Editor-in-Chief Hatice Kübra Elçioğlu Vice Editors Levent Kabasakal Esra Tatar Online ISSN 2630-6344 Publisher Marmara University Frequency Bimonthly (Six issues / year) Abbreviation J.Res.Pharm. Former Name Marmara Pharmaceutical Journal
Journal of Research in Pharmacy 2010 , Vol 14 , Num 2
Synthesis and cytotoxic activity of some 1,2,4-triazoline-3-thione and 2,5-disubstituted- 1,3,4-thiadiazole derivatives
Sevgi Karakuş1, Ufuk Çoruh2, Bilgehan Barlas-Durgun1, Ezequiel M. Vázquez-López3, Suna Özbaş-Turan1, Jülide Akbuğa1, Sevim Rollas1
1Marmara Üniversitesi, Eczacılık Fakültesi, İstanbul, Türkiye
2Ondokuz Mayıs Üniversitesi, Eğitim Fakültesi, Samsun, Türkiye
3Universidade de Vigo, Facultade de Ciencias- Química, Vigo, Galicia, İspanya
DOI : 10.12991/201014454

Summary

Çalışmada bir seri 5-[4-(4-florobenzoilamino)fenil]-2-sübstitüeamino-1,3,4-tiyadiazol (2a-g) ve 5-[4-(4-florobenzoilamino) fenil]-4-sübstitüe-2,4-dihidro-3H-1,2,4-triazol-3-tiyon (3a-f) türevi bileşik sentez edilmiş ve yapıları elementel analiz, UV-visibl, IR, 1H-NMR, MS spektroskopik yöntemlerle kanıtlanmıştır. Ayrıca madde 3b'nin X-ray kristalografisi ile yapısı aydınlatılmıştır. Sentez edilen bileşikler arasından prototip seçilen altı bileşiğin (2a, 2d, 2e, 3a, 3d ve 3e) sitotoksik aktiviteleri HeLa (ATCC CCL-2) ve normal hücre hattı kullanılarak MTT [3-(4,5-dimetiltiyazol-2-il)-2,5- difenil tetrazolyum bromür] yöntemine göre [Hücre Proliferasyon Kit I (MTT) Roche-Germany] incelenmiştir.

Introduction


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SCHEME 1: .

Substituted 1,3,4-thiadiazoles and 1,2,4-triazoles are important for pharmacological activity. These compounds show antibacterial[1-3], antifungal[4], antimicrobial[5-6], antimycobacterial[7-9], anti-inflammatory[10-11], antihypertensive[12], hypolipidemic[13] and anticancer[14-16] activity.

It has been reported that the thione form of 1,2,4-triazole-3-thiones were important for their antifungal activity[17]. Pospisil et al.[18] explained that tautomers are often disregarded in computer – aided molecular modeling applications. Tautomeric states of molecules are rarely registered in chemical databases.

As a continuation of our earlier study[19] on the 1-[4-(4-fluorobenzoyl-amino)benzoyl]-4-substituted thiosemicarbazides (1a-f), their cyclization products 1,3,4-thiadiazoles (2a-g) and 1,2,4-triazole- thiones (3a-f) were synthesized and characterized. In addition to the thiole↔thione tautomerism of 3b was explained by using X-ray crystallography. From the synthesized compounds (2a, 2d, 2e, 3a, 3d and 3e) were tested for their cytotoxic activities. Cell viability and cytotoxic activity profile of the compounds were analyzed using the MTT assay.

Results

The starting compounds, (1a-f) were synthesized according to the previously reported procedure[19]. 1,3,4-Thiadiazole (2a-g) were prepared from (1a-f) by reacting with concentrated sulphuric acid. Also 1,2,4-triazole derivatives (3a-f) were prepared from 1a-f by reacting with 2N sodium hydroxide. The synthetic route for compounds 2a-g and 3a-f is presented in Scheme 2.


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SCHEME 2: Synthetic route to 2a-g and 3a-f

All the synthesized compounds 2a-g and 3a-f were characterized by their melting points, elemental analysis and spectral data (UV, IR, 1H-NMR and MS). The UV spectra of 2a-g exhibited three absorption maxima at 204-208, 221-228 (shoulder) and 326-341 nm.

