! " # $ # % &" # # $! !" $ % # &(" # % & & ' % & & " # $ ) ' ! %& 6 &' * . & & ()* *' & & ' /'0 1 # # ./ ' 0 ./1 / 7& ) % * &%! 81 $ 233435 . 1 / 2333951 # ./1 # $ : + # 1 & + % *, & - & + # / 9 & ! # . 1 # / / 233 4351 7& / 81 2333951 . # A series of new nicotinyl rhodanine derivatives were synthesized by condensing various chloronicotinaldehydes with rhodanine and substituted rhodanines. The antitumor activity for these compounds was screened against MCF 7, A549 and HT29 human cancer cell lines. The results show that compounds 1, 3, 5, 7, 8 and 9 are more potent against MCF 7 cell lines; compounds 9 and 11 are more potent against A549 cell lines; compound 3 is more potent against HT29 cell lines amongst the 14 nicotinyl rhodanine compounds synthesized. The relationships between structure and antitumor activity were elucidated. Rhodanine Derivatives, Nicotinaldehydes, Synthesis, Antitumor Activity, Structure Activity Relations. UDP N acetylmuramate/L alanine ligase,27 antimalerials,28 HIV 1 Integrase inhibitors,29 aldose reductase,30 β lactamase,31 antidiabetic agents,32 HCV NS3 Protease inhibitor,33 and histidine decarboxylase,34 . Epalrestat, marketed drug as aldose reductase inhibitor, is an analogue of rhodanine and Rosiglitazone, PPAR γ agonist, is an isostere of rhodanine scaffold signifies its importance (Chart 1). On the other hand rhodanine derivatives also possess wide range of pharmacological action, especially as antitumor agents,22e p and an ability to inhibit the JNK stimulating phosphatase 1 inhibition. Tomasiae and Masie recently published a review on rhodanine as a privileged scaffold in drug discovery whose functionalization and appropriate modifications led to compounds endowed with various biological activities.35 It is also reported that, groups like pyridine and aryl substituted pyridine contribute to the biological activities,26 and these would result in highly potent and selective antitumor agents. The design, synthesis and biological study of new compounds with enhanced activity is an ongoing research project in our group.36 This article is the outcome of the intension to bring rhodanine pyridine (nicotinyl) based NCEs as antitumor agents and it deals with the synthesis, antitumor activity and structure activity relationships. Cancer is considered to be the most fatal disease and till today, cure of cancer is very challenging although there are several anticancer agents yet to be explored from literature and a lot are in clinical trials.1 Effective strategies in combating the disease include targeting proliferation pathways,2 and signal transduction mechanisms including Akt,1,3 apoptotic,4 JNK stimulation pathways,2,5 to accelerate cell demises. But the emergence of resistance,6 physiological destructive consequences of therapy,7 and toxicity of diversified anticancer agents leads to limitation of their use. These limitations have paved the way to search for biologically promising new chemical entities (NCEs),8 13 against deadly cancer disease. Major sources of bioactive NCEs are