Chemistry
Unless otherwise noted, materials were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA), Acros Organics (Fair Lawn, NJ, USA), and Merck Chemical Co. (Darmstadt, Germany), used without purification. Column chromatography was performed using silica gel 60 (Merck, 40-63 µm). For thin-layer chromatography, TLC aluminium sheets (Merck, silica gel 60 F254) were used. The spots were visualized under UV of 254–366 nm. All reactions were carried out in heat-dried glassware under a dry nitrogen atmosphere unless otherwise stated. All liquids transfer was conducted using standard syringe or cannula techniques. All spectral data were obtained on the following instruments: NMR spectra were obtained using Bruker Advance 500 (500 MHz for 1H NMR, 125 MHz for 13C NMR) spectrometer system (Bruker Bioscience, Billerica, MA, USA). Data were analyzed via Top Spin 3.6.1 software package. Chemical shifts were internally referred to as the solvent signals in CDCl3 (1H δ 7.26; 13C δ 77.0), acetone-d6 (1H δ 2.05; 13C δ 29.9 and 206.7), DMSO-d6 (1H δ 2.50; 13C δ 39.5), and tetramethylsilane (TMS) signal at 0.00 ppm. Chemical shift values are mentioned in δ (ppm) and coupling constants (J) are given in Hz. The mass spectra were recorded with a Waters Xevo QTOF MS (Waters Corporation, Milford, MA, USA), and it is reported in m/z. The infrared (IR) spectra were obtained through Perkin Elmer FT-IR spectrometer RX1 (Perkin Elmer, Waltham, MA, USA). Melting points were determined on the Buchi B-542 apparatus by an open capillary method and are uncorrected.
General procedure for the synthesis of acyl ethyl esters (2a-j).
The corresponding carboxylic acid (1a-j) (2 g) was refluxed in ethanol (10 mL) and concentrated H2SO4 (1.5 mL) for 8-10 hours. After completion of reaction (as evident from TLC profiles), the solvent was evaporated under reduced pressure. The mixture was extracted with ethyl acetate (5 mL × 3). The organic layer was dried over Na2SO4 and concentrated under reduced pressure to afford compounds (2a-j) in80-99% yields without purification.
Ethyl benzoate (2a).
White powder, 90% yield; mp 133-135 ºC; IR ʋ / cm-1 2984, 1713, 1440, 1269; 1H NMR (500 MHz, DMSO-d6) d 1.38 (t, J = 7.1 Hz, 3H, CH3, H9), 4.37 (q, J = 7.1 Hz, 2H, -OCH2, H8), 7.52 (t, J = 7.6 Hz, 2H, Ar-H3, H5), 7.64 (m, 1H, Ar-H4), 8.04 (dd, J = 1.3, 7.6 Hz, 2H, Ar-H2, H6); 13C NMR (125 MHz, DMSO-d6) d 13.7, 60.5, 128.5, 129.2, 130.6, 132.9, 165.8. All data were compared with the reported literature [21].
Ethyl-3-nitrobenzoate (2b).
Light purple powder, 92% yield; mp 145-147 ºC; IR ʋ / cm-1 3088, 2986, 1721, 1531, 1258; 1H NMR (500 MHz, DMSO-d6) d 1.36 (t, J = 7.1 Hz, 3H, -CH3, H9), 4.39 (q, J = 7.1 Hz, 2H, -OCH2, H8), 7.84 (t, J= 8.0 Hz, 1H, Ar-H5), 8.36 (d, J = 8.0 Hz, 1H, Ar-H6), 8.49 (d, J = 8.0 Hz, 1H, Ar-H4), 8.61 (s, 1H, Ar-H4); 13C NMR (125 MHz, DMSO-d6) d 14.5, 62.1, 124.0, 128.2, 131.2, 131.9, 135.7, 148.8, 164.6. All data were compared with the reported literature [22].
Ethyl 2-amino-3-chlorobenzoate (2c).
Brown crystal, 71% yield; mp 44-46 ºC; IR ʋ / cm-1 3404, 1700, 1362, 1224; 1H NMR (500 MHz, DMSO-d6) d 1.28 (t, J = 7.1 Hz, 3H, CH3, H9), 4.24 (q, J = 7.1 Hz, 2H, -OCH2, H8), 6.53 (t, J = 8.6 Hz, 1H, Ar-H5), 6.84 (d, J = 8.6 Hz, 1H, Ar-H4), 7.69 (d, J = 8.6 Hz, 1H, Ar-H6); 13C NMR (125 MHz, DMSO-d6) d 14.6, 60.6, 108.5, 115.3, 115.8, 133.02, 139.0, 152.6, 167.2. All data were compared with the reported literature [23].
