Materials and methods
The 1H NMR and 13C NMR spectra of the compounds were recorded in DMSO-d6 using an Agilent NMR VNMRS spectrometer at 400 MHz and 100 MHz, respectively. TMS was used as an internal standard. The IR spectra were measured in ATR using a Perkin Elmer FT-IR Spectrometer Frontier. The mass spectra were measured with a Thermo TSQ Quantum Access Max LC–MS/MS spectrometer. The elemental analysis of the compounds was performed using a LECO 932 CHNS device and the results were within ± 0.4% of the theoretical values. Melting points were recorded on a Thermo Scientific IA9000 series apparatus and were uncorrected. All of the chemicals were obtained from Sigma-Aldrich Chemicals.
General synthesis of 2-amino-1,3,4-thiadiazole derivatives (3,4)
In a round-bottomed flask, compounds 1 or 2 (0.075 mol) and thiosemicarbazide (0.100 mol) in trifluoroacetic acid (5 ml) at 60 °C were stirred for 3–5 h. After completion of the reaction, the reaction mixture was poured into 250 ml ice-water mixture and neutralized with diluted ammonia. The solution was filtered, and solid substance was obtained. The solid substance was washed with water, ethyl alcohol, and diethyl ether, respectively. The solid was recrystallized from the appropriate solvent. The pure substance is dried with P2O5 vacuum oven. Finally, the structures of the synthesized compounds were elucidated with FT-IR, 1H NMR, 13C NMR, mass spectroscopy, and elemental analysis. The spectral data and the physical properties of the products are listed below.
5-(2,6-Dichlorobenzyl)-1,3,4-thiadiazol-2-amine (3)
White solid, yield: 15.79 g (81%), m.p. 219–220 °C (DMF-EtOH, 1:5). IR (ATR, cm−1): 3258–3098 (–NH2), 3080 (Ar–CH), 2976 (Aliphatic CH), 1582 (C=N). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 4.40 (s, 2H, –CH2), Arom-H [7.49 (d, J = 7.8 Hz, 2H), 7.36 (t, J = 7.8 Hz, 1H)], 7.69 (bs, 2H, NH2). 13C NMR (400 MHz, DMSO-d6, δ ppm): 32.16 (–CH2), Arom-C [129.09 (CH), 130.49 (CH), 133.40 (C), 135.44 (C)], Thiadiazole-C [154.37 (C), 169.39 (C)]. Anal. Calcd. for C9H7Cl2N3S: C, 41.55; H, 2.71; N, 16.15. Found: C, 41.43; H, 2.76; N, 15.99. MS: m/z 260 (M+, 74); 261 (M + 1, 51).
5-(2-Chloro-6-fluorobenzyl)-1,3,4-thiadiazole-2-amine (4)
White solid, yield: 15.35 g (84%), m.p. 212–213 °C (DMF-EtOH, 1:6). IR (ATR, cm−1): 3261–3106 (–NH2), 3067 (Ar–CH), 2968 (Aliphatic CH), 1597 (C=N). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 4.25 (s, 2H, –CH2), Arom-H [7.35 (d, J = 12.0 Hz, 2H), 7.25 (t, J = 7.8 Hz, 1H)], 7.08 (bs, 2H, NH2). 13C NMR (400 MHz, DMSO-d6, δ ppm): 27.34 (–CH2), Arom-C [115.12 (CH), 123.98 (CH), 125.98 (C), 130.44 (C), 134.80 (CH), 160.02 (C)], Thiadiazole-C [154.52 (C), 169.21 (C)]. Anal. Calcd. for C9H7ClFN3S: C, 44.36; H, 2.90; N, 17.24. Found: C, 44.43; H, 2.87; N, 17.17. MS: m/z 243.73 (M+, 100).
General acylation reactions of 2-amino-1,3,4-thiadiazole derivatives (6a–n, 7a–n)
In a two-necked flask, compounds 3 or 4 (0.004 mol) were solved in dry benzene (40 ml) and added pyridine (1 ml) to this solution. Acyl chloride derivatives (5a–n) (0.004 mol) were added drop-wise to this solution at room temperature with the assistance of a dropping funnel. The mixture was then refluxed and stirred for 4–6 h. The progress of the reaction was monitored by TLC at appropriate time intervals. After completion of the reaction, the solution was filtered, and the solid matter was obtained. It was washed with deionized water, ethanol and diethyl ether, respectively. The solid matter was recrystallized from the appropriate solvent. All physical properties and spectral data derived from the obtained products are given below.
N-(5-(2,6-Dichlorobenzyl)-1,3,4-thiadiazol-2-yl)benzamide (6a)
White solid, yield: 1.14 g (78%), m.p. 279–280 °C (DMF-EtOH, 1:1). IR (ATR, cm−1): 3169 (–NH–), 3061 (Ar–CH), 2983 (Aliphatic CH), 1667 (C=O), 1582 (C=N). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 4.62 (s, 2H, –CH2), Arom-H [8.06 (d, J = 6.4 Hz, 2H), 7.61 (d, J = 6.0 Hz, 1H), 7.54 (bs, 2H), 7.52 (bs, 2H), 7.39 (t, J = 7.6 Hz, 1H)], 12.99 (s, 1H, NH). 13C NMR (400 MHz, DMSO-d6, δ ppm): 31.80 (–CH2), Arom-C [128.80 (CH), 129.08 (CH), 129.22 (CH), 130.64 (CH), 131.90 (CH), 133.41 (C), 134.64 (C), 135.52 (C)], Thiadiazole-C [159.86 (C), 160.84 (C)], 165.53 C=O. Anal. Calcd. for C16H11Cl2N3OS: C, 52.76; H, 3.04; N, 11.54. Found: C, 52.68; H, 3.00; N, 11.41. MS: m/z: 363.68 (M-1, 100), 365.85 (M + 1, 84).
