{Synthesis and assessment of the cytotoxic effect of some of 1,4-dihydropyridine derivatives which contain azole moiety} J. Serb. Chem. Soc. 86 (11) 1013–1021 (2021) Original scientific paper JSCS–5479 Published 1 October 2021 1013 Synthesis and assessment of the cytotoxic effect of some of 1,4-dihydropyridine derivatives which contain azole moiety SAEED GHORBANNEJAD1, KARIM AKBARI DILMAGHANI1* and ABBAS NIKOO2 1Department of Chemistry, Faculty of Science, Urmia University, Urmia, Iran and 2Shahid Bakeri High Education Center, Urmia University, Urmia, Iran (Received 18 August 2020, revised 5 August, accepted 10 August 2021) Abstract: A number of 1,4-dihydropyridine derivatives (9a–d, 10a–d and 11a–d) were designed and synthesized by the reaction of 1,3,4-oxadiazole-5-thiones and 1,2,4-triazole-5-thiones to 2,6-dibromomethyl-3,5-diethoxycarbonyl-4-(3- -nitrophenyl)-1,4-dihydropyridine. The synthesized compounds were charac- terized using FT-IR, 1H-NMR, 13C-NMR spectral data, ESI-MS and elemental analysis. The cytotoxicity of the synthesized compounds was evaluated in human breast cancer (MCF-7) cells based on the results of MTT assay. The results indicated that compound diethyl 4-(3-nitrophenyl)-2,6-bis[((5-(3-nitro- phenyl)-1,3,4-oxadiazol-2-yl)thio)methyl]-1,4-dihydro pyridine-3,5-dicarbo- xylate (9b) with (IC50 = 23±2.32 µM) was the most potent derivative against MCF-7 cells. Based on the results, the use of oxadiazole moiety in the C2 and C6 positions of 1,4-dihydropyridine ring system enhanced the cytotoxic potential of these derivatives. Therefore, some of the oxadiazole-substituted 1,4-DHPs may facilitate further modifications which result in the discovery of potent cytotoxic agents. Keywords: dimethylformamide; 3-nitrobenzaldehyde; 1,3,4-oxadiazole; 1,2,4-tri- azole. INTRODUCTION 1,4-Dihydropyridines (DHPs) have assumed considerable importance in the field of organic and medicinal chemistry due to their interesting pharmacological activities. The results of different studies have highlighted the fact that the DHPs are highly effective calcium antagonists and are used for treating various cardio- vascular system (CVS) activities.1–3 In addition to the CVS activities of DHPs, they possess a variety of biological activities including, cytotoxic activities,4–6 anti-proliferative activities,7 multidrug resistance activities8,9 and anti-tumour activities.10–13 However, synthesizing DHPs derivatives is an active and ongoing * Corresponding author. E-mail: k.adilmaghani@urmia.ac.ir https://doi.org/10.2298/JSC200818064G ________________________________________________________________________________________________________________________ (CC) 2021 SCS. Available on line at www.shd.org.rs/JSCS/ 1014 GHORBANNEJAD, DILMAGHANI and NIKOO research area. Chemotherapy is still one of the most effective methods for treat- ing cancer. The potential uses of the DHPs scaffolds in the chemotherapy are well-documented and stem from their suitability for reversing the drug resistance in the treatment of cancer.14,15 Calcium channel blockers like verapamil16 and nicardipine,17 among others have been reported to successfully overcome drug resistance. Notwithstanding, the introduction of calcium channel blockers to clinical use might pose a therapeutic problem which results from their strong vasodilator function.18 Consequently, a substance which has a strong capability to overcome anticancer drug resistance and does not lead to calcium antagonistic activity can be of great value in chemotherapy. Research has shown that, generally, DHPs display more cytotoxicity towards cancer cells in comparison with the non-cancer cells.19 The discovery of new DHP derivatives can encourage the development of novel and effective therapies for diverse pathologies, including