Journal of Research in Pharmacy

Summary

Bu çalışmada 1-(α,α-difenil-α-hidroksi)asetil-4-sübstitüetiyosemikarbazid (2a-k), [5-(sübstitüeamino) -1,3,4-tiyadiazol- 2-il] (difenil)metanol (3a-b) ve 3-[hidroksi(difenil)metil]-4-(nonsübstitüe/sübstitüe)-2,4-dihidro-5H-1,2,4-triazol-5- tiyon (4a-c) yapısındaki yeni bileşikler sentez edilmiş ve bileşiklerin sitotoksik etkinlikleri araştırılmıştır. Bu amaçla benzilik asit metil esterinin hidrazin hidrat ile etanollü ortamda reaksiyonu ile benzilik asit hidrazidi (1) elde edilmiş, 1'in uygun alkil/aril isotiyosiyanatlara katımı ile 1-(α,α-difenil-α-hidroksi)asetil-4-sübstitüetiyosemikarbazid (2a-k) kazanılmıştır. [5-(sübstitüeamino)-1,3,4-tiyadiazol-2-il](difenil)metanol türevi (3a-b) bileşikler, 2a ve 2c' nin derişik sülfürik asit ile siklizasyonu sonucu elde edilmiştir. 3-[hidroksi(difenil)metil]-4-(nonsübstitüe/sübstitüe)-2,4-dihidro-5H- 1,2,4-triazol-5-tiyon türevi (4a-c) bileşikler ise 2c, 2d ve 2g'nin 2N NaOH ile reaksiyonundan kazanılmıştır. Bileşiklerin yapıları elemental analiz, HPLC, UV, IR, 1H-NMR, 13C-NMR, HSQC ve MS verileri ile doğrulanmıştır. Bileşik 2a, 2c-k, 3b ve 4b HEK293 hücre hattı kullanılarak MTT yöntemiyle sitotoksik etkinlikleri araştırmak üzere seçilmiştir. Özellikle 1-(α,α-difenil-α-hidroksi)asetil-4-siklohekzilmetiltiyosemikarbazid (2e) bileşiğinde 10 mg/ml konsantrasyonda % 50.23 sitotoksik aktivite saptanmıştır.

Summary

Introduction

The use of thiosemicarbazide in organic synthesis has become a classical strategy for the synthesis of several heterocycles. Among the increasing number of heterocyclic sulphur and nitrogen containing compounds, which are being pursued in both industry and academia, 1,3,4-thiadiazole and 1,2,4-triazole derivatives are also interesting targets for drug design. Therefore, there have been intense investigations on 1,4-disubstitutedthiosemicarbazide, 1,3,4-thiadiazole and 1,2,4-triazole- thione compounds.

1,4-Disubstituted-thiosemicarbazide are biologically versatile compounds displaying a variety of biological effects which include anti-inflammatory[1], antimycobacterial[2,3], antimicrobial[4-6], antifungal[7], antibacterial[8,9] and antiviral[3] activities. 2,5-Disubstituted 1,3,4-thiadiazoles, synthesis of which frequently include the reaction of acylthiosemicarbazides with acidic reagents such as concentrated sulfuric acid, possess various biological properties such as anticonvulsant[10], antifungal[11,12], antituberculosis[13-15], antimicrobial (16, 17), anti-inflammatory (18), cytotoxic[19], and antiproliferative[20], antioxidant[21-22] activities. In addition, 1,2,4-triazole-thiones which possess important pharmacological activities such as anticonvulsant[23], anti-inflammatory[24-28], antibacterial[29], cytotoxic[30,31], antimicrobial[32], anticancer (33,34), and antiviral[35] have found wide use in medicinal chemistry as common structures.

The present communication deals with the synthesis of 1-(α,α-diphenyl-α-hydroxy) acetyl-4- substitutedthiosemicarbazide (2a-k), [5-(substi- tutedamino) -1,3,4-thiadiazole-2-yl] (diphenyl) methanol (3ab) and 3-[hydroxy (diphenyl) methyl]-4-(nonsusbtituted/ substituted)-2,4-dihydro-5H-1,2,4-triazole-5-thione derivatives (4a-c). Their structures were confirmed by means of UV, IR, 1H-NMR, Mass spectral data and elemental analysis. All the synthesized compounds were screened for their cytotoxic activities by using HEK293 cell line of MTT assay.

