General
Analytical grade chemicals and solvents were purchased from Acros, Alfa Aesar, Sigma-Aldrich, and Merck. Thin layer chromatography (TLC, Merck 60 F254) was used to monitor the chemical reactions. The melting points were examined using an SMP20 melting point apparatus and were uncorrected. The FTIR spectra were acquired using a Frontier spectrometer by attenuated total reflectance (ATR) apparatus (PerkinElmer, Waltham, Massachusetts, USA). Elemental analyses (CHNS) were performed on a Thermo Scientific Flash 2000 elemental analyzer (Finnigan MAT, USA). The 1H-NMR, 13C-NMR, COSY, and HMQC spectra were recorded on an Agilent Technologies 400 MHz NMR spectrometer (Agilent, USA). The cell proliferation BrdU ELISA kits were supplied by Roche (Roche Diagnostic GmbH, Germany). The FTIR and NMR data of compounds 1-24 are presented in the supplementary data section.
Synthesis
General synthetic procedure for compounds 1-12
2-Furoic acid hydrazide (0.01 mol) and substituted phenylisothiocyanate (0.01 mol) were dissolved in methanol and the mixture was refluxed overnight. After completion of the reaction, the mixture was left to cool down in order to precipitate the solids. Finally, ethanol was used to purify the precipitate [31].
1-[(furan-2-carbonyl)]-4-[phenyl]thiosemicarbazide (1) [32]
White solid. Yield: 73%; m.p. 205-206°C. FTIR νmax (cm-1): 3659, 3317, 3135 (NH stretching), 3057 (ArCH stretching), 1670 (C=O), 1294 (C-N), 1068 (C=S). 1H NMR (400 MHz, DMSO-d6): δ 6.88 (d, 1H, J1=3.2 Hz, J2=1.6 Hz, H-2), 7.16 (t, 1H, J=7.2 Hz, H-13), 7.25 (d, 1H, J=3.2 Hz, H-3), 7.33 (t, 2H, J=7.6 Hz, H-12), 7.45 (d, 2H, J=7.2 Hz, H-11), 7.92 (brs, 1H, H-1), 9.67 (brs, 1H, H-6), 9.82 (brs, 1H, H-9), 10.42 (brs, 1H, H-7). 13C NMR (150 MHz, DMSO-d6): δ 112.33 (C-2), 115.26 (C-3), 125.49 (C-13), 126.38 (C-11), 128.35 (C-12), 139.65 (C-10), 146.09 (C-1), 146.87 (C-4), 158.07 (C-5), 181.53 (C-8). Anal. calc. for (C12H11N3O2S): C, 55.16; H, 4.24; N, 16.08; S: 12.27%; found: C, 55.10; H, 4.16; N, 16.10; S: 11.98%.
1-[(furan-2-carbonyl)]-4-[4-chlorophenyl]thiosemicarbazide (2) [32]
White solid. Yield: 71%; m.p. 218-220°C. FTIR νmax (cm-1): 3671, 3293, 3180 (NH stretching), 2989 (ArCH stretching), 1668 (C=O), 1300 (C-N), 1090 (C=S), 1013 (C-Cl). 1H-NMR (400 MHz, DMSO-d6): δ 6.68 (dd, 1H, J1=3.6 Hz, J2=1.6 Hz, H-2), 7.25 (d, 1H, J=3.2 Hz, H-3), 7.38 (d, 2H, J=8.8 Hz, H-11), 7.49 (d, 2H, J=8.0 Hz, H-12), 7.92 (d, 1H, J=0.8 Hz, H-1), 9.77 (s, 1H, H-6), 9.86 (brs, 1H, H-9), 10.44 (s, 1H, H-7). 13C NMR (150 MHz, DMSO-d6): δ 112.36 (C-2), 115.35 (C-3), 127.93 (C-11), 128.26 (C-13), 129.51 (C-12), 138.66 (C-10), 146.15 (C-1), 146.81 (C-4), 158.09 (C-5), 181.53 (C-8). Anal. calc. for (C12H10ClN3O2S): C, 48.73; H.3.41; N, 14.21; S: 10.84%; found: C, 47.99; H, 3.40; N, 14.17; S: 10.75%.