cancer. The efficiency of oxadiazole and triazole derivatives has been assessed and proved for a wide range of pharmacological uses. The connection of 1,3,4-oxadi- azoles or 1,2,4-triazoles to the 1,4-DHPs core has produced a combination scaf- fold. The DHPs can be selectively functionalized in several positions. The syn- thesis and anticancer activities of bis(1,3,4-oxadiazole-2-thiol) and bis(4-amino- 1,2,4-triazole-3-thiole) derivatives of DHPs in the C3 and C5 have been rep- orted.20 Moreover, research has shown the synthesis and biological activities of 1,3,4-oxadiazole derivatives which are linked to N1 of DHP ring system.21 In spite of the highly developed chemistry of the DHPs, there is not adequate infor- mation about the synthesis of 1,4-DHPs bearing-substituents other than hydrogen atoms or alkyl groups in the C2 and C6. In our previous study, we reported the synthesis and antimicrobial assessment of 1,4-dihydropyridines with azole derivatives in the C2 and C6 positions of DHP ring.22 Based on the above-men- tioned gap in the related literature, the present study focuses on synthesizing the novel 1,4-dihydropyridine derivative, which are linked to triazole and oxadiazole moieties, and evaluating their cytotoxic activities. EXPERIMENTAL The chemicals of Sigma–Aldrich and Merck were used to produce the chemicals of this study. The solvents were purified based on the standard procedures before their use in the study. The thin-layer chromatography (TLC) analysis was performed in the case of the recoated silica gel (E-Merck kieselgel 60 F254 Aluminium sheets) plates. N-bromosuccinimide (NBS) and tetramethylsilane (TMS) were purchased from Merck. The melting points were determined on open capillaries using a digital melting point apparatus. The FT-IR spectra were recorded as KBr pellets on a thermo Nicolet Nexus 670 FT-IR. The 1H- and 13C-NMR spectra were recorded on the Bruker Avance AQS 300 MHz spectrometer at 300 and 100 MHz, respectively. The chemical shifts were measured in dimethyl sulfoxide (DMSO-d6) as solvent relative to TMS as the internal standard. These abbreviations were used to describe the multiplicities of signals in NMR spectra (s = singlet, d = doublet, t = triplet, q = quartet, dd = ________________________________________________________________________________________________________________________ (CC) 2021 SCS. Available on line at www.shd.org.rs/JSCS/ 1,4-DIHYDROPYRIDINES WITH CYTOTOXIC EFFECT 1015 doublet of doublets and br = broad signal). The mass spectra were recorded on a JEOL-JMS 600 (FAB MS) instrument and the ESI-MS spectra were recorded on an Agilent Technologies 5975C VL MSD mass spectrometer which operated at an ionization potential of 70 eV. The CHNS analysis was performed using CHNS-932 Leco analyser. The 3a–d23,24, 4a–d25, 5a–d26 and 6a–d27 compounds were produced according to the reviewed literature. Analytical and spectral data of the synthesized compound are given in Supplementary material to this paper. Procedure for preparing diethyl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-di- carboxylate (7) A mixture of ethylacetoacetate (0.02 mol, 2.60 g), ammonium acetate (0.015 mol, 1.16 g) and 3-nitrobenzaldehyde (0.01 mol, 1.51 g) in 50 % ethanol (50 mL) was mixed well under reflux for 12 h. The contests were cooled after the reaction (which was monitored by TLC via n-hexane/EtOAc (4:1) as eluent. The precipitate was filtered, washed with water and crystal- lized from ethanol.28,29 Procedure for synthesizing diethyl 2,6-bis(bromomethyl)-4-(3-itrophenyl)-1,4-dihydropyri- dine-3,5-dicarboxylate (8) NBS (0.02 mol, 3.56 g) was added to a solution of the diethyl 2,6-dimethyl-4-(3-nitro- phenyl)-1,4-dihydropyridine-3,5-dicarboxylate (7, 0.01 mol, 3.74 g) in methanol (100 