EXPERIMENTAL
Chemistry
All solvents and chemicals used in this study were supplied from Aldrich, Merck and Fluka and used without purification. Melting points (ºC) were measured using Schmelzpunktbestimmer SMP II melting point apparatus, uncorrected. The reactions were monitored on Merck pre-coated aluminium TLC plates 60F–254 and the products were visualized by UV-light using ethyl asetate and ether (50:50, v/v) as solvent system. The UV spectra were recorded on a Schimadzu UV-1601 spectrophotometer. The Infrared spectra were recorded on Schimadzu FTIR-8400S Spectrophotometer and expressed in wave number ν (cm^-1). ¹H-NMR spectra were recorded on Bruker AVANCE-DPX 400 and Varian Mercury Spectrometer at 400 MHz, using DMSO-d₆ as a solvent; tetramethylsilane (TMS) was used as internal standard. All NMR chemical shifts are reported as δ values in parts per million (ppm) and coupling constant (J) are given in hertz (Hz). Mass spectra were obtained by using Agilent 1100 LC-MS and Waters 2695 Alliance Micromass. ¹H-NMR and Mass analyses were provided by Faculty of Pharmacy Center Laboratory (Ankara University) and the Scientific and Technical Research Council of Turkey, TÜBİTAK (Ankara).

Preparation of benzilic acid hydrazide (1)
Hydrazine hydrate (0.03 mol, 80%) was added to benzilic acid methyl ester (0.01mol). The mixture was refluxed at 100°C for 30 minutes. After adding ethanol (10.0 mL), the mixture was heated in a water steam bath for two hour. The residue was filtered, washed with water and recrystallized from ethanol. mp: 171–172 °C, (lit. [36] mp. 169-170 °C).

General procedure for the synthesis of 1-(α,α-diphenyl-α- hydroxy)acetyl-4-(substituted)thiosemicarbazide (2a-k)

Equimolar amounts of benzilic acid hydrazide (0.01 mol) and appropriate alkyl/aryl isothiocyanates were refluxed in ethanol (30 mL) for 2-3 hours. The crystalline product was filtered and recrystallized from ethanol to obtain 1,4-disubstitutedthiosemicarbazides.

1-(α,α-diphenyl-α-hydroxy)acetyl-4-(2-chloroethyl)thiosemicarbazide (2a): White crystals, yield 86%, mp 260–262 oC; IR (ν_max, cm^-1): 3248 (NH), 1707 (C=O), 1244 (C=S). ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 3.51 (-CH₂Cl, protons are shadowed with solvent), 3.97 (2H, t, NHCH₂), 7.08 (1H, s, OH), 7.21–7.63 (10H, m, Ar-H), 10.80 (1H, N4-H), 11.09 (1H, s, N²-H), 12.00 (1H, s, N¹-H). Anal. Calcd for C₁₇H₁₈ClN₃O₂S (363.86): C, 56.12; H, 4.99; N, 11.55; S, 8.81%. Found: C, 56.23; H, 4.85; N, 11.48; S, 8.99%. (API-ES+, m/z, %): 328 [M⁺-35.5] (100), 310, 282, 210, 209, 208, 178, 150, 143.

1-(α,α-diphenyl-α-hydroxy)acetyl-4-(4-cyanophenyl)thiosemicarbazide (2b): White crystals, yield 85%, mp 169–173 oC; IR (ν_max, cm^-1): 3363 and 3300 (NH), 1687 (C=O), 1282 (C=S). ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 6.82 (1H, s, OH), 7.24– 7.78 (14H, m, Ar-H), 9.34 (1H, N2-H), 10.12 (1H, s, N¹-H), 10.50 (1H, s, N⁴-H). Anal. Calcd for C₂₂H₁₈N⁴O₂S (402.46): C, 65.65; H, 4.51; N, 13.92; S, 7.97%. Found: C, 65.33; H, 4.34; N, 14.08; S, 8.38%. (API-ES⁺, m/z, %): 403 [M⁺], 388, 386, 385 (100), 379, 366, 355, 347, 342, 307, 301, 294, 268, 264, 254, 246.

