fast adsorption at the initial stage was probably caused by available vacant active sites of the adsorbent (with func- tional groups of −OH, −COOH, and −COO−) and a high- er driving force between MB ions and the surface. How- ever, adsorption isotherms at three temperatures were not statistically different. This phenomenon is due to available active sites for adsorption, which previous studies used to modify plant materials for MB removal55,56. Meanwhile, decreased vacant sites and insufficient active sites of the adsorbent slowed down the adsorption rate and equilib- rium.56 Fig. 5. Pseudo-first and pseudo-second-order kinetics for MB ad- sorption by SA-RPB at different temperatures. The experiments were conducted by 1.0 g/L SA-RPB and 150 mg/L MB at 6.5 pH Two kinetic models (pseudo-first-order and pseu- do-second-order) were used to determine adsorption rate and analyze kinetic data. The calculated correlation coef- ficients (R2) and other data are listed in Table 3. The qe,cal and qe,exp values for each model at different temperatures slightly rose, whereas (and) k1 and k2 tended to increase at higher temperatures, indicating that adsorption kinetics was faster at higher temperatures. This shows that the ad- sorption is the endothermic, in which higher temperature is more favorable for dye adsorption. Regarding the second-order kinetic model as seen in Table 3, it fitted well with high correlation coefficients (R2 > 0.98). Moreover, slight differences between calculat- ed data (qe,cal) and experimental data (qe,exp) and its low χ2 values indicated the optimum adsorption at the equilib- rium. Therefore, the model better described experimental data indicating the adsorption highly depended on availa- ble active sites more than MB concentrations. Thus, it sat- isfactorily simulates MB adsorption onto modified cellu- lose fibers of P. australis.26,27 3. 3. 3. Intra-particle Diffusion and Film Diffusion Models Intra-particle diffusion was used to analyze kinetic adsorption at different MB concentrations. The multi-lin- ear (Fig. 6) shows three phases of the adsorption process. The first phase occurred within first 10 min, probably due to the adsorption on external surface of the adsorbent, or 818 Acta Chim. Slov. 2022, 69, 811–825 Nguyet et al.: Enhanced Adsorption of Methylene Blue by Chemically ... boundary layer diffusion of solute molecules (film diffu- sion).1,12 The electrostatic attraction between MB and the adsorbent might involve in this phase. The second phase was intra-particle diffusion at a gradual adsorption stage. The last one was equilibrium phase during which in- tra-particle diffusion occurred when MB concentration was reduced. Non-zero Ci intercepts (Table 4) showed that the in- tra-particle diffusion was not the only rate limiting step. Moreover, the fact that the first and second phases did not pass through the origin signifies the intra-particle diffu- sion. Ci enhanced when the temperature increased (Table 4), indicating that temperature promoted the boundary layer diffusion effect.1 The boundary layer diffusion rate constants of the first phase (kp1) were significantly higher than those of the second and third ones (Table 4). These results signify that mass transfer from bulk solution to exterior surface of SA-RPB was higher than that from exterior surface into its pores. Moreover, kp value at 313 K was higher than at 303 K, boosting MB diffusion rate. As shown in Fig. 8, calculated Bt values were plotted adsorption against time t (min). It denotes that linear lines for all MB initial concentrations did not pass through the origin. This indicates that MB adsorption on prepared SA- RPB is mainly governed by external mass transport, where particle diffusion is the rate limiting step. Boyd model was used to distinguish between film and intra-particle diffusions. Accordingly, Bt values plot- ted against time can be used to determine diffusion pro- cesses. If the lines are linear and pass through the origin, then intra-particle diffusion occurs. However, if they are Table 3. Kinetic parameters for MB adsorption by SA-RPB at different temperatures Temp. qe,exp First-order kinetic model Second-order kinetic model (K) (mg/g) k1 qe,cal R2 χ2 k2 qe,cal R2 χ2 (1/min) (mg/g) (1/min) (mg/g) 303 143.56 0.4047 139.36 0.807 4.084 0.0049 145.64 0.987 0.340 313 144.75 0.4192 140.35 0.849 3.841 0.0051 146.58 0.989 0.281 323 145.11 0.4275 141.79 0.828 4.241 0.0052 147.93 0.981 0.467 Table 4. Intra-particle diffusion model constant for MB adsorption by SA-RPB at different temperatures Temp. Intra-particle diffusion model (K) kp1 kp2 kp3 C1 C2 C3 (R1)2 (R2)2 (R3)2 (mg/gmin1/2) (mg/gmin1/2) (mg/gmin1/2) 303 22.065 2.607 0.220 58.823 125.330 141.099 0.996 0.862 0.939 313 22.114 3.000 0.342 60.732 123.992 141.161 0.994 0.926 0.953 323 21.584 2.520 0.470 63.361 128.449 141.652 0.999 0.940 0.870 Fig. 7. Boyd plots for MB adsorption by SA-RPB at 303, 313, and 323 K Fig. 6. Plots of intra-particle diffusion model for the adsorption of MB dye onto SA-RPB at 203; 313 and 323 K 819Acta Chim. Slov. 2022, 69, 811–825 Nguyet et al.: Enhanced Adsorption of Methylene Blue by Chemically ... linear but do not pass through the origin, or non-linear, then film diffusion controls the adsorption process. 15,16 Fig. 7 shows that the Boyd plots at all temperatures are non-linear, indicating the film diffusion occurred. This probably resulted from a mass transfer difference in the first and second phases. 3. 3. 4. Effects of Initial MB Concentration and Adsorption Isotherms The effects of initial MB concentration (from 125 to 300 mg/L) on adsorption were determined by 0.1 g/L SA- RPB, at pH 6.5 and 303 K. Adsorption isotherms were ana- lyzed at different initial MB concentrations, reflecting MB removal by SA-RPB (Fig. 8(a)). It clearly displays that the removal rate rapidly went up from 125 to 250 mg/L MB concentrations and gradually increased at higher concen- trations. More than 94% MB was absorbed at 125 and 150 mg/L concentrations, and the MB equilibrium adsorption capacity (qe) increased with higher initial dye concentra- tions (Fig. 8(a)). However, the initial MB concentration (from 125 to 300 mg/L) resulted in a decreased MB re- moval from 96.93% to 63.64% at 303 K (Fig. 8(a)). The lack of available active sites required for high initial MB concentrations accounted for these reductions. The ad- sorption isotherms, which revealed the interactive behav- iors between the adsorbate and adsorbent at liquid–solid interfaces, were analyzed. The obtained results show that Langmuir, Freun- dlich, Dubinin–Radushkevitch, and Temkin models sim- ulated MB adsorption on SA-RPB. The nonlinear plots at different concentrations are evident in Fig. 8(b), while Table 4 depicts their corresponding parameters. R2 and χ2 were used as indicators to analyze adsorption at equilibri- um. Langmuir model appeared to yield the best fit because of its higher R2 and lower χ2 than those of other models. The Langmuir isotherm model showed the homogeneous nature of the adsorbent surface and the monolayer cover of dye molecules formed on the outer surface of SA-RPB. Also, its isotherm RL values indicate that the fundamental features were higher than 0 and less than 1.0; thus, the ad- sorption was favorable within the evaluated concentration range.57 For the Freundlich model, the 1/n values (Table 5) were within the range of 0.1 < 1/n < 1.0, signifying phy- sisorption mechanism, and the adsorption process was considered favorable, rapid, and effective.58 The equilibri- um models provide insights on the adsorbent’s adsorption mechanism, surface properties, and affinity. Non-linear regression of the Temkin model fitted well with the experimental data with high R2(T) and low χ2(T) (Table 5). BT was 21.47 J/mol at 303K, reflecting the endothermic nature of adsorption. The R2(DR) value generated by Dubinin–Radushkevitch isotherm (Table 5) was significantly lower than those of other isotherms men- tioned above. This result shows that the MB adsorption by SA-RPB was not well-aligned with the Dubinin–Radush- kevitch isotherm. Moreover, mean energy of sorption (E) calculated from this model was 0.615 kJ/mol at 303 K, which proved the endothermic nature of adsorption.15 The NaOH-then-CA adsorbent exhibited its effec- tive MB removal with a maximum adsorption capacity of 191.49 mg/g at 150 mg/L MB concentration. This value is higher than those obtained in other studies using modified P. australis biomass and other modified plant materials listed in Table 6. For example, Kankılıç et al. reported that the maximum adsorption capacity of cellulose microfibrils of P. australis modified with NaOH was 54.9 mg/g at 400 mg/L.58 The treatment with citric acid increased the ab- sorption ability in our study. 3. 3. 5. Adsorption Thermodynamics Determining thermodynamic parameters is also conducted to better understand temperature effects on Fig. 8. (a) Effect of initial MB concentration on MB removal efficiency and adsorption capacity by SA-RPB at 303 K; (b) MB adsorption isotherm by SA-RPB based on Langmuir, Freundlich, Temkin, and Dubinin–Radushkevich isotherm models at 303 K. The experiments were conducted using 1.0 g/L SA-RPB at different initial MB concentrations and 6.5 pH value 820 Acta Chim. Slov. 2022, 69, 811–825 Nguyet et al.: Enhanced Adsorption of Methylene Blue by Chemically ... 3. 3. 6. Effect of Initial pH An increase in pH from 1.0 to 6.5 significantly en- hanced adsorption, but adsorption rates stayed almost constant at a higher pH value (Fig. 9). The adsorbent sur- face got more positively charged at low pH values, reduc- ing the attraction between the adsorbent and MB. A more negatively charged surface is available when pH increases, facilitating greater MB uptake.51 The pH effect on MB re- moval efficiency could be attributed to functional groups’ features on the surface and isoelectric point pHPZC of the SA-RPB adsorbent. The isoelectric point pHPZC value of SA-RPB (determined by the drift pH method) was 3.1 (as seen in Fig. 3(a) above). The hydroxyl (−OH) and carbox- yl (−COOH) groups were dominant on SA-RPB surface, which was deprotonated and became less charged, i.e. pHP- ZC < 3.1.61 When initial pH (pHin) was lower than pHPZC (3.1), the adsorbent surface was protonated and became more positive.61 In this case, the SA-RPB surface exhibited an electrostatic repulsion between SA-RPB surface and the MB−N+ cation in the solution, leading to poor adsorption efficiency.62 In contrast, when pH value was lower than pHPZC, functional groups on SA-RPB surface were depro- Table 5. Isotherm parameters for MB adsorption by SA-RPB at 303K Temp. qe,exp Langmuir isotherm Freundlich isotherm (K) (mg/g) qmax KL RL R2(L) χ2(L) KF (mg/g. nF R2(F) χ2(F) (mg/g) (L/mg) (L/mg)1/n) 303 190.94 191.49 0.404 0.0082 0.981 0.548 109.15 7.739 0.946 99.505 Temp. qe,exp Dubinin–Radushkevitch isotherm Temkin isotherm (K) (mg/g) qDR E KDR R2(DR) χ2(DR) AT (L/mg) BT R2(T) χ2(T) (mg/g) (kJ/mol) (mol2/kJ2) (J/mol) 303 190.94 177.62 0.615 −1.32 × 10-6 0.817 5.135 95.66 21.47 0.970 0.867 Table 7. Thermodynamic parameters for MB adsorption by SA-RPB Ea (kJ/mol) A (g/mg min) Temperature (K) ∆G° (kJ/mol) ∆H° (kJ/mol) ∆S° (kJ/mol K) 2.242 0.012 303 −28.63 16.82 0.155 313 −30.18 323 −31.73 Table 6. MB adsorbents’ capacities in comparison Absorbent Temperature (K) pH qmax (mg/g) P. australis treated with NaOH and citric acid 303 6.5 191.49 P. australis treated with NaOH26 298 7.0 54.9 Raw P. australis58 298 6.5 22.7 P. australis treated with organic compounds58 298 6.5 46.8 Raw Tunisian P. australis27 298 8.0 41.2 Peach stones modified with citric acid20 303 6.0 178.25 Lawny grass treated with citric acid28 298 5.7 301.1 Peanut shell modified with citric acid29 303 10.0 120.48 Activated carbon15 303 7 81.20 adsorption processes, applying Arrhenius equation (Eqs. (20)). Accordingly, a chemical reaction velocity is used for all predictive expressions of reaction-rate constants. (20) In Eqs. (20), A (g/mg.min) is the pre-exponential factor; Ea (kJ/mol) is the activation energy of absorption; R (8.314 J/mol K) is the gas constant, and T (K) is absolute temperature. Plots of lnK2 versus 1/T and lnKL versus 1/T were straight lines with R2 values of 0.99 and 0.98, respectively, from which Ea and A values were calculated (Table 7). The low values of activation energy (< 42 kJ/mol) obtained in this study indicated a diffusion-controlled process and a physisorption mechanism.59 The negative values of ΔG° at all temperatures reveal that the adsorption process was feasible and spontaneous. The obtained ΔS° was positive, showing the endothermic nature of adsorption, while the positive ΔS° spotted an increased randomness of the solid– liquid and adsorption medium interface over the process. The positive ΔSo value also indicated the adsorbent’s affinity and some structural changes in adsorbate and adsorbent.60 821Acta Chim. Slov. 2022, 69, 811–825 Nguyet et al.: Enhanced Adsorption of Methylene Blue by Chemically ... tonated and became more negative, inducing electrostatic attraction to MB−N+ and boosting the removal efficiency.62 Fig. 9. Initial pH effects on MB adsorption by SA-RPB (The tests performed using 1.0 g/L SA-RPB at 150 mg/L MB concentration) 3. 3. 7. Possible Mechanism of MB Adsorption onto SA-RPB Dye adsorption involves adsorbent-adsorbate inter- actions in the solution. Based on the result (ΔH° = 16.82 kJ/mol), adsorption was mainly induced by electrostatic and/or hydrogen bond forces.63 At a pH < 3.1 (pHPZC of SA-RPB), the protonated adsorbent surface became pos- itively charged (Fig. 3(a)). Therefore, the MB–N+ adsorp- tion was mainly attributed to physical interaction caused by capillary diffusion and weak hydrogen bonds. The sur- face of negatively charged adsorbent electrostatically inter- acted with MB–N+ at pH > 3.1, improving MB adsorption efficiency by SA-RPB, which reached a maximum value at the initial pH of the MB solution (6.5); hence, this pH value was selected to evaluate the adsorption mechanism. The FTIR analysis plots showed the spectra of MB, SA-RPB, and SA-RPB after MB adsorption and were used to describe their adsorption mechanisms. From the spec- tral peaks (Fig. 10), vibrations were revealed (Table 8). Based on the wavenumbers, it implies that pure MB had functional groups, that is, −OH, C=C, C=N, C=N+, C−N, C=S, C−S, and C−H.64,65 Variations in functional groups’ peak positions and the strength of SA-RPB dye complex indicate MB adsorption onto the SA-RPB surface. Differ- ences in the wavenumbers for C−H deformation in the benzene ring, C−N in the heterocycle, and C−N bonds connected with the benzene ring in the MB, C−H aromat- ic rings, C=C stretching vibrations in aromatic rings, and C–H asymmetric deformation in SA-RPB and SA-RPB dye complex (Table 8) corresponded to MB attachment to the adsorbent surface by π–π stacking between the aromatic backbone of MB and SA-RPB.50,64 This interaction was ev- idently reflected by adsorption peaks of MB and SA-RPB at 1599 and 1506 cm−1, respectively, disappearing in SA- RPB dye complex. Furthermore, SA-RPB peak at 897 cm−1 after MB adsorption (Fig. 10), attributed to the bending vi- bration of C−H in the aromatic ring, moved up to a higher intensity than the SA-RPB sample before MB adsorption. The peak ranges of 1340–1000 cm−1 of SA-RPB with oxygen-rich functional groups shifted, suggesting hydro- gen bonds created between SA-RPB and MB molecules. The band shifts occurred as N–CH3 stretching, Ar–N deformation vibration, C=S stretching vibration, and C–S stretching vibration. These phenomena signify that N in the −N(CH3)2 and Ar–N groups and S in the C=S and C–S groups might have been used as the hydrogen-bonding ac- ceptor and formed intramolecular hydrogen bonding with the hydrogen atom of the −OH and –COOH groups on the adsorbent surface.64 Hydrogen atoms in SA-RPB func- tional groups could also generate hydrogen bonds with N and S in MB functional groups. In addition, SA-RPB dye complex had a new adsorption peak at 1249 cm−1, owing to the Ar–N deformation vibration of the MB molecule; this verified MB adsorption onto the SA-RPB surface. Fig. 11. Possible adsorption mechanism of MB onto SA-RPB Fig. 10. FTIR spectra of SA-RPB (1) before and (2) after MB ad- sorption, and (3) pure MB 822 Acta Chim. Slov. 2022, 69, 811–825 Nguyet et al.: Enhanced Adsorption of Methylene Blue by Chemically ... These obtained results clearly show that MB adsorption by SA-RPB was attributed to four possible adsorption mech- anisms: electrostatic interaction, hydrogen bonding, π–π stacking interaction, and pores filling between MB and SA-RPB (Fig. 11) (i.e., adsorbent-adsorbate interactions). 3. 4. Reusability The reusable efficiency of adsorbents for wastewater treatment is economically and environmentally critical. The regeneration results showed that the adsorption effi- ciency decreased by approximately 11.69% after six des- orption–adsorption cycles compared with the first batch experiment (Fig. 12(a)). Ethanol was used to desorption MB from adsorbents in some previous reports.66,67 Addi- tionally, FTIR spectra of adsorbents showed similar spec- tra after six cycles (Fig. 12(b)), indicating the adsorbent stability during the adsorption process. This result sug- gests that the adsorbent is practically useful for treating real-time industrial effluent. Table 8. FTIR spectral features of MB and SA-RPB before and after MB adsorption MB SA-RPB Vibration Wavenumber (cm−1) Vibration Wavenumber (cm−1) Before ads. After ads. O–H or N–H stretching 3424 –OH stretching 3438 3427 –CH3 stretching 2939 C–H stretching vibration 2917 2915 C=N−C group 2360 –COOH stretching vibration 1735 1734 =N+(CH3)2 stretching 1661 –COO– stretching of carboxylate groups 1633 1601 with an aromatic ring C=N (and C=C) stretching in heterocycle 1599 C=C stretching vibrations in aromatic rings 1506 – C−H deformation in benzene ring 1492 C–H deformation in aromatic rings 1456 1489 C–N in heterocycle 1396 C=C stretching vibrations in aromatic rings 1431 1447 C–N bonds connected with benzene ring 1356 C–H asymmetric deformation 1384 1385 N–CH3 stretching 1340 –OH bending vibration in C–OH 1334 1355 C1–O vibrations in S derivatives 1321 1335 Ar–N deformation vibration 1252 – 1249 C=S stretching vibration 1183 C–O–C antisymmetric vibrations 1165 1164 C–S stretching vibration 1142 Anhydroglucose ring vibration 1111 1109 C–N stretching vibration 1066; 1038 C–O stretching vibration in cellulose, 1057; 1035 1056; 1034 hemicellulose, and lignin C–H axial deformation in aromataic rings 950–669 C–H rocking vibrations 898 897 C–S and C–N stretching 616–449 C–H bending in aromatic rings 875–500 875–500 Fig. 12. (a) Removal efficiency of MB onto SA-RPB in successive desorption–adsorption cycles; (b) FTIR spectra of SA-RPB after six desorption–ab- sorption cycles. The tests were done using 1.0 g/L SA-RPB at 150 mg/L MB concentration and 6.5 pH 823Acta Chim. Slov. 2022, 69, 811–825 Nguyet et al.: Enhanced Adsorption of Methylene Blue by Chemically ... 4. Conclusion This study demonstrates that chemically modified P. australis biomass can be used as an effective adsorbent for removing MB dye from aqueous solutions. Batch adsorp- tion test results show that materials treated with NaOH followed by citric acid boosted the removal compared with raw materials or those modified with only NaOH. The ini- tial pH of the solution, the adsorbent dosage, contact time, and initial MB concentrations significantly influenced the adsorption rates of SA-RPB. All SEM, FTIR, and BET anal- yses indicate significant modifications in the structure af- ter chemical treatments. Moreover, calculated adsorption energy denotes that MB adsorption by SA-RPB occurred through physical interactions at different temperatures when the removal process was endothermic and sponta- neous. The maximum MB adsorption capacity of SA-RPB was 191.49 mg/g, which was slightly decreased after four desorption–adsorption cycles. Four possible adsorp- tion mechanisms (i.e., electrostatic interaction, hydrogen bonding, π–π stacking interaction, and pores filling be- tween MB and SA-RPB) were spotted to functionally take place. This study shows that modified materials derived from reeds are expected to be highly economical and effi- cient for removing synthetic dyes in wastewater treatment. For practical uses, column experiments are underway for viable industrial scales and will be presented in the future. Acknowledgements This research is supported by the project SPD2020. 01.05. 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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 V raziskavi je bila biomasa iz vrste Phragmites australis kemično modificirana z NaOH in za tem še s citronsko kislino, da bi proizvedli učinkovit adsorbent, imenovan SA-RPB. Absorbent je bil karakteriziran z metodami XRD, SEM, BET in FT-IR. Dokazano je bilo, da adsorbent obstaja predvsem v obliki kristalov celuloze, vsebuje mikropore s povprečnim premerom 15,97 nm in ima veliko število hidroksilnih in karboksilnih skupin na površini. Adsorpcijski proces SA-RPB smo ovrednotili z adsorpcijo barvila metilen modrega (MB) v vodni raztopini. Kinetika adsorpcije je bila opisana z modelom psevdo-drugega reda. Adsorpcijska izoterma je skladna z Langmuirjevim modelom z največjo adsorpcijsko zmogljivostjo 191,49 mg/g pri 303 K. Ugotovitve kažejo, da je MB mogoče učinkovito odstraniti iz vodnih raztopin z uporabo adsorbenta, izdelanega iz surove biomase Phragmites australis, obdelane z NaOH in nato s citronsko kislino. 826 Acta Chim. Slov. 2022, 69, 826–836 Ambrož et al.: Assessment of the Capability of Magnetic Nanoparticles ... DOI: 10.17344/acsi.2022.7570 Scientific paper Assessment of the Capability of Magnetic Nanoparticles to Recover Neodymium Ions from Aqueous Solution Ana Ambrož,1 Irena Ban2 and Thomas Luxbacher1,3,* 1 University of Maribor, Faculty of Chemistry and Chemical Engineering, Laboratory for Water Biophysics and Membrane Processes, Smetanova 17, 2000 Maribor, Slovenia 2 University of Maribor, Faculty of Chemistry and Chemical Engineering, Laboratory for Inorganic Chemistry, Smetanova 17, 2000 Maribor, Slovenia 3 Anton Paar GmbH, Anton-Paar-Strasse 20, 8054 Graz, Austria * Corresponding author: E-mail: thomas.luxbacher@anton-paar.com Received: 10-03-2022 Abstract Magnetic nanoparticles (MNPs) have received increasing attention for various applications due to their fast synthesis, versatile functionalization, and recyclability by the application of a magnetic field. The high surface-to-volume ratio of MNP dispersions has suggested their use as an adsorbent for the removal of heavy metal ions. We investigated the ap- plicability of MNPs composed of a maghemite core surrounded by a silica shell functionalized with aminopropylsilane, γ-Fe2O3-NH4OH@SiO2(APTMS), for the removal of neodymium ions (Nd3+) from aqueous solution. The MNPs were characterized for their size, composition, surface functionality and charge. Despite of the promising properties of MNPs, their removal from the aqueous dispersion with an external magnet was not sufficient to reliably quantify the adsorption of Nd3+ by UV-Vis spectroscopy. Keywords: Rare earth elements; Maghemite; Nanoparticles; UV-Vis spectroscopy, Adsorption 1. Introduction The rare earth elements (REEs) are a group of 17 strongly related heavy elements that comprise scandium (Sc), yttrium (Y), lanthanum (La) and the f-block elements known as the lanthanide group, cerium (Ce) through lute- tium (Lu). In addition to being of great value to general geochemistry investigations, the REEs also have high com- mercial value and a wide scope of applications. They are used worldwide in various electronic and optical products, in advanced technologies, medical devices, military de- fence systems, as well as in the field of clean energy.1,2 RE- Es have been on the list of critical raw materials since 2010, and pressure on already limited resources is still increasing with the growth of the global population, industrializa- tion, and digitalization. With regard to high economic im- portance and high supply risk, neodymium (Nd), europi- um (Eu), terbium (Tb), dysprosium (Dy), and yttrium (Y) are considered most critical.3 With increasing demand and production, the amount of electronic waste containing REEs in various concentrations is also expanding. Sustain- ability in REE supply and proper treatment of end-of-life electrical and electronic compounds are crucial to achiev- ing climate neutrality.3 However, recycling of REEs pre- sents many challenges. Firstly, REEs are usually present in small amounts in tiny electronic parts of gadgets like mo- bile phones. In some materials like touch screens, these metals are evenly distributed making them much more difficult to extract.4 Secondly, due to the low yield and high cost of recycling processes, REEs are not recycled in large quantities, regardless of the end use. However, if REEs’ prices rise, recycling may become feasible.4,5 At present, the main focus is on the direct recycling of scrap and the urban mining, and subsequent recycling of end-of-life REE-containing products.6 The conventional processes for the separation and recovery of REEs mainly include precipitation, ion ex- change, coagulation, flocculation, liquid–liquid extrac- tion, biosorption, and adsorption. Among these methods, adsorption offers an efficient, environmentally friendly, and economical procedure for the removal of rare earth ions.7,8 Among different adsorbents, magnetic nanoparti- 827Acta Chim. Slov. 2022, 69, 826–836 Ambrož et al.: Assessment of the Capability of Magnetic Nanoparticles ... cles show a significant potential due to their high surface area and their response to an external magnetic field, which eases separation from a supernatant solution after completing the adsorption process.9–11 The chemical and physical stability, biocompatibility, ease of surface modifi- cation, low toxicity, straightforward synthesis, and low cost of iron oxide nanoparticles (IONPs) make them ideal for a variety of applications.12–15 Preparation methods and surface coating play a key role in determining the size distribution, morphology, magnetic properties, and surface chemistry of MNPs.16 Co-precipitation is the most widely used method for the synthesis of MNPs of controlled size and magnetic proper- ties. It is extensively used for biomedical applications of MNPs, because of the ease of preparation and the avoid- ance of harmful materials and procedures.16 Common problems in the preparation of magnetic nanoparticle dis- persions are agglomeration and oxidation, which may re- sult in the loss of dispersibility and magnetism. An addi- tional coating of the magnetic core helps to prevent particle agglomeration and aggregation.12 The coating method not only prevents agglomeration and oxidation of the particle, but also provides physical and chemical stability. The coat- ing provides an interface between the magnetic nanoparti- cle and the surrounding environment and offers the possi- bility for further functionalization. The properties of the coating may markedly differ from those of the nanoparti- cle core.15,17,18 Magnetic nanoparticles are commonly coated with organic (polymers or surfactants such as poly- ethylene glycol and dextran) or inorganic layers (gold, platinum, cobalt oxide, aluminium oxide, silica, activated carbon, etc.).19 The coating helps to obtain a specific affin- ity to target molecules, to increase dispersion stability, and to improve other physicochemical properties.9,20 The adsorption of heavy metal ions on MNPs com- bined with magnetic separation has been used extensively in water treatment and environmental clean-up.14,21 Func- tionalized magnetic nanoparticles act towards metal ions as a kind of “nano-sponges” and can easily be retrieved from solution with a magnet, thus they are excellent for the selective extraction of metal traces from wastewater or in- dustrial effluents. After the adsorbed ions are stripped, the nanoparticles can be reused, making this procedure a promising sustainable green technology.22,23 Core@shell MNPs composed of a maghemite core (γ-Fe2O3), and a functionalized silica coating have recently been applied for the adsorptive removal of Cu2+ and the rare earth ions Tb3+ and Dy3+.24,25 For the adsorption of Nd3+ from aqueous solutions, various magnetic nanoparticles have been synthesized. Ashour et al. synthesized magnetite nanoparticles func- tionalized with citric acid (CA@Fe3O4 NPs) or l-cysteine (Cys@Fe3O4 NPs) for the adsorption of La3+, Nd3+, Gd3+ and Y3+ from aqueous solution.26 Dupont et al. synthe- sized Fe3O4@SiO2(TMS-EDTA) nanoparticles for the ex- traction and separation of different rare-earth ions.27 Gal- houm et al. used Cysteine-functionalized chitosan magnetic nanoparticles for the sorption of La3+, Nd3+ and Yb3+ and hybrid chitosan magnetic nanoparticles func- tionalized by diethylenetriamine (DETA) for the recovery of Yb3+, Dy3+and Nd3+.28,29 Gok investigated batch adsorp- tion method as a green technology for removal and recov- ery of Nd and Sm using magnetic nano-hydroxyapatite adsorbent (MNHA).30 Li et al. prepared mesoporous mag- netic Fe3O4@mSiO2–DODGA nanomaterials for adsorp- tion and recycling of REEs. The surface of mesoporous Fe3O4 particles was modified with a diglycolamide li- gand.31 Liu et al. worked with magnetic bio-adsorbent Fe3O4-C18-chitosan-DETA (FCCD) composite to test the adsorption capacity of Dy3+, Nd3+, and Er3+.32 Miraoui et al. studied the sorption capacities of Nd3+ on magnetic na- noparticles grafted by poly(aminoethylene N-methyl 1-formic acid, 1-phosphonic acid) (PAEMFP).33 Molina et al. synthesized adsorbents based on functionalized mag- netite nanoparticles for the uptake of La3+, Pr3+ and Nd3+ from aqueous solutions.34 In this paper we extend the application of γ-Fe2O3- NH4OH@SiO2 nanoparticles functionalized with amino- propyl trimethly silane (APTMS) towards the adsorption of Nd3+ ions from dilute aqueous solutions. To the best of our knowledge, γ-Fe2O3-NH4OH@SiO2(APTMS) mag- netic nanoparticles have not been previously used for the removal of Nd3+ from aqueous solutions. The monitoring of the adsorption process of dis- solved heavy metal and rare-earth ions on MNPs requires the analytical detection of either the adsorbed ion concen- tration or the depletion of ions in solution. Aqueous solu- tions of Nd3+ appear strongly coloured and suggest the use of UV-Vis spectroscopy as a simple and reliable method for the determination of Nd3+ ion concentration in solu- tion. After separation of the MNPs with an external mag- net the remaining, Nd3+ ion concentration in the superna- tant is expected to reveal the REE removal efficiency at different adsorption time. Upon a re-dispersion of the MNPs, the adsorption process may continue thereby giv- ing fast and easy access to the characterization of the ki- netics and the optimization of the adsorption process. However, the polydisperse size distribution and the stabi- lization of the core@shell MNPs compete with the mag- netic force applied for their separation. We demonstrate that the additional separation processes of centrifugation and filtration are required for a complete removal of MNPs in order to obtain reliable information on the Nd3+ ion ad- sorption efficiency. These additional steps for MNP sepa- ration add complexity to the process and reduce the appar- ent benefits of REE recovery by the adsorption on MNPs. 2. Materials and Methods Iron(II) chloride tetrahydrate (FeCl2·4H2O), iron(I- II) chloride hexahydrate (FeCl3·6H2O), and tetraethyl or- 828 Acta Chim. Slov. 2022, 69, 826–836 Ambrož et al.: Assessment of the Capability of Magnetic Nanoparticles ... thosilicate (TEOS) were obtained from Merck. (3-amino- propyl)trimethoxysilane (APTMS, 97%) and 2-propanol ((CH3)2CHOH, ≥  99.8%) were obtained from Sigma Aldrich. Ethanol (C2H5OH) was obtained from Carlo Erba Reagents, ammonia solution (NH4OH, 25%) from Alka- loid AD, Skopje. Potassium chloride (KCl) and nitric acid (HNO3, ≥ 65%) were obtained from Kemika. All chemicals were used as received, without any further purification. Deionized water (dH2O) supplied by a water purification unit (MilliporeSigma, Burlington, USA) was used through- out the experiments. Neodymium(III) oxide (Nd2O3) ob- tained from Sigma Aldrich was used for the synthesis of neodymium(III) nitrate hexahydrate (Nd(NO3)3·6H2O). 2. 1. Synthesis of γ-Fe2O3-NH4OH@ SiO2(APTMS) For the preparation of the maghemite-silica core@ shell MNPs the protocol described by Kegl et al. was used.35 In brief, the γ-Fe2O3 magnetic nanoparticles were obtained by a co-precipitation method. To prepare 50 mL of 0.5 M Fe2+/Fe3+ solution in dH2O, FeCl2·4H2O and Fe- Cl3·6H2O were used in molar ratio 1:2. 25% ammonia solution (150 mL) was added to a round-bottomed flask and heated under reflux and constant stirring at 300 rpm. The temperature was maintained at 87 °C. Prepared 0.5 M Fe2+/Fe3+ solution (50 mL) was added instantaneously to the reaction mixture and kept for 1 h at 87 °C and pH 10.6. The obtained black coloured precipitate was then thor- oughly rinsed with dH2O and separated from the superna- tant using a permanent magnet. Rinsed γ-Fe2O3 particles were stabilized in 25 mL of 25% ammonia solution at 50 °C under constant stirring at 300 rpm for 24 h. The obtained particles were precipitated from the reaction mixture by a permanent magnet. The γ-Fe2O3-NH4OH particles were functionalized by SiO2 and APTMS. 2-propanol (66  mL), dH2O (15.42  mL), ammonia solution (1.7  mL, 25%), TEOS (0.324 mL, 99%) and APTMS (0.518 mL) were added to 4.93 mL aqueous dispersion of γ-Fe2O3-NH4OH. The re- action was carried out for 24 h in a closed vessel at room temperature and stirring at 500  rpm. The γ-Fe2O3- NH4OH@SiO2(APTMS) particles were precipitated from the reaction mixture by a permanent magnet and washed two times with ethanol and dH2O, respectively. 2. 2. Synthesis of Nd(NO3)3·6H2O Neodymium nitrate hexahydrate (Nd(NO3)3·6H2O) was synthesised from neodymium oxide (Nd2O3) powder. First, a small quantity of dH2O was added to cover the Nd2O3 powder, followed by HNO3 (≥  65%). Nd2O3 and HNO3 were used in molar ratio 1:2. The solution was heat- ed to 90 °C and mixed in a closed beaker to achieve a clear solution. When Nd2O3 was completely dissolved and the solution was clear, the liquid content of the mixture was evaporated at 110  °C. The collected light purple crystals were dried at room temperature. 2. 3. Adsorption Protocol Stock solutions of 0.05  M, 0.025  M and 0.01  M Nd(NO3)3·6H2O were prepared by dissolving an appropri- ate amount of obtained salt in dH2O. Adsorption experi- ments were conducted by mixing 5 mL of the stock solu- tion with 12  mg of magnetic nanoparticles (γ-Fe2O3- NH4OH@SiO2(APTMS)). Functionalized magnetic nano- particles were dispersed in the stock solution by placing the sample in an ultrasonic bath (Iskra PIO, Sonis 10) for 3 hours. After the reaction time, the adsorbent was separat- ed from the solution by an external magnet, centrifugation at 11000 rpm for 5 min (Eppendorf, centrifuge 5804 R), and filtration with 200 nm and/or 20 nm pore-size filters (Whatman, Anotop 25), to remove the remaining nanoad- sorbent. 2. 4. Characterization The thermal behaviour and stability of the magnetic nanoparticle samples was studied using a Mettler Toledo TGA/DSC1 thermogravimetric analyser in air and N2 at- mosphere, respectively, at a gas flow rate of 100 mL/min. The TGA curves with the weight pattern and heat flow were recorded as a function of temperature in the range of 25–600 °C with a heating rate of 10 °C/min, using alumina crucibles. The used MNPs were pre-dried for 24 hours at 80 °C. The presence of 6 mol of water in Nd(NO3)3·6H2O was confirmed using the same thermogravimetric analyser by direct heating in the range of 25–700 °C at a heating rate of 10 °C/min in N2, O2 and air atmosphere with 50 mL/ min flow rate. To confirm the formation of Nd2O3, X-ray diffrac- tion (XRD) analysis was conducted after TGA analysis us- ing an X’Pert PRO (PANalytical) X-ray diffractometer coupled with Cu Kα radiation with a wavelength of 0.15406 nm. The measurement was performed at room tempera- ture with a time step of 100 s in the angular range of 10° to 70° with a step size of 0.034°. Fully open (2.122 ) X’Celera- tor detector was used in the measuring protocol. The Brunauer, Emmet and Teller (BET) theory was used to determine the specific surface area of the nanopar- ticles by using the Micromeritics Tristar II 3020 Surface Area and Porosity system. The samples were degassed at 40°C for 24 hours prior to each measurement by using the Micromeritics FlowPrep 060 Gas Adsorption Sample Preparation Device. Specific surface area was determined in the relative pressure range of 0.05–0.3 in nitrogen gas and temperature of –195.8 °C. A Perkin Elmer Spectrum GX ATR-FTIR spectrom- eter was used to confirm the grafting of the organic ligands to the surface of the MNPs. Spectra were recorded over the 829Acta Chim. Slov. 2022, 69, 826–836 Ambrož et al.: Assessment of the Capability of Magnetic Nanoparticles ... range of 4000 cm–1 to 400 cm–1 in transmission mode at a resolution of 4 cm–1. Samples were dried for 24 h at 80 °C, ground into a fine powder, placed on the ATR crystal, and pressed into a thick film. Transmission electron microscopy (TEM) images were obtained to analyse the morphology and size distri- bution of the nanoparticles. TEM analyses were performed with a JEOL 2010F model transmission electron micro- scope operating at 200 kV. The sample was prepared in a water solution, dropped onto a carbon-copper grid, and dried at room temperature. Digital Micrograph and Orig- inPro 2015 software were used for image analysis. Zeta potential and size of the MNPs were measured using a Malvern Zetasizer Nano ZS with automatically chosen settings. Size characterization of the samples was made by dynamic light scattering (DLS) measurements with a 4  mW He–Ne laser operating at a wavelength of 633 nm, and a detection angle of 173° (backward scatter- ing). A disposable cuvette was filled up to 1 cm with the particle dispersion. The zeta potential was measured using a combination of electrophoresis and laser Doppler veloci- metry. For all measurements, a voltage of 50 V was applied. The attenuator index and measurement position are auto- matically adjusted by the software. For zeta potential measurements a folded capillary cell was used. Nanoparti- cle titration was performed using the Malvern MPT-2 Au- totitrator in parallel with the Zetasizer Nano ZS. A titra- tion from neutral to high pH using 0.1  M NaOH was performed to determine the isoelectric point (IEP). A sub- sequent titration from high to low pH with 0.1 M HCl was performed to determine the reversibility of the zeta poten- tial. The concentration of MNPs in dH2O was 0.01% V/V. UV-Vis absorption spectra of Nd3+ solutions were recorded in the range of 200–800 nm using a Varian Cary 1 UV–Vis spectrophotometer and a quartz cuvette. For the preparation of the reference sample, magnetic nanoparti- cles (12 mg) were added to 1 mM KCl (5 mL). After 3 hours the MNPs were separated from the solution by an external magnet, centrifugation (11000 rpm, 5 min), and filtration with 200 nm and/or 20 nm pore-size filters. As samples, 5 mL of Nd(NO3)3·6H2O stock solution of different con- centrations (0.05, 0.025, 0.01 M, respectively) and MNPs (12 mg) were used. 3. Results and Discussion The results of TGA of MNPs in air (blue curve) and nitrogen (red curve) atmosphere are displayed in Fig. 1, where the mass loss in percentages of γ-Fe2O3-NH4OH@ SiO2(APTMS) NPs by heating up to 600 °C is presented. The TGA curves show that the mass loss in both atmos- pheres occurs in one major step. In the range from 30 °C to 180 °C absorbed alcohol and water molecules evaporate from the MNPs surface. The interval from 280 °C to 600 °C is associated with the thermal decomposition of amino groups (NH2) and the removal of the alkyl chains of silanes from the silica coating.11,35,36 Around 550°C the transition of maghemite to hematite occurs without any mass change.37 Figure 1: Mass loss of γ-Fe2O3-NH4OH@SiO2(APTMS) in N2 and air atmosphere The mass loss in air atmosphere corresponds to 6.7% of the sample weight. In N2 atmosphere the mass loss pre- sents 7.3% of the sample weight. The difference in mass loss between both atmospheres is 0.6%. Both curves show the same trend for the evolution of the mass loss with tem- perature. We cannot see any additional effect of oxygen on MNPs and therefore no significant sign of oxidation. The thermal decomposition of Nd(NO3)3·6H2O is a complex step-wise process, which starts with the simulta- neous condensation of 6 mol of the initial monomer Nd(NO3)3·6H2O into the complex [Nd(NO3)3·6H2O]6. The main volatile products of the thermal decomposition are water, nitric acid, the azeotrope of 68% HNO3 and 32% H2O, nitrogen dioxide and oxygen.38 The results of TGA in air (blue curve), nitrogen (red curve) and oxygen (green curve) atmosphere are displayed in Fig. 2, where the mass loss in percentages of Nd(NO3)3·6H2O by heating up to 700 °C is presented. Figure 2: Mass loss of Nd(NO3)3· 6H2O in N2, O2 and air atmos- phere 830 Acta Chim. Slov. 2022, 69, 826–836 Ambrož et al.: Assessment of the Capability of Magnetic Nanoparticles ... The thermal decomposition of samples resulted in a mass loss of 58.4% in N2, 58.1% in O2 and 65.2% in air at- mosphere. The formation of Nd2O3 as the final product of TGA was confirmed with XRD analysis. The theoretical mass loss for the thermal decomposi- tion of Nd(NO3)3·6H2O is 61.6%.38 There are no significant changes between oxidizing and inert atmospheres. All three curves have similar trends for the evolution of mass loss with temperature. The de- composition occurs in multiple steps, regardless of the at- mosphere. The stepwise mass loss in N2 atmosphere is ex- plained in accordance with the experimental results.38 The first mass loss up to 62 °C reflects the evaporation of water during melting of the hexahydrate. It constitutes 2%, that corresponds to 3 mol of water out of 6 mol avail- able at the beginning of the decomposition process, which are eliminated during evaporation. In the 62–358  °C range, the mass loss is 21.1%, representing the removal of 14 mol of H2O and 5 mol of HNO3. The next mass loss is 23.3% and it takes place in the range of 358–436 °C with the removal of 1 mol HNO3, 5 mol of H2O and 10 mol of NO2. In the range of 436–513 °C 6 mol of H2O and 2 mol of NO2 are removed, constituting the mass loss of 7.6%. The final mass loss equals 4.4% and takes place in the range of 513–660 °C where 5 mol of H2O and 1 mol of O2 are removed. The remaining mass becomes constant at around 660 °C. The sum of the partial losses yields 58.4% and the presence of 6 mol of water in Nd(NO3)3·6H2O was confirmed. The BET analysis gave specific surface area of 78.27 m2/g for γ-Fe2O3-NH4OH@SiO2(APTMS). BJH adsorp- tion average pore width for γ-Fe2O3-NH4OH@SiO2 (APTMS) is 14.07 nm and desorption average pore width is 13.85 nm with total pore volume of 0.32 cm3/g. Infrared spectra of synthesized nanoparticles γ-Fe2O3-NH4OH (black spectrum), γ-Fe2O3-NH4OH@ SiO2(APTMS) (blue spectrum) and Nd3+/ γ-Fe2O3- NH4OH@SiO2(APTMS) (green spectrum) are shown in Fig. 3. The absorption peak at 3380.21 cm–1, attributed to N-H and O-H bonds, is observed in all spectra and it is the broadest band. By comparing the spectra of the coated and the uncoated MNPs, it is observed that the band at 3380.21 cm−1 is less pronounced and shallower for the coated nanoparticles than for the γ-Fe2O3- NH4OH nanoparticles, indicating the presence of the SiO2 shell. The presence of Nd3+ on coated MNPs is vis- ible by an even reduced intensity of the peak at 3380.21  cm–1, which indicates a successful adsorption of Nd3+ ions on MNPs. The absorption band observed at 1627.39 cm−1 is found in all three samples and corre- sponds to the N-H bending vibration. The peak found at 1020.92 cm–1 corresponds to the asymmetric stretch- ing vibration of the Si-O-Si bond and confirms the si- lanol functional groups grafted on the surface of γ-Fe2O3-NH4OH particles. The peak observed at 542.16 cm–1 belongs to the stretching vibration of Fe-O, which confirms the presence of the magnetic core. When comparing uncoated and coated MNPs this peak shifts from 540.46 cm–1 to 542.16 cm–1. When Nd3+ is complexed with γ-Fe2O3-NH4OH@ SiO2(APTMS) nanoparticles, a new peak at 1307.39 cm–1 could be observed in the spectrum. Due to the adsorption of Nd3+ two peaks shift to a higher wavenumber. The peak at 542.16 cm–1, which corresponds to the stretching vibra- tion of Fe-O, is shifted to 548.99  cm–1. The peak at 1020.92  cm–1, which corresponds to the asymmetric stretching vibration of the Si-O-Si bond, is shifted to 1022.44 cm–1. The ATR-FTIR absorption spectra confirm the composition of the synthesised magnetic nanoparti- cles, the successful coating with SiO2(APTMS), and the adsorption of Nd3+. The TEM photos in Fig. 4 A-C display the morphol- ogy of the MNPs with increasing magnification. The magnetic nanoparticles have a diameter ranging between 2.5 nm and 22.5 nm, with an average particle size of 9.5 ± 1.9 nm (Fig. 4 D). This value is an estimate of size, an- alysed from 81 particles, as it is difficult to determine the real size of individual particles due to agglomerates and blurred boundaries between particles. Aggregation of parti- cles results in wider size variation. None of the crystalline particles are ideally spherical, as their surface ends with a crystal plane, which is not curved. If nanoparticles are formed in a way that they can achieve an equilibrium struc- ture, they will assume an octahedral form. But since the pre- cipitation is an instantaneous process, we usually get incom- plete shapes, which are somewhere in between a sphere and an octahedron. As shown in Fig. 4 C the particle is octahe- dral, but because of orientation and 2D projection of TEM images it looks like a hexagon. In Fig. 4 B and C a surface layer of amorphous SiO2 is visible surrounding the Fe2O3 core, which confirms the successful coating of the MNPs. Figure 3: ATR-FTIR spectra of γ-Fe2O3-NH4OH (top curve, black), γ-Fe2O3-NH4OH@SiO2(APTMS) (middle curve, blue) and Nd3+/ γ-Fe2O3-NH4OH@SiO2(APTMS) (bottom curve, green) 831Acta Chim. Slov. 2022, 69, 826–836 Ambrož et al.: Assessment of the Capability of Magnetic Nanoparticles ... The measuring parameters and the specificity of each measurement technique often result in different sizes for the same sample. Dynamic light scattering (DLS) is com- pared with transmission electron microscopy (TEM) for the characterization of the size distribution of magnetic nanoparticles. TEM measures the geometric size of dry MNPs deposited on a support surface under ultrahigh vacuum conditions. The DLS technique measures the hy- drodynamic diameter that refers to how a particle diffuses within a liquid. Consequently, results obtained from DLS show a larger diameter than those from TEM. The signifi- cant difference in size between DLS and TEM results may be explained by an increased size in DLS due to the pres- ence of the dispersant and the formation of hydrate layers. The presence of bigger particles and aggregates enhances light scattering and can also contribute to larger size val- ues. Even though boundaries between particles in aggre- gates are not always obvious to recognize when analysing TEM images, the MNP size distribution observed by the TEM image-processing technique had a narrower range than that of DLS analysis. Fig. 6 shows the pH dependence of the particle size and the zeta potential for 0.01% V/V MNPs in dH2O. DLS reveals a hydrodynamic diameter of 250 nm at the current pH 7 of the MNP dispersion in dH2O. The TEM image in Fig. 4A already suggests larger aggregates of primary nan- oparticles, which maintain their assembly when dispersed in water. A pH titration was first performed from the na- tive pH 7 to pH 11 using 0.1 mol/L NaOH (measurement 1). Above pH 8, the particle size shows a sudden increase and approaches a steady diameter of 3 µm at pH 9. At higher pH we observe the onset of a trend towards smaller size, but the scatter of data disables a firm conclusion on a decreasing diameter. The direction of the pH change was then reversed by a titration towards the acidic range using 0.1 mol/L HCl. The diameter of 3 µm remains down to pH 8 followed by a sudden decrease and an approach of the initial diameter of 250 nm at pH 5. Below pH 3 we may assume again the onset of a trend towards a small growth of the particle aggregates. Although the changes in the hy- drodynamic diameter of the MNPs is obviously fully re- versible, we observe a hysteresis of the evolution of particle size with pH between the titrations to high and low pH, respectively. To understand the pH dependence of the particle size, the zeta potential of the MNPs was recorded in paral- Figure 4: TEM images of γ-Fe2O3-NH4OH@SiO2(APTMS) with increasing magnification (A: scale bar 100 nm, B: 20 nm, C: 5 nm) and size distri- bution of MNPs (D). 832 Acta Chim. Slov. 2022, 69, 826–836 Ambrož et al.: Assessment of the Capability of Magnetic Nanoparticles ... lel. The zeta potential is the key parameter that controls electrostatic interactions in particle dispersions, provides information about surface functionality and determines the dispersion stability. The zeta potential for particle dis- persions is calculated from the measurement of the elec- trophoretic mobility by electrophoretic light scattering (ELS). At pH 7 a positive zeta potential of ζ = +30 mV was observed, which indicates a significant positive charge density at the nanoparticle-water interface and confirm the presence of the amine functional groups of the outer- most APTMS coating of the core@shell maghemite nano- particles. The amine groups get protonated in water and assume a positive charge.39 When increasing the pH of the aqueous dispersion of the MNPs, the zeta potential de- creases and approaches the isoelectric point (IEP) at pH 9.8. Beyond the IEP the zeta potential assumes a negative sign indicating the charge reversal of the MNPs. The IEP 9.8 is indicative for the moderately basic character of the aminosilane.40 When continuing the pH titration from the alkaline to the acidic range, the zeta potential increases again and shows a charge reversal of the MNPs at a lower IEP 8.5. The electrokinetic charge density achieves a steady state indicated by the plateau value of the zeta potential of ζ = +35 mV below pH 5. We observe the same hysteresis for the pH dependence of the zeta potential and of the par- ticle size when repeating the titration from high to low pH. As a rule of thumb, a zeta potential below –25…– 30 mV or above +25…+30 mV describes a stable disper- sion where the aggregation of nanoparticles is suppressed by the electrostatic repulsion of particles with alike charge.41 This empirical observation is confirmed by the correlation of the hydrodynamic diameter and the zeta po- tential of MNPs shown in Fig. 5. As the initial zeta potential drops below ζ = +25 mV, the size of the MNPs starts to increase indicating the onset of the formation of larger aggregates. As the zeta potential approaches the IEP, the electrostatic repulsion between MNPs becomes weaker, and the average size of the particle aggregates, which remain suspended in dH2O, obtains its maximum diameter of 3 µm. Above the IEP 9.8, the nega- tive zeta potential steadily increases thereby introducing repulsive electrostatic forces between MNPs, which are now negatively charged. Since the zeta potential at pH 11 does not exceed the empirical threshold of ζ = ±25 mV, its effect on the disaggregation of particle assemblies remains rather small. In the opposite direction of the pH titration, the threshold value of ζ = +25 mV is observed at pH 6.5 where the hydrodynamic diameter returns to 250 nm, which is the average size of the MNP aggregates in dH2O. At very low pH, the significant volume of acid (0.1 mol/L HCl) added to decrease pH introduces a simultaneous increase in the ionic strength of the aqueous solution. Although the scatter of results in the range of pH 2.5–4 does not allow a conclusion on a decrease in the zeta potential, the electric double layer at the MNP-water interface gets suppressed at higher ionic strength, which again weakens the repulsive force between positively charged MNPs. The hysteresis observed when repeating the analysis of the pH dependence of the zeta potential and the shift of the IEP from pH 9.8 (for the first titration) to pH 8.5 (for the second titration) indicate a decrease in the average ba- sic strength of the functional coating of the MNPs. Obvi- ously, the stability of the silane coating on the silica shell of the MNPs at higher pH is limited. Figure 5: pH dependence of zeta potential and particle size for a dispersion of 0.01% V/V MNPs in dH2O 3. 