1-[(furan-2-carbonyl)]-4-[3,5-(dichloro)phenyl]thiosemicarbazide (3)
White solid. Yield: 90%; m.p. 211-213°C. FTIR νmax (cm-1): 3667, 3316, 3209 (NH stretching), 2988 (ArCH stretching), 1672 (C=O), 1298 (C-N), 1118 (C=S), 1015 (C-Cl). 1H-NMR (400 MHz, DMSO-d6): δ 6.68 (dd, 1H, J1=3.2 Hz, J2=1.6 Hz, H-2), 7.26 (d, 1H, J=3.2 Hz, H-3), 7.37 (brs, 1H, H-13), 7.71 (s, 2H, H-11), 7.94 (brs, 1H, H-1), 9.94 (brs, 1H, H-6), 10.02 (brs, 1H, H-9), 10.49 (brs, 1H, H-7). 13C NMR (150 MHz, DMSO-d6): δ 112.44 (C-2), 115.57 (C-3), 123.69 (C-13), 124.48 (C-11), 133.40 (C-12), 142.13 (C-10), 146.27 (C-1), 146.69 (C-4), 158.11 (C-5), 181.20 (C-8). Anal. calc. for (C12H9Cl2N3O2S): C, 43.65; H, 2.75; N, 12.73; S: 9.71%; found: C, 44.01; H, 2.70; N, 12.76; S: 9.88%.
1-[(furan-2-carbonyl)]-4-[{4-chloro-3-(trifluoromethyl)phenyl}]thiosemicarbazide (4)
White solid. Yield: 84%; m.p. 222-224°C. FTIR νmax (cm-1): 3671, 3315, 3211 (NH stretching), 3084 (ArCH stretching), 1672 (C=O), 1321 (C-N), 1111 (C-F), 1080 (C=S), 1015 (C-Cl). 1H-NMR (400 MHz, DMSO-d6): δ 6.70 (d, 1H, J=1.6 Hz, H-2), 7.27 (d, 1H, J=2.8 Hz, H-3), 7.68 (d, 1H, J=8.4 Hz, H-14), 7.87-7.96 (m, 2H, H-11 and H-15), 8.02 (d, 1H, J=0.8 Hz, H-1), 10.03 (s, 1H, H-6), 10.52 (s, 1H, H-9), 11.29 (s, 1H, H-7). 13C NMR (150 MHz, DMSO-d6): δ 112.45 (C-2), 112.89 (C-11), 113.33 (C-3), 122.42 (C-15), 130.79 (C-13), 131.59 (C-16), 132.96 (C-12), 139.08 (C-14), 146.28 (C-10), 146.72 (C-1), 151.82 (C-4), 159.13 (C-5), 181.41 (C-8). Anal. calc. for (C13H9ClF3N3O2S): C, 42.93; H, 2.49; N, 11.55; S: 8.82%; found: C, 42.86; H, 2.39; N, 11.62; S: 8.75%.
1-[(furan-2-carbonyl)]-4-[{5-chloro-2-(methoxy)phenyl}]thiosemicarbazide (5) [32]
White solid. Yield: 59%; m.p. 209-211°C. FTIR νmax (cm-1): 3672, 3264, 3143 (NH stretching), 2989 (ArCH stretching), 1668 (C=O), 1315 (C-N), 1086 (C=S), 1020 (C-Cl). 1H-NMR (400 MHz, DMSO-d6): δ 3.76 (s, 3H, H-16), 6.69 (d, 1H, J=1.3 Hz, H-2), 7.08 (d, 1H, J=7.8 Hz, H-12), 7.21 (d, 1H, J=7.8 Hz, H-13), 7.29 (d, 1H, J=3.3 Hz, H-3), 7.94 (brs, 2H, H-1 and H-15), 9.26 (brs, 1H, H-6), 9.93 (brs, 1H, H-9), 10.57 (brs, 1H, H-7). 13C NMR (150 MHz, DMSO-d6): δ 56.57 (C-16), 112.38 (C-2), 113.45 (C-12), 115.62 (C-3), 123.16 (C-15), 123.50 (C-13), 126.27 (C-14), 129.47 (C-10), 146.44 (C-1), 149.90 (C-4), 151.88 (C-11), 158.09 (C-5), 182.27 (C-8). Anal. calc. for (C13H12ClN3O3S): C, 47.93; H, 3.71; N, 12.90; S: 9.84%; found: C, 47.83; H, 3.61; N, 12.93; S: 9.82%.
1-[(furan-2-carbonyl)]-4-[2,4-(dimethoxy)phenyl]thiosemicarbazide (6)
Cream solid. Yield: 37%; m.p. 199-201°C. FTIR νmax (cm-1): 3285, 3186 (NH stretching), 2988 (ArCH stretching), 2836 (R-CH), 1647 (C=O), 1269 (C-N), 1027 (C=S). 1H-NMR (400 MHz, DMSO-d6): δ 3.74-3.83 (m, 6H, H-16 and H-17), 6.51 (d, 1H, J=8.4 Hz, H-14), 6.60 (brs, 1H, H-2), 6.67 (brs, 1H, H-12), 7.26 (d, 1H, J=2.8 Hz, H-3), 7.44 (d, 1H, J=8.4 Hz, H-15), 7.91 (brs, 1H, H-1), 9.11 (brs, 1H, H-6), 9.59 (brs, 1H, H-9), 10.43 (s, 1H, H-7). 13C NMR (150 MHz, DMSO-d6): δ 55.74 (C-16), 56.09 (C-17), 99.28 (C-12), 104.46 (C-14), 112.27 (C-2), 115.32 (C-3), 121.42 (C-15), 129.16 (C-10), 146.19 (C-1), 146.69 (C-4), 151.42 (C-11), 157.08 (C-13), 158.12 (C-5), 182.00 (C-8). Anal. calc. for (C14H15N3O4S): C, 52.33; H, 4.70; N, 13.08; S: 9.98%; found: C, 52.28; H, 4.69; N, 13.13; S: 9.95%.
