Chemistry All chemical compounds were purchased from commercial suppliers of Merck and Aldrich companies. The purity of the prepared compounds was proved by thin layer chromatography (TLC) using various solvents of different polarities. Merck silica gel 60 F
254 plates were applied for analytical TLC. Column chromatography was performed on Merck silica gel (70-230 mesh) for purification obtained compounds. 1H-NMR spectra were recorded using a Brucker 200 MHz spectrometer, and chemical shifts were expressed as
δ (ppm) with tetramethylsilane (TMS) as internal standard. The IR spectra were obtained on a Shimadzu 470 spectrophotometer (potassium bromide disks). Melting points were determined using electrothermal melting point analyzer apparatus and were uncorrected. The mass spectra were run on a Finigan TSQ-70 spectrometer (Finigan, USA) at 70 eV. All cell lines were purchased from the Pasteur Institute of Iran.
According to the
Figure 3, 5-amino- 1,3,4-thiadiazole-2-thiol (1 (
link)) was treated with 4-trifluoromethylphenylacetic acid for direct coupling of acid with amine. The reaction was carried out in the presence of EDC and hydroxybenzotriazole (HOBt) in acetonitrile as solvent. The termination of reaction was proved by thin layer chromatography (TLC). After completion, the solvent was evaporated using rotary evaporator apparatus and ethyl acetate and water were added. The aqueous phase was removed and the organic phase was washed two times by sodium bicarbonate 5%, diluted sulfuric acid and brine (16 (
link)-19 ). Anhydrous sodium sulfate was added for drying and filtration was done. Ethyl acetate was removed under reduced pressure and a yellow powder was obtained. The obtained product was used after crystallization from ethanol for the next step. Various benzyl chloride derivatives were reacted with compound 2 for obtaining the final appropriate products (3a-3l). The 1H NMR, IR and MS spectra were used to confirm the synthesized compounds.
Synthesis of N-(5-Mercapto-1,3,4-thiadiazol-2-yl)-2-(4-(trifluoromethyl)phenyl)acetamide (2)In a flask, equimolar amounts of 4-trifluoromethylphenylacetic acid, EDC and HOBtin acetonitrile solvent were stirred for 30 min and then equimolar quantity of 5-amino-1,3,4-thiadiazole-2-thiol was added. The stirring condition was continued for 24 h. The end point of the reaction was determined by thin layer chromatography(TLC). Acetonitrile was removed under reduced pressure and ethyl acetate/water was added. The aqueous layer was removed and organic layer was washed two times by sodium bicarbonate 5%, diluted sulfuric acid and brine. Anhydrous sodium sulfate was added for drying and then filtered. The ethyl acetate was evaporated using rotary evaporator apparatus. The obtained yellowish solid was washed by dry ether and used for the next step.
mp. 171°C, Yield: 65%,C
11H
8F
3N
3OS
2, MW: 319 g/mol,1H NMR (DMSO-d6, 200 MHz)
δ: 3.76 (s, 2H, -CH
2CO-), 3.95 (s, 1H, -SH), 7.58 (m, 2H,
J = 8Hz), 8.19 (m, 2H,
J = 8Hz, 4-trifluoromethylphenyl), 12.76 (brs, 1H, NH).IR (KBr, cm-1) ῡ: 3265, 1697, 1580, 1519, 1321, 1155, 1103, 1064, 821, 705.MS(m/z, %): M
++2: 322(10), M
+: 320(10), 279(45), 276(35), 167(80), 159(95), 149(100), 133(15), 109(15), 71(15), 57(20).
General procedure for synthesis of compounds 3a-3lEquimolar quantities of appropriate benzyl chloride derivative was treated with 2-(4-fluorophenyl)-
N-(5-mercapto-1,3,4-thiadiazol-2-yl)acetamide (2 (
link)). Equimolar amount of potassium hydroxide in absolute ethanol was added to convert the thiol moiety to the thiolate anion. Then, the related benzyl chloride derivative was added to the reaction medium and reflux condition was performed for 24 h. Crushed ice was added and the precipitate filtered, washed by cool water and purified by appropriate procedures such as crystallization or column chromatography (EtOAC/Petroleum ether: 3/2).