1. Adsorption of Nd3+ Ions To elucidate the efficiency of Nd3+ ion adsorption on MNPs, an appropriate analytical method for quantifica- tion of the adsorption process was selected. The concen- tration of Nd3+ ions adsorbed on the MNP surface or the depletion of Nd3+ ion concentration in the aqueous solu- tion may be determined. There have been many analytical techniques used for the determination of the REEs in solid and solution samples; flame or graphite furnace atomic ab- sorption spectrometry, atomic absorption with chemical vapor generation, X-ray fluorescence spectrometry (XRF), inductively coupled plasma optical emission spectrometry (ICP-OES), inductively coupled plasma mass spectrome- try (ICP-MS), high-performance liquid chromatography (HPLC) and neutron activation analysis (NAA).41–43 Among the methods presented above for the characteriza- tion of various properties of the MNPs (composition, functional groups, size, charge), ATR FTIR and ELS may be considered to exhibit changes in the IR spectrum and in the zeta potential, respectively, of MNPs before and after adsorption of Nd3+ ions. As shown in Fig. 3, the ATR FTIR spectrum of the core@shell MNPs after adsorption of Nd3+ ions indicates the presence of neodymium by the additional peak at 1307 cm–1. The intensity of this peak compares with the peak indicating the N-H bending vibration (at 1627 cm–1) and is likely assigned to a vibration of the assumed Nd-NH 833Acta Chim. Slov. 2022, 69, 826–836 Ambrož et al.: Assessment of the Capability of Magnetic Nanoparticles ... surface complex that shows responsible for the adsorption of Nd3+ ions on the positively charged γ-Fe2O3-NH4OH@ SiO2(APTMS) nanoparticles. Since the intensity of the peak at 1307 cm–1 is rather weak and a calibration protocol is thus not feasible to correlate peak intensity with Nd sur- face concentration, ATR FTIR is only applicable to qualita- tively confirm the presence of Nd. The zeta potential is occasionally applied to describe and to study the adsorption process of dissolved com- pounds such as surfactants, polymers (polyelectrolytes, polysaccharides), or proteins on material surfaces.44–46 It is therefore feasible to investigate the capability of ELS for monitoring the adsorption of Nd3+ ions on MNPs al- though the same positive sign of the charge reduces the sensitivity of zeta potential analysis for the characteriza- tion of adsorption processes. Table 1 shows the zeta potential of MNPs remaining in dispersion after an apparent separation by an external magnet followed by centrifugation and filtration with a 200 nm filter after adsorption of Nd3+ ions from aqueous solutions of Nd(NO3)3 with different bulk concentration (0.01, 0.025, 0.05 mol/L, respectively). The average zeta potential and the standard deviation were obtained from three repetitive measurements. A steady decrease in the positive zeta potential was found from ζ = +47.7 ± 4.2 mV after adsorption from a 0.01 mol/L Nd3+ to ζ = +23.4 ± 2.4 mV when the initial concentration of Nd3+ ions was 0.05 mol/L. In the same series, the conductivity of the corresponding solu- tions increased from 406 mS/m to 1366 mS/m. The de- pendence of the zeta potential on the ionic strength is therefore dominating the decrease in the magnitude of the positive zeta potential of remaining MNPs in dis- persion. Independent of the zeta potential and the ionic strength, the particle size remains at Dh = 80.5 ± 0.8 nm. This hydrodynamic diameter is significantly smaller than the size of Dh = 256 nm of the MNPs determined in the MNP stock solution in deionized water. The dif- ference in the particle size is explained by the removal of the larger fraction of MNPs by filtration with a 200 nm filter. The zeta potential of the bulk dispersion of MNPs in dH2O reads ζ = +28.2 ± 0.5 mV. This zeta po- tential is only 60% of the zeta potential determined in 0.01 mol/L Nd(NO3)3 (the lowest Nd3+ ion concentra- tion used in the series of adsorption experiments) al- though the conductivity of the MNP bulk dispersion (18.4 mS/m) is much lower than the corresponding conductivity (406 mS/m) of 0.01 mol/L Nd3+. If the di- minishingly small ionic strength of deionized water is considered, the Hückel approach for the calculation of the zeta potential can be used, which transfers ζ = +28.2 mV determined in the Smoluchowski limit to ζ = +42.3 mV and therefore closer to the zeta potential obtained in the 0.01 mol/L Nd3+ solution. However, the conduc- tivity of the bulk MNP dispersion of 18.4 mS/m is mul- tiple times higher than the conductivity expected for dH2O (theoretically 0.006 mS/m) indicating a signifi- cant contribution of the charged MNPs to the electric conductance of the aqueous dispersion. A conductivity of 18.4 mS/m corresponds to an ionic strength of ap- prox. 0.001 mol/L when considering a monovalent elec- trolyte such as NaCl or KCl. This ionic strength justifies the application of the Smoluchowski approach for the calculation of the zeta potential especially when consid- ering the average particle diameter of 256 nm. It can be concluded that either the influence of the particle size and/or the high concentration of MNPs in the bulk dis- persion affect the electrophoretic mobility and thus the zeta potential. In conclusion, the strong dependence of the zeta po- tential on the ionic strength does not qualify this method for the estimation of the surface concentration of adsorbed Nd3+ ions on the MNPs. Table 1: Zeta potential (ζ) and hydrodynamic diameter (Dh) of MNPs after adsorption of Nd3+ ions from aqueous solution with different bulk concentration of Nd(NO3)3. Nd3+ bulk Conductivity ζ Dh concentration (mS/m) (mV) (nm) (mol/L) 0 * 18.4 28.2 ± 0.5 256 0.01 406 47.7 ± 4.2 81.3 0.025 906 34.1 ± 3.4 80.3 0.05 1366 23.4 ± 2.4 79.8 * MNPs dispersed in dH2O, i.e., without separation For monitoring the depletion of Nd3+ ions in solu- tion upon adsorption on MNPs, the optical properties of aqueous solutions of this REE suggest the application of UV-Vis spectroscopy. Fig. 6 shows the UV-Vis spectrum of aqueous solutions of Nd(NO3)3 at different concentra- tion (0.01, 0.025, 0.05 mol/L, respectively) in the wave- length range of 540–780 nm. In this range we find two dis- tinct absorption peaks at 575 nm and 740 nm, which may be assigned to the electronic f-f transitions 4I9/2  2G(1)7/2 and 4G5/2 (575 nm, 17391 cm–1) and 4I9/2  4F7/2 (740 nm, 13514 cm–1).47 The absorbance recorded at these peaks was used to establish calibration curves for the estimation of the Nd3+ ion concentration from the measured absorb- ance after completing the adsorption process on MNPs. The corresponding calibration curves are shown in the in- set of Fig. 6. Coincidently at a given Nd3+ concentration, the peaks at 575 nm and 740 nm show almost the same absorbance. Fig. 6 also shows the UV-Vis spectra obtained after adsorption of Nd3+ on MNPs in solutions with the corre- sponding Nd3+ starting concentration (0.01, 0.025 and 0.05 mol/L, respectively) after separation of the MNPs by an external magnet, centrifugation and filtration using a 834 Acta Chim. Slov. 2022, 69, 826–836 Ambrož et al.: Assessment of the Capability of Magnetic Nanoparticles ... filter with 200 nm pores. We do not find any decrease in the absorbance for both f-f transition peaks, which sug- gests that adsorption of Nd3+ ions on γ-Fe2O3-NH4OH@ SiO2(APTMS) nanoparticles did not occur. However, up- on filtration of the supernatant obtained after centrifuga- tion of the MNP dispersion, that is remaining after apply- ing an external magnet, with a 20 nm filter, the absorbance decreases consistently for the peaks at both 575 nm and 740 nm. The evaluation of the depletion of Nd3+ from the stock solution using the UV-Vis calibration curves reveals a removal of 30% at pH 7 and after 3 h of interaction time. DLS and ELS measurements of the filtrate still show clear distributions of size and zeta potential with Dh = 51 nm and ζ = +17.8 ± 1.8 mV. We note that (i) small aggregates of primary MNPs pass the 20 nm filter and (ii) the filter shows a small rejection at the nominal threshold of 20 nm. The measured conductivity in the filtrate reads 1176 mS/m, which is 14% lower than the conductivity in the filtrate of the 200 nm filter. We may thus assume a larger zeta potential but observe the opposite. Obviously, the dif- ferent size fractions of MNPs exhibit different effective charge density represented by the difference in the zeta po- tential. The presence of MNPs in the filtrate of the 20 nm filter suggests a contribution of Nd3+ adsorbed on these MNPs to the UV-Vis absorption spectrum. The removal efficiency is therefore expected slightly higher than the es- timated 30%. From the change in the absorbance for MNPs filtered with a 20 nm filter after adsorption of 0.05  M Nd3+, we conclude that 0.035 M Nd3+ remain in solution. The ad- sorption efficiency after 3 hours is therefore 30%. 4. Conclusions Magnetic nanoparticles with a γ-Fe2O3 core, coated with SiO2 and functionalized with APTMS, were synthe- sized with the purpose of removing rare earth elements from aqueous solutions. The main reason for selecting Nd3+ as the source of REE is the fact that neodymium is one of the most important REE in terms of usage and ap- plicability. Furthermore, neodymium is among those RE- Es that show a lot of absorption peaks in the visible spec- trum, which makes UV-Vis absorption spectroscopy convenient for the determination of the Nd3+ concentra- tion in solution. The synthesized MNPs were character- ized in terms of size (TEM, DLS), charge (zeta potential; ELS), surface functionality (ATR-FTIR), and composition (TGA, XRD). The adsorption of Nd3+ on γ-Fe2O3- NH4OH@SiO2(APTMS) was performed in batch mode. The UV-Vis calibration curves revealed maximum adsorp- tion to be 30% at pH 7, with use of 12 mg of MNPs in 0.05 mol/L of neodymium solution. Based on the results, an obvious decrease in neodymium concentration becomes visible only after a reaction time of 3 hours, which makes this process uneconomical for large-scale use. The removal of γ-Fe2O3-NH4OH@SiO2(APTMS) from dispersion after Nd3+ adsorption requires multiple steps (removal with a magnet, centrifugation, and filtration) in order to measure the absorbance spectrum without a significant interfer- ence of MNPs remaining in the processed solution, which also requires time and adds additional steps to the process. Ban et al. reported the use of ultrafiltration to completely remove even the primary particles of MNPs.48 The slow Figure 6: Absorbance spectra of Nd3+ before and after adsorption on MNPs. The labels of spectra recorded after adsorption indicate the filter used for removing MNPs (pore size 20 nm, 200 nm). 835Acta Chim. Slov. 2022, 69, 826–836 Ambrož et al.: Assessment of the Capability of Magnetic Nanoparticles ... adsorption of Nd3+ ions on γ-Fe2O3-NH4OH@SiO2 (APTMS) suggests a coating by different chelating com- pounds, such as polyethylenimine or negatively charged MNPs.34 Acknowledgements The authors acknowledge dr. Julija Volmajer Valh for her help with ATR-FTIR, dr. Sašo Gyergyek for TEM anal- ysis, Edi Kranjc for XRD analysis, and dr. Karl Gatterer of Graz University of Technology for donating Nd2O3. 5. References 1. D. A. Atwood: The rare earth elements: Fundamentals and Applications, John Wiley & Sons Ltd, 2012, pp. 2–27. 2. J. H. L. Voncken: The Rare Earth Elements: An Introduction, Springer, 2016, pp. 1–11. DOI:10.1007/978-3-319-26809-5 3. U.S. department of Energy, Critical Materials Strategy, https://www.energy.gov/sites/prod/files/DOE_CMS2011_FI- NAL_Full.pdf, (accessed: February 18, 2022) 4. V. Balaran, Geosci. Front., 2019, 10, 1285–1303. DOI:10.1016/j.gsf.2018.12.005 5. C. E. D. 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Quarton, J. Chem. Phys., 1977, 66, 215–220. DOI:10.1063/1.433910 48. I. Ban, S. Markuš, S. Gyergyek, M. Drofenik, J. Korenak, C. Helix-Nielsen, I. Petrinić, J. Nanomater., 2019, 9, 1–17. DOI:10.3390/nano9091238 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 Magnetni nanodelci (MNP) so zaradi svoje hitre sinteze, vsestranske funkcionalizacije in možnosti recikliranja z upora- bo magnetnega polja deležni vse večje pozornosti za različne aplikacije. Visoko razmerje med površino in prostornino disperzij MNP predstavlja zmožnost njihove uporabe kot adsorbenta za odstranjevanje ionov težkih kovin. Raziskali smo uporabnost MNP, sestavljenih iz maghemitnega jedra in obdanih s kremenčevo lupino, funkcionalizirano z amino- propilsilanom, γ-Fe2O3-NH4OH@SiO2 (APTMS), za odstranjevanje neodimovih ionov (Nd3+) iz vodne raztopine. MNP so bili karakterizirani glede na velikost, sestavo, površinsko funkcionalnost in naboj. Kljub obetavnim lastnostim MNP pa njihova odstranitev iz vodne disperzije z zunanjim magnetom ni zadostovala za zanesljivo kvantificiranje adsorpcije Nd3+ z UV-Vis spektroskopijo. 837Acta Chim. Slov. 2022, 69, 837–847 Jahanbakhshi et al.: Ionic Liquid Supported on Magnetic Graphene Oxide ... DOI: 10.17344/acsi.2022.7593 Scientific paper Ionic Liquid Supported on Magnetic Graphene Oxide as a Highly Efficient and Stable Catalyst for the Synthesis of Triazolopyrimidines Azar Jahanbakhshi, Mahnaz Farahi* and Yeganeh Aghajani Department of Chemistry, Yasouj University, Yasouj 75918-74831, Iran * Corresponding author: E-mail: farahimb@yu.ac.ir Received: 05-25-2022 Abstract A novel sulfonic acid functionalized ionic liquid was prepared by anchoring 1-(propyl-3-sulfonate) vinylimidazolium hydrogen sulfate ([(CH2)3SO3HVIm]HSO4) on Fe3O4@GO. The prepared heterogeneous catalyst was characterized by XRD, FT-IR, EDX, SEM, VSM and TGA techniques. The results show that [(CH2)3SO3HVIm]HSO4 was successful- ly deposited on the surface of Fe3O4@GO and the prepared ionic liquid catalyst exhibited good thermal stability. The activity of the prepared catalyst was investigated in the synthesis of triazolo[1,5-a]pyrimidine derivatives by a one-pot three-component reaction of active methylene compound (malononitrile or ethyl cyanoacetate), 3-amine-1H-1,2,4-tri- azole and aryl aldehydes under solvent-free conditions. This catalyst could be rapidly separated by an external magnet and recycled seven times without significant loss of catalytic activity. Keywords: Ionic liquid; graphene oxide; triazolopyrimidines; Fe3O4 1. Introduction Ionic liquids (ILs) are increasingly recognized as environmentally friendly materials, alternative reaction media, and promising catalysts due to their unique prop- erties such as tunable acidity, selective solubility, negligi- ble vapor pressure, wide liquid range, and high thermal stability.1,2 Ionic liquids have been developed for various specialty applications such as catalysts, fossil fuel desul- furization reagents, lubricants, and as monomers for the synthesis of ionic polymers. They are also known as “ver- satile chemicals” in various fields of synthetic chemistry.3,4 Among them, the acidic ionic liquids functionalized with sulfonic acid groups with a hydrogen sulfate counteranion have been intensively studied as a class of dual acid func- tionalized ILs because both the SO3H functional groups and the hydrogen sulfate counteranion can increase their acidity. Despite the advantages of ionic liquids, their wide- spread use was still hindered by some disadvantages, such as intolerable viscosity, complex product isolation and cat- alyst recovery, high cost, and long reaction times.5,6 Im- mobilization of ionic liquids on various solid supports is one of the most efficient ways to overcome these problems. Ionic liquids on supports have the advantage of combining the properties of an ionic liquid with the typical advantag- es of immobilization, such as easy recycling and improved selectivity in applications with catalytic activity.7 Heterog- enization of ionic liquids on suitable porous supports,8–10 suitable magnetic nanoparticles,11–13 immobilized on solid supports, either by physical coating of the ionic liquids on Al2O3,14,15 SiO216,17 and TiO218,19 or by covalent bonding of the ionic liquids to the support surface, would be a feasible and attractive approach to prepare an efficient solid cata- lyst with superior activity and stability.20 While reasonable reusability was observed in most cases, the dispersion of the ionic liquid on the solid support was poor, and leaching of the ionic liquid occurred, resulting in loss of activity.21,22 To overcome these drawbacks, graphene oxide (GO) can be used as a support material for immobilization of the ionic liquid with better dispersion, excellent activity, co- valent bonding, and good reusability. In general, graphene oxide has some oxygen-containing functions (such as epoxy, carboxy and hydroxy groups). Therefore, it could be a promising candidate as an advantageous support for the immobilization of ILs.23–27 ILs on graphene oxide supports have advantages due to their physicochemical properties; nevertheless, it is the tedious and time-consuming separa- tion procedures that limit their practical applications. In this context, the development of magnetic graphene oxide as a support material has shown promise.28–31 838 Acta Chim. Slov. 2022, 69, 837–847 Jahanbakhshi et al.: Ionic Liquid Supported on Magnetic Graphene Oxide ... In recent years, green chemistry has become one of the most important aspects of experimental and industrial efforts of chemists. Due to their high atomic efficiency and significant diversity, multicomponent reactions (MCRs) have occupied an essential place in the world of green chemistry.32–34 The multicomponent reaction is a powerful synthetic strategy in modern chemistry in which three or more simple components are involved as starting reagents in a one-pot system to obtain new complex molecules with lower processing costs compared to the stepwise method, which usually produces few byproducts. Multicomponent reactions are an advantageous tool for the synthesis of critical heterocyclic compounds that have many biological activities.35–37 The pyrimidine family is the most impor- tant nitrogen-containing heterocycle because it is found in many natural and biologically active products. It is known that the condensation of triazole and pyrimidine leads to the formation of bicyclic heterocycles known as triazo- lopyrimidines, which exhibit a wide range of biological properties.38–40 Triazolopyrimidines can be used in a vari- ety of synthetic pharmacophores. In addition, they are val- uable building blocks for the structure of many herbicides such as penoxsulam, diclosulam, flumetsulam, azafenidin, and floransulan.41–45 Furthermore, triazolopyrimidines are synthetic analogs of purines and nucleosides. Also, [1,2,4]triazolo[1,5-a]pyrimidines, a subtype of bioisoster- ic purine analogs, have been reported to possess potential antitumor activities, particularly those bearing functional groups at C-5, C-6, or C-7 positions.46,47 Several synthet- ic strategies have been described for the preparation of triazolopyrimidine derivatives, most of which are based on a modification of the classical Biginelli reaction.48,49 Although some of these procedures are efficient, some of them have limiting factors, including long reaction times, side reactions, rigid workup, high-temperature conditions, and nonrecyclable reagents. Continuing our efforts to establish an environmentally friendly method for the synthesis of reusable catalysts,50–59 we report a novel magnetic graphene oxide supported by a doubly acidic ionic liquid and the catalytic activities in the synthesis of triazolo[1,5-a]pyrimidine derivatives. 2. Experimental All solvents, reagents, and chemicals were purchased from Sigma-Aldrich, Merck, and Fluka chemical com- panies. X-ray diffraction analyzes were recorded using a Philips X Pert Pro X diffractometer operated with a Ni-fil- tered Cu-Ka radiation source. XRD diffraction patterns were obtained from 2θ = 10–80°. The EDS was performed using the TESCAN-Vega model. Scanning electron mi- crographs were performed using a SEM: KYKY-EM3200 instrument. Thermogravimetric analysis was performed with a Perkin Elmer STA 6000 instrument in the temper- ature range 25–900 °C under air atmosphere. Vibration- al sample magnetometry of magnetic materials was per- formed using a Kavir Magnet VSM. The IR spectrum was recorded in the range 400–4000 cm–1 using a FT-IR JAS- CO 6300D instrument. 1H and 13C NMR spectra were re- corded with a Bruker Ultrashield spectrometer (400 MHz) using DMSO-d6 as solvent. Preparation of [(CH2)3SO3H-VIm]HSO4 (IL) First, a mixture of 1-vinylimidazole (9.4 g) and 1,4-butanesultone (12.4 g) was stirred at room temperature for 24 hours. Then the obtained white solid was collected, washed with diethyl ether and dried at 50 °C. The prepared material was dissolved in H2O (5 ml) in a 100-ml round bottom flask, and equimolar H2SO4 was added dropwise at 0 °C. After the addition was completed, the mixture was stirred at 50 °C for 12 h, during which the ionic liquid was formed. Finally, the ionic liquid was repeatedly washed with diethyl ether and dried in vacuo at 50 °C.2 Synthesis of graphene oxide (GO) Graphene oxide was synthesized using graphite powders by the modified Hummer method. In a typical synthesis procedure, graphite powder (3 g) was added to a mixture of concentrated H2SO4 (12 ml), K2S2O8 (2.5 g), and P2O5 (2.5 g) in a beaker containing 500 ml and stirred at 80 °C for 5 hours. The mixture was then cooled to room temperature, diluted with deionized water, and stirred at room temperature for 24 hours. The mixture was then fil- tered, washed with deionized water and ethanol, and dried. After drying, the resulting powder was dissolved in H2SO4 (120 ml) and KMnO4 (15 g) in an ice bath and stirred until complete dissolution. Deionized water (250 ml) was added slowly with stirring and then heated at 35 °C for 2 hours. The reaction was then stopped by adding deionized water (250 ml) and finally hydrogen peroxide (20 ml, 30%). The resulting mixture was washed several times with aqueous HCl solution (1:10) in a centrifuge and then dried in an oven at 60 °C. Finally, the graphite oxide was sonicated in deionized water for 1 h to obtain graphene oxide nano- sheets.60 Preparation of magnetic Fe3O4 nanoparticles (MNPs) To synthesize magnetic Fe3O4 nanoparticles, Fe- Cl3.6H2O (2.7 g, 10 mmol) and FeCl2.4H2O (1 g, 5 mmol) were first dissolved in deionized water (45 ml) under N2 atmosphere at 80 °C for 30 min. In the next step, NaOH solution (10 ml, 25%) was slowly added dropwise until the color darkened, and then stirred for 1 h. Finally, the black product of magnetic Fe3O4 nanoparticles was collected with an external magnet, washed with deionized water and ethanol, and dried.52 Synthesis of Fe3O4@GO nanocomposite The nanocomposite Fe3O4@GO was synthesized by the liquid self-assembly method. In a typical synthesis, GO (50 mg) was dispersed in DMF (5 ml) by ultrasonication 839Acta Chim. Slov. 2022, 69, 837–847 Jahanbakhshi et al.: Ionic Liquid Supported on Magnetic Graphene Oxide ... for two hours, and then Fe3O4 (40 mg) was slowly added dropwise in chloroform (10 ml). The mixture was soni- cated for 4 hours at room temperature. The black colored Fe3O4@GO nanocomposite was separated with an exter- nal magnet, washed with deionized water and ethanol, and finally dried.61 Preparation of Fe3O4@GO-Pr-SH For the functionalization of Fe3O4@GO nanocom- posites, 3-mercaptopropyltrimethoxysilane (2 ml) was added to Fe3O4@GO (1 g) in dry toluene (30 ml), and the mixture was stirred under N2 atmosphere and refluxed for 24 h. The resulting product (Fe3O4@GO-Pr-SH) was sep- arated by an external magnet, washed with distilled water and ethanol, and dried. Synthesis Fe3O4@GO-Pr-S/IL (1) To prepare an acidic ionic liquid on modified GO (nanocatalyst 1), a mixture of Fe3O4@GO-Pr-SH (1 g), [(CH2)3SO3H-VIm]HSO4 (5 ml), and azobisisobutyroni- trile (AIBN) (5 mol%) was refluxed in toluene (100 mL) for 30 h under N2 atmosphere. The catalyst was then sepa- rated with an external magnet, washed several times with diethyl ether, and then dried at 50 °C. General procedure for the synthesis of triazolopyrimi- dines 5 To a mixture of aldehyde (1 mmol), ethyl cyanoac- etate or malononitrile (1 mmol), and 3-amine-1H-1,2,4- triazole (1 mmol) was added catalyst 1 (0.004 g) under solvent-free conditions at 80 °C. The reaction progress was monitored by TLC (n-hexane/EtOAc). After completion of the process, EtOAc (10 ml) was added and the Fe3O4@ GO-Pr-S/IL-nanocatalyst was separated using an exter- nal magnet. Further purification of the product was per- formed by recrystallization from EtOH. General procedure for the recovery of nanocatalyst 1 Recovery of Fe3O4@GO-Pr-S/IL was carried out in the synthesis of pyrimidine derivatives. The mixture of benzaldehyde (1 mmol), ethyl cyanoacetate or malononi- trile (1 mmol) and 3-amine-1H-1,2,4-triazole (1 mmol) was stirred in the presence of catalyst 1 (0.004 g) under optimum conditions. After completion of the reaction, hot EtOAc (10 ml) was added and the nanocatalyst was sepa- rated from the reaction mixture using an external magnet. Then, the recovered catalyst was washed several times with EtOAc (10 ml) and deionized water (10 ml) and dried. Fi- nally, the recovered catalyst was reused seven times con- secutively under the same conditions. Selected spectral data Ethyl 5-amino-7-phenyl-1,7-dihydro-[1,2,4]triazolo [1,5-a]pyrimidine-6-carboxylate (5a). FT-IR (KBr, cm–1) ν 3396, 3375, 3326, 2746, 1687, 1565, 1490, 1110; 1H NMR (400 MHz, DMSO-d6) δ 9.25 (s, 1H), 8.08 (d, 1H, J = 8 Hz), 7.66 (s, 2H), 5.37 (s, 1H), 3.93 (q, 2H, J = 8 Hz), 0.84 (t, 3H, J = 8 Hz), 7.57–7.64 (m, 5H) ppm. Ethyl 5-amino-7-(4-chlorophenyl)-4,7-dihydro- [1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate (5b). FT-IR (KBr, cm–1) ν 3412, 3253, 3115, 2872, 1692, 1532, 1485, 1203; 1H NMR (400 MHz, DMSO-d6) δ 9.25 (s, 1H), 8.08 (d, 1H, J = 8 Hz), 7.65 (s, 2H), 5.41 (s, 1H), 4.38 (q, 2H, J = 7 Hz), 0.92 (t, 3H, J = 7 Hz), 7.45–7.58 (m, 5H); 13C NMR (100 MHz, DMSO-d6) δ 163.83, 160.63, 154.82, 134.82, 130.96, 129.87, 129.04, 110.16, 61.09, 57.01, 14.02. Ethyl 5-amino-7-(4-bromophenyl)-4,7-dihydro- [1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate (5c). FT-IR (KBr, cm–1) ν 3427, 3389, 3098, 2923, 1677, 1587, 1488, 1178; 1H NMR (400 MHz, DMSO-d6) δ 9.16 (s, 1H), 7.08 (d, 1H, J= 3.6 Hz), 8.39 (s, 2H), 5.21 (s, 1H), 3.98 (q, 2H, J =6.4 Hz), 1.33 (t, 3H, J = 6.6 Hz), 7.41–7.43 (m, 5H); 13C NMR (100 MHz, DMSO-d6): δ = 169.6, 156, 146, 141.99, 132.5, 129, 126.8, 62.1, 40.6, 27.5. Ethyl 5-amino-7-(4-nitrophenyl)-4,7-dihydro- [1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate (5d). FT-IR (KBr, cm–1) ν 3480, 3430, 3198, 2917, 1680, 1604, 1504, 1054; 1H NMR (400 MHz, DMSO-d6) δ 13.99 (s, 1H), 8.10 (d, 1H, J = 8 Hz), 7.83–7.91 (m, 4 H), 5.99 (s, 1H), 4.43 (q, 2H, J = 7.2 Hz), 1.03 (t, 1H, J = 3.2 Hz); 13C NMR (100 MHz, DMSO-d6) δ 163.62, 154.95, 149.60, 144.21, 134.01, 131.06, 130.42, 124.86, 124.46, 114.11, 63.57, 59.84, 14.00. Ethyl 5-amino-7-(3-nitrophenyl)-1,7-dihydro- [1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate (5e). FT-IR (KBr, cm–1) ν 3444, 3328, 3097, 2888, 1697, 1623, 1531, 1272; 1H NMR (400 MHz, DMSO-d6) δ 6.41 (s, 1H), 8.09 (d, 1H, J = 8 Hz), 7.80–7.83 (m, 5H), 5.69 (s, 1H), 4.03 (q, 2H, J = 8 Hz), 0.92 (t, 3H, J = 8 Hz); 13C NMR (100 MHz, DMSO-d6) δ 163.63, 154.55, 150.36, 147.93, 134.84, 131.17, 130.83, 125.30, 123.78, 123.13, 113.63, 63.19, 59.06, 13.86. Ethyl 5-amino-7-(2,4dichlorophenyl)-1,7-dihydro- [1,2,4]triazolo[1,5-a]pyrimidine-6-carboxylate (5f). FT-IR (KBr, cm–1) ν 3489, 3421, 3085, 2878, 1699, 1586, 1474, 1106; 1H NMR (400 MHz, DMSO-d6) δ 11.67 (s, 1H), 8.11 (d, 1H, J = 8 Hz), 7.87 (s, 2H), 4.16 (s, 1H), 2.48 (q, 2H, J = 8 Hz), 1.25 (t, 3H, J = 7 Hz), 7.26–7.77 (m, 5H); 13C NMR (100 MHz, DMSO-d6) δ 160.6, 153.6, 137.6, 130.2, 120.8, 127, 67.1, 40.4, 21.6. Ethyl 5-amino-7-(4-methylphenyl)-7,8-dihydro-[1, 2,4]-triazolo[4,3-a]pyrimidine-6-carbonitrile (5g). FT-IR (KBr, cm–1) ν 3347, 3262, 3185, 3118, 2921, 2192, 1660, 1633, 1531, 1482, 1363, 1286, 1214, 1157; 1H NMR (400 MHz, DMSO-d6) δ 2.28 (s, 3H), 5.29 (d, 1H, J = 2.4 Hz), 7.18 (s, 4H), 7.21 (s, 2H), 7.71 (s, 1H), 8.75 (d, 1H, J = 1.6 Hz); 13C NMR (100 MHz, DMSO-d6) δ 20.64, 53.70, 56.06, 119.06, 126.00, 129.18, 137.26, 140.24, 146.93, 151.83, 153.92. Ethyl 5-amino-7-(4-isopropylphenyl)-7,8-dihydro- [1,2,4]-triazolo[4,3-a]pyrimidine-6-carbonitrile (5h). FT-IR (KBr, cm–1) ν 3378, 3295, 3181, 3118, 2964, 2186, 1656, 1627, 1523, 1479, 1367, 1284, 1211, 1151; 1H NMR (400 MHz, DMSO-d6) δ 1.23 (d, 6H, J = 6.8 Hz), 2.92 (s, 1H), 840 Acta Chim. Slov. 2022, 69, 837–847 Jahanbakhshi et al.: Ionic Liquid Supported on Magnetic Graphene Oxide ... 5.33 (d, 1H, J = 2Hz), 7.24 (s, 4H), 7.31 (s, 2H), 7.76 (s, 1H), 8.80 (d, 1H, J = 1.6 Hz); 13C NMR (100 MHz, DM- SO-d6) δ 23.81, 33.11, 53.68, 55.97, 119.11, 125.99, 126.59, 140.71, 146.95, 148.21, 151.83, 153.91. 3. Results and Discussion The procedure for preparing the nanocatalyst Fe3O4@GO-Pr-S/IL (1) is shown in Scheme 1. Bronsted’s acidic ionic liquid [(CH2)3SO3HVIm]HSO4 synthesized by the reaction of 1-vinylimidazole with 1,4-butanesultone followed by treatment with sulfuric acid. Subsequently, a Fe3O4@GO-nanocomposite was prepared and function- alized with 3-mercaptopropyltrimethoxysilane (MPTMS) via covalent bonds to prepare Fe3O4@GO-Pr-SH. Finally, Fe3O4@GO-Pr-S/IL nanocatalyst 1 was obtained by the re- action of [(CH2)3SO3HVIm]HSO4 and Fe3O4@GO-Pr-SH in the presence of azobisisobutyronitrile (AIBN) in tolu- ene under N2 atmosphere. After successful synthesis of Fe3O4@GO-Pr-S/IL, the structure of this new nanocatalyst was characterized by XRD, EDS, FT-IR, VSM, SEM and TGA techniques. Scheme 1. Preparation of Fe3O4@GO-Pr-S/IL (1). Figure 1. XRD patterns of a) GO, b) Fe3O4, and c) Fe3O4@GO-Pr-S/ IL. 841Acta Chim. Slov. 2022, 69, 837–847 Jahanbakhshi et al.: Ionic Liquid Supported on Magnetic Graphene Oxide ... Figure 1 shows the X-ray diffraction spectrosco- py patterns of GO, Fe3O4, and Fe3O4@GO-Pr-S/IL. The peaks at 2θ = 10.5° and 43.5° could correspond to the (01) and (101) layers of GO, respectively.62,63 In Figure 1b, the peaks at 30.26°, 35.7°, 43.5°, 53.59°, 57.5°, and 63.26° relate to (220), (311), (400), (422), (511), and (440) free Fe3O4, respectively, which agrees well with the pattern of Fe3O4.64,65 After grafting various functional groups onto GO (Figure 1c), the 001 diffraction peak of GO complete- ly disappeared, indicating complete exfoliation of GO and preventing the aggregation of GO after partial reduction. Moreover, the Fe3O4@GO-Pr-S/IL nanocatalyst shows characteristic peaks at 2θ, which are consistent with the bare peaks of Fe3O4. Elemental analysis of Fe3O4@GO-Pr-S/IL was per- formed by EDX analysis (Figure 2). The EDX spectrum proves the presence of the common elements C, O, Fe, N, Si and S in the catalyst structure as shown. Fig. 2. EDS diagram of Fe3O4@GO-Pr-S/IL. In Figure 3a, three absorption peaks at about 645, 1050, and 1135 cm–1 are associated with the vibrations of the S-O, SO2, and C-S bands, respectively. In addition, the characteristic peaks of the imidazolium ring at 1563 cm–1 were observed (Figure 3a).66–68 The FT-IR spectra of GO are shown in Figure 3b. The peak at 1227 cm–1 is assigned to the stretching vibration of C-O of the epox- ide group, and the peak of C=O of the carboxyl and car- bonyl groups is at 1724 cm–1 (Figure 3b).69,70 The strong peak in all spectra at 3400 cm–1 corresponds to the OH group (Figure 3b-f). In Fe3O4@GO-Pr-S/IL this peak (OH group) is much more pronounced than in Fe3O4@ GO-Pr-SH due to the sulfonic acid groups. Moreover, the typical peak at 580 cm–1 in all spectra (Figure 3c-f) can be attributed to Fe-O stretching.71 In addition, sym- metric and asymmetric vibrations of the Si-O bond oc- cur at about 973 and 1140 cm–1, confirming the presence of MPTMS (Figure 3d).72 The presence of all these peaks indicates the successful association of the Bronsted ion- ic acid liquid with the Fe3O4@GO-Pr-SH via a radical reaction. Figure 3. FT-IR spectra of a) IL, b) GO, c) Fe3O4, d) Fe3O4@GO, e) Fe3O4@GO-Pr-SH, and f) Fe3O4@GO-Pr-S/IL. The magnetic property of Fe3O4 and Fe3O4@GO- Pr-S/IL nanocatalysts was determined by VSM at room temperature (Figure 4). The curves in Figure 4 show that the saturation magnetization values of Fe3O4@GO-Pr-S/ IL (20.55 emu/g) are lower than those of pure Fe3O4 na- noparticles (53.03 emu/g). The decrease in saturation magnetization of Fe3O4@GO-Pr-S/IL nanocatalyst might be related to the functionalized GO and the presence of an acid group on the surface of the ionic liquid associated with the support. Figure 4. VSM analysis of a) Fe3O4 and b) Fe3O4@GO-Pr-S/IL. The morphology and size of the nanocatalysts GO and Fe3O4@GO-Pr-S/IL were studied using SEM (Figure 5). GO has a 2D structure with a wrinkled edge and a large 842 Acta Chim. Slov. 2022, 69, 837–847 Jahanbakhshi et al.: Ionic Liquid Supported on Magnetic Graphene Oxide ... thickness (Figure 5a). However, after immobilization of the ionic liquid, the surface of GO is smooth (Figure 5b). According to this picture, the average particle size is 77–90 nm, and the surface of the nanocatalyst is almost uniform. The thermal stability of the Fe3O4@GO-Pr-S/IL na- nocatalyst was also investigated by thermogravimetric analysis (TGA) (Figure 6). The TGA curve of nanocatalyst 1 shows three stages of weight loss between 25 and 900 °C. In this curve, the first weight loss below 220 °C (18%) is due to desorption of chemisorbed and physically adsorbed solvents, organic solvents, and hydroxyl groups. The sec- ond weight loss occurs at about 220–550 °C (8%) and is due to decomposition of oxygenated groups into GO, or- ganic groups, acid groups, and amine groups. The final weight losses occur at 550–900 °C (10%). They may be due to the removal of these immobilized organic species on the surface of GO nanosheets and confirm the thermal stabil- ity of the prepared nanocatalyst. In the next step, Fe3O4@GO-Pr-S/IL was used as an effective nanocatalyst for the synthesis of triazolo[1,5-a] pyrimidine derivatives 5 (Scheme 2). Scheme 2. Synthesis of triazolo[1,5-a]pyrimidine derivatives 5 in the presence of nanocatalyst 1. Table 1 shows the effects of catalyst amount, tem- perature and different solvents on the reaction between benzaldehyde (1 mmol), ethyl cyanoacetate (1 mmol) and 3-amine-1H-1,2,4-triazole (1 mmol) as an example reac- Figure 5. SEM images of a) GO and b) Fe3O4@GO-Pr-S/IL. Figure 6. TGA curve of the Fe3O4@GO-Pr-S/IL nanocatalyst. 843Acta Chim. Slov. 2022, 69, 837–847 Jahanbakhshi et al.: Ionic Liquid Supported on Magnetic Graphene Oxide ... tion. The model reaction was carried out at different tem- peratures, and the best yield was obtained at 80 ºC. We optimized the amount of catalyst; the maximum yield of Table 1. The effect of catalyst loading, solvent and temperature in the synthesis of 5a.a Entry Catalyst 1 (g) Solvent Temp. (°C) Yield b (%) 1 0.002 – 25 62 2 0.002 – 60 70 3 0.002 – 80 80 4 0.001 – 80 65 5 0.004 – 80 95 6 0.006 – 80 90 7 0.008 – 80 90 8 0.004 EtOH 80 80 9 0.004 CH3CN 80 82 10 0.004 Toluene 80 70 11 0.004 H2O 80 65 12 0.004 H2O/EtOH (1:1) reflux 75 13 0.004 GO 80 37 14 0.004 Fe3O4 80 45 15 0.004 Fe3O4@GO 80 57 16 – – 80 trace a Reaction conditions: benzaldehyde (1 mmol), ethyl cyanoacetate (1 mmol), 3-amine-1H-1,2,4-triazole (1 mmol), time: 20 min. b Iso- lated yields. the product was obtained with 0.004 g of catalyst under solvent-free conditions. The yield of the product was high- est when the reaction proceeded under solvent-free con- ditions, and when EtOH, MeCN, toluene, H2O, and H2O/ EtOH (1:1) were used as solvents, the yield of the product decreased. When GO, Fe3O4 and Fe3O4@GO were used as catalysts under solvent-free conditions at 80 ºC, the product yield was much lower. The reaction did not pro- ceed without catalyst (Table 1, entry 16), indicating that the catalyst was necessary to promote the reaction. After optimizing the reaction conditions, the efficiency of the Fe3O4@GO-Pr-S/IL nanocatalyst was further tested in the synthesis of triazolo[1,5-a]pyrimidines using a series of aromatic aldehydes and malononitrile. The results are shown in Table 2, and it was found that almost all sub- strates gave excellent yields of the desired products in a short reaction time. A permissible mechanism for the three-component synthesis of triazolo[1,5-a]pyrimidines 5 based on previ- ous reports is shown in Scheme 3.73,74 First, the aldehyde and active methylene compounds (cyanoacetate or malo- nonitrile) are activated with the acidic surface of the cat- alyst, and a Knoevenagel condensation to intermediate I occurs. Subsequently, intermediate I reacts with 3-amine- 1H-1,2,4-triazole to give the intermediate II (via Michael addition). Finally, the desired product was synthesized af- ter intramolecular cyclization and tautomerization. Scheme 3. Proposed mechanism for the synthesis of 5 in the presence of Fe3O4@GO-Pr-S/IL as catalyst. 844 Acta Chim. Slov. 2022, 69, 837–847 Jahanbakhshi et al.: Ionic Liquid Supported on Magnetic Graphene Oxide ... Table 2. Synthesis of triazolo[1,5-a]pyrimidines in the presence of Fe3O4@GO-Pr-S/IL nanocatalyst.a Entry Aldehyde Product 5 Yield (%)b Mp (°C) 5a C6H4CHO 95 189–191c 5b 4-Cl C6H4CHO 97 190–19170 5c 4-Br C6H4CHO 93 184–18470 5d 4-NO2 C6H4CHO 95 194–19670 5e 3-NO2 C6H4CHO 90 190–192c 5f 2,4-Cl2 C6H3CHO 88 243–245c 5g 4-CH3 C6H4CHO 85 243–24571 5h 4-i-Pr C6H4CHO 84 218–220c 5i 4-NO2 C6H4CHO 90 245–24771 5j 2-Cl C6H4CHO 92 263–26671 5k 4-Br C6H4CHO 89 264–26671 5l 4-Cl C6H4CHO 93 257–25871 a Reaction conditions: aryl aldehyde (1 mmol), ethyl cyanoacetate or malononitrile (1 mmol), 3-amine-1H-1,2,4-tri- azole (1 mmol), catalyst 1 (0.004 g), solvent-free, 80 °C. b Isolated yields. c New compound. 845Acta Chim. Slov. 2022, 69, 837–847 Jahanbakhshi et al.: Ionic Liquid Supported on Magnetic Graphene Oxide ... A hot filtration test was also performed to study the leaching of the catalyst during the reaction. After about 50% of the process was complete, boiling EtOAc (5 ml) was added and the catalyst was separated with a magnet. The solvent was then removed and the reaction continued under optimized conditions. Nevertheless, no remarkable increase in product conversion was observed during the course of the reaction, indicating that the catalyst func- tioned as a heterogeneous method and the ionic liquid fraction was kept intact and efficient on the solid support. Moreover, the reusability of Fe3O4@GO-Pr-S/IL in the model reaction of benzaldehyde, ethyl cyanoacetate and 3-amine-1H-1,2,4-triazole was investigated under opti- mum conditions. After completion of the reaction, boil- ing EtOAc was added and the catalyst was separated by an external magnet, washed, and reused in subsequent runs. As shown in Figure 7, the synthesized catalyst can be re- covered and reused in at least 7 runs without significant decrease in its efficiency. In Figure 8, the FT-IR spectrum of the nanocatalyst after seven reaction cycles can be seen. This spectrum also confirms the stability of the structure of the recycled na- nocatalyst. The XRD pattern also indicates the structural stability of the catalyst after being reused (Figure 9). The position and relative intensities of all peaks confirm this well. 4. Conclusions In this work, Fe3O4@GO-Pr-S/IL was prepared as a new nanocatalyst and used for the synthesis of tri- azolo[1,5-a]pyrimidine derivatives under green condi- tions, and the products were obtained in high yield. This magnetic nanocatalyst was characterized by various tech- niques such as XRD, FT-IR, EDX, SEM, VSM and TGA. Fe3O4@GO-Pr-S/IL was used as an efficient catalyst with good durability, high selectivity and stability, mild reaction conditions and short reaction times. The heterogeneous Fe3O4@GO-Pr-S/IL catalyst was also successfully recov- ered seven times, maintaining its activity. 5. References 1. M. Tajbakhsh, H. 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Pripravljen heterogeni katalizator je okarakteriziran z XRD, FT-IR, EDX, SEM, VSM in TGA analiznimi tehnikami. Rezultati analiz kažejo, da se je [(CH2)3SO3HVIm]HSO4 uspešno vezal na površino Fe3O4@GO in da pripravljen katalizator kaže dobro termično stabilnost. Avtorji so aktivnost katalizatorja raziskali na primeru sinteze triazolo[1,5-a]pirimidinskih derivatov z enost- openjsko trikomponentno reakcijo aktivne metilenske spojine (malononitril ali etil cianoacetat), 3-amin-1H-1,2,4-tri- azola in aril aldehidov, brez uporabe topil. Katalizator je mogoče enostavno in hitro izločili iz reakcijske zmesi s pomočjo zunanjega magneta, ter do sedemkrat reciklirati, brez znatne izgube katalitske aktivnosti. 848 Acta Chim. Slov. 2022, 69, 848–862 Mustafa et al.: Hybrid Polymer Composite of Prussian Red Doped ... DOI: 10.17344/acsi.2022.7601 Scientific paper Hybrid Polymer Composite of Prussian Red Doped Polythiophene forAdsorptive Wastewater Treatment Application Mohd Mustafa,1 Shabnum Bashir,1 Syed Kazim Moosvi,2 Mohd. Hanief Najar,3 Mubashir Hussain Masoodi4, * and Masood Ahmad Rizvi1,* 1 Department of Chemistry, University of Kashmir, Hazratbal Srinagar, 190006, J&K, India. 2 Department of School Education, Government of Jammu and Kashmir, Srinagar, Jammu and Kashmir, India. 3 Department of Chemistry, Government College of Engineering & Technology, Safapora 193504, Jammu and Kashmir, India. 4 Department of Pharmaceutical Sciences, University of Kashmir, Hazratbal Srinagar, 190006, J&K, India. * Corresponding author: E-mail: masoodku2@gmail.com, mubashir@kashmiruniversity.ac.in Received: 08-22-2022 Abstract Coordination compounds as dopants to conducting polymers combine desirable properties of individual components for a synergistic effect. Prussian red (PR) a low spin iron (III) coordination compound was doped in polythiophene (PTP) matrix to explore propensity of this inorganic-organic hybrid composite material towards wastewater treatment. PR doping was observed to improve mechano, thermal, electrical, and photocatalytic attributes of pure PTP. PTP/PR composite characterization was attempted using the powder X-ray diffraction, TEM, TGA, FTIR, BET analysis and UV-Visible spectroscopy. Optimization of adsorption conditions, adsorbent regeneration, adsorption thermodynamics studies of PTP/PR were carried out using malachite green (MG) dye as a model system. Under optimized conditions 92% MG dye adsorption was observed over 20 mg PTP/PR nanocomposite in 20 minutes at pH 7. PTP/PR nanocomposite also demonstrated a complimentary performance with real wastewater samples. Thermodynamic studies indicate spon- taneous process with electrostatic attraction as the predominant noncovalent interaction. This study highlights designing catalysts capable of synergistic adsorption and photocatalytic activities for effective wastewater treatment. Keywords: Adsorption; Malachite Green; Prussian red; Hybrid Material, Nyquist plot; Kinetics; Isotherms 1. Introduction Water is the most significant substance for living be- ings, water scarcity is a global problem, and contamination further adds to it. Persistent contaminants from industrial effluents, domestic sewage, and agricultural practices make water unsafe to the aquatic ecosystem as well as hu- man life.1,2 Dyes as coloring agents have many applica- tions, limiting their usage is practically impossible, and their eventual disperse in the wastewater/environment is inevitable. Synthetic dyes bring toxicity risks as their inter- mediate metabolites have been identified to be mutagenic, teratogenic, or carcinogenic, posing serious health threats to ecosystems3 as well as aesthetic concerns. Malachite green is a triaryl methane cationic dye commonly used in pharmaceutics, paper, textile and printing industries. Treatment methods based on ozonation, nano-filtration, reverse osmosis, flocculation, electrochemical, photocata- lytic and advanced oxidation processes are in vogue for discoloration of colored water contaminants.4 Among wa- ter treatment methods such as advanced-oxidation, filtra- tion, flocculation, coagulation and microbial degrada- tion,5,6 adsorption based methods owing to their simplicity, non-invasive reactions, broader scale applicability, lower operational cost, good recyclability are interesting envi- ronmentally.7,8 Consequently, adsorption based methods are still desirable for safer and cost effective wastewater treatment application. 849Acta Chim. Slov. 2022, 69, 848–862 Mustafa et al.: Hybrid Polymer Composite of Prussian Red Doped ... Designing adsorbents with good adsorption capacity and selectivity is a major concern for effective adsorption processes. Natural adsorbents, although pleasing, have limitations of selectivity with lower adsorption capacity.9 Synthetic adsorbents can be desirably tuned for improved results under controlled conditions such as in sewage treatment plants. Among synthetic adsorbents, conduct- ing polymers (CP) having charge distribution over the en- tire polymeric surface offer many adsorption sites making these attractive materials towards adsorptive removal of water contaminants. CPs are significantly influenced in shape, porosity, charge separation etc. by doping with suit- able dopant. A well-engineered conducting polymer com- posite can desirably improve the adsorption capacity of the pristine conducting polymer.10 PR was selected as a dopant with the rationale of its good crystallinity, stability over en- vironmental pH range, high charge density, moderate par- amagnetism, redox behavior with relatively lower toxici- ty.11 Thus in continuation of our interests in applications of transition metal complexes,12–16 especially as photocata- lysts in organic synthesis,17,18 and as dopants in conduct- ing polymer matrix for electrical and environmental appli- cations,19–21 herein we envisaged the effect of Prussian red (PR) as a transition metal complex dopant to the polythio- phene conducting polymer matrix. We synthesized poly- thiophene (PTP) Prussian red (PR) nanocomposite PTP/ PR to envisage the effect of this versatile dopant on its pol- ymer properties. The effect of PR dopant on the polymer properties indicated an enhancement in the mechano, thermal, electrochemical and photocatalytic descriptors of pure PTP. The comparative studies of PR doping to PTP, relative to other organic dopants, revealed an increase in surface area, electrical conductivity, charge separation dy- namics, and electrochemical stability as desirable attrib- utes for sensing, photocatalytic activity, and device fabri- cation. The observed physicochemical properties of synthesized PTP/PR nanocomposite indicate its suitability as material for environmental applications, which was modelled using malachite green (MG) dye adsorption in aqueous phase. PTP/PR nanocomposite displayed signifi- cant adsorptive tendency towards decoloration of samples having up to 100 ppm of MG dye concentration under en- vironmentally viable conditions (pH 6–7). The effect of adsorbent dose, dye concentration, temperature, adsorp- tion time and pH effect were optimized for its develop- ment as adsorbent in real time water treatment plant appli- cation. The experimental data of MG dye adsorption over PTP/PR nanocomposite was also analyzed for parameters such as: kinetics, adsorption isotherms and thermody- namics. PTP/PR signified good propensity as hybrid mate- rial towards adsorptive handle of water treatment. The development of PTP/PR nanocomposite as an effective water treatment nanomaterial via synergistic adsorptive and photocatalytic elimination of MG dye was also estab- lished and its further extension to other persistent water contaminants is underway in our laboratory. 2. Experimental 2. 1. Materials All the reagents used in the study were of analytical grade and procured from Himedia and Merck India. Thio- phene was distilled prior to its use. Malachite green (MG), Potassium ferricyanide and anhydrous Ferric chloride were purchased from Merck India. Potassium ferricyanide was ground by ball milling for size reduction to nano di- mension (<10 nm). 2. 2. Synthesis of Nanocomposite The synthesis of PTP/PR nanocomposite was carried out via oxidative polymerization using FeCl3 as oxidant in a nonaqueous medium.21 In the optimized synthetic pro- cedure,180mL of 0.05 M FeCl3 solution in chloroform sol- vent were added (dropwise) to a magnetically stirred solu- tion of 0.022 M distilled thiophene monomer (in 70 mL of chloroform). The reaction mixture contained about 1 g of ground PR dopant (Scheme1). The polymerization reac- tion mixture was continuously stirred for 24 h, followed by filtering and washing the product several times with meth- anol to remove any oligomers and unreacted impurities. After repeated acetone washings and room temperature drying, the polymer composite was obtained as a brown powder which was subjected to structural characteriza- tion. 2. 3. Measurements The characterization of the synthesized composite was done using analytical methods and spectroscopic techniques. Infrared spectra were obtained over a Bruker Alpha FTIR spectrophotometer in range 4000–500 cm–1. The structural characterization of samples was performed by the powder X-ray diffraction (PXRD) method using a PW3050 diffractometer (CuKα radiations, λ = 1.5418 Å). The particle shape and size of PTP/PR nanocomposite was analyzed using transmission electron microscopy (TEM) measurements performed on Hitachi SU 8000 microscope at an accelerating voltage of 30 kV. For TEM studies the samples were prepared by spin-casting a THF solution of PTP/PR nanocomposite (3mg/L) on copper grids with carbon coating. The thermogravimetric analysis (TGA) of samples was performed on a SEIKO TG/DTA 6200 instru- ment. Thermal analysis was done from room temperature to 800°C at a heating rate of 10 °C min–1 under nitrogen environment. Adsorption measurements were performed using Double Beam Microprocessor UV-VIS Spectropho- tometer (Model:LI-2802). Zeta Potential (surface charge) of the adsorbent particles were determined using an An- ton Par Particle Size Analyser (Model:Litesizer 500). BET studies for surface area were attained at 77K using BET instrument, Quantachrome Austosorb IQ Station. The specific surface area and pore size distribution was calcu- 850 Acta Chim. Slov. 2022, 69, 848–862 Mustafa et al.: Hybrid Polymer Composite of Prussian Red Doped ... lated using a multiple point BET method and non-local density functional theory (NLDFT) equilibrium model. Electrochemical experiments were carried out on Bio-Log- ic SAS Potentiostat (Model SP 150) using three electrode system. 2. 4. Adsorption Experiments For adsorption studies, 20 mg of PTP/PR nanocom- posite was added to a 30mL solution of 10ppm MG dye and the suspension was magnetically stirred. Under the optimized reaction conditions, at the regular time inter- vals aliquots were taken, the adsorbent was separated via centrifugation at 1000 rpm for 20 min and the left-over concentration of dye in the supernatant was measured by a UV–visible spectrophotometer at λmax = 617 nm (corre- sponding to MG dye). The adsorptive capacity of PTP/PR hybrid material was optimized under influencing parame- ters like, adsorbent dose, contact time, initial MG dye con- centration and temperature range of 25–45 °C. The equa- tions 1, 2 were used to calculate adsorption capacity of PTP/PR nanocomposite and % dye removal efficiency.22 (1) (2) Where, C0 and Ct (mg/L) are the initial and final (af- ter adsorption) concentrations of MG dye solution respec- tively, m(g) is the weight of PTP/PR nanocomposite, and V (L) is the initial volume of dye solution. 2. 5. Adsorption Studies of M.G Dye in Real Water Samples To test the adsorption efficiency of PTH/PR in real samples, the analysis was performed on collected industri- al effluent from local textile dye shop (Hazratbal, Srina- gar). The dye sample of (1 mg/mL) concentration was pre- pared in 100 mL deionised water to which required quantities of fixing agent and mordant were also added. To 30 mL of this prepared dye solution 20 mg of PTP/PR composite was added, samples were magnetically stirred and subjected for left over MG dye estimation over differ- ent time intervals using UV-visible spectrophotometry. Furthermore, analysis of MG dye adsorption propensity of samples prepared in different water samples (Tap water and deionised water) was also explored under optimized conditions for comparative studies. 