Ethyl 5-bromo-2-hydroxybenzoate (2d).
White powder, 96% yield; mp 144-146 ºC; IR ʋ / cm-1 3004, 2584, 1700, 1196, 1360; 1H NMR (500 MHz, CDCl3) d 1.34 (t, J = 7.1 Hz, 3H, CH3, H9), 4.33 (q, J = 7.1 Hz, 3H, OCH2, H8), 6.80 (d, J = 8.9 Hz, 1H, Ar-H3), 7.43 (dd, J = 2.3, 8.9 Hz, 1H, Ar-H4), 7.86 (d, J = 2.3 Hz, 1H, Ar-H6); 13C NMR (125 MHz, CDCl3) d 9.0, 56.8, 105.6, 108.9, 114.3, 126.9, 133.1, 155.4, 163.9. All data were compared with the reported literature [24].
Ethyl 4-chlorobenzoate (2e).
White powder, 57% yield; mp 71-73 °C; IR ʋ / cm-1 3004, 2928, 1659, 1180, 865, 657; 1H NMR (500 MHz, DMSO-d6) d 1.32 (t, J = 7.1 Hz, 3H, -CH3, H9), 4.31 (q, J = 7.1 Hz, 2H, -OCH2, H8), 7.56 (d, J = 8.6 Hz, 2H, Ar-H2, H6), 7.94 (d, J = 8.6 Hz, 2H, Ar-H3, H5); 13C NMR (125 MHz, DMSO-d6) d 14.5, 61.5, 129.2, 130.1, 131.6, 138.3, 166.9. All data were compared with the reported literature [22].
Ethyl 3-(4′-nitrophenyl)propanoate (2f).
Light yellow powder, 88% yield; mp 123-125 °C; IR ʋ / cm-1: 3061, 1606, 1522. 1H NMR (500 MHz, DMSO-d6) d 1.35 (t, J = 7.2, Hz, 3H, CH3, H5), 2.39 (t, J = 7.6 Hz, 2H, -CH2, H2), 2.96 (t, J = 7.6 Hz, 2H, -CH2, H3), 4.35 (q, J = 7.2 Hz, 2H, -OCH2, H4), 7.48 (d, J = 8.6 Hz, 2H, Ar-H3′, H5′), 8.13(d, J = 8.6 Hz, 2H, Ar-H2′, H6′); 13C NMR (125 MHz, DMSO-d6) d 14.9, 31.2, 34.7, 64.1, 123.8, 130.0, 146.4, 150.1, 170.8. All data were compared with the reported literature [25].
Ethyl (E)-2-methyl-3-phenylacrylate (2g).
Colourless liquid, 80% yield; mp 35-38 °C; IR ʋ / cm-1 3342, 2472, 2070, 1119; 1H NMR (500 MHz, DMSO-d6) d 1.36 (t, J = 7.1 Hz, 3H, CH3, H5), 4.31 (q, J = 7.1 Hz, 2H, -OCH2, H4) ,2.04 (d, J = 1.5 Hz, 3H, CH3-H6), 7.22 (s, 1H, CH, H3), 7.37 (m, 1H, Ar-H4′), 7.38 (t, J = 7.3 Hz, 2H, Ar-H3′, H5′), 7.49 (d, J = 7.3 Hz, 2H, Ar-H2′, H6′); 13C NMR (125 MHz, DMSO-d6) d 14.1, 21.1, 60.7, 128.7, 129.5, 129.6, 129.8, 136.2, 136.5, 167.1. All data were compared with the reported literature [26].
Ethyl 1H-indole-2-carboxylate (2h).
Purple powder, 97% yield; mp 104-106 ºC; IR ʋ / cm-1 3324, 2983, 1701, 1241, 1199; 1H NMR (500 MHz, DMSO-d6) d 1.35 (t, J = 7.2, Hz, 3H, CH3-H11), 4.35 (q, J = 7.2 Hz, 2H, -OCH2-H10), 7.10 (dt, J = 1.0, 7.5 Hz, 1H, Ar-H2), 7.15 (dd, J = 0.8, 1.3 Hz, 1H, Ar-H6), 7.26 (dt, J = 1.1, 7.1 Hz, 1H, Ar-H7), 7.46 (dd, J = 0.9, 7.5 Hz, 1H, Ar-H5), 7.66 (d, J = 8.0 Hz, 1H, Ar-H4), 11.86 (s, 1H, -NH); 13C NMR (125 MHz, DMSO-d6) d 14.8, 60.9, 108.1, 113.0, 120.6, 122.5, 125.1, 127.2, 127.8, 137.8, 161.8 All data were compared with the reported literature [27].
