Bangladesh Journal of Pharmacology Research Article Studies of Studies of Studies of in vitro in vitro in vitro antiantianti---prostate prostate prostate cancer potential of newer 1,2,4cancer potential of newer 1,2,4cancer potential of newer 1,2,4--- triazolotriazolotriazolo---1,3,41,3,41,3,4---thiadiazines with thiadiazines with thiadiazines with different heteroaromaticsdifferent heteroaromaticsdifferent heteroaromatics BJP Introduction Cancer therapeutics works upon inducing apoptosis and newer strategies to enhance apoptosis in cancerous cells upon targeting apoptosis regulators can be the vital factor in the further growth of anti-cancer regimen. Also, such newly invented scaffolds may combat drug resistant phenomenon towards the formally used chemotherapeutics (Earnshaw et al, 1999). In fact, now- a-days, in clinics there is no selective cytotoxic agents available and current arsenal like hormone-therapy, radical prostatectomy and radiotherapy are successful in destroying androgen-dependent, localized prostate cancer growth, whereas, therapy of hormone-refractory prostate cancer remains restricted by unavoidable development of resistance to the first-line treatment with docetaxel drug. Consequently, novel medication is needed to create on immediate foundation to battle against extensive of this dangerous illness (Feldman and Feldman, 2001). Heterocyclic compounds have long been known to exert desirable potency against several different diseases in terms of effective medications. Quinoline (Solomon and Lee, 2011), coumarin (Kostova, 2005) and benz-imidazole (EL Rashedy, 2013) moieties as heterocyclic pharmacophore play a vital role in cancer drug discovery process. Moreover, the 1,2,4-triazole entity and their derivatives emerge rapidly with advance of modern heterocyclic chemistry, promising a variety of medical applications (Clemons et al, 2004; Wakale et al 2013). Furthermore, 1,3,4-thiadiazine ring system also contributed to the significant biological potential, in particular anti-cancer efficacies (Abdel- Rahman, 2006). Hence, in this study we have attempted the construction of new 1,3,4-triazolo-1,3,4-thiadiazines and screened their potential against prostate cancer cell lines. Materials and Methods TLC was performed with Merck precoated TLC plates, and the compounds were made visible using a A Journal of the Bangladesh Pharmacological Society (BDPS) Bangladesh J Pharmacol 2015; 10: 308-315 Journal homepage: www.banglajol.info Abstracted/indexed in Academic Search Complete, Agroforestry Abstracts, Asia Journals Online, Bangladesh Journals Online, Biological Abstracts, BIOSIS Previews, CAB Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Google Scholar, HINARI (WHO), International Pharmaceutical Abstracts, Open J-gate, Science Citation Index Expanded, SCOPUS and Social Sciences Citation Index ISSN: 1991-0088 Abstract This study was aimed to evaluate anti-cancer potential of newer synthesize 1,2,4-triazolo[3,4-b]-1,3,4-thiadiazines and its derivatives. All newly furnished scaffolds were subjected to screening for their in vitro anti-cancer potential against DU-145 and PC-3 prostate cancer cell lines using SRB and MMT bioassays. The structures of final compounds were confirmed with the aid of FT-IR, 1H NMR, 13C NMR spectroscopy and CHN analysis. Bioassay studies suggested that all thiadiazines were promising cytotoxic agents with % cytotoxicity ranging