2. 6. Cyclic Voltammetry Experiments The cyclic voltammetry experiments were carried out using three electrode system with Ag/AgCl as reference, Scheme 1. Schematic depiction of PTP/PR nanocomposite synthesis via FeCl3 oxidative polymerization. 851Acta Chim. Slov. 2022, 69, 848–862 Mustafa et al.: Hybrid Polymer Composite of Prussian Red Doped ... glassy carbon as working, and platinum wire as a counter electrode with 0.1M KNO3 as supporting electrolyte. The solid samples of PTP/PR and PR each were dispersed in 0.5mL ethanol solution to which 15 μL of nafion was added as binder. The suspensions were kept on ultrasonication for about 30 min to make the slurries of each suspension. The obtained slurries were drop-casted onto glassy carbon elec- trodes and left for open air drying for 40 min. 3. Results and Discussion A well-planned dopant to the polymer matrix can generate composites with unique properties for aimed ap- plications. PR turned out to be one such interesting dopant to the polythiophene matrix in terms of modulating essen- tial parameters of a polymer composite system for potential applications. These included enhancement in the mechano, thermal, electrochemical, and photocatalytic attributes of pure PTP. The comparative studies of PR doping to PTP relative to organic dopants revealed an increased surface area, conductivity, and electrochemical stability desirable for electrochemical sensing and device fabrication. The shifting of band gap towards visible region and prevention of charge carrier recombination after PR doping were im- portant attributes for its development as photocatalyst. The synergistic effect of adsorption and photocatalysis was also explored for effective degradation of MG dye. 3. 1. Characterization Analysis 3. 1. 1. IR Spectral Analysis The FT-IR spectra of synthesized composite were compared with pure reactant forms and are as shown in Figure 1. The broad band around 1630 cm–1 and 3300– 3400 cm–1 is attributed to O-H stretching vibration of ad- sorbed water on PTP surface, and the range of 600–1500 cm–1 reflects the fingerprint region of PTP.23,24 Further- more, the C-S stretching vibration of PTP and C-H out of plane deformation modes are designated by weak absorp- tion bands around 690–1300 cm–1. From the Figure 1, the black line corresponding to pure PR shows a specific ab- sorption band around 2100 cm–1 that designates CN stretching and the peak at around 600 cm–1 is due to Fe- CN vibrations. Moreover, the PTP/PR nanocomposite spectrum exhibits peaks from both the constituents (green line). The intensity of pure PR vibrations is shifted and ob- served at 2078 cm–1 and 497 cm–1 in the nanocomposite FT-IR spectrum. Thus, from the peak pattern analysis (shifting of the major stretching frequencies of PTP and PR) the inclusion of PR nanoparticles particles in PTP ma- trix can be expected. 3. 1. 2. Thermal Analysis Thermal stability is an essential parameter for mate- rial applications, and thermo-gravimetric analysis (TGA) is used to examine this important property via % weight change over the programmed heating. TGA curves of pure PTP and PTP/PR nanocomposite are shown in Figure 2. It is evident from TGA plots that incorporation of PR into PTP matrix has enhanced its thermal stability noticeably. The 33% enhancement in thermal stability of PTP/PR composite compared to pristine PTP at higher tempera- ture indicates stronger interaction between PR dopant and PTP matrix possibly via noncovalent supramolecular type forces. The observed initial weight loss at a lower tempera- ture in case of PTP/PR composite can be attributed to the evaporation of the solvent and clinging low molecular mass volatile compounds. Furthermore, the improved mechano strength of PTP post PR doping is evident from the less sharp slope of % weight loss with temperature in the PTP/PR TGA plot. At 600oC the amount of weight loss in the case of PTP/PR composite is only 60% compared to almost 100% for pristine PTP. Thus it can be inferred from the thermograms that PR doping introduces interactions between dopant and polymer matrix which modulates the mechanical strength of pristine PTP polymer leading to its enhanced thermal stability. A comparative analysis of the dopant effect on the thermal stability of PTP composites with varied dopants indicates the improved thermal stabil- ity in the presence of PR dopant.25,26 The changes in pure PTP structure and function post PR doping were also ana- lyzed using PXRD, Microscopy, and BET analysis. Figure 1. PTP/PR composite characterization (from selected peak picking) using FTIR spectra. 3. 1. 3. XRD Analysis X-ray Diffraction patterns of PTP and PTP/PR na- nocomposite are shown in Figure 3a. Evidently, PTP shows an amorphous halo around Bragg angles of 20–30°, thus exhibiting its amorphous nature.27 However, the X-ray dif- fraction pattern of PTP/PR nanocomposite clearly re- vealed strong intense peaks at various Bragg angles, illus- 852 Acta Chim. Slov. 2022, 69, 848–862 Mustafa et al.: Hybrid Polymer Composite of Prussian Red Doped ... trating its crystalline nature. The crystallinity developed in nanocomposite can be attributed to the incorporation of PR in PTP matrix. This corroborates the successful synthe- sis of prepared nanocomposite, which is also evidenced from TEM micrographs. This is expected as polymers, be- ing larger molecules, are capable of stronger Van der- Waals and other types of possible non-covalent interac- tions with dopant molecules. These polymer-dopant supra interactions lead to a fair degree of order and compactness in the nanocomposite material.24 The characteristic peaks were indexed using Powder X software which revealed monoclinic structure with FCC lattice. For crystallite size determination, Scherrer equation (equation 3) has been used.28–29 (3) Where D is the average crystallite size, k the shape factor (0.94), λ the wavelength used (Cu Kα = 1.54 Å), β the FWHM (full width at half maximum) and θ is the Bragg angle. The average crystallite size as determined from above equation has been found to be 7.94 nm. This is almost similar to that obtained from TEM micrographs (8.2 nm), which is indicative of the fact that PR nanoparti- cle might represent a single grain or crystallite. Thus XRD analysis corroborate with the FT-IR and thermal analysis results. Figure 2. Thermogravimetric analysis plots showing PR enhanced thermal stability of PTP polymer. 3. 1. 4. TEM Analysis The TEM image of PTP/PR nanocomposite is shown in Figure 3b. The image clearly reveals spherical/granular shape of PR nanoparticles which are dispersed in PTP ma- trix indicating the formation of nanocomposite. PR nano- particles are seen to are dispersed in PTP matrix in discrete (inset-1) as well as agglomerated units depending upon their particle size. The agglomeration might result owing to the Van der Waals interaction of smaller particles for bigger sized particles. The particle size distribution of these nano- particles has been carried out by Image J software followed by Gaussian fitting that revealed the average particle size of around 8.2 nm (inset-2), revealing a mesoporous character. From TEM results, it is clear that PTP/PR is a nanocom- posite. Thus, owing to the presence of small sized particles, the nanocomposite is expected to show enhanced surface area, which is confirmed from BET analysis. 3. 1. 5 Structural and Textural Characterization Adsorption propensity is greatly influenced by the sur- face area and pore size of the catalyst, BET analysis was at- tempted to determine surface characteristics of the synthe- Figure 3. X-ray diffraction pattern of PTP & PTP/PR nanocompos- ite (A). TEM image of PTP/PR nanocomposite (B) spherical shaped PR nanoparticles of PTP/PR nanocomposite (inset-1). Gaussian histogram fitting plot for particle size distribution (inset-2) 853Acta Chim. Slov. 2022, 69, 848–862 Mustafa et al.: Hybrid Polymer Composite of Prussian Red Doped ... sized PTP/PR nanocomposite. The N2 adsorption-desorption isotherms and pore diameter distributions of PTP/PR nano- composite, PR, pure PTP are as shown in Figure 4. The meas- ured surface area for PTP/PR nanocomposite was found to be 81.018 m2 g− 1, which is higher than both PTP (25.560 m2/g) and PR (15.453 m2/g) respectively. Besides, the N2 ad- sorption/desorption isotherm portrays combined type-II and IV isotherm behavior which is typical of mesoporous structure based on the IUPAC classification.30,31 Pure PTP has a granular texture with lesser inter-particle distance, which is evident from its lower porosity and smaller surface area. However, on addition of PR, the inter particle space gets increased, which is evident from increased porosity, in case of PTP/PR nanocomposite. The observed increase in poros- ity can be the reason for the enhanced surface area of PTP/ PR nanocomposite compared to undoped PTP.32 The major pore size distribution of the PTP/PR nanocomposite (2.769 nm) is within the range of 1–3nm, which is a characteristic of smaller pore volume systems. Thus, mesoporous structure with enhanced surface area gives PTP/PR nanocomposite a good adsorptive capacity desirable for an adsorption-based wastewater treatment application. The good surface area and visible region bandgap also makes PTP/PR a possible photo- catalyst. The comparative analysis of PTP/PR nanocompos- ite with other closely related PTP nanocomposite systems indicates a significant increase in surface area for adsorption in case of PTP/PR Table 1. Table 1. Comparative effect of dopants on the surface area of PTP composites PTP Composite Surface area Reference (m 2 /g) PTP/Fe(CN) 3 (NO)(bpy).4H 2 O 18.9 33 PTP /Fe 3 O 4 19.4 27 PTP/Cu 20.88 34 PTP/CuFe2O4 30.9 35 PTP/PR 81.018 Present work 3. 2. Modeling Adsorptive Propensity of PTP/ PR Towards Industrial Dye Malachite Green (MG) The higher surface area coupled with negative zeta po- tential value around pH 7 range indicates selectivity of PTP/ PR nanocomposite towards cationic water contaminants. With this rationale, we used Malachite green as a model cat- ionic dye for exploring the adsorptive propensity of PTP/PR nanocomposite under environmentally viable conditions. 3. 2. 1. Effects of Contact Time and Initial MG Solution Concentration The effects of initial MG dye concentration and its contact time on PTP/PR nanocomposite adsorption ca- pacity were observed around 20οC ± 2οC under fixed pH and MG dye concentrations from 10 to 30 mg/L. The ad- sorption capacity of MG dye on PTP/PR nanocomposite under different initial concentrations with contact time is shown in Figure S1a. At lower dye concentrations, the re- moval rate was relatively fast and equilibrium was reached within 20 min. which shifted to 25 min. at higher concen- trations. The adsorption rate was high for first 20 min. and then progressively saturated with increasing contact time (after 60 min). This observation can be corroborated with the progressive saturation of the available surface sites with increasing contact time. Since many of the adsorption sites get occupied initially and as process proceeds the number of vacant sites gets decreased and occupying re- maining sites becomes difficult to occupy due to repulsive forces between the adsorbate molecules on the surface and bulk phases.22 The effect of initial concentration of MG dye on equilibrium adsorption capacity is shown in Figure S1b. From Figure S1b it can be seen that the equilibrium capacities of MG dye under different initial concentrations showed an increasing trend from 14.187 to 34.5 mg/g sig- nifying that MG dye concentration gradient offers a stronger driving force to reduce the mass transfer resist- ance of the MG dye. The removal efficiency of MG by PTP/ PR displayed relatively higher % removal at lower concen- trations with decreasing trend towards high concentra- tions. This can be corroborated with the fixed adsorption capacity of the 20 mg of PTP/PR sample. Thus from ad- sorption capacities and removal efficiency studies, 10mg/L was observed as optimized concentration of MG dye in the subsequent experiments. 3. 2. 2. Effect of Adsorbent Dosage The effect of PTP/PR nanocomposite dosage on ad- sorption properties was studied by adding its increasing amounts (10 to 30 mg) to 30mL of 10 mg/L MG dye solu- tion at 298 K with stirring for 160 min. with the corre- sponding results presented in Figure S2. The removal effi- ciency of MG dye increases from 93.8 to 95.94% on increasing adsorbent dosage from 10 to 30 mg. This behav- ior can be attributed to the fact that there are more acces- sible active adsorption sites for MG dye over increasing adsorbent dosage, which increases its % removal efficien- cy. However, the adsorption capacity reduces from 28.2 to 9.59 mg/g with increasing PTP/PR dose which can be at- tributed to the aggregate formation at high concentra- tion.36 The aggregation of adsorbent particles at their high- er concentrations was supported by Dynamic Light Scattering technique. DLS data indicates that with increase in adsorbent dosage, particle diameter increases and is shown in Figure S3. The aggregated particles at higher ad- sorbent dosage create diffusion resistance for MG dye to- wards the adsorbent surface, thereby decreasing the ad- sorption capacity. Thus from adsorbent dosage studies of 10 mg to 30 mg PTP/PR, 20 mg gives 94.58% removal effi- 854 Acta Chim. Slov. 2022, 69, 848–862 Mustafa et al.: Hybrid Polymer Composite of Prussian Red Doped ... sorbed at any time t (min) and at equilibrium, respectively; k1 (min–1) and k2 (g/mg/min) are the rate constants of pseudo first order and pseudo-second-order kinetic mod- els respectively. The kinetic parameter counting correla- tion coefficients (R2), k1, k2 and qe (cal) values were deter- mined by linear regression. The ‘α’ in the equation 6 denotes initial adsorption rate (mg/g/min) and ‘β’ is the desorption constant (g/mg). From the plot of qt vs lnt, Elovich constants (α, β) were determined from slope and intercept values and the values are presented in Table 2. It is seen from table 2, that the R2 value (0.9999) for the pseu- do-second-order kinetic model was much higher than that of pseudo first-order as well as Elovich kinetic models. In addition, the values of qe,cal calculated under pseudo-sec- ond-order model were found close to the experimental qe,exp. Based on these observations, the pseudo-second-or- der kinetic model can be predicted to be the suitable to quantify the adsorption kinetics of MG onto PTP/PR na- nocomposite. As is evident from calculated descriptors in table 2, the experimental data do not have an acceptable fitting to pseudo first order and Elovich models. The pseu- do second order kinetic model better describes adsorption behavior of MG onto PTP/PR nanocomposite. The pseudo second-order kinetic model broadly involves adsorption, including external film diffusion, intraparticle diffusion and surface adsorption.22 Typically, intraparticle diffusion is considered as a possible rate-limiting step for a batch ciency under an adsorption capacity of 14.2 mg/g. Further increase in PTP/PR dose was not observed to significantly increase the removal efficiency. Therefore, from the adsor- bent dose studies 20 mg was observed as optimum dose for subsequent experiments. 3. 3. Adsorption Kinetic Analysis Adsorption as a physicochemical process involves the mass transfer of a solute from bulk liquid to the adsor- bent surface. Kinetic investigations of such a transfer pave a significant understanding of the adsorption parameters. The kinetics of MG dye adsorption on the PTP/PR nano- composite was explored by using three kinetic models: pseudo-first-order, pseudo second-order and Elovich models Figure 5 A-C. These kinetic models can be stated in their linear form as equations (4–6). 37,38 (4) (5) (6) where, qt (mg/g) and qe (mg/g) are the amounts of MG ad- Figure 4. N2 adsorption-desorption isotherms of PR, PTP and PTP/PR nanocomposite. Pore size distribution curves, Effect of PR wt% on the sur- face area, pore volume of PTP/PR 855Acta Chim. Slov. 2022, 69, 848–862 Mustafa et al.: Hybrid Polymer Composite of Prussian Red Doped ... reaction. To envisage the rate-limiting step of the MG dye adsorption process on PTP/PR nanocomposite, the proba- bility of intraparticle diffusion was tested by using Weber– Morris equation 7.38 (7) Here, Kid (mg/g/min–1/2) is the intraparticle diffu- sion rate constant, C (mg/g) is the intercept related to the thickness of the boundary layer. Figure 5D displays the plot of qt against t0.5, and the corresponding kinetic pa- rameters are listed in table S1. It is seen that the regression of qt versus t0.5 was prompted to be linear, and the plots did not pass through the origin, interpreting that besides in- traparticle diffusion, film diffusion may also be involved in the rate-controlling step. The Figure 5D shows two stages, an initial stage showing a faster adsorption rate of MG dye from solution, and flat portion depicting decreased ad- sorption rate due to unavailability of active sites.39,40 The smaller slope of intra-particle stage than that of the film diffusion stage illustrates lower adsorption is occurring at intra-particle stage.41 Besides the more significant inter- cept of the second segment, which gives the thickness of the boundary layer indicates that surface adsorption is also involved in the rate limiting step.42 Thus, adsorption of MG dye over PTP/PR nanocomposite involves film disper- sion and intraparticle diffusion as possible mechanistic Figure 5. Kinetic models for data fitting of MG (10, 15, 20, 25 mg/L) adsorption onto PTP/PR adsorbent (20mg in 30mL solution at 298K): (A) Pseudo-first order (B) Pseudo-second order (C) Elovich kinetic models (D) Intraparticle diffusion model processes, with the intraparticle diffusion as a dominant adsorption mechanism. 3. 4. Adsorption Isotherms In general, adsorption isotherm marks the relation- ship between the amount of adsorbate adsorbed per unit mass of an adsorbent at a given temperature. Adsorption isotherms are essential descriptors for analyzing and de- veloping any adsorption system.43 The equilibrium ad- sorption isotherm unfolds the interaction between the ad- sorbate and adsorbent. In view of this, MG, PTP/PR system was tested for Langmuir, Freundlich, Temkin iso- therm models Figure 6. The Langmuir isotherm model considers monolayer adsorption with all the sorption sites identical and energetically equivalent.44 The Freundlich isotherm is based on multilayer adsorption on the hetero- geneous surface, and there are interactions between the adsorbed molecules.45Temkin model considers adsorption on heterogeneous surfaces, effects of indirect adsorbate/ adsorbent interactions and assumes heat of adsorption of all molecules decreases linearly with an increase in surface coverage.44 The linear forms of the three isotherms are giv- en by the following equations (8–10). (8) 856 Acta Chim. Slov. 2022, 69, 848–862 Mustafa et al.: Hybrid Polymer Composite of Prussian Red Doped ... RL indicated the type of the isotherm to be either irrevers- ible (RL=0), favorable (0 < RL< 1), unfavorable (RL> 1) or linear (RL = 1). The calculated value of RL was found in the range of 0.01 – 0.1 in this study,48 indicating the adsorp- tion of MG on PTP/ PR is favorable, and as such, it can be a good adsorbent material for MG removal from aqueous solution. In addition, the calculated R2 value of 0.994, for the Freundlich isotherm model signifies a good degree of fit and the heterogeneity factor (n) was calculated to be 1.304. This also confirms a conducive adsorption process- es as a reaction is classified as favorable if 1 < n < 10.49 The fitting of observed adsorption data with regression values of R2 > 0.99 to both Langmuir and Freundlich isotherms models indicate that adsorption of MG dye over PTP/PR nanocomposite includes combined physical as well as chemical adsorption with monolayer/multilayer adsorp- tion.50,51 The possible forces involved in adsorbate adsor- bent interaction include π-π stacking, hydrogen-bond in- teraction, and electrostatic interaction. 3. 5. Effect of Temperature To assess effect of temperature on adsorption capac- ity of PTP/PR nanocomposite for MG, batch adsorption experiments in the temperature range of 25–45 °C were carried out. The adsorption propensity of the PTP/PR na- nocomposite improved slightly with increase of system temperature52,53 (Figure 7A). This can be attributed to in- creased thermal motion of MG molecules at higher tem- perature allowing more MG molecules to interact with adsorption sites on PTP/PR nanocomposite. Increasing temperature also decreases viscosity and enhances diffu- sion of MG dye molecules due to adsorbent surface.54 An important observation to note was that adsorption capaci- ties of PTP/PR for MG dye remained at relatively high val- ue of above 14.5 mg/g over the entire investigated temper- ature range, specifying its ability towards MG dye removal under varied temperature conditions. Here, qe (mg/g) is the amount of MG adsorbed at equilibrium time, Ce (mg/L) is equilibrium concentration, qm (mg/g) is the maximum adsorption capacity and KL is the Langmuir constant. The parameters of Langmuir mod- el can be calculated from the slope and intercept of the lin- ear plot 1/qe vs 1/Ce. (9) KF and n are Freundlich constants (10) Where β = RT/bT, bT (kJ/mol) is Temkin constant gives heat of adsorption, T is the absolute temperature (K), R is the universal gas constant (equal to 8.314 J/mol), and AT is equilibrium binding constant (L/g). The value of Temkin constant indicates the possible mechanism (phys- ical or chemical adsorption) on the adsorbent surface. If bT > 40 kJ/mol, chemisorption occurs and if bT < 40 kJ/ mol, physisorption proceeds.46 The quantities KF, KL, n, q0, b and qt were calculated, and the values are summarized in Table 3. The correlation coefficient for Langmuir isotherm has been found to be higher (0.997) and the adsorption capacity calculated from Langmuir model (34.48 mg/g) is found to be close to the experimental value of 34.5 mg/g. This suggests that the Langmuir isotherm could well ex- plain the adsorption characteristic of PTP/PR nanocom- posite. In addition, the separation factor or equilibrium parameter (RL) of Langmuir adsorption isotherm, which evaluates the feasibility of adsorption on adsorbent was calculated by equation (11). 47 (11) where, KL was the Langmuir equilibrium constant and C0 (mg/L) was the initial MG dye concentration. The value of Table 2. Kinetic models for the adsorption of MG on PTP/PR nanocomposite at 298 K Pseudo-1st-order Pseudo-2nd-order Elovich model C0 qeexp k1 qecal R2 k2 qecal R2 α β R2 (mg/L) (mg/g) (min–1) (mg/g) (g /mg/min) (mg/g) (mg/g/min) (g/mg) 10 13.92 0.05 1.042 0.50 0.18 14.14 0.99 7.787 0.368 0.821 15 20.81 0.046 1.218 0.51 0.22 21.28 0.99 22.606 0.248 0.813 20 27.45 0.045 17.95 0.78 0.06 28.17 0.99 51.779 0.185 0.831 25 34.15 0.031 6.184 0.86 0.116 34.5 0.99 164.130 0.151 0.814 Table 3. Isotherm parameters for the adsorption of MG onto PTP/PR nanocomposite at 298 K Langmuir Freundlich Temkin KL(L/mg) qm(mg/g) RL R2 Kf(L/g) n R2 bT (kJ/mol) AT R2 1.14 34.48 0.08 0.997 18 1.304 0.994 0.145 12.5 0.98 857Acta Chim. Slov. 2022, 69, 848–862 Mustafa et al.: Hybrid Polymer Composite of Prussian Red Doped ... ∆S (54.95 J/mol K) means that the randomness increased at the solid–liquid interface during the adsorption of MG dye in the aqueous solution on the PTP/PR nanocompos- ite. 3. 7. Effect of pH The pH is a vital parameter to consider adsorptive propensity as it influences surface charge of the adsorbent, degree of ionization of different adsorbates, protonation/ deprotonation of functional groups at the adsorbent active sites as well as adsorbate. Moreover, for a significant real time application the adsorption shall occur in the environ- mentally viable pH range.55 Accordingly, pH effect for MG dye adsorption on PTP/PR nanocomposite was studied. Figure 8A depicts comparative pH influence towards MG dye adsorption on PTP/PR nanocomposite. Noticeably, adsorption capacities of PTP/PR nanocomposite for MG were relatively higher at pH 7 (14.187 mg/g) and progres- sively decreased from pH 7 to 4 from 14.187 to 13.53 mg/g. The higher adsorption of MG dye by PTP/PR nanocom- Figure 6. Adsorption isotherms of MG on PTP/PR nanocomposite; (A) Langmuir (B) Freundlich (C) Temkin. Figure 7 (A) Temperature effect on MG (10 mg/L, 30 mL) adsorption on 20 mg PTP/PR composite (B) Van’t Hoff plot 3. 6. Thermodynamic Analysis The thermodynamic parameters (change in Gibbs free energy (∆G), enthalpy (∆H) and entropy (∆S)) were assessed from the effect of temperature on the adsorption of MG dye onto PTP/PR using the equations (12,13) (12) (13) where, KL (L/mol) is the Langmuir equilibrium constant; T(K) is system temperature and R (8.314 J/(mol K)) is the molar gas constant. The negative values of ∆G calculated using equation 12 inferred that adsorption of MG onto PTP/PR nanocomposite is thermodynamically feasible. ∆H and ∆S calculated from slope and intercept of the van’t Hoff plot log(KL) versus 1/T shown in Figure 7B. The cal- culated thermodynamic parameters of MG dye onto PTP/ PR are presented in Table 4. Furthermore, the positive val- ue of ∆H (9.03 kJ/mol) specified that the adsorption pro- cess is endothermic in nature. Also, the positive value of 858 Acta Chim. Slov. 2022, 69, 848–862 Mustafa et al.: Hybrid Polymer Composite of Prussian Red Doped ... posite from the neutral solutions was an encouraging re- sult from the wastewater treatment point of view. Under acidic pH, the surface of the PTP/PR gets positively charged due to protonation of sulphur atom of the thio- phene units in the polymer matrix,56 thereby repelling the cationic MG dye from polymer matrix. For pH lower than 7.0, MG dye gradually takes positive charge due to proto- nation on its nitrogen atoms57 which decrease adsorption capacities with decreasing solution pH. Around pH 7 de- protonation of sulphur atoms of polymer matrix is com- plete which attracts cationic MG dye towards PTP/PR sur- face leading to increased adsorption capacity at environmentally viable neutral pH. The pH effect results were supported by zeta potential studies of PTP/PR nano- composite over a pH scan Figure 8B. From the zeta poten- tial studies, the pH corresponding to point of zero charge (pHpzc) of pristine PTP58 and PTP/PR nanocomposite were calculated to be 4.3 and 3.79 respectively. PTP/PR na- nocomposite has a point of zero charge (pHpzc) at pH of 3.79 which also marks its transient pH. Above this pH its surface is negatively charged and below it positively charged. The lowered value of pHpzc corroborates with the presence of negative surface charge in case of PTP/PR nanocomposite above pH 3.8. In pH range 0–3 surface of the nanocomposite is expected to have positive zeta poten- tial, which progressively changes with increasing pH and acquires a maximum negative zeta potential value around pH 7 Figure 8B. From zeta potential studies repulsive in- teraction at lower pH as well as attractive interaction near neutral pH between cationic MG dye and adsorbent sur- face can hence be confirmed. The negative surface charge under the environmental pH range of 6 to 8 makes, PTP/ PR nanocomposite an excellent adsorbent for cationic contaminants in wastewater. The adsorptive propensity of PTP/PR nanocomposite for cationic MG dye prepared in tap, deionized waters and also with anionic dyes was at- tempted for comparative analysis. Although pH and zeta potential studies indicate dominance of electrostatic inter- action for MG dye adsorption onto PTP/PR nanocompos- ite but the possibility of other interactional forces such as pi–pi stacking and van der Waals type interactions cannot be ruled out. Table 4. Thermodynamic parameters for adsorption of MG (10 mg/L, 30 mL) onto (20 mg/L) PTP/PR nanocomposite. T (K) ΔG° (kJ/mol) ΔH° (kJ/mol) ΔS° (J/mol K) 298 –3.194 9.03 54.95 308 –3.446 9.03 54.95 318 –3.663 9.03 54.95 3. 8. Adsorption Mechanism IR Spectrum To gain some insights about the adsorption mecha- nism, FTIR spectral analysis of PTP/PR nanocomposite before and after MG dye adsorption was carried out and are as shown in Fig. S4. The FTIR bands of PTP/PR nano- composite at 490–498 cm–1 corresponding to –C-S-C- ring deformation are decreased in intensity and the absorption bands corresponding to –C-H-, -C-C-, O-H stretch get slightly shifted to lower wave numbers post adsorption with MG dye. These changes indicate role of sulphur site -S- in MG dye adsorption process. A prominent absorp- tion band at 2090 cm–1 characteristic of -C-N- stretching frequency due to PR particles also gets decreased in inten- sity after MG dye adsorption indicates ionic type interac- tion with -C-N-group. Furthermore, the red shift in ab- sorption frequencies can be attributed to electrostatic attraction resulting from lone pair on -S- and cationic MG dye. In addition, pi-pi stacking interaction could be possi- Figure 8. (A) Effects of solution pH on adsorption of MG (10 mg/L, 30 mL) onto the 20 mg of PTP/PR nanocomposite at 298 K.(B) Zeta potential studies of PTP/PR over pH range of 2-12. 859Acta Chim. Slov. 2022, 69, 848–862 Mustafa et al.: Hybrid Polymer Composite of Prussian Red Doped ... ble between aromatic rings of MG dye and polythiophene matrix. The possible adsorption mechanism and probable interactions are shown in Figure S5. 3. 9 Adsorption Studies of MG Dye in Real Time Samples The real time efficiency of PTP/PR nanocomposite was examined via dye removal efficiency in textile indus- try wastewater samples. The good adsorptive removal of MG dye from textile industry effluents offers the possibili- ty of using PTP/PR nanocomposite in environmental wastewater treatment application.59 The efficacy of PTP/ PR nanocomposite towards MG dye removal in samples, prepared in normal tap water and deionized water depict- ed more or less similar % adsorption under optimized conditions Figure 9A. The viability of selective adsorption using PTP/PR were studied by taking Rhodamine as cati- onic dye, Congo red and Methyl orange as anionic dyes. It was found that the synthesized PTP/PR exhibited higher adsorption capacity towards cationic dyes than anionic dyes Figure 9B. This can be ascribed to the negative surface charge on the PTP/PR nanocomposite under an environ- mentally viable pH range. Figure 9. Adsorptive efficiency of PTP/PR towards: (A) real time samples (B) representative dyes from different dye classes. 4. Adsorbent Recovery and Recycling Adsorption process being a benign and non-invasive method for the widespread wastewater treatment applica- tions is often limited by adsorbent recovery and regenera- tion post treatment. Regarding regeneration and reusabil- ity of adsorbent post treatment, recovery is one of the crucial parameters, magnetic separation is a very helpful method to recover magnetically active adsorbents for an improved economic feasibility. PTP/PR nanocomposite has PR as a magnetic core in the adsorbent nanocompos- ite, bringing the chances of easy magnetic separation. PTP/PR nanocomposite adsorbent was recovered using magnetic separation any undesired change for better recy- clability. In each regeneration cycle, the used adsorbent was suspended in distilled water and magnetically stirred overnight followed by resuspending in 0.1 M HNO3 and 0.1M NaOH. The adsorption capacity of PTP/PR nano- composite decreased overall by around 20% after five con- secutive cycles post. However, adsorption equilibrium time of 30 minutes progressively increased to 50 minutes after 5 cycles. 4. 1. Synergism of Photocatalysis and Adsorption for Removal of MG Dye by PTP/PR Nanocomposite. In our attempt to investigate the PR based modula- tion of the PTP matrix, we calculated the band gap of PTP/ PR nanocomposite using Tauc Method.60 The calculated band gap of 8 wt% of PTP/PR was found to be 2.4 eV rela- tive to the bandgap of 3.0 eV for pure PTP matrix. This PR lowering of band gap has made the PTP/PR nanocompos- ite as visible light absorbing semiconductor. The larger surface area of 81m2/g and visible region band gap of PTP/ PR nanocomposite are desirable features for the photocat- alytic activity. To examine the PR effect on the photo gen- erated hole electron pair recombination of PTP/PR nano- composite cyclic voltammetry (CV) technique was used. CV experiments in 0.1 M KNO3 supporting electrolyte at varying scan rates were carried out using PTP/PR nano- composite and PR modified Glassy carbon electrodes (GCE). The changes in the voltammograms of modified GCE with increasing scan rates from 20 to 100 mV/s can be ascribed to the modified rate of the PR electron transfer in the form of PTP/PR nanocomposite. From Figure 10, it was seen that peak currents increased with scan rate and can be linearly correlated with square root of scan rates suggesting diffusion-controlled process. The shifting of peak potentials on increasing scan rate corroborates with the overall quasi reversibility of redox process. The electro- chemical impedance spectroscopy (EIS) analysis of the PTP/PR modified electrode was observed in 0.1M KNO3. The results of EIS are represented as Nyquist plots depict- ing semicircular and linear regions. The semicircular part portraying high frequency zone describes electron-trans- fer resistance (Rct), while the lower frequency linear posi- tion indicates diffusion-controlled operation. The higher resistance of PR/GCE electrode can be evidenced from the larger diameter of semicircle which is typically under high Rct condition. The comparatively lower semi-circular arc diameter (Nyquist plot inset of Figure 10) in case of PTP/ PR confirms attenuation of Rct on composite formation with PR dopant. The lowering of Rct on composite forma- tion with PTP, indicates that hybrid material (PTP/PR) possesses lower charge transfer resistance indicating that charge carriers (photogenerated electron-hole pairs) are more separated with higher mobility and lower recombi- nation tendency. These attributes are highly desirable for photocatalytic activity.61With such desirable properties of PTP/PR nanocomposite towards photocatalytic activity, we envisaged a synergistic effect of photocatalysis and ad- sorption by PTP/PR nanocomposite towards the MG dye attenuation. The synergistic effect of photocatalysis and 860 Acta Chim. Slov. 2022, 69, 848–862 Mustafa et al.: Hybrid Polymer Composite of Prussian Red Doped ... adsorption could be observed within 30 minutes under the irraddiation from 150 W CFL. The results of synergistic effect are summarized in the Fig.11. Figure 11. The synergistic effect of photocatalysis and adsorption for MG dye attenuation by PTP/PR nanocomposite 5. Conclusion Prussian red (PR) on account of its unique proper- ties was envisaged as an inorganic dopant towards polyth- iophene (PTP) conducting polymer matrix. Oxidative po- lymerization reaction via solvothermal route was utilized for incorporating ball milled PR in the polythiophene ma- trix. The interaction of PR with PTP matrix was observed from the spectral changes in the FTIR and PXRD patterns. TEM imaging data identified PTP/PR hybrid material as a nanocomposite system. The observed thermal, electro- chemical and photocatalytic descriptors of PTP/PR over pristine PTP suggested a desirable enhancement in these properties for the possible applications as water treatment nano-material. Zeta potential and pH studies suggest good adsorptive propensity towards cationic dyes under envi- ronmental conditions. An adsorption capacity of 35mg/g for removal of cationic dye Malachite Green (MG) was ob- served in the real time effluents from dye industry. From structural analysis, electrostatic, and pi-pi type interac- tions have been predicted to be predominant noncovalent forces involved in adsorption of MG dye over PTP/PR na- nocomposite. Adsorption data fitted Langmuir and Freun- dlich adsorption isotherms suggesting monolayer as well as multi-layer adsorption process, besides kinetic studies revealed pseudo-second order model for the adsorption of MG dye over nanocomposite. Negative value of free ener- gy indicated thermodynamic spontaneity, recyclability with good regeneration was observed even after five cycles. Taken together PR as novel dopant in the PTP matrix has desirably enhanced its polymeric properties and as PTP/ PR nanocomposite, it has increased the potency of con- ducting polymer towards wastewater treatment applica- tion. Further development of PTP/PR nanocomposite for Figure 10. Comparative Cyclic Voltammograms of PR and PTP/PR at constant and increasing scan rates (A, B, D), (C) Comparative Nyquist plots for PR and PTP/PR nanocomposite. 861Acta Chim. Slov. 2022, 69, 848–862 Mustafa et al.: Hybrid Polymer Composite of Prussian Red Doped ... a wider water treatment application involving combined effect of adsorptive and photocatalytic elimination of per- sistent water contaminants is underway in our laboratory. Acknowledgements The authors are grateful to Head Dept. of Chemistry NIT Srinagar for allowing the BET studies. MM acknowl- edges the University Grants Commission GOI, for the award of JRF fellowship. Conflict of interest The authors declare that they have no conflict of interest and no competing financial interest. 6. References 1. T. H. Y. Lee, J. Chuah, S. A. Snyder, ACS EST Water 2022, 2, 907–931. DIO:10.1021/acsestwater.1c00453 2. W. Ahmad, R. D. Alharthy, M. Zubair, M. Ahmed, A. Hameed, S. Rafque, Sci. Rep. 2021, 11, 17006. DIO:10.1038/s41598-021-94616-4 3. A. Tkaczyk, K. Mitrowska, A. Posyniak, Sci. Total. Environ. 2020, 717, 137222. DIO:10.1016/j.scitotenv.2020.137222 4. R. Al-Tohamy, S. S. Ali, F. Li, K. M. Okasha, A. G. Mahmoud, T. Elsamahy, H. Jiao, Y. Fu, J. Sun. Ecotoxicol. Environ. Saf. 2022, 231, 113160. DIO:10.1016/j.ecoenv.2021.113160 5. Z. Peng, D. Wu, W. Wang, F. Tan, T. Ng, J. Che, X. Qiao, P. Wong. Appl. Surf. Sci. 2017, 396, 19–25. DIO:10.1016/j.apsusc.2016.11.026 6. M. Shaban, M. R. Abukhadra., Environ. Earth Sci. 2017, 76, 2–16. DIO:10.1007/s12665-017-6636-3 7. S. 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Koordinacijska spojina prusko rdeča (PR) z železom (III) je bila dopirana v matriko politiofena (PTP) z namenom raziskati nagnjenost tega anorgansko-organskega hibridnega kompozitnega materiala k čiščenju od- padne vode. Dopiranje PR izboljša mehanske, toplotne, električne in fotokatalitske lastnosti čistega PTP. Karakterizacija kompozita PTP/PR je bila opravljena z rentgensko difrakcijo prahu, TEM, TGA, FTIR, BET analizo in UV-vidno spek- troskopijo. Optimizacija adsorpcijskih pogojev, regeneracija adsorbenta, študije adsorpcijske termodinamike PTP/PR so bile izvedene z uporabo barvila malahitno zeleno (MG). Pri optimiziranih pogojih je bila dosežena 92% adsorpcija barvila MG pri 20 mg nanokompozita PTP/PR v 20 minutah pri pH 7. Nanokompozit PTP/PR je prav tako dokazal kom- plementarno učinkovitost v primeru realnih vzorcev odpadne vode. Termodinamične študije kažejo na spontan proces z elektrostatično privlačnostjo kot prevladujočo nekovalentno interakcijo. 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DOI: 10.17344/acsi.2022.7614 Scientific paper Synthesis and Biological Evaluation of Some Hydrazide-Hydrazone Derivatives as Anticancer Agents Kadriye Akdağ,1 Fatih Tok,1 Sevgi Karakuş,1,* Ömer Erdoğan,2 Özge Çevik2 and Bedia Koçyiğit-Kaymakçıoğlu1 1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Marmara University, 34854, Istanbul, Turkey 2 Department of Biochemistry, School of Medicine, Aydin Adnan Menderes University, 09010, Aydın, Turkey * Corresponding author: E-mail: skarakus@marmara.edu.tr Received: 10-04-2022 Abstract In this study, a series of hydrazide-hydrazone derivatives (3a-3u) were synthesized and evaluated for their anticancer activities against prostate cancer cell line (PC-3), breast cancer cell line (MCF-7), colon cancer cell line (HT-29) and hu- man umbilical vein endothelial cells (HUVEC) using MTT assay. In particular, compound 3h having a pyrrole ring was found to be the most potent derivative with IC50 = 1.3, 3.0, 1.7 µM against PC-3, MCF-7, HT-29 cancer cell lines respec- tively using paclitaxel as a standard compound. Furthermore, compound 3h was subjected to further biological studies such as caspase-3 activity and Annexin-V assay to evaluate their inhibitory potentials. The activity results displayed that compound 3h increased caspase-3 activation and the number of cells to early apoptosis. The additional studies like pharmacokinetics, bioavailability scores and drug-likeness properties were also evaluated. The in silico pharmacokinetics predictions displayed that the bioavailability of these compounds may be high. Keywords: Hydrazone, anticancer, apoptosis, drug-likeness, MCF-7. 1. Introduction Cancer is a disease characterized by the uncontrolled proliferation and spread of the body’s cells. It is the second leading cause of death in developed countries after cardio- vascular diseases. It was reported that one out of every two people born after 1960 will get cancer. The distribution of cancer types according to gender differs.1 While breast cancer is more common in women than other types of cancer, prostate cancer is more common in men. Lung cancer and colorectal cancer highly affect both men and women.2 There are different approaches to can- cer treatment such as surgery, radiotherapy, chemotherapy and hormone therapy. Despite their severe toxicity, chemo- therapy is the main approach for the treatment of cancer worldwide.3 Detailed analyses of pathways and mecha- nisms and structures of antitumor compounds have led to significant developments in the prevention and treatment of cancer. Therefore, there is still a need for new anticancer compounds with higher potency and less toxicity, as well as less toxic to non-cancerous cells.4 Researchers working in the field of discovering new drugs seek to synthesize simple compounds having vari- ous pharmacological activities such as anticancer, antivi- ral, antibacterial, antioxidant.5 Hydrazide-hydrazone de- rivatives are molecules containing a highly reactive group (CO-NH-N=CH) and are considered to be a good candi- date for the development of a new drug.6-8 There are many studies in the literature that hy- drazide-hydrazones have anticancer activity. Saini et. al. presented useful information about the mechanisms of anticancer activity of hydrazide-hydrazones.9 Hydrazone structures can act by inhibiting topoisomerases, protein kinases and induce apoptosis pathways.10,11 Abou-Seri et al. designed and synthesized potent hydrazones as poten- tial inhibitors of VEGFR2.12 Taha et al. synthesized a new morpholine hydrazone scaffold due to potential antican- cer activity.13 In addition, the benzothiazole-hydrazone derivatives were reported as potent anticancer agents.14 In another study, the hydrazone derivatives bearing the pyridine ring synthesized and assessed the anticancer activity against MCF-7. In this study, some of the tested compounds displayed higher anticancer activity than cis- platin.15 Hydrazone structures play an important role in anticancer-related processes, as mentioned in the exam- ples above. 864 Acta Chim. Slov. 2022, 69, 863–875 Akdağ et al.: Synthesis and Biological Evaluation of Some ... In light of the above information, we synthesized some hydrazone derivatives and investigated them for their anticancer activity against prostate cancer cell line (PC-3), breast cancer cell line (MCF-7), colon cancer cell line (HT- 29) and human umbilical vein endothelial cells (HUVEC). 2. Experimental All chemicals were purchased from Merck Company and Sigma-Aldrich. Melting points were determined by using a SMP II melting point apparatus. IR spectra were recorded on a Shimadzu FTIR-8400S spectrophotome- ter. 1H-NMR and 13C-NMR spectra were obtained on a Bruker Avance DPX-400 spectrometer. Tetramethylsilane as the internal standard and DMSO-d6 as the solvent was used for NMR spectrums. Elemental analyses were per- formed with GmbH varioMICRO CHNS. 2. 1. Synthesis 2. 1. 1. General procedure for the synthesis of ethyl 4-[(4-methoxybenzoyl)amino] benzoate (1) Ethyl 4-aminobenzoate (1mmol) was dissolved in ether (10 mL). 1 mmol of 4-methoxybenzoyl chloride was added dropwise to this solution. It was stirred for 2 hours on a magnetic stirrer. The precipitate was washed with wa- ter, filtered and dried.16 2. 1. 2. General procedure for the synthesis of N-[4-(hydrazinylcarbonyl)phenyl]-4- methoxybenzamide (2) Ethyl 4-[(4-methoxybenzoyl)amino]benzoate (1 mmol) was heated with hydrazine monohydrate (3 mL) in ethanol (15 mL) for 10 hours at 100 °C. After TLC control, the precipitate was filtered and crystallized with ethanol.16 2. 1. 3. General procedure for the synthesis of hydrazide-hydrazones (3a-3u) In a solution of N-(4-(hydrazinecarbonyl)phe- nyl)-4-methoxybenzamide (1 mmol) in 10 mL ethanol was added 1 mmol substituted aldehyde derivatives. The mix- ture was refluxed for 8 hours. After TLC control, the pre- cipitate was filtered and dried. The product was crystallized with ethanol.17 Compounds 3j, 3l, 3m, 3n, 3r and 3u were reported in the literature.18-20 The other hydrazone deriva- tives were synthesized for the first time in this article. N-{4-[2-(2,4-Dichlorobenzylidene)hydrazinecarbonyl] phenyl}-4-methoxybenzamide (3a) Yield: 68%, M.p. = 298-299 °C, FTIR (ν, cm-1): 3271, 3173 (N-H), 3010 (=C-H), 2983 (C-H), 1641 (C=O), 1606 (C=N), 1589, 1444 (C=C), 852 (=C-H). 1H-NMR (400 MHz, DMSO-d6, ppm): δ 3.85 (s, 3H, OCH3), 7.07-8.04 (m, 11H, Ar-H), 8.83 (s, 1H, =CH-), 10.37 (s, 1H, -CONH-), 12.08 (s, 1H, -CONHN=). 13C-NMR (100 MHz, DMSO-d6, ppm): δ 55.44 (OCH3), 113.66, 119.42, 126.56, 128.00, 128.48, 129.35, 129.74, 130.80, 133.76, 134.94, 142.09, 142.80, 162.12, 162.59 (C=O), 165.18 (C=O). Anal. Calcd for C22H17Cl2N3O3: C 59.74, H 3.87, N 9.50. Found: C 59.56, H 3.90, N 9.42 %. N-(4-(2-(3,4-Dichlorobenzylidene)hydrazinecarbonyl) phenyl)-4-methoxybenzamide (3b) Yield: 63%, M.p. = 280-281 °C, FTIR (ν, cm-1): 3315, 3238 (N-H), 3072 (=C-H), 2935 (C-H), 1641 (C=O), 1602 (C=N), 1589, 1543 (C=C), 840 (=C-H). 1H-NMR (400 MHz, DMSO-d6, ppm): δ 3.83 (s, 3H, OCH3), 7.05–7.98 (m, 11H, Ar-H), 8.41 (s, 1H, =CH-), 10.34 (s, 1H, -CONH- ), 11.95 (s, 1H, -CONHN=). 13C-NMR (100 MHz, DMSO-d6, ppm): δ 55.44 (OCH3), 113.64, 119.41, 126.56, 127.49, 129.74, 131.07, 131.69, 132.09, 135.28, 142.73, 144.52, 162.10, 162.71 (C=O), 165.17 (C=O). Anal. Cal- cd for C22H17Cl2N3O3: C 59.74, H 3.87, N 9.50. Found: C 59.49, H 3.91, N 9.44 %. N-{4-[2-(4-Nitrobenzylidene)hydrazinecarbonyl]phe- nyl}-4-methoxybenzamide (3c) Yield: 57%, M.p. = 334-335 °C, FTIR (ν, cm-1): 3302, 3213 (N-H), 3068 (=C-H), 2837 (C-H), 1643 (C=O), 1602 (C=N), 1587, 1537 (C=C), 833 (=C-H). 1H-NMR (400 MHz, DMSO-d6, ppm): δ 3.85 (s, 3H, OCH3), 7.07-8.33 (m, 12H, Ar-H), 8.56 (s, 1H, =CH-), 10.41 (s, 1H, -CONH-), 12.09 (s, 1H, -CONHN=). 13C-NMR (100 MHz, DMSO-d6, ppm): δ 55.91 (OCH3), 114.11, 119.87, 124.52, 127.00, 128.35, 128.99, 130.20, 141.21, 148.21, 162.57 (C=O), 165.63 (C=O). Anal. Calcd for C22H18N4O5: C 63.15, H 4.34, N 13.39. Found: C 62.96, H 4.33, N 13.31 %. N-{4-[2-(3-Nitrobenzylidene)hydrazinecarbonyl]phe- nyl}4-methoxybenzamide (3d) Yield: 58%, M.p. = 294 °C, FTIR (ν, cm-1): 3302, 3228 (N-H), 3074 (=C-H), 2845 (C-H), 1637 (C=O), 1602 (C=N), 1525, 1506 (C=C), 842 (=C-H). 1H-NMR (400 MHz, DMSO-d6, ppm): δ 3.83 (s, 3H, OCH3), 7.04–8.26 (m, 12H, Ar-H), 8.54 (s, 1H, =CH-), 10.35 (s, 1H, -CONH-), 12.04 (s, 1H, -CONHN=). 13C-NMR (100 MHz, DMSO-d6, ppm): δ 55.44 (OCH3), 113.65, 119.41, 120.80, 124.11, 126.55, 128.51, 129.75, 130.43, 133.32, 136.30, 142.76, 144.83, 148.23, 162.11, 162.77 (C=O), 165.17 (C=O). Anal. Calcd for C22H18N4O5: C 63.15, H 4.34, N 13.39. Found: C 62.99, H 4.35, N 13.33 %. N-{4-[2-(3,5-Dichloro-2-hydroxybenzylidene)hydrazi- necarbonyl]phenyl}-4-methoxybenzamide (3e) Yield: 61.%, M.p. = 331-332 °C, FTIR (ν, cm-1): 3340, 3219 (N-H), 3039 (=C-H), 2970 (C-H), 1645 (C=O), 1602 (C=N), 1587, 1500 (C=C), 840 (=C-H). 1H-NMR 865Acta Chim. Slov. 2022, 69, 863–875 Akdağ et al.: Synthesis and Biological Evaluation of Some ... (400 MHz, DMSO-d6, ppm): δ 3.86 (s, 3H, OCH3), 7.08- 8.01 (m, 10H, Ar-H), 8.58 (s, 1H, =CH-), 10.40 (s, 1H, -CONH-), 12.26 (s, 1H, -CONHN=), 12.83 (s, 1H, OH). 13C-NMR (100 MHz, DMSO-d6, ppm): δ 55.44 (OCH3), 113.66, 119.41, 126.55, 127.37, 128.01, 128.47, 129.74, 130.80, 133.76, 142.09, 142.79, 162.11, 162.57 (C=O), 165.17 (C=O). Anal. Calcd for C22H17Cl2N3O4: C 57.66, H 3.74, N 9.17. Found: C 57.47, H 3.75, N 9.14 %. N-{4-[2-(Pyridine-4-ylmethylene)hydrazinecarbonyl] phenyl}-4-methoxybenzamide (3f) Yield: 75%, M.p. = 311-312 °C, FTIR (ν, cm-1): 3327, 3263 (N-H), 3032 (=C-H), 2910 (C-H), 1645 (C=O), 1602 (C=N), 1525, 1496 (C=C), 842 (=C-H). 1H-NMR (400 MHz, DMSO-d6, ppm): δ 3.85 (s, 3H, OCH3), 7.08-8.65 (m, 12H, Ar-H), 8.67 (s, 1H, =CH-), 10.38 (s, 1H, -CONH-), 12.06 (s, 1H, -CONHN=). 13C-NMR (100 MHz, DMSO-d6, ppm): δ 55.49 (OCH3), 113.70, 119.45, 120.98, 126.56, 128.59, 129.80, 141.60, 142.88, 144.85, 150.31, 162.15, 162.82 (C=O), 165.24 (C=O). Anal. Calcd for C21H18N4O3: C 67.37, H 4.85, N 14.96. Found: C 67.14, H 4.82, N 14.94 %. N-{4-[2-(Pyridine-3-ylmethylene)hydrazinecarbonyl] phenyl}-4-methoxybenzamide (3g) Yield: 65%, M.p. = 287 °C, FTIR (ν, cm-1): 3336, 3273 (N-H), 3043 (=C-H), 2982 (C-H), 1647 (C=O), 1602 (C=N), 1589, 1471 (C=C), 844 (=C-H). 1H-NMR (400 MHz, DMSO-d6, ppm): δ 3.85 (s, 3H, OCH3), 7.07–8.62 (m, 12H, Ar-H), 8.86 (s, 1H, =CH-), 10.37 (s, 1H, -CONH-), 11.96 (s, 1H, -CONHN=). 13C-NMR (100 MHz, DMSO-d6, ppm): δ 55.49 (OCH3), 113.70, 119.46, 124.06, 126.58, 128.49, 129.79, 130.35, 133.41, 142.73, 144.57, 148.73, 150.66, 162.14, 162.67 (C=O), 165.21 (C=O). Anal. Calcd for C21H18N4O3: C 67.37, H 4.85, N 14.96. Found: C 67.07, H 4.83, N 14.92 %. N-{4-[2-(1H-Pyrrol-3-yl-methylene)hydrazinecarbon- yl]phenyl}-4-methoxybenzamide (3h) Yield: 78%, M.p. = 273 °C, FTIR (ν, cm-1): 3308, 3194 (N-H), 3047 (=C-H), 2964 (C-H), 1645 (C=O), 1604 (C=N), 1525, 1500 (C=C), 842 (=C-H). 1H-NMR (400 MHz, DMSO-d6, ppm): δ 3.83 (s, 3H, OCH3),4.65 (s, 1H, NH), 7.11-7.87 (m, 11H, Ar-H), 8.33 (s, 1H, =CH-), 10.28 (s, 1H, -CONH-), 11.65 (s, 1H, -CONHN=).13C-NMR (100 MHz, DMSO-d6, ppm): δ 55.48 (OCH3), 113.67, 119.39, 126.69, 127.60, 127.91, 129.74, 141.91, 162.07, 165.15 (C=O), 165.57 (C=O). Anal. Calcd for C20H18N4O3: C 66.29, H 5.01, N 15.46. Found: C 66.17, H 5.04, N 15.39 %. N-{4-[2-(Thiophen-2-ylmethylene)hydrazinecarbonyl] phenyl}-4-methoxybenzamide (3i) Yield: 70%, M.p. = 250-251 °C, FTIR (ν, cm-1): 3304, 3192 (N-H), 3012 (=C-H), 2966 (C-H), 1645 (C=O), 1604 (C=N), 1581, 1491 (C=C), 842 (=C-H). 1H-NMR (400 MHz, DMSO-d6, ppm): δ 3.82 (s, 3H, OCH3), 7.04–7.97 (m, 11H, Ar-H), 8.41 (s, 1H, =CH-), 10.32 (s, 1H, -CONH-), 11.79 (s, 1H, -CONHN=). 13C-NMR (100 MHz, DMSO-d6, ppm): δ 55.36 (OCH3), 113.56, 119.32, 126.46, 127.75, 128.23, 128.73, 129.65, 130.68, 139.13, 142.37, 161.99, 162.30 (C=O), 165.06 (C=O). Anal. Cal- cd for C20H17N3O3S: C 63.31, H 4.52, N 11.07. Found: C 63.40, H 4.53, N 11.13 %. N-{4-[2-(5-Nitrofuran-2-yl-methylene)hydrazinecar- bonyl]phenyl}-4-methoxybenzamide (3j) Yield: 64%, M.p. = 297-298°C. Ref. lit. M.p= 292 °C.18 N-{4-[2-(2-Chloroquinolin-3-yl-methylene)hydrazine- carbonyl]phenyl}-4-methoxybenzamide (3k) Yield: 58%, M.p. = 302 °C, FTIR (ν, cm-1): 3281, 3227 (N-H), 3030 (=C-H), 2989 (C-H), 1641 (C=O), 1604 (C=N), 1591, 1537 (C=C), 840 (=C-H). 1H-NMR (400 MHz, DMSO-d6, ppm): δ 3.86 (s, 3H, OCH3), 7.08–8.26 (m, 13H, Ar-H), 8.94 (s, 1H, =CH-), 10.39 (s, 1H, -CONH-), 12.21 (s, 1H, -CONHN=). 13C-NMR (100 MHz, DMSO-d6, ppm): δ 55.36 (OCH3), 113.57, 119.34, 126.16, 126.44, 126.80, 127.24, 127.53, 127.76, 128.47, 128.92, 129.67, 131.65, 135.49, 142.22, 142.77, 147.01, 148.41, 162.03, 162.55 (C=O), 165.09 (C=O). Anal. Calcd for C25H19ClN4O3: C 65.43, H 4.17, N 12.21. Found: C 65.29, H 4.21, N 12.14 %. N-[4-(2-Benzylidenehydrazinecarbonyl)phenyl]-4- methoxybenzamide (3l) Yield: 65%, M.p. = 292-293 °C. Ref. lit. M.p = 282 °C.19 N-{4-[2-(4-Hydroxybenzylidene)hydrazinecarbonyl] phenyl}-4-methoxybenzamide (3m) Yield: 78%, M.p. = 284-285 °C. Ref. lit. M.p= 289 °C.19 N-{4-[2-(4-Hydroxy-3-methoxybenzylidene)hydrazine- carbonyl]phenyl}-4-methoxybenzamide (3n) Yield: 67%, M.p. = 264-265 °C. Ref. lit. M.p= 270 °C.19 N-{4-[2-(3-(4-Chlorophenoxybenzylidene)hydrazine- carbonyl]phenyl}-4-methoxybenzamide (3o) Yield: 74%, M.p. = 252-253 °C, FTIR (ν, cm-1): 3329, 3259 (N-H), 3005 (=C-H), 2837 (C-H), 1641 (C=O), 1604 (C=N), 1573, 1508 (C=C), 839 (=C-H). 