mL) portion-wise at ambient temperature. The reaction mixture was stirred at room temperature for 24 h. The pale yellow precipitate was filtered and washed with water. The precipitate was crystallized from ethanol.29 General procedure for synthesizing the 9a–d, 10a–d and 11a–d compounds A mixture of 3a–d, 4a–d or 6a–d (0.02 mol), NaOH (0.02 mol, 0.8 g) and DMF/H2O (50/50, 50 mL) was stirred at room temperature for 1 h. Moreover, diethyl 2,6-bis(bromo- methyl)-4-(3-nitrophenyl)-1,4-dihydropyridine-3, and 5-dicarboxylate (8, 0.01 mol, 5.32 g) were added to it and it was stirred at room temperature for 8–12 h. The reaction mixture was poured into water (100 mL) and the residue was extracted with CH2Cl2. The organic layer was washed with water, dried over Na2SO4 and evaporated and recrystallized from ethanol. Biological assessment Reagent and chemical. (RPMI-1640) and fetal bovine serum (FBS) were purchased from (Gibco, USA). 3-(4,5-Dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Sigma. Penicillin/streptomycin was purchased from Invitrogen (San Diego, CA, USA). Dimethyl sulphoxide (DMSO) were obtained from Merck. Cell culture. The human breast cancer cells (MCF-7) were purchased from National Cell Bank of Iran (Pasteur, Tehran, Iran). These cells were cultured in Roswell Park Memorial Ins- titute 1640 (RPMI-1640) (Gibco, USA) medium which was enriched with 10 % fetal bovine serum (FBS, Gibco, USA), 100 unit/mL penicillin and 100 mg/mL streptomycin and was maintained under 37 °C and 5 % CO2 conditions. The cells, which reached the 70 % conflu- ence, were sub cultured and were used for conducting the experiments. Cell viability assay (MTT). In order to determine the cytotoxic effect of various com- pounds on the viability of MCF-7 cells, the MTT reduction assay was performed as described previously.30-32 First MCF-7 cells were plated in 96-well microplates at a density of 1×104 cells per well and were maintained overnight at 37 °C to allow them to attach to the bottom of the wells. After cell attachment, the medium was removed and cells were treated with various compounds (9a–d, 10a–d and 11a–d) at the concentrations which ranged from 10 to 100 µM. ________________________________________________________________________________________________________________________ (CC) 2021 SCS. Available on line at www.shd.org.rs/JSCS/ 1016 GHORBANNEJAD, DILMAGHANI and NIKOO All of the compounds were dissolved in DMSO and were diluted in medium in a way that the maximum concentration of DMSO in the wells did not exceed 0.5 %. Cells were further inc- ubated for 48 h. Thirdly, the fresh medium, which contained 500 µg/mL MTT powder, was added to each well and plates were incubated for another 4 h time at 37 °C. Then MTT and media mixtures were removed and formazan crystals which formed by the mitochondrial dehydrogenase activity of vital cell were solubilized in 200 µl DMSO and was put on an orbi- tal shaker for 20 min. Finally, the absorbance of each plate was measured at 570 nm with the background correction at 620 nm using an Elisa plate reader (Statefax, USA). Effects of the drug cell viability were calculated using cells treated with DMSO as control. Cell survival was calculated using the formula: Survival, % = [(absorbance of treated cells – absorbance of cul- ture medium)/(absorbance of untreated cells – absorbance of culture medium)]×100. The experiment was done in triplicate and the inhibitory concentration (IC) values were calculated from a dose-response curve. Graph Pad Prism software 6.01 was used to calculate IC50 values. IC50 is the concentration in µM required for 50 % inhibition of cell growth as compared to that of the untreated control. IC50 values were determined from the linear portion of the curve by calculating the concentration