N-(5-(2-Fluorobenzylthio)-1,3,4-thiadiazol-2-yl)-2-(4-(trifluoromethyl)phenyl)acetamide (3a)mp. 201 °C, Yield: 42%,C
18H
13F
4N
3OS
2, MW: 427 g/mol,1H NMR (DMSO-d
6, 200 MHz)
δ: 4.00 (s, 2H, -CH
2CO-), 4.53 (s, 2H, -CH
2S-), 7.16-7.54 (m, 2-fluorobenzyl), 7.59 (d, 2H,
J = 8 Hz, 4-trifluoromethylphenyl), 7.76 (d, 2H,
J = 8 Hz, 4-trifluoromethylphenyl), 13.00 (s, NH). IR(KBr, cm
-1) ῡ: 3158, 3050, 2898, 1690, 1560, 1493, 1456, 1421, 1402, 1337, 1301, 1236, 1168, 1117, 1071, 1019, 972, 844, 757, 693, 655.MS(m/z, %): M
+: 427(95), 377(60), 325(35), 297(30), 236(25), 236(15), 159(75), 109(100).
N-(5-(3-Fluorobenzylthio)-1,3,4-thiadiazol-2-yl)-2-(4-(trifluoromethyl)phenyl)acetamide (3b)mp. 190 °C, Yield: 58%,C
18H
13F
4N
3OS
2, MW: 427 g/mol,1H NMR (DMSO-d
6, 200 MHz) δ: 3.93 (s, 2H, -CH
2CO-), 4.53 (s, 2H, -CH
2S-), 7.01-7.47 (m, 3-fluorobenzyl), 7.58 (d, 2H,
J = 8 Hz, 4-trifluoromethylphenyl), 7.75 (d, 2H,
J = 8 Hz, 4-trifluoromethylphenyl), 12.98 (s, NH).IR(KBr, cm
-1) ῡ: 3448, 3156, 2916, 1699, 1562, 1488, 1358, 1326, 1172, 1110, 1068, 837.MS(m/z, %): M
+: 427(85), 377(80), 325(40), 297(25), 236(20), 159(70), 109(100).
N-(5-(4-Fluorobenzylthio)-1,3,4-thiadiazol-2-yl)-2-(4-(trifluoromethyl)phenyl)acetamide (3c)mp. 166 °C, Yield: 40%,C
18H
13F
4N
3OS
2, MW: 427 g/mol,1H NMR (DMSO-d
6 , 200 MHz)
δ: 3.99 (s, 2H, -CH
2CO-), 4.51 (s, 2H, -S-CH
2-), 7.24 (t, 2H, 4-fluorobenzyl), 7.48 (t, 2H, 4-fluorobenzyl), 7.58 (d, 2H,
J = 8 Hz, 4-trifluoromethylphenyl), 7.75 (d, 2H,
J = 8 Hz, 4-trifluoromethylphenyl), 12.97 (s, NH).IR(KBr, cm-1) ῡ: 3370, 3030, 2920, 2829, 1701, 1558, 1508, 1323, 1219, 1160, 1109, 1060, 835.MS(m/z, %): M+: 427(90), 377(75), 325(40), 297(30), 236(25), 159(75), 109(100).
N-(5-(2-Chlorobenzylthio)-1,3,4-thiadiazol-2-yl)-2-(4-(trifluoromethyl)phenyl)acetamide (3d)mp. 208 °C, Yield: 54%,C
18H
13ClF
3N
3OS
2, MW: 443 g/mol,1H NMR (DMSO-d6, 200 MHz)
δ: 4.00 (s, 2H, -CH
2CO-), 4.59 (s, 2H, -CH2S-), 7.35 (m, 2-chlorophenyl), 7.48 (d, 2H,
J = 12 Hz, 4-trifluoromethylphenyl), 7.55 (m, 2-chlorophenyl), 7.97(d, 2H,
J = 12 Hz, 4-trifluoromethylphenyl), 13.00 (s, NH). IR(KBr, cm
-1) ῡ: 3447, 3165, 2922, 1700, 1570, 1445, 1326, 1171, 1109, 1067, 868, 759.MS(
m/z, %): M
+:443(10), 410(55), 409(85), 159(40), 127(50), 125(100), 109(10), 89(10).
N-(5-(3-Chlorobenzylthio)-1,3,4-thiadiazol-2-yl)-2-(4-(trifluoromethyl)phenyl)acetamide (3e)mp. 198 °C, Yield: 57%,C
18H
13ClF
3N
3OS
2, MW: 443 g/mol,1H NMR (DMSO-d6 , 200 MHz)
δ: 4.00 (s, 2H, -CH
2CO-), 4.59 (s, 2H, -S-CH2-), 7.33-7.40 (m, 3-chlorobenzyl), 7.43 (d, 2H,
J = 8 Hz, 4-trifluoromethylphenyl), 7.56 (m, 3-chlorobenzyl), 7.75 (d, 2H,
J = 8 Hz, 4-trifluoromethylphenyl), 13.00 (s, NH).IR(KBr, cm
-1) ῡ: 3427, 3165, 2914, 2730, 1700, 1569, 1446, 1356, 1326, 1301, 1170, 1112, 1066, 868, 839, 759.MS(
m/z, %): M
+:443(15), 410(60), 409(65), 282(10), 159(55), 127(35), 125(100), 89(10).