1H-NMR (400 MHz, DMSO-d6, ppm): δ 3.82 (s, 3H, OCH3), 7.04-7.97 (m, 16H, Ar-H), 8.42 (s, 1H, =CH-), 10.33 (s, 1H, -CONH-), 11.81 (s, 1H, -CONHN=). 13C-NMR (100 MHz, DMSO-d6, ppm): δ 55.35 (OCH3), 113.56, 115.85, 119.31, 120.44, 122.95, 126.46, 127.44, 128.31, 129.66, 129.92, 130.60, 136.48, 142.56, 146.35, 155.23, 156.71, 162.00, 162.48 (C=O), 165.07 (C=O). Anal. Calcd for C28H22ClN3O4: C 67.27, H 4.44, N 8.40. Found: C 67.15, H 4.42, N 8.36 %. N-{4-[2-(2-Phenylethylidene)hydrazinecarbonyl]phe- nyl}-4-methoxybenzamide (3p) Yield: 68%, M.p. = 305-306 °C, FTIR (ν, cm-1): 3296, 3223 (N-H), 3037 (=C-H), 2841 (C-H), 1651 (C=O), 1602 (C=N), 1548, 1504 (C=C), 840 (=C-H). 1H-NMR 866 Acta Chim. Slov. 2022, 69, 863–875 Akdağ et al.: Synthesis and Biological Evaluation of Some ... (400 MHz, DMSO-d6, ppm): δ 3.60 (d, 2H, -CH2), 3.82 (s, 3H, OCH3), 7.04-8.02 (m, 13H, Ar-H), 8.42 (s, 1H, =CH-), 10.29 (s, 1H, -CONH-), 11.47 (s, 1H, -CONHN=). 13C-NMR (100 MHz, DMSO-d6, ppm): δ 55.37 (OCH3), 113.55, 119.27, 125.73, 126.52, 127.91, 128.15, 128.60, 128.75, 129.64, 136.83, 142.33, 149.99, 161.99 (C=O), 165.04 (C=O). Anal. Calcd for C23H21N3O3: C 71.30, H 5.46, N 10.85. Found: C 71.07, H 5.42, N 10.92 %. N-{4-[2-(Furan-2-ylmethylene)hydrazinecarbonyl]phe- nyl}-4-methoxybenzamide (3q) Yield: 85%, M.p. = 279°C, FTIR (ν, cm-1): 3304, 3234 (N-H), 3016 (=C-H), 2887 (C-H), 1641 (C=O), 1602 (C=N), 1521, 1494 (C=C), 840 (=C-H). 1H-NMR (400 MHz, DMSO-d6, ppm): δ 3.85 (s, 3H, OCH3), 6.64–8.01 (m, 11H, Ar-H), 8.34 (s, 1H, =CH-), 10.36 (s, 1H, -CONH-), 11.73 (s, 1H, -CONHN=). 13C-NMR (100 MHz, DMSO-d6, ppm): δ 55.36 (OCH3), 112.09, 113.20, 113.56, 119.32, 126.47, 127.60, 128.27, 129.66, 137.02, 142.51, 145.01, 149.43, 162.01, 162.38 (C=O), 165.08 (C=O). Anal. Calcd for C20H17N3O4: C 66.11, H 4.72, N 11.56. Found: C 66.26, H 4.77, N 11.48 %. N-{4-[2-(4-Methoxybenzylidene)hydrazinecarbonyl] phenyl}-4-methoxybenzamide (3r) Yield: 74%, M.p. = 295 °C. Ref. lit. M.p= 298 °C.20 N-{4-[2-(3-Fluorobenzylidene)hydrazinecarbonyl]phe- nyl}-4-methoxybenzamide (3s) Yield: 83%, M.p. = 286°C, FTIR (ν, cm-1): 3333, 3255 (N-H), 3037 (=C-H), 2964 (C-H), 1645 (C=O), 1602 (C=N), 1575, 1537 (C=C), 840 (=C-H). 1H-NMR (400 MHz, DMSO-d6, ppm): δ 3.82 (s, 3H, OCH3), 7.04–8.00 (m, 12H, Ar-H), 8.46 (s, 1H, =CH-), 10.35 (s, 1H, -CONH-), 11.89 (s, 1H, -CONHN=). 13C-NMR (100 MHz, DMSO-d6, ppm): δ 55.36 (OCH3), 112.74, 112.96, 113.57, 116.52, 119.33, 123.30, 126.48, 127.51, 128.37, 129.67, 130.78, 136.89, 142.61, 145.82, 161.12, 162.02, 162.57, 163.54 (C=O), 165.10 (C=O). Anal. Calcd for C22H18FN3O3: C 67.51, H 4.64, N 10.74. Found: C 67.33, H 4.60, N 10.79%. N-{4-[2-(4-Cyanobenzylidene)hydrazinecarbonyl]phe- nyl}-4-methoxybenzamide (3t) Yield: 68%, M.p. = 306°C, FTIR (ν, cm-1): 3338, 3253 (N-H), 3070 (=C-H), 2978 (C-H), 1645 (C=O), 1600 (C=N), 1539, 1494 (C=C), 839 (=C-H). 1H-NMR (400 MHz, DMSO-d6, ppm): δ 3.86 (s, 3H, OCH3), 7.08–8.02 (m, 12H, Ar-H), 8.52 (s, 1H, =CH-), 10.38 (s, 1H, -CONH-), 12.04 (s, 1H, -CONHN=). 13C-NMR (100 MHz, DMSO-d6, ppm): δ 55.36 (OCH3), 111.66, 113.57, 118.59, 119.33, 126.45, 127.47, 128.43, 129.67, 132.66, 138.81, 142.71, 145.18, 162.03, 162.62 (C=O), 165.10 (C=O). Anal. Calcd for C23H18N4O3: C 69.34, H 4.55, N 14.06. Found: C 69.15, H 4.53, N 14.19 %. N-{4-[2-(4-Bromobenzylidene)hydrazinecarbon- yl]phenyl}-4-methoxybenzamide (3u) Yield: 65%, M.p. = 312-313°C. Ref. lit. M.p= 316°C.18 2. 2. Biological activity 2. 2. 1. MTT assay Cytotoxicity tests of the synthesized compounds were performed in a human prostate cancer cell line (PC- 3, ATCC CRL-1435), human breast cancer cell line (MCF- 7, ATCC-HTB-22), human colon cancer cell line (HT-29, ATCC-HBT-38) and human umbilical vein endothelial cells (HUVEC, ATCC-CRL-1730). MCF-7 and HT-29 cells were cultured in DMEM medium, PC-3 cells were cultured in RPMI-1640 medium and HUVEC cells were cultured in F-12K medium supplemented with 10% fetal bovine serum FBS), 100 U/mL penicillin and 100 μg/mL streptomycin and 2 mM L-glutamine. The cells were incubated at 37˚C in a humidified atmosphere with 5% CO2. Cells were seeded in 96 well plates at a density of 1x104 and treated with com- pounds synthesized in different (0.1-1000 µM) concentra- tion ranges. We prepare a 10 mM main stock, dilute them with DMSO and add not exceeding the highest concentra- tion of DMSO (0.5%) (for example, 1 mg compound can be dissolved in about 500 µL DMSO). The compounds we put in the medium are added by dilution in the cell medium when necessary so that they do not exceed the DMSO limit. Paclitaxel was used at the same concentrations as a positive control. The MTT test was performed after the cells were incubated with the synthesized compounds for 24 hours. After incubation, cells were washed once with PBS, and a fresh medium was added. Then, 10 µL of MTT dye (0.5 mg/ mL) was placed in each well of the plate and incubated at 37 °C for 4 hours. Finally, DMSO was added and incubated for 30 minutes to dissolve the formazan crystals. Color chang- es were measured at a wavelength of 570 nm. IC50 values were analyzed using the Graphpad Prism 7.00 program. All experiments were performed with triple biological rep- licates, and data were given as mean±standard deviation. 2. 2. 2. AO/EB Staining MCF-7, PC-3 and HT-29 cells were seeded in 12 well plates and were incubated with the compound 3h (IC50 concentration) for 24 hours.21 After incubation, PBS washed cells and incubated them with AO/EB staining solution (100 μg/ml acridine orange and 100 μg/mL ethid- ium bromide) in PBS. Images of cells were taken under an inverted fluorescent microscope (Zeiss AxioVert1, Ger- many). In the analysis, green staining shows viable cells, and red staining shows dead or destructed cells, so the in- tensity was calculated by taking the green/red ratio. All ex- periments were performed with triple biological replicates, and data were given as mean±standard deviation. 2. 2. 3. Annexin-V assay MCF-7, PC-3 and HT-29 cells were seeded in 6 well plates. After the cells reached a density of 1 × 106, they were incubated with compound 3h (IC50 concentration) for 24 867Acta Chim. Slov. 2022, 69, 863–875 Akdağ et al.: Synthesis and Biological Evaluation of Some ... hours.22 After the incubation period was complete, the cells were washed with PBS and removed using trypsin-EDTA. Cells were centrifuged at 600xg for 5 minutes and washed once with PBS. Cells were suspended with a fresh medium contain- ing 10% FBS and performed according to the manufacturer’s protocol using the Annexin V & Dead Cell Assay kit (Muse- MCH100105, MilliporeSigma, CA, USA ). Cells were incubat- ed in the dark for 30 minutes with the binding buffer and dyes in the kit. Assay results were measured using the Muse Cell Analyzer. All experiments were performed with triple biologi- cal replicates, and data were given as mean±standard deviation. 2. 2. 4. Caspase-3 activity MCF-7, PC-3, and HT-29 cells were seeded in 12 well plates. Cells were incubated with compound 3h (IC50 concentration) for 24 hours and then washed with PBS. Caspase-3 activity in cells was performed using a commer- cial kit (CASP-3-C, Sigma-Aldrich). After the cells were treated with 100 µl of cell lysis buffer, they were centrifuged at 10.000xg for 10 minutes at 4°C. The supernatant was taken, and measurements were made colorimetrically at a wavelength of 405 nm (Epoch, Biotek) using Ac-DEVD- pNA substrate. The results were calculated as µmol pNA/ min/mL, and the protein values of the cells were calculated and used as nmol pNA/min/µg protein. All experiments were performed with triple biological replicates, and data were given as mean±standard deviation. 2. 3. In silico ADME analysis Physicochemical, pharmacokinetic and drug-like- ness properties of all compounds were predicted through SwissAdme online server (http://www.swissadme.ch/). 3. Results and Discussion 3. 1. Synthesis In this study, some hydrazide-hydrazone derivatives were synthesized as given in Scheme 1. Firstly, the amide functional group was prepared from the reaction of ethyl p-aminobenzoate and 4-methoxybenzoyl chloride in ether. In the second step, hydrazide structure was obtained by heating ethyl 4-(4-methoxybenzamido)benzoate with hy- drazine monohydrate in an ethanolic medium. Finally, hydrazone structures were successfully synthesized by the treatment with hydrazide and different substituted alde- hydes in ethanol. The structures of the hydrazone deriv- atives were elucidated by FTIR, 1H-NMR, and 13C-NMR spectroscopic methods and elemental analysis. IR spectra of hydrazone compounds (3a-3u), the C=O stretching bands of compounds were observed at 1637-1653 cm-1. The NH stretching bands of amide and hydrazone structures were detected at 3173-3340 cm- 1. The characteristic strong band in the 1600–1606 cm–1 region, attributed to a C=N stretching, confirmed the hy- drazone feature of all derivatives. In the 1H-NMR spec- tra, the NH peak of amide structure gave a peak between 10.28 ppm and 10.41 ppm as a singlet. The proton of the imine (-N=CH-) group resonated at 8.33–8.94 ppm as a singlet. The proton of the –CONHN= group was detected at 11.47-12.26 ppm as a singlet. In addition, the disappear- ance of the proton peaks of the hydrazide amino group is evidence of hydrazone synthesis. In the 13C-NMR spec- tra, carbonyl peaks were observed at 161.99-165.63 ppm. The carbon of the imine (-N=CH-) group was detected at 144.52-149.99 ppm. 3. 2. Biological activity The cytotoxicity studies of synthesized compounds were performed on prostate cancer cell line (PC-3), breast cancer cell line (MCF-7), colon cancer cell line (HT-29), and human umbilical vein endothelial cells (HUVEC) com- pared to the Paclitaxel as a standard compound (Table 1). Compounds carrying 4-nitrophenyl (3c), phenyl (3l), benzyl (3p), 4-bromophenyl (3u) and 3-pyrrole (3h) against the PC3 cancer cell line; compounds carrying 4-nitrophe- nyl (3c), 5-nitro-2-furyl (3j), 4-hydroxyphenyl (3m), ben- zyl (3p), 2-furyl (3q) and 3-pyrrole (3h) against the MCF-7 cancer cell line; compounds carrying 3-nitrophenyl (3d), 2-furyl (3q) and 3-pyrrole (3h) structures against the HT- 29 cancer cell line exhibited significant cytotoxic activity. Scheme 1. The synthetic pathways of target compounds. Reagents: (i) ether, 25 °C, 2h, yield: 75%; (ii) hydrazine monohydrate, ethanol, 100 °C, 10h, yield: 80%; (iii) substituted aldehydes, ethanol, reflux, 8h, yield: 57–85%. 868 Acta Chim. Slov. 2022, 69, 863–875 Akdağ et al.: Synthesis and Biological Evaluation of Some ... In particular, the compound having the 3-pyrrole ring (3h) was found to have higher cytotoxicity against all cancer cell lines than the standard compound. In ad- dition, the toxicity of this compound against HUVEC is very low. The selectivity index (SI = IC50 for normal cell line HUVEC/IC50 for cancerous cell line) of compound 3h was found at 181.0 for PC3, 78.4 for MCF-7, and 138.4 for HT-29. As a result, the compound 3h has been detected that has a selective cytotoxic effect against cancer cell lines. While synthesizing the hydrazone structure in order to compare the structure-activity relationship; different ar- omatic and heteroaromatic rings were selected. The ability of electron-donating or electron-withdrawing groups at different positions on these rings to affect the interactions with cancer cells was also investigated. Among the com- pounds carrying nitro group, compound 3c showed high cytotoxic effect against PC3 and MCF7, compound 3d against HT29, and compound 3j against MCF7 due to the strong electron withdrawing and resonance properties of the nitro group. Moreover, of these compounds, 3j showed the least cytotoxic activity against HUVEC normal cell. Many studies showed that that the nitro group has high antiproliferative activity. Nitro compounds can release NO due to redox reactions inside the cell. For these reasons, cytotoxic effects occur due to disruption of oxidative stress mechanisms. Nitro substituents can also increase the inhi- bition of target biomolecules such as proteins or enzymes due to its electron withdrawing property favoring inter- action with some amino acids such as threonine and glu- tamine. It has been determined that the nitro group of the compounds at the para and meta position shows higher ef- ficiency than the ortho position due to the steric effect.23, 24 On the other hand, compound 3h has a pyrrole ring as a heteroaromatic ring in its structure and showed the highest cytotoxic activity against all cancer cell lines selec- tively, regardless of the above-mentioned structure-activ- ity relationship. AO/EB, a dye that allows it to appear under a fluo- rescent microscope to identify changes in cell membranes during cell death, helps us understand the process of ap- optosis. We qualitatively and quantitatively analyzed the changes of compound 3h (IC50  value) on MCF-7, PC-3, and HT-29 cells using the AO/EB staining method. In AO/ EB staining of MCF-7 cells, when compound 3h was com- pared with the control group, it was observed that the ratio of live cells to dead cells decreased significantly (p<0.001, Figure 1a-b). In PC3 cells, compound 3h was compared with the control group, and it was seen that the viable/ dead (green/red) ratio of the cells decreased significantly (p<0.001, Figure 2a-b). Finally, in HT-29 cells, the viable/ dead ratio of cells in the compound 3h group was signifi- cantly reduced (p<0.001, Figure 3a-b). Annexin-V is a protein that binds to the cell mem- brane lipid phosphatidylserine from the onset of apoptosis. Annexin-V binding was measured in MCF-7, PC-3, and HT-29 cells after 24 hours of incubation with compound 3h. In MCF-7 cells, compound 3h significantly increased the number of cells to early apoptosis, late apoptosis, and dead cells and decreased the number of live cells compared to the control group (p<0.05, p<0.001, Figure 4a-b). In Table 1. The IC50 values of synthesized compounds. Comp. R PC-3* MCF-7* HT-29* HUVEC* 3a 2,4-dichlorophenyl 137.2±1.2 210.4±12.4 174.2±8.0 218.0±9.1 3b 3,4-(dichloro)phenyl 204.7±0.1 112.3±8.2 102.5±6.7 213.8±8.6 3c 4-nitrophenyl 42.2±1.3 30.1±6.0 54.2±4.0 65.8±5.2 3d 3-nitrophenyl 67.7±2.1 61.0±3.2 36.3±2.9 206.3±1.1 3e 2-hydroxy-3,5-(dichloro)phenyl 81.3±1.1 74.1±2.1 91.0±3.5 154.2±7.3 3f pyridin-4-yl 178.6±6.3 128.3±7.2 94.2±2.1 302.6±4.1 3g pyridin-3-yl 321.2±11.0 216.4±9.1 300.1±11.1 314.5±6.1 3h pyrrol-3-yl 1.3±0.1 3.0±0.1 1.7±0.2 235.3±6.5 3i thiophen-2-yl 86.4±0.6 165.4±6.0 143.5±3.9 330.3±12.0 3j 5-nitro-2-furyl 88.9±4.1 23.7±3.2 78.2±2.1 538.1±14.7 3k 2-chloroquinolin-3-yl 194.3±3.5 128.2±2.1 205.4±8.7 337.4±4.2 3l phenyl 46.2±1.0 58.4±5.0 69.1±5.0 84.3±6.0 3m 4-hydroxyphenyl 66.9±6.4 27.1±3.9 86.2±2.2 70.6±3.4 3n 4-hydroxy-3-methoxyphenyl 200.3±8.1 111.1±8.7 170.6±3.1 248.5±8.0 3o 3-(4-chlorophenoxy)phenyl 218.4±9.4 154.1±7.3 204.3±4.6 407.1±11.0 3p benzyl 14.0±2.0 26.3±2.1 56.1±2.0 67.1±3.3 3q 2-furyl 78.24±8.6 32.2±1.4 44.1±4.2 74.3±2.1 3r 4-methoxyphenyl 103.0±11.1 100.2±5.1 115.4±9.5 233.5±10.9 3s 3-fluorophenyl 258.3±4.0 301.2±12.1 195.3±8.4 374.2±8.7 3t 4-cyanophenyl 145.4±8.0 162.0±4.3 184.1±2.2 388.6±6.2 3u 4-bromophenyl 49.0±5.0 79.2±4.1 62.1±4.3 54.7±1.1 Paclitaxel 2.4±1.4 5.5±1.1 12.0±2.1 74.4±3.5 *:µM, mean±SD 869Acta Chim. Slov. 2022, 69, 863–875 Akdağ et al.: Synthesis and Biological Evaluation of Some ... PC-3 cells, compound 3h significantly increased the num- ber of cells going to early apoptosis and late apoptosis, and decreased the number of viable cells compared to the con- trol group (p<0.05, p<0.001, Figure 5a-b). In HT-29 cells, it was observed that compound 3h significantly increased only the number of cells going to late apoptosis and de- creased the number of viable cells compared to the control group (p<0.001, Figure 6a-b). Caspase-3 is an enzyme that is important in the ap- optotic pathway. Caspase-3 is a marker of both intrinsic and extrinsic apoptosis. In the apoptosis of the cell, amino acids in the structure of proteins are targeted by caspase-3 activity, the peptide bonds of the proteins are cut, and the protein that has lost its function cannot perform its function. In this study, caspase-3 activities of compound 3h were measured enzymatically in MCF-7, PC-3, and HT-29 cells. Compound 3h increased caspase-3 enzyme activity in MCF-7 cells compared to the control group (p<0.001, Figure 4c). In PC-3 cells, compound 3h in- creased caspase-3 enzyme activity in 24 hours (p<0.001, Figure 5c). Similarly, compound 3h increased caspase-3 enzyme activity in HT-29 cells compared to the control group (p<0.001, Figure 6c). In this study, among the synthesized compounds, 3h bearing pyrrole ring showed the highest cytotoxic activity against different cancer cell lines such as breast, colon and prostate, it did not show cytotoxic activity against human umbilical vein endothelial cells. Therefore, compound 3h, which can show selective activity against cancer cell lines in this study, was obtained. Figure 1. MCF-7 cells treated with 3.00 µM concentration of compound 3h, a) AO/EB staining in floresance imaging b) AO/EB staining ratio (*** p < 0.001 vs control) Figure 2. PC-3 cells treated with 1.30 µM concentration of compound 3h, a) AO/EB staining in floresance imaging b) AO/EB staining ratio (*** p < 0.001 vs control) 870 Acta Chim. Slov. 2022, 69, 863–875 Akdağ et al.: Synthesis and Biological Evaluation of Some ... In addition to the effectiveness of a compound in the fight against cancer, it is also very important that it exhibits selective activity. There are many examples in the literature where the non-selective compound showing ac- tivity against the cancer cell line was not included in fur- ther studies.25,26 Pena-Moran et al. reported that the com- pound with an SI value ≥ 10 belongs to a selected potential compound that can be investigated further.27 On the other hand, Weerapreeyakul et al. suggested a lower SI value (≥ 3) for classification of a possible anti-cancer compound.28 Figure 3. HT-29 cells treated with 1.70 µM concentration of compound 3h, a) AO/EB staining in floresance imaging b) AO/EB staining ratio (*** p < 0.001 vs control) Figure 4. Apoptosis profile for MCF-7 cells treated with compound 3h (3.00 µM) a) Annexin-V binding b) The percentage of live, early and late apoptosis/dead cells by MUSE cell analyzer c) Caspase‐3 activity (* p < 0.05, *** p < 0.001 vs control) 871Acta Chim. Slov. 2022, 69, 863–875 Akdağ et al.: Synthesis and Biological Evaluation of Some ... Evaluation of the anti-cancer activity of a compound us- ing only malignant cell lines without SI determination has been reported to be a poor predictor for further (clinical) study.29 For example colchicine exhibits very high cytotox- ic effects. Unfortunately, colchicine is too toxic to be used as an antitumor agent.30 As a result, the selectivity index of compound 3h was found at 181.0 for PC3, 78.4 for MCF-7, and 138.4 for HT-29. The selectivity index of standard drug (Paclitaxel) was found at 31.0 for PC3, 13.5 for MCF-7, and 6.2 for HT-29. The selectivity index of compound 3h was high- er than standard drug. In addition, caspase-3 activity and Annexin-V assay were performed to explain the activity mechanism of compound 3h. Dose response curves of five compounds (3c, 3d, 3h, 3l, 3p and 3q) were also added to the Supporting Information (Figure S1). 3. 3. In silico ADME analysis A promising compound must pass in silico analysis before it can be taken up for further studies.31 Therefore, we evaluated the pharmacokinetics, drug-likeness and physicochemical properties of hydrazone derivatives. The drug-likeness was established based on the physicochemi- cal properties to obtain oral drug candidates.32 All data for the calculation were shown in Tables 2 and 3. The SwissAdme web tool allows assessing the prob- ability of a molecule to become an oral drug with respect to bioavailability. There are five different rule-based filters, also defined below, which can be calculated with the SwissAdme pro- gram:33 (i) Lipinski’s rule of five includes molecular weight ≤ 500, MLOGP (lipophilicity) ≤ 4.15, hydrogen bond donors ≤ 5 and hydrogen bond acceptors ≤ 10. (ii) Ghose’s filter includes 160 ≤ molecular weight ≤ 480, −0.4 ≤ WLOGP (lipophilicity) ≤ 5.6, 40 ≤ the molar refractivity ≤ 130, and 20 ≤ number of atoms ≤ 70. (iii) Veber’s rule includes the number of rotatable bonds ≤ 10 and the total polar surface area ≤ 140. (iv) Egan’s filter includes WLOGP (Lipophilicity) ≤ 5.88 and the total polar surface area ≤ 131.6. (v) Muegge’s filter includes 200 ≤ molecular weight ≤ 600, −2 ≤ XLOGP (lipophilicity) ≤ 5, the total polar surface area ≤ 150, the number of rings ≤ 7, the number of car- bon > 4, the number of heteroatoms > 1, the number of rotatable bonds ≤15, the hydrogen bond acceptors ≤ 10, and the hydrogen bond donors ≤ 5. Figure 5. Apoptosis profile for PC-3 cells treated with compound 3h (1.30 µM) a) Annexin-V binding b) The percentage of live, early and late apop- tosis/dead cells by MUSE cell analyzer c) Caspase‐3 activity (* p < 0.05, *** p < 0.001 vs control) 872 Acta Chim. Slov. 2022, 69, 863–875 Akdağ et al.: Synthesis and Biological Evaluation of Some ... The result of the drug-likeness evaluation of hydra- zones (3a–u) was shown in Table 3, and we can conclude that: (i) Lipinski, Veber, Ghose, Egan and Muegge filters of all compounds were within the accepted range except compounds 3j and 3o. (ii) The bioavailability score is 0.55 for all compounds, meaning good bioavailability. On the other hand, solubility is an another important property related to drug absorption by assessing whether the drug is soluble or moderately soluble. Furthermore, one of the prerequisites for the compound to show activ- ity is that it should have good solubility. 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Karasungur, M. Simsir, L. C. Kumruoglu, I. Karakaya, J Sustain Metall, 2021, 7, 1224–1240. DOI:10.1007/s40831-021-00406-7 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 Raziskava se osredotoča na določitev ekonomsko najbolj optimalnih pogojev za pridobitev polistirena z različnimi cil- jnimi molekulskimi masami z ultrazvočno podprto emulzijsko polimerizacijo. Šaržne polimerizacije stirena z ultraz- vočno podprtim postopkom emulzijske polimerizacije so bile izvedene pri različnih sestavah reakcijskih komponent. Hitrost polimerizacije je bila izračunana na osnovi pretvorbe monomera pri različnih reakcijskih časih. Molekulske mase sintetiziranih polimerov in Mark-Houwinkove konstante so bile določene z meritvami intrinzične viskoznosti in gelske izključitvene kromatografije. Ugotovljeno je bilo, da je indeks polidisperznosti polimerov v razponu od 1,2 do 1,5, povprečne molekulske mase viskoznosti pa so med 100. 000–1.500.000 g/mol, odvisno od reakcijskih pogojev. Na koncu so bile razvite tudi modelne enačbe za določitev glavnih spremenljivk, najbolj ekonomični načini za doseganje različnih ciljnih molekulskih mas pa so bili razloženi z metodologijo odzivne površine, ki temelji na optimizaciji več ciljev. 896 Acta Chim. Slov. 2022, 69, 896–904 Xu et al.: The Paramagnetic or Spin Crossover Iron(III) ... DOI: 10.17344/acsi.2022.7680 Scientific paper The Paramagnetic or Spin Crossover Iron(III) Complexes Based-on Pentadentate Schiff Base Ligand: Crystal Structure, and Magnetic Property Investigation Zhijie Xu, Shuo Meng, Tong Cao, Yu Xin, Mingjian Zhang, Xiaoyi Duan, Zhen Zhou and Daopeng Zhang* College of Chemical and Chemical Engineering, Shandong University of Technology, Zibo 255049, PR China * Corresponding author: E-mail: dpzhang73@126.com Received: 07-12-2022 Abstract A series of bi- or mononuclear hexacoordinate iron(III) complexes, [Fe(L)][Fe(bpb)(CN)2]·CH3OH·0.5H2O (1), [Fe(L)] [Co(bpb)(CN)2]·CH3OH (2) [(Fe (L))2(4,4’-bipy)](BPh4)2 (3), [Fe(L)(py)](BPh4) (4) and [Fe(L)(dmap)](BPh4) (5) (bpb = 1,2-bis(pyridine-2-carboxamido)benzenate, L = N,N’-bis(2-hydroxybenzyliden)-1,7-diamino-4-azaheptane, dmap = 4-dimethylaminopyridine), have been prepared with the pentadentate Schiff base iron(III) compound as assemble pre- cursor and characterized by element analysis, IR and X-ray diffraction. Single crystal structural determination revealed the neutral cyanide-bridged binuclear entity for complexes 1 and 2 and the cationic di- or mononuclear structure for complexes 3–5 with the positive charge(s) balanced by BPh4– ion(s). The experimental study and theoretical simulation of the magnetic property discovered the ferromagnetic coupling between the Fe(III) ions bridged by cyanide group in complex 1 and the always high spin state of the Fe(III) ion coordinated to the Schiff base ligand in both complexes 1 and 2. The temperature dependent magnetic susceptibility investigation over complexes 3–5 showed the occurrence of the thermo-induced gradual complete spin crossover (SCO) property at about 115, 170 and 200 K, respectively. Keywords: Cyanide-bridged, Crystal structure, Ferromagnetic coupling, Spin crossover Introduction Since the 21st century, the synthesis of materials with special functions and applications based on the coordina- tion chemistry has been one of the important directions of current chemical research.1–10 Molecular materials with spin crossover behavior have broad application prospects in the fields of nano device, spintronics, information stor- age, sensors, digital display, and so on.11–18 The use of spin crossover complexes as switches or sensors depent on spin transition distinguished by different magnetic, optical and structural characteristics, which included structural chang- es between two different spin states and could be realized under the suitable coordination field by external stimuli such as temperature, light irradiation and the guest solvent molecules.19–25 It was generally found that the metal centers with the 3d4–3d7 electronic configurations and involved in octahedral coordination surroundings could readily occur spin transition between the different spin states.26–28 Among 3d4–3d7 metal ions, the strong interest has always been devoted to the switchable molecular materials centered with iron(II) ion. During the past several dec- ades, a great deal of the Fe(II)-based complexes with vari- ous structure types from 0D clusters, 1D infinite chains to 2–3D networks and interesting SCO properties have been reported. However, with comparison to the intensely stud- ied divalent Fe(II) in SCO field, research on Fe(III) ion- based SCO material is comparatively limited,29–33 as is also with the SCO possibility between the high spin S = 5/2 and low spin S = 1/2. The pentadentate Schiff base ligands (Scheme 1), which can encapsulate the hexa-coordinated metal with sixth position weakly bonded to other ligand, are a type of precursor for preparation function complex- es.34–36 In fact, some previous reports have proven that the above pentadentate Schiff base based iron(III) compounds are suitable candidates for assembling SCO materials un- der the help of the pyridine-like ligands37,38 and some of the obtained complexes have been structurally character- ized and experimentally magnetic investigated. Taking into account that the cyanometallic precursors with tuna- ble coordination field, such as [M(CN)4]2– (M = Ni, Pd, 897Acta Chim. Slov. 2022, 69, 896–904 Xu et al.: The Paramagnetic or Spin Crossover Iron(III) ... Pt), [M’(CN)2]– (M’ = Cu, Ag, Au) and other polycyano- metallates, have been widely employed to construct SCO materials, especially for those ones with Hoffman topolog- ic structures,39–42 the reactions of the pentadentate Schiff base iron(III) compound with the trans-dicyanoiron(III)/ cobalt(III) building blocks (Scheme 1), for the latter which has been extensively used to prepare cyanide-bridged mo- lecular magnetic materials by our group,43–46 were investi- gated, resulting in two new binuclear homo- or heterome- tallic complexes formulated as [Fe(L)][Fe(bpb)(CN)2] CH3OH 0.5H2O (1), [Fe(L)][Co(bpb)(CN)2] CH3OH (2) (bpb = 1,2-bis(pyridine-2-carboxamido)benzenate, L = saldptn = N,N’-bis(2-hydroxybenzyliden)-1,7-diamino- 4-azaheptane).47,48 At the same time, by using 4,4’-bipy,49 py or dmap (dmap = 4-dimethylaminopyridine) as ancil- lary ligand, three monometallic Fe(III) complexes with the formula [(Fe(L))2(4,4’-bipy)](BPh4)2 (3), [Fe(L)(py)] (BPh4) (4) and [Fe(L)(dmap)](BPh4) (5) have been suc- cessfully obtained. The synthesis, crystal structures and magnetic properties for the reported complexes will be de- tailed described in this paper. tum Design MPMS SQUID magnetometer. The experi- mental susceptibilities were corrected for the diamag- netism of the constituent atoms (Pascal’stables). General procedures and materials. All the reac- tions were carried out under air atmosphere and all chem- icals and solvents used were reagent grade without further purification. H2L, [Fe(L)]Cl, K[Fe(bpb)(CN)2] and K[Co(bpb)(CN)2] were prepared to accord to experimen- tal methods already described.50–52 Caution! KCN is hypertoxic and should be handled in small quantities with great care. 2. 1. The Preparation of the Complexes 1 and 2 A solution containing K[Fe(bpb)(CN)2] (0.10 mmol, 46.5 mg) or K[Co(bpb)(CN)2] (0.10 mmol, 46.7 mg) dis- solved in methanol (10 mL) was slowly added into the methanol-water (10 mL, 4:1 v/v) solution of [Fe(L)]Cl (43 mg, 0.10 mmol) under the stirring. The mixture was stirred at room temperature for several minutes and filtered to re- move any insoluble substances. Then, the filtrate was al- lowed for slow evaporation without interference for about two weeks. Dark brown crystals suitable for X-ray diffrac- tion were collected by filtration, washed with cold metha- nol and dried in air. Complex 1: Yield: 52.9 mg, 61.7%. Anal. Calcd. For C41H40Fe2N9O5: C, 57.36; H, 4.70; N, 14.68. Found: C, 57.47; H, 4.62; N, 14.79. Main IR bands (cm–1): 2160, 2120 (s, νC–N), 1630 (vs νC=N), 3056, 2846, 2660 (w, νC–H), 1444, 1401, 1279 (s, νC–O). Complex 2: Yield 53.9 mg, 63.3%. Anal. Calcd. For C41H39CoFeN9O5: C, 57.76; H, 4.61; N, 14.79. Found: C, 57.87; H, 4.52; N, 14.89. Main IR bands (cm–1): 2158, 2122 (s, νC–N), 1630 (vs νC=N), 3059, 2855, 2656 (w, νC–H), 1458, 1387, 1275 (s, νC–O). 2. 2. The Preparation of Complexes 3–5 To a methanol solution of [Fe(L)]Cl (43 mg, 0.10 mmol) was added 4,4’-bipy(7 mg, 0.05 mmol) or py (8 mg, 0.1 mmol) or dmap (12 mg, 0.1 mmol). The mixture was stirred for about ten minutes at 60 °C before an excess of sodium tetraphenylborate (855 mg, 2.5 mmol) was added. After the insoluble substances were filtered out, the filtrate was partial evaporated and the crystals obtained was col- lected and washed with methanol and ether. Complex 3: Yield 48.0 mg, 60.7 %. Anal. Calcd. for C98H94B2Fe2N8O4: C, 74.44; H, 5.99; N, 7.09. Found: C, 74.52; H, 5.93; N, 7.15. Main IR bands (cm–1): 1633 (vs νC=N), 732, 708 (νB–C), 3057, 2978 (w, νC–H), 1251 (s, νC–O). Anal. Complex 4: Yield 42.0 mg, 53.1 %. Anal. Calcd. for C49H48BFeN4O2: C, 74.34; H, 6.11; N, 7.08. Found: C, 74.44; H, 6.01; N, 7.15. Main IR bands (cm–1): 1628 (vs νC=N), 735, 706 (νB–C), 3054, 2977 (w, νC–H), 1252 (s, νC–O). Anal. Scheme 1. Structure of the Schiff base iron(III) precursor and the trans-dicyano building blocks. 2. Experimental Section Elemental analyses (C, H and N) were carried out with a VarioEl element analyser. IR spectroscopic analysis on KBr pellets was performed on a Magna-IR 750 spectro- photometer in the 4000–400 cm–1 region. Variable-tem- perature magnetic susceptibility and field-dependent magnetization measurements were performed on a Quan- 898 Acta Chim. Slov. 2022, 69, 896–904 Xu et al.: The Paramagnetic or Spin Crossover Iron(III) ... Complex 5: Yield 42.6 mg, 51.2 %. Anal. Calcd. for C51H52BFeN5O2: C, 73.48; H, 6.29; N, 8.40. Found: C, 73.54; H, 6.19; N, 8.54. Main IR bands (cm–1): 1630 (vs νC=N), 733, 707 (νB–C), 3050, 2975 (w, νC–H), 1245 (s, νC–O). Anal. 2. 3. X-ray Data Collection and Structure Refinement Single crystals of all the complexes for X-ray diffrac- tion analyses with suitable dimensions were mounted on a glass rod and the crystal data were collected on a Bruker SMART CCD diffractometer with a Mo Kα sealed tube (λ = 0.71073 Å) at 293 K, using a ω scan mode. For complex- es 3 and 4, their structures have been further measured at about 120 K. The structures were solved by direct methods and expanded to use Fourier difference techniques with the SHELXTL-97 program package. The non-hydrogen at- oms were refined anisotropically, while hydrogens were introduced as fixed contributors. All non-hydrogen atoms were refined with anisotropic displacement coefficients. Hydrogens were assigned isotropic displacement coeffi- cients U(H) = 1.2U(C) or 1.5U(C), and their coordinates were allowed riding on their respective carbons using SHELXL-2018, except some hydrogens of solvent mole- cules, which were refined isotropically with fixed U values and the DFIX command was used to rationalize the bond parameters. CCDC 2169018–2169022 for complexes 1–5 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc. cam.ac.uk/data_request/cif.Details of the crystal parame- ters, data collection, and refinement are summarized in tables 1 and 2. Table 1. Crystallographic data for complexes 1, 2 and 5. 1 (293k) 2 (293k) 5 (293k) Chemical formula C41H40Fe2N9O5.5 C41H39CoFeN9O5 C51H53BFeN5O2 Fw 858.52 852.59 834.64 Crystal system Monoclinic Monoclinic Monoclinic Space group P21/c P21 P21/n a/Å 10.8753(10) 10.875(7) 18.3588(16) b/Å 10.0446(9) 10.065(6) 14.5766(13) c/Å 36.595(3) 18.331(11) 18.5738(16) α/deg 90.0 90.0 90 β/deg 92.349(2) 95.586(11) 117.698(2) γ/deg 90.0 90.0 90 Z 2 2 4 V/Å3 3994.2(6) 1997(2) 4400.9(7) F(000) 1780.0 882.0 1764.0 GOF 1.030 1.028 1.024 R1 [I >2σ(I)] 0.1241 0.073 0.1180 wR2 (all data) 0.1435 0.1901 0.1455 Table 2. Crystallographic data for complexes 3 and 4. 3 (293k) 3 (120k) 4 (293k) 4 (120k) Chemical formula C49H48BFeN4O2 C49H48BFeN4O2 C98H94B2Fe2N8O4 C98H94B2Fe2N8O4 Fw 791.57 791.57 1581.13 1581.13 Crystal system Monoclinic Monoclinic Monoclinic Monoclinic Space group P21/c P21/c C2/c C2/c a/Å 18.373(2) 18.295(4) 33.480(2) 33.1757(16) b/Å 11.9535(13) 11.880(2) 16.7773(11) 16.7010(6) c/Å 21.064(2) 20.678(4) 16.3933(11) 16.0077(6) α/deg 90.0 90.0 90.0 90.0 β/deg 114.530(2) 114.60(3) 117.094(1) 116.567(5) γ/deg 90.0 90.0 90.0 90.0 Z 4 4 4 4 V/Å3 4208.6(8) 4086.5(4) 8197.6(10) 7932.9(6) F(000) 1668.0 1668.0 3328.0 3328.0 GOF 1.006 1.028 1.005 1.027 R1 [I >2σ(I)] 0.0415 0.0462 0.1159 0.0607 wR2 (all data) 0.1086 0.0793 0.1394 0.1503 899Acta Chim. Slov. 2022, 69, 896–904 Xu et al.: The Paramagnetic or Spin Crossover Iron(III) ... 3 Results and Discussion 3. 1. Crystal Structure Description 3. 1. 1 Crystal Structures of Complexes 1 and 2 The selected important structural parameters for complexes 1 and 2 are collected in Table 3. The neutral bi- nuclear and the 1D supramolecular single chain structure constructed by the hydrogen bond interactions for these two complexes are shown in Figure 1. The structures of complexes 1 and 2, crystallizing in space group P21/c and P21 containing four and two inde- pendent units in the unit cell, respectively, belong to neu- tral binuclear entity. The [Fe/Co(bpb)(CN)2]- anion acting as monodentate ligand connects the [Fe(L)]+ cation through one cyanide group with the other trans one termi- nal, therefore forming neutral binuclear dimer. The FeIII/ CoIII in the cyano precursors with low spin state is coordi- nated by an equatorial N4 unit from bpb and two carbons of cyanide groups in the trans position. The FeIII/CoIII–N bond lengths were slightly longer than the FeIII/CoIII–Ccya- nide ones (1.923(8)–1.976(5) Å), revealing a slightly distort- ed octahedral geometry around the metal ions. As listed in Table 3, the bond angles of FeIII/CoIII–C–N very close to Figure 1. The neutral binuclear structures of complexes 1 and 2 (top) and the 1D supramolecular chain constructed by H-bond interactions (bot- tom). All the H atoms except one used to form H-bond and the solvent content have been omitted for clarity. Table 3. Selected bond lengths (Å) and angles (°) for complexes 1 and 2. Complexes Fe Co1 Fe2–O1 1.911(3) 1.918(5) Fe2–O2 1.927(3) 1.900(5) Fe2–N1 2.134(4) 2.141(7) Fe2–N7 2.098(4) 2.087(7) Fe2–N8 2.201(4) 2.202(7) Fe2–N9 2.108(4) 2.115(8) Fe1/Co1–C1 1.976(5) 1.923(8) Fe1/Co1–C2 1.951(5) 1.893(8) Fe1/Co1–N3 1.879(4) 1.894(6) Fe1/Co1–N4 1.891(4) 1.885(6) Fe1/Co1–N5 1.933(4) 1.960(5) Fe1/Co1–N6 2.004(4) 1.963(6) C1–Fe1/Co1–N6 90.56(16) 88.0(3) O1–Fe2–N9 89.66(14) 95.1(3) N1–C1–Fe1/Co1 172.5(4) 173.9(6) C2–Fe1/Co1–C1 168.93(19) 174.3(3) O2–Fe2–N1 179.01(15) 179.2(3) N2–C2–Fe1/Co1 175.2(5) 174.1(7) N3–Fe1/Co1–N4 83.26(19) 82.4(3) N4–Fe1/Co1–C1 91.47(11) 90.0(3) 900 Acta Chim. Slov. 2022, 69, 896–904 Xu et al.: The Paramagnetic or Spin Crossover Iron(III) ... 180° clearly indicated that the three atoms were with pre- fect linear conformation. The coordination sphere of FeIII ion in [Fe(L)]+ is six-coordinated octahedron, in which the four equatorial positions are occupied by three N atoms and one O atom from the Schiff base ligand and the two axial ones coordi- nated by one O atom of the Schiff base ligand and one N atom of the bridge cyanide group. The averaged Fe–Ncya- nide and Fe–NSchiff base bond lengths in complexes 1 and 2 are 2.134(4), 2.141(7), 2.136(4) and 2.147(7) Å, respective- ly, longer than the averaged Fe–OSchiff base bond with the values of 1.919(3) and 1.909(5) Å, demonstrating the obvi- ously distorted octahedral geometry around the Fe(III) ion in [Fe(L)]+ unit. These bond lengths are in good agree- ment with the corresponding bond lengths around the high spin Fe(III) ion found in the reported complexes,53 indicative of the high spin state of Fe(III) ions involved in the Schiff base precursor in these two complexes. Different from the perfect linear FeIII/CoIII–C–N unit, the C–N–FeI- II/CoIII bond angles are some bent with the values of 172.2(5) and 169.7(6)°, respectively. With the help of the intermolecular N–H···N hydrogen bond interactions, the binuclear entity can be further constructed into supramo- lecular 1D infinite structure. 3. 1. 2 Crystal Structures of Complexes 3–5 Some important bond parameters for complexes 3–5 are given in Table 4. The cationic bi- or mononuclear Figure 3. The cell packing diagram of complex 3 along b axial. All the H atoms have been omitted for clarity. Figure 2. The cationic binuclear structure of complex 3. All the H atoms and the balanced anions have been omitted for clarity. Table 4. Selected bond lengths (Å) and angles (°) for complexes 3–5 at room and low temperature Complex 3 (293K) (120K) Fe1–O1 1.889(5) 1.8725(5) Fe1–O2 1.895(5) 1.8827(5) Fe1–N1 2.094(7) 2.012(7) Fe1–N2 1.999(7) 1.952(7) Fe1–N3 2.083(6) 2.01(6) Fe1–N4 2.007(6) 1.961(6) O1–Fe1–N1 88.34(9) 87.42(11) O1–Fe1–O2 174.20(10) 175.91(11) O2–Fe1–N2 92.72(11) 87.87(13) O2–Fe1–N3 91.66(11) 93.16(12) O2–Fe1–N4 88.58(12) 91.57(13) N2–Fe1–N4 168.15(12) 173.79(13) N3–Fe1–N1 177.43(10) 178.29(13) Complex 4 (293K) (120K) Fe1–O1 1.889(5) 1.8725(17) Fe1–O2 1.895(5) 1.8827(18) Fe1–N1 2.094(7) 2.012(2) Fe1–N2 1.999(7) 1.961(2) Fe1–N3 2.083(6) 2.020(2) Fe1–N4 2.007(6) 1.952(2) O1–Fe1–N1 88.2(3) 88.77(8) O2–Fe1–O1 175.3(2) 176.04(8) O2–Fe1–N2 92.2(3) 91.26(8) O2–Fe1–N3 90.7(2) 90.53(8) O2–Fe1–N4 89.3(2) 89.47(8) N3–Fe1–N1 177.6(3) 177.42(9) N4–Fe1–N1 94.3(3) 91.27(8) Complex 5 (293K) Fe1–O1 1.883(2) Fe1–O2 1.901(2) Fe1–N1 2.011(3) Fe1–N3 1.996(3) Fe1–N4 2.060(4) Fe1–N5 1.985(3) O1–Fe1–N1 92.09(11) O1–Fe1–N5 90.63(12) O1–Fe1–O2 179.06 N1–Fe1–N4 179.95(19) O2–Fe1–N3 91.31(12) O2–Fe1–N4 91.30(13) N3–Fe1–N1 91.52(12) N3–Fe1–N5 176.01(13) C24–N4–Fe1 116.8(4) C25–N4–Fe1 117.6(4) 901Acta Chim. Slov. 2022, 69, 896–904 Xu et al.: The Paramagnetic or Spin Crossover Iron(III) ... structures are presented in Figure 2 and the cell packing diagram with complex 3 as representative is shown in Fig- ure 3. As can be found, the three complexes, which crystal- lizes in C2/c, P21/c and P21/n space group, respectively, are composed by cationic bi- or mononuclear entity and the balanced BPh4– anion(s). For complex 3, the asymmetric unit contains only half the dinuclear molecule. In com- plexes 3–5, the coordination geometry of the Fe(III) ion is octahedron, in which the equatorial plane is connected by the N2O2 unit from the Schiff base ligand, different from that in complexes 1 and 2, and the two axial positions are occupied by the N atom of the Schiff base ligand and the N atom of the pyridine ligand. It should be pointed out that the configuration of the Schiff base ligand in these three complexes are obviously different from that in complexes 1 and 2, implying maybe the different spin state of the Fe(I- II) ion. Additionally, there exist weak π-π stacking interac- tions in complex 3 between the pyridine ring and the ben- Figure 4. The cationic mononuclear structure of complexes 4 (top) and 5 (bottom). All the H atoms and the balanced anion have been omitted for clarity. zene ring of the BPh4– with center-center distance of 3.74(7) Å. To further confirm the spin transition, the structures of complexes 3 and 4 have been measured at low tempera- ture (120 K). As tabulated in Table 4, the cell volume of these two complexes in the low temperature contracted obviously from 8197.68 to 7932.85 Å3 for 3 and 4208.58 to 4086.34 Å3 for 4, respectively. The averaged Fe–N and Fe–O bond lengths at low temperature are 1.984(7) Å, 1.993(5) Å, 1.878(5) Å, 1.867(7) Å, which are conspicu- ously shorter than the room temperature ones with the values 2.045(7) and 2.099(2) Å, indicating different spin state of the Fe(III) ion in these complexes. The compara- tively smaller difference for complex 4 can be attributed to the mixed spin state of Fe(III) ion at room temperature, proved also by the magnetic property (vide infra). 3.2 Magnetic Properties of Complexes 1–5. The temperature dependent magnetic susceptibili- ties of the five complexes were measured by using the cor- responding single crystal with quality about 10–20 mg in the range of 2–300 K under an external magnetic field of 2000 Oe. The room temperature χmT values of complexes 1 and 2 (Figure 5) are 4.78 and 4.29 emu K mol–1, respec- tively, which are basically consistent with the spin only val- ue 4.75 and 4.375 emu K mol–1 for the free low spin Fe(III) and high spin Fe(III) ion or the only high spin Fe(III) ion (the cyanide precursor in complex 2 is diamagnetic), re- spectively. For complex 1, the χmT value increases with very low speed and attain the highest peak about 5.23 emu K mol–1 with the temperature decreasing to about 10 K, and then decreases at a high speed and reaches the value of about 3.85 emu K mol–1 at 2 K. The χmT-T change tenden- cy can preliminarily confirm the ferromagnetic coupling between the high spin and low spin Fe(III) ion through the cyanide bridge. For complex 2, with temperature decreas- ing from 300 K, the χmT value keeps always constant until the sample is cooled to about 20 K, and since then the χmT began to decrease obviously and reaches the lowest value about 3.23 emu K mol–1 at 2 K, demonstrating the always high spin state of the Fe(III) ion in this complex. The ferromagnetic coupling observed in complex 1 can be understood by the orthogonality between the t2g and eg magnetic orbital of high and low spin Fe(III) ions through the bridging cyanide group. On the basis of the binuclear model, the magnetic susceptibility of complex 1 can be fitted accordingly by the following expression de- rived from the exchange spin Hamiltonian Ĥ = –JŜFe(HS) ŜFe(LS): (1) By using the above model, the susceptibility over the temperature range of 10–300 K was simulated, giving the best-fit parameters J = 1.47(1), g = 2.01(2), R = ∑(cobsdT – 902 Acta Chim. Slov. 2022, 69, 896–904 Xu et al.: The Paramagnetic or Spin Crossover Iron(III) ... ccaldT)2/∑(cobsdT)2 = 4.37 × 10–5. The small positive J value indicates also the ferromagnetic interaction in complex 1. To further confirm the magnetic coupling nature in com- plex 1, the field-dependent magnetization measured up to 50 kOe at 2 K was carried out. The experimental M-H curve (Inset of Figure 5) are basically consistent with the calculated Brillouin function corresponding to the ferro- magnetic coupled low spin Fe(III) (S = 1/2) ion and high spin Fe(III) (S = 5/2) ion with g = 2.0 at 2 K. The saturated magnetization value is about 5.89 Nβ, which is very close to the expected theoretical value (6.0 Nβ), indicating fur- ther the existed overall weak ferromagnetic interactions between the adjacent Fe(III) ions. Figure 5. Temperature dependence of χmT of 1 (top) and 2(bottom). Inset: Field dependence of magnetization at 2 K of complex 1(the solid Brillouin curve is the ferromagnetic coupled one low spin Fe(III) ion and one high spin Fe(III) ion for complex 1). The χmT-T curves of complexes 3-5 are shown in Figure 6 and Figure S1 (Support Information). The room temperature χmT value for complex 3 is about 7.4 emu K mol–1, indicating the two Fe(III) ions bridged by the bi- pyridine ligand are with almost complete high spin state. For complexes 4 and 5, the χmT values at 300 K are only 1.82 and 1.41 emu K mol–1, providing clear information that the stable high spin state of the Fe(III) ions in these two complexes outclass the room temperature. With the temperature lowering, the χmT for all the three complexes decreases with a rapid speed to about 1.33, 0.61 and 0.55 cm3 K mol–1 at about 115, 170 and 200 K, respectively, showing the occurrence of a gradually almost complete spin transition. After that, the χmT values keep almost con- stant with the temperature cooling to 2 K, indicating the stable low spin state at low temperature for these three complexes. Such types of the χmT-T change tendency can also be found in the reported examples based on the simi- lar pentadentate Schiff base Fe(III) precursor and pyri- dine-like ligands. Figure 6. Temperature dependence of χmT of 3 4. Conclusion In summary, for the purpose of preparation of Fe(I- II)-based spin crossover molecular magnetic materials, the reactions of pentadentate Schiff base based Fe(III) com- pounds with the trans-dicyanometallates or the pyri- dine-type ligands were investigated, leading to cyano pre- cursor or 4,4’-bipy bridged binuclear and pyridine coordinated mononuclear complexes. The study over the temperature dependence of the magnetic susceptibility and the field-dependent magnetization revealed the ferro- magnetic coupling in cyanide-bridged Fe(III)(low spin)-Fe(III)(high spin) complex. For the pyridine-like li- gand involved mono- or binuclear Fe(III) complexes, the thermo-induced gradual complete spin crossover behavior occurring at different temperatures could be found, re- vealed the important role of the auxiliary ligand with dif- ferent coordination fields, which provided meaningful in- formation for the future design of SCO magnetic material. Acknowledgement This work was supported by the Natural Science Foundation of China (22171166). All authors disclosed no relevant relationships. 903Acta Chim. Slov. 2022, 69, 896–904 Xu et al.: The Paramagnetic or Spin Crossover Iron(III) ... 5. References 1. P. Wang, Y. Xu, Q. Lin and M. Lu, Chem. Soc. Rev. 2018, 20, 7522–7538. DOI:10.1039/C8CS00372F 2. P. He, J. G. Zhang, X. Yin, J. T. Wu, L. Wu, Z. N. Zhou and T. L. Zhang, Chem. Eur. J. 2016, 22, 7670–7685. DOI:10.1002/chem.201600257 3. D. K. Mahapatra, S. K. Bharti, V. Asati and S. K. Singh, Eur. J. Med. Chem. 2019, 174, 142–158. DOI:10.1016/j.ejmech.2019.04.032 4. J. 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DOI:10.1021/ic2027708 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 Serija dvo- ali enojedernih heksakoordiniranih kompleksov železa(III), [Fe(L)][Fe(bpb)(CN)2]·CH3OH·0.5H2O (1), [Fe(L)][Co(bpb)(CN)2]·CH3OH (2) [(Fe(L))2(4,4‘-bipy)](BPh4)2 (3), [Fe(L)(py)](BPh4) (4) in [Fe(L)(dmap)](BPh4) (5) (bpb = 1,2-bis(piridin-2-karboksamido)benzenat, L = N,N‘-bis(2-hidroksibenziliden)-1, 7-diamino-4-azaheptan, dmap = 4-dimetilaminopiridin), je bila pripravljena iz železovega(III) kompleksa s pentadentatno Schiffovo bazo kot prekurzorjem ter okarakterizirana z elementno analizo, IR in rentgensko difrakcijo. Strukturna analiza monokristalov je razkrila nevtralno cianidno vezano dvojedrno zvrst za kompleksa 1 in 2 ter kationsko dvo- ali enojedrno strukturo za komplekse 3–5 s pozitivnim nabojem ter BPh4– protiionom. Eksperimenti in teoretične simulacije magnetnih lastnosti razkrijejo feromagnetno sklopitev med Fe(III) ioni, ki jih povezuje cianidna skupina v kompleksu 1, in visokospinsko stanje Fe(III) iona, koordiniranega z ligandom Schiffove baze v kompleksih 1 in 2. Raziskava magnetne susceptibilnosti kompleksov 3–5 v odvisnosti od temperature je pokazala, da se pri približno 115, 170 in 200 K pojavi toplotno-induciran popoln spinski prehod. 905Acta Chim. Slov. 2022, 69, 906–912 Ali et al.: Zinc(II) Complex Containing Oxazole Ring: Synthesis, ... DOI: 10.17344/acsi.2022.7682 Scientific paper Zinc(II) Complex Containing Oxazole Ring: Synthesis, Crystal Structure, Characterization, DFT Calculations, and Hirshfeld Surface Analysis Karwan Omer Ali,1,* Hikmat Ali Mohamad,2 Thomas Gerber3 and Eric Hosten3 1 Department of Physics, College of Science, University of Halabja, Halabja 46018, Iraq 2 Department of Chemistry, College of Education, Salahaddin University, Erbil 44001, Iraq 3 Department of Chemistry, Faculty of Science, Nelson Mandela University, Port Elizabeth 6031, South Africa * Corresponding author: E-mail: karwan.ali@uoh.edu.iq Phone No. 009647503849284 Received: 07-12-2022 Abstract A new complex of Zn(II), with 5-chloro-2-methylbenzoxazole ligand (L), has been synthesized by the reaction of zinc dichloride with the ligand (L= C8H6ClNO) in ethanol solution: dichloridobis(5-chloro-2-methyl-1,3-benzoxaz- ole)-zinc(II), C16H12Cl4N2O2Zn. The synthesized complex has been fully characterized by elemental analysis, molar con- ductivity, FT-IR, UV-Vis, and single-crystal X-ray diffraction (XRD). The XRD analysis reveals that the complex has a 1:2 metal-to-ligand ratio. The zinc(II) complex has a distorted tetrahedral geometry with two coordinated nitrogen atoms from the ligand. Density Functional Theory (DFT) calculations were performed at the B3LYP level of theory using the LANL2DZ basis set for metal complex and the 6–31G(d) basis set for non-metal elements to determine the optimum ge- ometry structure of the complex, and the calculated HOMO and LUMO orbital energies were presented. A natural bond orbital (NBO) analysis was carried out on the molecules to analyze the atomic charge distribution before and after the complexation of the ligand. The Hirshfeld surface mapped over dnorm, shape index, and curvature exhibited strong H... Cl/Cl...H and H...H intermolecular interactions as the principal contributors to crystal packing. Keywords: Zn(II), Benzoxazole, Distorted tetrahedral geometry, Hirshfeld surface analysis, NBO analysis, DFT calcu- lations 1. Introduction Benzoxazole is a bicyclic heterocyclic compound containing both oxygen and nitrogen atoms in which the benzene ring is fused to a 1,3-oxazole ring at po- sitions 4 and 5.1 It is one of the most common heter- ocyclics in industry and scientific research.2 Transition metal ions have different binding forces with N and O atoms.3 Commonly, N-donor oxazole groups have demonstrated excellent coordination ability with the first-row transition metal ions.4 Due to the variety of coordination modes and configurations, N-heterocy- clic ligands are typically used as neutral ligands in the synthesis of metal complexes.5,6 Counterions are used to balance the total charge when studying the neutral ligand, which not only affects the coordination modes of the metal ions but also the entire geometry of the met- al complex.7,8 Most Zn(II) complexes show tetrahedral and distorted tetrahedral coordination geometries, in agreement with a d10 electronic configuration.9,10 Thus, the strategy of using neutral mono and bidentate lig- ands with metal halides to force tetrahedral geometry has been widely used for stabilizing Zn(II) complexes. Similarly, benzoxazole derivatives have also been used to stabilize zinc in a +2 oxidation state.11 Because of their good emission properties and inexpensive cost com- pared to other d10 metal complexes, zinc (II) complexes have been shown to be important candidates for elec- troluminescent applications. For example, the Zinc(II) complex of [(2-(2-hydroxyphenyl)benzoxazole)(2-me- thyl-8-hydroxyquinoline)] has been recognized as a blue-emitting zinc complex to fabricate stacked organic light-emitting diodes.12 Changes in the intermolecular interactions between metal ions and ligands that are 906 Acta Chim. Slov. 2022, 69, 906–912 Ali et al.: Zinc(II) Complex Containing Oxazole Ring: Synthesis, ... studied by the Hirshfeld surface analysis and DFT cal- culations can be seen in a series of Zinc(II) complexes with 2-(4-imidazolyl)-4-methyl-1,2-quinazoline-N3-ox- ide,13 2-cetylpyridinenicotinichydrazone,14 and sul- famethoxazole,15 etc. Nonetheless, data on X-ray crystal structures and theoretical studies of related complexes containing an oxazole ring are scarce. The current work reports the synthesis and characterization of a new zinc(II) complex based on a 5-chloro-2-methylbenzox- azole ligand (L), which was characterized by elemental analysis, molar conductivity, FT-IR, UV-Vis, and X-ray analysis. Furthermore, the crystal structure was verified using Hirshfeld surface analysis, and it is helpful for un- derstanding the intermolecular forces in crystal pack- ing. In addition, DFT calculations were done to predict the electronic and geometrical structure of the complex. 