1-[(furan-2-carbonyl)]-4-[2,5-(dimethoxy)phenyl]thiosemicarbazide (7)
White solid. Yield: 77%; m.p. 204-206°C. FTIR νmax (cm-1): 3658, 3211, 3117 (NH stretching), 2997 (ArCH stretching), 2832 (R-CH), 1668 (C=O), 1280 (C-N), 1012 (C=S). 1H-NMR (400 MHz, DMSO-d6): δ 3.70 (s, 6H, H-16 and H-17), 6.70 (d, 1H, J=2.4 Hz, H-2), 6.71 (d, 1H, J=9.2 Hz, H-13), 6.96 (d, 1H, J=9.2 Hz, H-12), 7.29 (d, 1H, J=3.2 Hz, H-3), 7.78-7.94 (m, 2H, H-1 and H-15), 9.17 (brs, 1H, H-6), 9.86 (brs, 1H, H-9), 10.55 (brs, 1H, H-7). 13C NMR (150 MHz, DMSO-d6): δ 55.81(C-17), 56.75 (C-16), 110.45 (C-13), 111.94 (C-15), 112.38 (C-2), 112.69 (C-12), 115.59 (C-3), 128.90 (C-10), 146.38 (C-1), 146.48 (C-4), 148.45 (C-11), 152.87 (C-14), 158.09 (C-5), 180.63 (C-8). Anal. calc. for (C14H15N3O4S): C, 52.33; H, 4.70; N, 13.08; S: 9.98%; found: C, 52.30; H, 4.72; N, 13.11; S: 9.93%.
1-[(furan-2-carbonyl)]-4-[3,4-(dimethoxy)phenyl]thiosemicarbazide (8)
White solid. Yield: 66%; m.p. 200-202°C. FTIR νmax (cm-1): 3629, 3310, 3125 (NH stretching), 2957 (ArCH stretching), 2836 (R-CH), 1686 (C=O), 1280 (C-N), 1023 (C=S). 1H-NMR (400 MHz, DMSO-d6): δ 3.73 (s, 3H, H-16) , 3.75 (s, 3H, H-17), 6.68 (dd, 1H, J1=3.6 Hz, J2=1.6 Hz, H-2), 6.89-6.86 (m, 2H, H-14 and H-15), 7.08 (brs, 1H, H-11), 7.25 (d, 1H, J=3.2 Hz, H-3), 7.92 (d, 1H, J=0.8 Hz, H-1), 9.58 (brs, 1H, H-6), 9.70 (brs, 1H, H-9), 10.38 (s, 1H, H-7). 13C NMR (150 MHz, DMSO-d6): δ 55.89 (C-17), 56.05 (C-16), 110.98 (C-11), 111.40 (C-2), 112.32 (C-14), 115.24 (C-3), 118.36 (C-15), 132.65 (C-10), 146.07 (C-1), 146.70 (C-13), 146.87 (C-4), 148.25 (C-12), 158.10 (C-5), 181.44 (C-8). Anal. calc. for (C14H15N3O4S): C, 52.33; H, 4.70; N, 13.08; S, 9.98%; found: C, 52.26; H, 4.61; N, 13.35; S, 9.86%.
1-[(furan-2-carbonyl)]-4-[3,5-(dimethoxy)phenyl]thiosemicarbazide (9)
White solid. Yield: 65%; m.p. 203-205°C. FTIR νmax (cm-1): 3654, 3320, 3145 (NH stretching), 2961 (ArCH stretching), 2813 (R-CH), 1689 (C=O), 1264 (C-N), 1057 (C=S). 1H-NMR (400 MHz, DMSO-d6): δ 3.72 (s, 6H, H-14), 6.31 (brs, 1H, H-2), 6.65 (s, 1H, H-13), 6.79 (brs, 2H, H-11), 7.25 (d, 1H, J=2.4 Hz, H-3), 7.92 (brs, 1H, H-1), 9.69 (s, 2H, H-6 and H-9), 10.40 (s, 1H, H-7). 13C NMR (150 MHz, DMSO-d6): δ 55.63 (C-14), 97.32 (C-13), 104.00 (C-11), 112.34 (C-2), 115.31 (C-3), 141.23 (C-10), 146.13 (C-1), 146.82 (C-4), 158.10 (C-5), 160.15 (C-12), 181.01 (C-8). Anal. calc. for (C14H15N3O4S): C, 52.33; H, 4.70; N, 13.08; S, 9.98%; found: C, 52.31; H, 4.76; N, 13.09; S, 9.94%.