N-(5-(2,6-Dichlorobenzyl)-1,3,4-thiadiazol-2-yl)-4-nitrobenzamide (6b)
White solid, yield: 1.21 g (74%), m.p. 320–321 °C (DMF-EtOH, 1:15). IR (ATR, cm−1): 3133 (–NH–), 3044 (Ar–CH), 2930 (Aliphatic CH), 1677 (C=O), 1596 (C=N). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 4.62 (s, 2H, –CH2), Arom-H [8.29 (d, 4H), 7.53 (bs, 2H), 7.39 (bs, 1H)], 13.40 (s, 1H, NH). 13C NMR (400 MHz, DMSO-d6, δ ppm): 31.80 (–CH2), Arom-C [124.09 (CH), 126.64 (CH), 129.23 (CH), 130.41 (CH), 133.57 (C), 135.51 (C), 139.82 (C), 150.33 (C)], Thiadiazole-C [159.88 (C), 160.81 (C)], 165.40 C=O. Anal. Calcd. for C16H10Cl2N4O3S: C, 46.96; H, 2.46; N, 13.69. Found: C, 46.88; H, 2.59; N, 13.562. MS: m/z: 408.93 (M+, 100), 410.96 (M + 1, 93).
N-(5-(2,6-Dichlorobenzyl)-1,3,4-thiadiazol-2-yl)-4-(methylthio)benzamide (6c)
White solid, yield: 1.13 g (69%), m.p. 282–283 °C (DMF-EtOH, 1:1). IR (ATR, cm−1): 3132 (–NH–), 3035 (Ar–CH), 2921 (Aliphatic CH), 1674 (C=O), 1595 (C=N). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 2.48 (t, 3H, –CH3), 4.61 (s, 2H, –CH2), Arom-H [8.00 (d, J = 8.0 Hz, 2H), 7.54 (d, J = 8.4 Hz, 2H), 7.37 (m, 3H)], 12.90 (s, 1H, NH). 13C NMR (400 MHz, DMSO-d6, δ ppm): 14.95 (CH3), 31.79 (–CH2), Arom-C [125.32 (CH), 127.54 (CH), 129.22 (CH), 130.64 (CH), 133.66 (C), 135.52 (C), 139.63 (C), 145.70 (C)], Thiadiazole-C [160.46 (C), 163.66 (C)], 165.63 C=O. Anal. Calcd. for C17H13Cl2N3OS2: C, 49.76; H, 3.19; N, 10.24. Found: C, 49.68; H, 3.06; N, 10.12. MS (ESI–m/z): 411.38 (M + 1, 76), 413.34 (M + 2, 48).
N-(5-(2,6-Dichlorobenzyl)-1,3,4-thiadiazol-2-yl)-3,4-dichlorobenzamide (6d)
White solid, yield: 1.25 g (73%), m.p. 295–296 °C (DMF-EtOH, 1:15). IR (ATR, cm−1): 3142 (–NH–), 3084 (Ar–CH), 2967 (Aliphatic CH), 1682 (C=O), 1560 (C=N). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 4.62 (s, 2H, –CH2), Arom-H [8.30 (s, 1H), 7.99 (d, J = 8.0 Hz, 1H), 7.80 (d, J = 8.4 Hz, 1H), 7.53 (d, J = 8.0 Hz, 2H), 7.38 (t, J = 8.0, 7.2 Hz, 1H)], 13.15 (s, 1H, NH). 13C NMR (400 MHz, DMSO-d6, δ ppm): 31.84 (–CH2), Arom-C [129.05 (CH), 129.22 (CH), 129.97 (CH), 130.68 (CH), 130.76 (CH), 131.42 (C), 131.98 (C), 133.54 (C), 135.51 (C), 136.16 (C)], Thiadiazole-C [159.85 (C), 160.96 (C)], 166.16 C=O. Anal. Calcd. for C16H9Cl4N3OS: C, 44.37; H, 2.09; N, 9.70. Found: C, 44.23; H, 2.01; N, 9.57. MS (ESI–m/z): 433.71 (M + 1, 100).
N-(5-(2,6-Dichlorobenzyl)-1,3,4-thiadiazol-2-yl)-3,4-difluorobenzamide (6e)
White solid, yield: 1.30 g (80%), m.p. 304–305 °C (DMF-EtOH, 1:15). IR (ATR, cm−1): 3142 (–NH–), 3087 (Ar–CH), 2941 (Aliphatic CH), 1663 (C=O), 1563 (C=N). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 4.61 (s, 2H, –CH2), Arom-H [8.13 (t, 1H), 7.95 (s, 1H), 7.60 (q, 1H), 7.53 (d, J = 8.4 Hz, 2H), 7.38 (t, J = 8.0, 8.0 Hz, 1H)], 13.09 (s, 1H, NH). 13C NMR (400 MHz, DMSO-d6, δ ppm): 31.82 (–CH2), Arom-C [118.32 (CH), 126.65 (CH), 129.21 (CH), 130.65 (CH), 133.54 (CH), 135.51 (C), 148.33 (C), 150.91 (C), 151.47 (C), 154.11 (C)], Thiadiazole-C [159.80 (C), 161.90 (C)], 163.80 C=O. Anal. Calcd. for C16H9Cl2F2N3OS: C, 48.02; H, 2.27; N, 10.50. Found: C, 48.11; H, 2.12; N, 10.36. MS (ESI–m/z): 400.00 (M+, 94), 402.26 (M + 2, 54).