2. Experimental 2. 1. Materials and General Methods The solvents used in this study (Ethanol 99% and dimethyl sulfoxide 99.8%) were purchased from Alfa Aesar and used without further purification. 5-Chloro-2-methylbenzoxazole produced by Sigma Aldrich was used without purification. Single-crystal X-ray structure measurement was performed at 200 K using a Bruker Kappa Apex II diffractometer with a ra- diation wavelength of (λ = 0.71073 Å). C, H, N, and O percentages were determined by EURO EA 300 CHNS analyzer. A Shimadzu FT-IR-8400S spectrophotometer was used to record infrared spectra in the 4000–400 cm–1 range as KBr discs and the 600–200 cm–1 range as CsI discs. The UV–visible spectra in DMSO were measured on the AEUV1609 LTD Shimadzu spectrophotometer. The molar conductivities were measured on Meter CON 700 Benchtop conductivity meter using 10–3 M solutions of the complex and ligands in DMSO at room tempera- ture. The melting point was measured using scientific Stu- art SMP3 melting point equipment. 2. 2. Synthesis of [Zn(L)2Cl2] A solution of ZnCl2 (0.284 g, 2.0 mmol) in ethanol (25 mL) was added dropwise under stirring to a solution of 5-chloro-2-methylbenzoxazole (L=C8H6ClNO) ligand (0.670 g, 4.0 mmol) in ethanol (25 mL). Subsequently, the mixture was stirred for 5 hrs. at room temperature with the formation of a clear solution, which was then evap- orated slowly at room temperature to yield pale-yellow crystalline products within one week. m.p. 208–209 °C. Yield: 0.915 g (96%). Anal. Calcd. for C16H12Cl4N2O2Zn: C 41.10, H 2.56, N 5.94, O 6.78. Found: C 41.05, H 2.70, N 5.97, O 6.79. Molar conductivity: 9.11 × 10–5 S cm2 mol–1. FT-IR (KBr) 3091, 3062, 1600, 1562, 1452, 1303, 445, 343 cm–1. UV–Vis data in DMSO [λ/nm, (cm–1)]: 426(23474), 287(34843), 280(35714). 2. 3. X-ray Crystal Structure Determination X-ray diffraction measurement for the Zn(II) com- plex was performed on a Bruker Kappa Apex II X-ray diffractometer equipped with graphite monochromated Mo-Ka radiation (λ = 0.71073 Å) at 200 K. The structure was solved by a dual-space algorithm using SHELXT-2018 and refined by least-squares procedures using the SHELXL-2018/3 crystallographic software.16,17 All C, N, Cl, O, and Zn atoms were anisotropically resolved. The hy- drogen atoms attached to C atoms were allowed to rotate geometrically and treated as a riding model with a C-H distance of  0.95 Å (aromatic) and 0.98 Å (-CH3 group) with Uiso(H) = 1.2 Ueq(C ).18 The crystal data and struc- ture refinement details for the complex are summarized in (Table 1). Table 1. Crystal data and structure refinement of the complex Formula C16H12Cl4N2O2Zn Formula weight 471.47 Temperature, K 200 Wavelength, nm 0.71073 Crystal system Monoclinic Space group P21/n Crystal size, mm 0.52 × 0.53 × 0.67 a / Å 7.5590(5) b / Å 7.1873(5) c / Å 33.842(2) α / ° 90 β / ° 95.101(3) γ / ° 90 V / Å3 1831.3(2) Z 4 Dc / g cm–3 1.710 Absorption coefficient, mm–1 1.937 θ range for data collection, ° 2.4, 28.3 Dataset –10:9; –9:9; –43:45 F 000 944 No. of reflections 4539 No. of parameters 228 Rint 0.027 R1, wR2 0.0405, 0.0921 S 1.28 [I >2σ(I)] 4325 Δρmin, Δρmax / eÅ–3 –0.83, 0.47 2. 4. Computational Details To better understand the structure of the zinc com- plex, the Gaussian 09 software package was used for den- sity functional theory (DFT) calculations. The frontier molecular orbitals of the ligand and the Zn(II) complex were helped by the Gauss View 6.0 software at the B3LYP level of theory. In particular, the LANL2DZ basis set for the zinc metal atom19 and the 6-31G(d)20 basis set for 907Acta Chim. Slov. 2022, 69, 906–912 Ali et al.: Zinc(II) Complex Containing Oxazole Ring: Synthesis, ... non-metal elements (C, H, N, O, and Cl) were both treat- ed. The neutral bond orbital (NBO) analysis of the com- plex was done using the Gaussian 09 program at the same level of theory.21 2. 5. Hirshfeld Surface A crystallographic information file (CIF) obtained from single-crystal X-ray diffraction analysis was used as an input file for Hirshfeld surface visualization of the zinc complex. To generate the Hirshfeld surface analysis and a better understanding of the intermolecular interactions in the complex crystal structure, the program Crystal Explor- er 21.5 was used.22 Hirshfeld surface visualization, pres- entation of results as dnorm, shape index, and curvedness, and calculation of 2D fingerprint plots with dean de and di distances were produced using the same software.23 4. Results and Discussion The zinc (II) complex is formed by the complexation of Zn(II) chloride with the 5-chloro-2-methylbenzoxazole ligand, as shown in (Scheme 1). The complex is stable un- der atmospheric conditions. The complex was produced as pale-yellow crystals at a good yield suitable for sin- gle-crystal X-ray structure analysis. At room temperature, the complex is soluble in common organic solvents such as dimethyl sulfoxide, dimethylformamide, and chloroform, but not in ethanol, acetone, methanol, and petroleum ether. Scheme 1. Synthetic route of the Zn(II) complex. 3. 1. The Crystal Structure Description of the Zn(II) Complex The crystal molecular structure of the [Zn(L)2(Cl)2] complex was depicted in (Figure 1). Relevant bond lengths and bond angles from X-ray diffraction are summarized in (Table 2). Through two nitrogen atoms from the oxazole ring and two chlorine atoms, the Zn(II) metal is located on a crystal lattice center and achieves a slightly distorted tetrahedral coordination geometry. Bond angles of the in- ternal coordination sphere of the complex, which are dif- ferent from the ideal angle of 109° for a perfect tetrahedral geometry, are [(Cl3-Zn1-Cl4) 120.52(3)°, (Cl3-Zn1-N1) 108.88(7)°, (Cl3-Zn1-N2) 104.79(7)°, (Cl4-Zn1-N2) 109.70(8)°, and (Cl4-Zn1-N1) 104.99(7)°]. The Zn-Cl bond distances range between 2.1986(7) and 2.2799(14) Å, and the bond angles involving the Zn(II) atom range between 97.5(13)° and 114.87(11)° are comparable to those found in the literature.24,25 The bond distances between Zn1-N1 (2.068(3)) and Zn1-N2 (2.042(3)) are shorter than those between Zn1-Cl3 (2.2154(9)) and (Zn-Cl4 2.2227(9)), in- dicating that the interaction between Zn(II) metal center and N atom is stronger than that between Zn(II) and Cl atom. The Torsion/Dihedral Angles of N2-Zn1-N1-C17, N1-Zn1-N2-C27, Cl4-Zn1-N1-C11, and Zn1-N2- C21-C26 are 63.0(3)°, 48.9(3)°, 111.0(2)°, and 6.7(5)° re- spectively, which results in a steric interaction between methyl groups on the benzoxazole rings and electron re- pulsion of chlorine atoms.26 Consequently, crystallograph- ically generated centroids Cg(1) to Cg(4) were related to the various aromatic rings around the Zn(II) centers. The distance between the adjacent ring centroids for  Cg(1)... Cg(1), Cg(2)...Cg(2), Cg(3)...Cg(3),  and  Cg(4)...Cg(4)  are 4.7676(17), 4.5328(18), 5.4387(19), and 3.7652(18) Å re- spectively. As shown in Figure 1, the electron density around C18 clearly shows that this methyl group has the rotational disorder, whereas C28 has not. Each hydrogen for the C18 group has half occupancy, so the total number of hydrogens is three. 3. 2. FT-IR Spectra In the free ligand (Fig. S1), the bands at 3093 cm−1 and 1166 cm−1 are attributed to C-H and C-Cl stretching vibrations, respectively.27 Free 5-chloro-2-methylbenzox- azole shows strong intensity bands at 1608 cm–1 and 1253 cm−1, with the two bands being assigned to the C=N and Table 2. Selected bond distances (Å) and bond angles (°) of the complex. Zn1-Cl3 2.2154(9) Cl3-Zn1-Cl4 120.52(3) Zn1-Cl4 2.2227(9) Cl3-Zn1-N1 108.88(7) Zn1-N1 2.068(3) Cl3-Zn1-N2 104.79(7) Zn1-N2 2.042(3) Cl4-Zn1-N1 104.99(7) N1-C11 1.421(4) Cl4-Zn1-N2 109.70(8) N1-C17 1.302(4) N1-Zn1-N2 107.41(11) N2-C21 1.412(4) C12-O1-C17 105.0(2) 908 Acta Chim. Slov. 2022, 69, 906–912 Ali et al.: Zinc(II) Complex Containing Oxazole Ring: Synthesis, ... C-O stretching vibrations of the oxazole group, respective- ly.28,29 The C=N band in the Zn(II) complex (Fig. S2) is shifted to lower wavenumber (1600 cm–1) indicating the participation of benzoxazole nitrogen in coordination with the Zn(II) ion.30 The presence of a new weak band at 445 cm–1 in the spectrum of the complex corresponding to the (Zn-N) vibration band also confirms the bonding between ligand and Zinc metal.31 In the IR spectrum of the zinc complex (Fig. S3), the weak band at 343 cm–1 was matching to Zn-Cl vibration.32 3. 3. Electronic Spectra and Conductivity Properties The electronic absorption spectra of 5-chloro-2- methylbenzoxazole (L) Ligand and their complex were measured at room temperature in 10–3 mol. L–1 DMSO solution (Figure 2). The free ligand 5-chloro-2-methylben- zoxazole shows high energy absorption bands at 280 and 287 nm, which are attributed to ligand π–π* transitions of the benzene ring and C=N bond respectively.33,34 These absorption bands that remain unchanged in the spectrum of the complex indicate the coordination of the ligand to the Zn(II) metal center. Additionally, a new absorption peak in the complex was observed at 426 nm (23474 cm–1), which is attributed to the ligand‐to‐metal charge transfer (LMCT) that is characteristic of the zinc metal complex.35 The synthesized complex containing chlorinated ligand had a very low molar conductivity value in DMSO (9.11 10–5 S. cm2. mol–1), showing that it is non-electrolyte in nature.36 3. 4. DFT Studies The frontier molecular orbitals were used to inves- tigate the electronic properties of the ligand (L) and com- plex. The energies of the highest occupied molecular orbit- al (EHOMO) and the lowest unoccupied molecular orbital (ELUMO) are used to estimate the HOMO-LUMO energy gap (ΔE = ELUMO– EHOMO). The EHOMO, ELUMO, and ΔE en- ergy gaps of the ligand and Zn(II)complex are shown in (Table 3) and (Figure 3). The free 5-chloro-2-methylben- zoxazole ligand has an energy gap (ΔE) of 3.526 eV, while the Zn(II) complex has an ΔE of 2.423 eV. According to DFT calculations, Beheshti et al. reported the synthesis of a pyrazolyl-based mononuclear zinc(II) complex with a 4.59 eV HOMO-LUMO energy gap.37 The HOMO-LUMO energy gap value of the zinc(II) complex based on the ben- zoyl hydrazone ligand was calculated by the DFT method to be 3.76 eV in 2017.38 As a result, the synthesized zinc(II) complex in this study is less stable than those that have been previously described. The HOMO orbital primari- ly acts  as an electron donor, whereas the LUMO orbital mainly acts as an electron acceptor. A metal complex that has a large HOMO-LUMO energy gap is more stable than one that has a small HOMO-LUMO energy gap. The calcu- lated NBO atomic charges of atoms for the free ligand and its complex are collected in Table 3. The calculated charge on the zinc metal (+0.993) is lower than the formal charge of the zinc ion (+2), suggesting electron transfer from the ligand to the metal center.39 The NBO data shows that the N2 atom in the complex has a greater negative charge than the N1 atom. This result supports X-ray results showing Figure 1. Single-crystal x-ray molecular structure of the complex. ellipsoids with a 30% of probability Figure 2. Electronic absorption spectra of ligand (L) and Zn(II) complex 909Acta Chim. Slov. 2022, 69, 906–912 Ali et al.: Zinc(II) Complex Containing Oxazole Ring: Synthesis, ... the Zn1-N2 bond distance is shorter than the Zn1-N1 bond distance and suggests that N2 is coordinated to the metal center more strongly than N1.40 Table 3. HOMO-LUMO orbital energies (eV) and NBO Charges (e) of ligand (L) and its complex Parameter Ligand Zn complex EHOMO –5.672 –6.381 ELUMO –2.146 –3.958 ΔE 3.526 2.423 The NBO charge of ligand and Zin(II) complex Atom Ligand Atom Zn complex Cl 0.009 Zn 0.993 N –0.541 Cl4 –0.608 O –0.505 Cl3 –0.605 N1 –0.697 N2 –0.737 3. 5. Hirshfeld Surfaces Analysis (HAS) The Hirshfeld surface analyses (HSA) and the fin- gerprints of the zinc complex were achieved with Crystal Explorer 21.5  program.22 Fingerprint plots of the com- plex is displayed in (Figure 4). Similarly, Hirshfeld sur- face (HS)  of the complex is shown in (Figure 5). Figure 5 exposes surfaces that were mapped across dnorm, shape index, and curvature. The dnorm surface has been mapped over a range of –0.0490 to 1.3232 Å while shape index and curvedness are mapped over the ranges –1.0000 to 1.0000 Å and –4.0000 to 0.4000 Å, respectively. As illustrated in (Figure 4), the 2D fingerprint plots reveal that the major intermolecular interactions in the zinc complex are H...Cl/ Cl...H, H...H, H...C/C...H, C...Cl/Cl...C, C...O/O...C, C...C, H...O/O...H, and O…Cl/Cl...O. The highest contribution to the overall Hirshfeld surface occurs due to H…Cl/ Cl...H close contacts with 39.1%. The percentages of H...H, H...C/C...H, C...Cl/Cl...C, C...O/O...C, C...C, H...O/O...H, O..Cl/Cl...O, Cl…Cl, H…N/N…H, C…N/N…C, Zn… H/H…Zn, and Cl…N/N…Cl interactions are 21.7, 7.7, 7.0, 5.7, 4.7, 4.8, 3.4, 3.4, 1.8, 0.3, 0.3, and 0.1 % of the com- plex surface, respectively. The  dnorm  Hirshfeld  surface  of the complex shows red and white spots, which indicate the presence of C-H...Cl and C-H..H intermolecular interac- tion in the crystal structure of the zinc complex respective- ly. The shape index and curvedness of HS can be used to investigate π∙∙∙π stacking interaction, where blue triangles represent convex regions of the compound inside the sur- face and red triangles represent concave regions above the Figure 3. Surface plots of HOMO and LUMO orbitals of ligand (L) and zinc complex 910 Acta Chim. Slov. 2022, 69, 906–912 Ali et al.: Zinc(II) Complex Containing Oxazole Ring: Synthesis, ... surface due to the π∙∙∙π stacked compound’s phenyl carbon atoms.41 Green flat regions on the curvedness surface also indicate the presence of π∙∙∙π interaction in the complex. 4. Conclusion In summary, a new zinc(II) complex was synthe- sized with a 5-chloro-2-methylbenzoxazole ligand. The spectroscopic method and crystallographic data indicated the formation of a mononuclear zinc complex with a ben- zoxazole ligand acting as a monodentate ligand in neutral form. Structural analysis showed a tetracoordinate envi- ronment via two nitrogen and two chloride anions of the complex with distorted tetrahedral geometry. The elec- tronic spectrum of the complex displayed a peak at 23474 cm–1, which corresponded to the ligand‐to‐metal charge transfer (LMCT). The  DFT  study reveals that the zinc complex was less stable and more reactive than the ligand. The NBO analysis showed that the charge on the zinc met- al surrounded by the nitrogen atoms of the ligand is 0.993 e found to be lower than the formal charge of the zinc ion Figure 4: Fingerprint plots for the zinc(II) complex show the percentages of major contacts contributed to the total Hirshfeld surface analysis (HAS). 911Acta Chim. Slov. 2022, 69, 906–912 Ali et al.: Zinc(II) Complex Containing Oxazole Ring: Synthesis, ... (+2). The Hirshfeld surface and 2D fingerprint plots analy- sis showed various H... Cl/Cl ...H (39.1%), H ... H (21.7%), and H...C/C... H (7.7%) noncovalent interactions are the driving force in stable crystal packing. Supplementary material Crystallographic data for the Zin(II) complex have been deposited with the Cambridge crystallographic data Center (CCDC), with the deposit number 2097040. The  data can be obtained free of charge via http://www. ccdc.cam.ac.uk/conts/retrieving.html. Acknowledgments The authors would like to acknowledge the depart- ment of chemistry, college of education, University of Sa- lahaddin and the department of physics, college of science, University of Halabja for providing the required assistance to complete the present study. We acknowledge Nelson Mandela University for single-crystal X-ray diffraction analysis. The authors would also like to thank Mr. Mzgin Ayoob for assisting us with the FT-IR measurement. Funding The authors would like to acknowledge the college of science, University of Halabja in Kurdistan region, Iraq for providing the financial funding throughout this study. 5. References 1. D. F. Back, G. M. de Oliveira, M. A. Ballin, V. A. Corbel- lini, Inorg. Chim. Acta. 2010, 363, 807–812. DOI:10.1016/j.ica.2009.11.033 2. Y. Xu, S. Mao, K. Shen, X. Shi, H. Wu, X. Tang, Inorg. Chim. Acta. 2018, 471, 17–22. DOI:10.1016/j.ica.2017.10.023 3. A. Altun, S. Dursun, N. M. Aghatabay, Vib.  Spectrosc. 2015, 81, 1–12. DOI:10.1016/j.vibspec.2015.09.001 4. L. Wang, H. Zhou, J. Wu, Y. Tian, Dalton Trans. 2015,  44, 9921–9926. DOI:10.1039/C5DT01090J 5. Y. X. Sun, W. Y. Sun, Cryst. Eng. Comm. 2015, 17, 4045–4063. DOI:10.1039/C5CE00372E 6. H. Wu, J. Zhang, C. Chen, H. Zhang, H. Peng, F. Wang, Z. Yang, New J. Chem. 2015, 39, 7172–7181. DOI:10.1039/C5NJ01684C 7. M. M. Kimani, D. Watts, L. A. 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DOI:10.1002/aoc.5368 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 Z reakcijo med cinkovim dikloridom in ligandom 5-kloro-2-metilbenzoksazol (L= C8H6ClNO) v etanolni raztopini smo sintetizirali nov kompleks Zn(II): dikloridobis(5-kloro-2-metil-1,3-benzoksazol)-cink(II), C16H12Cl4N2O2Zn. Spojino smo karakterizirali z elementno analizo, meritvami molarne prevodnosti, FTIR, UVVis in monokristalno rentgensko analizo (XRD). Rentgenska analiza je pokazala, da se v kompleksu kovina in ligand povezujeta v razmerju 1 : 2. Spoji- na ima popačeno tetraedrično geometrijo z dvema vezanima dušikovima atomoma iz liganda. Izvedli smo izračune z DFT metodo in uporabo B3LYP funkcije z naborom osnov LANL2DZ za kovinski kompleks in 6-31G(d) za nekovinske elemente. Izračunali in predstavili smo optimalno geometrijsko strukturo kompleksa in orbitalne energije HOMO in LUMO. Opravili smo analizo naravnih veznih orbital (NBO) z namenom analize razporeditve naboja pred in po kompl- eksaciji liganda. Hirshfeldova analiza opravljena na dnorm je pokazala da močne medmolekulske interakcije H...Cl/Cl...H in H...H predstavljajo glavni prispevek k pakiranju kristalov. 913Acta Chim. Slov. 2022, 69, 913–919 Lin et al.: Synthesis, Crystal Structures and Antibacterial Activities ... DOI: 10.17344/acsi.2022.7749 Scientific paper Synthesis, Crystal Structures and Antibacterial Activities of N,N’-Ethylene-bis(3-bromosalicylaldimine) and Its Copper(II) and Cobalt(III) Complexes Xue-Song Lin,*Yong-Gang Huang, Rui-Fa Jin and Ya-Li Sang College of Chemistry and Life Science, Chifeng University, Chifeng 024000, P.R. China * Corresponding author: E-mail: xuesong_lin@126.com Received: 08-17-2022 Abstract A bis-Schiff base N,N’-ethylene-bis(3-bromosalicylaldimine) (H2L) was prepared from 3-bromosalicylaldehyde and ethane-1,2-diamine. With H2L as ligand, a new copper(II) complex [CuL] (1) and a new cobalt(III) complex [CoL(NCS) (DMF)] (2) were prepared and characterized by physico-chemical methods and single crystal X-ray analysis. X-ray analysis indicates that the Cu atom in complex 1 is in square planar coordination, and the Co atom in complex 2 is in octahedral coordination. The compounds were tested in vitro for their antibacterial activities on Bacillus subtilis, Staph- ylococcus aureus, Escherichia coli and Pseudomonas fluorescens. Both complexes have effective activities on the bacteria. Keywords: Schiff base; copper complex; cobalt complex; crystal structure; antibacterial activity 1. Introduction Schiff bases bearing typical group C=N are impor- tant ligands in coordination chemistry, which are readily prepared by the condensation reaction of carbonyl con- taining compounds with organic amines. Schiff bases with donor atoms (N, O, S) have structure similarities with nat- ural biological systems and due to the presence of imine group, are utilized in biological systems.1 A great number of literature reported that they have interesting antibacte- rial, antifungal and antitumor activities.2 It is well known that some biological activities, when administered as met- al complexes, are being increased.3 Bis-Schiff bases derived from ethane-1,2-diamine, propane-1,3-diamine, pro- pane-1,2-diamine and cyclohexyl-1,2-diamine with salic- ylaldehyde and its analogues are widely used tetradentate ligands.4 It was reported that salicylaldehyde derivatives with halo atoms in the aromatic ring showed variety of bi- ological activities, especially antibacterial activities.5 Schiff base copper and cobalt complexes have shown remarkable antibacterial properties.6 In addition, thiocyanate anion is a co-ligand in most Schiff base cobalt complexes.7 In the present work, two new copper(II) and cobalt(III) com- plexes, [CuL] (1) and [CoL(NCS)(DMF)] (2), where L is the dianionic form of N,N’-ethylene-bis(3-bromosalicy- laldimine) (H2L), are prepared and characterized. The an- tibacterial activities against Bacillus subtilis, Staphylococ- cus aureus, Escherichia coli and Pseudomonas fluorescens, were studied. Scheme 1. The bis-Schiff base H2L 2. Experimental 2. 1. Materials and Measurements 3-Bromosalicylaldehyde and ethane-1,2-diamine with AR grade were obtained from Aldrich and used as received. Copper nitrate and cobalt nitrate were purchased from TCI. Ammonium thiocyanate was purchased from Aladin Chemical Co. Ltd. Elemental analyses were per- formed using a Perkin-Elmer 240C analytical instrument. Infrared spectra were recorded on a Nicolet 5DX FT-IR spectrophotometer with KBr pellets. UV-Vis spectra were 914 Acta Chim. Slov. 2022, 69, 913–919 Lin et al.: Synthesis, Crystal Structures and Antibacterial Activities ... recorded on a Lambda 35 spectrometer. Molar conduct- ance was measured with a Shanghai DDS-11A conduc- tometer. 2. 2. Synthesis of H2L 3-Bromosalicylaldehyde (0.40 g, 2.0 mmol) dis- solved in methanol (30 mL) was reacted with ethane-1,2- diamine (0.030 g, 1.0 mmol) diluted by methanol (10 mL). The mixture was stirred at reflux for 1 h and with three quarter of the solvent removed by distillation, to give yel- low product. Yield: 0.37 g (88%). Anal. Calcd. for C16H14Br2N2O2 (%): C, 45.10; H, 3.31; N, 6.57. Found: C, 44.92; H, 3.40; N, 6.71. IR data (KBr, cm–1): 3438 (OH), 1628 (C=N), 1217 (Ar–O). UV-Vis data in methanol [λmax (nm), ε (L mol–1 cm–1)]: 215, 22350; 260, 8940; 328, 2980; 415, 1935. Diffraction quality yellow single crystals were ob- tained by slow evaporation of the methanol solution of the product. 2. 3. Synthesis of [CuL] (1) H2L (42 mg, 0.10 mmol) and copper nitrate trihy- drate (24 mg, 0.10 mmol) were dissolved in methanol (20 mL). A brown solution was formed immediately. After 30 min stirring, the solution was filtered and the filtrate was kept for slow evaporation. The diffraction quality brown single crystals that deposited over a period of 5 days were collected by filtration and washed with methanol. Yield: 22 mg (45%). Anal. Calcd. for C16H12Br2CuN2O2 (%): C, 39.41; H, 2.48; N, 5.74. Found: C, 39.26; H, 2.37; N, 5.85. IR data (KBr, cm–1): 1632 (C=N), 1180 (Ar–O). UV-Vis data in methanol [λmax (nm), ε (L mol–1 cm–1)]: 270, 16720; 368, 6270. ΛM (10–3 mol L–1 in DMSO/H2O): 41 Ω–1 cm2 mol–1. 2. 4. Synthesis of [CoL(NCS)(DMF)] (2) H2L (42 mg, 0.10 mmol) and cobalt nitrate hexahy- drate (29 mg, 0.10 mmol) were dissolved in methanol (20 mL) and DMF (5 mL). A deep red solution was formed immediately. After 30 min stirring, the solution was fil- tered and the filtrate was kept for slow evaporation. The diffraction quality red single crystals that deposited over a period of 27 days were collected by filtration and washed with methanol. Yield: 18 mg (29%). Anal. Calcd. for C20H19Br2CoN4O3S (%): C, 39.11; H, 3.12; N, 9.12. Found: C, 39.27; H, 3.21; N, 9.03. IR data (KBr, cm–1): 1641 (C=O), 1633 (C=N), 1183 (Ar–O). UV-Vis data in methanol [λmax (nm), ε (L mol–1 cm–1)]: 267, 15560; 380, 3315. ΛM (10–3 mol L–1 in DMSO/H2O): 33 Ω–1 cm2 mol–1. 2. 5. X-Ray Crystallography Suitable single crystals of the complexes were select- ed and mounted on a Bruker Smart 1000 CCD area-detec- tor diffractometer with graphite monochromatized Mo- Kα radiation (λ = 0.71073 Å). Diffraction data for the compounds were collected by ω scan mode at 298(2) K. Data reduction and cell refinement were performed by the SMART and SAINT programs.8 Empirical absorption cor- rection was applied by using SADABS.9 The structures were solved by direct methods and refined with the full-matrix least-squares technique using SHELXL.10 The non-H atoms in the structures were subjected to refined anisotropic refinement. The hydrogen atoms were located Table 1. Crystallographic data and refinement details for the compounds H2L 1 2 Molecular formula C16H14Br2N2O2 C16H12Br2CuN2O2 C20H19Br2CoN4O3S Molecular weight 426.11 487.64 614.20 Crystal system Monoclinic Orthorhombic Monoclinic Space group P21/n Pbca P21/c a, Å 8.3320(5) 10.6159(8) 11.1597(6) b, Å 10.1997(7) 12.6984(9) 16.6043(8) c, Å 9.5717(7) 24.0006(12) 13.3480(7) β, º 101.332(2) 90 109.9890(10 V, Å3 797.58(9) 3235.4(4) 2324.4(2) Z 2 8 4 ρcalcd, g cm–3 1.774 2.002 1.755 μ, mm–1 5.090 6.299 4.295 Reflections collected 7634 16247 26163 Unique reflections 1730 3009 4330 Observed reflections (I ≥ 2σ(I)) 1344 2320 3243 Data/restraints/parameters 1730/0/101 3009/0/208 4330/0/282 Rint 0.0329 0.0538 0.0365 GOOF on F2 1.055 1.035 1.015 R1, wR2 (I ≥ 2σ(I)) 0.0328, 0.0724 0.0272, 0.0552 0.0360, 0.0766 R1, wR2 (all data) 0.0496, 0.0812 0.0446, 0.0605 0.0571, 0.0861 915Acta Chim. Slov. 2022, 69, 913–919 Lin et al.: Synthesis, Crystal Structures and Antibacterial Activities ... in geometrically and treated with the riding mode. Crys- tallographic data and experimental details for the com- pounds are summarized in Table 1. 2. 6. Antibacterial Assay Antibacterial activities of the compounds were test- ed in vitro against Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas fluorescens using MH medium (Mueller–Hinton medium: casein hydrolysate 17.5 g, soluble starch 1.5 g, beef extract 1000 mL). The minimum inhibitory concentrations (MIC) of the test compounds were determined by a colorimetric method using the dye MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-di- phenyltetrazolium bromide).11 A solution of the com- pound (50 μg mL–1) in DMSO was prepared and graded quantities of the assayed compounds were incorporated in specified quantity of sterilized liquid MH medium. A specified quantity of the medium containing the com- pound was poured into microtitration plates. Suspension of the microorganism was prepared to contain about 105 colony forming units cfu mL–1 and applied to micro-titra- tion plates with serially diluted compounds in DMSO to be tested and incubated at 37 °C for 24 h. After the MICs were visually determined on each of the micro-titration plates, 50 μL of PBS (Phosphate Buffered Saline 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 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 isopropyl alcohol containing 5% 1.0 mol L–1 HCl was added to extract the dye. After 12 h of incubation at room temperature, the optical density (OD) was measured with a micro-plate reader at 550 nm. 3. Results and Discussion 3. 1. Chemistry The bis-Schiff base H2L was prepared by 2:1 conden- sation reaction of 3-bromosalicylaldehyde with ethane-1,2- diamine in methanol (Scheme 2). Complexes 1 and 2 were facile prepared by the reaction of the bis-Schiff base with copper nitrate trihydrate and cobalt nitrate hexahydrate, re- spectively, in methanol. As usually observed for the prepara- tion of cobalt complexes, CoII in complex 2 underwent aerial oxidation to CoIII in the synthetic route. The molar conductivities of the complexes 1 and 2 measured in DM- SO/H2O (V:V = 1:9) at concentration of 10–3 mol L–1 are 41 and 33 Ω–1 cm2 mol–1, respectively, indicating the non-elec- trolytic nature of both complexes in such solution.12 3. 2. IR and Electronic Spectra In the infrared spectrum of H2L, the weak absorption at 3438 cm–1 is assigned to the O–H vibration of the phenol group, and the characteristic imine stretching is observed at 1628 cm–1 as a strong signal.13 The spectra of complexes 1 and 2 show imine stretching at 1632–1633 cm–1. The Schiff base ligands coordination is substantiated by the phenolic C–O stretching bands at 1217 cm–1 for H2L, while 1180–1183 cm–1 for the complexes.14 The intense absorp- tion band at 2117 cm–1 in the spectrum of complex 2 can be assigned to the thiocyanate ligand.15 Coordination of the Schiff base ligands is further confirmed by the appearance of weak bands in the low wave numbers 400–600 cm–1, cor- responding to ν(Cu/Co–N) and ν(Cu/Co–O). In the electronic spectra of H2L and the complexes, the bands at 260–270 nm and 328 nm are attributed to the n–π* transitions.16 The bands at 360–380 nm in the com- plexes can be attributed to the ligand to metal charge transfer transition (LMCT).17 3. 3. Crystal Structure Description of H2L Molecular structure of H2L is shown in Fig. 1. Select- ed bond lengths and angles for the compound are listed in Table 2. All the bond lengths in the compound are within normal ranges,18 and comparable to those of the similar bis-Schiff bases.19 The bond length of C7−N1 confirms it as a double bond. The two benzene rings form a dihedral angle of 0° due to the centrosymmetric symmetry. In the crystal structure of the compound, the molecules are linked through intermolecular hydrogen bonds of C– H∙∙∙O (C7–H7 = 0.93 Å, H7∙∙∙O1i = 2.41 Å, C7∙∙∙O1i = Scheme 2. The synthetic procedure of the complexes Fig. 1. Molecular structure of H2L with thermal ellipsoids of 30% probability level. 916 Acta Chim. Slov. 2022, 69, 913–919 Lin et al.: Synthesis, Crystal Structures and Antibacterial Activities ... 3.328(3) Å, C7–H7∙∙∙O1i = 170º; symmetry code for i: ½ + x, ½ – y, ½ + z), to form two-dimensional sheets parallel to the bc plane (Fig. 2). Moreover, there are π–electron ring– –π-electron ring interactions (4.209(3) Å) in the packing structure of the compound. 3. 4. Crystal Structure Description of Complex 1 Molecular structure of the mononuclear copper(II) complex 1 is shown in Fig. 3. Selected bond lengths and angles for the compound are listed in Table 2. The Cu atom is coordinated by two phenolate oxygen and two imino ni- trogen of the bis-Schiff base ligand, forming square planar coordination. The square planar geometry is slightly dis- torted from ideal model, as evidenced by the bond angles. The angles in the coordination are in the ranges of 84.78(11)–92.96(10)° and 176.27(10)–177.64(10)°, respec- tively. The Cu–O and Cu–N bond lengths are comparable to those observed in Schiff base copper complexes.20 The two benzene rings form a dihedral angle of 5.7(5)°. Fig. 3. Molecular structure of complex 1 with thermal ellipsoids of 30% probability level. 3. 5. Crystal Structure Description of Complex 2 Molecular structure of the mononuclear cobalt(III) complex 2 is shown in Fig. 4. Selected bond lengths and angles for the compound are listed in Table 2. The Co atom is coordinated by two phenolate oxygen and two imino ni- trogen of the bis-Schiff base ligand, one thiocyanate nitro- gen and one oxygen of a DMF ligand, forming octahedral coordination. The equatorial plane of the octahedral coor- dination is defined by the four donor atoms of the Schiff base ligand, and the axial positions are occupied by the donor atoms of thiocyanate and DMF ligands. The octahe- Fig. 2. Molecular packing diagram of H2L, viewed along the c axis. Hydrogen bonds are shown as dashed lines. Table 2. Selected bond distances (Å) and angles (º) for the com- pounds Bond Å Bond Å H2L C7–N1 1.269(4) C8–N1 1.463(3) C2–O1 1.319(3) 1 C7–N1 1.279(4) C8–N1 1.474(4) C10–N2 1.279(4) C9–N2 1.471(4) C2–O1 1.301(3) C12–O2 1.312(3) Cu1–N1 1.940(2) Cu1–N2 1.930(2) Cu1–O1 1.896(2) Cu1–O2 1.908(2) O1–Cu1–N1 92.96(10) O1–Cu1–N2 177.64(10) O1–Cu1–O2 89.74(8) N1–Cu1–N2 84.78(11) N1–Cu1–O2 176.27(10) N2–Cu1–O2 92.49(10) 2 C7–N1 1.271(5) C8–N1 1.474(5) C10–N2 1.276(5) C9–N2 1.464(5) C2–O1 1.302(4) C12–O2 1.297(4) Co1–O1 1.887(2) Co1–N1 1.889(3) Co1–N2 1.891(3) Co1–O2 1.896(2) Co1–N3 1.893(3) Co1–O3 1.937(3) O1–Co1–N1 94.70(12) O1–Co1–N2 176.99(12) O1–Co1–O2 87.30(11) N1–Co1–N2 85.47(13) N1–Co1–O2 177.45(12) N2–Co1–O2 92.45(12) N3–Co1–O1 92.68(13) N3–Co1–O2 92.28(13) N3–Co1–N1 89.21(13) N3–Co1–N2 90.32(14) O3–Co1–O1 87.40(11) O3–Co1–O2 89.96(11) O3–Co1–N1 88.56(12) O3–Co1–N2 89.60(12) N3–Co1–O3 177.76(13) Fig. 4. Molecular structure of complex 2 with thermal ellipsoids of 30% probability level. 917Acta Chim. Slov. 2022, 69, 913–919 Lin et al.: Synthesis, Crystal Structures and Antibacterial Activities ... dral geometry is distorted from ideal model, as evidenced by the bond angles. The bond angles in the coordination are in the ranges of 85.47(13)–94.70(12)° and 176.99(12)– 177.76(13)°, respectively. The Co–O and Co–N bond lengths are comparable to those observed in Schiff base cobalt complexes.21 3. 6. Antibacterial Activities The compounds were screened in vitro for antibacte- rial activities against Bacillus subtilis, Staphylococcus au- reus, Escherichia coli and Pseudomonas fluorescens by the MTT method. The MICs of the compounds against the bacteria are presented in Table 3. Penicillin was used as a reference. Table 3. Antibacterial activities (MIC (μg mL–1)) Bacillus Escherichia Pseudomonas Staphylococcus subtilis coli fluorescens aureus H2L 12.5 25 25 6.25 1 0.78 3.12 12.5 1.56 2 6.25 6.25 3.12 3.12 Penicillin 1.3 > 100 > 100 2.1 The bis-Schiff base H2L shows medium to weak ac- tivities against the bacteria. In general, both complexes have stronger activities against the bacteria than H2L. Complex 1 showed strong activity against Bacillus subtilis, Escherichia coli and Staphylococcus aureus, and medium activity against Pseudomonas fluorescens. The complex has better activity against Bacillus subtilis than the polynuclear copper complex with the Schiff base ligand 2-hy- droxy-5-methylbenzaldehyde oxime.22 Complex 2 showed strong activity against Pseudomonas fluorescens and Staph- ylococcus aureus, and medium activity against Bacillus sub- tilis and Escherichia coli. Complex 1 has stronger activities against the bacteria than complex 2, except for Pseu- domonas fluorescens. Interestingly, complex 1 has stronger activities against the bacteria than Penicillin. The trends in the present work are in accordance with those in the liter- atures that metal complexes usually have stronger antibac- terial activities than their corresponding ligands.23 The copper complex has stronger activity against Bacillus sub- tilis, Staphylococcus aureus and Escherichia coli than the copper complex with the Schiff base 2-(2-(2,4-dinitrophe- nyl)hydrazono)-1,2-diphenylethanone.24 The cobalt com- plex has stronger activity against Staphylococcus aureus than the cobalt complexes with the Schiff bases 4-X-2-{[2- (2-pyridine-2-yl-ethylsulfanyl)ethylimino]methyl}phenol (X = methoxy, phenylazo, bromo, nitro).25 This enhanced antibacterial activity of the complexes can be explained by the coordination of the metal ions with the azomethine groups of the Schiff base ligands.26 According to chelating theory,27 chelation could enhance the lipophilic character of the central metal ions, which subsequently favor their permeation through the lipid layers of the cell membrane and blocking the metal binding sites on enzymes of micro- organism. On chelation, the polarity of the metal ions de- creases to a greater extent, due to the overlap of the ligand orbital and partial sharing of their positive charge with donor groups. In addition, it improves the π-electron delo- calization on the whole chelating ring which affects the li- pophilicity of the complexes.28 4. Conclusion Two new copper(II) and cobalt(III) complexes have been prepared and characterized. The bis-Schiff base li- gand coordinates to the metal atoms through phenolate oxygen and imino nitrogen. Structures of H2L and the complexes were characterized by spectroscopic methods and confirmed by single crystal X-ray determination. The antibacterial activities of the bis-Schiff base and the com- plexes were assayed. The results indicated that both com- plexes are potential antibacterial agents. Supplementary Material CCDC reference numbers 969428 (H2L), 2195359 (1) and 2195360 (2) contain the supplementary crystallo- graphic data for this paper. These data can be obtained free of charge at www.ccdc.cam.ac.uk, or from Cambridge Crystallographic Data Center, 12, Union Road, Cambridge CB2 1EZ, UK; Fax: +44 1223 336 033; e-mail: deposit@ ccdc.cam.ac.uk. Acknowledgments We gratefully acknowledge the financial support by the Natural Science Research Project of Chifeng City (SZR2022015), the Research Program of Science and Technology at Universities of Inner Mongolia Autono- mous Region (NJZY21139), and Inner Mongolia Key Lab- oratory of Photoelectric Functional Materials. 5. References 1. (a) E. Keskioglu, A.B. Gunduzalp, S. Cete, F. Hamurcu, B. Erk, Spectrochim. Acta A, 2008, 70, 634–640. DOI:10.1016/j.saa.2007.08.011 (b) J.Z. Wu, L. Yuan, J. Inorg. Biochem. 2004, 98, 41–45. DOI:10.1016/j.jinorgbio.2003.08.011 2. (a) H. Liu, Z.-W. Chu, D.-G. Xia, H.-Q. Cao, X.-H. Lv, Bioorg. Chem. 2020, 99, 103807. DOI:10.1016/j.bioorg.2020.103807 (b) N. Caliskan, A. Usta, F.S. Beris, N. Baltas, E. Celik, Lett. Org. Chem. 2020, 17, 631–638. 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Proc. Phys. Soc. 1961, 78, 1174–1181; DOI: 10.1088/0370-1328/78/6/311 28. P. Gull, M.A. Malik, O.A. Dar, A.A. Hashmi, J. Mol. Struct. 2017, 1134, 734–741. DOI:10.1088/0370-1328/78/6/311 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 Iz 3-bromosalicilaldehida in etan-1,2-diamina smo pripravili bis-Schiffovo bazo N,N’-etilen-bis(3-bromosalicilaldimin) (H2L). Z H2L kot ligandom smo pripravili nova kompleksa bakra(II) [CuL] (1) in kobalta(III) [CoL(NCS)(DMF)] (2), ki smo ju okarakterizirali s fizikalno-kemijskimi metodami in monokristalno rentgensko analizo. Rentgenska analiza kaže, da je atom Cu v kompleksu 1 v kvadratni planarni koordinaciji, atom Co v kompleksu 2 pa v oktaedrični koordinaciji. Spojine so bile testirane in vitro na antibakterijsko delovanje na Bacillus subtilis, Staphylococcus aureus, Escherichia coli in Pseudomonas fluorescens. Oba kompleksa izkazujeta učinkovito delovanje na bakterije. 920 Acta Chim. Slov. 2022, 69, 920–927 Sabouri et al.: Synthesis and In Vitro Cytotoxicity of Novel Halogenated ... DOI: 10.17344/acsi.2022.7754 Scientific paper Synthesis and In Vitro Cytotoxicity of Novel Halogenated Dihydropyrano[3,2-b]Chromene Derivatives Salehe Sabouri,1,2 Ehsan Faghih-Mirzaei3 and Mehdi Abaszadeh*,4 1 Herbal & Traditional Medicine Research Center, Kerman University of Medical Sciences, Kerman, Iran. 2 Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran. 3 Department of Medicinal Chemistry, Faculty of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran 4 Pharmaceutics Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran. * Corresponding author: E-mail: abaszadeh@kmu.ac.ir Received: 08-20-2022 Abstract Lung and breast cancers are among the most common cancers. In the present work, initially, 6-bromo-; and 6-chloro-3-hy- droxychromone compounds were prepared. In the next step, a series of 8-bromo-; and 8-chloro-dihyropyrano[3,2-b] chromene derivatives were synthesized by one-pot three component reaction of these two compounds, aromatic alde- hydes, and ethyl cyanoacetate in the presence of triethylamine in EtOH at reflux conditions. The synthesized compounds were tested for their in vitro cytotoxic activity against A549 (lung cancer) and MCF-7 (breast cancer) cell lines. It was found that some compounds have high to moderate cytotoxicity, which makes them potential candidates for further studies. This study can be the basis for further studies to design and synthesis potent anticancer compounds and inves- tigating their mechanism of action. Keywords: Chromone derivatives; 6-bromo-3-hydroxychromone; 6-chloro-3-hydroxychromone; Three-component re- actions; Cytotoxicity; Cancer cell line 1. Introduction Compounds with a fused benzene and 4-pyrone ring are called 4H-1-benzopyran-4-one, 4H-chromen-4-one or chromone. Chromones are a group of naturally occurring compounds that are ubiquitous in nature, especially in plants.1,2 They are present in various flavonoids as a core structure. For instance, 3-hydroxyflavone (a type of hy- droxyflavone) is a compound with a phenyl group in the 2-position and a hydroxyl group in the 3-position in the pyrone ring of chromone scaffold. Moreover, the chro- mone derivatives with a hydroxyl group in the 3-position in the pyrone ring are called 3-hydroxychromone. Chro- mone derivatives display a wide range of biological activi- ties including antifungal,3 antimicrobial,4 antiobesity,5 an- tiviral,6,7 anti inflammatory,8 anticancer,9,10 antioxidant,11 and protein kinase inhibitory.12 In addition, chromones are good bidentate ligands able to coordinate metal ions. These compounds are widely investigated as fluorescent membrane probes and fluorescent chelators.13,14 Cancer, a worldwide health problem, is the second cause of death. Lung cancer is the leading cause of cancer death in males and females. Breast cancer is also one of the most common cancers that ranks as the second cause of deaths from cancer among females.15 In spite of the exist- ence of several strategies for cancer therapy, searching for new chemotherapeutic agents continues. It is because of the complex nature of cancer and occurring drug resist- ance in cancerous cells.16 Herein, we have synthesized 6-bromo-; and 6-chloro- 3-hydroxychromone (3a,b) at first step. Then, we prepared 8-halopyrano[3,2-b]chromen-10(4H)-one derivatives (6a- j) by one-pot three component reactions of (3a,b), aro- matic aldehydes, and ethyl cyanoacetate in the presence of triethylamine in ethanol as solvent and at reflux condi- tions. Biological evaluation was also carried out for screen- ing the potential cytotoxic activity of the compounds by MTT assay. 921Acta Chim. Slov. 2022, 69, 920–927 Sabouri et al.: Synthesis and In Vitro Cytotoxicity of Novel Halogenated ... 2. Experimental 2. 1. General Methods All chemicals and reagents used in current study were obtained from commercial sources and used without further purification. All melting points were determined on Electrothermal-9100 apparatus and are uncorrected. IR spectra were recorded on a Bruker FTIR (Alpha model) spectrophotometer using KBr pallets. 1H NMR (300 MHz) and 13C NMR (75 MHz) spectra were recorded on a Bruk- er AVANCE III 300 MHz spectrometer in DMSO-d6, with TMS as an internal standard. Chemical shifts (δ) are given in parts per million (ppm) and coupling constants (J) are given in Hertz (Hz). Reactions were monitored by thin lay- er chromatography (TLC) on the Aluminium-backed sili- ca gel sheets (GF254) and visualized in UV light (254 nm). Elemental analyses were performed using a Heraeus CHN-O-Rapid analyzer. 2. 2. Preparation of 6-bromo-; and 6-chloro-3- hydroxychromone (3a,b) These compounds were prepared according to litera- ture procedures which presented by Spadafora and et al. and represented in Scheme 1.13 The isolated products were crystallised from ethanol. 6-Bromo-3-hydroxy-4H-chromen-4-one (3a) Pale yellow powders; yield: 88%; mp 205–206 °C; IR (KBr) ν 3101 (OH), 1623 (C=O), 1601 cm–1 (C=C); 1H NMR (300 MHz, DMSO-d6) δppm: 9.35 (br, 1H, OH), 8.27 (s, 1H, CH), 8.17 (d, 1H, J=3 Hz, ArH), 7.89 (dd, 1H, J=9 Hz, 3 Hz, ArH), 7.62 (d, 1H, J=9 Hz, ArH); 13C NMR (75 MHz, DMSO-d6) δppm: 171.96 (C=O), 154.60 (C3), 142.50 (C2), 141.70, 136.38, 127.40, 124.72, 121.59, 117.35; Anal. calcd. for C9H5BrO3: C, 44.85; H, 2.09%. Found: C, 44.64; H, 1.96%. 6-Chloro-3-hydroxy-4H-chromen-4-one (3b). White powders; yield: 91%; mp 218–219˚C (lit.17 216 °C); IR (KBr) ν 3296 (OH), 1629 (C=O), 1605 cm–1 (C=C); 1H NMR (300 MHz, DMSO-d6) δppm: 9.36 (br, 1H, OH), 8.26 (s, 1H, CH), 8.05 (d, 1H, J=3 Hz, ArH), 7.80 (dd, 1H, J=9 Hz, 3 Hz, ArH), 7.71 (d, 1H, J=9 Hz, ArH); 13C NMR (75 MHz, DMSO-d6) δppm: 172.10 (C=O), 154.26 (C3), 142.45 (C2), 141.73, 133,74, 129.49, 124.29, 124.25, 121.44; Anal. calcd. for C9H5ClO3: C, 54.99; H, 2.56%. Found: C, 54.83; H, 2.41%. 2. 3. General Procedure for the Preparation of Dihydropyrano[3,2-b]chromene derivatives (6a-j) A mixture of 6-bromo-; or 6-chloro-3-hydroxychro- mone (3a,b) (2 mmol), aromatic aldehydes (4a-j) (2 mmol) and ethyl cyanoacetate (5) (2.1 mmol), and three drops of triethylamine in ethanol (10 mL) were added to a 50 mL round bottomed flask equipped with a magnetic stirring bar and a reflux condenser. It was stirred and re- fluxed for 1 h. The progress of the reaction was monitored by TLC using hexane/ethyl acetate as an eluent. After com- pletion of the reaction, the mixture was cooled and the ob- tained crude product was filtered, washed with ethanol and crystallized from ethanol to give the pure solid sample for analysis. Ethyl 2-amino-8-bromo-10-oxo-4-phenyl-4,10-dihy- dropyrano[3,2-b]chromene-3-carboxylate (6a) white powders; yield: 90%; mp 200–201 °C; IR (KBr) ν 3431, 3315 (NH2), 3027 (CH, aromatic), 2982 (CH, ali- phatic), 1686, 1650 (2C=O), 1611 (C=C), 1193 cm–1 (C-O); 1H NMR (300 MHz, DMSO-d6) δppm: 8.13 (d, 1H, J=3 Hz, ArH), 7.89 (br, 2H, NH2), 7.85 (dd, 1H, J=9 Hz, 3 Hz, ArH), 7.50 (d, 1H, J=9 Hz, ArH), 7.36–7.21 (m, 5H, ArH), 4.93 (s, 1H, CH), 3.97 (q, 2H, J=6 Hz, OCH2CH3), 1.04 (t, 3H, J=6 Hz, OCH2CH3); 13C NMR (75 MHz, DM- SO-d6) δppm: 167.99 (C-10, CO), 167.75 (ester CO), 160.04 (C-2), 153.95, 153.51, 143.36, 137.28, 133.35, 129.01, 128.22, 127.71, 125.23, 121.31 (C-8), 118.21, 75.38 (C-3), 59.42 (OCH2CH3), 41.03 (C-4), 14.57 (OCH2CH3); Anal. calcd. for C21H16BrNO5: C, 57.03; H, 3.65; N, 3.17%. Found: C, 56.79; H, 3.49; N, 3.21%. Ethyl 2-amino-8-bromo-4-(4-chlorophenyl)-10-oxo- 4,10-dihydropyrano[3,2-b]chromene-3-carboxylate (6b) white powders; yield: 92%; mp 239–241 °C; IR (KBr) ν 3468, 3333 (NH2), 3062 (CH, aromatic), 2984 (CH, ali- phatic), 1708, 1671 (2C=O), 1622 (C=C), 1192 cm–1 (C-O); 1H NMR (300 MHz, DMSO-d6) δppm: 8.13 (d, 1H, J=3 Hz, ArH), 7.93 (br, 2H, NH2), 7.87 (dd, 1H, J=6 Hz, 3 Hz, ArH), 7.52 (d, 1H, J=9 Hz, ArH), 7.40–7.33 (m, 4H, ArH), 4.96 (s, 1H, CH), 3.97 (q, 2H, J=6 Hz, OCH2CH3), 1.04 (t, 3H, J=6 Hz, OCH2CH3); 13C NMR (75 MHz, DM- SO-d6) δppm: 167.87 (C-10, CO), 167.80 (ester CO), 159.99 (C-2), 153.97, 152.81, 142.34, 137.34, 133.40, 132.29, 130.14, 128.96, 127.73, 125.24, 121.32 (C-8), 118.24, 74.99 (C-3), 59.48 (OCH2CH3), 40.48 (C-4), 14.60 (OCH2CH3); Anal. calcd. for C21H15BrClNO5: C, 52.19; H, 3.17; N, 2.94%. Found: C, 52.22; H, 2.98; N, 2.69%. Ethyl 2-amino-8-bromo-10-oxo-4-(p-tolyl)-4,10-dihy- dropyrano[3,2-b]chromene-3-carboxylate (6c) white powders; yield: 87%; mp 209–210 °C; IR (KBr) ν 3396, 3291 (NH2), 3023 (CH, aromatic), 2984 (CH, ali- phatic), 1680, 1661 (2C=O), 1615 (C=C), 1196 cm–1 (C-O); 1H NMR (300 MHz, DMSO-d6) δppm: 8.14 (d, 1H, J=3 Hz, ArH), 7.90–7.86 (m, 3H, NH2, ArH), 7.53 (d, 1H, J=9 Hz, ArH), 7.16 (q, 4H, J=9 Hz, ArH), 4.89 (s, 1H, CH), 3.97 (q, 2H, J=6 Hz, OCH2CH3), 2.24 (s, 3H, CH3), 1.07 (t, 3H, J=6 Hz, OCH2CH3); 13C NMR (75 MHz, DMSO-d6) δppm: 168.02 (C-10, CO), 167.76 (ester CO), 160.01 (C-2), 153.96, 153.78, 140.42, 137.29, 136.88, 133.29, 129.59, 922 Acta Chim. Slov. 2022, 69, 920–927 Sabouri et al.: Synthesis and In Vitro Cytotoxicity of Novel Halogenated ... 128.06, 127.73, 125.24, 121.34 (C-8), 118.21, 75.49 (C-3), 59.44 (OCH2CH3), 40.61 (C-4), 21.07 (CH3), 14.62 (OCH2CH3); Anal. calcd. for C22H18BrNO5: C, 57.91; H, 3.98; N, 3.07%. Found: C, 58.02; H, 3.71; N, 2.89%. Ethyl 2-amino-8-bromo-4-(4-fluorophenyl)-10-oxo-4,10- dihydropyrano[3,2-b]chromene-3-carboxylate (6d) cream powders; yield: 90%; mp 214–215 °C; IR (KBr) ν 3466, 3381 (NH2), 3044 (CH, aromatic), 2991 (CH, aliphatic), 1707, 1684 (2C=O), 1659 (C=C), 1194 cm–1 (C-O); 1H NMR (300 MHz, DMSO-d6) δppm: 8.13 (d, 1H, J=3 Hz, ArH), 7.91–7.86 (m, 3H, NH2, ArH), 7.52 (d, 1H, J=9 Hz, ArH), 7.38–7.34 (m, 2H, ArH), 7.15 (t, 2H, J=9 Hz, ArH), 4.96 (s, 1H, CH), 3.97 (q, 2H, J=6 Hz, OCH2CH3), 1.04 (t, 3H, J=6 Hz, OCH2CH3); 13C NMR (75 MHz, DMSO-d6) δppm: 167.92 (C-10, CO), 167.80 (ester CO), 163.33 (C-2), 160.12, 159.97, 153.97, 153.11, 139.52, 137.32, 133.33, 130.22, 130.11, 127.73, 125.24, 121.32 (C- 8), 118.22, 115.89, 115.61, 75.26 (C3), 59.44 (OCH2CH3), 40.55 (C-4), 14.58 (OCH2CH3); Anal. calcd. for C21H15BrF- NO5: C, 54.80; H, 3.29; N, 3.04%. Found: C, 54.59; H, 2.98; N, 3.11%. Ethyl 2-amino-8-bromo-4-(4-methoxyphenyl)-10-oxo- 4,10-dihydropyrano[3,2-b]chromene-3-carboxylate (6e) yellow powders; yield: 86%; mp 211–213 °C; IR (KBr) ν 3423, 3297 (NH2), 3069 (CH, aromatic), 2983 (CH, aliphatic), 1686, 1656 (2C=O), 1632 (C=C), 1189 cm–1 (C-O); 1H NMR (300 MHz, DMSO-d6) δppm: 8.13 (d, 1H, J=3 Hz, ArH), 7.89–7.85 (m, 3H, NH2, ArH), 7.52 (d, 1H, J=9 Hz, ArH), 7.21 (d, 2H, J=9 Hz, ArH), 6.87 (d, 2H, J=9 Hz, ArH), 4.87 (s, 1H, CH), 3.98 (q, 2H, J=6 Hz, OCH2CH3), 3.71 (s, 3H, OCH3), 1.07 (t, 3H, J=6 Hz, OCH2CH3); 13C NMR (75 MHz, DMSO-d6) δppm: 168.05 (C-10, CO), 167.75 (ester CO), 159.98 (C-2), 158.84, 153.96, 153.85, 137.26, 135.39, 133.22, 129.22, 127.72, 125.24, 121.31 (C-8), 118.19, 114.38, 75.64 (C-3), 59.42 (OCH2CH3), 55.48 (OCH3), 40.17 (C-4), 14.64 (OCH2CH3); Anal. calcd. for C22H18BrNO6: C, 55.95; H, 3.84; N, 2.97%. Found: C, 55.98; H, 3.56; N, 2.73%. Ethyl 2-amino-8-chloro-10-oxo-4-phenyl-4,10-dihy- dropyrano[3,2-b]chromene-3-carboxylate (6f) white powders; yield: 91%; mp 190–192 °C; IR (KBr) ν 3466, 3411 (NH2), 3028 (CH, aromatic), 2982 (CH, aliphatic), 1687, 1662 (2C=O), 1612 (C=C), 1193 cm–1 (C-O); 1H NMR (300 MHz, DMSO-d6) δppm: 8.03 (t, 1H, J=3 Hz, ArH), 7.90 (br, 2H, NH2), 7.82–7.77 (m, 1H, ArH), 7.63 (q, 1H, J=3 Hz, ArH), 7.37–7.22 (m, 5H, ArH), 4.95 (s, 1H, CH), 3.97 (q, 2H, J=6 Hz, CH2), 1.05 (t, 3H, J=6 Hz, CH3); 13C NMR (75 MHz, DMSO-d6) δppm: 168.00 (C-10, CO), 167.92 (ester CO), 160.06 (C-2), 153.61, 143.38, 134.63, 133.35, 130.34, 129.04 (C-8), 128.22, 127.71, 124.89, 124.64, 121.22, 75.41 (C-3), 59.43 (OCH2CH3), 41.03 (C-4), 14.58 (OCH2CH3); Anal. calcd. for C21H16ClNO5: C, 63.40; H, 4.05; N, 3.52%. Found: C, 63.27; H, 3.85; N, 3.49%. Ethyl 2-amino-8-chloro-4-(4-chlorophenyl)-10-oxo-4,10- dihydropyrano[3,2-b]chromene-3-carboxylate (6g) cream powders; yield: 93%; mp 238–240 °C; IR (KBr) ν 3470, 3335 (NH2), 3065 (CH, aromatic), 2984 (CH, aliphatic), 1706, 1672 (2C=O), 1654 (C=C), 1194 cm–1 (C-O); 1H NMR (300 MHz, DMSO-d6) δppm: 8.01 (d, 1H, J=3 Hz, ArH), 7.93 (br, 2H, NH2), 7.79 (dd, 1H, J=9 Hz, 3 Hz, ArH), 7.62 (d, 1H, J=9 Hz, ArH), 7.41–7.33 (m, 4H, ArH), 4.97 (s, 1H, CH), 3.97 (q, 2H, J=6 Hz, OCH2CH3), 1.05 (t, 3H, J=6 Hz, OCH2CH3); 13C NMR (75 MHz, DMSO-d6) δppm: 167.93 (C-10, CO), 167.88 (ester CO), 159.99 (C-2), 153.60, 152.83, 142.35, 134.66, 133.38, 132.29, 130.36, 130.16, 128.97 (C-8), 124.86, 124.62, 121.18, 75.00 (C-3), 59.48 (OCH2CH3), 40.47 (C-4), 14.60 (OCH2CH3); Anal. calcd. for C21H15Cl2NO5: C, 58.35; H, 3.50; N, 3.24%. Found: C, 58.11; H, 3.37; N, 3.19%. Ethyl 2-amino-8-chloro-10-oxo-4-(p-tolyl)-4,10-dihy- dropyrano[3,2-b]chromene-3-carboxylate (6h) yellow powders; yield: 88%; mp 196–197 °C; IR (KBr) ν 3460, 3313 (NH2), 3049 (CH, aromatic), 2995 (CH, aliphatic), 1687, 1660 (2C=O), 1607 (C=C), 1191 cm–1 (C-O); 1H NMR (300 MHz, DMSO-d6) δppm: 8.00 (d, 1H, J=3 Hz, ArH), 7.87 (br, 2H, NH2), 7.74 (dd, 1H, J=9 Hz, 3 Hz, ArH), 7.57 (d, 1H, J=9 Hz, ArH), 7.15 (q, 4H, J=9 Hz, ArH), 4.87 (s, 1H, CH), 3.97 (q, J=6 Hz, 2H, OCH2CH3), 2.23 (s, 3H, CH3), 1.06 (t, J=6 Hz, 3H, OCH2CH3); 13C NMR (75 MHz, DMSO-d6) δppm: 168.02 (C-10, CO), 167.85 (ester CO), 160.01 (C-2), 153.73, 153.55, 140.42, 136.87, 134.55, 133.26, 130.31, 129.57 (C-8), 128.05, 124.82, 124.59, 121.12, 75.47 (C-3), 59.43 (OCH2CH3), 40.61 (C-4), 21.05 (CH3), 14.60 (OCH2CH3); Anal. calcd. for C22H18ClNO5: C, 64.16; H, 4.41; N, 3.40%. Found: C, 64.23; H, 4.17; N, 3.39%. Ethyl 2-amino-8-chloro-4-(4-fluorophenyl)-10-oxo-4,10- dihydropyrano[3,2-b]chromene-3-carboxylate (6i) white powders; yield: 89%; mp 208–210 °C; IR (KBr) ν 3469, 3334 (NH2), 3068 (CH, aromatic), 2985 (CH, aliphatic), 1707, 1672 (2C=O), 1653 (C=C), 1194 cm–1 (C-O); 1H NMR (300 MHz, DMSO-d6) δppm: 7.98 (d, 1H, J=3 Hz, ArH), 7.91 (br, 2H, NH2), 7.75 (dd, 1H, J=9 Hz, 3 Hz, ArH), 7.58 (d, 1H, J=9 Hz, ArH), 7.38–7.33 (m, 2H, ArH), 7.19–7.12 (m, 2H, ArH), 4.95 (s, 1H, CH), 3.96 (q, 2H, J=6 Hz, OCH2CH3), 1.04 (t, 3H, J=6 Hz, OCH2CH3); 13C NMR (75 MHz, DMSO-d6) δppm: 167.92 (C-10, CO), 167.89 (ester CO), 163.34 (C-2), 160.12, 159.98, 153.57, 153.08, 139.56, 139.52, 134.59, 133.30, 130.21 (C-8), 124.83, 124.60, 121.11, 115.88, 115.60, 75.25 (C-3), 59.43 (OCH2CH3), 40.55 (C-4), 14.57 (OCH2CH3); Anal. calcd. for C21H15ClFNO5: C, 60.66; H, 3.64; N, 3.37%. Found: C, 60.39; H, 3.67; N, 3.13%. Ethyl 2-amino-8-chloro-4-(4-methoxyphenyl)-10-oxo- 4,10-dihydropyrano[3,2-b]chromene-3-carboxylate (6j) yellow powders; yield: 87%; mp 207–208 °C; IR (KBr) ν 3419, 3295 (NH2), 3072 (CH, aromatic), 2984 (CH, ali- 923Acta Chim. Slov. 2022, 69, 920–927 Sabouri et al.: Synthesis and In Vitro Cytotoxicity of Novel Halogenated ... phatic), 1686, 1656 (2C=O), 1632 (C=C), 1192 cm–1 (C-O); 1H NMR (300 MHz, DMSO-d6) δppm: 8.00 (d, 1H, J=3 Hz, ArH), 7.86 (brs, 2H, NH2), 7.76 (dd, 1H, J=9 Hz, 3 Hz, ArH), 7.59 (d, 1H, J=9 Hz, ArH), 7.22 (d, 2H, J=9 Hz, ArH), 6.88 (d, 2H, J=9 Hz, ArH), 4.87 (s, 1H, CH), 3.98 (q, 2H, J=6 Hz, OCH2CH3), 3.70 (s, 3H, OCH3), 1.07 (t, 3H, J=6 Hz, OCH2CH3); 13C NMR (75 MHz, DMSO-d6) δppm: 168.05 (C-10, CO), 167.86 (ester CO), 159.98 (C-2), 158.84, 153.84, 153.57, 135.40, 134.55, 133.19, 130.30, 129.22 (C-8), 124.84, 124.60, 121.13, 114.37, 75.63 (C-3), 59.42 (OCH2CH3), 55.47 (OCH3), 40.17 (C-4), 14.63 (OCH2CH3); Anal. calcd. for C22H18ClNO6: C, 61.76; H, 4.24; N, 3.27%. Found: C, 61.51; H, 4.29; N, 3.01%. 