According to IR spectra of compounds 2a-g the bands between 1642-1670 cm-1 were assigned to the C=O group. The content of 1H-NMR spectrum of compound 2c includes a singlet at 10.40 ppm which was assigned to the –CONH- signal. The 1HNMR spectrum of 2g exhibited a singlet at 10.40 ppm that was attributable to –NH- and –CONH- groups. The molecular ion peaks at m/z 328 (m.w.: 328.3), m/z 342 (m.w.: 342.4), m/z 356 (m.w.: 356.43), m/z 354 (m.w.: 354.41), m/z 396 (m.w.: 396.49) and m/z 390 (m.w.: 390.44) were obtained from MS spectra of compounds 2a, 2b, 2c , 2d, 2e and 2g respectively. Within the context of MS spectra of compounds 2b, 2c and 2g the main fragmentation pattern existed as the removal of 4-fluorobenzoyl moiety in conformity with m/z 123 peak. The second fragmentation way is the removal of the substituent which was adjacent to amine moiety. The main MS fragmentation patterns of 2b are shown in Scheme 3.


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SCHEME 3: MS fragmentation patterns of compound 2b

The UV spectra of 3a-f showed three absorption maxima at 204-208, 243-260 and 263-293 nm. In the 1H-NMR spectra of 1,2,4-tiazole-3-thione derivatives, signals for triazole NH proton were observed between the ranges 13.65-13.82 ppm. MS spectra of compounds 3a, 3b and 3f gave molecular ion peaks at m/z 328 (m.w.: 328.39), m/z 342 (m.w.: 342.40) and m/z 390 (m.w.: 390.54) respectively. The main MS fragmentation patterns of 3a are shown in Scheme 4.


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SCHEME 4: MS fragmentation patterns of compound 3a

3,4,5-trisubstituted-1,2,4-triazoles derived from thiosemicarbazide possess two possible tautomeric forms[20]. UV and 1HNMR spectra were used to investigate the tautomerism of 3a-f.

Solvent effects on the UV spectra of compounds 3a-g were studied. The UV spectra of compound 3f exhibited maximum absorbtions at 280, 281, 286 and 281 nm in ethanol, cyclohexane, sodium hydroxide and phosphate buffer (pH:7.4) respectively. The spectrum of compound 3f that was measured in nonpolar solvent cyclohexane is similar to the other spectra evaluated by using the other solvents. This observation suggest that the compound exists in thione form (Table 1). Our suggestion constitutes a precedent of the literature by Kubota and Uda[21] that paraphrases; the band at 280 nm determined by measuring ethanolic solution of 1-methyl-3-phenyl-1,2,4-triazoline-5-thione belongs to thiocarbonile group of the compound.

TABLE 1: UV data of 3a-f

The UV spectra of compounds 3a-e were recorded in phosphate buffer (pH:7.4) and absorption maximas at 261, 257, 264, 254 and 281 nm were determined respectively. Consequently, compounds 3a-e may exist in thione↔thiole tautomeric forms in biological fluids.

The presence of the peaks due to the NH function of the triazoline ring of compounds 3a-f at 13.82, 13.69, 13.65 and 13.74 ppm supported the thione form[22,23].

These results indicated that the thione form of compounds 3a-f was the predominant tautomer in both solid and solution states. The substituent effect on tautomeric forms of these compounds was investigated. The thione form of compound 3f, which has a phenyl substituent, was attended as the most stable tautomer. These results were in good agreement with the fact that certain of thiol ↔ thione tautomerism exists predominantly in the thione forms[20,21].

Following the crystallization from ethanol the melting point of compound 3b was detected as 245-246 ºC hereafter the compound recrystallized from DMF in order to be analysed by Xray and its melting point rised to 281 ºC.

X-ray analysis showed that 5-[4-(4-fluorobenzoylamino) phenyl]-4-ethyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (3b) (Fig 1) contains three planar rings namely two benzene rings [C1-C6 (A) and C8-C13 (B)] and a triazole ring (C). The structure that was formed by all these rings involves a substitued DMF molecule. DMF was not used in the process of compound 3b synthesis, but, DMF might have been involved with the main structure during crystallization period.