from 44.39-71.24%, whereas potent GI50 level in the range 11.96-32.51 µg/mL and results were comparable to the potencies of control drugs adriamycin and doxorubicin. Variation of heterocyclic pharmacophores along with the C-5 position of 1,2,4-triazole in terms of quinoline, quinazoline, coumarin and pyridine lead to the different SAR predictions in which quinoline and benzimidazole moieties found most promising. Article Info Received: 28 February 2015 Accepted: 18 March 2015 Available Online: 9 April 2015 DOI: 10.3329/bjp.v10i2.22424 Cite this article: Fan MC, Han GY, Zhang XJ, Xi HF. Studies of in vitro anti-prostate cancer potential of newer 1,2,4-triazolo-1,3,4- thiadiazines with different heteroaro- matics. Bangladesh J Pharmacol. 2015; 10: 308-15. This work is licensed under a Creative Commons Attribution 3.0 License. You are free to copy, distribute and perform the work. You must attribute the work in the manner specified by the author or licensor. Studies of in vitro anti-prostate cancer potential of newer 1,2,4- triazolo-1,3,4-thiadiazines with different heteroaromatics Mao-Chuan Fan1, Guang-Ye Han1, Xin-Jun Zhang1 and Hui-Fang Xi2 1Department of Urology, The First Affiliated Hospital of Xinxiang Medical University, Henan 453 100, China; 2Department of Pediatric Surgery, The First Affiliated Hospital of Xinxiang Medical University, Henan 453 100, China. fluorescent inspection lamp and iodine vapor. Gravity chromatography was done with Merck silica gel 60 (70- 230 mesh). The melting points were recorded in open capillary tubes and are uncorrected. FT-IR spectra were recorded on FTIR Shimadzu (potassium bromide pellets) spectrometer (4000-400 cm-1). 1H and 13C NMR spectra were determined as DMSO solution at 400 and 100 MHz using TMS as an internal standard. Elemental analyses were performed on a Perkin Elmer EAL 240 elemental analyzer. All new compounds yielded spectral data consistent with the proposed structure and microanalysis within ± 0.4% of the theoretical values. All hydrazide derivatives were synthesized according to the procedure reported in the literature for 2-(1H- benzo[d]imidazol-1-yl) acetohydrazide (1a) (Ansari et al., 2009; Patel et al., 2012), 2-oxo-2H-chromene-3-carbo- hydrazide (1b) (Bhat et al., 2008., Patel et al., 2013), quinoline-6-carbohydrazide (1c) (Saitoh et al 2009; Patel et al., 2012), 2-(6-bromoquinolin-4-yloxy) aceto hydra- zide (1d), 2-(1-methyl-2-oxo-1,2-dihydroquinolin-4- yloxy)acetohydrazide (1e), 2-(quinazolin-4-yloxy) aceto- hydrazide (1f) (Patel et al., 2012) and isoniazid or isonicotinohydrazide (1g) obtained commercially. Moreover, the formation of 3-(2-bromoacetyl)-2H- chromen-2-one via Knoevenagel condensation was also performed. All analytical data were in accordance with the literature data (Naik et al., 2013). Synthetic procedure for synthesis of potassium aryldithio-carbazates (2a-2g) 0.2 Mol of derivatives 1a-1j was treated with a solution of 0.30 mol of KOH in 50 mL methanol at 0-5°C under N2 atmosphere with stirring. To this mixture, 0.30 mol of CS2 was added slowly and reaction mixture was allowed to stirred at RT overnight. The precipitated potassium aryldithiocarbazate was collected by filtration and washed with anhydrous ether and dried under vacuo. Potassium aryldithiocarbazate thus obtained was directly used in the next chemical procedure. Synthetic procedure for synthesis of 4-amino-5- (substituted