2. 4. Cell Culture All the cell lines were purchased from the Iranian Bio- logical Resource Center (IBRC, Tehran, Iran) and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Biosera, France) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco, USA) and antibiotics (100 U/ml penicil- lin and 100 μg/ml streptomycin, Biosera, France). The cells were incubated at 37 °C, 5% CO2, and 95% relative humidity. 2. 4. 1. The Cytotoxic Assay The synthesized compounds were dissolved in DMSO to prepare stock solutions. Afterwards, different concentra- tions (from 0.2 to 1000 μM) were prepared by diluting the appropriate amounts of the stock solutions in DMEM (with- out FBS). The final amounts of DMSO were kept below 1%. The cells with a confluency of about 80% were har- vested with trypsin-EDTA (Biosera, France); and 1 × 104 cells were cultured in each well of a 96-well culture mi- croplate. The microplates were then incubated in the same conditions mentioned above for 24 h. The next day, the me- dia was removed from the wells and replaced with 100 µL of the prepared concentrations of the synthesized com- pounds or doxorubicin (as the standard). At least 3 wells of the microplate were used for each concentration. The cell controls were treated with DMEM containing the same percent of DMSO without the compounds. The microplates were further incubated for 24 h. Finally, 10 µL of the MTT solution (5 mg/ml, Melford, England) was added to the wells, the microplates were incubated for 3 h protected from light, formed formazan crystals were solubilized in 100 μL DMSO, and the absorbance was measured at 570 nm in a multiplate reader. The experiment was repeated 3 times. GraphPad® Prism version 5 was used to calculate the IC50 values from the mean percent of viable cells.16 3. Results and Discussions To prepare (6a-j) derivatives, 3-hydroxychromone derivatives were used as substrates (Scheme 1). The con- densation of 5’-bromo-; or 5’-chloro-2’-hydroxyacetophe- none (1a,b) with N,N-dimethylformamide-dimethylac- etal (DMF-DMA) was irradiated under microwave conditions, then refluxed with concentrated HCl. This re- action led to the production of 6-bromo-; and 6-chloro-chromone derivatives (2a,b), which formed epoxy chromones upon treatment with H2O2/NaOH in methylene chloride. This undergoes ring opening with concentrated HCl afforded 6-bromo-; and 6-chloro-3-hy- droxychromones (3a,b) in good yields.13 These com- pounds were fully characterized by standard spectroscopic techniques (IR, 1H and 13C NMR) and elemental analyses. Following our previous works on multi-component reactions to reach potentially bioactive scaffolds,18,19 we have synthesized a novel one-pot three component reac- tion for the synthesis of 8-halopyrano[3,2-b]chromen- 10(4H)-one (6a-j) including 6-bromo-; and 6-chloro-3-hy- droxychromones (3a,b), aromatic aldehydes (4a-j), and ethyl cyanoacetate (5) in the presence of three drops of Et3N in ethanol as the solvent and at reflux conditions. Af- ter completion of the reaction, the crude product was pu- rified by recrystallization and a series of ethyl 8-ha- lo-4,10-dihydropyrano[3,2-b]chromene-3-carboxylate derivatives (6a-j) were prepared in 86–93% yields (Scheme 2). The structures of compounds (6a-j) were determined on the basis of their elemental analyses, 1H and 13C NMR and IR spectroscopic data. In similar studies heteroannel- ation of cyclic ketones were conducted by use of catalysts like sodium carbonate, sodium saccharine and poly(4-vi- nylpyridine); 20–22 however in the present study we man- Scheme 1. Synthesis of 6-bromo-; and 6-chloro-3-hydroxychromone. Reagents and conditions: (i): 1) DMF-DMA/ MW; 2) HCl (con.), CH2Cl2, reflux; (ii): 1) H2O2, NaOH, CH2Cl2, ice-bath; 2) HCl (con.), reflux. 924 Acta Chim. Slov. 2022, 69, 920–927 Sabouri et al.: Synthesis and In Vitro Cytotoxicity of Novel Halogenated ... aged to synthesized halogenated dihydropyranochromene by one pot three component reaction without any catalyst in good yields. 3. 1. The Cytotoxic Assay To evaluate the cytotoxic activity of the synthesized compounds, MTT assay was used after treating MCF-7 (breast cancer) and A549 (lung cancer) cell lines. The com- pounds had three or four aromatic rings, and based on Na- gai et. al, having more than two aromatic rings leads to a higher tumor specificity.9 The IC50 values range from 36 µM (compound 6f) to 631 µM (compound 6j) for MCF-7 and 56 µM (compound 6f) to 558 µM (compound 6j) for A549 cells (Table 1). The most potent compound is com- pound 6f (Fig.1). Compared to compound 6a, which has a Br atom in C8, 6f (the compound with a Cl atom in this position) is about 11 and 7 times stronger on MCF-7 and A549, respectively. Placing any moiety on para position of aromatic aldehydes (R) leads to a decrease in the cytotox- icity when X is a chlorine atom, however, when X is a bro- mine atom, adding an R moiety results in a more potent compound especially when the R group is a chlorine atom (compound 6b). In our previous study, a halogen group substitution on the carbon 4 of the phenyl ring decreased the cytotoxic activity of the compound except when it was Cl, interestingly it is also understood that chromenes bear- ing chlorine and bromine have quite much cytotoxic effect in comparison with ones that lack Cl and Br.19 Since com- pound 6f can be a promising candidate as a cytotoxic agent, it was tested on SW480 (a colorectal cancer cell line) and HUVEC (Human Umbilical Vein Endothelial Cells). The IC50 values were 10.8 ± 1.5 and 57.2 ± 3.2 µM, respec- tively. By comparison of our compounds with benzo[h] chromene derivatives, it is revealed that fused ring at 2,3-position may boost antitumor activity of compound.23 So a rationale in our future work is to fuse ring at 2,3 posi- tion and making new compounds which may have strong- er cytotoxic activity. One study showed that inclusion of thienyl group next to chromene ring make these com- pounds more potent and also selective against prostate cancer.22 In another study it is found that a chlorophenyl moiety on central dihyrobenzo[h]pyrano[3,2-c]chromene ring has a positive impact on cytotoxicity, which is also effective in our compounds.24 It is also revealed that elec- tron withdrawing group at the four position of the phenyl ring at the 1-position of 1H-benzo[f]chromene enhance the antitumor activity of the compounds.25 This trend is also in accordance with our finding. Generally, it seems that modifications at the C-4 and C-6 positions of Scheme 2. Synthesis of dihydropyrano[3,2-b]chromene derivatives. Reagents and conditions: (i): triethylamine, EtOH, reflux. 925Acta Chim. Slov. 2022, 69, 920–927 Sabouri et al.: Synthesis and In Vitro Cytotoxicity of Novel Halogenated ... chromenes have impacts on anticancer activity of these compounds.26 4. Conclusion The present study describes the synthesis and inves- tigation of cytotoxic activities of a series of novel ha- lopyranochromene derivatives. We have synthesized some derivatives by one-pot three component reactions of 6-bromo-; or 6-chloro-3-hydroxychromone, aromatic al- dehydes, and ethyl cyanoacetate in the presence of trieth- ylamine in ethanol as solvent and at reflux conditions. Some of the compounds showed moderate cytotoxicity on the two cancer cell lines (A549 and MCF-7). The best com- Fig. 1. The viability percent of the cells (MCF-7 and A549) after 24 hours treatment with different concentrations of the synthesized compounds. Table 1. The IC50 (µM) values of the compounds on two cancer cell lines measured by MTT assay method (mean ± standard error of mean). Compd. NO. A549 MCF-7 6a 414.8 ± 2.1 389.0 ± 18 6b 120.5 ± 0.3 115.5 ± 1.2 6c 155 ± 1.2 169.1 ± 0.5 6d 142.3 ± 0.5 168.5 ± 1.5 6e 150.5 ± 8.9 175.8 ± 1.9 6f 56 ± 0.3 35.8 ± 3.8 6g 107.8 ± 6.4 179.8 ± 5.8 6h 272.3 ± 8.9 232.1 ± 3 6i 230.6 ± 4.8 328.8 ± 16.6 6j 558.2 ± 8.7 631.2 ± 8.5 Doxorubicin 7. 9 ± 0.2 6.4 ± 0.1 926 Acta Chim. Slov. 2022, 69, 920–927 Sabouri et al.: Synthesis and In Vitro Cytotoxicity of Novel Halogenated ... pound had a Cl atom (C8) and a phenyl ring (C4) of pyranochromene scaffold (compound 6f). It is concluded that these compounds can be lead compounds for synthe- sizing anticancer agents. Abbreviations EtOH: ethanol; MTT: (3-(4,5-dimethylthi- azol-2-yl)-2,5-diphenyltetrazolium bromide; DMF-DMA: N,N-dimethylformamide-dimethylacetal; HUVEC: Hu- man Umbilical Vein Endothelial Cells; IC50: 50% Inhibi- tion concentration; KBr: Potassium bromide; ppm: parts per million; TLC: thin layer chromatography. Supplementary Information The online version contains supplementary material available at https://doi.…. Acknowledgements The authors express their great appreciation to Phar- maceutics Research Center, Institute of Neuropharmacol- ogy, Kerman University of Medical Sciences for support- ing this investigation. Authors’ contributions Ehsan Faghih-Mirzaei and Mehdi Abaszadeh, de- signed, synthesized and performed experiments, analysed data and wrote the paper. Salehe Sabouri, designed and performed the biologic assay and data analysis and con- tributed in writing the manuscript. All authors were in- volved in revising the content, agree to take accountability for the integrity and accuracy of the work, and have read and approved the final manuscript. Funding This research was supported by a grant from Kerman University of Medical Sciences [No.97000675]. The funders had no role in the research design, data collection, analysis, and the decision to publish. Disclosure statement No potential conflict of interest was reported by the authors. 5. References 1. G. P. Ellis. Chemistry of Heterocyclic Compounds: Chromenes, Chromanones, and Chromones, vol. 31. New York: John Wiley & Sons, Inc.; 1977, pp. 1–10. DOI:10.1002/9780470187012.ch1 2. A. Gaspar, M. J. Matos, J. Garrido, E. Uriarte, F. Borges. Chem Rev. 2014, 114, 4960–92. DOI:10.1021/cr400265z 3. S. Yang, J. Zhou, D. Li, C. Shang, L. Peng, S. Pan. Food Chem. 2017, 224, 26–31. DOI:10.1016/j.foodchem.2016.12.001 4. T. P. Cushnie, A. J. Lamb. Int J Antimicrob Agents. 2005, 26, 343–56. DOI:10.1016/j.ijantimicag.2005.09.002 5. S. Lee, W. K. Yang, J. H. Song, Y. M. Ra, J. H. Jeong, W. Choe, I. Kang, S. S. Kim, J. Ha. Biochem Pharmacol. 2013, 85, 965– 76. DOI:10.1016/j.bcp.2012.12.023 6. H. Zakaryan, E. Arabyan, A. Oo, K. Zandi. Arch Virol. 2017, 162, 2539–51. DOI:10.1007/s00705-017-3417-y 7. P. Ninfali, A. Antonelli, M. Magnani, E. S. Scarpa. Nutrients. 2020, 12, 2534. DOI:10.3390/nu12092534 8. H. P. Kim, K. H. Son, H. W. Chang, S. S. Kang. J Pharmacol Sci. 2004, 96, 229–45. DOI:10.1254/jphs.CRJ04003X 9. J. Nagai, H. Shi, N. Sezaki, N. Yoshida, K. Bandow, Y. Uesawa, H. Sakagami, M. Tomomura, A. Tomomura, K. Takao, Y. Su- gita. Anticancer Res. 2019, 39, 6479–88. DOI:10.21873/anticanres.13862 10. J. Baby, A. R. Devan, A. R. Kumar, J. N. Gorantla, B. Nair, T. S. Aishwarya, L. R. Nath. J Food Biochem. 2021, 45, e13761 (1–23). 11. N. Phosrithong, W. Samee, P. Nunthanavanit, J. Ungwitaya- torn. Chem Biol Drug Des. 2012, 79, 981–89. DOI:10.1111/j.1747-0285.2012.01368.x 12. J. Lee, T. Park, S. Jeong, K. Kim, C. Hong. Bioorg Med Chem Lett. 2007, 17, 1284–87. DOI:10.1016/j.bmcl.2006.12.011 13. M. Spadafora, V. Y. Postupalenko, V. V. Shvadchak, A. S. Klymchenko, Y. Mély, A. Burger, R. Benhida. Tetrahedron. 2009, 65, 7809–16. DOI:10.1016/j.tet.2009.07.021 14. A. M. Verdan, H. C. Wang, C. R. Garcia, W. P. Henry, J. L. Brumaghim. J Inorg Biochem. 2011, 105, 1314–22. DOI:10.1016/j.jinorgbio.2011.07.006 15. R. L. Siegel, K. D. Miller, H. E. Fuchs, A. Jemal. Cancer J Clin. 2021, 71, 7–33. DOI:10.3322/caac.21654 16. M. Abaszadeh, A. Ebrahimi, S. Sabouri. Biointerface Res Appl Chem. 2021, 11, 10987–95. DOI:10.33263/BRIAC113.1098710995 17. P. Rao, G. Srimannarayana. Synth Commun. 1987, 17, 1507– 12. DOI:10.1080/00397918708057776 18. M. Abaszadeh, M. Seifi. Lett Org Chem. 2015, 12, 271–76. DOI:10.2174/1570178612666150203004727 19. S. Sabouri, M. Abaszadeh. Polycyclic Aromat. Compd. 2021, 41, 467–77. DOI:10.1080/10406638.2019.1597381 20. R. Keshavarz, M. Farahi, B. Karami. Acta Chim. Slov. 2021, 68, 332–340. DOI:10.17344/acsi.2020.6266 21. L. Moradi, M. Aghamohammad Sadegh. Acta Chim. Slov. 2017, 64, 506–512. DOI:10.17344/acsi.2017.3417 22. J. Albadi, A. Mansournezhad, F. Akbari Balout-Bangan. Acta Chim. Slov. 2014, 61, 185–190. 23. R. M. Okasha, F. F. Alblewi,  T. H. Afifi,  A. Naqvi,  A. M. Fouda, A. A. M. Al- Dies, A. M. El-Agrody. Molecules. 2017, 22, 479. DOI:10.3390/molecules22030479 24. N. Esmati, M. Foroughian, M. Saeedi, M. Mahdavi, M. Khoshneviszadeh, O. Firuzi, N. Tanideh et al. J. Heterocycl. Chem. 2015, 52, 97–104. DOI:10.1002/jhet.1991 927Acta Chim. Slov. 2022, 69, 920–927 Sabouri et al.: Synthesis and In Vitro Cytotoxicity of Novel Halogenated ... 25. H. M. Mohamed, A. M. Fouda, E. S. Khattab, A. M. El-Agro- dy, and T. H. Afifi. Zeitschrift für Naturforschung C. 2017, 72, 161–71. 26. V. Raj, J. Lee. Front. Chem. 2020, 8, 623. DOI:10.3389/fchem.2020.00623 Povzetek Pljučni rak in rak dojk sta med najpogostejšimi raki. V okviru dela smo najprej pripravili 6-bromo- in 6-kloro-3-hidrok- sikromonske spojine. V naslednjem koraku je bila sintetizirana serija derivatov 8-bromo- in 8-kloro-dihidropirano[3,2-b] kromena s hkratno trikomponentno reakcijo teh dveh spojin, aromatskih aldehidov in etil cianoacetata v prisotnosti trietilamina v EtOH pri pogojih povratnega toka. Sintetizirane spojine so bile testirane za in vitro citotoksično delovanje na celičnih linijah A549 (pljučni rak) in MCF-7 (rak dojke). Ugotovljeno je bilo, da imajo nekatere spojine visoko do zmerno citotoksičnost, zato so potencialni kandidati za nadaljnje študije. Ta študija je lahko podlaga za nadaljnje študije za načrtovanje in sintezo močnih proti-rakavih spojin ter raziskovanje njihovega mehanizma delovanja. Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License 928 Acta Chim. Slov. 2022, 69, 928–936 Han et al.: Syntheses, Crystal Structures and Xanthine Oxidase ... DOI: 10.17344/acsi.2022.7817 Scientific paper Syntheses, Crystal Structures and Xanthine Oxidase Inhibitory Activity of Aroylhydrazones Yong-Jun Han,1 Xue-Yao Guo2 and Ling-Wei Xue1,* 1 School of Chemical and Environmental Engineering, Pingdingshan University, Pingdingshan Henan 467000, P.R. China 2 Pingdingshan Ecological Environment Monitoring Center, Henan 467000, P.R. China * Corresponding author: E-mail: pdsuchemistry@163.com Received: 09-21-2022 Abstract A series of hydrazones, (E)-N’-(4-hydroxy-3-methoxybenzylidene)-4-nitrobenzohydrazide (1), (E)-4-(dimethylami- no)-N’-(4-hydroxy-3-methoxybenzylidene)benzohydrazide (2), N’-(2-hydroxy-5-methylbenzylidene)-4-nitrobenzohy- drazide (3) and 2-fluoro-N’-(2-hydroxy-5-methylbenzylidene)benzohydrazide (4), were prepared and structurally char- acterized by elemental analysis, IR and 1H NMR spectra, and X-ray single crystal determination. The xanthine oxidase inhibitory activities of the compounds were investigated. Among the compounds, N’-(3-methoxybenzylidene)-4-ni- trobenzohydrazide (1) showed the strongest activity. Docking simulations were performed to insert the compounds into the crystal structure of xanthine oxidase at the active site and to investigate the probable binding modes. Keywords: Hydrazone; xanthine oxidase; inhibition; crystal structure; molecular docking study. 1. Introduction Xanthine oxidase (XO; EC 1.17.3.2), a molybdenum hydroxylase, catalyzes the hydroxylation of hypoxanthine and xanthine to form uric acid and superoxide anions. These superoxide anions are associated with postischemic tissue damage and edema, as well as vascular permeabili- ty.1 XO can oxidize the synthetic purine drug 6-mercapto- purine, causing it to lose its pharmacologic properties. XO is also associated with diseases such as liver and kidney damage, atherosclerosis, chronic heart failure, hyperten- sion, and sickle cell anemia because it produces reactive oxygen species (ROS) in addition to uric acid.2 Therefore, control of XO activity may be helpful in the therapy of some diseases. Allopurinol is a widely recognized XO in- hibitor used to treat gout.3 However, given its side effects, toxicity, and inability to prevent free radical formation by the enzyme,4 there is a need to investigate new and effi- cient XO inhibitors. A number of compounds of different Scheme 1. The aroylhydrazones. 929Acta Chim. Slov. 2022, 69, 928–936 Han et al.: Syntheses, Crystal Structures and Xanthine Oxidase ... types, such as carboxylic acids and pyrimidines,5 pyrimid- inones and 3-cyanoindoles,6 amides,7 pyrazoles,8 thiobar- biturates,9 hydrozingerones,10 have been described with XO inhibitory activities. Schiff bases have long been of great interest in biological chemistry.11 Leigh and cowork- ers have described some Schiff bases as novel XO inhibi- tors.12 However, studies on hydrazones are limited, and rational structure-activity relationships have not yet been established. As an extension of work exploring effective XO inhibitors in conjunction with Schiff bases, a series of hydrazone-like Schiff bases, (E)-N’-(4-hydroxy-3-methox- ybenzylidene)-4-nitrobenzohydrazide (1), (E)-4-(dimeth- ylamino)-N’-(4-hydroxy-3-methoxybenzylidene)benzo- hydrazide (2), N’-(2-hydroxy-5-methylbenzylidene)-4- nitrobenzohydrazide (3), and 2-fluoro-N’-(2-hydroxy-5- methylbenzylidene)benzohydrazide (4), were synthesized and structurally characterized. The XO inhibitory activi- ties of the compounds were investigated by both experi- mental and molecular docking studies. 2. Experimental 2. 1. Materials and Methods Starting materials, reagents, and solvents of AR grade were purchased from commercial suppliers and used without further purification. Elemental analyzes were performed using a Perkin-Elmer 240C elemental analyzer. IR spectra were recorded as KBr pellets in the 4000-400 cm-1 range using a Jasco FT/IR−4000 spectrometer. 1H NMR data were recorded using a Bruker 300 MHz instru- ment and X-ray diffraction was performed with a Bruker SMART 1000 CCD area diffractometer. 2. 2. General Method for the Synthesis of the Compounds The compounds were prepared according to the lit- erature method.13 Equimolar amounts (1.0 mmol each) of the hydrazides and aldehydes were dissolved in methanol (30 ml) and stirred at room temperature for 30 minutes to give a clear solution. X-ray quality single crystals were formed by slowly evaporating the solution in air over sev- eral days. 2. 2. 1. N’-(3-Methoxybenzylidene)-4- nitrobenzohydrazide (1) 4-Hydroxy-3-methoxybenzaldehyde and 4-nitro- benzo hydrazide. Yield: 87%. Anal. calcd. for C31H32N6O12: C, 54.7; H, 4.7; N, 12.3; Found: C, 54.5; H, 4.8; N, 12.4%. IR (KBr, cm–1) ν 3318 (m, O-H), 3217 (m, N-H), 1653 (s, C=O), 1621 (s, C=N). 1H NMR (300 MHz, CDCl3) δ 12.07 (s, 1H, -NH), 10.12 (s, 1H, -OH), 8.57 (s, 1H, CH=N), 8.08–8.50 (m, 4H, ArH), 6.87–7.53 (m, 3H, ArH), 3.79 (s, 3H, -OCH3). 2. 2. 2. N’-(4-Hydroxy-3-methoxybenzylidene)-4- dimethylaminobenzohydrazide (2) 4-Hydroxy-3-methoxybenzaldehyde and 4-dime- thylaminobenzohydrazide. Yield: 91%. Anal. calcd. for C17H21N3O4: C, 61.6; H, 6.4; N, 12.7; Found: C, 61.5; H, 6.4; N, 12.6%. IR data (KBr, cm–1) ν 3351 (m, O-H), 3208 (m, N-H), 1649 (s, C=O), 1623 (s, C=N). 1H NMR (300 MHz, CDCl3) δ 12.23 (s, 1H, -NH), 10.10 (s, 1H, -OH), 8.56 (s, 1H, CH=N), 6.90–7.63 (m, 7H, ArH), 3.79 (s, 3H, -OCH3), 3.02 (s, 6H, -N(CH3)2). 2. 2. 3. N’-(2-Hydroxy-5-methylbenzylidene)-4- nitrobenzohydrazide (3) 5-Methylsalicylaldehyde and 4-nitrobenzohydrazide. Yield: 89%. Anal. calcd. for C15H13N3O4: C, 60.2; H, 4.4; N, 14.0. Found: C, 60.1; H, 4.5; N, 14.2%. IR data (KBr, cm–1) ν 3403 (w, O-H), 3186 (w, N-H), 1650 (s, C=O), 1606 (s, C=N), 1565 (m, NO2), 1334 (s, NO2). 1H NMR (300 MHz, CDCl3) δ 12.17 (s, 1H, -NH), 11.23 (s, 1H, -OH), 8.75 (s, 1H, CH=N), 8.39 (d, 2H, ArH), 8.15 (d, 2H, ArH), 7.51 (s, 1H, ArH), 7.12 (d, 1H, ArH), 6.89 (d, 1H, ArH), 2.32 (s, 3H, CH3). 2. 2. 4. 2-Fluoro-N’-(2-hydroxy-5- methylbenzylidene)benzohydrazide (4) 5-Methylsalicylaldehyde and 2-fluorobenzohy- drazide. Yield: 92%. Anal. calcd. for C15H13FN2O2: C, 66.2; H, 4.8; N, 10.3. Found: C, 66.0; H, 4.7; N, 10.2%. IR data (KBr, cm–1) ν 3411 (w, O-H), 3183 (w, N-H), 1653 (s, C=O), 1608 (s, C=N). 1H NMR (300 MHz, CDCl3) δ 12.21 (s, 1H, -NH), 11.16 (s, 1H, -OH), 8.75 (s, 1H, CH=N), 8.01 (m, 2H, ArH), 7.50 (s, 1H, ArH), 7.41 (m, 2H, ArH), 7.12 (d, 1H, ArH), 6.89 (d, 1H, ArH), 2.32 (s, 3H, CH3). 2. 3. Measurement of the XO Inhibitory Activity XO activities with xanthine as substrate were meas- ured spectrophotometrically, based on the procedure re- ported by L. D. Kong et al (with modifications).14 Xanthine oxidase activity is measured by the formation of uric acid at 295 nm. The assay was performed in a final volume of 1 mL 50 mmol L–1 K2HPO4 pH 7.8 in a quartz cuvette. The reaction mixture contains 200 µL of 84.8 µg·mL–1 xanthine in 50 mmol L–1 K2HPO4, 50 L of the tested compounds at various concentrations. The reaction is started by adding 66 µL 37.7 mU mL–1 xanthine oxidase. The reaction is monitored at 295 nm for 6 min, and the product is ex- pressed as µmol of uric acid per minute. The reaction ki- netics were linear during this 6-minute monitoring. 2. 4. Docking Simulations The molecular docking study of the compounds to the 3D X-ray structure of XO (entry 1FIQ in the Protein Data 930 Acta Chim. Slov. 2022, 69, 928–936 Han et al.: Syntheses, Crystal Structures and Xanthine Oxidase ... Bank) was performed using AutoDock version 4.2. First, the AutoGrid component of the program precomputes a 3D grid with interaction energies based on the macromolecular tar- get using the force field AMBER. The cubic grid box with 60 × 80 × 66 Å3 points in x, y, and z directions with a spacing of 0.375 Å and grid maps were created representing the catalyt- ically active target region in which the native ligand was em- bedded. Automated docking studies were then performed to determine the free energy of the inhibitor within the macro- molecules. The search algorithm GALS (genetic algorithm with local search) was chosen to search for the best conform- ers. Parameters were set using ADT software (AutoDock- Tools package, version 1.5.4) at PC in conjunction with Au- toDock 4.2. Default settings were used with an initial population of 100 randomly placed individuals, a maximum number of 2.5 × 106 energy ratings, and a maximum number of 2.7 × 104 generations. A mutation rate of 0.02 and a cross- over rate of 0.8 were chosen. Considering the most favorable free energy of the bidding and majority clusters, the results were selected as the most likely complex structures. 2. 5. Data Collection, Structural Determination and Refinement Diffraction intensities for the compounds were re- corded at 298(2) K using a Bruker D8 VENTURE PHO- TON diffractometer with Mo Kα radiation (λ = 0.71073 Å). Collected data were reduced using SAINT,15 and mul- ti-scan absorption corrections were performed using SADABS.16 Structures were solved using direct methods and refined against F2 using full matrix least squares methods with SHELXTL.17 All non-hydrogen atoms were refined anisotropically. The amino, hydroxyl, and water H atoms were localized using Fourier difference maps and refined isotropically, with N-H, O-H, and H···H distances constrained to 0.90(1), 0.85(1), and 1.37(2) Å, respectively. All other H atoms were placed in idealized positions and confined to their parent atoms. Crystallographic data for the compounds are summarized in Table 1. Information on the hydrogen bonds can be found in Table 2. 3. Results and Discussion 3. 1. Chemistry The compounds were readily synthesized by reacting aldehydes in a 1:1 molar ratio with hydrazides in methanol at room temperature with high yield and purity. Single crys- tals suitable for X-ray diffraction were obtained by slowly evaporating the solutions containing the compounds in air. The compounds were characterized by elemental analyzes and IR spectra. The structures of the compounds were also confirmed by single crystal X-ray crystallography. Table 1. Crystallographic and experimental data for the compounds. Compound 1 2 3 4 × ½ H2O × H2O × ½ CH3OH Formula C31H32N6O12 C17H21N3O4 C15H13N3O4 C15H10F3N3O5 Mr 680.6 331.4 299.3 369.3 T (K) 298(2) 298(2) 298(2) 298(2) Crystal system Triclinic Monoclinic Monoclinic Monoclinic Space group P-1 P21/n P21/c P21/c a (Å) 7.655(1) 8.243(1) 10.257(2) 9.753(2) b (Å) 12.638(2) 21.573(2) 15.190(2) 10.505(2) c (Å) 17.213(1) 10.106(2) 9.181(3) 14.251(30 α (°) 77.350(2) 90 90 71.257(2) β (°) 80.122(2) 106.749(2) 94.912(2) 84.879(3) γ (°) 88.953(2) 90 90 81.267(3) V (Å3) 1600.4(3) 1720.8(5) 1425.2(6) 1365.5(5) Z 2 4 4 4 Dc (g cm–3) 1.412 1.279 1.395 1.324 µ (Mo-Kα) (mm–1) 0.110 0.092 0.104 0.099 F(000) 712 704 624 568 Reflections collected 11674 8178 5818 10030 Unique reflections 5874 3178 2597 4990 Observed reflections (I ≥ 2σ(I)) 2665 1655 1315 4077 Parameters 458 230 205 371 Restraints 36 4 2 2 Goodness-of-fit on F2 0.988 0.993 0.941 1.069 R1, wR2 [I ≥ 2σ(I)]a 0.0679, 0.1664 0.0586, 0.1236 0.0618, 0.1111 0.0482, 0.1460 R1, wR2 (all data)a 0.1516, 0.2129 0.1261, 0.1543 0.1313, 0.1334 0.0614, 0.1718 Large diff. peak and hole (eÅ–3) 0.244, –0.528 0.186, –0.177 0.157, –0.203 0.509, –0.329 aR1 = Fo – Fc/Fo, wR2 = [ w(Fo2 – Fc2)/∑ w(Fo2)2]1/2 931Acta Chim. Slov. 2022, 69, 928–936 Han et al.: Syntheses, Crystal Structures and Xanthine Oxidase ... 3. 2. Structure Description of the Compounds 1 and 2 The structures of compounds 1 and 2 and the num- bering scheme of the atoms are shown in Figures 1 and 2, respectively. The asymmetric unit of 1 contains two carbo- hydrazone molecules, one methanol molecule, and one water molecule. The asymmetric unit of 2 contains one carbohydrazone molecule and one water molecule. The carbohydrazone molecules adopt an E configuration around the C=N bonds. The dihedral angles between the C1–C6 and C9–C14 in benzene rings, and C16–C21 and C24–C29 in 1, and C1–C6 and C9–C14 in 2 are 5.8(3), 5.2(3), and 10.4(3)°, respectively. The bond distances C7– N1 (1.285(4) Å) and C22–N4 (1.258(4) Å) in 1 and C7–N1 (1.259(3) Å) in 2 correspond to C=N double bonds and are comparable to the previously reported analogs of carbohy- drazones.18 The bond distances C8–N2 (1.330(5) Å) and C23–N5 (1.339(4) Å) in 1 and C8–N2 (1.339(4) Å) in 2 are shorter than the typical values for C-N single bonds, indi- cating the presence of conjugation in the carbohydrazone molecules. In the crystal structures of the two compounds, the carbohydrazone molecules and the solvent molecules are connected via N–H∙∙∙O, O–H∙∙∙N, and O–H∙∙∙∙O intermo- lecular hydrogen bonds to form 3D networks (Figure 3 for 1 and Figure 4 for 2). The water and methanol mole- cules in the compounds act as both acceptors and donors in the hydrogen bonds. In addition, weak π···π interac- tions are observed in 1, ranging from 3.6461 to 3.9636 Å. In 2, the centroid to centroid distances are in the range of 4.8621–5.5703 Å, which are far from the π···π interac- tions. Table 2. Hydrogen bond distances (Å) and bond angles (°) for the compounds. D–H∙∙∙A d(D–H) d(H∙∙A) d(D∙∙∙A) Angle(D–H∙∙∙A) 1 O11–H11A∙∙∙N1 0.85(1) 2.32(2) 3.092(5) 153(4) O11–H11A∙∙∙O1 0.85(1) 2.31(3) 2.861(5) 124(3) O11–H11B∙∙∙O7#1 0.85(1) 2.25(3) 3.011(5) 150(4) N5–H5A∙∙∙O4#2 0.90(1) 2.13(2) 3.003(4) 165(4) N2–H2A∙∙∙O12 0.90(1) 1.99(2) 2.870(5) 168(4) O12–H12A∙∙∙O11#3 0.82 1.89 2.709(7) 177 O8–H8∙∙∙O1#1 0.82 2.04 2.813(4) 156 O3–H3∙∙∙O6#4 0.82 1.91 2.718(4) 169 2 O1–H1∙∙∙O4#5 0.82 1.79 2.608(3) 172 N2–H2∙∙∙O1#6 0.90(1) 2.25(1) 3.141(3) 173(3) O4–H4A∙∙∙O3#7 0.85(1) 1.88(1) 2.734(3) 175(3) O4–H4B∙∙∙O3#6 0.85(1) 2.07(2) 2.850(3) 152(3) O4–H4B∙∙∙N1#6 0.85(1) 2.49(2) 3.170(3) 137(2) 3 O1–H1∙∙∙N1 0.85(1) 1.90(2) 2.655(3) 147(3) N2–H2∙∙∙O2#8 0.90(1) 2.04(2) 2.911(3) 163(3) 4 N4–H4∙∙∙O2#9 0.90(1) 2.05(1) 2.920(2) 163(2) N2–H2∙∙∙O4 0.90(1) 1.99(1) 2.872(2) 168(2) O3–H3∙∙∙N3 0.82 1.89 2.609(2) 145 O1–H1∙∙∙N1 0.82 1.92 2.619(2) 143 Symmetry codes: #1: 1 – x, 1 – y, 1 – z; #2: x, y, –1 + z; #3: x, 1 – y, 1 – z; #4: –1 + x, 1 + y, z; #5: x, y, –1 + z; #6: –1/2 + x, 1/2 – y, –1/2 + z; #7: 3/2 – x, –1/2 + y, 1/2 – z; #8: x, 1/2 – y, –1/2 + z; #9: 1 + x, y, z. Figure 1. A perspective view of the molecular structure of 1 show- ing the atomic labeling scheme. The thermal ellipsoids are drawn at the 30% probability level. The hydrogen bonds are shown as a dashed line. 932 Acta Chim. Slov. 2022, 69, 928–936 Han et al.: Syntheses, Crystal Structures and Xanthine Oxidase ... Figure 2. A perspective view of the molecular structure of 2 show- ing the atomic labeling scheme. The thermal ellipsoids are drawn at the 30% probability level. Figure 3. The packing diagram of 1. Hydrogen bonding interac- tions are shown as dashed lines. Figure 4. The packing diagram of 2. Hydrogen bonding interac- tions are shown as dashed lines. 3. 3. Structure Description of the Compounds 3 and 4 The structures of compounds 3 and 4 are shown in Figures 5 and 6, respectively, along with the atom number- ing scheme. The asymmetric unit of compound 3 consists of two independent molecules. All molecules of the com- pounds adopt the trans configuration with respect to the C=N methylidene units. The distances of the methylidene bonds, ranging from 1.26 to 1.28 Å, confirm that they are typical double bonds. The shorter than usual distances of the C–N bonds and the longer than usual distances of the C=O bonds for the –C(O) –NH units indicate the presence of conjugation effects in the molecules. It is noteworthy that the C8=N1 bond in 3 is much shorter than that of the C=N double bonds in 4, which could be due to the elec- tron-withdrawing effects of the nitro groups. The other bond lengths in the compounds are comparable with each other and are within normal values.18 The dihedral angles between the two aromatic rings are 4.8(3)° for 3 and 31.1(3)° and 52.4(3)° for 4. In each of the compounds, an intramolecular O–H···N hydrogen bond (Table 2) forms an S(6) ring motif.19 In the crystal structure of 3, the molecules are linked by intermolecular N–H···O hydrogen bonds (Table 2) and form 1D chains running along the c axis (Figure 7). In the crystal structure of 4, the molecules are linked by intermo- lecular N–H···O hydrogen bonds (Table 2) and form 1D chains running along the c-axis (Figure 8). In addition, weak π···π interactions are observed in the compounds, ranging from 3.692 to 4.025 Å for 3 and from 4.018 to 4.833 Å for 4. 3. 4. Infrared and 1H NMR Spectra The broad and intermediate bands at 3318 cm–1 (1) and 3351 cm–1 (2) are due to the O–H stretching vibra- tions of the water and hydroxyl groups. The sharp bands at 3217 cm–1 (1), 3208 cm–1 (2), 3186 cm–1 (3) and 3183 cm– 1 (4) are due to the N–H stretching vibrations. The com- pounds exhibit strong absorptions at 1621–1623 cm–1 for 1 and 2, and 1606–1608 cm–1 for 3 and 4, which can be attributed to the C=N vibrations.20 The bands originating from the stretching vibrations of the C=O groups are ob- served at 1649–1653 cm–1 for the compounds. The bands Figure 5. A perspective view of the molecular structure of 3 show- ing the atomic labeling scheme. The thermal ellipsoids are drawn at the 30% probability level. 933Acta Chim. Slov. 2022, 69, 928–936 Han et al.: Syntheses, Crystal Structures and Xanthine Oxidase ... indicative of the νas(NO2) and νs(NO2) vibrations are ob- served at 1565 and 1334 cm–1 for compound 1.20 In 1H NMR, the absence of NH2 signals and the ap- pearance of peaks for NH protons in the range δ = 12.07– 12.23 ppm and imine CH protons in the range δ = 8.56– 8.75 ppm confirm the synthesis of the hydrazine compounds. The signals of aromatic protons were found in their respective ranges with different multiplicities, con- firming their relevant substitution pattern. Figure 6. A perspective view of the molecular structure of 4 show- ing the atomic labeling scheme. The thermal ellipsoids are drawn at the 30% probability level. Hydrogen bonds are shown as dashed lines. Figure 7. Molecular packing diagram of 3, viewed along the b axis. Hydrogen bonds are shown as dashed lines. 3. 5. Pharmacology Measurement of XO inhibitory activity was per- formed for three parallel time points. The percentages of inhibition at a concentration of 100 μmol·L–1 and the IC50 values for the compounds against XO are summarized in Table 3. Allopurinol served as a reference with a percent in- hibition of 80.7 ± 4.3 and an IC50 value of 8.7 ± 2.3 μmol·L–1. Compound 1 showed the strongest activity with a percent inhibition of 82.3 ± 3.0 and an IC50 value of 7.6 ± 1.8 μmol·L–1, which is better than allopurinol. The other three compounds showed intermediate activity with a per- cent inhibition value of less than 50%. Although the num- ber of compounds tested is limited, some structural fea- tures important for the inhibitory effect on xanthine oxidase can be deduced. Compound 1 containing NO2 group has significantly higher activity than compound 2 containing an NMe2 group, suggesting that NO2 is a pre- ferred group for the inhibition process. Comparing com- pounds 1 and 2 with compound 3, it can be seen that the other substituent groups such as OH and OMe also con- tribute to the inhibition. And interestingly, it is not diffi- cult to find out from the results of compounds 3 and 4 that NO2 is a better group than F for XO inhibition. These re- sults are consistent with reports in the literature that the presence of electron-withdrawing groups in the benzene rings can enhance the activities21 and are also similar to the fact that the presence of a bulky ethyl group has a stronger activity than a methyl group.22 3. 6. Molecular Docking Study To explain and understand the strong inhibitory ef- fect observed in the experiment, a molecular docking Figure 8. Molecular packing diagram of 4, viewed along the b axis. Hydrogen bonds are shown as dashed lines. Table 3. Inhibition of XO by the compounds tested. Compounds Percent of Inhibitionb IC50 (μmol·L–1) 1 82.3 ± 3.0 7.6 ± 1.8 2 45.4 ± 2.7 – 3 39.5 ± 2.6 – 4 35.7 ± 2.2 – Allopurinol 80.7 ± 4.3 8.7 ± 2.3 b The concentration of the tested material is 100 μmol·L–1. 934 Acta Chim. Slov. 2022, 69, 928–936 Han et al.: Syntheses, Crystal Structures and Xanthine Oxidase ... study was performed to investigate the binding effects be- tween compound 1 and the active sites of XO (entry 1FIQ in the Protein Data Bank). Allopurinol was used to test the docking model, which gave satisfactory results. Figure 9 shows the binding model for compound 1 at the active site of enzyme XO. The docking score is –9.83. In comparison, the docking score for allopurinol is –6.27. From the docking results, the molecule of compound 1 fits well into the active pocket of XO. The molecule of 1 is attached to the enzyme via four hydrogen bonds with ALA1079, PHE1008, THR1010, and ARG880. In addition, there are hydrophobic interactions between the com- pounds and the active sites of the enzyme. The results of the molecular docking study may explain the effective in- hibitory effect of compound 1 on XO. 4. Conclusion The present study reports the synthesis, crystal struc- tures, and XO inhibitory activities of a series of hydra- zones. The compounds were characterized by elemental analysis, IR and 1H NMR spectra, as well as single crystal X-ray diffraction. Among the compounds, N’-(3-methox- ybenzylidene)-4-nitrobenzohydrazide (1) has effective XO inhibition with an IC50 value of 7.6 ± 1.8 μmol·L–1, which can be used as a potential XO inhibitor and deserves fur- ther investigation. The molecule can be well filled and combined with hydrogen bonds in the active pocket of XO. Supplementary Material CCDC – 859725 for 1, 859726 for 2, 902484 for 3, and 902485 for 4 contain the supplemental crystallograph- ic data for this article. These data are available free of charge at http://www.ccdc.cam.ac.uk/const/retrieving. html or from the Cambridge Crystallographic Data Center (CCDC), 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44(0)1223-336033 or e-mail: deposit@ccdc.cam.ac.uk. 5. References 1. 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DOI:10.1016/j.ejmech.2010.02.013 936 Acta Chim. Slov. 2022, 69, 928–936 Han et al.: Syntheses, Crystal Structures and Xanthine Oxidase ... 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 V prispevku je opisana priprava štirih hidrazonov: (E)-N’-(4-hidroksi-3-metoksibenziliden)-4-nitrobenzohidrazida (1), (E)-4-(dimetilamino)-N’-(4-hidroksi-3-metoksibenziliden)benzo-hidrazida (2), N’-(2-hidroksi-5-metilbenzi- liden)-4-nitrobenzohidrazida (3) in 2-fluoro-N’-(2-hidroksi-5-metilbenziliden)benzohidrazida (4), ter njihova struktur- na karakterizacija z elementarno analizo, IR in 1H NMR spektroskopijo, ter rentgensko analizo monokristalov. Raziskane so bile tudi inhibitorne aktivnosti pripravljenih spojin na ksantin oksidazo. Med njimi je N’-(3-metoksibenziliden)-4-ni- trobenzohidrazid (1) pokazal največjo aktivnost. Izvedene so bile tudi simulacije prileganja spojin v kristalno strukturo aktivnega mesta ksantin oksidaze, ter verjetni načini njihove vezave. 937Acta Chim. Slov. 2022, 69, 937–943 Imieje et al.: In vitro Assessment of Antiprotozoal and Antimicrobial ... DOI: 10.17344/acsi.2022.7762 Scientific paper In vitro Assessment of Antiprotozoal and Antimicrobial Activities of Fractions and Isolated Compounds from Pallenis hierochuntica Vincent O. Imieje,1 Abiodun Falodun1 and Ahmed A. Zaki2 1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Benin, Benin City, 300001, Nigeria. 2 Pharmacognosy Department, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt. * Corresponding author: E-mail: vincent.imieje@uniben.edu; +2348024118853 Received: 08-23-2022 Abstract The antiprotozoal and antimicrobial properties of the extract and fractions of the whole plant of Pallenis hierochuntica were investigated against a panel of pathogenic organisms. Fractionation of the methanol extract of the whole plant of P. hierochuntica using reverse-phase chromatography gave 28 fractions and led to the isolation of 2 new bisabolone hydroperoxides, 6,10β,11-trihydroxybisabol-2-ene-1-one (1a), 6,10α,11-trihydroxybisabol-2-ene-1-one (1b) and also 6,10β-dihydroxybisabol-2,11-diene-1-one (2). They were characterised by extensive spectrometric analysis. Anti-infec- tive investigations of the fractions revealed that fractions 22 to 26 possessed significant antimalarial activity against the D6 and W2 strains of Plasmodium falciparum with IC50 = 7.62–9.91 µg/mL and 5.49–6.08 µg/mL, respectively, and SI > 6.0 on average. Fractions 7, 16 to 24 exhibited good activity against Leishmania donovani promastigotes (IC50 = 6.71– 18.77 µg/mL). Fractions 25 to 28 were active against Trypanosoma brucei trypomastigotes, fraction 25 being the most potent (IC50 = 4.13 µg/mL). Only fractions 11 to 13 were active against Aspergillus fumigatus (IC50 = 13.406 µg/mL). Compounds 1a and 2 were not promising against the organisms tested. 1a and 1b were characterised for the first time. Keywords: Pallenis hierochuntica, leishmaniasis, antimalarial, characterization, spectrometry 1. Introduction Medicinal plants have been a major reservoir of unique and chemically diverse molecules and a large pool of novel drug leads. Plants are known to synthesise potent molecules that exhibit anticancer, anti-infective, anti-in- flammatory, antiviral and antiprotozoal activities. Today, many drugs in clinical use are either directly obtained from natural sources or natural products derived. A study conducted by Newman et al. revealed that over 35% of drugs approved by the United States in the past four dec- ades are either natural products or their derivatives.1 The genus Pallenis (synonym: Asteriscus) is known to express biologically valuable compounds, especially those with humulene and bisabolone skeletons. Extracts, frac- tions and isolated compounds from members of this ge- nus have been shown to exhibit different pharmacological activities: antibacterial and antileishmanial,2,3 anticancer and phytotoxicity,4 and antioxidant activity.5 Phytochem- ical studies of some species of this genus have resulted in the isolation and characterisation of bioactive humulene skeleton sesquiterpene lactones: asteriscunolides A–D6–9, steriscanolide and aquatolide sesquiterpene lactones,9 fla- vonoids, bisabolone hydroperoxides and farsenol deriva- tives.10,3,11 Others include sesquiterpene alcohol, germac- rane and deoxygenated germacrane,12,13 and naupliolide, having a novel tetracyclic skeleton.14 This study investigated the antiprotozoal and an- timicrobial activity of fractions of Pallenis  hierochunti- ca (Michon) Greuter, family Asteraceae. Herein we report the isolation, characterisation, and structure elucidation of three compounds (two of these are new) from the metha- nol extract of the whole plant and their antiprotozoal and antimicrobial activities. 938 Acta Chim. Slov. 2022, 69, 937–943 Imieje et al.: In vitro Assessment of Antiprotozoal and Antimicrobial ... 2. Materials and Methods 2. 1. General Experimental The acquisition of the 1D and 2D NMR spectra were done on Bruker Avance III 500 and 400 MHz spectrom- eter. The compounds were dissolved in CD3OD (13C and 1H NMR data at 125 and 500 MHz, respectively). Chem- ical shift values are reported in ppm and referenced to the residual protons of the solvent (CD3OD). Mass spec- tra were acquired on an Agilent Technologies 6200 series mass spectrometer. Isolations and purifications of all com- pounds were performed by column chromatography (CC), over normal silica gel (32–63 μ, Dynamic adsorbents Inc.), and reversed-phase C-18 silica Polar Plus (J. T. Baker®). Analytical TLC was conducted on precoated silica gel F254 aluminum sheets (0.25 mm, Sorbtent Tech.) or Silica 60 RP–18 F254 aluminum sheets (20 × 20 cm, Merck). Spots were visualized by observing under UV at 254 nm and 365 nm light and by spraying with 1% vanillin (Sigma) in conc. H2SO4/EtOH mixture (1:9) followed by heating with a heat gun. All isolation and purification procedures were done by using analytical grade solvents (Fisher chemi- cals). Pentamidine and amphotericin B (Sigma-Aldrich, St Louis, MO) were used as standard antileishmanial agents. Chloroquine and artemisinin (Sigma-Aldrich, MO) were used as drug controls in the antimalarial assay. Flucona- zole, amphotericin B, ciprofloxacin, vancomycin, methi- cillin, cefotaxime and meropenem were used as positive control antibacterial and antifungi agents. 2. 2. Plant Material The whole plant of Pallenis hierochuntica was collect- ed from the Mediterranean coastal area of Egypt in 2015, and the plant was identified at the Pharmacognosy De- partment, Mansoura University, Egypt, where a voucher specimen (AH-14-PD) was deposited. 2. 3. Extraction and Isolation The dried whole plant of Pallenis hierochuntica was grounded to powder. The powdered plant material (500 g) was macerated with methanol (98%) by percolation (4 L × 4) for 48 h at room temperature. The solvent was removed with a rotary evaporator at 40 °C to give 35 g of crude ex- tract (7% as yield). The extract (33 g) was mixed with 30 g RP-18 silica gel and applied to a VLC over RP-18 silica (30 cm × 3.5 cm, 500 g) and eluted with gradients of H2O/ MeOH (90:10–0:100) and acetone to give 28 fractions (AH- 1 to AH-28). Fraction AH-25 (1.8 g) was subjected to col- umn chromatography (SiO2, EtOAc:CHCl3:MeOH:H2O (15:8:4:1; 10:6:4:1; 8:2:0.25; 7:3:0.5; MeOH 100%)) to give 45 sub-fractions AH1A–AH45A. Fractions with similar Rf were pooled to get 7 fractions (H1–H7). Fraction H6 (200 mg) was processed over column chromatography with normal silica gel (3 × 65 cm) eluted with hexane:EtOAc (4:1, 7:3, 3:2) to give 8 fractions (G1–G8). Repeated col- umn chromatographic purification of fraction G8 (40 mg) with hexane:EtOAc (4:1–3:2) yielded AH6 (2.4 mg) as fine needles. Compounds 1a (0.8 mg) and 1b (0.7 mg) were purified from AH6, and 2 was purified from PTLC (20 × 20 cm, 500 µm pore size) of AH5 (14.9 mg) with elution system of hexane:chloroform (1:4) (50 mL) to give 0.8 mg of white solid. 2. 4. Antiprotozoal and Antimicrobial Assays 2. 4. 1. Antileishmanial Assay The fractions and isolated compounds 1a and 2 were evaluated against Leishmania donovani promastigote, L. donovani axenic amastigote, and L. donovani amastigote in THP1 according to the protocol described by Jain et al.15 which uses the Alamar Blue colourimetric assay method.16 Pentamidine and amphotericin B standard antileishmani- al drugs were used as positive controls. The IC50 and IC90 values were computed from response curves using XLFit®. 2. 4. 2. Antimalarial Assay The in vitro antiplasmodial activity of the fractions and compounds 1a and 2 was measured by a colouri- metric assay that determines the parasites lactate dehy- drogenase (pLDH) activity.17,18 Included in this assay are two strains of Plasmodium falciparum (Sierra Leone D6 (chloroquine-sensitive) and Indochina W2 (chloroquine resistant) obtained from the Walter Reed Army Institute of Research, Silver Spring, MD. The effects of the fractions and test compounds on plasmodial LDH activity were de- termined using Malstat reagent (Flow Inc, Portland, OR). DMSO (0.25%) and chloroquine/artemisinin were includ- ed in each assay which serves as vehicle and positive con- trol drugs, respectively. 2. 4. 3. Cytotoxicity Assay The cytotoxicity of the test samples was determined against transformed human monocytic (THP1) cells. The assay method previously described by Jain et al. was adopt- ed. This experiment used a 4 days old culture of THP1 cells in the experimental phase diluted with RPMI medium to 2.5 ∙ 105 cells/mL. To achieve the parasite cells transforma- tion to the adherent macrophages, Phorbol 12-myristate 13-acetate (PMA) was added to the culture at a concen- tration of 25 ng/mL. The THP1 cell culture treated with PMA was seeded into 96 well plates with 200 μL culture (2.5 ∙ 105 cells/mL) in each well and incubated overnight at 37 °C in a 5% CO2 incubator. The medium in plates with THP1 cells was replaced with a fresh medium. The test samples (fractions and compounds) and standards diluted with RPMI medium in separate plates were added to these plates and then incubated in a 5% CO2 incubator at 37 °C 939Acta Chim. Slov. 2022, 69, 937–943 Imieje et al.: In vitro Assessment of Antiprotozoal and Antimicrobial ... for 48 h. After the incubation period, each well received 10 μL of Alamar Blue solution (AbDSerotec, catalogue number BUF012B), and the plates were incubated further overnight. Again, standard fluorescence was measured on a Fluostar Galaxy fluorometer (BMG LabTechnologies) at 544 nm excitation, 590 nm emission wavelengths. The half-maximal concentration IC50 and IC90 values were computed from the dose-response growth inhibition curve by XLfit version 5.2.2.15 The selectivity indices (SI) were computed by measuring the cytotoxicity of the test compounds against Vero cell lines (monkey fibroblast).15 2. 4. 4. In vitro Antimicrobial Activity Extracts, fractions, and isolated compounds of P. hi- erochuntica were subjected to in vitro susceptibility testing against a panel of pathogenic organisms: the fungi include Candida albicans (ATCC 90028), Candida krusei (ATCC 6258), Candida glabrata (ATCC 90030), Cryptococcus ne- oformans (ATCC 90113), Aspergillus fumigatus (ATCC 204305); while the bacteria include methicillin-resistant bacterium Staphylococcus aureus (MRSA; ATCC 33591), Escherichia coli (ATCC 35218), Klebsiella pneumonia (ATCC 43816), vancomycin-resistance Enterococcus fae- cium (49532) and Mycobacterium intracellulare (ATCC 23068) using a modified version of the NCCLS methods.19 On the other hand, that against M. intracellulare was done using the modified Alamar Blue procedure previously de- scribed.20 The fungi and bacteria used in this experiment were obtained from the American Type Culture Collection (ATCC), Manassas, VA. All the test samples were dissolved in DMSO (0.25%), which also acted as a negative control agent. They were all diluted with 0.9% saline serially and transferred in duplicate to the 96-well microtitre plates. The final microbial inoculums were prepared after com- parison of the absorbance at 630 nm of cell suspensions to the 0.5 McFarland standard and diluting the suspensions in broth (Sabouraud dextrose and cation-adjusted Müller– Hinton (Difco) for the fungi and bacteria, respectively, and 5% Alamar Blue (BioSource International) in Middlebrook 7H9 broth to afford recommended inocula. Microbial in- ocula were added to the diluted samples to realize a final volume of 200 μL. The microtitre plates were read at either 630 nm or 544ex/590em before and after incubation. IC50 values relative to controls were obtained using XL fit 4.2 software (IDBS, Alameda, CA). 3. Results and Discussion 3. 1. Characterisation of Compounds 1a and 1b Compounds 1a and 1b were purified from com- pound 1 (2.4 mg) which showed a clear molecular ion peak [M+Na]+ at m/z 293.1696, corresponding to the molecular formula C15H26O4Na (calcd 293.1729) from its HRESIMS spectrum. However, careful examination of the 1H and 13C NMR spectra of 1 (Figure S18), revealed the presence of double peaks, suggesting a mixture of two bisabolone-type sesquiterpenoids3, a feature usually associated with closely related compounds (mixtures). Figure 1. Molecular structures of compounds 1a, 1b and 2 As such, compound 1 was subjected to further pu- rification to give two compounds of the same molecu- lar weight, 1a (0.8 mg) and 1b (0.7 mg). Compound 1a (6,10β,11-trihydroxybisabol-2-ene-1-one), white needles from sub-fraction AH6 (0.8 mg), HRESIMS [M+Na]+ at m/z 293.1694, corresponding to the molecular formula C15H26O4Na (calcd 293.1729). The 1H NMR spectrum of 1a in CD3OD (Table 1) displayed an olefinic proton signal at δ 5.84 (1H, dq, J = 2.4, 1.1 Hz), a doublet at δ 1.01 (3H, d, J = 6.7 Hz), a singlet at δ (6H, s, 1.12), a singlet at δ 1.99 (3H, s) and a methine proton at δ 3.15 (dd, J = 9.3, 2.4 Hz). The 13C NMR spectrum (Figure S2) revealed the presence of characteristic signals: a carbonyl carbon at δ 204.1, ole- finic methine at δ 124.5 and quaternary olefinic carbon at δ 165.5, together with the olefinic proton signal at δ 5.84 (1H, dq, J = 2.4, 1.1 Hz), indicated the presence of a mono- substituted α,β-unsaturated carbonyl groups. The DEPT experiment (Supplementary material, Figure S4) showed the presence of four methylene carbons at δ 31.6, 29.1, 28.9 and 29.3. The HMQC (Supplementary material, Figure S6) revealed their connectivities with the proton signals at δ (2.24, 2.28), (1.29, 1.40), (1.06, 1.27), and (2.42, 2.44), re- spectively. The correlations observed in HMBC spectrum (Supplementary material, Figure S7) between the proton at δ 2.36 with carbon signals at δ 125.0, 165.5, 32.9, and 78.1 (quaternary carbon), together with the correlations be- 940 Acta Chim. Slov. 2022, 69, 937–943 Imieje et al.: In vitro Assessment of Antiprotozoal and Antimicrobial ... tween the protons at δ 2.24 with 78.1 (quaternary carbon), 202.6 and 29.1, and between the proton at δ 5.84 with 78.1 and 29.1, are consistent with the cyclic (six-carbon ring) unsaturated ketone with branching at the oxygenated qua- ternary carbon (δ 78.1). The side chain consists of eight carbon atoms discriminated by DEPT experiment into three methyls (δ 24.3, 23.5 and 12.6), two methylene (δ 28.7.0 and 27.4), one quaternary (δ 72.4), and one methine (δ 78.8). Careful examination of all correlations in 1H–1H COSY, HMQC and HMBC confirmed the proposed iden- tity of the structure, which is a bisabolone-skeleton with an OH at C-10 (δ 78.8) and C-11 (δ 72.4) (Figure 1). The relative configuration of 1a was established through the analysis of cross-peaks observed in the NOESY spectrum (Supplementary material, Figure S8), which displayed a correlation of H-15 (s, 1.99, 3H) with both H-14 (d, 1.01, 3H) and H-4 (2.44), correlation of H-10 (dd, 3.16) with H-14 (d, 1.01, 3H), indicated the β-oriented H-14 and H-10. Therefore, the structure of compound 1a was de- termined to be 6,10β,11-trihydroxybisabol-2-ene-1-one. The other compound 1b (6,10α,11-trihydroxybisab- ol-2-ene-1-one), white needles from sub-fraction AH6 (0.7 mg), HRESIMS [M+Na]+ at m/z 293.1694, corresponding to the molecular formula C15H26O4Na (calcd 293.1729), was of the same structure as 1a with a slight deviation in the NMR signals (Table 1, supplemental material, Figures S11–S17). The differences resulted from the hydroxy group orientation at C-10. Upon careful analysis of the NOESY (Supplementary material, Figure S16) spectrum of 1b, the correlation of H-15 (s, 1.99, 3H) with both H-14 (d, 1.02) and H-4 (2.44), and the correlation of H-10 (dd, 3.15) with H-7 (m, 1.91), indicated that the 10-OH is α-oriented. The structure of 1b was concluded to be 6,10α,11-trihydroxy- bisabol-2-ene-1-one. 3. 