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FIGURE 1: X-ray analysis of 5-[4-(4-fluorobenzoylamino)phenyl]-4-ethyl-2,4-dihydro- 3H-1,2,4-triazole-3-thione (3b)

The crystal structure is stabilized by intramolecular hydrogen bonds and intermolecular hydrogen bond, C-H…π and π…π type interactions. The hydrogen bonding details can be seen in Table 6. The C-H…π interaction involves triazole ring of a symmetry related molecule at (2-x, -y, 1-z)[C17…Cg1=3.580(5)Ǻ, H17C…Cg1=2.84Ǻ and C17-H17C…Cg1=134o; Cg1 is the centroid of ring C at (2-x, -y, 1-z)]. Finaly, the π…π stacking interaction occurs between ring C at (x, y, z) and ring A at (x, y-1, z) [ring A…Ring B=3.717(2)Ǻ] (Fig. 2, DMF molecules are excluded for clarity, Ct01 and Ct02 represent ring C and ring A, respectively).


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FIGURE 2: Intramolecular hydrogen bonds of 5-[4-(4-fluorobenzoylamino) phenyl]-4-ethyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (3b)

Experimental
Melting points were determined by using a Büchi–530 melting point apparatus. Elemental analysis were performed on a Carlo Erba 1106. UV spectra were determined on a Shimadzu UV 2100 S spectrophotometer. IR spectra were run by a Perkin Elmer 5100 spectrophotometer as KBr pellets. 1H–NMR spectra were obtained on a Bruker AC 200L spectrometer at 200 MHz using TMS as the internal reference. MS spectra were determined at 70 eV on a Kratos MS –9/50 spectrometer.

General procedure for the preparation of 5-[4-(4-fluorobenzoylamino) phenyl]-2-substitutedamino-1,3,4-thiadiazoles (2a-f)
To 0.001 mol of compounds 1a-f, concentrated sulphuric acid (1ml) was added dropwise. The mixture was stirred for 30 min. The reaction content was poured into ice-water mixture. The precipitate was washed with sodium carbonate solution and water. The crude product was dried and recrystallized from ethanol[19].

General procedure for the preparation of 5-[4-(4-fluorobenzoylamino) phenyl]-4-substituted-2,4-dihydro-3H-1,2,4-triazole-3-thiones (3a-f).
To 0.01 mol of compounds 1a-f, 2N NaOH (30 mL) was added and the mixture was heated at reflux for 1h. The reaction mixture was neutralized with hydrochloric acid. The precipitate was filtered, washed with water and recrystallized from ethanol[17].

5-[4-(4-Fluorobenzoylamino)phenyl]-2-methylamino-1,3,4- thiadiazole (2a)
This compound was obtained as pale yellow powder (ethyl alcohol), yield 0.26 g (78%); mp 267-268 ºC; UV: λ max 208 nm (ε 26532), 228 nm (ε 21344), 326 nm (ε 50733); IR (KBr): 3320, 3170, 1642, 1600, 1530, 1225, 820 cm-1 ; MS (70 eV, electron impact) m/z: 328 (M+), 327, 311, 293, 125, 100. Anal. Calcd. for C16H13FN4OS : C, 58.52; H, 3.98; N, 17.06. Found: C, 59.17; H, 4.01; N, 16.63.

5-[4-(4-Fluorobenzoylamino)phenyl]-2-ethylamino-1,3,4-thiadiazole (2b)
This compound was obtained as cream coloured needles (ethyl alcohol), yield 0.15g (64 %); mp 254-258 ºC; UV: λmax 208 nm (ε 33452), 226 nm (ε 27392), 266 nm (ε 16880), 328 nm (ε 59338); IR (KBr): 3320, 3178, 1650, 1570, 1230, 820 cm-1; 1H-NMR (DMSOd6): δ 0.94 (t, 3H, -CH2CH2CH3), 1.6 (m, 2H, -CH2CH2CH3), 3.27 (-CH2CH2CH3 was over shadow by DMSO peak), 7.35- 8.05 (m, 9H, aromatic protons and –NH-CH2CH2CH3), 10.40 (s, 1H, -CONH-); MS (70 eV, electron impact) m/z: 342 (M+), 326, 313, 241, 219, 117, 102, 95, 77, 69, 44, 28. Anal. Calcd. for C17H15FN4OS : C, 59.63; H, 4.42; N, 16.36. Found : C, 59.32; H, 4.43 ; N, 16.31.