Ethyl 1′-hydroxy-2′-naphthoate (2i).
Light purple powder, 91% yield; mp 68-70 °C; IR ʋ / cm-1 3184, 2984, 3151, 1678, 1214; 1H NMR (500 MHz, Acetone-d) d 1.45 (t, J = 7.2 Hz, 3H, -CH3, H3), 4.49 (q, J = 7.2 Hz, 2H, -OCH2, H2), 7.31 (s, 1H, Ar-H3′), 7.35 (t, J = 7.5 Hz, 1H, Ar-H7′), 7.53 (t, J = 7.0 Hz, 1H, Ar-H8′), 7.75 (d, J = 8.5 Hz, 1H, Ar-H4′), 7.90 (d, J = 8.5 Hz, 1H, Ar-H6′), 8.54 (s, 1H, Ar-H9′), 10.5 (s, 1H, -OH, H1′); 13C NMR (125 MHz, Acetone-d) d 13.5, 61.8, 111.2, 114.5, 124.0, 126.1, 127.1, 129.2, 132.3, 137.9, 156.0, 169.8. All data were compared with the reported literature [28].
Ethyl 2-(1′-H-indol-3-yl)acetate (2j).
Purple crystal, 82 % yield; mp 33-37 °C; IR ʋ / cm-1 3403, 2977, 1705, 1361, 1224; 1H NMR (500 MHz, CDCl3-d) d 1.23 (t, J = 7.1 Hz, 3H, CH3, H4), 3.77 (s, 2H, CH2-H2), 4.13 (q, J = 7.1 Hz, 2H, -OCH2, H3), 7.1 (t, J = 8 Hz, 1H, Ar-H8′), 7.13 (t, J = 7.6 Hz, 1H, Ar-H7′), 7.29 (s, 1H, Ar-H2′), 7.41 (d, J = 8.1, 1H, Ar-H6′), 7.61 (d, J = 7.9 Hz, 1H, Ar-H5′); 13C NMR (125 MHz, CDCl3-d) d 14.2, 31.4, 60.8, 108.6, 111.2, 118.9, 119.6, 122.2, 123.0, 128.6, 136.1, 172.4. All data were compared with the reported literature [29].
General procedure for the synthesis of acid hydrazide derivatives (3a-j).
Hydrazine monohydrate (5 mmol) was added dropwise to a solution of the corresponding ethyl ester (2a-j) (1 mmol) in EtOH (5 mL). The reaction mixture was stirred in room temperature for 12−48 h until completion, as determined by TLC profiles. After completion of reaction, the solvent was evaporated under reduced pressure. The acid hydrazide derivatives (3a-j) of 42-99% yield obtained were kept for further reactions without purification.
Benzohydrazide (3a).
White powder, 98% yield; mp 115-117 °C; IR ʋ / cm-1 3531, 1659, 1385; 1H NMR (500 MHz, Acetone-d) d 7.46 (t, 2H, J = 7.6 Hz, Ar-H3, H5), 7.53 (t, 1H, J = 7.6 Hz, Ar-H4), 7.89 (d, 2H, J = 7.6 Hz, Ar-H2, H6), 9.92 (s, 1H, -NH); 13C NMR (125 MHz, Acetone-d) d 127.5, 128.4, 131.3, 134.3, 159.2. All data were compared with the reported literature [30].
3-Nitrobenzohydrazide (3b).
Brown powder, 99% yield; mp 100-102 °C; IR ʋ / cm-1 3503, 2930, 1655, 1501, 1255; 1H NMR (500 MHz, DMSO-d6) d 7.77 (t, J= 8.0 Hz, 1H, Ar-H5), 8.27 (dd, J = 1.1, 8.0 Hz, 1H, Ar-H6), 8.37 (dd, 1H, J = 1.1, 8.0 Hz, Ar-H4), 8.64 (t, J = 1.1 Hz, 1H, Ar-H2); 13C NMR (125 MHz, DMSO-d6) d 124.0, 128.2, 131.2, 131.9, 135.7, 148.8, 164.6. All data were compared with the reported literature [31].