N-(5-(2,6-Dichlorobenzyl)-1,3,4-thiadiazol-2-yl)-3,4-dimethoxybenzamide (6f)
White solid, yield: 1.07 g (64%), m.p. 252–253 °C (DMF-EtOH, 1:1). IR (ATR, cm−1): 3155 (–NH–), 3047 (Ar–CH), 2941 (Aliphatic CH), 1661 (C=N), 1587 (C=N). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 3.82 (s, 6H, –OCH3), 4.60 (s, 2H, –CH2), Arom-H [7.71 (s, 2H), 7.52 (s, 2H), 7.37 (s, 1H), 7.08 (s, 1H)], 12.82 (s, 1H, NH). 13C NMR (400 MHz, DMSO-d6, δ ppm): 31.76 (–CH2), 56.10 (–OCH3), 56.17 (–OCH3), Arom-C [111.59 (CH), 122.85 (CH), 123.66 (CH), 129.19 (CH), 130.59 (C), 133.68 (CH), 135.51 (C), 148.81 (C), 153.17 (C), 160.13 (C)], Thiadiazole-C [160.59 (C), 162.74 (C)], 164.73 C=O. Anal. Calcd. for C18H15Cl2N3O3S: C, 50.95; H, 3.56; N, 9.90. Found: C, 50.78; H, 3.46; N, 9.78. MS (ESI–m/z): 423.91 (M+, 100), 425.87 (M + 2, 66).
N-(5-(2,6-Dichlorobenzyl)-1,3,4-thiadiazol-2-yl)-3,5-dimethoxybenzamide (6g)
White solid, yield: 1.00 g (59%), m.p. 204–205 °C (DMF-EtOH, 1:1). IR (ATR, cm−1): 3138 (–NH–), 3032 (Ar–CH), 2959 (Aliphatic CH), 1683 (C=O), 1596 (C=N). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 3.76 (s, 6H, –OCH3), 4.39 (s, 2H, –CH2), Arom-H [7.49 (d, J = 8.0 Hz, 2H), 7.34 (t, J = 8.0, 7.6 Hz, 1H), 7.04 (s, 2H), 6.71 (s, 1H)], 13.01 (bs, 1H, NH). 13C NMR (400 MHz, DMSO-d6, δ ppm): 31.76 (–CH2), 56.09 (–OCH3), 56.16 (–OCH3), Arom-C [107.89 (CH), 109.73 (CH), 129.17 (CH), 130.59 (CH), 133.64 (C), 135.53 (C), 146.41 (C), 160.48 (C)], Thiadiazole-C [153.18 (C), 162.75 (C)], 164.33 C=O. Anal. Calcd. for C18H15Cl2N3O3S: C, 50.95; H, 3.56; N, 9.90. Found: C, 50.79; H, 3.50; N, 9.81. MS (ESI–m/z): 423.91 (M+, 100).
N-(5-(2,6-Dichlorobenzyl)-1,3,4-thiadiazol-2-yl)-3,5-difluorobenzamide (6h)
White solid, yield: 1.18 g (74%), m.p. 270–271 °C (DMF-EtOH, 1:1). IR (ATR, cm−1): 3147 (–NH–), 3044 (Ar–CH), 2928 (Aliphatic CH), 1679 (C=O), 1595 (C=N). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 4.62 (s, 2H, –CH2), Arom-H [7.76 (s, 2H), 7.54 (bd, 3H), 7.39 (t, 1H)], 13.16 (s, 1H, NH). 13C NMR (400 MHz, DMSO-d6, δ ppm): 31.84 (–CH2), Arom-C [108.80 (CH), 112.24 (CH), 112.43 (C), 129.22 (CH), 130.68 (CH), 133.51 (C), 135.51 (C), 163.80 (C)], Thiadiazole-C [161.35 (C), 161.48 (C)], 164.93 C=O. Anal. Calcd. for C16H9Cl2F2N3OS: C, 48.02; H, 2.27; N, 10.50. Found: C, 47.96; H, 2.20; N, 10.56. MS (ESI–m/z): 399.80 (M-1, 100), 401.83 (M + 1, 74).
N-(5-(2,6-Dichlorobenzyl)-1,3,4-thiadiazol-2-yl)-3,5-dichlorobenzamide (6i)
White solid, yield: 1.33 g (77%), m.p. 260–261 °C (DMF-EtOH, 1:1). IR (ATR, cm−1): 3147 (–NH–), 3070 (Ar–CH), 2969 (Aliphatic CH), 1663 (C=O), 1563 (C=N). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 4.62 (s, 2H, –CH2), Arom-H [8.06 (s, 2H), 7.89 (s, 1H), 7.54 (d, J = 8.0 Hz, 2H),7.39 (t, J = 8.0, 7.6 Hz, 1H)], 13.16 (s, 1H, NH). 13C NMR (400 MHz, DMSO-d6, δ ppm): 31.87 (–CH2), Arom-C [127.60 (CH), 129.22 (CH), 130.69 (C), 132.50 (CH), 133.50 (C), 134.90 (C), 135.47 (C), 135.52 (C)], Thiadiazole-C [160.08 (C), 162.54 (C)], 163.93 C=O. Anal. Calcd. for C16H9Cl4N3OS: C, 44.37; H, 2.09; N, 9.70. Found: C, 44.22; H, 2.18; N, 9.52. MS (ESI–m/z): 431.79 (M-1, 76).