5-[4-(4-Fluorobenzoylamino)phenyl]-2-propylamino-1,3,4- thiadiazole (2c)
This compound was obtained as yellow powder (ethyl alcohol), yield 0.32g (89 %); mp 233-237 ºC; UV: λmax 207 nm (ε 21564), 226 nm (ε 15148), 329 nm (ε 38102); IR (KBr): 3280, 3180, 2958, 2868, 1650, 1600, 1550, 1500, 1230, 830 cm-1 ;1H-NMR (DMSO-d6): δ 0.94 (t, 3H, -CH2CH2CH3), 1.6 (m, 2H, -CH2CH- 2CH3), 3.27 (-CH2CH2CH3 was over shadow by DMSO peak), 7.35-8.05 (m, 9H, aromatic protons and –NH-CH2CH2CH3), 10.40 (s, 1H, -CONH-); MS (70 eV, electron impact) m/z: 356 (M+), 341, 327, 314, 241, 123, 119, 117, 95, 65, 63. Anal. Calcd. for C18H17FN4OS : C, 60.65; H, 4.81; N, 15.72. Found : C, 60.56; H, 4.86; N, 15.30.

5-[4-(4-Fluorobenzoylamino)phenyl]-2-allylamino-1,3,4-thiadiazole (2d)
This compound was obtained as yellow powder (ethyl alcohol), yield 0.26 g (72 %); mp 219-220 ºC; UV λmax 205nm (ε 14885), 226nm (ε 9923), 326nm (ε 27998); IR (KBr): 3270, 3030, 2920, 2840, 1650, 1600, 1550, 1500, 1230, 834 cm-1; MS (70 eV, electron impact): m/z 354 (M+), 353, 339, 325, 311, 127, 124. Anal. Calcd. for C18H15FN4OS . 3/2.H2O: C, 56.68; H, 4.36. Found : C, 57.23; H, 4.35.

5-[4-(4-Fluorobenzoylamino)phenyl]-2-cyclohexylamino- 1,3,4-thiadiazole (2e)
This compound was obtained as pale yellow powder (ethyl alcohol), yield 0.36 g (69 %); mp 251 ºC; UV λmax 208nm (ε 25415), 226nm (ε 18833), 330 nm (ε 41354); IR (KBr): 3500-3000, 2922, 2843, 1670, 1600, 1525, 1500, 1230, 832 cm-1; MS (70 eV, electron impact) m/z: 396 (M+), 395, 346, 345, 339, 337, 325, 319, 318, 311, 269, 255, 217, 165, 135, 128. Anal. Calcd. for C21H21FN4OS: C, 63.61; H, 5.33; N, 14.13. Found: C, 63.67; H, 5.46; N, 13.72.

5-[4-(4-Fluorobenzoylamino)phenyl]-2-phenylamino-1,3,4- thiadiazole (2f)
This compound was obtained as yellow powder (ethyl alcohol), yield 0.25g (68 %); mp 293 ºC; UV λmax 207nm (ε 29947), 221nm (ε 21981), 341nm (ε 43651 ); IR (KBr): 3330, 3180, 3040, 1642, 1600, 1570, 1500, 1230, 840, 750 cm-1; 1H-NMR (DMSOd6): δ 7.02 (t, 1H, the proton of para position according to secondary amine), 7.36-8.10 (m, 12H, aromatic protons), 10.40 (s, 2H, -NH- and –CONH-); MS (70 eV, electron impact) m/z 390 (M+), 389, 267, 256, 150, 124, 123, 119, 95, 77, 32. Anal. Calcd. for C21H15FN4OS: C, 64.61; H, 3.93; N, 14.11. Found: C, 64.60; H, 3.87; N, 14.35.

5-[4-(4-Fluorobenzoylamino)phenyl]-2-phenylethylamino- 1,3,4-thiadiazole (2g)
This compound was obtained as yellow powder (ethyl alcohol), yield 0.09 g (75 %); mp 185 ºC; UV λmax 204 nm (ε 32391), 227 nm (ε 15275), 328 nm (ε 28960 ); IR (KBr): 3280, 3180, 3040, 2922, 2860, 1650, 1600, 1560, 1500, 1235, 840, 750 cm-1. Anal. Calcd. for C23H19FN4OS.3/2.H2O: C, 62.00; H, 4.63; N, 12.57. Found: C, 61.47; H, 4.50; N, 12.05.