of agent that reduced absorbance in treated cells, compared to control cells, by 50 %. Evaluation is based on mean values from three independent experi- ments, each comprising at least six micro cultures per concentration level. IC50 Values repre- sent the mean of triplicate determination (n = 3) ± SD with (95 %) confidence interval. The cytotoxicity of the synthesized derivatives was not compared to standard drugs.33 RESULTS AND DISCUSSION The 3a–d, 4a–d, 5a–d and 6a–d derivatives were prepared according to the method which was described in the relevant literature.23–27 It is well-known that the thiol–thione tautomeric equilibrium exists in 3a–d, 4a–d and 6a–d com- pounds. On the basis of 1H-NMR and FT-IR experimental findings, it is argued that the thione tautomer is more stable than thiol in the solution. 1H-NMR spectra of these compounds exhibited the NH signal as a broad peak in the δ 12–14 ppm range which supports the proposed thione structure. The appearance of a C=S absorption peak in the 1248–1278 cm–1 region indicated that the oxadiazoles and triazoles were in their thione form.34 Diethyl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxy- late (7) was synthesized using the classical Hantzsch three-component reaction method. This method includes the cyclocondensation of 3-nitrobenzaldehyde with two equivalents of ethyl acetoacetate in the presence of a nitrogen donor such as ammonia or ammonium acetate (based on the procedure reported in the literature).33 We needed a quick entry into 1,4-dihydropyridine-3,5-dicarboxylic acid diesters in which the 2,6-methyl groups were altered by a range of different groups. Allylic bromination is the replacement of a hydrogen on a carbon adjacent to a double bond. Allylic bromination in dihydropyridines was performed by NBS. The synthesis of 2,6-dibromomethyl-3,5-diethoxycarbonyl-1,4-dihydropyri- dine (8) was achieved as a result of the bromination of the corresponding 2,6-di- methyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (7) by NBS in methanol according to the procedure which was described in the literature.29 The ________________________________________________________________________________________________________________________ (CC) 2021 SCS. Available on line at www.shd.org.rs/JSCS/ 1,4-DIHYDROPYRIDINES WITH CYTOTOXIC EFFECT 1017 bromine atoms in compound 8 can be replaced with the other substituents (Scheme 1). Scheme 1. Synthesis of compound 8. The replacement of the bromines of compound 8 with 1,2,4-triazole-5-thi- ones 3a–d, 1,3,4-oxadiazole-5-thiones 4a–d and 4-amino-3-mercapto-1,2,4-triaz- oles 6a–d was carried out in the presence of sodium hydroxide as a base in DMF in order to afford the corresponding coupled 1,4- DHPs (9a–d, 10a–d and 11a–d) (Scheme 2). Scheme 2. The synthesis of compounds 9a–d, 10a–d and 11a–d. ________________________________________________________________________________________________________________________ (CC) 2021 SCS. Available on line at www.shd.org.rs/JSCS/ 1018 GHORBANNEJAD, DILMAGHANI and NIKOO The structures of 9a–d, 10a–d and 11a–d compounds were identified using spectroscopic methods. In the IR spectra the disappearance of the C=S absorption peak in the 1248–1278 cm–1 region and the absence of NH peak at δ 12–14 ppm supported the connection of oxadiazole and triazoles to 1,4-DHP ring. The CH2X protons in the C2 and C6 positions of symmetrically substituted 1,4-dihydropyri- dine ring became diastereotopic and provided an AB system in the corresponding 1H-NMR spectra. The extent of the observed anisotropy of the methylene protons must have been influenced by the spatial conformation between the ester groups and the formation of a CH…O=C intramolecular hydrogen bonding.35 The increase in concentration resulted in a