1-(α,α-diphenyl-α-hydroxy)acetyl-4-benzoylthiosemicarbazide (2c): White crystals, yield 42%, mp 189–190 oC; IR (νmax, cm- 1): 3232 (NH), 1674 (C=O), 1251 (C=S). ¹H NMR (400 MHz, DMSO- d6) δ (ppm): 7.28 (1H, s, OH), 7.13–8.04 (15H, m, Ar-H), 10.86 (1H, N⁴-H), 11.93 (1H, s, N²-H), 13.17 (1H, s, N¹-H). Anal. Calcd for C₂₂H₁₉N₃O₃S (405.46): C, 65.17; H, 4.72; N, 10.36; S, 7.91%. Found: C, 64.91; H, 4.60; N, 10.36; S, 7.16%. (API-ES⁺, m/z, %): 407 (M⁺+H)⁺, 389 (100), 388, 338, 276, 247, 229, 199, 157.

1-(α,α-diphenyl-α-hydroxy)acetyl-4-(4-trifluoromethoxyphenyl) thiosemicarbazide (2d): White crystals, yield 48%, mp 145–146 ºC; IR (ν_max, cm^-1): 3300 and 3142 (NH), 1653 (C=O), 1261 (C=S). 1H NMR (400 MHz, DMSO-d₆) δ (ppm): 6.78 (1H, s, OH), 7.65–7.14 (12H, m, Ar-H), 9.28 (1H, N⁴-H), 9.97 (1H, s, N2-H), 10.50 (1H, s, N¹-H). Anal. Calcd for C₂₂H₁₈F- ₃N₃O₃S (461.45): C, 57.26; H, 3.93; N, 9.11; S, 6.95%. Found: C, 57.37; H, 4.00; N, 9.08; S, 6.63%. (API-CI⁺, m/z, %): 462 [M⁺], 446, 445, 444 (100), 252, 225, 223, 208, 79.

1-(α,α-diphenyl-α-hydroxy)acetyl-4-cyclohexylmethylthiosemicarbazide (2e): White crystals, yield 94%, mp 194–196 oC; IR (νmax, cm-1): 3162 (NH), 1687 (C=O), 1294 (C=S). ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 0.65–1.84 (11H, m,cyclohexyl), 3.28 (2H, m, –CH₂), 6.76 (1H, s, OH), 7.09–7.59 (11H, m, Ar-H and N4-H), 9.34 (1H, s, N2-H), 10.26 (1H, s, N¹-H). Anal. Calcd for C₂₂H₂₇N₃O₂S (397.53): C, 66.47; H, 6.85; N, 10.57; S, 8.07%. Found: C, 66.32; H, 6.84; N, 10.51; S, 8.04%. (API-ES⁺, m/z, %): 398 [M⁺], 381, 342, 304, 292, 264, 263, 225, 208, 188, 173.

1-(α,α-diphenyl-α-hydroxy)acetyl-4-(3-pyridyl)thiosemicarbazide (2f): White crystals, yield 85%, mp 184–186 ºC; IR (ν_max, cm^-1): 3182 (NH), 1651 (C=O), 1257 (C=S). ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 6.75 (1H, s, OH), 7.17–7.38 ve 7.45–7.60 (10H, m, Ar-H), 7.40 (1H, dd, J=8.07 Hz, 8.08 Hz, pyridine-H5), 7.93 (1H, d, pyridine-H4), 8.36 (1H, d, pyridine-H6), 8.53 (1H, s, pyridine-H2) 9.33 (1H, N⁴-H), 10.02 (1H, s, N2-H), 10.56 (1H, s, N¹-H). Anal. Calcd for C₂₀H₁₈N₄O₂S (378.44): C, 63.47; H, 4.79; N, 14.80; S, 8.47%. Found: C, 63.26; H, 4.83; N, 14.58; S, 8.15%. (API-CI⁺, m/z, %): 379 [M⁺] (100), 243, 226, 225, 197, 139, 138, 137.