5-[4-(4-Fluorobenzoylamino)phenyl]-4-methyl-2,4-dihydro- 3H-1,2,4-triazole-3-thione (3a)
This compound was obtained as cream powder (ethyl alcohol), yield 0.45 g (87 %); mp 250-253 ºC; UV λmax 204 nm (ε 33109), 260 nm (ε 24132), 293 nm (ε 23208); IR (KBr): 3286, 3197, 3098, 2937, 1641, 1603, 1505, 1480, 1447, 1439, 1241, 1181 cm-1. 1H-NMR (DMSO-d6): δ 3.48 (s, 3H, -NH-CH3), 7.38-8.10 (m, 8H, aromatic protons), 10.49 (s, 1H, -CONH-), 13.82 (s, 1H, triazole NH); MS (70 eV, electron impact) m/z 328 (M+), 205, 133, 123, 117, 95, 89, 73. Anal. Calcd. for C16H13FN4OS . H2O: C, 55.48; H, 4.36; N, 16.17. Found: C, 55.16; H, 4.25; N, 16.13.

5-[4-(4-Fluorobenzoylamino)phenyl]-4-ethyl-2,4-dihydro- 3H-1,2,4-triazole-3-thione (3b)
This compound was obtained as dark yellow powder (ethyl alcohol), yield 0.28 g (80 %); mp 245-246 ºC; UV λmax 208 nm (ε 33106), 265 nm (ε 68049); IR (KBr): 3205, 3093, 2931, 1615, 1518, 1490, 1435, 1190. MS (70 eV, electron impact) m/z 342 (M+), 341, 339, 337, 326, 325, 323, 311, 309, 297, 293, 265, 255, 253, 185, 145, 139, 125, 116 cm-1. Anal. Calcd. for C17H15FN4OS. H2O: C, 56.65; H, 4.75; N, 15.54. Found: C, 56.41; H, 4.67; N, 15.38.

5-[4-(4-Fluorobenzoylamino)phenyl]-4-propyl-2,4-dihydro- 3H-1,2,4-triazole-3-thione (3c)
This compound was obtained as dark yellow powder (ethyl alcohol), yield 0.53 g (67 %); mp 201-202 ºC; UV λmax 204 nm (ε 43435 ), 243 nm (ε 20669 ), 288 nm (ε 31790 ); IR (KBr): 3245, 3095, 2923, 2846, 1609, 1498, 1476, 1456, 1409, 1258, 1185 cm-1. 1H-NMR (DMSO-d6): δ 0.78 (t, 3H, -CH2CH2CH3), 1.53 (m, 2H, -CH2CH2CH3), 4.00 (q, 2H, -CH2CH2CH3), 6.65-8.12 (m, 8H, aromatic protons), 10.54 (s, 1H, -CONH-), 13.69 (widespread, 1H, triazole NH). Anal. Calcd. for C18H17FN4OS . H2O: C, 57.74; H, 5.11; N, 14.96. Found: C, 57.49; H, 4.92; N, 14.65.

5-[4-(4-Fluorobenzoylamino)phenyl]-4-allyl-2,4-dihydro-3H- 1,2,4-triazole-3-thione (3d)
This compound was obtained as dark yellow powder (ethyl alcohol), yield 0.51 g (91 %); mp 207-208 ºC; UV λmax 204 nm (ε 38136), 245 nm (ε 17839), 287 nm (ε 26405); IR (KBr): 3228, 3115, 3095, 2952, 2974, 1634, 1608, 1516, 1487, 1471, 1443, 1418, 1196 cm-1; 1H-NMR (DMSO-d6) δ: 4.68 (d, 2H, -CH2), 4.89 (d, 1H, allyl=CH, trans, J:17.1Hz ), 5.15 (d, 1H, allyl=CH, cis, J:10.3 Hz), 5.76-5.95 (m, 1H, CH=), 6.61-8.08 (m, 8H, aromatic protons), 10.45 (s, 1H, -CONH-), 13.65 (s, 1H, triazole NH). Anal. Calcd. for C18H15FN4OS . H2O : C, 57.75; H, 5.12; N, 14.97. Found: C, 57.73; H, 4.44; N, 14.95.