N-(5-(2,6-Dichlorobenzyl)-1,3,4-thiadiazol-2-yl)-4-ethylbenzamide (6j)
White solid, yield: 1.02 g (65%), m.p. 275–276 °C (DMF-EtOH, 1:1). IR (ATR, cm−1): 3184 (–NH–), 3059 (Ar–CH), 2964 (Aliphatic CH), 1662 (C=O), 1576 (C=N). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 1.17 (t, 3H, –CH3), 2.65 (q, 2H, –CH2–), 4.61 (s, 2H, –CH2), Arom-H [7.99 (d, J = 8.0 Hz, 2H), 7.54 (d, J = 8.4 Hz, 2H), 7.36 (m, 3H)], 12.87 (s, 1H, NH). 13C NMR (400 MHz, DMSO-d6, δ ppm): 15.61 (–CH3), 28.58 (–CH2–), 31.79 (–CH2), Arom-C [128.47 (CH), 128.95 (CH), 129.01 (CH), 129.21 (CH), 130.62 (C), 133.66 (C), 135.52 (C), 149.83 (C)], Thiadiazole-C [159.65 (C), 160.72 (C)], 165.40 C=O. Anal. Calcd. for C18H15Cl2N3OS: C, 55.11; H, 3.85; N, 10.71. Found: C, 55.02; H, 3.79; N, 10.63. MS (ESI–m/z): 393.77 (M + 1, 93).
N-(5-(2,6-Dichlorobenzyl)-1,3,4-thiadiazol-2-yl)-4-(trifluoromethyl)benzamide (6k)
White solid, yield: 1.07 g (62%), m.p. 274–275 °C (DMF-EtOH, 1:1). IR (ATR, cm−1): 3152 (–NH–), 3041 (Ar–CH), 2943 (Aliphatic CH), 1680 (C=O), 1531 (C=N). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 4.63 (s, 2H, –CH2), Arom-H [8.23 (d, J = 8.0 Hz, 2H), 7.90 (d, J = 8.4 Hz, 2H), 7.54 (d, J = 8.4 Hz, 2H), 7.39 (t, J = 7.6, 7.6 Hz, 1H)], 13.26 (s, 1H, NH). 13C NMR (400 MHz, DMSO-d6, δ ppm): 31.84 (–CH2), 122.84 (CF3), Arom-C [125.56 (CH), 125.99 (CH), 126.03 (CH), 129.22 (CH), 129.78 (C), 130.68 (C), 133.56 (C), 135.52 (C)], Thiadiazole-C [159.86 (C), 160.84 (C)], 165.53 C=O. Anal. Calcd. for C17H10Cl2F3N3OS: C, 47.24; H, 2.33; N, 9.72. Found: C, 47.36; H, 2.26; N, 9.65. MS (ESI–m/z): 431.86 (M-1, 82).
N-(5-(2,6-Dichlorobenzyl)-1,3,4-thiadiazol-2-yl)-4-cyanobenzamide (6l)
White solid, yield: 1.26 g (81%), m.p. 334–335 °C (DMF-EtOH, 1:1). IR (ATR, cm−1): 3142 (–NH–), 3094 (Ar–CH), 2921 (Aliphatic CH), 2235 (CN), 1684 (C=O), 1542 (C=N). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 4.63 (s, 2H, –CH2), Arom-H [8.18 (d, J = 8.4 Hz, 2H), 8.01 (d, J = 8.0 Hz, 2H), 7.54 (d, J = 8.4 Hz, 2H), 7.38 (t, J = 8.0, 8.0 Hz, 1H)], 13.27 (s, 1H, NH). 13C NMR (400 MHz, DMSO-d6, δ ppm): 31.86 (–CH2), 118.56 (CN), Arom-C [115.40 (C), 125.54 (CH), 129.23 (CH), 129.61 (CH), 130.70 (CH), 133.03 (C), 133.53 (C), 135.52 (C)], Thiadiazole-C [159.88 (C), 160.86 (C)], 165.80 C=O. Anal. Calcd. for C17H10Cl2N4OS: C, 52.45; H, 2.59; N, 14.39. Found: C, 52.36; H, 2.46; N, 14.50. MS (ESI–m/z): 385.22 (M-4, 100).
N-(5-(2,6-Dichlorobenzyl)-1,3,4-thiadiazol-2-yl)acetamide (6m)
White solid, yield: 1.03 g (83%), m.p. 284–285 °C (DMF-EtOH, 1:15). IR (ATR, cm−1): 3158 (–NH–), 3052 (Ar–CH), 2976 (Aliphatic CH), 1698 (C=O), 1563 (C=N). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 2.12 (s, 3H, –CH3), 4.56 (s, 2H, –CH2), Arom-H [7.59 (d, J = 7.6 Hz, 2H), 7.36 (t, J = 8.4, 8.0 Hz, 1H)], 13.26 (s, 1H, NH). 13C NMR (400 MHz, DMSO-d6, δ ppm): 22.77 (CH3), 31.75 (–CH2), Arom-C [129.15 (CH), 130.56 (CH), 133.63 (C), 135.48 (C)], Thiadiazole-C [159.99 (C), 160.26 (C)], 169.01 C=O. Anal. Calcd. for C11H9Cl2N3OS: C, 43.72; H, 3.00; N, 13.91. Found: C, 43.76; H, 3.09; N, 13.86. MS (ESI–m/z): 302.01 (M+, 85).