heteroaryl)-4H-1,2,4-triazole-3-thiols (3a- 3g) To a mixture of 0.1 mol of above potassium aryldithiocarbazates (2a-2g) in 10 mL of water, 0.3 mol of 99% hydrazine hydrate was added and reaction mixture was allowed to reflux for 8-11 hours until complete evolution of H2S gas and the color of mixture change to green and finally it became homogeneous. The reaction mixture was then cooled to RT and diluted with 100 mL of water and acidified with conc. HCl. The mixture was then extracted in EtOAC, washed with water, brine solution and dried over anhydrous Na2SO4. Pure 3a-3g were obtained using column chromatography with 5-6% EtOAc-petrolium ether as eluent as yellow solids. Synthetic procedure for synthesis of title compounds 1,2,4-triazolo[3,4-b]-1,3,4-thiadiazines (1-10) A mixture of 4-amino-5-(substituted heteroaryl)-4H- 1,2,4-triazole-3-thiols (3a-3g, 1 mmol) and 3-(2- bromoacetyl)-2H-chro-men-2-one (1.1 mmol) in ethanol (5 mL) was refluxed for 6-13 hours. Upon completion of the reaction as monitored by TLC until no staring material left, the reaction mixture was concentrated and the residue thus obtained was diluted with water, extracted in EtOAc (75 mL). The combined extract was washed with 10% NaHCO3 solution, water followed by brine solution and dried over anhydrous Na2SO4. The crude product obtained was purified by column chromatography using 0.75-1.0% methanol in dichloromethane to afford the title compounds 1-10 (Puthiyapurayil et al., 2012). The Scheme 1 outlines chemical reaction sequences and conditions required to furnish final compounds 1-10. 3-(3-((1H-benzo[d]imidazol-1-yl)methyl)-7H-[1,2,4] triazolo[3,4-b][1,3,4]thiadiazin-6-yl)-2H-chromen-2- one (1) Yield 71%, m.p. 256-258°C, IR (KBr, cm–1): 3049 (Ar-H), 2988, 2919, 2845 (C-H), 1729 (lactone C=O), 1640 (C=N), 1389 (N=C-S), 1327 (C-N), 1317 (N-N=C), 679 (C-S-C bend-ing); 1H NMR (400 MHz, DMSO–d6): δ 8.49 (s, 1H, –N=CH, benzimidazole), 8.21 (s, 1H, H–4, coumarin), 7.83-7.67 (m, 2H, Ar-H), 7.35-7.43 (m, 2H, coumarin), 7.32-7.17 (m, 2H, Ar-H), 7.05-7.12 (m, 2H, coumarin), 4.21 (s, 2H, S-CH2, thiadiazine), 3.71 (s, 2H, –N–CH2); 13C NMR (100 MHz, DMSO–d6): δ 168.1 (1C, C=O, coumarin), 165.2, 162.6 and 160.1 (3C, C-3, C-6 and C-9, triazolothiadiazine), 153.2 (1C, C-9, coumarin), 152.7 (1C, C–2, benzimidazole), 145.2-116.8 (13C, Ar-C), 43.8 (1C, N–CH2), 41.2 (1C, C-7, CH2, triazolothiadiazine). Anal Calcd for C21H14N6O2S: C, 60.86; H, 3.40; N, 20.28; Found: C, 60.71; H, 3.58; N, 20.16. 3,3'-(7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine-3,6- diyl)bis(2H-chromen-2-one) (2) Yield 68%, m.p. 245-257°C, IR (KBr, cm–1): 3058 (Ar-H), 2988, 2937, 2840 (C-H), 1734 (lactone C=O), 1633 (C=N), 1380 (N=C-S), 1331 (C-N), 1324 (N-N=C), 681 (C-S-C bending); 1H NMR (400 MHz, DMSO–d6): δ 8.34 (s, 1H, H–4, coumarin), 8.37 (s, 1H, H–4, coumarin), 7.29-7.46 (m, 4H, coumarin), 7.04-7.15 (m, 4H, coumarin), 4.27 (s, 2H, S-CH2, thiadiazine); 13C NMR (100 MHz, DMSO– d6): δ 169.1 (1C, C=O, coumarin), 167.8 (1C, C=O, coumarin), 166.1, 163.3 and 159.8 (3C, C-3, C-6 and C-9, triazolothiadiazine), 153.4 (1C, C-9, coumarin), 152.8 (1C, C-9, coumarin), 145.2-125.6 (14C, Ar-C), 41.9 (1C, C -7, CH2, triazolothiadia-zine). Anal Calcd for C22H12N4O4S: C, 61.68; H, 2.82; N, 13.08; Found: C, 61.81; H, 2.97; N, 13.21. 