1-[(furan-2-carbonyl)]-4-[3,4,5-(trimethoxy)phenyl]thiosemicarbazide (10)
White solid. Yield: 50%, m.p. 222-224°C. FTIR νmax (cm-1): 3748, 3241, 3136 (NH stretching), 2988 (ArCH stretching), 2842 (R-CH), 1681 (C=O), 1263 (C-N), 1013 (C=S). 1H-NMR (400 MHz, DMSO-d6): δ 3.65 (s, 3H, H15), 3.75 (s, 6H, H14), 6.68 (dd, 1H, J1=3.2 Hz, J2=1.6 Hz, H2), 6.89 (brs, 2H, H11), 7.26 (d, 1H, J=3.2 Hz, H3), 7.92 (brs, 1H, H1), 9.66 (brs, 2H, H6 and H9), 10.38 (brs, 1H, H7). 13C NMR (150 MHz, DMSO-d6): δ 55.25 (C14), 60.51 (C15), 103.58 (C11), 112.35 (C2), 115.34 (C3), 135.06 (C10), 135.38 (C13), 146.14 (C1), 146.81 (C4), 152.45 (C12), 158.16 (C5), 181.02 (C8). Anal. calc. for (C15H17N3O5S): C, 51.27; H, 4.88; N, 11.96; S: 9.13%; found: C, 51.23; H, 4.84; N, 11.90; S: 9.10%.
1-[(furan-2-carbonyl)]-4-[4-(dimethylamino)phenyl]thiosemicarbazide (11)
White solid. Yield: 67%; m.p. 214-216°C. FTIR νmax (cm-1): 3658, 3351, 3132 (NH stretching), 2961 (ArCH stretching), 2805 (R-CH), 1683 (C=O), 1263 (C-N), 1056 (C=S). 1H-NMR (400 MHz, DMSO-d6): δ 2.88 (s, 6H, H-14), 6.67-6.69 (m, 3H, H-2 and H-11), 7.17 (d, 2H, J=8.8 Hz, H-12), 7.24 (d, 1H, J=3.2 Hz, H-3), 7.91 (d, 1H, J=0.8 Hz, H-1), 9.45 (s, 1H, H-6), 9.59 (s, 1H, H-9), 10.35 (s, 1H, H-7). 13C NMR (150 MHz, DMSO-d6): δ 40.79 (C-14), 112.17 (C-12), 112.26 (C-2), 115.12 (C-3), 127.36 (C-11), 128.77 (C-10), 146.00 (C-1), 146.93 (C-4), 148.69 (C-13), 158.05 (C-5), 181.71 (C-8). Anal. calc. for (C14H16N4O2S): C, 55.25; H, 5.30; N, 18.41; S, 10.53%; found: C, 55.21; H, 5.28; N, 18.35; S, 10.42%.
1-[(furan-2-carbonyl)]-4-[4-(diethylamino)phenyl]thiosemicarbazide (12)
White solid. Yield: 62%; m.p. 215-217°C. FTIR νmax (cm-1): 3672, 3346, 3203 (NH stretching), 3114 (ArCH stretching), 2967 (R-CH), 1686 (C=O), 1265 (C-N), 1079 (C=S). 1H-NMR (400 MHz, DMSO-d6): δ 1.08 (t, 6H, J=6.8 Hz, H-15), 3.30 (q, 4H, H-14), 6.60 (d, 2H, J= 8.8 Hz, H-11), 6.70 (dd, 1H, J1=3.2 Hz, J1=1.6 Hz, H-2), 7.12 (d, 2H, J=8.4 Hz, H-12), 7.23 (d, 1H, J=3.2 Hz, H-3), 7.90 (brs, 1H, H-1), 9.42 (s, 1H, H-6), 9.55 (s, 1H, H-9), 10.34 (s, 1H, H-7). 13C NMR (150 MHz, DMSO-d6): δ 12.88 (C-15), 44.16 (C-14), 111.25 (C-2), 112.26 (C-12), 115.10 (C-3), 127.63 (C-11), 127.70 (C-10), 145.68 (C-13), 146.00 (C-1), 146.93 (C-4), 158.02 (C-5), 181.55 (C-8). Anal. calc. for (C16H20N4O2S): C, 57.81; H, 6.06; N, 16.85; S, 9.65%; found: C, 57.86; H, 6.03; N, 16.77; S, 9.62%.