1-(α,α-diphenyl-α-hydroxy)acetyl-4-(3,5-bistrifluoromethylphenyl) thiosemicarbazide (2g): White crystals, yield 29%, mp 166–168 oC; IR (ν_max, cm^-1): 3252 and 3186 (NH), 1651 (C=O), 1273 (C=S). ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 6.79 (1H, s, OH), 7.22–7.75 (10H, m, Ar-H), 7.88 (1H, s, Ar-H4), 8.26 (2H, s, Ar-H2, Ar-H6), 9.64 (1H, s, N²-H), 10.27 (1H, s, N¹-H), 10.55 (1H, N⁴-H). Anal. Calcd for C₂₃H₁₇F₆N₃O₂S (513.45): C, 53.80; H, 3.34; N, 8.18; S, 6.24%. Found: C, 54.44; H, 3.39; N, 8.29; S, 6.24%. (APCI^-, m/z, %): 512 (M⁺-H)+, 511 (100), 286, 252.

1-(α,α-diphenyl-α-hydroxy)acetyl-4-(2-chloro-5-trifluoromethylphenyl) thiosemicarbazide (2h): White crystals, yield 58%, mp 152–154 ºC; IR (ν_max, cm^-1): 3302 and 3180 (NH), 1693 (C=O), 1261 (C=S). 1H NMR (400 MHz, DMSO-d₆) δ (ppm): 6.65 (1H, s, OH), 7.27–7.86 (13H, m, Ar-H), 10.75 (1H, N⁴-H), 9.06 (1H, s, N²-H), 10.09 (¹H, s, N¹-H). Anal. Calcd for C₂₂H₁₇ClF₃N₃O₂S (479.90): C, 55.06; H, 3.57; N, 8.76; S, 6.68%. Found: C, 55.21; H, 3.58; N, 8.72; S, 6.82%.

1-(α,α-diphenyl-α-hydroxy)acetyl-4-(2-trifluoromethylphenyl) thiosemicarbazide (2i): White crystals, yield 64%, mp 168–170 ºC; IR (ν_max, cm^-1): 3271 and 3194 (NH), 1651 (C=O), 1278 (C=S). ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 6.64 (1H, s, OH), 7.14–7.82 (14H, m, Ar-H), 9.02 (1H, N⁴-H), 9.94 (1H, s, N2-H), 10.55 (1H, s, N¹-H). Anal. Calcd for C₂₂H₁₈F₃N₃O₂S (445.45): C, 59.32; H, 4.07; N, 9.43; S, 7.20%. Found: C, 58.36; H, 4.51; N, 8.73; S, 6.80%. (API-ES⁺, m/z, %): 446 [M⁺] (100), 428, 368, 302, 296, 279, 257, 225, 192.

1-(α,α-diphenyl-α-hydroxy)acetyl-4-(2-methylsulphanylphenyl) thiosemicarbazide (2j): White crystals, yield 63%, mp 164–166 ºC; IR (ν_max, cm^-1): 3325 and 3261 (NH), 1651 (C=O), 1255 (C=S). ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.34 (3H, s, S-CH₃), 6.72 (1H, s, OH), 7.11–7.60 (14H, m, Ar-H), 9.00 (1H, N4-H), 9.82 (1H, s, N2-H), 10.53 (1H, s, N¹-H). Anal. Calcd for C₂₂H₂₁N₃O₂S₂ (423.55): C, 62.39; H, 5.00; N, 9.92; S, 15.14%. Found: C, 62.21; H, 4.93; N, 9.89; S, 14.92%.