Ethyl 5-(2,6-Dichlorobenzyl)-1,3,4-thiadiazol-2-ylcarbamate (6n)
White solid, yield: 0.76 g (56%), m.p. 217–218 °C (DMF-EtOH, 1:5). IR (ATR, cm−1): 3160 (–NH–), 3022 (Ar–CH), 2982 (Aliphatic CH), 1720 (C=O), 1569 (C=N). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 1.23 (t, 3H, –CH3), 4.17 (q, 2H, –OCH2–), 4.41 (s, 2H, –CH2), Arom-H [7.51 (d, 2H), 7.36 (t, 1H)], 12.08 (s, 1H, NH). 13C NMR (400 MHz, DMSO-d6, δ ppm): 14.65 (–CH3), 27.00 (–CH2–), 62.60 (–OCH2–), Arom-C [129.20 (CH), 130.37 (CH), 133.83 (C), 135.42 (C)], Thiadiazole-C [160.63 (C), 161.48 (C)], 162.49 C=O. Anal. Calcd. for C12H11Cl2N3O2S: C, 43.39; H, 3.34; N, 12.65. Found: C, 43.33; H, 3.27; N, 12.56. MS (ESI–m/z): 331.14 (M-1, 93).
N-(5-(2-Chloro-6-fluorobenzyl)-1,3,4-thiadiazol-2-yl)benzamide (7a)
White solid, yield:1.13 g (81%), m.p. 258–259 °C (DMF-EtOH, 1:2). IR (ATR, cm−1): 3174 (–NH–), 3054 (Ar–CH), 2977 (Aliphatic CH), 1670 (C=O), 1580 (C=N). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 4.49 (s, 2H, –CH2), Arom-H [8.06 (d, J = 7.6 Hz, 2H), 7.62 (t, J = 6.8 Hz, 1H), 7.52 (t, J = 7.2 Hz, 2H), 7.41 (bs, 2H), 7.30 (bd, 1H)], 12.99 (s, 1H, NH). 13C NMR (400 MHz, DMSO-d6, δ ppm): 26.95 (–CH2), Arom-C [115.27 (CH), 123.79 (CH), 126.14 (C), 128.80 (CH), 129.07 (CH), 130.63 (CH), 131.92 (CH), 133.41 (C), 134.82 (C), 161.13 (C)], Thiadiazole-C [160.08 (C), 162.54 (C)], 165.55 C=O. Anal. Calcd. for C16H11ClFN3OS: C, 55.25; H, 3.19; N, 12.08. Found: C, 55.31; H, 3.21; N, 12.11. MS: m/z: 347.79 (M+, 100), 348.81 (M + 1, 32).
N-(5-(2-Chloro-6-fluorobenzyl)-1,3,4-thiadiazol-2-yl)-4-nitrobenzamide (7b)
White solid, yield:1.19 g (76%), m.p. 257–258 °C (DMF-EtOH, 1:3). IR (ATR, cm−1): 3116 (–NH–), 3039 (Ar–CH), 2924 (Aliphatic CH), 1679 (C=O), 1604 (C=N). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 4.50 (s, 2H, –CH2), Arom-H [8.34 (d, J = 7.6 Hz, 2H), 8.26 (d, J = 8.0 Hz, 2H),7.38 (d, 2H), 7.31 (bd, 1H)], 13.39 (s, 1H, NH). 13C NMR (400 MHz, DMSO-d6, δ ppm): 27.03 (–CH2), Arom-C [115.28 (CH), 124.09 (CH), 126.12 (CH), 126.16 (C), 130.41 (CH), 130.70 (CH), 130.89 (C), 134.82 (C), 147.08 (C), 160.08 (C)], Thiadiazole-C [150.26 (C), 162.54 (C)], 167.10 C=O. Anal. Calcd. for C16H10ClFN4O3S: C, 48.92; H, 2.57; N, 14.26. Found: C, 48.88; H, 2.59; N, 14.22. MS: m/z: 392.90 (M+, 100), 394.72 (M + 2, 54).
N-(5-(2-Chloro-6-fluorobenzyl)-1,3,4-thiadiazol-2-yl)-4-(methylthio) benzamide (7c)
White solid, yield:1.21 g (77%), m.p. 275–276 °C (DMF-EtOH, 1:2). IR (ATR, cm−1): 3158 (–NH–), 3043 (Ar–CH), 2949 (Aliphatic CH), 1658 (C=O), 1591 (C=N). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 2.51 (s, 3H, –CH3), 4.48 (s, 2H, –CH2), Arom-H [8.01 (d, J = 6.8 Hz, 2H), 7.37 (d, J = 6.8 Hz, 2H), 7.35 (s, 2H), 7.29 (bs, 1H)], 12.90 (s, 1H, NH). 13C NMR (400 MHz, DMSO-d6, δ ppm): 14.39 (CH3), 26.95 (–CH2), Arom-C [115.20 (CH), 123.89 (CH), 125.32 (C), 126.12 (CH), 130.63 (CH), 130.73 (CH), 134.88 (C), 139.69 (C), 145.70 (C), 161.14 (C)], Thiadiazole-C [160.09 (C), 162.54 (C)], 165.48 C=O. Anal. Calcd. for C17H13ClFN3OS2: C, 51.84; H, 3.33; N, 10.67. Found: C, 51.79; H, 3.36; N, 10.62. MS (ESI–m/z): 411.38 (M + 1, 96), 413.34 (M + 2, 48).
N-(5-(2-Chloro-6-fluorobenzyl)-1,3,4-thiadiazol-2-yl)-3,4-dichloro benzamide (7d)
White solid, yield:1.27 g (76%), m.p. 214–215 °C (DMF-EtOH, 1:11). IR (ATR, cm−1): 3092 (–NH–), 3010 (Ar–CH), 2928 (Aliphatic CH), 1674 (C=O), 1591 (C=N). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 4.82 (s, 2H, –CH2), Arom-H [8.30 (s, 1H), 7.99 (d, J = 8.4 Hz, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.38 (d, 2H), 7.29 (t, 1H)], 13.15 (s, 1H, NH). 13C NMR (400 MHz, DMSO-d6, δ ppm): 27.00 (–CH2), Arom-C [115.26 (CH), 123.66 (CH), 126.11 (C), 126.14 (CH), 129.03 (CH), 130.66 (CH), 130.76 (CH), 131.39 (C), 131.99 (C), 134.82 (C), 136.17 (C), 160.08 (C)], Thiadiazole-C [159.85 (C), 162.53 (C)], 164.12 C=O. Anal. Calcd. for C16H9Cl3FN3OS: C, 46.12; H, 2.18; N, 10.08. Found: C, 46.17; H, 2.15; N, 10.12. MS (ESI–m/z): 417.60 (M + 1, 100).