3-(3-(quinolin-6-yl)-7H-[1,2,4]triazolo[3,4-b][1,3,4] thiadiazin-6-yl)-2H-chromen-2-one (3) Yield 64%, m.p. 288-290°C, IR (KBr, cm–1): 3042 (Ar-H), Bangladesh J Pharmacol 2015; 10: 308-315 309 2971, 2919, 2871 (C-H), 1724 (lactone C=O), 1648 (C=N), 1382 (N=C-S), 1324 (C-N), 1316 (N-N=C), 666 (C-S-C bending); 1H NMR (400 MHz, DMSO–d6): δ 9.28 (dd, J = 2.1, 4.4 Hz, 1H, H-2, quinoline), 8.45–8.52 (m, 2H, H-3 and H-4, quinoline), 8.28 (s, 1H, H–4, coumarin), 8.09 (d, J = 1.9 Hz, 2H, H-5 and H-8, quinoline), 7.61 (dd, J = 4.8, 9.3 Hz, 1H, H-7, quinoline), 7.34-7.41 (m, 2H, coumarin), 7.08-7.17 (m, 2H, coumarin), 4.19 (s, 2H, S- CH2, thiadiazine); 13C NMR (100 MHz, DMSO–d6): δ 169.2 (1C, C=O, coumarin), 165.7, 161.9 and 160.3 (3C, C -3, C-6 and C-9, triazolothiadiazine), 154.7 (1C, C-9, couma-rin), 152.8, 151.1 (2C, C2 and C9, quinoline), 143.9 –121.6 (14C, Ar–C), 43.7 (1C, C-7, CH2, triazolothiadiazine). Anal Calcd for C22H13N5O2S: C, 64.22; H, 3.18; N, 17.02; Found: C, 64.09; H, 3.37; N, 16.89. 3-(3-((6-bromoquinolin-4-yloxy)methyl)-7H-[1,2,4] triazolo[3,4-b][1,3,4]thiadiazin-6-yl)-2H-chromen-2- one (4) Yield 59%, m.p. 277-280°C, IR (KBr, cm–1): 3045 (Ar-H), 2966, 2946, 2850 (C-H), 1730 (lactone C=O), 1644 (C=N), 1376 (N=C-S), 1325 (C-N), 1321 (N-N=C), 674 (C-S-C bending); 1H NMR (400 MHz, DMSO–d6): δ 8.76 (d, J = 2.7 Hz, 1H, H-5, quinoline), 8.67 (d, J = 9.2 Hz, 1H, N=CH, H-2, quinoline), 8.31 (s, 1H, H-3, quinoline), 8.25 (s, 1H, H–4, coumarin), 8.21 (d, J = 7.5 Hz, 1H, H-8, quinoline), 8.15 (s, 1H, H-5, quinoline), 8.09 (dd, J = 8.3, 1.9 Hz, 1H, H-7, quinoline), 7.37-7.45 (m, 2H, couma- rin), 7.05-7.11 (m, 2H, coumarin), 5.12 (2H, s, O-CH2), 4.26 (s, 1H, S-CH2, thiadiazine); 13C NMR (100 MHz, DMSO–d6): δ 167.6 (1C, C=O, coumarin), 166.3, 163.1 and 161.6 (3C, C-3, C-6 and C-9, triazolo-thiadiazine), 152.8 (1C, C-9, coumarin), 152.2, 151.9 (2C, C2 and C9, quinoline), 151.3 (1C, O-CH2), 144.9-118.6 (14C, Ar-C), 39.9 (1C, C-7, CH2, triazolothiadiazine). Anal Calcd for C23H14BrN5O3S: C, 53.09; H, 2.71; N, 13.46; Found: C, 52.96; H, 2.89; N, 13.63. 1-methyl-4-((6-(2-oxo-2H-chromen-3-yl)-7H-[1,2,4] triazolo[3,4-b][1,3,4]thiadiazin-3-yl)methoxy)quinolin- 2(1H)-one (5) Yield 73%, m.p. 293-296°C, IR (KBr, cm–1): 3055 (Ar-H), 2969, 2933, 2864 (C-H), 1728 (lactone C=O), 1638 (C=N), 1380 (N=C-S), 1329 (C-N), 1318 (N-N=C), 684 (C-S-C bending); 1H NMR (400 MHz, DMSO–d6): δ 8.21 (s, 1H, H–4, coumarin), 8.07 (d, J = 6.9 Hz, 1H, C-8 proton of quinoline), 7.72 (t, J = 8.2 Hz, 1H, C-7 proton of qui- noline), 7.56 (d, J = 8.8 Hz, 1H, C-5 proton of quinoline), 7.47 (t, J = 6.6 Hz, 1H, C-6 proton of quinoline), 7.40 (s, 1H, C-3 proton of quinoline), 7.30-7.37 (m, 2H, couma- rin), 7.04-7.09 (m, 2H, coumarin), 5.23 (2H, s, O-CH2), 4.22 (s, 2H, S-CH2, thiadiazine), 3.79 (s, 3H, N-CH3); 13C NMR (100 MHz, DMSO–d6): δ 168.5 (1C, C=O, couma- rin), 166.8 (1C, C=O, quinoline), 164.8, 161.9 and 159.5 (3C, C-3, C-6 and C-9, triazolo-thiadiazine), 155.0 (1C, C -9, coumarin), 151.9 (1C, O-CH2), 146.8-122.2 (16C, Ar- C), 42.6 (1C, C-7, CH2, triazolothiadiazine). Anal Calcd for C24H17N5O4S: C, 61.14; H, 3.63; N, 14.85; Found: C, 61.31; H, 3.51; N, 15.02. 