identified from or inspired by natural products,14,15 marine metabolites,16,17 and random screening of chemical library.18 20 In discovering small antitumor molecules, a notable role is played by heterocyclic structures,21 and among these, a growing attention focuses on the synthesis and study of the biological properties of compounds containing various combinations of pyridine and /or rhodanine moieties.22 A wide spectrum of pharmacological activities has been reported for these compounds. These include fungal protein mannosyl transferase 1 inhibitors,23 PDE4 inhibitors,24 Protease inhibitors,25 JNK stimulating phosphatase 1 (JSP 1) inhibitors,26 6 Chart 1 CH3 N O O N CH3 S NH COOH S Epalrestat: Aldose Reductase Inhibitor Chemistry: Substituted nicotinaldehydes 17 23 were prepared via reported procedures [37]. Nicotinyl rhodanines 1 14 were synthesized in good yields by treating rhodanines 15 16 with various nicotinaldehydes 17 23 using acetic acid and sodium acetate under reflux conditions and the corresponding route is presented in Scheme 1. All the synthesized N O S O Rosiglitazone: PPAR receptors compounds are well characterized by 1H NMR, 13 C NMR, Mass, IR etc. There are two sets of final products w. r. t nitrogen of rhodanine, one being secondary amide in ring ( NH ) and other is substituted with acetic acid (tertiary). Structure activity relationship studies focused primarily on two regions of inhibitors: substitution on pyridine and rhodanine skeleton. Scheme 1 General synthetic strategy of nicotinyl rhodanine derivatives (1 14) diphenyl tetrazolium bromide] (MTT) assays, of respective cell lines, for reach compound were conducted in triplicates at 10 4 M and 10 5 M concentrations and the results of these studies are represented in Table 1. Biological Activity: All compounds synthesized were evaluated for their activity against three human tumor cell lines MCF 7 (Breast cancer), A549 (Lung cancer), HT 29 (Colon cancer). [3 (4,5 dimethylthiazol 2 yl) 2,5 Table 1 The in vitro cell growth inhibitory effect of nicotinyl rhodanine derivatives (1 14) in various cell lines with MTT assays. R3 R5 S S R4 N N R2 O R1 1 14 Compound No. R1 1 H 2 CH2COOH 3 H 4 CH2COOH 5 H 6 CH2COOH 7 H 8 CH2COOH 9 H 10 CH2COOH 11 H 12 CH2COOH 13 H 14 CH2COOH a R2 Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl H H R3 H H H H H H H H H H H H Ph Ph R4 CH3 CH3 CH2CH3 CH2CH3 CH2 CH2CH3 CH2 CH2CH3 CH(CH3)2 CH(CH3)2 Ph Ph H H CN CN R5 H H H H H H H H H H COOCH3 COOCH3 Cl Cl MCF 44 130 18 82 14 109 53 36 46 87 70 141 104 191 IC50 ("g/ml) 7 A549 HT 29 106 115 a NA NAa 97 27 187 136 125 136 174 84 101 NAa 193 NAa 41 184 131 NAa 42 84 164 134 69 NAa 108 89 NA IC50 values are beyond 200 'g/ml It has been observed from the results presented in Table 1 that most of the compounds are active against three cell lines. Some of these compounds have been found to be highly cell line specific and show appreciable inhibition of a particular cell line at low concentration. Free –NH in rhodanine series of compounds show good activity than