5-[4-(4-Fluorobenzoylamino)phenyl]-4-cyclohexyl-2,4-dihydro- 3H-1,2,4-triazole-3-thione (3e)
This compound was obtained as dark yellow powder (ethyl alcohol), yield 0.35 g (70 %); mp 232-235 ºC; UV λmax 206 nm (ε 30121), 263 nm (ε 50017); IR (KBr): 3233, 3109, 2927, 2850, 1608, 1506, 1489, 1456, 1404, 1254, 1190 cm-1; 1H-NMR (DMSO-d6) δ: 0.94-2.38 (m, 10H, cyclohexyl protons), 4.25 (t, 1H, cyclohexyl proton), 6.63-7.97 (m, 8H, aromatic protons), 10.52 (s, 1H, -CONH-), 13.74 (singlet, 1H, triazole NH). Anal. Calcd. for C21H21FN4OS . H2O : C, 60.85; H, 5.59; N, 13.52. Found : C, 60.61; H, 5.55; N, 13.63.

5-[4-(4-Fluorobenzoylamino)phenyl]-4-phenyl-2,4-dihydro- 3H-1,2,4-triazole-3-thione (3f)
This compound was obtained as pale yellow powder (ethyl alcohol), yield 0.27 g (86 %); mp 258-260 ºC; UV λmax 204 nm (ε 36949), 280 nm (ε 22801); IR (KBr): 3105, 3090, 2929, 2887, 1652, 1627, 1606, 1581, 1436, 1410, 1244, 1183 cm-1; 1H-NMR (DMSOd6) δ: 7.04-8.10 (m, 13H, aromatic protons), 10.49 (s, 1H, -CONH-); MS (70 eV, electron impact) m/z 390 (M+), 389, 373, 273, 272, 253, 245, 243, 237, 235, 233, 229, 227, 223, 219, 213, 191, 189, 183, 181, 176, 173, 169, 151, 135, 128, 127, 126, 117, 115, 114, 109, 105, 100. Anal. Calcd. for C21H15FN4OS. H2O : C, 61.76; H, 4.19; N, 13.72. Found : C, 62.00; H, 4.11; N, 13.69.

X-ray crystallography analysis
The structure of title compound, 5-[4-(4-fluorobenzoylamino) phenyl]-4-ethyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (3b) is shown in Fig 1 and crystal data are given in Table 2, together with refinement details. After, single crystals were selected and mounted on the tip of the glass fiber, preliminary examination and data collection were performed with Mo Kα radiation (λ=0.71073Ǻ) on an Enraff-Nonius CAD4 kappa axis difractometer operating in ω/2θ scanning mode. The structure was determined by direct methods ( SHELXS-97[24] ) and refined by full covariance matrix methods ( SHELXL-97[25] ).

TABLE 2: Crystal data and summary of data collection and structure refinement

The fractional coordinates and mean temperature factors with estimated Standard deviations for non-hydrogen atoms are listed in Table 3 and selected bond lengths are given in Table 4, selected bond angles are given in Table 5. The hydrogen bonding details are shown in Table 6. The geometric calculations and preparing material for pablication were performed using the programs SHELX-97, PARST[26] and WinGX[27].

TABLE 3: The fractional coordinates and mean temperature factors with estimated standart deviations for non-hydrogen atoms

TABLE 4: Selected bond lenghts (Å)

TABLE 5: Selected bond angles(o)

TABLE 6: Lengths (Å) and angles (o) of the hydrogen bonds

Symmetry transformations used to generate equivalent atoms: (none) x, y,z; (i) x, y-1, z; (ii) –x+1, -y, -z+1.

Sıx of the synthesized compounds (2a, 2d, 2e, 3a, 3d and 3e) were tested for their cytotoxic activities. Cell viability and cytotoxic activity profile of the compounds were analyzed using the MTT assay. MTT is cleaved to formazan by the “succinatetetrazolium reductase” system (EC 1.3.99.1) which belongs to the mitochondrial respiratory chain and is active only in viable cells[28] .

Two cell lines (normal and cancerous) were used for the determination of cytotoxic activity. HeLa cell line was used for the determination of cytotoxic activity. HeLa cells are an immortal cell line used in scientific research and were purchased from the American Type Culture Collection (ATCC CCL-2, Rockville, MD). The MTT metabolic assay was carried out in 96-well flatbottom cell culture plates seeded with 1x104 cells/well HeLa cells in 200μL MEM (Minumum Essential Medium) with 10% FBS (Fetal Bovine Serum). The following day, media was aspirated and the compounds were solved in DMSO and diluted with broth before they were added to the cell culteres at the concentrations of 5μg/mL and 10μg/mL. Cells were incubated for 48 hrs at 37 ºC, 5% CO2. After the incubation period, 10μL of the MTT labelling reagent (final concentration 0.5 mg/mL) was added to each well. After the incubation of the microplate for 4-12 hrs in a humidified atmosphere (e.g. 37 ºC, 5.0 % CO2 ) 100μL of the solubilization solution was added into each well. Following the plate was allowed to stand overnight in the incubator in a humidified atmosphere (e.g. 37 ºC, 5.0 % CO2 ) the formazan precipitates become soluable. Absorbance of formazan product was measured spectrophotometrically at 550 and 690 nm.