3-(3-((quinazolin-4-yloxy)methyl)-7H-[1,2,4]triazolo [3,4-b][1,3,4]thiadiazin-6-yl)-2H-chromen-2-one (6) Yield 66%, m.p. 259-261°C, 3039 (Ar-H), 2990, 2944, 2849 (C-H), 1722 (lactone C=O), 1640 (C=N), 1377 (N=C -S), 1334 (C-N), 1326 (N-N=C), 670 (C-S-C bending); 1H NMR (400 MHz, DMSO–d6): δ 8.32 (s, 1H, H–4, coumarin), 8.22 (s, 1H, N–CH–N, quinazoline), 8.09 (dd, J = 10.2, 8.3 Hz, 2H, quinazoline ring), 7.33-7.41 (m, 2H, coumarin), 7.36-7.23 (m, 3H, Ar-H), 7.01-7.07 (m, 2H, coumarin), 5.17 (2H, s, O-CH2), 4.19 (s, 1H, S-CH2, thiadiazine); 13C NMR (100 MHz, DMSO–d6): δ 167.1 (1C, C=O, coumarin), 165.7, 162.1 and 160.8 (3C, C-3, C- 6 and C-9, triazolothiadiazine), 158.2, 157.7, 156.6 (3C, quinazo-line), 153.8 (1C, C-9, coumarin), 144.6-124.7 (13C, Ar-C), 41.9 (1C, C-7, CH2, triazolothiadiazine). Anal Calcd for C22H14N6O3S: C, 59.72; H, 3.19; N, 18.99; Found: C, 59.87; H, 3.33; N, 19.13. 3-(3-(pyridin-4-yl)-7H-[1,2,4]triazolo[3,4-b][1,3,4] thiadiazin-6-yl)-2H-chromen-2-one (7) Yield 70%, m.p. 252-254°C, IR (KBr, cm–1): 3055 (Ar-H), 2977, 2917, 2848 (C-H), 1733 (lactone C=O), 1649 (C=N), 1391 (N=C-S), 1330 (C-N), 1320 (N-N=C), 681 (C-S-C bending); 1H NMR (400 MHz, DMSO–d6): δ 8.87 (2H each, 2d, J = 5.8 Hz, 5.9 Hz, pyridine), 8.27 (s, 1H, H–4, coumarin), 7.38-7.44 (m, 2H, coumarin), 7.08-7.15 (m, 2H, couma- rin), 4.21 (s, 2H, S-CH2, thiadiazine); 13C NMR (100 MHz, DMSO–d6): δ 169.0 (1C, C=O, coumarin), 164.9, 161.8 and 161.0 (3C, C-3, C-6 and C-9, triazolothiadia- zine), 154.5 (1C, C-9, coumarin), 150.3 (2C, pyridine), 143.7-123.9 (10C, Ar-C), 40.4 (1C, C-7, CH2, triazolothia- diazine). Anal Calcd for C18H11N5O2S: C, 59.82; H, 3.07; N, 19.38; Found: C, 59.69; H, 3.22; N, 19.25. In vitro evaluation of anti-cancer activity Sulforhodamine B (SRB) assay Cancerous cell lines were grown using 2 mM L- glutamine and 10% fetal bovine serum under RPMI 1640 medium and incubation was carried out using 96- well microtiter plates having 90 µL at plating densities with regard to the doubling time of individual cell lines. At first, incubation of microtiter plates was performed at 95% air, 5% CO2, 100% relative humidity and 37 ± 1° C temperature for 24 hours before introducing the compounds and after inoculation. After incubation, for each line of cells, cell population was recorded on the time title compounds’ appropriate concentrations (prepared using DMSO at 400-fold) were supplied (Tz) fixing one plate of each cell line with TCA. To the appropriate well 100 mL of each screen compound was inserted which already hold 100 mL of medium containing the cells which is to be incubated at 37°C for 48 hours (Skehn et al., 1990). Cells were fixed in situ by 310 Bangladesh J Pharmacol 2015; 10: 308-315 the soothing inclusion of 50 mL of cold 10% w/v TCA and incubated for 60 min at 4°C and then plated were cleaned properly and air dried. Then, 50 µL of 0.4% w/ v SBR solution in 1% CH3COOH was added to each well and incubated for 20 min at 25°C and after incubation, plates were washed gently and air dried. Trizma base (10 mM) was used to dissolve the bound stain and the absorbance was read on an Elisa plate reader (690 nm reference wavelength) at a wavelength of 515 nm. %Growth was observed with reference to the control well for each plate and determined as the ratio of average absorbance of the test well to the average absorbance of the control wells X 100. Using the six absorbance measurements [time zero (Tz), control growth (C), and analyze development in the existence of drug at the four concentration levels (Ti), the percentage growth was calculated at each of the drug concentration levels. Percentage growth inhibition was calculated as: [(Ti-Tz)/(C-Tz)] x 100 for concentrations for which Ti ≥ Tz and [(Ti-Tz)/Tz] x 100 for concentrations for which Ti