General synthetic procedure for compounds 13-24
A mixture of thiosemicarbazides 1-12 (0.45 mmol) and 2 N NaOH solution (10 mL) was refluxed for 4 h. The reaction mixture was filtered, allowed to cool, and then brought to pH 5.0-6.0 with a dilute solution of HCl. The crude product was dried, washed with water, and recrystallized from ethanol [31].
5-(2-furyl)-4-phenyl-2,4-dihydro-1,2,4-triazole-3-thione (13) [32]
Cream solid. Yield: 67%; m.p. 209-211°C. FTIR νmax (cm-1): 3094 (NH stretching), 2986 (ArCH stretching), 1272 (C-N), 1015 (C=S). 1H-NMR (400 MHz, DMSO-d6): δ 5.89 (d, 1H, J=3.6 Hz, H-3), 6.50 (dd, 1H, J1=3.6 Hz, J2=1.6 Hz, H-2), 7.43-7.45 (m, 2H, H-12), 7.59-7.61 (m, 3H, H-11 and H-13), 7.81 (d, 1H, J=1.2 Hz, H-1), 14.15 (s, 1H, H-7). 13C NMR (100 MHz, DMSO-d6): δ 112.07 (C-2), 113.02 (C-3), 129.09 (C-11), 130.40 (C-13), 134.81 (C-12), 140.31 (C-10), 143.61 (C-1), 145.74 (C-4), 158.84 (C-5), 169.18 (C-8). Anal. calc. for (C12H9N3OS): C, 59.24; H, 3.73; N, 17.27; S, 13.18%; found: C, 59.21; H, 3.75; N, 17.12; S, 13.11%.
5-(2-furyl)-4-(4-chlorophenyl)-2,4-dihydro-1,2,4-triazole-3-thione (14) [32]
White solid. Yield: 52%; m.p. 224-226°C. FTIR νmax (cm-1): 3092 (NH stretching), 2982 (ArCH stretching), 1265 (C-N), 1091 (C=S), 1024 (C-Cl). 1H-NMR (400 MHz, DMSO-d6): δ 6.11 (d, 1H, J=3.6 Hz, H-3), 6.54 (dd, 1H, J1=3.6, J2=1.2 Hz, H-2), 7.51 (d, 2H, J=8.8 Hz, H-11), 7.67 (d, 2H, J=8.8 Hz, H-12), 7.82 (d, 1H, J=1.2 Hz, H-1), 14.02 (s, 1H, H-6). 13C NMR (100 MHz, DMSO-d6): δ 112.25 (C-2), 113.21 (C-3), 130.13 (C-11), 131.17 (C-12), 133.71 (C-13), 135.12 (C-10), 140.17 (C-1), 143.45 (C-4), 145.89 (C-5), 168.97 (C-8). Anal. calc. for (C12H8ClN3OS): C, 51.90; H, 2.90; N, 15.13; S, 11.55%; found: C, 51.82; H, 2.81; N, 15.16; S, 11.57%.
5-(2-furyl)-4-(3,5-dichlorophenyl)-2,4-dihydro-1,2,4-triazole-3-thione (15)
Cream solid. Yield: 85%; m.p. 217-219°C. FTIR νmax (cm-1): 3056 (NH stretching), 2971 (ArCH stretching), 1278 (C-N), 1104 (C=S), 980 (C-Cl). 1H-NMR (400 MHz, DMSO-d6): δ 6.28 (d, 1H, J=3.6 Hz, H-3), 6.58 (dd, 1H, J1=3.6 Hz, J2=1.6 Hz, H-2), 7.73 (d, 1H, J=1.6 Hz, H-1), 7.83 (brs, 1H, H-13), 7.87-7.90 (m, 2H, H-11), 14.22 (s, 1H, H-7). 13C NMR (100 MHz, DMSO-d6): δ 112.38 (C-2), 113.30 (C-3), 128.71 (C-13), 134.93 (C-11), 137.05 (C-12), 140.09 (C-10), 143.20 (C-1), 145.97 (C-4), 154.28 (C-5), 168.86 (C-8). Anal. calc. for (C12H7Cl2N3OS): C, 46.17; H, 2.26; N, 13.46; S. 10,27%; found: C, 46.23; H, 2.34; N, 13.35 ; S, 10.29%.