that of substituted derivatives (substitution on NH with CH2COOH group) except very few compounds. This is quite interesting because one may expect more interactions with – CH2COOH group than secondary amide. In MCF 7 cell lines, compound 1 exhibited good anti tumor activity with an IC50 value of 44 Gg/mL. Presence of ethyl and propyl group on 5th position of pyridine ring, 3 and 5 respectively, is more potent compared to methyl group 1. Substitution of isopropyl group in place of propyl group 7 on pyridine gave diminished activity and it may be the consequence of steric hindrance caused by bulkiness (volume) of isopropyl group. This shows that the presence of linear alkyl chain is essential to have very good activity. At same position, replacement of isopropyl with phenyl group 9 exhibited similar activity as 1 and 7. Other compounds 11 and 13 are less potent compared to the rest of the compounds in the same series. In this particular cell lines, presence of alkyl groups on pyridine ring helped to have better antitumor activity compared to other groups. Further, presence of CH2COOH group on rhodanine nitrogen in most of the compounds also leads to less activity. SAR of rhodanine derivatives against A549 cell lines is different than that of MCF 7 cell lines. Alkyl substituted rhodanine derivatives 1, 3, 5 and 7 has moderate activity with IC50 value range of 125 – 97 Gg/mL. Compound 9 where phenyl group has been substituted in place of alkyl group has better activity with IC50 value of 41 Gg/mL. Compound 11 has similar activity as 9 where phenyl on 5th position and hydrogen on 6th position of pyridine is replaced with hydrogen and COOCH3, electron with drawing group, respectively. Compound 13, where positions of phenyl, CN (electron with drawing group), and chlorine are present at altered positions, exhibited moderate activity. In this cell lines, presence of phenyl groups, and or electron with drawing groups on pyridine ring help to have better antitumor activity compared to alkyl groups. On the other hand, all the compounds bearing CH2COOH group has lower activity compared to its counterpart, secondary amide in rhodanine. Compounds possessing free –NH group on rhodanine skeleton, have better antitumor activity against HT 29 cell lines except for 5 and 13. Compound 3, where 5th position of pyridine ring is substituted with ethyl, is found to have better activity than its methyl counterpart 1. This is the potent compound amongst the screened compounds against HT29 cell lines. Increment in chain length from ethyl 3 to either linearly to propyl 5 or with branching to isopropyl 7, exhibited lower activity. Substitution of phenyl group and or electron withdrawing group, like results against MCF 7 cell lines, demonstrated lower activity compared to ethyl substitution. Therefore, a suitable combination of the substituents and their appropriate position in the molecule significantly control the activity/function of the molecule. In conclusion, novel nicotinaldehyde attached rhodanine derivatives were synthesized in