N-(5-(4-Chlorobenzylthio)-1,3,4-thiadiazol-2-yl)-2-(4-(trifluoromethyl)phenyl)acetamide (3f)mp. 198 °C, Yield: 34%,C
18H
13ClF
3N
3OS
2, MW: 443 g/mol,1H NMR (DMSO-d
6, 200 MHz)
δ: 3.99 (s, 2H, -CH
2CO-), 4.52 (s, 2H, -S-CH
2-), 7.38-7.48 (m, 4-chlorobenzyl), 7.58 (d, 2H,
J = 8 Hz, 4-trifluoromethylphenyl), 7.78 (d, 2H,
J = 8 Hz, 4-trifluoromethylphenyl), 12.97 (s, NH).IR(KBr, cm
-1) ῡ: 3440, 3130, 3040, 2877, 1690, 1556, 1334, 1163, 1118, 1068, 1020, 846, 745, 702.MS(
m/z, %): M++1: 444(10), M
+:443(10), 410(60), 409(80), 282(12), 178(12), 159(40), 127(50), 125(100), 109(10), 89(10).
N-(5-(3-Methoxybenzylthio)-1,3,4-thiadiazol-2-yl)-2-(4-(trifluoromethyl)phenyl)acetamide (3g)mp. 160 °C, Yield: 32%,C19H16F3N3O2S2, MW: 439 g/mol,1H NMR (DMSO-d
6 , 200 MHz)
δ:1H NMR (DMSO-d
6 , 200 MHz) δ: 3.80 (s, 3H, -OCH
3), 3.99 (s, 2H, -CH
2CO-), 4.48 (s, 2H, -S-CH
2-),7.06(m, 3H, 3-methoxybenzyl), 7.32(m, 1H, 3-methoxybenzyl), 7.58(d, 2H,
J = 8 Hz, 4-trifluoromethylphenyl), 7.75 (d, 2H,
J = 8 Hz, 4-trifluoromethylphenyl), 12.97 (s, NH).IR(KBr, cm-1) ῡ:3167, 3038,2946, 1735, 1702, 1607, 1577, 1488, 1438, 1358, 1325, 1302, 1271, 1158, 1113, 1068, 839, 777, 736.MS(
m/z, %): M
+: 439(40), 159(75), 122(45), 121(100), 109(35).
N-(5-(4-Methoxybenzylthio)-1,3,4-thiadiazol-2-yl)-2-(4-(trifluoromethyl)phenyl)acetamide (3h)mp. 219 °C, Yield: 36%,C
19H
16F
3N
3O
2S
2, MW: 439 g/mol,1H NMR (DMSO-d6 , 200 MHz)
δ: 3.76 (s, 3H, -OCH3), 3.99 (s, 2H, -CH
2CO-), 4.46 (s, 2H, -S-CH
2-), 6.92 (d, 2H,
J = 8 Hz, 4-methoxybenzyl), 7.35 (d, 2H,
J = 8 Hz, 4-methoxybenzyl), 7.58 (d, 2H,
J = 8 Hz, 4-trifluoromethylphenyl), 7.75 (d, 2H,
J = 8 Hz, 4-trifluoromethylphenyl), 12.96 (s, NH).IR(KBr, cm
-1) ῡ: 3265, 3045, 2870, 1691, 1554, 1510, 1400, 1332, 1298, 1170, 1122, 1107, 1066, 827. MS(
m/z, %): M
++2: 441(15), M
++1: 440(20), M
+: 439(25), 159(60), 122(45), 121(100), 109(30).
N-(5-(2-Nitrobenzylthio)-1,3,4-thiadiazol-2-yl)-2-(4-(trifluoromethyl)phenyl)acetamide (3i)mp. 146 °C, Yield: 35%,C
18H
13F
3N
4O
3S
2, MW: 454 g/mol,1H NMR (DMSO-d
6, 200 MHz)
δ: 4.00 (s, 2H, -CH
2CO
-), 4.79 (s, 2H, -CH
2S-), 7.49 (m, aromatic), 8.11 (m, aromatic), 8.21 (m, aromatic), 13.00 (s, NH). IR(KBr, cm
-1) ῡ: 3447, 3165, 2923, 1698, 1612, 1573, 1527, 1443, 1335, 1168, 1106, 1066, 830, 827, 702.MS(
m/z,%): M
+: 454(15), 270(35), 242(65), 225(15), 195(100), 179(65), 165(85), 136(70), 106(45), 90(50), 78(35).