5-(2-furyl)-4-(4-chloro-3-(trifluoromethyl)phenyl)-2,4-dihydro-1,2,4-triazole-3-thione (16)
White solid. Yield: 72%; m.p. 229-231°C. FTIR νmax (cm-1): 3080 (NH stretching), 2971 (ArCH stretching), 1275(C-N), 1131 (C-F), 1073 (C-Cl), 1071 (C=S). 1H-NMR (400 MHz, DMSO-d6): δ 6.30 (d, 1H, J=3.6 Hz, H-3), 6.56 (dd, 1H, J1=3.6, J2=1.6 Hz, H-2), 7.78-8.01 (m, 2H, H-11 and H-15), 7.97 (d, 1H, J=8.4 Hz, H-14), 8.14 (d, 1H, J=2.4 Hz, H-1), 14.21 (brs, 1H, H-7). 13C NMR (100 MHz, DMSO-d6): δ 112.35 (C-2), 113.31 (C-3), 124.17 (C-11), 129.31 (C-13), 132.70 (C-16), 134.33 (C-15), 135.27 (C-12), 140.19 (C-14), 142.34 (C-10), 143.27 (C-1), 145.91 (C-4), 155.48 (C-5), 168.90 (C-8). Anal. calc. for (C13H7ClF3N3OS): C, 45.16; H, 2.04; N, 12.15; S, 9.27%; found: C, 45.07; H, 2.11; N, 12.01; S, 9.30%.
5-(2-furyl)-4-(5-chloro-2-methoxyphenyl)-2,4-dihydro-1,2,4-triazole-3-thione (17) [32]
White solid. Yield: 88%; m.p. 216-218°C. FTIR νmax (cm-1): 3094 (NH stretching), 2971 (ArCH stretching), 2773 (R-CH), 1281 (C-N), 1028 (C=S), 980 (C-Cl). 1H-NMR (400 MHz, DMSO-d6): δ 3.69 (s, 3H, H-16), 6.14 (d, 1H, J=3.6 Hz, H-3), 6.54 (dd, 1H, J1=3.6, J2=1.6 Hz, H-2), 7.30 (d, 1H, J=8.8 Hz, H-13), 7.61-7.66 (m, 2H, H-12 and H-15), 7.81 (d, 1H, J=1.2 Hz, H-1), 14.02 (brs, 1H, H-7). 13C NMR (150 MHz, DMSO-d6): δ 56.96 (C-16), 112.10 (C-2), 112.21 (C-3), 115.16 (C-12), 124.39 (C-15), 124.58 (C-13), 130.56 (C-10), 131.87 (C-14), 140.49 (C-11), 143.70 (C-1), 145.69 (C-4), 154.63 (C-5), 169.22 (C-8). Anal. calc. for (C13H10ClN3O2S): C, 50.73; H, 3.28; N, 13.65; S, 10.42%; found: C, 50.59; H, 3.23; N, 13.58; S, 10.29%.
5-(2-furyl)-4-(2,4-dimethoxyphenyl)-2,4-dihydro-1,2,4-triazole-3-thione (18)
Brown solid. Yield: 65%; m.p. 205-207°C. FTIR νmax (cm-1): 3286 (NH stretching), 2918 (ArCH stretching), 1211 (C-N), 1048 (C=S). 1H-NMR (400 MHz, DMSO-d6): 3.67 (s, 3H, H-17), 3.86 (s, 3H, H-16), 5.96 (brs, 1H, H-3), 6.51 (brs, 1H, H-2), 6.70 (d, 1H, J=7.2 Hz, H-14), 6.79 (brs, 1H, H-12), 7.29 (d, 1H, J=8.0 Hz, H-15), 7.81 (brs, 1H, H-1), 14.02 (s, 1H, H-7). 13C NMR (150 MHz, DMSO-d6): δ 56.10 (C-16), 56.54 (C-17), 100.20 (C-12), 106.14 (C-14), 111.97 (C-2), 112.19 (C-3), 115.83 (C-15), 131.22 (C-10), 144.21 (C-1), 145.67 (C-4), 156.36 (C-11), 162.28 (C-5), 169.46 (C-8). Anal. calc. for (C14H13N3O3S): C, 55.43; H, 4.32; N, 13.85; S, 10.57%; found: C, 55.40; H, 4.31; N, 13.76; S, 10.55%.
5-(2-furyl)-4-(2,5-dimethoxyphenyl)-2,4-dihydro-1,2,4-triazole-3-thione (19)
Cream solid. Yield: 85%; m.p. 210-212°C. FTIR νmax (cm-1): 3082 (NH stretching), 2913 (ArCH stretching), 2776 (R-CH), 1224 (C-N), 1043 (C=S). 1H-NMR (400 MHz, DMSO-d6): δ 3.61 (s, 3H, H-16), 3.75 (s, 3H, H-17), 5.94 (d, 1H, J=3.6 Hz, H-3), 6.50 (q, 1H, H-2), 7.03 (d, 1H, J=2.8 Hz, H-15), 7.13 (d, 1H, J=9.2, H-13), 7.19 (d, 1H, J=9.2 Hz, H-12), 7.79 (brs, 1H, H-1), 14.00 (s, 1H, H-7). 13C NMR (150 MHz, DMSO-d6): δ 56.30 (C-17), 56.97 (C-16), 111.51 (C-2), 112.07 (C-3), 114.67 (C-15), 116.58 (C-13), 116.87 (C-12), 124.17 (C-10), 140.92 (C-11), 149.63 (C-14), 143.91 (C-1), 145.32 (C-4), 153.76 (C-5), 169.27 (C-8). Anal. calc. for (C14H13N3O3S): C, 55.43; H, 4.32; N, 13.85; S, 10.57%; found: C, 55.40; H, 4.39; N, 13.75; S, 10.36%.