good yields for their antitumor activity. Compounds possessing alkyl chain on pyridine ring, in particular increment of chain length linearly rather than branched, demonstrated the better activity against the MCF 7 cell lines. In contrast, presence of phenyl or electron with drawing group on pyridine and secondary amide of rhodanine skeleton exhibits increased antitumor activity in A549 cell lines. Compound 3, where 5th position of pyridine ring is substituted with ethyl, is most potent amongst the screened compounds against HT29 cell lines. Either increment or decrease of alkyl chain length exhibited relatively lower activity. Presence of – CH2COOH group on rhodanine skeleton showed least activity except few cases against three cell lines. Present investigation highlight the role of various groups along with the position in the molecule toward their tumor cell growth inhibitory properties and could be useful in the further tailoring of the molecules for improving their antitumor activity. All reactions were carried out under an open atmosphere. Rhodanine and rhodanine 3 acetic acid from Sigma Aldrich and the solvent were obtained from commercial suppliers and used without further purification. Melting points were determined on a Mel apparatus and are uncorrected. The 1H NMR and 13C NMR spectra were recorded on Varian inova 400 MHz spectrometer with tetramethylsilane (TMS) as internal standard. Chemical shifts values are given in (δ) ppm, coupling constants ( ) are in hertz, and splitting patterns are designated as follows: s, Singlet; d, doublet; dd, doublet of doublets; t, triplet; q, quartet; m, multiplet. HRMS (ESI) data were recorded on a QSTAR XL high resolution mass spectrometer. IR spectra were taken on a Thermo Nicolet nexus 670 FT IR spectrophotometer. General procedure for synthesis of Rhodanine derivatives 0.01 Mole (1 equivalent) of chloronicotinaldehyde, 0.01 mole (1equivalent), of rhodanine derivatives, 0.03 mole (3 equivalents) of freshly fused sodium acetate in 25 ml. of acetic acid, to which 0.008 moles (0.8 equivalents) of acetic anhydride had been added, was refluxed for 3 hours, and allowed to cool. Water (200 mL) was then added to the solution where precipitate was formed. The precipitate was collected via filtration, re crystallized from methanol and dried to give the desired compound. Yields (75 80%) 5 [(Z) 1 (2 chloro 5 methyl 3 pyridyl) methylidene] 2 thioxo 1,3 thiazolan 4 one (1): Light brown solid (2.04g, 76%); mp 245 247 °C; IR (KBr) 3204, 2814, 2660, 1719, 1595, 1470, 1387, 1222, 1166, 1052 cm 1; 1H NMR (400 MHz, DMSO ): δ 2.37 (s, 3H), 7.59 (s, 1H), 7.70 (d, = 1.8 Hz, 1H), 8.31 (d, = 1.8 Hz, 1H ); 13C NMR (75 MHz, DMSO ): δ 17.0, 124.4, 127.0, 130.7, 133.7, 137.7, 148.1, 150.6, 168.9, 195.0; MS (ESI, ve): m/z: 269 [M H]– ; HRMS: m/z [M H]– Calcd for C10H6N2OS2Cl: 268.9610, found: 268.9617. 