2. Characterisation of Compound 2 Compound 2 (Figure 1) was obtained as fine white needles. HRESIMS showed a clear molecular ion peak [M+Na]+ at m/z 275.1594 (calcd C15H24O3Na, 275.1623). The 1H and 13C NMR spectra of 2 revealed the same skel- eton as compound 1a, a bisabolone-type sesquiterpene, with the disappearance of the one methyl and oxygenated quaternary carbon at δ 72.5 which was assigned for C-11 of 1a and 1b, and the appearance of one olefinic methyl- ene group and quaternary olefinic carbon. The 1H NMR spectrum of 2 showed two olefinic protons at δ 4.86 (p, J = 1.6 Hz) and 4.87 (dt, J = 1.9, 0.9 Hz) (Table S1) as- signed to C-12 explaining the absence of a methyl group. The 13C NMR displayed a hydroxylation at C-10, the same as compound 1a, and the H-10 chemical shift at δ 3.94 together with the coupling value (t, J = 6.7 Hz) is in full agreement with the β-oriented C-10 hydroxyl group. The chemical structure of 2 was deduced as 6,10β-dihydrox- ybisabol-2,11-diene-1-one (Figure 1). Compound 2 was previously reported as a reduction product of 10-peroxy derivatives,3 but this is the first time it was isolated directly from a natural source. 3. 3. In vitro Antiparasitic Screening of Fractions and Isolated Compounds The results of the antimalarial screening of the frac- tions against the two strains of Plasmodium falciparum (D6 and W2) are shown in Table S2 (Supplementary material). Similarly, the antileishmanial and antitrypanosomal re- sults of the fractions are shown in Table S3 (Supplementa- ry material), and the antimicrobial activity of the fractions is reported in Table S4. Results of the in vitro antimalarial, antileishmanial, antitrypanosomal and antimicrobial ac- tivities of compounds 1a and 2 are presented in Tables S5 to S7 (Supplementary material). According to the WHO report of 2019, it was esti- mated that there were 229 million cases of malaria world- wide, resulting in 409,000 deaths, most of whom are chil- dren under five, especially in Sub-Saharan Africa, making malaria a major global health problem.21 Several research- ers have reported the antimalarial effects of plant extracts and fractions.22–24 According to the WHO, about 80% of people depend on herbal products as their primary health- care source(s).21 The result of the antimalarial screening of the frac- tions of Pallenis hierochuntica is shown in Table S2 (Sup- plementary material). In the primary screening experi- ment, the two strains (D6 and W2) of P. falciparum were tested against the fractions at concentrations range of 47.6–5.28 μg/mL. Only fractions that showed antimalar- ial activity (≥ 50%) in this screening were investigated in the secondary antimalarial screening to determine their IC50 values. From the table, fractions AH11–AH12 and AH14–AH28 exhibited significant antimalarial activity against the chloroquine-sensitive (D6) and resistant (W2) strains of P. falciparum with IC50 values ranging from 7.62–30.33 μg/mL and 5.49–25.49 μg/mL, respectively. Fractions AH23–AH27 were particularly effective against the two strains of PF with IC50 = 5.49–9.19 μg/mL. As such, these fractions are classified as having promising an- timalarial activity. Their IC50 values were, however, higher than those obtained for artemisinin and chloroquine (IC50 <0.026–0.202 μg/mL), standard antimalarial drugs used as positive control drugs. They also showed better selectivity indices (SI = > 5.2 – > 8.7). Our report is the first on the in vitro antimalarial activity of this plant. There is a lack of information on the antimalarial activity of the Pallenis genus representatives in general. Similarly, leishmaniasis and trypanosomiasis (sleep- ing sickness) affect humans and livestock in the tropical and subtropical countries of Africa, Asia and South Amer- ica. It has been estimated that over 70 million and 350 mil- lion people worldwide are at risk of trypanosomiasis and leishmaniasis, respectively, with attendant annual deaths of 14,000 to 70,000.25 In our continued investigation of Table 1. 13C and 1H NMR data for compound 1a and 1b (CD3OD at 125 and 400 MHz, respectively). Carbon No. 1a 1b δH (ppm), Multiplicity, J (Hz) 13C DEPT δH (ppm), Multiplicity, J (Hz) 13C DEPT 1 – 204.1 C – 204.0 C 2 5.84 (dq, J = 2.4, 1.1 Hz) 123.2 CH 5.83 (dq, J = 2.4, 1.1 Hz) 124.6 CH 3 – 163.9 C – 165.2 C 4 2.42 2.36 2.44 29.3 CH2 2.44 30.5 CH2 2.24(ddd, J = 5.0, 3.3, 1.7Hz) 5 2.28 31.6 CH2 2.23(ddd, J = 5.0, 3.3, 1.7 Hz)33.1 CH2 2.28 6 – 78.1 C – 78.5 C 7 1.91, m 35.4 CH 1.91, m 37.3 CH 1.06 8 1.27 28.9 CH2 1.07 1.26 29.0 CH2 9 1.29 1.40 29.1 CH2 1.29 1.40 30.8 CH2 10 3.15 (dd, J = 9.3, 2.4 Hz) 78.8 CH 3.15 (dd, J = 9.3, 2.4 Hz) 80.3 CH 11 – 72.5 C – 74.0 C 12 1.12, s 24.3 CH3 1.14, s 25.9 CH3 13 CH3 CH3 14 1.01(d, J = 6.7 Hz) 12.5 CH3 1.02 (d, J = 6.7 Hz) 14.1 CH3 15 1.99, s 23.5 CH3 1.97, s 23.9 CH3 941Acta Chim. Slov. 2022, 69, 937–943 Imieje et al.: In vitro Assessment of Antiprotozoal and Antimicrobial ... medicinal plants for antiprotozoal metabolites, fractions of Pallenis hierochuntica were subjected to in vitro screening against Leishmania donovani (promastigotes, axenic amas- tigotes, and intracellular amastigotes in THP1 cells) and blood-stage promastigotes of Trypanosoma brucei. The result of this screening is shown in Table S3 (Supplemen- tary material). From the result, fractions AH-1 and AH-3 showed activity against T. brucei, IC50 = 11.33 and 12.50 μg/mL, respectively. Fraction AH-7 was active against L. donovani promastigotes and axenic amastigotes with IC50 and IC90 values of 12.03–19.47 μg/mL and also against T. brucei (IC50 = 13.6 μg/mL). Fractions AH-12, AH-16 to AH-18, and AH-24 were also effective against the promas- tigotes of L. donovani. While fractions AH-25 to AH-28 were active against T. brucei blood-stage trypomastigotes with IC50 values of 4.13–13.48 μg/mL and IC90 values of 11.46–19.28 μg/mL. All the fractions at 20–8 μg/mL test concentrations did not show activity against intracellu- lar amastigotes in THP1 cells. The positive control drugs, pentamidine and DMFO, possess better activity against these protozoa except fraction AH-25, which showed bet- ter activity (IC50 = 4.13 μg/mL) than DMFO (IC50 = 6.25 μg/mL) against T. brucei. Several studies have highlighted the activity of plant extracts and fractions against leish- maniasis and trypanosomiasis.26,25,27 Further purification of these extracts and fractions has led to the isolation of potent compounds exhibiting significant leishmanicidal and trypanocidal effects against these pathogenic pro- tozoal.28–30 In a related study, the ethyl acetate extract of Asteriscus graveolens exhibited potent activity against both promastigote and amastigote forms of L. infantum and L. major with IC50 value of 22.93 ± 0.39 µg/mL and 131.6 ± 0.21 µg/mL against L. infantum. Also, the hydroethanol- ic extract of the plant inhibited L. major and L. infantum parasites with IC50 = 33.64 ± 0.46 µg/mL and 143.4 ± 0.28 µg/mL, respectively.2 An in vitro antiprotozoal activity of crude methanol extract of Pallenis hierochuntica (Aster- iscus hierochuntica) was reported by Zaki et al.,31 in which the extract showed promising and good antitrypanosomal activity against the promastigotes of T. brucei with IC50 and IC90 values being 1.18 and 1.89 µg/mL, respectively. Our study is the first report of the antileishmanial and an- titrypanosomal activity of the fractions of Pallenis hiero- chuntica. In the antimicrobial screening experiment, 28 frac- tions (AH-1 to AH-28) were subjected to in vitro antimi- crobial evaluation against a panel of pathogenic micro- organisms (fungi and bacteria) (Table S4). The fractions were tested at a 200–8 µg/mL concentration range. From the results of our study, only fractions AH-11 to AH-13 exhibited significant activity against Aspergillus fumiga- tus with IC50 values of 13.406 (AH-11), 88.607 (AH-12) Table 1. 13C and 1H NMR data for compound 1a and 1b (CD3OD at 125 and 400 MHz, respectively). Carbon No. 1a 1b δH (ppm), Multiplicity, J (Hz) 13C DEPT δH (ppm),Multiplicity, J (Hz) 13C DEPT 1 -- 204.1 C -- 204.0 C 2 5.84 (dq, J = 2.4, 1.1 Hz) 123.2 CH 5.83 (dq, J = 2.4, 1.1 Hz) 124.6 CH 3 -- 163.9 C -- 165.2 C 4 2.422.44 29.3 CH2 2.36 2.44 30.5 CH2 5 2.24(ddd, J = 5.0, 3.3, 1.7Hz)2.28 31.6 CH2 2.23(ddd, J = 5.0, 3.3, 1.7 Hz) 2.28 33.1 CH2 6 -- 78.1 C -- 78.5 C 7 1.91, m 35.4 CH 1.91, m 37.3 CH 8 1.061.27 28.9 CH2 1.07 1.26 29.0 CH2 9 1.291.40 29.1 CH2 1.29 1.40 30.8 CH2 10 3.15 (dd, J = 9.3, 2.4 Hz) 78.8 CH 3.15 (dd, J = 9.3, 2.4 Hz) 80.3 CH 11 -- 72.5 C -- 74.0 C 12 1.12, s 24.3 CH3 1.14, s 25.9 CH313 CH3 CH3 14 1.01(d, J = 6.7 Hz) 12.5 CH3 1.02 (d, J = 6.7 Hz) 14.1 CH3 15 1.99, s 23.5 CH3 1.97, s 23.9 CH3 942 Acta Chim. Slov. 2022, 69, 937–943 Imieje et al.: In vitro Assessment of Antiprotozoal and Antimicrobial ... and 130.228 µg/mL (AH-13), respectively. Other studies reported the antimicrobial activities of members of the genus Asteriscus. Ramdane et al. reported the antimi- crobial activity of the ethyl acetate fractions of A. grave- olens against L. monocytogenes (MIC = 0.312 mg/mL), S. aureus and B. cereus (MIC = 0.625 mg/mL), but the fractions show no activity against E. coli (ATCC 35214) and P. aeruginosa (ATCC 27853).2 Also, Medimagh et al. evaluated the root oil of Asteriscus maritimus (L.) for antimicrobial activity against some pathogenic fungi, in- cluding Aspergillus flavus, A. niger, Botrytis cinerea and Penicillium sp. The zones of inhibition range from 8.3 mm to 10.3 mm. However, the oil was not active against the bacteria isolates tested.32 The oil of A. graveolens was also reported to significantly (p <0.05) inhibit the my- celial growth of some pathogenic fungi (Alternaria sp., P. expansum, and R. stolonifer) at different concentra- tions.33 3. 4. In vitro Antimalarial and Antimicrobial Activities of Compounds 1a and 2 The in vitro antimalarial, antileishmanial, antitryp- anosomal and antimicrobial screening of compounds 1a and 2 are reported in Tables S5–S7 (Supplementary mate- rial). They were screened against the different pathogen- ic organisms mentioned in section 2.4 above. The study results showed that the compounds exhibited no signifi- cant activity against tested organisms at the concentrations tested. 4. Conclusion In the present study, two new compounds with bis- abolone skeleton and a known compound were isolated and identified as 6,10β,11-trihydroxybisabol-2-ene-1-one, 6,10α,11-trihydroxybisabol-2-ene-1-one, and 6,10β-dihy- droxybisabol-2,11-diene-1-one, respectively. These com- pounds hold no promising antiprotozoal and antimicro- bial activities. The fractions of Pallenis hierochuntica show significant activity against Plasmodium falciparum, Leish- mania donovani promastigotes, axenic amastigotes and trypomastigotes of Trypanosoma brucei. This plant holds potential for further investigation for lead compounds as antiprotozoal agents. Acknowledgements This work was in part supported by USAID/HED grant 153 – 6200BF A15 – 01 to one of the authors. We also acknowledge the University of Benin as a shared cost partner in this grant, and the National Center for Natural Product Research (NCNPR), School of Pharmacy, Univer- sity of Mississippi, for the use of their laboratory for part of this work. 5. References 1. D. J. Newman, G. M. Cragg, K. M. Snader. Nat.  Prod.  Rep. 2000, 17, 215–234. DOI:10.1039/a902202c 2. F. Ramdane, R. Essid, K. Mkadmini, M. Hammami, N. Fares, M. H. Mahammed, D. El Ouassis, O. Tabbene, F. Limam, M. D. O Hadj. Process Biochem. 2017, 56, 186–192. DOI:10.1016/j.procbio.2017.03.004 3. T. Sarg, S. El-Dahmy, A. 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Frakcioniranje metanolnega ekstrakta iz rastline P. hierochuntica s pomočjo reverznofazne kromatografije je dalo 28 frakcij in omogočilo izolacijo dveh novih bisabolonskih hidroperoksidov: 6,10β,11-trihidroksibisabol-2-en-1-ona (1a) ter 6,10α,11-trihidroksibisabol-2-en-1-ona (1b) in tudi 6,10β-dihidroksibisabol-2,11-dien-1-ona (2). Vse tri spojine smo karakterizirali z obširno spektroskopsko analizo. Izkazalo se je, da imajo frakcije 22 do 26 občutno antimalarijsko delovanje proti sevoma Plasmodium falciparum D6 (z IC50 vrednostmi 7.62–9.91 µg/mL) in W2 (z IC50 vrednostmi 5.49–6.08 µg/mL); indeks selektivnosti je bil v pov- prečju večji od 6.0. Frakcije 7 in 16 do 24 so izkazale dobro aktivnost proti Leishmania donovani promastigotom (IC50 = 6.71–18.77 µg/mL). Frakcije 25 do 28 so bile aktivne proti Trypanosoma brucei tripomastigotom, od katerih se je frakcija 25 izkazala kot najbolj učinkovita (IC50 = 4.13 µg/mL). Proti Aspergillus fumigatus so bile učinkovite zgolj frakcije 11 do 13 (IC50 = 13.406 µg/mL). Izkazalo se je, da spojini 1a in 2 nista obetavni učinkovini proti testiranim organizmom. Karakterizaciji spojin 1a in 1b v literaturi še nista bili opisani. 944 Acta Chim. Slov. 2022, 69, 944–947 Author Index / Kazalo avtorjev Abaszadeh Mehdi ..................................920 Abdassalam Aesha FSH .........................187 Abdo Nadia Y. Megally ..........................700 Aghajani Yeganeh ...................................837 Akdağ Kadriye ........................................863 Akyıldız Hasan .......................................... 39 Al-Asafi Omar Jamal Mahdi .................519 Al-Dhalemi Dhuha Mohsin ..................681 Aldulaim Ahmed Kareem Obaid .........681 Ali Karwan Omer ...................................905 Ali Saqib ...................................................405 Alimuddin ...............................................681 Alkan Leman ...........................................316 Alruwaili Nabil K ....................................483 Ambrož Ana ............................................806 Arenas Luis Andres Barboza .................681 Arif Saira ..................................................200 Asaadi Negin ............................................. 30 Asiltürk Erol .............................................. 60 Aslan Nazife ............................................638 Atakol Orhan ..........................................147 Ayad Magda Mohamed ..........................507 Aydogdu Seyda .......................................647 Azam Mohammed Afzal .......................393 Azizi Seyed Naser ...................................458 Bahar Mehmet Refik ..............................281 Bai Su-Zhen .............................................787 Bakhouch Mohamed ..............................489 Bálint Erika ..............................................735 Ban Irena .................................................826 Bano Saeeda ............................................405 Barka Noureddine ..................................536 Bártová Iveta ...........................................371 Basha Mubarak Ali Muhamath ................ 1 Bashir Shabnum ......................................848 Bavec Aljoša ............................................478 Baviskar Shweta ......................................437 Belaidi Salah ............................................489 Boh Podgornik Bojana ...........................167 Božnar Alič Elizabeta .............................564 Bren Urban ..............................................378 Brontowiyono Widodo ..........................681 Cai Zhi-qiang ..........................................227 Çalışkan Eray ..........................................281 Cao Tong ..................................................896 Carmona-Alvarado Idalia Francisca ...... 49 Cavazos-Rocha Norma ............................ 49 Çelik İsmail .............................................419 Cerc Korošec Romana ...........................217 Çesko Cengiz ..........................................665 Çetinkaya Zeynep ..................................... 39 Çevik Özge ..............................................293 Çevik Özge ..............................................863 Chen Ruo-nan .........................................227 Chtita Samir ............................................489 Ciuffreda Pierangela ...............................571 Coskun Demet .......................................... 73 Coskun Mehmet Fatih ............................. 73 Černič Tina .............................................448 Dalvand Kolsoum ...................................322 Dascalu Izabella ......................................331 de Torres Noemi Waksman ..................... 49 Deniz Nahide Gulsah .............................187 Dere Nurşen ............................................108 Dilmaghani Karim Akbari ....................619 Dinçer Barbaros ......................................604 Doğan Hacer ...........................................281 Dolničar Danica......................................167 Drtil Miloslav ..........................................657 Duan Xiaoyi ............................................896 Duc Ha Danh ..........................................811 Dural Turan .............................................604 Elmetwally Amira Mohamed .................. 13 El-Sayed kh. .............................................722 Elshoky Hisham Ali ...............................722 ElZorkany Heba Elsayed .......................722 Emirik Mustafa .......................................604 Enache Mirela .........................................331 Erden Fuat ...............................................884 Erdoğan Ömer ................................293, 863 Erol Rabia .................................................. 81 Eryılmaz Müjde ......................................419 Eshwaraiah Latha Haraluru Kamalamma .......................................116 Fabjan Teja ...............................................564 Faghih-Mirzaei Ehsan ............................920 AUTHOR INDEX Acta Chimica Slovenica Year 2022, Vol. 69 No. 1–4 945Acta Chim. Slov. 2022, 69, 944–947 Author Index / Kazalo avtorjev Falodun Abiodun ...................................937 Farah Mahnaz .........................................837 Farahi Mahnaz .......................................... 30 Farajian Fereshte .....................................714 Fatima Nasreen .......................................405 Fei-fei Li ...................................................227 Feng Xinhui .............................................674 Ferk Savec Vesna .....................................167 Findik Serap ....................................336, 552 Fırat Özge .................................................. 81 Fırat Özgür ................................................ 81 Frlan Rok .................................................261 Fuchs Godec Regina ...............................378 Gaál Enikő Éva ........................................796 Gamaan Marwa Soliman ......................... 13 Garza-Juarez Aurora de Jesús ................. 49 Gerber Thomas .......................................905 Ghiasvand Alireza ..................................322 Gilani Neda Salek ...................................458 Glamočlija Una .......................................243 Gökmen Uğur .........................................638 Golični Marko .........................................478 Görgülü Ahmet Orhan ..........................281 Gruden Evelin .........................................S95 Gu Yuqing ................................................674 Gunduz Bayram ........................................ 73 Guo Xue-Yao ...........................................928 Gürpınar Kübra ......................................147 Gürpınar Suna Sibel ...............................419 Halenova Tetiana ....................................584 Hamdi Amin ...........................................322 Hamrahjou Nasrin ................................... 98 Han Yong-Jun ..................................385, 928 Hanuljaková Hana ..................................657 Haraluru Lalithamba Shankraiah .........116 Hashemi Payman ....................................714 Hassan Mohamed A. ..............................722 Hatipoglu Arzu .......................................647 Heidari Nahid .........................................322 Hemmesi Leila ........................................876 Hosny Mervat Mohamed.......................507 Hosten Eric ..............................................905 Hrast Martina ..........................................261 Hu Yanhong.............................................133 Hu Ze .......................................................133 Huang Qiu-chen .....................................227 Huang Yong-Gang ..................................913 Hung Nguyen Van ..................................811 Hussein Shaymaa Abed .........................681 Hussien Emad Mohamed ......................507 Ibrahim Rehab Ali .................................... 13 Imad Saima ..............................................405 Imreová Zuzana ......................................657 Imieje O. Vincent ...................................937 İnal Emine Kübra ...................................147 Iqbal Sadaf ...............................................405 Iqbal Samina ............................................405 Ishfaq Shazia ...........................................405 Islas Jose Francisco ................................... 49 Ivanova Iliana A. .....................................722 Izzat Samar Emad ...................................681 Jahanbakhshi Azar .................................837 Jamil Waqas .............................................772 Jayaram Unni ..........................................393 Jeran Marko ............................................S95 Jereb Matjaž .............................................564 Jiang Jian ..................................................629 Jin Rui-Fa .................................................913 Karakoyun Gülen Önal ............................ 60 Karakuş Sevgi ..........................................863 Karami Bahador........................................ 30 Karlovská Ines .........................................657 Kešelj Dragana ........................................803 Khieu Dinh Quang .................................811 Kilcigil Gülgün ........................................419 Kızıl Demet .............................................604 Klečková Marta .......................................371 Kobliha Zbyněk ......................................125 Koca Murat ..............................................466 Koçyiğit-Kaymakçıoğlu Bedia ......293, 863 Koran Kenan ...........................................281 Koraqi Hyrije...........................................665 Koren Monika .........................................448 Korkut Ibrahim .......................................884 Kostadinova Anelyia s............................722 Kožárová Bibiána ....................................657 Kshash Abdullah Hussein .....................519 Kucukguzel Sukriye Guniz ....................526 Kulabas Necla ..........................................526 Kumer Kristina .......................................564 Kurt Adnan ..............................................466 Kuzmič Samo ..........................................261 Lazarevic Jelena S ...................................571 Lazić Dragica...........................................803 Lei Yan ......................................................235 Leng Xinyu ..............................................779 Li Fen-Fang .............................................596 Li Shi-Tong ..............................................385 Li Wei .......................................................227 Li Xiaoyan ...............................................674 Liang Peng ...............................................629 Lin Xue-Song ..........................................913 Lisjak Darja .............................................448 Liu Cheng ................................................157 Liu Chengguo..........................................779 Liu Peng ...................................................133 946 Acta Chim. Slov. 2022, 69, 944–947 Author Index / Kazalo avtorjev Liu Shu-Juan ............................................694 Liu Zhaogang ..........................................133 Ljubijankić Nevzeta ................................243 Lobotka Martin .......................................125 Lukić Milica .............................................564 Luo Xiao-Qiang ......................................385 Luxbacher Thomas .................................826 Mahadevaiah Raghavendra ...................116 Mahani Nosrat Madadi ............................ 91 Mahdi Ahmed B. ....................................681 Mahmood Khalid ...................................200 Majaron Boris .........................................448 Makovec Darko .......................................756 Malik Sabaahatul Ain .............................200 Markovic Ana..........................................571 Masoodi Mubashir Hussain ..................848 Matoh Lev ................................................217 Meng Shuo ...............................................896 Metias Youstina Mekhail .......................507 Mihovec Katja .........................................796 Mohamad Hikmat Ali ............................905 Mohamed Ahmed Said ..........................489 Mohareb Rafat Milad .......................13, 700 Molčanov Krešimir .................................243 Moosvi Syed Kazim ................................848 Moradi Somayeh .....................................349 Moradian Mohsen ..................................349 Mostaghni Fatemeh .................................. 91 Mustafa Mohd .........................................848 Mustafa Yasser Fakri ..............................681 Naeimi Hossein ..............................349, 876 Najar Mohd. Hanief ...............................848 Nangare Sopan ........................................437 Nath Kaushik ..........................................304 Nazır Hasan .............................................147 Nghi Nguyen Huu ..................................811 Nguyet Bui Thi Minh .............................811 Nisar Shazia .............................................405 Ocak Sema Bilge .....................................638 Osmanović Amar ...................................243 Osredkar Joško ........................................564 Ouassaf Mebarka ....................................489 Ozbay Salih ..............................................884 Ozyurek Mustafa ....................................187 Panda Dibya Sundar ...............................483 Parra Rosario Mireya Romero ..............681 Patil Ashwini ...........................................437 Patil Pravin ..............................................437 Patra Indrajit ...........................................681 Pattnaik Satyanarayan ............................483 Pavlova Elitsa L. ......................................722 Penedo Medina Margarita .....................536 Petrič Boštjan ..........................................478 Petrović Zoran .......................................803 Pitschmann Vladimír .............................125 Ponikvar-Svet Maja ................................448 Popovics-Tóth Nóra ...............................735 Pourali Ali Reza ......................................271 Pourkazemi Arezoo .................................. 30 Pucko Sara ...............................................564 Qais Faizan Abul .....................................489 Qazimi Bujar ...........................................665 Qiu Xiao-Yang ........................................694 Reli Martin ..............................................217 Riverón Aymara Ricardo .......................536 Rizvi Masood Ahmad ............................848 Rodriguez Maria Martin .......................564 Roškar Robert .........................................796 Rusek Martin ...................................359, 371 Sabouri Salehe .........................................920 Sadat-Mansouri Seyedeh Nazanin ......... 98 Salazar-Cavazos Maria de la Luz ............ 49 Salih Hanaa Kaen ...................................519 Salihoglu Huseyin...................................187 Salihović Mirsada ...................................243 Sandal Süleyman .....................................281 Sang Ya-Li ...............................................913 Santaniello Enzo .....................................571 Sarveahrabi Yasin ...................................619 Savchuk Olexii ........................................584 Sayil Cigdem ...........................................187 Şen Hasan Tahsin ...................................419 Senkardes Sevil .......................................526 Serbest Kerim ..........................................604 Shafiekhani Homa .................................... 91 Shafqat Syed Salman ..............................200 Shahzaman Muhammad........................405 Shuai Xiao-min .......................................227 Siirilä Joonas............................................251 Sirka Lütfiye .............................................281 Sirotek Vladimír .....................................371 Smelcerovic Andrija ...............................571 Solangi Sorath .........................................772 Srinivasan Sathiya ....................................... 1 Stasevych Maryna ...................................584 Stojanovic Gordana ................................571 Sumrra Sajjad Hussain ...........................200 Sun Deyun ...............................................133 Sun Tao ....................................................227 Svoboda Ingrid........................................147 Swain Kalpana .........................................483 Škapin Andrijana Sever Škapin ............217 Špirtović-Halilović Selma ......................243 Štrofová Jitka ...........................................371 947Acta Chim. Slov. 2022, 69, 944–947 Author Index / Kazalo avtorjev Štukovnik Zala ........................................378 Tabari Sonia .............................................271 Taghvaei-Ganjali Saeed ............................ 98 Taha Muhammad ...................................772 Tang Mao .................................................133 Tekin Suat ................................................281 Temova Rakuša Žane .............................796 Tenhu Heikki...........................................251 Tien Nguyen Anh ...................................811 Tilami Salma Ehsani ..............................458 Toader Ana Maria ..................................331 Tok Fatih ..........................................293, 863 Torres Lidia Naccha ................................. 49 Toshkovska Radostina D. ......................722 Trajkovska Petkoska Anka ....................665 Tramšek Melita .......................................S95 Tuncer Yaprak Gürsoy ...........................147 Uslu Harun ..............................................281 Vaghela Naresh R ....................................304 Vargas Armando Rojas ..........................536 Vaskevych Alla ........................................584 Veljović Elma ..........................................243 Vives Alba González ..............................536 Vojíř Karel .......................................359, 371 Vovk Mykhaylo .......................................584 Wang Chu-Yi...........................................694 Wang Jing.................................................674 Wu Jinxiu .................................................133 Xin Yu ......................................................896 Xu Zhijie ..................................................896 Xue Ling-Wei ........................ 928, 385, 787 Yang Ting .................................................674 Yaqoob Muhammad ...............................772 Yaremkevych Olena................................584 Ye Fei ........................................................779 Yesil Emin Ahmet ...................................187 Yeşilçayır Elif ...........................................419 Yıldırım Şeküre ......................................... 39 Yılmaz Nurdane ......................................147 Yocheva Lyubomira D. ...........................722 Yolcu Murat .............................................108 Yolcu Zuhal .............................................108 You Zhonglu ....................................629, 674 Yu Huiyuan ..............................................629 Zadmard Reza ........................................... 98 Zafar Wardha ..........................................200 Zahid Kanwal ..........................................405 Zahmatkesh Karim .................................619 Zakhar Ronald ........................................657 Zaki A. Ahmed .......................................937 Zang Qi-qi ...............................................227 Zare Ehsan Nazarzadeh .........................271 Zarnegaryan Ali ........................................ 30 Zengin Ali ................................................604 Zhang Daopeng ......................................896 Zhang Li ...................................................674 Zhang Mingjian ......................................896 Zhang Wei ...............................................227 Zhao Xiao-Jun .........................................787 Zhou Gao-Qi ...........................................694 Zhou Zhen ...............................................896 Zvarych Viktor ........................................584 Žener Boštjan ..........................................217 S93Acta Chim. Slov. 2022, 69, (4), Supplement Društvene vesti in druge aktivnosti DRUŠTVENE VESTI IN DRUGE AKTIVNOSTI SOCIETY NEWS, ANNOUNCEMENTS, ACTIVITIES Vsebina Trideset let delovanja Šole eksperimentalne kemije na Institutu »Jožef Stefan«: trideset let motiviranja mladih generacij in utrjevanja poti naravoslovnega izobraževanja ............................................................................................................................ S95 Slavnostna akademija ob 70-letnici Slovenskega kemijskega društva ............................ S98 Koledar važnejših znanstvenih srečanj s področja kemije in kemijske tehnologije ....... S107 Navodila za avtorje ............................................................................................................... S110 Contents Thirty years of the School of Experimental Chemistry at the “Jožef Stefan” Institute: thirty years of motivating young generations and strengthening the path of scientific education ............................................................................................................ S95 Ceremonial Academy on the Occasion of the 70th Anniversary of the Slovenian Chemical Society ....................................................................................... S98 Scientific meetings – Chemistry and chemical engineering .............................................. S107 Instructions for authors ....................................................................................................... S110 S94 Acta Chim. Slov. 2022, 69, (4), Supplement Društvene vesti in druge aktivnosti S95Acta Chim. Slov. 2022, 69, S95–S97 Tramšek et al.: Trideset let delovanja Šole eksperimentalne kemije ... DOI: 10.17344/acsi.2022.7868 Chemical education Trideset let delovanja Šole eksperimentalne kemije na Institutu »Jožef Stefan«: trideset let motiviranja mladih generacij in utrjevanja poti naravoslovnega izobraževanja Melita Tramšek, Evelin Gruden in Marko Jeran* Odsek za anorgansko kemijo in tehnologijo, Institut »Jožef Stefan«, Jamova cesta 39, Ljubljana, Slovenija * Corresponding author: E-mail: marko.jeran@ijs.si Tel.: +386 1 477 33 28 Received: 11-01-2022 Abstract Pred 30. leti, natančneje spomladi leta 1992, je bila, v okviru Odseka za anorgansko kemijo in tehnologijo Instituta »Jožef Stefan«, ustanovljena Šola eksperimentalne kemije. Zaradi razvoja znanosti in interdisciplinarnih pristopov, je njen glavni namen približevanje kemije mladim generacijam in prikazovanje njene širše uporabe v vsakdanjem življenju. Šola eksperimentalne kemije tako ustvarja pomemben most med raziskovanjem in izobraževanjem ter aktivno prispeva k popularizaciji predmetnega področja v šolah. Ključne besede: Šola eksperimentalne kemije; izobraževanje; raziskovanje; povezovanje; popularizacija; kemija Kemija se v modernem času ukvarja z odgovori na pomembna vprašanja, kot so na primer kemijske osnove mišljenja, razvojev procesov življenja, okoljska problemati- ka, sinteza različnih materialov in na vse zadnje tudi z od- krivanjem novih (alternativnih) virov energije1. Omenjena področja zajemajo izrazit interdisciplinarni pristop, zato je potreba po povezovanju ključna. Kemija prav tako pred- stavlja gonilno silo različnih panog industrije, kjer se mora- jo strokovnjaki prav tako spoprijemati z najrazličnejšimi praktičnimi izzivi. Če pomislimo, je kemija postala del na- šega življenja1. Zaradi omenjenih razlogov in razvoja inter- disciplinarnih pristopov v znanosti, je vselej nujno mlajše generacije opremiti z znanjem in veščinami, ki jih bodo lahko tekom študija in razvoja profesionalne poti tudi ople- menitili. Še kako pomembno je navduševati mlade genera- cije učencev in dijakov za povezovanje v naravoslovju skozi različne dogodke in vsebine, in nenazadnje tudi za komu- nikacijo znanstvenih vsebin s splošno javnostjo. Ker je kemija nekoč veljala za splošno pust in težak predmet, kar je posledično vplivalo tudi na njeno prilju- bljenost, je bila pred 30. leti, natančneje spomladi leta 1992, v okviru Odseka za anorgansko kemijo in tehnologi- jo Instituta »Jožef Stefan«, ustanovljena Šola eksperimen- talne kemije2,3,4. Njen začetnik, prof. dr. Andrej Šmalc, je že ob njeni 15. obletnici poudaril, da so kemijski eksperi- menti tisto, s čimer je mogoče pouk kemije bistveno pope- striti, ga narediti zanimivega in privlačnega2. V izvedben- em pogledu so prav kemijski poskusi, v primerjavi s fizikalnimi, bolj zahtevni in potrebujejo posebej za to ure- jene prostore. Po večini se v šolskih kemijskih učilnicah izvajajo zgolj demonstracijski tipi poskusov, med tem ko so možnosti za individualno eksperimentalno delo učencev dokaj omejene2. Šola eksperimentalne kemije je namenjena učencem in dijakom, ki želijo znanja kemije poglobiti še na eksperi- mentalni ravni, predvsem takim, ki jih veseli samostojno eksperimentiranje. Poskusi, ki so zanimivi, zabavni in postavljeni v kon- tekstualni okvir vsakdanjega življenja, se hkrati skladajo z vsebinami temeljnega kurikuluma in udeležence spodbu- jajo k poglobljenemu razmišljanju2. Pri eksperimentiranju udeleženci na primer spoznajo naravne pojave in jih na preprost način razložijo s kemijskimi poskusi (primer na- stanek in oddajanje svetlobe)5,6, pojasnjujejo vlogo in upo- rabo naravnih barvil7, povezujejo kemijo z drugimi teh- niškimi disciplinami, kot sta elektrotehnika in razvoj senzorjev8, in podobno (Slika 1). Med delom v laboratoriju si udeleženci pridobijo os- novne eksperimentalne veščine in spoznajo ukrepe za var- no delo. Skozi delo v skupinah se učijo medsebojnega so- delovanja ter skozi predstavitve in demonstracije poskusov urijo svoje govorniške spretnosti. S96 Acta Chim. Slov. 2022, 69, S95–S97 Tramšek et al.: Trideset let delovanja Šole eksperimentalne kemije ... Obiski učencev in dijakov se lahko povežejo tudi z ogledom laboratorijev Odseka za anorgansko kemijo in te- hnologijo. Posamezniki se tako seznanijo z dejansko upora- bo nekaterih metod pri raziskovalnem delu, katerih osnove so spoznali pri poskusih. V tem primeru lahko v živo vidijo in vsaj na kratko občutijo utrip raziskovalnega okolja. Šola eksperimentalne kemije je bila sprva zasnovana v obliki enotedenskih tečajev v skupnem trajanju 32 šol- skih ur, ki potekajo v t.i. šolskem laboratoriju institutskega odseka. Tečaj vodi mentor, ki udeležencem pripravi ustre- zen program, gradivo in udeležence seznani z varnostno kulturo ter z ravnanjem z odpadki po izvedenem posku- su2. Skozi leta je Šola eksperimentalne kemije postala pomemben akter promocije znanosti na različnih dogodkih. Predvsem je bila dobro sprejeta na vsakoletnih dogodkih Festivala znanosti, ki poteka pod okriljem Slo- venske znanstvene fundacije (SZF)9. Člani ekipe vsako leto aktivno promoviramo znanost z različnimi demonstracij- skimi nastopi po osnovnih in srednjih šolah ter vrtcih. Od leta 2018, izvajalci delavnice Šola eksperimentalne kemije v okviru Evropske noči raziskovalcev, aktivno sodelujemo pri projektu »Noč ima svojo moč«, kjer skupaj s partnerji (poleg Instituta »Jožef Stefan« še: Ustanova Hiša eksperi- mentov, Kemijski inštitut, Tehniški muzej Slovenije, Geo- loški zavod Slovenije ter Botanični vrt Univerze v Ljublja- ni) poudarjamo pomen znanosti za širšo družbo10. Evropski dogodek noči raziskovalcev med drugim, preko različnih predavanj in delavnic, omogoča neposredno ko- munikacijo raziskovalcev s posamezniki. Na ta dan sode- lavci Instituta »Jožef Stefan« sodelujemo pri večeru od- prtih vrat z najrazličnejšimi delavnicami, in dogajanje popestrimo s »showi« eksperimentov (Slika 2). Slika 1: Odzivi udeležencev poletne Šole eksperimentalne kemije (a, b) in primera umetniških vtisov po obisku delavnice Šole eksperimentalne kemije na eni izmed šol (c, d) (Vir: osebni arhiv M. Tramšek). Slika 2: »Show« eksperimentov, ki je potekal v okviru dogodka »Noč ima svojo moč 2022«, na Institutu »Jožefa Stefana« (Foto: M. Verč, Institut »Jožef Stefan). (a) Difuzija in fluorescenca barvila fluorescein v vodnem stolpcu (angl. vortex); (b) s kromovim(III) oksidom katalizirana oksidacija amonijaka, pri kateri nastanejo iskre oz. t.i. »kresničke«; (c) vključevanje naravnih barvil v natrijev alginat in preučevanje lastnosti gela v tekočem dušiku. (Foto: M. Verč, Institut »Jožef Stefan«) S97Acta Chim. Slov. 2022, 69, S95–S97 Tramšek et al.: Trideset let delovanja Šole eksperimentalne kemije ... Glavni namen Šole eksperimentalne kemije temelji na približevanju kemije mladim generacijam in prikazo- vanju njene širše uporabe v vsakdanjem življenju ter s tem prispevati k njeni popularizaciji. Ob enem pa naj bi šola tistim učencem, ki čutijo večje nagnjenje do naravoslovja, ustvarila željo po študiju kemije in morda tudi po razisko- valnem delu na tem področju. »Življenje je potovanje, ne cilj.« Ralph Waldo Emerson Izvajalci programa Šole eksperimentalne kemije smo počaščeni, da smo lahko del programa s trideset letno tra- dicijo. Nadvse nas navdušuje, da lahko svoje poslanstvo širimo med mlade in jih tako motiviramo za raziskovanje naravoslovnih znanosti. Zahvala Avtorji prispevka se iskreno zahvaljujemo začetni- kom Šole eksperimentalne kemije: prof. dr. Andreju Šmal- cu, prof. dr. Borisu Žemvi in mag. Tomažu Ogrinu, ter njihovim sodelavcem in prostovoljcem, ki so s svojim en- tuziazmom in predanostjo pripomogli k častitljivi obletni- ci. Prav tako hvala tudi doc. dr. Gašperju Tavčarju, vodji Odseka za anorgansko kemijo in tehnologijo Instituta »Jo- žef Stefan«, za podporo in motivacijo pri nadaljnjih kora- kih. Velika zahvala gre tudi Javni agenciji za raziskovalno dejavnost Republike Slovenije (ARRS) in raziskovalnemu programu P1-0045 za podporo pri ohranjanju povezav in- stituta s celotno izobraževalno vertikalo. Literatura 1. A. Godec, Kemija v šoli in družbi 2007, 19, 4, 27–33. 2. A. Šmalc, M. Tramšek, Novice IJS 2007, januar, 129, 20–22. 3. T. Ogrin, Novice IJS 2002, april, 95, 14–15. 4. E. Gruden, M. Tramšek, Novice IJS 2022, 202, 12–14. 5. M. Jeran, S. Cvar, A. Podgoršek Berke, Kemija v šoli in družbi 2012, 24, 4, 10–16. 6. M. Jeran, Proteus 2016, 78, 5, 205–214. 7. M. Orel (ur.), M. Jeran (ur.); Skozi mavrico kemijskih spre- memb: kemijski poskusi, Gimnazija Moste in Mako R, Lju- bljana, 2017. https://www.gimoste.si/images/datoteke/Skozi_ mavrico_kemijskih_sprememb.pdf (obiskano: 13. 10. 2022) 8. M. Kovačič; Telefoncek.si, Šola eksperimentalne kemije, Lju- bljana, 2018. https://telefoncek.si/2018/07/04/sola-eksperi- mentalne-kemije/ (obiskano: 10. 10. 2022) 9. Ustanova Slovenska znanstvena fundacija; Evropski festival znanosti, Ljubljana, 2021. https://www.u-szf.si/evropski-fes- tival-znanosti/ (obiskano: 15. 10. 2022) 10. Evropska noč raziskovalcev; Noč ima svojo moč, Konzor- cij partnerjev: Ustanova Hiša eksperimentov, Institut »Jožef Stefan«, Kemijski inštitut, Tehniški muzej Slovenije, Ge- ološki zavod Slovenije ter Botanični vrt Univerze v Ljublja- ni, Ljubljana, 2022. https://www.nocmoc.eu/#predstavitev (obiskano: 15. 10. 2022) Except when otherwise noted, articles in this journal are published under the terms and conditions of the  Creative Commons Attribution 4.0 International License S98 Acta Chim. Slov. 2022, 69, (4), Supplement Društvene vesti in druge aktivnosti Dr. Peter Venturini, predsednik SKD: Spoštovani visoki gostje, dragi prijatelji! Biti kemik je velik privilegij in hkrati odgovornost. Je pos- lanstvo, ki ga z veseljem izpolnjujemo. Pri raziskovalnem delu odkrivamo nova znanja, ki so osnova za razvoj no- vih izdelkov in tehnologij, ki vodijo v napredek industrije in družbe kot celote ter smo pri tem zavezani varovanju okolja in zdravja ljudi. Prav nova spoznanja kemikov, ke- mijskih tehnologov in kemijskih inženirjev so bistveno prispevala k vse boljši kvaliteti življenja v Sloveniji in po svetu. Prvi namen ustanovitve Slovenskega kemijskega društva je pospeševanje napredka kemije v najširšem pomenu be- sede. Zavedanje, da je za razvoj stroke ključnega pomena dobro sodelovanje med strokovnjaki v Sloveniji in tesna vpetost v mednarodno okolje, vodi delo članov društva in vse naše aktivnosti. Člani društva aktivno sodelujemo pri mnogih mednarodnih znanstvenih in strokovnih pobu- dah- na primer pri organizaciji konferenc, kot uredniki pri mednarodnih publikacijah in smo aktivni člani mno- gih sorodnih mednarodnih združenj. Ob mnogih aktivnostih našega društva tokrat izpostav- ljam dve: • Vsakoletno srečanje poimenovano Slovenski kemijski dnevi in • Izdajanje revije Acta Chimica Slovenia. Dr. Peter Venturini, SCS President: Distinguished guests, dear friends! Being a chemist is both a privilege and a responsibility. It is a mission that we are happy to fulfil. Our research work uncovers new knowledge that forms the basis for the de- velopment of new products and technologies that lead to progress in industry and society as a whole, with a commit- ment to protecting the environment and human health. It is the new discoveries of chemists, chemical technologists and chemical engineers that have contributed significantly to the increasing quality of life in Slovenia and around the world. The initial purpose of the founding of the Slovenian Chem- ical Society was to promote the progress of chemistry in the broadest sense of the word. The understanding that good cooperation between experts in Slovenia and close involvement in the international environment are crucial for the development of the profession guides the work of the members of the Society and all our activities. Members of the Society actively participate in many internation- al scientific and professional initiatives – for example, in the organisation of conferences, as editors of international publications and as active members of many related inter- national associations. Among the many activities of our Society, I would like to highlight two: V letu 2021 je Slovensko kemijsko društvo praznovalo 70 –letnico ustanovitve. Zaradi epidemiološke situacije smo bili primorani Slavnostno akademijo pre- ložiti na letošnje leto- potekala je 22. 9. 