Based on the gained data evaluated from normal cell line procedure, four compounds which belong to our set of six compounds demonstrated inhibition between 1-10 % whereas the other two compounds demonstrated inhibition between 10-20 %.

Based on the gained data evaluated from cancerous cell line procedure, three compounds which belong to our set of six compounds demonstrated inhibition between 1-10 % whereas the other three compounds demonstrated inhibition between 10-30 %.

The highest inhibition was confirmed as 18.63 % and 16.97 % for the compounds 5-[4-(4-fluorobenzoylamino)phenyl]-2-cy- clohexylamino-1,3,4-thiadiazole (2e) and 5-[4-(4-fluorobenzoylamino) phenyl]-2-allylamino-1,3,4-thiadiazole (2d) respectively at the end of the experiments on normal cell line.

The highest inhibition was confirmed as 29.70 % and 21.97 % for the compunds 5-[4-(4-fluorobenzoylamino)phenyl]-2-cyclohexylamino- 1,3,4-thiadiazole (2e) and 5-[4-(4-fluorobenzoylamino) phenyl]-2-allylamino-1,3,4-thiadiazole (2d) respectively as a result of the experiments by using cancerous cell line.

The cytotoxic activity of the selected compounds towards cancerous cell line is higher than normal cell line. In normal cell line, the cytotoxic activity of 5-[4-(4-fluorobenzoylamino) phenyl]-2-allylamino-1,3,4-thiadiazole (2d) were found to be invariable according to the the means of dose variation. In cancerous cell line cytotoxic activity increases as a positive function of the increase in doses.

ACKNOWLEDGEMENTS
This work was supported by the Research Fund of Marmara University. Project Number: SAG-65/1998.


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FIGURE 3: Absorbance values of after MTT assay formazan crystalls (a)


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FIGURE 4: Absorbance values of after MTT assay formazan crystalls (b)


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FIGURE 5: The effect of substances on cell viability

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4) Collin X, Sauleau A, Coulon J. 1,2,4-Triazolo mercapto and aminonitriles as potent antifungal agents. Bioorg Med Chem Lett, 13: 2601-2605, 2003.

5) Doğan HN, Duran A, Rollas S, Şener G, Uysal MK, Gülen D. Synthesis of new 2,5-disubstituted-1,3,4-thiadiazoles and preliminary evaluation of anticonvulsant and antimicrobial activities. Bioorg Med Chem, 10: 2893-2898, 2002.

6) Rollas S, Yılmaz N, Erdeniz H, Kiraz M. Antimicrobial activities of some 5-(4-nitro/aminophenyl)-1, 2, 4- triazoline- 3-thione derivatives. Med Sci Res, 26: 83-84,1998.

7) Oruç E, Rollas S, Kandemirli F, Shvets N, Dimoglo AS. 1,3,4-Thiadiazole Derivatives Synthesis, Structure Elucidation and Structure-Antitüberculosis Activity Relationships Investigations. J Med Chem, 47: 6760-6767, 2004.

8) Shiradkar MR, Murahari KK, Gangadasu HR, Suresh T, Kalyan CA, Panchal D, Kaur R, Burange P, Ghogare J, Mokale V, Raut M. Bioorg Med Chem, 15: 3997-4008, 2007.

9) Küçükgüzel I, Tatar E, Küçükgüzel ŞG, Rollas S, Clercq ED. Synthesis of some novel thiourea derivatives obtained from 5-[(4-aminophenoxy)methyl]-4-alkyl/aryl- 2,4-dihydro-3H-1,2,4-triazole-3-thiones and evaluation as antiviral/anti-HIV and anti-tuberculosis agents. Eur J Med Chem, 43: 381- 392, 2008.