5-(2-furyl)-4-(3,4-dimethoxyphenyl)-2,4-dihydro-1,2,4-triazole-3-thione (20)
Cream solid. Yield: 93%; m.p. 206-208°C. FTIR νmax (cm-1): 3276 (NH stretching), 2974 (ArCH stretching), 2770 (R-CH), 1221 (C-N), 1043 (C=S). 1H-NMR (400 MHz, DMSO-d6): δ 3.72 (s, 3H, H-17), 3.85 (s, 3H, H-16), 5.92 (d, 1H, J=2.8 Hz, H-3), 6.51 (dd, 1H, J1=3.2, J2=2.8 H-2), 6.95 (d, 1H, J=8.4, H-14), 7.07-7.13 (m, 2H, H-11 and H-15), 7.82 (brs, 1H, H-1), 14.05 (brs, 1H, H-7). 13C NMR (150 MHz, DMSO-d6): δ 56.28 (C-17), 56.50 (C-16), 112.43 (C-2), 112.11 (C-11), 112.91 (C-3), 113.06 (C-14), 121.48 (C-15), 127.34 (C-10), 140.51 (C-13), 149.74 (C-12), 143.92 (C-1), 145.57 (C-4), 150.35 (C-5), 169.37 (C-8). Anal. calc. for (C14H13N3O3S): C, 55.43; H, 4.32; N, 13.85; S, 10.57%; found: C, 55.42; H, 4.30; N, 13.81; S, 10.48%.
5-(2-furyl)-4-(3,5-dimethoxyphenyl)-2,4-dihydro-1,2,4-triazole-3-thione (21)
Yellow solid. Yield: 91%; m.p. 209-211°C. FTIR νmax (cm-1): 3218 (NH stretching), 2970 (ArCH stretching), 2771 (R-CH), 1207 (C-N), 1159 (C=S). 1H-NMR (400 MHz, DMSO-d6): δ 3.76 (s, 6H, H-14), 6.03 (d, 1H, J=3.2 Hz, H-3), 6.53 (dd, 1H, J1=3.2, J2=1.6 Hz, H-2), 6.63 (d, 2H, J=2.0 Hz, H-11), 6.71 (d, 1H, J=2.0, H-13), 7.83 (d, 1H, J= 1.6 Hz, H-1), 13.97 (brs, 1H, H-7). 13C NMR (100 MHz, DMSO-d6): δ 56.15 (C-14), 102.07 (C-13), 112.21 (C-2), 112.88 (C-3), 107.54 (C-11), 136.38 (C-10), 140.29 (C-1), 143.51 (C-4), 145.69 (C-5), 161.35 (C-12), 168.82 (C-8). Anal. calc. for (C14H13N3O3S): C, 55.43; H, 4.32; N, 13.85; S, 10.57%; found: C, 55.40; H, 4.28; N, 13.81; S, 10.51%.
5-(2-furyl)-4-(3,4,5-trimethoxyphenyl)-2,4-dihydro-1,2,4-triazole-3-thione (22)
Cream solid. Yield: 47%; m.p. 228-230°C. FTIR νmax (cm-1): 3242 (NH stretching), 2972 (ArCH stretching), 2769 (R-CH), 1235 (C-N), 1128 (C=S). 1H-NMR (400 MHz, DMSO-d6): δ 3.74 (s, 6H, H-14), 3.77 (s, 3H, H-15), 5.98 (d, 1H, J=3.6 Hz, H-3), 6.54 (dd, 1H, J1=3.6 Hz, J2=1.6 Hz, H-2), 6.84 (brs, 2H, H-11), 7.92 (d, 1H, J=1.2 Hz, H-1), 14.00 (s, 1H, H-7). 13C NMR (100 MHz, DMSO-d6): δ 56.81 (C-15), 60.69 (C-14), 112.28 (C-2), 112.95 (C-3), 107.06 (C-11), 130.18 (C-10), 138.93 (C-13), 140.31 (C-1), 143.37 (C-4), 145.67 (C-12), 153.80 (C-5), 168.99 (C-8). Anal. calc. for (C15H15N3O4S): C, 54.04; H, 4.54; N, 12.60; S, 9.62%; found: C, 54.07; H, 4.56; N, 12.57; S, 9.58%.