2-amino-3-chlorobenzohydrazide (3c).
Light brown crystal, 71% yield; mp 46-48 °C; IR ʋ / cm-1 3404, 2992, 1700, 1362, 865; 1H NMR (500 MHz, Acetone-d) d 6.57 (dd, J = 2.1, 8.7 Hz, 1H, Ar-H5), 6.86 (d, J = 2.1 Hz, 1H, Ar-H4), 7.80 (d, J = 8.7 Hz, 1H, Ar-H6); 13C NMR (125 MHz, Acetone-d) d 108.5, 115.3, 115.8, 133.0, 139.0, 152.6, 167.2.
5-bromo-2-hydroxybenzohydrazide (3d).
Light brown powder, 42% yield; mp 130-132 °C; IR ʋ / cm-1 3485, 3327, 3004, 1657, 1385, 1255, 658; 1H NMR (500 MHz, CDCl3) d 6.80 (d, J = 8.9 Hz, 1H, Ar-H3), 7.43 (dd, J = 2.3, 8.9 Hz, 1H, Ar-H4), 7.86 (d, J = 2.3 Hz, 1H, Ar-H6); 13C NMR (125 MHz, CDCl3) d 105.6, 108.9, 114.3, 127.0, 133.1, 155.4, 163.9.
4-chlorobenzohydrazide (3e).
Cream powder, 98% yield; mp 142-144 °C; IR ʋ / cm-1 3408, 3318, 3004, 1659, 1385, 1089, 865, 657; 1H NMR (500 MHz, DMSO-d6) d 7.56 (d, J = 8.6 Hz, 2H, Ar-H2, H6), 7.94 (d, 2H, J = 8.6 Hz, Ar-H3, H5); 13C NMR (125 MHz, DMSO-d6) d 128.9, 129.3, 132.5, 136.4, 165.3. All data were compared with the reported literature [32].
3-(4′-nitrophenyl)propanehydrazide (3f).
Light brown powder, 89% yield; mp 120-122 °C; IR ʋ / cm-1 3488, 2929, 1655, 1590, 1503, 1438, 1385, 657; 1H NMR (500 MHz, DMSO-d6) d 2.39 (t, J = 7.6 Hz, 2H, -CH2, H2), 2.96 (t, J = 7.6 Hz, 2H, -CH2, H3), 7.48 (d, J = 8.8 Hz, 2H, Ar-H3′, H5′), 8.13(d, J = 8.8 Hz, 2H, Ar-H2′, H6′); 13C NMR (125 MHz, DMSO-d6) d 31.2, 34.7, 123.8, 130.0, 146.4, 150.1, 170.8. All data were compared with the reported literature [33].
(E)-2-Methyl-3-phenylacrylohydrazide (3g).
Dark brown liquid, 91% yield; mp 33-38 °C; IR ʋ / cm-1 3251, 3061, 1731, 1495, 1453, 1257; 1H NMR (500 MHz, DMSO-d6) d 2.04 (d, J = 1.5 Hz, 3H, CH3, H10), 7.22 (s, 1H, CH-H3), 7.37 (s, 1H, Ar-H7), 7.38 (t, J = 8.3 Hz, 2H, Ar-H6, H8), 7.49 (d, J = 8.3 Hz, 2H, Ar-H5, H9); 13C NMR (125 MHz, DMSO-d6) d 21.1, 128.7, 129.5, 129.6, 129.8, 136.2, 136.5, 167.1.
1′-H-Indole-2-carbohydrazide (3h).
Dark purple powder, 90% yield; mp 120-122 °C; IR ʋ / cm-1 3525, 2929, 1655, 1385, 1255, 1090, 658; 1H NMR (500 MHz, DMSO-d6) d 7.00 (dt, J = 1.1, 7.5 Hz, 1H, Ar-H6′), 7.15 (s, 1H, Ar-H3′), 7.21 (dt, J = 1.1, 7.5 Hz, 1H, Ar-H7′), 7.46 (dd, J = 1.1, 7.5 Hz, 1H, Ar-H5′), 7.66 (d, J = 7.5 Hz, 1H, Ar-H8′), 11.86 (s, 1H, -NH); 13C NMR (125 MHz, DMSO-d6) d 102.1, 112.0, 120.6, 121.5, 123.1, 127.2, 130.9, 136.9, 161.8. All data were compared with the reported literature [34].