1-(α,α-diphenyl-α-hydroxy)acetyl-4-(1-adamantyl)thiosemicarbazide (2k): White crystals, yield 83%, mp 160–162 oC; IR (ν_max, cm^-1): 3313 and 3180 (NH), 1651 (C=O), 1282 (C=S). 1H NMR (400 MHz, DMSO-d₆) δ (ppm): 1.06 (3H, t,–CH₃ of ethanol), 1,48–2.59 (16H, m, adamantyl-H), 3.40–3.53 (2H, m,–CH₂ of ethanol), 4.36 (1H, t, OH of ethanol), 6.12 (1H, N⁴-H), 6.92 (1H, s, OH), 7.23–7.48 (10H, m, Ar-H), 9.13 (1H, s, N²-H), 10.26 (1H, s, N¹-H). ¹³C NMR (100 MHz, DMSO-d₆) δ (ppm): 19.21, 29.64, 36.58, 39.51, 39.72, 39.93, 40.14, 40.35, 40.55, 40.77, 41.54 (adamantyl), 53.68 (solvent), 56.76 (NH-adamantyl carbon), 81.05 (C-OH), 128.10, 128.32 (Ar), 144.21 (C=O), 180.00 (C=S). Anal. Calcd for C₂₅H₂₉N₃O₂S.C₂H₅OH (481.65): C, 67.33; H, 7.32; N, 8.72; S, 6.66%. Found: C, 67.14; H, 7.07; N, 8.79; S, 6.51%. (API-CI⁺, m/z, %): 436 [M⁺] (100), 418, 226, 225, 243.

General procedure for the synthesis of [5-(substitutedamino)- 1,3,4-thiadiazole-2-yl](diphenyl)methanol (3a-b)
A sample of corresponding 1,4-disubstituted-thiosemicarbazides (0.001 mol) was treated with concentrated sulphuric acid at room temperature with constant stirring for 1-2 h. The reaction mixture was poured into ice water. The product was precipitated, filtered and washed with water to afford 3a-b in quantitative yield.

[5-(ethenylamino)-1,3,4-thiadiazole-2-yl](diphenyl)methanol (3a): White powder, yield 81%, mp 202–203 ºC; IR (νmax, cm- 1): 3257 (NH), 1614 (C=N). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 3.66 (t, 2H, =CH2, J= 6.86 Hz, 13.77 Hz), 4.76 (s, 1H, OH), 6.79 (d, 1H, -CH=, J=7.70 Hz), 6.93–7.42 (m, 10 H, ArH), 7.81 ve 8.18 (2s, 1H, NH). Anal. Calcd for C17H15N3OS (309.38): C, 66.00; H, 4.89; N, 13.58; S, 10.36%. Found: C, 65.12; H, 4.86; N, 13.11; S, 10.13%. (API-ES+, m/z, %): 310 [M+] (100), 256, 208, 192.

[5-(benzoylamino)-1,3,4-thiadiazole-2-yl](diphenyl)methanol (3b): Yellow crystals, yield 87%, mp 206–208 oC; IR (ν_max, cm^-1): 3250 and 3165 (NH), 1600 (C=N). ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 7.04–8.17 (m,16 H, ArH ve OH), 12.44 (s, 1H, -NHCO). Anal. Calcd for C₂₂H₁₇N₃O₂S (387.45): C, 68.20; H, 4.42; N, 10.85; S, 8.28%. Found: C, 68.20; H, 4.61; N, 10.91; S, 8.90%.

General procedure for the synthesis of 3-[hydroxy(diphenyl) methyl]-4-(nonsusbtituted/substituted)-2,4-dihydro-5H- 1,2,4-triazole-5-thione (4a-c)
Sodium hydroxide (2N, 15-20 mL) was added to corresponding 1,4-disubstituted-thiosemicarbazides and refluxed on water bath for 4h. The reaction mixture was neutralized with hydrochloric acid (10%). The precipitate was filtered, washed with water and crystallized from ethanol.

3-[hydroxy(diphenyl)methyl]-2,4-dihydro-5H-1,2,4-triazole- 5-thione (4a): White crystals, yield 49%, mp 258 ºC; IR (ν_max, cm^-1): 3107 (NH), 1577, 1487 (C=N, NH), 1260 (C-N), 1180 (C=S), 1H NMR (400 MHz, DMSO-d₆) δ (ppm): 6.95 (1H, s, OH), 7.11–7.58 (10H, m, Ar-H), 13.20 (1H, s, NH), 13.40 (1H, s, NH). Anal. Calcd for C₁₅H₁₃N₃OS.1/2 H₂O (292.35): C, 61.62; H, 4.83; N, 14.37; S, 10.97%. Found: C, 60.97; H, 4.60; N, 13.84; S, 10.64%.