decrease in the viability of cells for all of the compounds and indicated that the cytotoxicity of all of the com- pounds depended on the concentration. Some of the compounds including 10a and c did not display a high level of cytotoxicity towards MCF-7 cells. None- theless, a number of the other compounds including 9b and d displayed a high level of cytotoxicity towards these cells at a concentration which was confirmed by their IC50 values. According to the in vitro MTT assay, the IC50 represents the concentration of the newly synthesized compounds that is required for 50 % inhi- bition of the human breast cancer cell (MCF-7) viability. The IC50 value for each compound was calculated and summarized in Table I. As shown in Table I, based on the IC50 value, the most cytotoxic compound was 9b. Therefore, it can be suggested that, this compound is a potent cytotoxic agent. TABLE I. Cytotoxic activity of the synthesized 1,4-DHP derivatives assessed by the MTT assay Compound R Molecular weight IC50 / µM MCF-7 IC50 / μg mL-1 MCF-7 9a C6H5– 726 45±2.72 32.67 9b 3-NO2–C6H4– 816 23±2.32 18.76 9c 2-HO–C6H4– 758 40±4.65 30.32 9d 2-Cl–C6H4– 794 33±1.55 26.20 10a C6H5– 876 63±3.10 55.12 10b 3-NO2–C6H4– 966 57±4.27 55.06 10c 2-OH–C6H4.– 908 >100 10d 2-Cl–C6H4– 944 49±3.10 46.25 11a C6H5– 754 54±3.10 40.71 11b 3-NO2–C6H4– 844 44±1.8 36.16 11c 2-HO–C6H4– 786 51±4.6 39.16 11d 2-Cl–C6H4– 822 47±4.65 38.63 CONCLUSION In the present study, diethyl-2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyri- dine-3 and 5-dicarboxylate derivatives were coupled with 1,3,4-oxadiazole-5-thi- ones and 1,2,4-triazole-5-thiones in the C2, C6 positions of 1,4-dihydropyridine ring system in order to produce compounds with greater cytotoxicity. The syn- thesized compounds were characterized using FT-IR, 1H-NMR, 13C-NMR spec- ________________________________________________________________________________________________________________________ (CC) 2021 SCS. Available on line at www.shd.org.rs/JSCS/ 1,4-DIHYDROPYRIDINES WITH CYTOTOXIC EFFECT 1019 tral data, ESI mass and elemental analysis. The cytotoxic effects of the new compounds on human breast cancer (MCF-7) cells were investigated using MTT assay. The results of the MTT assay showed that a number of compounds inc- luding 9b and d displayed good cytotoxicity at a certain concentration. This find- ing was confirmed by their IC50 values. The highest potency was observed in the case of MCF-7 cells (9b: IC50 = 23±2.32 μM). Based on the results, the 10c com- pound did not have a cytotoxic effect on the tested cancer cell line due to its bulky scaffold and the steric hindrance in its site of action. These preliminary en- couraging results of the biological screening of the tested compounds may offer an excellent framework for the discovery of potent cytotoxic agents in this field. SUPPLEMENTARY MATERIAL Additional data and information are available electronically at the pages of journal website: https://www.shd-pub.org.rs/index.php/JSCS/article/view/9811, or from the corres- ponding author on request. Acknowledgements. The authors are grateful to Urmia University which provided them with a fellowship for the present study. Our grateful thanks go to Dr. Vahid Shafiei-Irannejad and Morteza Molaparast (from Cellular and Molecular Medicine Institute, Urmia University of Medical Sciences) who investigated the cytotoxicity of the compounds and to Prof. Dr. Joachim Thiem (from Hamburg University) and Prof. Dr. Abdolali Alizadeh (from Tarbiat Modares University) for the ESI-MS. И З В О Д СИНТЕЗА И ОДРЕЂИВАЊЕ ЦИТОТОКСИЧНОГ ЕФЕКТА ДЕРИВАТА 1,4-ДИХИДРОПИРИДИНА КОЈИ САДРЖЕ АЗОЛСКУ СТРУКТУРУ SAEED GHORBANNEJAD1, KARIM AKBARI DILMAGHANI1 и ABBAS NIKOO2 1Department of Chemistry, Faculty of Science, Urmia University, Urmia, Iran