N-(5-(3-Nitrobenzylthio)-1,3,4-thiadiazol-2-yl)-2-(4-(trifluoromethyl)phenyl)acetamide (3j)mp. 132 °C, Yield: 45%,C
18H
13F
3N
4O
3S
2, MW: 454 g/mol,1H NMR (DMSO-d
6, 200 MHz)
δ: 3.84 (s, 2H, -CH
2CO-), 4.51 (s, 2H, -CH
2S-), 7.71(m, 5H, aromatic), 8.16(m, 3H, aromatic), 13(brs, NH).IR(KBr, cm-1) ῡ: 3318, 3154, 2850, 1692, 1629, 1562, 1527, 1508, 1487, 1348, 1329, 1163, 1117, 1073, 810, 747.MS(
m/z,%): M
++1: 455(7), M
+: 454(15), 270(30), 242(75), 195(100), 179(70), 165(90), 136(60), 106(35), 90(45), 78(60).
N-(5-(4-Nitrobenzylthio)-1,3,4-thiadiazol-2-yl)-2-(4-(trifluoromethyl)phenyl)acetamide (3k)mp. 198 °C, Yield: 54%,C
18H
13F
3N
4O
3S
2, MW: 454 g/mol,1H NMR (DMSO-d
6, 200 MHz)
δ: 3.99 (s, 2H, -CH
2CO-), 4.66 (s, 2H, -S-CH
2-),7.55(m, 2H, aromatic), 7.77(m, 4H, aromatic), 8.25(m, 2H, aromatic), 12.99(brs, NH).IR(KBr, cm-1) ῡ:3265, 3153, 3040, 2912, 2852, 1697, 1554, 1517, 1342, 1323, 1155, 1103, 1064, 1020, 960, 830.MS(
m/z,%): M
++1: 455(10), M
+: 454(10), 270(40), 242(75), 225(40), 195(100), 179(95), 165(85), 136(60), 106(50), 90(50), 78(60).
N-(5-(Benzylthio)-1,3,4-thiadiazol-2-yl)-2-(4-(trifluoromethyl)phenyl)acetamide (3l)mp. 203 °C, Yield: 47%,C
18H
14F
3N
3OS
2, MW: 409 g/mol,1H NMR (DMSO-d
6 , 200 MHz)
δ: 3.99 (s, 2H, -CH
2CO-), 4.52 (s, 2H, -S-CH
2-), 7.30-7.49 (m, 5H, benzyl), 7.58 (d, 2H,
J = 8 Hz, 4-trifluoromethylphenyl), 7.75 (d, 2H,
J = 8 Hz, 4-trifluoromethylphenyl), 12.97 (s, NH).IR(KBr, cm
-1) ῡ: 3375, 3040, 1701, 1556, 1508, 1323, 1294, 1220, 1159, 1107, 1066, 1020, 827, 700.MS(
m/z, %): M
+: 410(100), 409(95), 408(95), 159(75), 148(60), 91(75).
MTT assayDiverse derivatives of 1,3,4-thiadiazole (compounds 3a-3l) were tested for cytotoxic activity at 0.1-250 μg/mL concentration in three human cancer cell lines of PC3 cell (prostate cancer), U87 (gliobalstoma) and MDA (breast cancer). Cells from different cell lines were seeded in 96-well plates at the density of 8000–10,000 viable cells per well and incubated for 48 h to allow cell attachment.The cells were then incubated for another 48-96 h (depends to cell cycle of each cell line) with various concentrations of compounds 3a-3l. Cells were then washed inPBS, and 20 μL of MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide solution (5 mg/mL) were added to each well. An additional 4 h of incubation at 37°C were done, and then the medium was discarded. Dimethyl sulfoxide (60 μL) was added to each well, and the solution was vigorously mixed to dissolve the purple tetrazolium crystals. The absorbance of each well was measured by plate reader (Anthous 2020; Austria) at a test wavelength of 550 nm against a standard reference solution at 690 nm. The amount of produced purple formazan is proportional to the number of viable cells (16 (
link)).
Aliabadi A., Hasanvand Z., Kiani A, & Mirabdali S.S. (2013). Synthesis and In-vitro Cytotoxicity Assessment of N-(5-(Benzylthio)-1,3,4- thiadiazol-2-yl)-2-(4-(trifluoromethyl)phenyl)acetamide with Potential Anticancer Activity. Iranian Journal of Pharmaceutical Research : IJPR, 12(4), 687-693.