10) Sharma S, Srivastava VK, Kumar A. Newer N-substituted anthranilic acid derivatives as potent anti-inflammatory agents. Eur J Med Chem, 37: 689-697, 2002.

11) Palaska E, Şahin G, Kelicen P, Durlu NT, Altınok G. Synthesis and anti-inflammatory activity of 1-acylthiosemicarbazides, 1,3,4-oxadiazoles, 1,3,4-thiadiazoles and 1,2,4-triazole-3-thiones. Farmaco, 57: 101-107, 2002.

12) Vio L, Mamolo MG, Laneve A. Synthesis and antihypertensive activity of some 1,3,4-thiadiazole derivatives. Farmaco, 44: 165-172, 1988.

13) Idrees GA, AlyGamal OM, El-Din A.A. Abuo-Rahma G, Radwan MF. Design, synthesis and hypolipidemic activity of novel 2-(naphtalen-2-yloxy)propionic acid derivatives as desmethyl fibrate analogs. Eur J Med Chem, 44: 3973-3980, 2009.

14) Mavrova AT, Wesselinova D, Tsenov YA, Denkova P. Synthesis, cytotoxicity and effects of some 1,2,4-triazole and 1,3,4-thiadiazole derivatives on immunocompetent cells. Eur J Med Chem, 44: 63-69, 2009.

15) Doğan HN, Duran A, Rollas S. Synthesis and preliminary anticancer activity of new 1H-4,5-dihydro-3-(3-hydroxy- 2-naphthyl)-4-substituted-1,2,4-triazoline-5-thiones. Part II. Indian J Chem, 44B: 2301-2307, 2005.

16) Holla BS, Veerendra B, Shivananda MK, Poojary B. Synthesis characterization and anticancer activity studies on some Mannich bases derived from 1,2,4-triazoles. Eur J Med Chem, 38: 759-767, 2003.

17) Rollas S, Kalyoncuoğlu N, Sür-Altıner D, Yeğenoğlu Y. 5-(4-Aminophenyl)-4- substituted-2,4-dihydro-3H-1,2,4- triazole-3-thiones : Synthesis and antibacterial and antifungal activities. Pharmazie, 48: 308-309, 1993.

18) Pospisil P, Ballmer P, Scapozza L, Folkers G. Tautomerism in computer-aided drug design. J Recept Signal Transduct Res, 23: 361-371, 2003.

19) Durgun BB, Rollas S, Apaydın S, Öztürk R. Synthesis and antimicrobial activity of some new 1-[4-(4-fluorobenzoylamino) benzoyl]-4-substitutedthiosemicarbazides. Drug Metab and Drug Interact, 12: 145-150, 1995.

20) Sharba AHK, Al-Bayati RH, Rezki N, Aouad MR. Synthesis of thiadiazoles and 1,2,4-triazoles derived from cyclopropane dicarboxylic acid. Molecules, 10: 1153-1160, 2005.

21) Kubota S, Uda M. 1,2,4-Triazoles II. The tautomerism of 3-a-pyridyl-1,2,4-triazoline-5-thione and its methyl derivatives. Chem Pharm Bull, 20: 2096-2101, 1972.

22) Dobosz M, Pitucha M, Wujec M. The reactions of cyclization of thiosemicarbazide derivatives to 1,2,4-triazole or 1,3,4-thiadiazole system. Acta Pol. Pharm, 53: 31-38, 1996.

23) Rollas S. Synthesis and spectroscopic data of some 1,3,4-thiadiazoles. J Fac Pharm Istanbul, 17: 155-163, 1981.

24) Sheldrick GM. SHELXS-97, Program for solution of crystal structures, University of Göttingen, 1997.

25) Sheldrick GM. SHELXL-97, Program for solution of crystal structures, University of Göttingen, 1997.

26) Nardelli M. A system of Fortran routines for calculating molecular structure parameters from the results of crystal structure analyses. J Appl Cryst, 28: 659, 1995.

27) Farrugia LJ. WinGX suite for small-molecule single-crystal crystallography. J Appl Cryst, 32: 837, 1999.

28) Sgouras D, Duncan R. Methods for the Evaluation of Biocompatibility of Soluble Synthetic Polymer Which have potential for Biomedical Use: 1-Use of the Tetrazolium- Based Colorimetric assay (MTT) As a Preliminary Screen for Evaluation of In Vitro Cytotoxicity. J Mater Sci Mater Med, 1: 61-68, 1990.

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