и 2Shahid Bakeri High Education Center, Urmia University, Urmia, Iran Синтетисана је серија деривата 1,4-дихидропиридина (9a–d, 10a–d и 11a–d) реак- цијом 1,3,4-оксадиазол-5-тиона или 1,2,4-триазол-5-тиона са 2,6-дибромметил-3,5-ди- етоксикарбонил-4-(3-нитрофенил)-1,4-дихидропиридином. Синтетисана једињења су окарактерисана помоћу FT-IR, 1 H-NMR, 13 C-NMR и ESI-MS спектара и елементалном анализом. Испитана је цитотоксичност добијених деривата према ћелијама хуманог канцера дојке (MCF-7) МТТ есејом. Резултати указују да једињење диетил 4-(3-ни- трофенил)-2,6-бис[((5-(3-нитрофенил)-1,3,4-оксадиазол-2-ил)тио)метил]-1,4-дихидро- пиридин-3,5-дикарбоксилат (9b) са (IC50 = 23 ± 2,32 μM) има највећу активност према MCF-7 ћелијама. На основу добијених резултата оксадиазолски део структуре на C2 и C6 положајима 1,4-дихидропиридинског система повећава цитотоксични потенцијал ових деривата. Из тога произилази да би неки 1,4-DHP деривати који садрже оксадиазолске супституенте омогућили припрему нових активнијих једињења. (Примљено 18. августа 2020, ревидирано 5. августа, прихваћено 10. августа 2021) REFERENCES 1. D. J. Triggle, Biochem. Pharmacol. 74 (2007) 1 (http://dx.doi.org/10.1016/j.bcp.2007.01.016) ________________________________________________________________________________________________________________________ (CC) 2021 SCS. Available on line at www.shd.org.rs/JSCS/ 1020 GHORBANNEJAD, DILMAGHANI and NIKOO 2. C. Bladen, M. G. Gündüz, R. Şimşek, C. Şafak, G. W. Zamponi, Pflugers Arch – Eur. J. Physiol. 466 (2014) 1355 (http://dx.doi.org /10.1007/s00424-013-1376-z) 3. R. Bansal, G. Narang, C. Calle, R. Carron, K. Pemberton, A. L. Harvey, Drug Dev. Res. 74 (2013) 50 (http://dx.doi.org/10.1002/ddr.21056) 4. N. Razzaghi-Asl, R. Miri, O. Firuzi, Iran. J. Pharm. Sci. 15 (2016) 413 (http://dx.doi.org/10.22037/IJPR.2016.1870) 5. A. Ahamed, I. A. Arif, M. Mateen, R. S. Kumar, A. Idhayadhull, Saudi J. Biol. Sci. 25 (2018) 1227 (http://dx.doi.org/10.1016/j.sjbs.2018.03.001) 6. J. Marín-Prida, G. L. P. Andreu, C. P. Rossignoli, M. G. Durruthy, E. O. Rodríguez, Y. V. Reyes, R. F. Acosta, S. A. Uyemura, L. C. Alberici, Toxicol. In Vitro 42 (2017) 21 (http://dx.doi.org/10.1016/j.tiv.2017.03.011) 7. D. Viradiya, S. Mirza, F. Shaikh, R. Kakadiya, A. Rathod, N. Jain, R. Rawal, A. Shah, Anticancer Agents Med. Chem. 17 (2017) 1003 (http://dx.doi.org/10.2174/1871520616666161206143251) 8. F. Shekari, H. Sadeghpour, K. Javidnia, L. Saso, F. Nazari, O. Firuzi, R. Miri, Eur. J. Pharmacol. 746 (2015) 233 (http://dx.doi.org/10.1016/j.ejphar.2014.10.058) 9. S. Tasaka, H. Ohmori, N. Gomi, M. Iino, T. Machida, A. Kiue, S. Naito, M. Kuwano, Bioorganic Med. Chem. Lett. 11 (2001) 275 (http://dx.doi.org/10.1016/S0960- 894X(00)00651-X) 10. H. Engi, H. Sakagami, M. Kawase, A. Parecha, D. Manvar, H. Kothari, P. Adlakha, A. Shah, N. Motohashi, I. Ocsovszki, J. Molnar, In Vivo 20 (2006) 637 (https://www.researchgate.net/publication/6705056) 11. M. F. Mohamed, A. F. Darweesh, A. H. M. Elwahy, I. A. Abdelhamid, RSC Adv. 6 (2016) 40900 (http://dx.doi.org/10.1039/c6ra04974e) 12. O. Firuzi, K. Javidnia, E. Mansourabadi, L. Saso, A.R. Mehdipour, R. Miri, Arch. Pharm. Sci. Res. 36 (2013) 1392 (http://dx.doi.org/10.1007/s12272-013-0149-8) 13. M. G. Pavani, M. Nunez, P. Brigidi, B. Vitali, R. Gambari, Bioorg. Med. Chem. 10 (2002) 449 (http://dx.doi.org/10.1016/S0968-0896(01)00294-2) 14. R. Miri, A. Mehdipour, Bioorg. Med. Chem. 16 (2008) 8329 (http://dx.doi.org/10.1016/j.bmc.2008.07.025) 15. A. Zarrin, A. R. Mehdipour, R. Miri, Chem. Biol. Drug. Des. 76 (2010) 369 (http://dx.doi.org/10.1111/j.1747-0285.2010.01025.x) 16. J. R. Warr, F. Brewer, M. Anderson, J. Fergusson, Cell Biol. Int. Rep. 10 (1986) 389 (http://dx.doi.org/10.1016/0309-1651(86)90011-1) 17. T. Tsuruo, H. Kawabata, N. Nagumo, H. lida, Y. Kitatani, S. Tsukagoshi, Y. Sakurai, Cancer Chemother. Pharmacol. 