N-(5-(2-Chloro-6-fluorobenzyl)-1,3,4-thiadiazol-2-yl)-3,4-difluorobenz- amide (7e)
White solid, yield:1.21 g (79%), m.p. 278–279 °C (DMF-EtOH, 1:10). IR (ATR, cm−1): 3157 (–NH–), 3063 (Ar–CH), 2979 (Aliphatic CH), 1667 (CvO), 1608 (C=N). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 4.49 (s, 2H, –CH2), Arom-H [8.14 (t, 1H), 7.95 (bs, 1H), 7.62 (q, 1H), 7.39 (bs, 2H), 7.29 (t, 1H)], 13.09 (s, 1H, NH). 13C NMR (400 MHz, DMSO-d6, δ ppm): 26.99 (–CH2), Arom-C [115.26 (CH), 118.33 (CH), 123.69 (CH), 126.11 (CH), 126.71 (CH), 130.66 (C), 134.82 (CH), 148.45 (C), 150.78 (C), 151.59 (C), 154.12 (C), 160.08 (C)], Thiadiazole-C [157.63 (C), 161.78 (C)], 162.55 C=O. Anal. Calcd. for C16H9ClF3N3OS: C, 50.07; H, 2.36; N, 10.95. Found: C, 50.11; H, 2.33; N, 10.97. MS (ESI–m/z): 383.78 (M+, 70), 385.15 (M + 2, 100).
N-(5-(2-Chloro-6-fluorobenzyl)-1,3,4-thiadiazol-2-yl)-3,4-dimethoxy benzamide (7f)
White solid, yield:1.11 g (68%), m.p. 248–249 °C (DMF-EtOH, 1:4). IR (ATR, cm−1): 3178 (–NH–), 3088 (Ar–CH), 2940 (Aliphatic CH), 1661 (C=N), 1588 (C=N). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 3.82 (s, 6H, –OCH3), 4.47 (s, 2H, –CH2), Arom-H [7.73 (s, 2H), 7.39 (s, 2H), 7.30 (d, 1H), 7.08 (d, 1H)], 12.82 (s, 1H, NH). 13C NMR (400 MHz, DMSO-d6, δ ppm): 26.89 (–CH2), 56.10 (–OCH3), 56.17 (–OCH3), Arom-C [111.58 (CH), 115.03 (CH), 115.25 (CH), 122.86 (CH), 123.65 (CH), 123.83 (C), 126.14 (C), 130.70 (CH), 134.82 (C), 148.82 (C), 153.17 (C), 160.08 (C)], Thiadiazole-C [160.97 (C), 162.54 (C)], 164.70 C=O. Anal. Calcd. for C18H15ClFN3O3S: C, 53.01; H, 3.71; N, 10.30. Found: C, 52.97; H, 3.74; N, 10.28. MS (ESI–m/z): 407.81 (M+, 100), 409.91 (M + 2, 37).
N-(5-(2-Chloro-6-fluorobenzyl)-1,3,4-thiadiazol-2-yl)-3,5-dimethoxy benzamide (7g)
White solid, yield: 0.93 g (57%), m.p. 252–254 °C (DMF-EtOH, 1:1). IR (ATR, cm−1): 3268 (–NH–), 3109 (Ar–CH), 2947 (Aliphatic CH), 1624 (C=O), 1580 (C=N). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 3.76 (s, 6H, –OCH3), 4.39 (s, 2H, –CH2), Arom-H [7.75 (d, 1H), 7.34 (t, 1H), 7.04 (s, 2H), 6.71 (s, 2H)], 13.03 (bs, 1H, NH). 13C NMR (400 MHz, DMSO-d6, δ ppm): 26.93 (–CH2), 56.10 (–OCH3), 56.17 (–OCH3), Arom-C [111.56 (CH), 115.31 (CH), 122.86 (CH), 123.65 (CH), 126.14 (C), 130.76 (CH), 134.87 (C), 148.82 (C), 160.08 (C), 160.97 (C)], Thiadiazole-C [153.25 (C), 162.54 (C)], 164.70 C=O. Anal. Calcd. for C18H15ClFN3O3S: C, 53.01; H, 3.71; N, 10.30. Found: C, 53.05; H, 3.69; N, 10.29. MS (ESI–m/z): 409.09 (M + 1, 96).
N-(5-(2-Chloro-6-fluorobenzyl)-1,3,4-thiadiazol-2-yl)-3,5-difluoro benzamide (7h)
White solid, yield: 1.24 g (81%), m.p. 261–262 °C (DMF-EtOH, 1:2). IR (ATR, cm−1): 3154 (–NH–), 3096 (Ar–CH), 2932 (Aliphatic CH), 1677 (C=O), 1597 (C=N). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 4.49 (s, 2H, –CH2), Arom-H [7.77 (s, 2H), 7.58 (t, 1H), 7.40 (bs, 2H), 7.30 (t, 1H)], 13.16 (s, 1H, NH). 13C NMR (400 MHz, DMSO-d6, δ ppm): 26.99 (–CH2), Arom-C [108.64 (CH), 112.16 (CH), 112.44 (CH), 115.28 (CH), 123.68 (C), 126.13 (CH), 130.69 (C), 134.82 (C), 160.08 (C), 163.81 (C)], Thiadiazole-C [161.35 (C), 162.54 (C)], 163.94 C=O. Anal. Calcd. for C16H9ClF3N3OS: C, 50.07; H, 2.36; N, 10.95. Found: C, 50.05; H, 2.38; N, 10.99. MS (ESI–m/z): 383.87 (M+, 100), 385.83 (M + 2, 53).