3-[hydroxy(diphenyl)methyl]-4-[4-(trifluoromethoxy) phenyl]-2,4-dihydro-5H-1,2,4-triazole-5-thione (4b): White crystals, yield 84%, mp 235–237 ºC; IR (ν_max, cm^-1): 3169 (NH), 1510, 1558 (C=N, NH), 1271 (C-N), 1155 (C=S). ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 6.91 (2H, d, Ar-H, J= 8.9 Hz), 7.00 (1H, s, OH), 7.15 (2H, d, Ar-H, J= 8.2 Hz), 7.18–7.35 (10H, m, Ar-H), 13.49 (1H, s, NH). Anal. Calcd for C₂₂H₁₆F₃N₃O₂S.H₂O (461.46): C, 57.26; H, 3.93; N, 9.11; S, 6.95%. Found: C, 58.04; H, 4.03; N, 9.05; S, 6.34%. (API-ES⁺, m/z, %): 444 [M⁺], 443, 426, 425, 388, 314, 261, 79.

4-[3,5-bis(trifluoromethyl)phenyl)]-3-[hydroxy(dipenyl) methyl]-2,4-dihydro-5H-1,2,4-triazole-5-thione (4c): White crystals, yield 78%, mp 215–216 oC; IR (ν_max, cm^-1): 3365 (NH), 1556, 1492 (C=N, NH),1276 (C-N), 1120 (C=S). 1H NMR (400 MHz, DMSO-d₆) δ (ppm): 7.12 (1H, s, OH), 7.18–7.34 (10H, m, Ar-H), 7.52 (2H, s, triazole ArC₂-H, ArC₆-H), 8.07 (1H, s, triazole ArC₄-H), 14.14 (1H, s, NH). Anal. Calcd for C₂₃H₁₅F₆N₃OS (495.44): C, 55.76; H, 3.05; N, 8.48; S, 6.47%. Found: C, 55.86; H, 2.86; N, 8.55; S, 6.19%.

Cytotoxic Activity
The synthesized compounds were tested for their cytotoxic activities. Cell viability and cytotoxic activity profile of the compounds were analyzed using the Cell Proliferation Kit I (MTT) [Roche, Germany]. MTT [3-(4,5-dimethylthiazole-2-yl)-2,5- diphenyltetrazolium bromide] is cleaved to formazan crystals by the “succinate-tetrazolium reductase” system which belongs to the mitochondrial respiratory chain and is active only in viable cells[37-38]. HEK293 cell line was used for the determination of cytotoxic activity. The MTT metabolic assay was carried out in 96-well flat-bottom cell culture plates seeded with 5x103 cells/well. HEK293 cells in 100mM containing LGlutamine without antibiotic Eagle’s MEM (Minimum Essential Medium) and RPMI 1640MEM with 10% FBS (Fetal Bovine Serum).

The following day, media was aspirated and the compounds were solved in DMSO and diluted with medium before they were added to the cell cultures at the concentrations of 5.0 μg/ mL and 10.0 μg/mL. Cells were incubated for 48 hrs at 37ºC, 5.0% CO₂. After the incubation period add 10 μL of the MTT labeling reagent (final concentration 0.5 mg/mL) to each well. Incubate the microplate for 4-12 hrs in a humidified atmos phere (e.g. 37ºC, 5.0% CO₂) and add 100 μL of the solubilization solution into each well. Allow the plate to stand overnight in the incubator in a humidified atmosphere (e.g. 37ºC, 5.0% CO₂), the formazan crystals solubilized. Absorbance of formazan product was measured with spectrophotometrically at 550 and 690 nm.

Introduction

Conclusion

A series of 1,4-disubstituted-thiosemicarbazides, 2,5-disubstituted- 1,3,4-thiadiazoles and 1,2,4-triazole-5-thiones were synthesized and screened for their cytotoxic activities against HEK293 cell line. The cytotoxicity screening indicated that among the tested compounds 1-(α,α-diphenyl-α-hydroxy) acetyl-4-cyclohexylmethylthiosemicarbazide (2e) exhibited cytotoxic activity.

ACKNOWLEDGEMENTS
This study was financially supported by the Marmara University Scientific Research Committee (Project No: SAG-CYLP- 090909-0286).

Conclusion