2022 v Grand Hotelu Bernardin v Portorožu. Dogodka se je udeležilo več kot 310 povabljenih gostov iz 13 držav, med drugim tudi predsednika hrvaškega in slovaškega kemijskega društva ter predstavniki ECTN in EuChemS. Slavnostni govorci so bili prof. dr. Tamara Lah Turnšek, akad. prof. dr. Branko Stanovnik in prof. dr. Venčeslav Kaučič, katerih govore si lahko preberete v nadaljevanju. Slovensko kemijsko društvo je podelilo priznanja 8 častnim članom, 9 zaslu- žnim članom, 11 zaslužnim inštitucijam; priznanja za sodelovanje pri uredni- štvu znanstvene revije Acta Chimica Slovenica pa je prejelo 30 sodelavcev. Vsem se želim še enkrat zahvaliti za odlično delo v Slovenskem kemijskem društvu. Na koncu gre zahvala tudi sponzorjem Slavnostne akademije, ki so omogočili proslavo ob tako pomembni obletnici- hvala torej podjetjem Cinkarna Celje, Salonit Anhovo, Knauf Insulation, Melamin, Aquafil, Novartis, Belinka Perke- mija, Krka, Kemomed, Mettler Toledo in Primalab za podporo in sodelovanje. Dr. Peter Venturini, predsednik Slovenskega kemijskega društva S99Acta Chim. Slov. 2022, 69, (4), Supplement Društvene vesti in druge aktivnosti Velika zahvala gre vsem članom in prijateljem Slovenskega kemijskega društva, ki ste pripomogli k odlični kakovo- sti in mednarodni prepoznavnosti obeh. V zadnjih letih je Kemijske dneve zelo uspešno razvijal organizacijski od- bor pod vodstvom profesorja Albina Pintarja s Kemijske- ga inštituta. Zelo smo ponosni tudi na visoko odmevnost naše revije Acta Chimica Slovenica. Revija sledi sodobnim trendom odprte znanosti pri objavi znanstvenih člankov s prostim dostopom do vseh objav. Uredniški odbor revije že vrsto let uspešno vodi glavna urednica profesorica Kse- nija Kogej s Fakultete za kemijo in kemijsko tehnologijo, Univerze v Ljubljani. Lepa hvala spoštovana Ksenija in Al- bin za izjemno opravljeno delo. Že daljši čas je pomembna usmeritev pri našem delu traj- nostni razvoj in ponosen sem, da bomo v okviru tokratnih Slovenskih kemijskih dnevov prvič podelili tudi nagrade študentom za najboljša dela s tega področja. Pomembno delo za navduševanje mladih za kemijo ter njihovo ozaveščanje za okoljsko odgovorno obnašanje op- ravijo naši kolegi učitelji v osnovnih, srednji šolah in na univerzah za kar jim gre vse priznanje. V Sloveniji smo lahko ponosni na izredno visok inte- res in nivo znanja na različnih področjih kemije na vseh stopnjah šolanja. Odličen je tudi nivo znanosti v visoko- šolskih ustanovah in na raziskovalnih inštitutih. Mnogi slovenski strokovnjaki sodijo med vodilne v svetu, kar je temelj za to, da so tudi mnoga slovenka podjetja zelo uspe- šna v mednarodnem okolju. V Slovenskem kemijskem društvu se bomo tudi v prihod- nje trudili za pospeševanje napredka kemije, kemijske teh- nologije in kemijskega inženirstva s ciljem pridobivanja ključnih znanj za naslednje korake pri trajnostnem razvoju in dvigu kvalitete življenja v Sloveniji in širše. Na koncu se želim zahvaliti vsem članom društva, pred- stavnikom podjetij, raziskovalnih in izobraževalnih usta- nov ter javne uprave za odlično sodelovanje in partnerstvo ter tudi finančno podporo, ki nam omogoča izvedbo vseh naših aktivnosti. Iskrene čestitke in velika zahvala gre vsem današnjim nagrajencem, ki ste pomembno prispevali k razvoju Slo- venskega kemijskega društva. Vsem želim prijetno in zanimivo druženje, ter veliko no- vih poznanstev in izmenjav znanj v okviru konference Slo- venski kemijski dnevi. • the annual meeting known as Slovenian Chemical Days and • the publication of Acta Chimica Slovenica. A very special thank you to all the members and friends of the Slovenian Chemical Society who have contributed to the excellent quality and international visibility of both. In recent years, the Chemical Days have been developed very successfully by the organising committee led by Professor Albin Pintar from the National Institute of Chemistry. We are also very proud of the high visibility of our journal Acta Chimica Slovenica. The journal follows the current trends of open science in publishing scientific articles with open access to all publications. The Editorial Board of the journal has been successfully led for a number of years by the Editor-in-Chief, Professor Ksenija Kogej from the Fac- ulty of Chemistry and Chemical Technology, University of Ljubljana. Thank you very much, Ksenija and Albin, for your outstanding work. Sustainable development has been an important focus of our work for quite some time and I am proud that for the first time at this year’s Slovenian Chemical Days, we will also be awarding prizes for the best work in this field to students. The work our fellow teachers in primary and secondary schools and at universities are doing is essential for getting young people excited about chemistry and to raise their awareness of environmentally responsible behaviour, for which they are to be commended. Slovenia can be proud of the extremely high level of inter- est and knowledge in various areas of chemistry at all lev- els of education. The level of science in higher education institutions and research institutes is also excellent. Many Slovenian experts are among the leading in the world, which is why a number of Slovenian companies are also highly successful in the international environment. The Slovenian Chemical Society will continue its efforts to promote the advancement of chemistry, chemical tech- nology and chemical engineering in order to accumulate key competencies for the next steps in sustainable devel- opment and improving the quality of life in Slovenia and beyond. Finally, I would like to thank all the members of the Socie- ty, representatives of companies, research and educational institutions and public administration for their excellent cooperation and partnership, as well as the financial sup- port that makes all our activities possible. Heartfelt congratulations and a big thank you to all of today’s award winners who have made a significant con- tribution to the development of the Slovenian Chemical Society! I wish you an enjoyable and eventful time and I hope that you form many new acquaintances and knowledge ex- changes during the Slovenian Chemical Days. S100 Acta Chim. Slov. 2022, 69, (4), Supplement Društvene vesti in druge aktivnosti Prof. dr. Tamara Lah Turnšek, Predsednica znanstveno- raziskovalnega razreda IAS: »Spoštovanim tovarišem kemikom spročamo, da je bil dne 15. februarja 1951 ustanovni občni zbor »Slovenskega ke- mijskega društva« ki bo združevalo vse kemike v LR Slove- niji. S tem se je spremenila dosedanja organizacijska oblika kemične sekcije Društva inženirjev in tehnikov LRS in razši- rila v samostojno strokovno in znanstveno društvo«. Tako je bil oznanjen zgodovinski dogodek ustanovitve Slovenskega Kemijskega Društva - SKD pred dobrim sto- letjem. Seveda segajo začetki kemije na slovenskem kar precej dlje! V različnih oblikah se je kemija poučevala na sre- dnjih, višjih in visokih šolah in predhodnih formalnih oblik današnje Fakulte za kemijo in kemijski tehnologijo Univerze v Ljubljani Iz tega (zgodovinskega) prepleta znanj je izšel tudi prvi slovenski Nobelov nagrajenec, dr. Friderik Fritz Pregl, ro- jen v Ljubljani in leta 1894 promoviran za doktorja vsega zdravilstva (medicum universum) na univerzi v Gradcu. Njegovo delo bi danes sodillo na področje biokemije, saj so njegova temeljna spoznanja v organski kemiji obrav- navala predvsem aminokisline, ogljikove hidrate  in puri- ne, kar bi sodilo danes na podroje biokemije! Nobelono nagrado je prejel 1923 za razvoj, na milijoninko grama natančne, tehtnice za kvantitativno organsko mikroanali- zo v sodelovanju s podjetjem Kuhlmane in je omogočil hiter napredek organske kemije v 20. stoletju. V svoji raz- iskovalni vnemi se že kot priznani znanstvenik udinjal kot vajenec pri nekem mizarju, pri ključavničarju in steklopi- haču. Tako je sam zaradi svoje »dodatne izobrazbe«  izdelal zamišljene aparature od prvega grobega osnutka do zadnje fine obdelave. S tem pa je postavil tudi enega pommemb- nih temeljnih kamnov slovenskega inženirstva! To dejstvo se dotika drugega pomemebnega dogodka leta 1995, ko je na podlagi več kot stoletja inženirskega dela Skupina slovenskih znanstvenikov s področja tehniških in naravoslovnih znanosti ter uglednih inženirjev, članov društva SATENA, ustanovilo Inženirsko akademijo Slove- nije (IAS) z namenom, da se ustvari platforma za politiko in formiranje razvojnih perspektiv proizvodnih industrij, znanstvenih in tehnoloških raziskav ter kvalitetnega inže- nirskega in poslovnega študija v Sloveniji, v letu 2006 je bila IAS tudi zakonsko ustanovljena na državni ravni. Trdimo lahko, da prav edina »slovenska« Nobelova na- grada kaže na tesno povezanost med poslanstvom Sloven- skega kemijskega društva in IAS. Na to smo lahko prav na današnji slavnosti ponosni! Ob tem se zavedam globokih korenin znanj, inovatitivega Prof. Dr. Tamara Lah Turnšek, President of the Scientific-Research Group of the IAS: “Esteemed fellow chemists, we would like to inform you that the founding general assembly of the ‘Slovenian Chemical Society’, which will unite all the chemists in the People’s Re- public of Slovenia, was held on 15 February 1951. The cur- rent organisational form of the Chemical Section of the So- ciety of Engineers and Technicians of the PRS has thus been changed and expanded to form an independent professional and scientific society”. This was how the historic event of the founding of the Slo- venian Chemical Society (SCS) was announced nearly a century ago. Of course, the beginnings of chemistry in Slovenia go back much further! Chemistry was taught in various forms at secondary and higher education institutions, and at the previous organisational forms of today’s Faculty of Che- mistry and Chemical Technology of the University of Lju- bljana. This (historical) intertwining of knowledge produced the first Slovenian Nobel Prize winner, Dr. Friderik Fritz Pregl, born in Ljubljana and promoted to Doctor of Me- dicine (medicum universum) at the University of Graz in 1894. His work would today be a part of biochemistry, as his fundamental findings in organic chemistry mainly de- alt with amino acids, carbohydrates and purines! He won the 1923 Nobel Prize for developing a balance, precise to the millionth of a gram, for quantitative organic microa- nalysis in collaboration with the company Kuhlmann, and thereby enabled the rapid progress of organic chemistry in the 20th century. In his eagerness for research, he served, when he was already a renowned scientist, as an apprenti- ce to a carpenter, a locksmith and a glass-blower. Thanks to his “additional education”, he could make the envisaged apparatus for himself from the first rough draft to the last finishing touches. In doing so, he also laid one of the most important cornerstones of Slovenian engineering! This fact touches upon another significant event in 1995, when, on the basis of more than a century of engineering work, a group of Slovenian scientists, working in technical and natural sciences, and prominent engineers, members of the SATENA association, founded the Slovenian Aca- demy of Engineering (IAS) with the aim of creating a plat- form for policies and shaping the development perspecti- ves of the manufacturing industries, scientific and tech- nological research, and quality engineering and business studies in Slovenia; in 2006, the IAS was also established by law at the national level. It could be argued that the only “Slovenian” Nobel Prize S101Acta Chim. Slov. 2022, 69, (4), Supplement Društvene vesti in druge aktivnosti duha in stremljenj vseh mojih predhodnikov v slovenski zgodovni slovenske kemijske znanosti in inženirstva, ki so danes pripeljali obe vedi do uspehov, ki jih naši razisko- valci dosegajo doma in v svetu! S ponosom sem in smo danes lahko hvaležni, da smo imeli priložnost vskravati vse te dobrine tekom svojega učenja in delovanja. Danes imam torej čast, da kot članica, in obenem inženirka or- ganske oz biokemijske smeri, v imenu predsednika in vseh članov IAS čestitam SKD o jubileju društva. Obenem bi želela poudarit, da imata obe instutuciji zelo podobna strmljenja in poslanstva: da pospešujeta napredek kemije, kemijske tehnologije in kemijskega inženirstva v najširšem pomenu; da skrbita za rast strokovnega znanja svojega članstva in da sodelujeta z vsemi organizacijami, ki se ukvarjajo s kemijsko, tudi bi- okemijski stroko in bioinženirstvom, z njimi izmenjujeta izkušnje in jim pomagata Nadalje pa si članice in čani IAS želimo tesnejšega sode- lovnja, da skupaj z SKD opažamo pereče probleme stroke, jih rešujemo in odločevalcem skupaj predlagamo aktivno- sti in najbolj kakovostne reštitve; tudi za relevante širše družbene probleme, ki vključujejo in potrebujejo naša znanja in izkušnje. Čisto nazadnje in sploh ne najmanj pomembno, je omeni- ti prispevek ženskih kolegic k delu, uspehu in ugledu obeh institucij. Čeprav je danes diplomantk in doktorandk na področju kemije in kemijskega inženirstva v Sloveniji vsaj enako ali celo več, kar se delno odseva tudi članstvu SKD, so ženske dokaj slabo, le z nekaj odstotki zastopane v aka- demijah in med dobitniki najvišjih strokovnih priznanj. Če se vrnem na začetek nagovora – k Nobelovi nagradi – je bilo nagrajenk v 121. letih je samo pet, začenši z Marie Sklodowksi Curie v letu 1911 in Jeniffer Doudna v letu 2021! Zato seveda potrebujemo podporo skupnosti in tudi ko- legov! shows how closely linked the missions of the Sloveni- an Chemical Society and the IAS really are. This is truly something we can be proud of at today’s celebration! I am also aware of the deep roots of knowledge, the inno- vative spirit and the ambitions of all my predecessors in the history of Slovenian chemical science and engineering, which have led both sciences to the successes that our researchers are today achieving at home and around the world. I, and we, can be proud and thankful today that we have had the opportunity to absorb all these benefits in the course of our studies and work. Today, therefore, I have the honour, as a member and as an organic chemistry or biochemical engineer, to congratulate the SCS on its anni- versary on behalf of the President and all the members of the IAS. At the same time, I would like to point out that both insti- tutions have very similar ambitions and missions: to promote the advancement of chemistry, chemical tech- nology and chemical engineering in the broadest sense; to foster the professional development of its members and to work with all the organisations involved in the chemical and biochemical profession and bioengineering by sharing experience and assisting them. Furthermore, IAS members would like to work more clo- sely together with the SCS to identify pressing problems in the profession, solve them and jointly propose actions and the best solutions to decision makers; including for any re- levant broader societal problems that involve and need our knowledge and experience. Last but by no means least, it is important to note the contribution of female colleagues to the work, success and reputation of both institutions. Although the number of female graduates and PhD students in chemistry and chemical engineering in Slovenia today is at least equal or even higher, which is partly reflected in the member- ship of the SCS, women are rather poorly represented, i.e. only a few percent, in academies and among the winners of the highest professional awards. If I may return to the beginning of my address – to the Nobel Prize – there have been only five female Nobel Prize winners in 121 years, starting with Marie Skłodowska-Curie in 1911 and Jenni- fer Doudna in 2021! So we obviously need the support of the community and also of our male colleagues! S102 Acta Chim. Slov. 2022, 69, (4), Supplement Društvene vesti in druge aktivnosti Akad. prof. dr. Branko Stanovnik, predstavnik Slovenske akademije znanosti in umetnosti: Spoštovani gospod predsednik SKD Dr. Peter Venturini, Spoštovani ugledni gostje, kolegice in kolegi, dragi prijatelji V prijetno dolžnost mi je, da vas najprej pozdravim v ime- nu presednika Slovenske akademije znanosti in umetnosti akadenika Petra Štiha, ki se zaradi drugih obveznosti ni mogel udležiti današnje slovesnsoti, in izročim njegove pozdrave in čestitke SKD in vam ob tem pomembnem ju- bileju za vse imenitne dosežke, ki jih je društvo doseglo v svoji zgodovini. Obenem želi SKD še naprej veliko uspe- hov, predsedniku pa uspešno vodenje društva še naprej. Slovenska akademija znanosti in umetnosti je ponosna, da je bil prvi predsednik SKD član akademije in v mednaro- dnem svetu ugledni znanstveni Maks Samec. 2. 9. 1945 je bil Samec odstranjem z univerze. Njegovo na- daljne delovanje je povezano z gradnjo instituta za kemijo, ki je bil ustanovljenn hkrati s fizikalnim institutom Jožef Stefan in elektroinstitutom Milana Vidmarja v okviru Slo- venske akademije znanosti in umetnosti. Od 1. 10. 1946 do 1959 je bil upravnik kemijskega laboratorija AZU (pozneje SAZU), ki se je 12. 6. 1953 preimenoval v Kemični institut Borisa Kidriča. Maks Samecje odigral pionirsko vlogo na področju oraniziranja Slovenskega kemijskega društva, ki je bilo ustanovljeno 15. 12. 1951. Bil je njegov prvi predse- dnik 1951–1962 in častni predsednik 1963/64. Ker je Komite za šolstvo in znanost Federativne republike Jugoslavije v Beogradu zahteval, da se naj znanstvena dela najprej objavijo v domaačih strokovnih revijah, šele nato v tujini je Samec ustanovil najprej Zbornik in nato Vestnik Slovenskega kemijskega društva. Zgodovina SKD na spletni strani je razmeroma skromna. Zato naj dodam še nekaj glede tega. 1. Uredništvo Vestnika Slovenskega kemijskega društva in preimenovaje v Acta Chimica Slovenica Prof. Dušan Hadži me je kot predsednik SKD 1976 zapro- sil, da bi prevzel uredništvo Vestnika, ki je do takrat zelo neredno izhajal. Kot novi urednik sem si zadal dve nalogi: 1. da bo Vestnik izhajal redno štirikrat na leto in 2. predla- gal sem, da bi Vestnik preimenovali v Acta Chimica Slove- nica. Glede rednega izhajanja mi je stvar uspela ob izdatni pomoči Marka Razingerja kot tehničnega urednika. Ve- stniku je bil kmalu priznam faktor vpliva. Glede preime- novanja pa je moj predlog leta 1976 popolnoma pogorel. Eksplicitno sta bila proti prof. Hadži in prof. Dolar. Nisem se hotel prepirati, sklenil sem počakati na ugodnejše čase. Počakal sem do razglasitve samostojne države. Potem, ko Akad. prof. dr. Branko Stanovnik, representative of the Slovenian Academy of Sciences and Arts: Honourable President of the SCS, Dr. Peter Venturini, distinguished guests, colleagues, dear friends, It is my pleasant duty first of all to greet you on behalf of the President of the Slovenian Academy of Sciences and Arts, Academician Peter Štih, who was unable to attend to- day’s ceremony due to other commitments, and to convey his greetings and congratulations to the SCS and to you on this special anniversary for all the outstanding achieve- ments that the Society has accomplished in its history. He also wishes the SCS many successes in the future and the President continued successful leadership of the Society. The Slovenian Academy of Sciences and Arts is proud that the first President of the SCS was a member of the Acad- emy and internationally renowned scientist Maks Samec. On 2 September 1945 Samec was removed from the uni- versity. His further activities were linked to the construc- tion of the Institute of Chemistry, which was founded at the same time as the Jožef Stefan Institute and the Milan Vidmar Electric Power Research Institute under the aus- pices of the Slovenian Academy of Sciences and Arts. From 1 October 1946 to 1959, he was the manager of the chemical laboratory of the Academy of Sciences and Arts (later the Slovenian Academy of Sciences and Arts), which was renamed the Boris Kidrič Institute of Chemistry on 12 June 1953. Maks Samec had a pioneering role in the organisation of the Slovenian Chemical Society, which was founded on 15 December 1951. He was its first President from 1951 to 1962 and Honorary President in 1963/64. Because the Committee for Education and Science of the Federal Republic of Yugoslavia in Belgrade demanded that scientific works should first be published in domestic pro- fessional journals, and then abroad, Samec founded first the Zbornik and then the Vestnik Slovenskega kemijskega društva. There is relatively little written about the history of the SCS on its website. Allow me to add something to this topic. 1. The Editorial Board of the Vestnik Slovenskega kem- ijskega društva and the renaming to Acta Chimica Slovenica. In 1976, Prof. Dušan Hadži, then President of the SCS, asked me to take over the editorship of the Vestnik, which until then had been published very irregularly. As the new editor, I set myself two tasks: 1. that the Vestnik would be published regularly four times a year, and 2. I proposed that the Vestnik be renamed Acta Chimica Slovenica. As regards regular publication, I succeeded with the substan- S103Acta Chim. Slov. 2022, 69, (4), Supplement Društvene vesti in druge aktivnosti je bilo SKD sprejeto 1992 v Federacijo evropskih kemijskih društev, sem kot urednik Vestnika predlagal spremembo naslova. Tokrat je bil predlog brez pripomb soglasno spre- jet in leta 1993 je začel izhajati kot Acta Chimica Slovenica. Uredništvo so prevzeli mlajši ljudje, ki so ga ustrezno pre- oblikovali in digitalizirali in že v nekaj letih so Acta Chi- mica Slovenica postala mednarodno prepoznaven časopis. 2. Sprejem Slovenskega kemijskega društva v Federacijo evropskih kemijskih društev. Prvo mednarodno pri- zanje strokovnega društva v mednarodni rganizaciji v samostojni Sloveniji. V osemdesetih in devetdesetih letih prejšnjega stoletja sem bil najprej kot predstavnik Unije jugoslovanskih kemijskih društev, pozneje pa kot predstavnik Slovenskega kemij- skega drušva član Sveta Federacije evropskih kemijskih društev. Seja Sveta Federacije EKD je bila v Londonu 24. in 25. junija 1991. Ker je bilo takrat že jasno, kakšna bo usoda Jugoslavije, sem situacijo pojasnil in se dogovoril s predsednikom Sveta Federacije evropskih kemijskih dru- štev dr. Gowom, generalnim skretarjem Royal Society of Chemistry in drugimi člani Sveta za sprejem Slovenskega kemijskega društva v Federacijo evropskih kemijskih dru- štev. To ustno soglasje je bilo sprejeto nekaj ur pred razgla- sitvijo samostojne Slovenije. Ko sem zgodaj popoldne pri- šel na letališče v Londonu in sem se hotel prijaviti za let v Ljubljano, mi povedo, da je Adriin let odpovedan, namesto tega pa leti letalo Air France. Izkazalo se je, da je Adrii- no letalo letelo pod francosko zastavo in tako smo varno pristali v Ljubljani na predvečer razglastitve samostojne Slovenije. To je bilo zadnje letalo, ki je pristalo v Ljubljani pred desetdnevno vojno za Slovenijo. Tisto noč so tanki odpeljali iz vrhniške kasarne proti Brniku. Letališče je bilo nato nekaj mesecev zaprto. Glede na to, da sem prinesel iz London ustno zagotovilo o priznanju Slov. kem. društva, je prof. Ljubo Golič, tedanji predsednik Slovenskga kemijskega društva, pripravil pisno vlogo na Federacijo, ki je bila obravnavana na Seji Sveta Federacije evr. kem društev v Varšavi na sedežu Polske akademije znanosti 22. junija 1992. Slovenija je bila spre- jeta z aplavzom brez kakršne koli pripombe ali komentar- ja. To je prvo mednarodno priznanje nekega strokovnega društva v mednarodni asociaciji. Hvala lepa tial support of Marko Razinger as technical editor. The Vestnik was soon recognised as having an impact factor. As for the renaming, my proposal in 1976 was complete- ly rejected. Prof. Hadži and Prof. Dolar were explicitly against it. As I did not want to argue, I decided to wait for better times. I waited until the country declared inde- pendence. After the SCS was admitted to the Federation of European Chemical Societies in 1992, I, as editor of the Vestnik, proposed a change of its name. This time the pro- posal was unanimously adopted without objections and in 1993 the journal started to be published as Acta Chimica Slovenica. The Editorial Board was taken over by younger people, who reorganised and digitised it accordingly, and within a few years, Acta Chimica Slovenica became an in- ternationally renowned journal. 2. The admission of the Slovenian Chemical Society to the Federation of European Chemical Societies. The first international recognition of a professional society by an international organisation in independent Slovenia. In the 1980s and 1990s, I was a member of the Council of the Federation of European Chemical Societies, first as a representative of the Union of Yugoslav Chemical Socie- ties, and later as a representative of the Slovenian Chem- ical Society. The Council of the Federation of European Chemical Societies met in London on 24 and 25 June 1991. At the time, it was already clear what the fate of Yugosla- via would be, so I explained the situation and agreed with the President of the Council of the Federation of Euro- pean Chemical Societies, Dr. Gow, the General Secretary of the Royal Society of Chemistry and other members of the Council that we would admit the Slovenian Chemical Society to the Federation of European Chemical Societies. This oral consent was given a few hours before the decla- ration of Slovenia’s independence. When I arrived at the airport in London in the early afternoon to check in for my flight to Ljubljana, I was told that Adria’s flight had been cancelled and that Air France was flying instead. It turned out that Adria’s plane was flying under the French flag, which allowed us to land safely in Ljubljana on the eve of Slovenia’s declaration of independence. This was the last plane to land in Ljubljana before the Ten-Day War for Slo- venia. That night, tanks started rolling out of the Vrhnika barracks towards Brnik. The airport was closed for several months after that. As I had brought from London a verbal assurance that the Slovenian Chemical Society would be recognised, Prof. Lju- bo Golič, then President of the Slovenian Chemical Society, prepared a written application for the Federation, which was discussed at the Warsaw meeting of the Council of the Fed- eration of European Chemical Societies at the headquarters of the Polish Academy of Sciences on 22 June 1992. Slove- nia was admitted with applause and without any remarks or comments. This is the first international recognition of a professional society by an international association. Thank you. S104 Acta Chim. Slov. 2022, 69, (4), Supplement Društvene vesti in druge aktivnosti Prof. dr. Venčeslav Kaučič, častni predsednik SKD: Spoštovane in drage kolegice in kolegi, prisrčno pozdrav- ljeni. Posebej želim pozdraviti, jim čestitati in se jim zahvaliti za njihov doprinos k delovanju in uspehom društva da- našnjim slavljencem, novim častnim in novim zaslužnim članom društva. I also warmly welcome our dear guests from abroad, with whom we were meeting and cooperated closely for many years. Today we thank them for their contribution to the professional growth of our society and for their help in in- tegrating our society into international professional orga- nizations and international connections by awarding them the title of honorary member of the Slovenian Chemical Society. Zelo na kratko želim navesti nekaj utrinkov o razvoju in delovanju društva v preteklih sedemdesetih letih. Kot smo že slišali je bil pobudnik ustanovitve in prvi predsednik Prof. Maks Samec. Za njim so ga vodili izjemni, svetov- no znani znanstveniki in inženirji: prof. Roman Modic (od 1963 do 1974), akad. prof. Dušan Hadži (od 1974 do 1986), akad. prof. Ljubo Golič (od 1986 do 1996) in v no- vejšem času prof. Venčeslav Kaučič (od 1996 do 2017), prof. Albin Pintar (od 2017 do 2021) in od 2021 ga vodi dr. Peter Venturini. Zanimiva je bila moja včlanitev in nato 50 let delovanja v društvu in v mednarodnih združenjih kemikov. Na po- delitvi visokošolske diplome leta 1973 sem dobil vabilo za včlanitev v SKD. Še isti dan sem šel v pisarno društva na Kemijskem inštitutu, kjer sem srečal takratnega tajnika društva prof. Marcela Žorgo, ki me je vpisal v društvo pod vpisno št. 305. Kupil sem še Laboratorijski priročnik, ki ga je izdalo društvo in mi je bil v tistem času, ko je bilo na razpolago malo strokovnih knjig v slovenskem in v tujih jezikih, v veliko pomoč. Leta 1986 sem bil izvoljen za tajnika društva in v tem svojstvu deloval do leta 1996, ko sem bil na občnem zboru, ki je potekal na konferenci Slovenski kemijski dnevi v Ma- riboru, izvoljen za predsednika društva. To je bilo na dan, ko se je Prof. Miha Japelj veselil rojstva svoje prve vnučke in sva imela razlog več za proslavljanje. Prof. Japelj se tega zagotovo dobro spominja. Funkcijo predsednika društva sem z veseljem in spošto- vanjem opravljal do leta 2017. Posebej sem si prizadeval za članstvo društva v uglednih mednarodnih strokov- nih združenjih in aktivno delovanje naših članov v njih. Prepričan sem, da so naši člani s svojim strokovnim delo- vanjem primerno promovirali slovensko znanost v med- narodni znanstveni srenji. Prof. dr. Venčeslav Kaučič, Honorary President of the SCS: Ladies and gentlemen, dear colleagues, a warm welcome. I would also like to extend a special welcome, congratu- lations and thanks for their contribution to the operation and success of the Society to today’s honorees, the new Honorary Members and the new Emeritus Members of the Society. I also warmly welcome our dear guests from abroad, with whom we have been meeting and cooperating closely for many years. Today, we thank them for their contribution to the professional growth of our Society and for their help in integrating our Society into international professional organisations and international connections by award- ing them the title of Honorary Member of the Slovenian Chemical Society. I would like to give you, very briefly, some highlights from the development and activities of the Society over the past 70 years. As we have already heard, the initiator and first President was Professor Maks Samec. After him, the So- ciety was led by outstanding, world-renowned scientists and engineers: Prof. Roman Modic (from 1963 to 1974), Acad. Prof. Dušan Hadži (from 1974 to 1986), Acad. Prof. Ljubo Golič (from 1986 to 1996) and more recently Prof. Venčeslav Kaučič (from 1996 to 2017), Prof. Albin Pintar (from 2017 to 2021), and since 2021 it has been led by Dr. Peter Venturini. My becoming a member and then 50 years of service in the Society and in international associations of chemists was interesting. I received an invitation to join the SCS at my university graduation ceremony in 1973. On the same day, I went to the Society’s office at the National Institute of Chemistry, where I met the then Secretary of the Society, Prof. Marcel Žorga, who enrolled me in the Society un- der registration number 305. I also bought the Laboratory Manual published by the Society, which was a great help to me at that time, because there were few scientific books available in Slovenian or in foreign languages. In 1986, I was elected Secretary of the Society and served in this capacity until 1996, when I was elected President at the general assembly held at the Slovenian Chemical Days in Maribor. This was on the day Prof. Miha Japelj was celebrating the birth of his first granddaughter and we had an additional reason to celebrate. I am sure Prof. Japelj remembers this well. I have served as President of the Society with pleasure and respect until 2017. It was always my goal that the Socie- ty would become a member of renowned international professional associations and that our members would be S105Acta Chim. Slov. 2022, 69, (4), Supplement Društvene vesti in druge aktivnosti Društvo aktivno sodeluje tudi s številnimi društvi na bila- teralni ravni, z društvi iz naše bližnje okolice, v evropskem prostoru in tudi z društvi iz drugih kontinentov. Leta 2017 sem bil izvoljen za častnega predsednika Sloven- skega kemijskega društva, na kar sem ponosen. Od 1986 do 2017 (mandat tajnika in nato predsednika) je več kot 30 let in prof. Pejovnik mi je večkrat rekel, da zanj priimek Kaučič hkrati pomeni kemijsko društvo in obratno, da ke- mijsko društvo hkrati pomeni Kaučič. Hvala ti, Stane. Morda omenim še, da sem bil preko 30 let aktiven v IU- PAC-u. Letos je izšel IUPAC-ov periodni sistem, preveden v 26 svetovnih jezikov. Na svetu obstaja med 6000 in 7000 jezikov. Ponosno povem, da je izšel tudi v slovenskem jezi- ku (eden od 26 prevodov). Zahvaljujem se za vsestransko podporo društvu s strani pomembnih industrijskih partnerjev, univerz, znanstve- nih in strokovnih inštitutov ter ministrstva za šolstvo, zna- nost in šport in Javne agencije za raziskovalno dejavnost Republike Slovenije. Hkrati si želim, da društvo strokovno raste še naprej in da z dejavnostmi aktivno nadaljuje v naslednjih letih ter jih tudi nadgrajuje. Hvala za vašo pozornost, po koncu te slovesnosti pa vam želim veselo, prijazno in sproščujoče druženje. active in them. I am confident that our members have ad- equately promoted Slovenian science in the international scientific community through their professional activities. The Society also actively and bilaterally cooperates with a number of societies, with societies in our immediate sur- roundings, in the European area and also with societies from other continents. In 2017, I was elected Honorary President of the Slovenian Chemical Society, which I am proud of. The period from 1986 to 2017 (my service as Secretary and then President) was longer than 30 years, and Prof. Pejovnik told me sever- al times that, for him, the surname Kaučič is synonymous with the Chemical Society and vice versa, that the Chem- ical Society also means Kaučič. Thank you for that, Stane. I would also like to mention that I have been active in the IUPAC for over 30 years. This year, the IUPAC Periodic Table was published, translated into 26 world languages. There are between 6000 and 7000 languages in the world. I am proud to say that it has also been published in Sloveni- an (one of 26 translations). For their comprehensive support provided to the Society, I would like to extend my thanks to the important industrial partners, universities, scientific and professional institutes, the Ministry of Education, Science and Sport and the Slo- venian Research Agency. At the same time, I would like to see the Society continue to grow professionally and to actively continue and build on its activities in the coming years. Thank you for your attention and have a happy, pleasant and relaxing time after this ceremony. S106 Acta Chim. Slov. 2022, 69, (4), Supplement Društvene vesti in druge aktivnosti Častni člani: 1. Prof. dr. Peter Glavič 2. Akademik prof. dr. Branko Stanovnik 3. Zasl. prof. dr. Marjan Veber 4. Prof. dr. Jean Marie Lehn 5. Prof. dr. Wolfram Koch 6. Prof. dr. Pavel Drašar 7. Prof. dr. Andrej Šmalc 8. Prof. dr. Leiv K. Sydnes Zaslužni člani: 1. Dr. Vida Hudnik 2. Prof. dr. Darinka Brodnjak Vončina 3. Prof. dr. Majda Žigon 4. Mag. Alenka Gogala 5. Mija Marin, dipl. inž. kem. tehnologije 6. Prof. dr. Miha Japelj 7. Zasl. prof. dr. Stane Pejovnik 8. Prof. dr. Aleksander Pavko 9. Prof. dr. Lucija Zupančič Kralj Zaslužne inštitucije: 1. Kemijski inštitut 2. Institut „Jožef Stefan“ 3. Fakulteta za kemijo in kemijsko tehnologijo, UL 4. Fakulteta za kemijo in kemijsko tehnologijo, UM 5. Cinkarna Celje, d.d. 6. Krka, d.d., Novo mesto 7. Kemomed, d.o.o. 8. AquafilSLO d.o.o. 9. Mettler Toledo d.o.o. 10. MIKRO+POLO d.o.o. 11. DONAU Lab d.o.o. Priznanja za delo v uredništvu Acta Chimica Slovenica: 1. Janez Košmrlj 2. Aleksander Pavko 3. Ksenija Kogej 4. Marija Bešter-Rogač 5. Matija Strlič 6. Mladen Franko 7. Alojz Demšar 8. Andrej Petrič 9. Bert VW Maes 10. Janez Cerkovnik 11. Barbara Malič 12. Krištof Kranjc 13. Damjana Rozman 14. Primož Šegedin 15. Boris Pihlar 16. Johannes T. Van Elteren 17. Michael Beeston 18. Irena Vovk 19. Helena Prosen 20. Melita Tramšek 21. Franc Perdih 22. Aleš Podgornik 23. Alen Albreht 24. Aleš Berlec 25. Mirela Dragomir 26. Matjaž Kristl 27. Vinko Vovk 28. Stanislav Oražem 29. Olga Gorše 30. Marjana Gantar Albreht Seznam nagrajencev S107Acta Chim. Slov. 2022, 69, (4), Supplement Društvene vesti in druge aktivnosti 2023 January 2023 1 EUROPEAN FOOD CHEMISTRY CONGRESS XXI – EuroFoodChem XXI Belgrade, Serbia Information: http://horizon2020foodentwin.rs/eurofoodchemxxi/ February 2023 8 – 11 EMBO WORKSHOP IN SITU STRUCTURAL BIOLOGY: FROM CRYO-EM TO MULTI- SCALE MODELLING Heidelberg, Germany Information: https://www.embl.org/about/info/course-and-conference-office/events/iss23-01/ March 2023 20 – 23 VIII INTERNATIONAL CONGRESS “ENGINEERING, ENVIRONMENT AND MATERIALS IN PROCESS INDUSTRY Jahorina, Bosnia and Hercegovina Information: https://eem.tfzv.ues.rs.ba/ April 2023 16 – 21 HTCC 5 Dubrovnik, Croatia Information: https://htcc5.org/ May 2023 16 – 17 EUROPEAN CONFERENCE ON CO2 CAPTURE, STORAGE & REUSE 2023 Copenhagen, Denmark 21 – 25 PPEPPD 2023 Tarragona, Spain Information: https://ppeppd.org/ppeppd2023/ 29 – June 2 15TH MEDITERRANEAN CONGRESS OF CHEMICAL ENGINEERING – MECCE Barcelona, Spain Information: https://www.mecce.org/ 31 – June 2 8TH EUROPEAN PROCESS INTENSIFICATION CONFERENCE Warsaw, Poland Information: https://www.epic2023.pw.edu.pl/index/ KOLEDAR VAŽNEJŠIH ZNANSTVENIH SREČANJ S PODROČJA KEMIJE IN KEMIJSKE TEHNOLOGIJE SCIENTIFIC MEETINGS – CHEMISTRY AND CHEMICAL ENGINEERING S108 Acta Chim. Slov. 2022, 69, (4), Supplement Društvene vesti in druge aktivnosti July 2023 2 – 6 FEZA 2023 – 9TH CONFERENCE OF THE FEDERATION OF THE EUROPEAN ZEOLITE ASSOCIATIONS Portorož-Portorose, Slovenia Information: https://feza2023.org/en/ 2 – 7 XV POSTGRADUATE SUMMER SCHOOL ON GREEN CHEMISTRY Venice, Italy Information: https://www.greenchemistry.school/ 9 – 14 38TH INTERNATIONAL CONFERENCE ON SOLUTION CHEMISTRY Belgrade, Serbia Information: https://icsc2023.pmf.uns.ac.rs/ 7 – 11 9TH EUCHEMS CHEMISTRY CONGRESS (ECC9) Dublin, Ireland S109Acta Chim. Slov. 2022, 69, (4), Supplement Društvene vesti in druge aktivnosti S110 Acta Chim. Slov. 2022, 69, (4), Supplement Društvene vesti in druge aktivnosti Sub mis sions Submission to ACSi is made with the implicit under- standing that neither the manuscript nor the essence of its content has been published in whole or in part and that it is not being considered for publication else- where. All the listed authors should have agreed on the content and the corresponding (submitting) au- thor is responsible for having ensured that this agree- ment has been reached. The acceptance of an article is based entirely on its scientific merit, as judged by peer review. There are no page charges for publishing articles in ACSi. The authors are asked to read the Author Guidelines carefully to gain an overview and assess if their manuscript is suitable for ACSi. Additional information • Citing spectral and analytical data • Depositing X-ray data Sub mis sion ma te rial Typi cal sub mis sion con sists of: • full manuscript (PDF file, with title, authors, ab- stract, keywords, figures and tables embedded, and references) • supplementary files – Full manuscript (original Word file) – Statement of novelty (Word file) – List of suggested reviewers (Word file) – ZIP file containing graphics (figures, illustra- tions, images, photographs) – Graphical abstract (single graphics file) – Proposed cover picture (optional, single graphics file) – Appendices (optional, Word files, graphics files) Incomplete or not properly prepared submissions will be rejected. Sub mis sion pro cess Before submission, authors should go through the checklist at the bottom of the page and prepare for submission. Submission process consists of 5 steps. Step 1: Star ting the sub mis sion • Choo se one of the jour nal sections. • Con firm all the re qui re ments of the chec klist. • Ad di tio nal plain text com ments for the edi tor can be pro vi ded in the re le vant text field. Step 2: Up load sub mis sion • Up load full ma nus cript in the form of a Word fi­ le (with tit le, aut hors, ab stract, key words, fi gu res and tab les em bed ded, and re fe ren ces). Step 3: En ter me ta da ta • First na me, last na me, con tact email and af lia tion for all aut hors, in re le vant or der, must be pro vi ded. Cor res pon ding aut hor has to be se lec ted. Full po- stal ad dress and pho ne num ber of the cor res pon- ding aut hor has to be pro vi ded. • Tit le and ab stract must be pro vi ded in plain text. • Key words must be pro vi ded (max. 6, se pa ra ted by se mi co lons). • Data about con tri bu tors and sup por ting agen cies may be en te red. • Re fe ren ces in plain text must be pro vi ded in the re le vant text fi led. Step 4: Up load sup ple men tary fi les • Original Word file (original of the PDF uploaded in the step 2) • List of suggested reviewers with at least five re- viewers with two recent references from the field of submitted manuscript must be uploaded as a Word file. At the same time, authors should declare (i) that they have no conflict of interest with suggest- ed reviewers and (ii) that suggested reviewers are experts in the field of the submitted manuscript. • All grap hics ha ve to be up loa ded in a sin gle ZIP fi le. Grap hics should be na med Fi gu re 1.jpg, Fi gu re 2.eps, etc. • Grap hi cal ab stract ima ge must be uploaded separately • Pro po sed co ver pic tu re (op tio nal) should be up- loa ded se pa ra tely. • Any ad di tio nal ap pen di ces (optional) to the paper may be uploaded. Appendices may be published as a supplementary material to the paper, if accepted. • For each uploaded file the author is asked for addi- tional metadata which may be provided. Depending of the type of the file please provide the relevant title (Statement of novelty, List of suggested re- viewers, Figures, Graphical abstract, Proposed cov- er picture, Appendix). Step 5: Con fir ma tion • Fi nal con fir ma tion is re qui red. Ar tic le Types Feature Articles are contributions that are written on Editor’s invitation. They should be clear and concise summaries of the author’s most recent work written with the broad scope of ACSi in mind. They are intend- ed to be general overviews of the authors’ subfield of research but should be written in a way that engages and informs scientists in other areas. They should con- tain the following (see also general guidelines for arti- cle structure below): (1) an introduction that acquaints readers with the authors’ research field and outlines the important questions for which answers are being sought; (2) interesting, novel, and recent contributions of the author(s) to the field; and (3) a summary that presents possible future directions. Manuscripts should normally not exceed 40 pages of one column format (font size 12, 33 lines per page). Generally, experts who have made an important contribution to a specific field in recent years will be invited by the Editor to contrib- ute a Feature Article. Individuals may, however, send a proposal (of no more than one page) for a Feature Article to the Editor-in-Chief for consideration. Acta Chimica Slovenica Author Guidelines S111Acta Chim. Slov. 2022, 69, (4), Supplement Društvene vesti in druge aktivnosti Scien ti fic ar tic les should report significant and inno- vative achievements in chemistry and related scienc- es and should exhibit a high level of originality. They should have the following structure: 1. Tit le (max. 150 cha rac ters), 2. Aut hors and af lia tions, 3. Ab stract (max. 1000 cha rac ters), 4. Key words (max. 6), 5. Intro duc tion, 6. Experimental, 7. Re sults and Dis cus sion, 8. Conc lu sions, 9. Acknowledgements, 10. Re fe ren ces. The sections should be arranged in the sequence gen- erally accepted for publications in the respective fields and should be successively numbered. Short com mu ni ca tions generally follow the same order of sections as Scientific articles, but should be short (max. 2500 words) and report a significant as- pect of research work meriting separate publication. Editors may decide that a Scientific paper is catego- rized as a Short Communication if its length is short. Tech ni cal ar tic les report applications of an already described innovation. Typically, technical articles are not based on new experiments. Pre pa ra tion of Sub mis sions Text of the submitted articles must be prepared with Microsoft Word. Normal style set to single column, 1.5 line spacing, and 12 pt Times New Roman font is recommended. Line numbering (continuous, for the whole document) must be enabled to simplify the re- viewing process. For any other format, please consult the editor. Articles should be written in English. Correct spelling and grammar are the sole responsibility of the author(s). Papers should be written in a concise and succinct manner. The authors shall respect the ISO 80000 standard [1], and IUPAC Green Book [2] rules on the names and symbols of quantities and units. The Système International d’Unités (SI) must be used for all dimensional quantities. Grap hics (figures, graphs, illustrations, digital imag- es, photographs) should be inserted in the text where appropriate. The captions should be self-explanatory. Lettering should be readable (suggested 8 point Arial font) with equal size in all figures. Use common pro- grams such as MS Excel or similar to prepare figures (graphs) and ChemDraw to prepare structures in their final size. Width of graphs in the manuscript should be 8 cm. Only in special cases (in case of numerous data, visibility issues) graphs can be 17 cm wide. All graphs in the manuscript should be inserted in relevant places and aligned left. The same graphs should be provid- ed separately as images of appropriate resolution (see below) and submitted together in a ZIP file (Graphics ZIP). Please do not submit figures as a Word file. In graphs, only the graph area determined by both axes should be in the frame, while a frame around the whole graph should be omitted. The graph area should be white. The legend should be inside the graph area. The style of all graphs should be the same. Figures and illustrations should be of sufcient quality for the printed version, i.e. 300 dpi minimum. Digital images and photographs should be of high quality (minimum 250 dpi resolution). On submission, figures should be of good enough resolution to be assessed by the refer- ees, ideally as JPEGs. High­resolution figures (in JPEG, TIFF, or EPS format) might be required if the paper is accepted for publication. Tab les should be prepared in the Word file of the pa- per as usual Word tables. The captions should appear above the table and should be self-explanatory. Re fe ren ces should be numbered and ordered se- quentially as they appear in the text, likewise meth- ods, tables, figure captions. When cited in the text, reference numbers should be superscripted, follow- ing punctuation marks. It is the sole responsibility of authors to cite articles that have been submitted to a journal or were in print at the time of submission to ACSi. Formatting of references to published work should follow the journal style; please also consult a recent issue: 1. J. W. Smith, A. G. 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- S112 Acta Chim. Slov. 2022, 69, (4), 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). These studies must be approved by an appropriate institutional review board or commit- tee, and informed consent must be obtained from subjects. The Methods section of the paper must include: 1) a statement of protocol approval from an institutional review board or committee and 2), a statement that informed consent was obtained from the human subjects or their representatives. • Pub lis hed Sta te ment of Hu man and Ani mal Rights.When reporting experiments on human subjects, authors should indicate whether the procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and na- tional) and with the Helsinki Declaration of 1975, as revised in 2008. If doubt exists whether the research was conducted in accordance with the Helsinki Declaration, the authors must explain the rationale for their approach and demonstrate that the institutional review body explicitly ap- proved the doubtful aspects of the study. When reporting experiments on animals, authors should indicate whether the institutional and national guide for the care and use of laboratory animals was followed. • To avoid conflict of interest between authors and referees we expect that not more than one referee is from the same country as the corresponding au- thor(s), however, not from the same institution. • 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. It is made available on the Journal’s web site and is linked to the article in the Journal’s Web edition. The use of supporting information is particular- ly appropriate for presenting additional graphs, spectra, tables and discussion and is more likely to be of interest to specialists than to general readers. When preparing supporting information, authors should keep in mind that the supporting information files will not be edited by the editorial staff. In addition, the files should be not too large (upper limit 10 MB) and should be provided in common widely known file formats to be accessible to readers without difculty. All files of supplementary materials are loaded separately during the submission process as supplementary files. Pro po sed Co ver Pic tu re and Grap hi cal Ab stract Image Grap hi cal con tent: an ideally full-colour illustration of resolution 300 dpi from the manuscript must be proposed with the submission. Graphical abstract pic- tures are printed in size 6.5 x 4 cm (hence minimal resolution of 770 x 470 pixels). Cover picture is print- ed in size 11 x 9.5 cm (hence minimal resolution of 1300 x 1130 pixels) Authors are encouraged to submit illustrations as can- didates for the journal Cover Picture*. The illustration must be related to the subject matter of the paper. Usually both proposed cover picture and graphical ab- stract are the same, but authors may provide different pictures as well. * The authors will be asked to contribute to the costs of the cover picture production. Sta te ment of no velty Statement of novelty is provided in a Word file and submitted as a supplementary file in step 4 of sub- mission process. Authors should in no more than 100 words emphasize the scientific novelty of the present- ed research. Do not repeat for this purpose the con- tent of your abstract. List of sug ge sted re vie wers List of suggested reviewers is a Word file submitted as a supplementary file in step 4 of submission pro- cess. Authors should propose the names, full afliation (department, institution, city and country) and e­mail addresses of five potential referees. Field of expertise and at least two references relevant to the scientif- ic field of the submitted manuscript must be provid- ed for each of the suggested reviewers. The referees should be knowledgeable about the subject but have no close connection with any of the authors. In addi- tion, referees should be from institutions other than (and countries other than) those of any of the authors. Authors declare no conflict of interest with suggested reviewers. Authors declare that suggested reviewers are experts in the field of submitted manuscript. How to Sub mit Users registered in the role of author can start sub- mission by choosing USER HOME link on the top of the page, then choosing the role of the Author and follow the relevant link for starting the submission process. Prior to submission we strongly recommend that you familiarize yourself with the ACSi style by browsing the journal, particularly if you have not submitted to the ACSi before or recently. S113Acta Chim. Slov. 2022, 69, (4), Supplement Društvene vesti in druge aktivnosti Cor res pon den ce All correspondence with the ACSi editor regarding the paper goes through this web site and emails. Emails are sent and recorded in the web site database. In the correspondence with the editorial ofce please provide ID number of your manuscript. All emails you receive from the system contain relevant links. Please do not answer the emails directly but use the embed­ ded links in the emails for carrying out relevant actions. Alternatively, you can carry out all the ac- tions and correspondence through the online system by logging in and selecting relevant options. Proofs Proofs will be dispatched via e-mail and corrections should be returned to the editor by e­mail as quick- ly as possible, normally within 48 hours of receipt. Typing errors should be corrected; other changes of contents will be treated as new submissions. Sub mis sion Pre pa ra tion Chec klist As part of the submission process, authors are required to check off their submission’s compliance with all of the following items, and submissions may be returned to authors that do not adhere to these guidelines. 1. The submission has not been previously published, nor is it under consideration for publication in any other journal (or an explanation has been provid- ed in Comments to the Editor). 2. 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. Name of author(s) denoted as the corresponding author(s), together with their e­mail address, full postal ad- dress and telephone/fax numbers are given. 7. The ab stract sta tes the ob jec ti ve and conc lu­ sions of the re search con ci sely in no mo re than 150 words. 8. Keywords (minimum three, maximum six) are provided. 9. Sta te ment of no velty (maximum 100 words) clearly explaining new findings reported in the manuscript should be prepared as a separate Word file. 10. The text adheres to the stylistic and bibliographic requirements outlined in the Aut hor gui de li nes. 11. Text in normal style is set to single column, 1.5 line spacing, and 12 pt. Times New Roman font is recommended. All tables, figures and illustrations have appropriate captions and are placed within the text at the appropriate points. 12. Mathematical and chemical equations are provided in separate lines and numbered (Arabic numbers) consecutively in parenthesis at the end of the line. All equation numbers are (if necessary) appropri- ately included in the text. Corresponding numbers are checked. 13. Tables, Figures, illustrations, are prepared in cor- rect format and resolution (see Aut hor gui de li­ nes). 14. The let te ring used in the fi gu res and graphs do not vary greatly in si ze. The re com men ded let te ring si ze is 8 point Arial. 15. Separate files for each figure and illustration are prepared. The names (numbers) of the separate files are the same as they appear in the text. All the figure files are packed for uploading in a single ZIP file. 16. Aut hors ha ve read spe cial no tes and ha ve ac cor- dingly pre pa red their ma nus cript (if ne ces sary). 17. Re fe ren ces in the text and in the Re fe ren ces are cor rectly ci ted. (see Aut hor gui de li nes). All ref- erences mentioned in the Reference list are cited in the text, and vice versa. 18. 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 S114 Acta Chim. Slov. 2022, 69, (4), 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 www.krka.biz We will ensure top results and good interpersonal relationships by following our mission and taking responsible steps. Happy 2023! 342202-2023 NY 2023 Ad 205x276 Acta Chimica SI EN.indd 2 25. 11. 2022 12:33:00 www.helios-group.eu Znanje, kreativnost zaposlenih in inovacije so ključnega pomena v okolju, kjer nastajajo pametni premazi skupine KANSAI HELIOS. Z rešitvami, ki zadostijo široki paleti potreb, kontinuiranim razvojem ter s kakovostnimi izdelki, Helios predstavlja evropski center za inovacije in poslovni razvoj skupine Kansai Paint. Razvoj in inovacije za globalno uspešnost 4 n Year 2022, Vol. 69, No. 4 ActaChimicaSlovenica ActaChimicaSlovenica ActaChimicaSlovenica ActaChimicaSlovenica SlovenicaActaChim A cta C him ica Slovenica 69/2022 Pages 733–947 Pages 733–947 n Year 2022, Vol. 69, No. 4 http://acta.chem-soc.si 4 69/2022 4 ISSN 1580-3155 The significance of nitrogen- and oxygen-heterocycles in many areas of life is well-known, however, the preparation of new derivatives remains important. The multicomponent synthesis of potentially biologically active heterocycles containing a phosphonate or a phosphine oxide moiety was performed (page 735).