N-(5-(2-Chloro-6-fluorobenzyl)-1,3,4-thiadiazol-2-yl)-3,5-dichloro benzamide (7i)
White solid, yield:1.25 g (75%), m.p. 277–278 °C (DMF-EtOH, 1:2). IR (ATR, cm−1): 3140 (–NH–), 3082 (Ar–CH), 2913 (Aliphatic CH), 1677 (C=O), 1569 (C=N). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 4.62 (s, 2H, –CH2), Arom-H [8.06 (s, 2H), 7.89 (s, 1H), 7.40 (m, 2H),7.29 (m, 1H)], 13.17 (s, 1H, NH). 13C NMR (400 MHz, DMSO-d6, δ ppm): 27.00 (–CH2), Arom-C [115.28 (CH), 123.64 (CH), 126.12 (C), 127.60 (CH), 130.68 (CH), 130.78 (CH), 132.51 (C), 134.81 (C), 134.87 (C), 134.91 (C)], Thiadiazole-C [160.07 (C), 162.54 (C)], 164.21 C=O. Anal. Calcd. for C16H9Cl3FN3OS: C, 46.12; H, 2.18; N, 10.08. Found: C, 46.09; H, 2.16; N, 10.11. MS (ESI–m/z): 417.10 (M+, 90).
N-(5-(2-Chloro-6-fluorobenzyl)-1,3,4-thiadiazol-2-yl)-4-ethylbenzamide (7j)
White solid, yield: 1.11 g (74%), m.p. 243–244 °C (DMF-EtOH, 1:1). IR (ATR, cm−1): 3182 (–NH–), 3056 (Ar–CH), 2966 (Aliphatic CH), 1662 (C=O), 1580 (C=N). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 1.20 (t, 3H, –CH3), 2.66 (q, 2H, –CH2–), 4.48 (s, 2H, –CH2), Arom-H [7.99 (d, J = 8.0 Hz, 2H), 7.39 (m, 4H), 7.29 (m,1H)], 12.89 (s, 1H, NH). 13C NMR (400 MHz, DMSO-d6, δ ppm): 15.60 (–CH3), 26.92 (–CH2–), 28.57 (–CH2CH3), Arom-C [115.28 (CH), 123.63 (CH), 126.11 (C), 128.47 (CH), 128.95 (CH), 129.34 (CH), 130.62 (C), 134.87 (C), 149.84 (C), 160.09 (C)], Thiadiazole-C [161.07 (C), 162.55 (C)], 165.56 C=O. Anal. Calcd. for C18H15ClFN3OS: C, 57.52; H, 4.02; N, 11.18. Found: C, 57.49; H, 4.05; N, 11.21. MS (ESI–m/z): 375.02 (M-1, 91).
N-(5-(2-Chloro-6-fluorobenzyl)-1,3,4-thiadiazol-2-yl)-4-(trifluoromethyl)-benzamide (7k)
White solid, yield: 1.18 g (71%), m.p. 273–274 °C (DMF-EtOH, 1:1). IR (ATR, cm−1): 3144 (–NH–), 3052 (Ar–CH), 2933 (Aliphatic CH), 1677 (C=O), 1581 (C=N). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 4.49 (s, 2H, –CH2), Arom-H [8.23 (d, J = 8.0 Hz, 2H), 7.90 (d, J = 8.4 Hz, 2H), 7.39 (m, 2H), 7.29 (m, 1H)], 13.27 (s, 1H, NH). 13C NMR (400 MHz, DMSO-d6, δ ppm): 26.97 (–CH2), 122.84 (CF3), Arom-C [115.27 (CH), 123.51 (CH), 125.98 (C), 126.15 (CH), 129.77 (CH), 130.66 (CH), 132.66 (C), 132.98 (C), 134.82 (C), 160.09 (C)], Thiadiazole-C [161.12 (C), 162.55 (C)], 164.96 C=O. Anal. Calcd. for C17H10ClF4N3OS: C, 49.11; H, 2.42; N, 10.11. Found: C, 49.07; H, 2.45; N, 10.13. MS (ESI–m/z): 415.90 (M+, 93).
N-(5-(2-Chloro-6-fluorobenzyl)-1,3,4-thiadiazol-2-yl)-4-cyanobenzamide (7l)
White solid, yield: 1.25 g (84%), m.p. 327–328 °C (DMF-EtOH, 1:2). IR (ATR, cm−1): 3149 (–NH–), 3071 (Ar–CH), 2926 (Aliphatic CH), 2243 (CN), 1679 (C=O), 1581 (C=N). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 4.50 (s, 2H, –CH2), Arom-H [8.19 (d, J = 8.4 Hz, 2H), 8.01 (d, J = 8.4 Hz, 2H), 7.40 (m, 2H), 7.30 (m, 1H)], 13.25 (s, 1H, NH). 13C NMR (400 MHz, DMSO-d6, δ ppm): 27.02 (–CH2), 118.56 (CN), Arom-C [115.07 (CH), 115.30 (C), 126.14 (CH), 126.17 (CH), 129.62 (CH), 130.69 (CH), 130.79 (CH), 133.04 (C), 134.82 (C), 160.08 (C)], Thiadiazole-C [154.73 (C), 162.54 (C)], 165.79 C=O. Anal. Calcd. for C17H10ClFN4OS: C, 54.77; H, 2.70; N, 15.03. Found: C, 54.78; H, 2.68; N, 15.07. MS (ESI–m/z): 372.97 (M+, 92).