15 (1985) 16 (http://dx.doi.org/10.1007/BF00257287) 18. T. Godfraind, J. Cardiovasc. Pharmacol. Ther. 19 (2014) 501 (http://dx.doi.org/10.1177/1074248414530508) 19. B. Laupeze, L. Amiot, N. Bertho, J. M. Grosset, G. Lehne, R. Fauchet, O. Fardel, Hum. Immunol. 62 (2001) 1073 (http://dx.doi.org/10.1016/S0198-8859(01)00307-X) 20. R. Surendrakumar, A. Manilal, A. J. Abdul Nasser, B. Merdekios, X. Chen, A. Idhayadhulla, J. Pharmacol. Toxicol. 9 (2014) 119 (https://dx.doi.org/10.3923/jpt.2014.119.128 ) 21. A. B. Archana, D. R. Dinesh, S. G. Paraag, Y. S. Prabhakar, Int. J. Pharm. Chem. 4 (2014) 62 (http://dx.doi.org/10.7439/ijpc.v4i2.75) 22. M. Ziaie, K. Akbari Dilmaghani, A. Tukmechi, Acta Chim. Slov. 64 (2017) 895 (http://dx.doi.org/10.17344/acsi.2017.3506) ________________________________________________________________________________________________________________________ (CC) 2021 SCS. Available on line at www.shd.org.rs/JSCS/ 1,4-DIHYDROPYRIDINES WITH CYTOTOXIC EFFECT 1021 23. R. M. Shaker, ARKIVOC IX (2006) 59 (https://dx.doi.org/10.3998/ark.5550190.0007.904) 24. A. A. Aly, A. A. Hassan, M. M. Makhlouf, S. Brase, Molecules 25 (2020) 3036 (http://dx.doi.org/10.3390/molecules25133036) 25. A. A. Othman, M. Kihel, S. Amara, Arab. J. Chem. 12 (2019) 1660 (http://dx.doi.org/10.1016/j.arabjc.2014.09.003) 26. K. M. Dawood, A. M. Farag, H. A. Abdel-Aziz, Heteroat. Chem. 16 (2005) 621 (http://dx.doi.org/10.1002/hc.20162) 27. J. Shneine, Y. H. Alaraji, IJSR 5 (2016) 1411 (https://www.ijsr.net/get_abstract.php?paper_id=NOV161902) 28. S. D. Bajaj, O. A. Mahodaya, P. V. Tekade, V. B. Patil, S. D. Kukade, Russ. J. Gen. Chem. 87 (2017) 546 (http://dx.doi.org/10.1134/S1070363217030264) 29. V. Palermo, A. G. Sathicq, T. Constantieux, J. Rodriguez, P. G. Vazquez, G. P. Romanelli, Catal. Lett. 146 (2016) 1634 (http://dx.doi.org/10.1007/s10562-016-1784-8) 30. D. Viradiya, S. Mirza, F. Shaikh, R. Kakadiya, A. Rathod, N. Jain, R. Rawal, A. Shah, Anti-Cancer Agents Med. Chem. 17 (2017) 1003 (http://dx.doi.org/10.2174/1871520616666161206143251) 31. N. Razzaghi-Asl, R. Miri, O. Firuzi, Iran. J. Pharm. Res. 15 (2016) 413 (http://dx.doi.org/10.22037/ijpr.2016.1870) 32. R. Surendra kumar, A. Idhayadhulla, A. Jamal Abdul Nasser, K. Murali, Indian J. Chem., B 50 (2011) 1140 (http://nopr.niscair.res.in/handle/123456789/12520) 33. R. Sarkhosh Inanlou, M. Molaparast, A. Mohammadzadeh, V. Shafiei Irannejad, Chem. Biol. Drug. Des. 95 (2019) 215 (http://dx.doi.org/10.1111/cbdd.13621) 34. K. H. Chikhalia, D. B. Vashi, M. J. Patel, J. Enzyme Inhib. Med. Chem. 24 (2009) 617 (http://dx.doi.org/10.1080/14756360802318936) 35. M. Petrova, R. Muhamadejev, B. Vigante, B. Cekavicus, A. Plotniece, G. Duburs, E. Liepinsh, Molecules 16 (2011) 8041 (http://dx.doi.org/10.3390/molecules16098041). ________________________________________________________________________________________________________________________ (CC) 2021 SCS. Available on line at www.shd.org.rs/JSCS/ << /ASCII85EncodePages false /AllowTransparency false /AutoPositionEPSFiles true /AutoRotatePages /None /Binding /Left /CalGrayProfile (Dot Gain 20%) /CalRGBProfile (sRGB IEC61966-2.1) /CalCMYKProfile (U.S. Web Coated \050SWOP\051 v2) /sRGBProfile (sRGB IEC61966-2.1) /CannotEmbedFontPolicy /Error /CompatibilityLevel 1.4 /CompressObjects /Tags /CompressPages true /ConvertImagesToIndexed true /PassThroughJPEGImages true /CreateJobTicket false /DefaultRenderingIntent /Default /DetectBlends true /DetectCurves 0.0000 /ColorConversionStrategy /CMYK /DoThumbnails false /EmbedAllFonts true /EmbedOpenType false /ParseICCProfilesInComments true /EmbedJobOptions true /DSCReportingLevel 0 /EmitDSCWarnings false /EndPage -1 /ImageMemory 