1′-hydroxy-2′-naphthohydrazide (3i).
Light yellow powder, 98% yield; mp 168-170 ºC; IR ʋ / cm-1 3285, 3158, 3049, 1655, 1590; 1H NMR (500 MHz, DMSO-d6) d 7.29 (s, 1H, Ar-H3′), 7.32 (t, J = 8.5 Hz, 1H, Ar-H7′), 7.48 (t, J = 8.5 Hz, 1H, Ar-H8′), 7.73 (d, J = 8.5 Hz, 1H, Ar-H6′), 7.83 (d, J = 8.1 Hz, 1H, Ar-H4′), 8.47 (s, 1H, Ar-H9′), 8.61 (s, 1H, -OH); 13C NMR (125 MHz, DMSO-d6) d 111.1, 118.6, 124.1, 126.2, 127.1, 128.6, 129.1, 129.5, 136.3, 155.4, 167.4. All data were compared with the reported literature [35].
2-(1H-indol-3-yl)acetohydrazide (3j).
Dark brown liquid, 80% yield; mp 34-35 °C; IR ʋ / cm-1 3267, 3057 , 1705, 1361; 1H NMR (500 MHz, Acetone-d) d 3.51 (s, 2H, CH2, H10), 6.99 (t, J = 8.2 Hz, 1H, Ar-H5), 7.1 (t, J = 8.2 Hz, 1H, Ar-H6), 7.22 (s, 1H, Ar-H2), 7.38 (d, J = 8.2 Hz, 1H, Ar-H4), 7.58 (d, J = 8.2 Hz, 1H, Ar-H7); 13C NMR (125 MHz, Acetone-d) d 31.4, 108.6, 111.2, 118.9, 119.6, 122.2, 123.0, 128.6, 136.1, 172.4. All data were compared with the reported literature [36].
General procedure for the synthesis of 5-substituted-1,3,4-Oxadiazole-2-thiol derivatives (4a-j)
A mixture of the corresponding acid hydrazide 3a-j (1.0 mmol) and carbon disulfide (3.0 mmol) in DMF (2.0 mL) was stirred for 15 min at room temperature. The reaction mixture was then refluxed for 8-24 h until the ring closure reaction was completed. The reaction progress was checked by TLC (ethyl acetate: n-hexane [1:2]). After cooling to room temperature, the reaction mixture was added dropwise into an ice-cold water (15 mL) and the precipitate formed was collected by filtration, washed with water, recrystallized from EtOH/water and dried to give compounds 4a-j (40–95% yields).
5-Phenyl-1,3,4-oxadiazole-2-thiol (4a).
Cream powder, 33% yield; mp 119-121 °C; IR ʋ / cm-1 3192, 3063, 1654, 1517, 1250, 1078; 1H NMR (500 MHz, Acetone-d6) d 7.46 (t, J = 7.6 Hz, 2H, Ar-H3′, H5′), 7.53 (t, J = 7.6 Hz, 1H, Ar-H4′), 7.89 (d, J = 7.6 Hz, 2H, Ar-H2′, H6′); 13C NMR (125 MHz, Acetone-d6) d 123.1, 126.1, 129.3, 132.2, 160.9, 178.5. All data were compared with the reported literature [37].
5-3′-Nitrophenyl-1,3,4-oxadiazole-2-thiol (4b).
Yellow powder, 80% yield; mp: 108-110 °C; IR ʋ / cm-1 2992, 1655, 1501, 1255; 1H NMR (500 MHz, DMSO-d6) d 7.77 (t, J= 8.0 Hz, 1H, Ar-H5′), 8.27 (dd, J = 1.1, 8.0 Hz, 1H, Ar-H6′), 8.37 (dd, J = 1.1, 8.0 Hz, 1H, Ar-H4′), 8.64 (t, J = 1.1 Hz, 1H, Ar-H2); 13C NMR (125 MHz, DMSO-d6) d 120.3, 125.6, 125.8, 131.6, 134.3, 148.7, 160.1, 179.7. All data were compared with the reported literature [31].
5-(2′-Amino-3′-chlorophenyl)-1,3,4-oxadiazole-2-thiol (4c).