2 5 [(Z) 1 (2 chloro 5 methyl 3 pyridyl) methylidene] 4 oxo 2 thioxo 1, 3 thiazolan 3 ylacetic acid (2) Light brown solid (2.46g, 75%); mp 211 213 o C; IR (KBr) 3426, 3040, 1722, 1594, 1411, 1330, 1206, 1107, 1057 cm 1; 1H NMR (500 MHz, DMSO ): δ 2.43 (s, 3H), 4.75 (s, 2H), 7.66 (s, 1H), 7.91 (s, 1H), 8.24 (s, 1H); 13C NMR (75 MHz, DMSO ): δ 17.4, 44.6, 95.7, 126.7, 127.0, 127.2, 132.8, 137.6, 148.9, 150.4, 166.6, 191.4; MS (ESI, ve): m/z: 327 [M H]– ; HRMS: m/z [M H]– Calcd for C12H8N2O3S2Cl: 326.9664, found: 326.9678. 5 [(Z) 1 (2 chloro 5 ethyl 3 pyridyl) methylidene] 2 thioxo 1,3 thiazolan 4 one (3) Light brown solid (2.27g, 80%); mp 222 224 o C; IR (KBr) 3004, 2935, 2711, 2494, 1712, 1616, 1402, 1330, 1222, 1195 cm 1; 1H NMR = 8.1 Hz, (400 MHz, DMSO ): δ 1.23 (t, 3H), 2.73 (q, = 8.1 Hz, 2H), 7.63 (s, 1H), 7.75 (d, = 2.7 Hz, 1H), 8.38 (d, = 2.7 Hz, 1H). C NMR (75 MHz, DMSO ): δ 14.9, 24.3, 124.6, 127.4, 131.0, 136.8, 139.5, 148.2, 150.0, 169.0, 195.1; MS (ESI, ve): m/z: 283 [M H]–; HRMS: m/z [M H]– Calcd for C11H8N2OS2Cl: 282.9766, found: 282.9779. 13 2 5 [(Z) 1 (2 chloro 5 ethyl 3 pyridyl) methylidene] 4 oxo 2 thioxo 1,3 thiazolan 3 ylacetic acid (4) Yellow solid (2.7g, 79%); mp 223 225 oC; IR (KBr) 3415, 2970, 2821, 1723, 1594, 1460, 1396, 1219, 1170, 1059 cm 1; 1H NMR (400 = 7.8 Hz, 3H), MHz, DMSO ): δ 1.23 (t, 2.73 (q, = 7.8 Hz, 2H), 4.75 (s, 2H), 7.84 (s, 1H), 7.85 (d, = 2.7 Hz, 1H), 8.40 (d, = 2.7 Hz, 1H); 13C NMR (75 MHz, DMSO ): δ 15.0, 24.4, 45.1, 127.3, 127.4, 137.2, 139.7, 148.2, 148.3, 150.5, 165.8, 167.2, 192.7; MS (ESI, ve): m/z: 341 [M H]–; HRMS: m/z [M H]– Calcd 340.9821, found: for C13H10N2O3S2Cl: 340.9835. 5 [(Z) 1 (2 chloro 5 propyl 3 pyridyl) methylidene] 2 thioxo 1,3 thiazolan 4 one (5) Light brown solid (2.2g, 77%); mp 196 198 oC; IR (KBr) 2959, 2871, 1705, 1650, 1539, 1393, 1334, 1198, 1110, 1060 cm 1; 1H NMR (400 MHz, DMSO ): δ 0.88 (t, = 6.8 Hz,3H), 1.54 1.61 (m, = 7.6 Hz, =6.8 Hz, 2H), 2.53 (t, = 7.6 Hz, 2H), 7.14 (s, 1H), 7.45 (s, 1H), 7.79 (s, 1H); 13C NMR (75 MHz, DMSO ): δ 13.3, 23.6, 32.9, 124.8, 127.4, 131.0, 137.4, 138.0, 148.3, 150.5, 168.9, 195.2; MS (ESI, ve): m/z: 297 [M H]–; HRMS: m/z [M H]– Calcd 296.9923, found: for C12H10N2OS2Cl: 296.9930 2 5 [(Z) 1 (2 chloro 5 propyl 3 pyridyl) methylidene] 4 oxo 2 thioxo 1,3 thiazolan 3 ylacetic acid (6) Light brown solid (2.77g, 78%); mp 158 159 o C; IR (KBr) 3420, 2960, 1706, 1650, 1540, 1394, 1335, 1200, 1109, 1061 cm 1; 1H NMR (400 MHz, DMSO ): δ 0.87 (t, = 7.6 Hz, 3H), 1.50 1.54 (m, = 7.6 Hz, 2H), 2.36 (t, = 7.6 Hz, 2H), 4.69 (s, 2H), 7.48 (s, 1H), 7.62 (s, 1H), 7.86 (s, 1H); 13C NMR (75 MHz, DMSO ): δ 13.2, 23.2, 32.1, 44.9, 120.1, 121.3, 122.3, 131.6, 137.5, 148.3, 160.2, 166.9, 167.4, 196.2; MS (ESI, ve): m/z: 355 [M H]–; HRMS: m/z [M H]– Calcd for C14H12N2O3S2Cl: 354.9977, found: 354.9976. 5 [(Z) 1 (2 chloro 5 isopropyl 3 pyridyl) methylidene] 2 thioxo 1,3 thiazolan 4 one (7) Light brown solid (2.26g, 76%); mp 243 245 o C; IR (KBr) 2960, 2832, 1724, 1647, 1595, 1445, 1215, 1172, 1061 cm 1; 1H NMR (400 MHz, DMSO ): δ 1.27 (d, = 6.3 Hz, 6H), 3.05 3.12 (m, = 6.3 Hz, 1H), 7.64 (s, 1H), 7.75 (d, = 1.8 Hz, 1H), 8.43 (d, = 1.8 Hz, 1H); 13C NMR (75 MHz, DMSO ): δ 23.2, 30.3, 125.0, 127.6, 131.2, 135.5, 143.9, 148.3, 149.3, 168.9, 195.1; MS (ESI, ve): m/z: 297 [M H]–; HRMS: m/z [M H]– Calcd for C12H10N2OS2Cl: 296.9923, found: 296.9933. 