N-(5-(2-Chloro-6-fluorobenzyl)-1,3,4-thiadiazol-2-yl)acetamide (7m)
White solid, yield: 0.99 g (87%), m.p. 262–263 °C (DMF-EtOH, 1:8). IR (ATR, cm−1): 3158 (–NH–), 3038 (Ar–CH), 2908 (Aliphatic CH), 1699 (C=O), 1558 (C=N). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 2.13 (s, 3H, –CH3), 4.44 (s, 2H, –CH2), Arom-H [7.38 (m, 2H), 7.28 (m, 1H)], 12.45 (s, 1H, NH). 13C NMR (400 MHz, DMSO-d6, δ ppm): 22.78 (CH3), 26.88 (–CH2), Arom-C [115.23 (CH), 123.79 (CH), 126.11 (C), 130.69 (CH), 134.84 (C), 160.07 (C)], Thiadiazole-C [159.08 (C), 162.51 (C)], 169.03 C=O. Anal. Calcd. for C11H9ClFN3OS: C, 46.24; H, 3.17; N, 14.71. Found: C, 46.26; H, 3.14; N, 14.69. MS (ESI–m/z): 286.11 (M + 1, 95).
Ethyl 5-(2-Chloro-6-fluorobenzyl)-1,3,4-thiadiazol-2-ylcarbamate (7n)
White solid, yield: 0.80 g (63%), m.p. 192–193 °C (DMF-EtOH, 1:5). IR (ATR, cm−1): 3160 (–NH–), 3037 (Ar–CH), 2982 (Aliphatic CH), 1720 (C=O), 1569 (C=N). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 1.22 (t, 3H, –CH3), 4.16 (q, 2H, –OCH2–), 4.41 (s, 2H, –CH2), Arom-H [7.36 (m, 2H), 7.25 (m, 1H)], 12.09 (s, 1H, NH). 13C NMR (400 MHz, DMSO-d6, δ ppm): 14.65 (–CH3), 26.96 (–CH2–), 62.61 (–OCH2–), Arom-C [115.18 (CH), 123.53 (CH), 126.06 (C), 130.55 (CH), 134.77 (C), 160.03 (C)], Thiadiazole-C [154.34 (C), 161.47 (C)], 162.49 CvO. Anal. Calcd. for C12H11ClFN3O2S: C, 45.65; H, 3.51; N, 13.31. Found: C, 45.56; H, 3.48; N, 13.20. MS (ESI–m/z): 315.16 (M+, 96).
Crystallographic analysis
The X-ray fraction data of the compound 7n examined in this study was collected using the MoKα ray at 293(2)K degree using a ‘Bruker APEX-II CCD diffractometer. The structures of the crystals were solved using direct methods in ShelXT [43] software. During the process, in order to determine the positions of the atoms, except for hydrogen, the refinement procedure was conducted using the ShelXL [44] software [45] that used the full-matrix least-squares method. After the structure solution and refinement procedures were finished, olex2 and MERCURY software were used in molecular drawings and calculations.
Biological activity studies
Fungi culture
Monilia fructigena, Fusarium oxysporum f. sp. lycopersici and Alternaria solani plant pathogens were used for the tests. The pathogens were grown on a PDA (potato dextrose agar) medium at 22 ± 2 °C for about 7 days.
In vitro antifungal activity
Antifungal activity studies were determined using disk diffusion method [46]. The compounds were dissolved in dimethyl sulphoxide (DMSO). In the laminar flow cabin, Whatman no. 1 sterile filter paper discs (6 mm) were impregnated with 50 μl of the compounds (corresponding to 500 and 1000 μg/ml of compounds) and allowed to dry at room temperature (for 4 h) [47]. Then the compound impregnated paper discs were placed in a PDA medium in 90 mm sterile petri plates. Mycelium discs (5 mm diameter) of 7-day-old culture of test fungi were inoculated to the centre of the Petri plate. All fungi were incubated at 22 ± 2 °C. There is a 25 mm distance between the mycelium discs and paper discs. The obtained inhibition zones were recorded. As negative control, only DMSO was impregnated to discs. In the positive control, 80% thiram (3000 μg/ml) was used against the test fungi at the recommended dose. All antifungal activity values were determined by measuring inhibition zone distance between pathogen and paper disc [24].
Percent inhibition was calculated according to the following formula:
$${\text{\% Inhibition}} = {\text{Inhibition zone in treatment}}/{\text{Control*}} \times 1 0 0$$
*Control: Inhibition zone of positive control.
Lethal doses (LD50), minimum fungicidal concentration (MFC) and minimum inhibitory concentration (MIC)
Minimum inhibitory concentration (MIC) and Minimum fungicidal concentration (MFC) of the compounds were tested by the twofold serial dilution method. The test compound was dissolved in DMSO to obtain 1000 µg/ml stock solutions. For the MIC and MFC assay, each compound was prepared in the concentrations of 1000, 500, 250, 125, 62.5 and 31.25 µg/ml. Fifty microliter of concentrations of the compound was transferred on to paper disc. Then the same methods as described in the in vitro antifungal activity studies were applied. The MIC was defined as the lowest test concentration that allowed no detectable mycelium growth. The MFC was defined as the lowest test concentration that allows no mycelium growth of the organism on agar [48]. In addition, LD50 values were calculated. Six different doses used in the calculation of the MIC were calculated using the results of the inhibition zones. Lethal doses (LD) estimates for LD50 were determined with Polo Plus (LeOra software).