5-(2-furyl)-4-(4-(dimethylamino)phenyl)-2,4-dihydro-1,2,4-triazole-3-thione (23)
Cream solid. Yield: 89%; m.p. 221-223°C. FTIR νmax (cm-1): 3196 (NH stretching), 2972 (ArCH stretching), 2770 (R-CH), 1327 (C-N), 1230 (C=S). 1H-NMR (400 MHz, DMSO-d6): δ 3.00 (s, 6H, H-14), 5.86 (d, 1H, J=3.2 Hz, H-3), 6.51 (dd, 1H, J1=3.6, J2= 3.6 Hz, H-2), 6.82 (d, 2H, J=8.8 Hz, H-11), 7.15 (d, 2H, J=8.8 Hz, H-12), 7.83 (brs, 1H, H-1), 14.02 (brs, 1H, H-7). 13C NMR (100 MHz, DMSO-d6): δ 40.72 (C-14), 112.05 (C-2), 112.56 (C-3), 112.81 (C-12), 122.59 (C-11), 129.32 (C-10), 140.64 (C-13), 144.14 (C-1), 145.56 (C-4), 151.32 (C-5), 169.51 (C-8). Anal. calc. for (C14H14N4OS): C, 58.72; H, 4.93; N, 19.57; S, 11.20%; found: C, 58.66; H, 4.95; N, 19.48; S, 11.14%.
5-(2-furyl)-4-(4-(diethylamino)phenyl)-2,4-dihydro-1,2,4-triazole-3-thione (24)
Cream solid. Yield: 80%; m.p. 219-220°C. FTIR νmax (cm-1): 3076 (NH stretching), 2977 (ArCH stretching), 2767 (R-CH), 1270 (C-N), 1190 (C=S). 1H-NMR (400 MHz, DMSO-d6): δ 1.15 (t, 6H, J=6.8 Hz, H-15), 3.41 (q, 4H, H-14), 5.87 (d, 1H, J=3.6 Hz, H-3), 6.53 (dd, 1H, J1=3.6, J2=1.6 Hz, H-2), 6.77 (d, 2H, J=9.2 Hz, H-11), 7.12 (d, 2H, J=9.2 Hz, H-12), 7.85 (d, 1H, J=0.8 Hz, H-1), 14.02 (s, 1H, H-7). 13C NMR (100 MHz, DMSO-d6): δ 12.81 (C-15), 44.22 (C-14), 111.82 (C-2), 112.07 (C-3), 112.84 (C-12), 121.58 (C-11), 129.55 (C-10), 140.64 (C-13), 144.25 (C-1), 145.56 (C-4), 148.73 (C-5), 169.57 (C-8). Anal. calc. for (C16H18N4OS): C, 61.12; H, 5.77; N, 17.82; S, 10.20%; found: C, 61.10; H, 5.65; N, 17.71; S, 10.24%.
Biological studies
Antiproliferative activity assay
The antiproliferative assessment of the compounds was conducted on HeLa (cervical) cells using the BrdU ELISA [33-36]. The results of this in vitro investigation were given as means ± SEM of six parallel measurements (p<0.01). IC50 values were calculated using the online software ED50 plus v1.0.
Antioxidant activity assays
Solutions of thiosemicarbazides 1-12 and 1,2,4-triazole 23-24 were prepared at four different concentrations (25, 50, 100, 200 µM) in DMSO. Activity was compared using control (DMSO) and antioxidant standards (BHA and α-TOC). The results were presented as 50% inhibition versus concentration (IC50) for the ABTS˙+ scavenging activity, β-carotene-linoleic acid, and DPPH˙ assays, while in the CUPRAC assay the results were expressed as 0.5 absorbance versus concentration (A0.5). As given in the literature, ABTS˙+ scavenging activity [37], lipid peroxidation inhibitory activity (β-carotene-linoleic acid assay) [38], cupric reducing antioxidant capacity (CUPRAC assay) [39], and DPPH radical scavenging activity [40] assays were used to determine the antioxidant activity of the thiosemicarbazides 1-12 and 1,2,4-triazoles 23-24.
In silico ADME prediction
Computational studies of the synthesized compounds 1-24 were performed to predict molecular properties using the SwissADME online server [41]. The molecular volume (Mv), molecular weight (Mw), logarithm of partition coefficient (milog P), number of hydrogen-bond donors (HBDs), number of hydrogen-bond acceptors (HBAs), topological polar surface area (TPSA), number of rotatable bonds (Nrotbs), and Lipinski’s rule of five of the synthesized compounds were determined.
Statistical analysis
The antiproliferative and antioxidant activity data were given as the means of six and three parallel measurements, respectively. All biological activity assays were carried out at four different concentrations, and the results were presented as IC50 values. The data were recorded as mean ± SEM (standard error of the mean); p<0.01.