2 5 [(Z) 1 (2 chloro 5 isopropyl 3 pyridyl) methylidene] 4 oxo 2 thioxo 1,3 thiazolan 3 ylacetic acid (8) Yellow solid (2.81g, 79%); mp 163 165 oC; IR (KBr) 3411, 2966, 2714, 2506, 1852, 1714, 1615, 1328, 1198, 1055 cm 1; 1H NMR (400 MHz, DMSO ): δ 1.28 (d, = 7.2 Hz, 6H), 3.07 3.13 (m, = 7.2 Hz, 1H), 4.76 (s, 2H), 7.86 (s, 1H), 7.87 (d, = 1.8 Hz, 1H), 8.4 (d, = 1.8 Hz, 1H); 13C NMR (75 MHz, DMSO ): δ 23.0, 30.2, 45.0, 127.4, 127.5, 135.8, 135.9, 143.9, 148.1, 149.6, 165.7, 167.1, 192.6; MS (ESI, ve): m/z: 355 [M H]–; HRMS: m/z [M H]– Calcd for C14H12N2O3S2Cl: 354.9977, found: 354.9976. 5 [(Z) 1 (2 chloro 5 phenyl 3 pyridyl) methylidene] 2 thioxo 1,3 thiazolan 4 one (9) Light brown solid (2.49g, 75%); mp 246 248 o C; IR (KBr) 3029, 2822, 1701, 1591, 1443, 1386, 1222, 1167, 1056 cm 1; 1H NMR (400 MHz, DMSO ): δ 7.56 7.67 (m, 3H), 7.75 (s, 1H), 7.84 (d, = 7.1 Hz, 2H), 8.15 (d, = 2.3 Hz, 1H), 8.86 (d, =2.3 Hz, 1H); 13C NMR (75 MHz, DMSO ): δ 124.4, 126.9, 127.9, 128.9, 129.1, 129.3, 31.6, 134.7, 135.2, 135.4, 148.2, 149.3, 168.7, 194.8, 206.4; MS (ESI, ve): m/z: 331 [M H]–; HRMS: m/z [M H]– Calcd for C15H8N2OS2Cl: 330.9766, found: 330.9760. 2 5 [(Z) 1 (2 chloro 5 phenyl 3 pyridyl) methylidene] 4 oxo 2 thioxo 1,3 thiazolan 3 ylacetic acid (10) Light brown solid (2.92g, 75%); mp 236 238 o C; IR (KBr) 3420, 2936, 1720, 1596, 1390, 1333, 1207, 1178, 1110, 1058 cm 1; 1H NMR (400 MHz, DMSO ): δ 4.76 (s, 2H), 7.48 (t, = 7.2 Hz, 2H), 7.55 (t, = 7.2 Hz, 2H), 7.78 (d, = 7.2 Hz, 2H), 7.87 (s, 1H), 8.17 (d, = 2.7 Hz, 1H), 8.22 (d, =2.7 Hz, 1H); 13C NMR (75 MHz, DMSO ): δ 45.0, 127.0, 127.1, 127.9, 128.0, 128.9, 129.3, 129.6, 134.6, 135.5, 135.6, 148.6, 149.3, 165.6, 167.1, 192.5, 206.4; MS (ESI, ve): m/z: 389 [M H] –; HRMS: m/z [M H] – Calcd for C17H10N2O3S2Cl: 388.9821, found: 388.9820. Methyl 6 chloro 5 [(4 oxo 2 thioxo 1, 3 thiazolan 5 yliden) methyl] 2 pyridinecarboxylate (11) Light brown solid (2.44 g,78%); mp 227 229 C; IR (KBr) 3176, 1717, 1595, 1426, 1322, 1237, 1198, 1143, 1064 cm 1; 1H NMR (400 MHz, DMSO ): δ 3.87 (s, 3H), 7.57 (s, 1H), 8.06 (d, = 8.05 Hz, 1H), 8.11 (d, = 8.05 Hz, 1H); 13C NMR (75 MHz, DMSO ): δ 52.8, 123.0, 124.6, 131.2, 133.0, 139.1, 146.8, 150.5, 163.2, 168.9, 194.8; MS (ESI, ve): m/z: 313 [M H]– ; HRMS: m/z [M H]– calcd for C11H6N2O3S2Cl: 312.9508, found: 312.9506. o 5 (Z) 1 [2 chloro 6 (methoxy carbonyl) 3 pyridyl] methylidene 4 oxo 2 thioxo 1, 3 thiazolane 3 carboxylic acid (12) Light brown solid (2.28 g, 76%); mp 214 216 o C; IR (KBr) 3477, 3021, 2939, 1722, 1554, 1404, 1311, 1212, 1109, 1048 cm 1; 1H NMR (400 MHz, DMSO ): δ 3.88 (s, 3H), 4.73 (s, 2H), 7.82 (s, 1H), 8.14 (d, = 8.1 Hz, 1H), 8.19 (d, = 8.05 Hz, 1H); 13C NMR (75 MHz, DMSO ): δ 45.0, 52.9, 124.6, 125.7, 129.1 131.1, 139.5, 147.2, 150.5, 163.2, 165.7, 167.0, 192.3; MS (ESI, ve): m/z: 371 [M H]–; HRMS: m/z [M H]– calcd for C13H8N2O5S2Cl: 370.9563, found: 370.9577. 