1048576 /LockDistillerParams false /MaxSubsetPct 100 /Optimize true /OPM 1 /ParseDSCComments true /ParseDSCCommentsForDocInfo true /PreserveCopyPage true /PreserveDICMYKValues true /PreserveEPSInfo true /PreserveFlatness true /PreserveHalftoneInfo false /PreserveOPIComments true /PreserveOverprintSettings true /StartPage 1 /SubsetFonts true /TransferFunctionInfo /Apply /UCRandBGInfo /Preserve /UsePrologue false /ColorSettingsFile () /AlwaysEmbed [ true ] /NeverEmbed [ true ] /AntiAliasColorImages false /CropColorImages true /ColorImageMinResolution 300 /ColorImageMinResolutionPolicy /OK /DownsampleColorImages true /ColorImageDownsampleType /Bicubic /ColorImageResolution 300 /ColorImageDepth -1 /ColorImageMinDownsampleDepth 1 /ColorImageDownsampleThreshold 1.50000 /EncodeColorImages true /ColorImageFilter /DCTEncode /AutoFilterColorImages true /ColorImageAutoFilterStrategy /JPEG /ColorACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /ColorImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000ColorACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000ColorImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasGrayImages false /CropGrayImages true /GrayImageMinResolution 300 /GrayImageMinResolutionPolicy /OK /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /GrayImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000GrayACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000GrayImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasMonoImages false /CropMonoImages true /MonoImageMinResolution 1200 /MonoImageMinResolutionPolicy /OK /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 1200 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict << /K -1 >> /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile () /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False /CreateJDFFile false /Description << /ARA /BGR /CHS /CHT /CZE /DAN /DEU /ESP /ETI /FRA /GRE /HEB /HRV (Za stvaranje Adobe PDF dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. Stvoreni PDF dokumenti mogu se otvoriti Acrobat i Adobe Reader 5.0 i kasnijim verzijama.) /HUN /ITA /JPN /KOR /LTH /LVI /NLD (Gebruik deze instellingen om Adobe PDF-documenten te maken die zijn geoptimaliseerd voor prepress-afdrukken van hoge kwaliteit. De gemaakte PDF-documenten kunnen worden geopend met Acrobat en Adobe Reader 5.0 en hoger.) /NOR /POL /PTB /RUM /RUS /SKY /SLV /SUO /SVE /TUR /UKR /ENU (Use these settings to create Adobe PDF documents best suited for high-quality prepress printing. Created PDF documents can be opened with Acrobat and Adobe Reader 5.0 and later.) >> /Namespace [ (Adobe) (Common) (1.0) ] /OtherNamespaces [ << /AsReaderSpreads false /CropImagesToFrames true /ErrorControl /WarnAndContinue /FlattenerIgnoreSpreadOverrides false /IncludeGuidesGrids false /IncludeNonPrinting false /IncludeSlug false /Namespace [ (Adobe) (InDesign) (4.0) ] /OmitPlacedBitmaps false /OmitPlacedEPS false /OmitPlacedPDF false /SimulateOverprint /Legacy >> << /AddBleedMarks false /AddColorBars false /AddCropMarks false /AddPageInfo false /AddRegMarks false /ConvertColors /ConvertToCMYK /DestinationProfileName () /DestinationProfileSelector /DocumentCMYK /Downsample16BitImages true /FlattenerPreset << /PresetSelector /MediumResolution >> /FormElements false /GenerateStructure false /IncludeBookmarks false /IncludeHyperlinks false /IncludeInteractive false /IncludeLayers false /IncludeProfiles false /MultimediaHandling /UseObjectSettings /Namespace [ (Adobe) (CreativeSuite) (2.0) ] /PDFXOutputIntentProfileSelector /DocumentCMYK /PreserveEditing true /UntaggedCMYKHandling /LeaveUntagged /UntaggedRGBHandling /UseDocumentProfile /UseDocumentBleed false >> ] >> setdistillerparams << /HWResolution [2400 2400] /PageSize [612.000 792.000] >> setpagedevice