2 chloro 5 [(4 oxo 2 thioxo 1, 3 thiazolan 5 yliden) methyl] 4 phenyl 3 pyridyl cyanide (13) Yellow solid (2.82g, 79%); mp 290 292 oC; IR (KBr) 3157, 3055, 2992, 2853, 2235, 1701, 1611, 1544, 1435, 1360, 1229, 1068 cm 1; 1H NMR (400 MHz, DMSO ): δ 7.00 (s, 1H), 7.50 7.63 (m, 5H), 8.79 (s, 1H); 13C NMR (75 MHz, DMSO ): δ 110.9, 114.1, 123.7, 128.2, 128.7, 128.8, 129.2, 129.3, 130.5, 131.4, 132.7, 150.8, 152.1, 155.8, 168.4, 195.0; MS (ESI, ve): m/z: 356 [M H] –; HRMS: m/z [M H] – Calcd for C16H7N3OS2Cl: 355.9719, found: 355.9720. 5 [(Z) 1 (6 chloro 5 cyano 4 phenyl 3 pyridyl) methylidene] 4 oxo 2 thioxo 1,3 thiazolane 3 carboxylic acid (14) Yellow solid (3.32g, 80%); mp 172 174 oC; IR (KBr) 3517, 3279, 3037, 2942, 2554, 2233, 1718, 1607, 151, 1324, 1197, 1051 cm 1; 1H NMR (400 MHz, DMSO ): δ 4.70 (s, 2H), 7.23 (s, 1H), 7.49 (d, = 6.6 Hz, 2H), 7.61 7.63 (m, = 6.6 Hz, 3H), 8.85 (s, 1H); 13C NMR (75 MHz, DMSO ): δ 54.6, 120.6, 123.7, 135.8, 136.2, 137.3, 137.7, 138.5, 138.8, 140.2, 142.4, 160.7, 162.1, 165.6, 175.0, 176.6, 202.3; MS (ESI, ve): m/z: 414 [M H]– ; HRMS: m/z [M H] – Calcd for C18H9N3O3S2Cl: 413.9773, found: 413.9775. Biology: HT 29 (Colon cancer), A549 (Lung cancer), MCF 7 (Breast cancer) cell line was obtained from National center for Cell science (NCCS), Pune, India. DMEM (Dulbeccos Modified Eagles Medium), MTT [3 (4,5 dimethylthiazol 2 yl) 2,5 diphenyl tetrazolium bromide], Trypsin, EDTA were purchased from Sigma Chemicals Co (st.Louis, MO), Fetal bovine serum were purchased from Arrow labs,96 well flat bottom tissue culture plates were purchased from Tarson. Method a) Maintenance of cell lines. HT 29 (Colon cancer), A549 (Lung cancer), MCF 7 (Breast cancer), all cell lines were grown as adherent in DMEM media supplemented with 10% fetal bovine serum, 100 Gg / ml penicillin, 200 Gg/ml streptomycin, 2mM L glutamine, and culture was maintained in a humidified atmosphere with 5% CO2. b) Preparation of samples for cytotoxicity Stock solution of 10mg/ml stock solution in DMSO, from the above stock various dilutions was made with sterile water to get required concentration. c) MTT assay 1. HT 29 (Colon cancer), A549 (Lung cancer), MCF 7 (Breast cancer) cell lines were seeded at a density of 1x 104 cells (cell number was determined by Trypan blue exclusion dye method) per each well in 100Gl of DMEM supplemented with 10% FBS 2. 24 hrs after seeding, above media was replaced with fresh DMEM supplemented with 10% FBS then 10Gl sample from above stock solutions were added to each well in triplicates which gives final concentration of 200, 100, 50,10 Gg/well. 3. The above cells were incubated for 48 hrs at at 37 0C with 5% CO2 4. After 48 hrs, incubation the above media was replaced with 100 Gl of fresh DMEM without FBS and to this 10 Gl of MTT (5mg dissolved in 1ml of PBS) was added and incubated for 3 hrs at 370C with 5% CO2. 5. After 3 hrs incubation the above media was removed with multi channel pipette, then 200 Gl of DMSO was added to each well and the incubated at 37 0C for 15min. 6. 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