Dark brown solid, 47% yield; mp 47-49 °C; IR ʋ / cm-1 2927, 1619, 1255, 1089, 865; 1H NMR (500 MHz, Acetone-d) d 6.52 (d, J = 8.6 Hz, 1H, Ar-H4′), 6.8 (t, J = 8.6 Hz, 1H, Ar-H5′), 7.67 (d, J = 8.6 Hz, 1H, Ar-H6′); 13C NMR (125 MHz, Acetone-d) d 108.4, 115.2, 115.7, 124.5, 128.3, 139.0, 145.5, 152.7, 167.2.
5′-Bromo-2′-hydroxy-1,3,4-oxadiazole-2-thiol (4d).
Cream powder, 56% yield; mp: 134-136 °C; IR ʋ / cm-1 3395, 3004, 1619, 1453, 1342, 1107, 748, 696; 1H NMR (500 MHz, CDCl3) d 6.80 (d, J = 8.2 Hz, 1H, Ar-H3′), 7.43 (dd, J = 2.4, 8.2 Hz, 1H, Ar-H4′), 7.86 (d, J = 2.4 Hz, 1H, Ar-H6′), 9.99 (s, 1H, -OH-H2′); 13C NMR (125 MHz, CDCl3) d 105.6, 108.9, 114.3, 127.0, 133.1, 155.4, 163.9, 164.1.
5-(4′-Chlorophenyl)-1,3,4-oxadiazole-2-thiol (4e).
Cream powder, 95% yield; mp: 132-134 ºC; IR ʋ / cm-1 3120, 1619, 1255, 1089, 865; 1H NMR (500 MHz, DMSO-d6) d 7.66 (d, J = 8.7 Hz, 2H, Ar-H2′, H6′), 7.90 (d, J = 8.7 Hz, 2H, Ar-H3′, H5′); 13C NMR (125 MHz, DMSO-d6) d 121.9, 128.4, 130.1, 137.4, 160.2, 178.0. All data were compared with the reported literature [38].
5-(4′-Nitrophenethyl)-1,3,4-oxadiazole-2-thiol (4f).
Cream powder, 66% yield; mp 128-130 °C; IR ʋ / cm-1 3058, 2931, 2749, 1690, 1604, 1507, 1339, 1250, 1175, 740; 1H NMR (500 MHz, Acetone-d) d 3.20 (t, J = 7.4 Hz, 2H, -CH2-H7′), 3.29 (t, J = 7.4 Hz, 2H, -CH2-H8′), 7.64 (d, J = 8.5 Hz, 2H, Ar-H3′, H5′), 8.21 (d, J = 8.5 Hz, 2H, Ar-H2′, H6′); 13C NMR (125 MHz, Acetone-d) d 26.3, 30.8, 123.5, 129.7, 147.0, 147.6, 163.1, 178.9.
(E)-5-(1′-Phenylprop-1-en-2′-yl)-1,3,4-oxadiazole-2-thiol (4g).
Colourless liquid, 65% yield; IR ʋ / cm-1 2935, 1656, 1181, 1063, 852; 1H NMR (500 MHz, DMSO-d6) d 2.04 (d, J = 1.5 Hz, 3H, CH3-H3′), 7.22 (s, 1H, CH-H1′), 7.37 (t, J = 7.3 Hz, 1H, Ar-H7′), 7.38 (t, J = 7.3 Hz, 2H, Ar-H6′, H8′), 7.49 (d, J = 7.3 Hz, 2H, Ar-H5′, H9′); 13C NMR (125 MHz, DMSO-d6) d 21.1, 128.7, 129.5, 129.6, 129.8, 136.2, 136.5, 168.2, 177.9.
5-(1′-H-indol-2′-yl)-1,3,4-oxadiazole-2-thiol (4h).
Light yellow powder, 32% yield; mp 122-124 ºC; IR ʋ / cm-1 3306, 3120, 2765, 1688, 1309, 1248, 1176, 739; 1H NMR (500 MHz, DMSO-d6) d 7.11 (t, J = 7.5 Hz, 1H, Ar-H6′), 7.18 (s, 1H, Ar-H3′), 7.26 (t, J = 7.5 Hz, 1H, Ar-H7′), 7.46 (d, J = 7.5 Hz, 1H, Ar-H5′), 7.66 (d, J = 7.5 Hz, 1H, Ar-H8′), 12.18 (s, 1H, -NH); 13C NMR (125 MHz, DMSO-d6) d 105.9, 112.8, 120.7, 120.9, 121.9, 124.9, 127.8, 138.6, 156.5, 177.9. All data were compared with the reported literature [39].
5-(2′-naphthalen-1′-ol)-1,3,4-oxadiazole-2-thiol (4i).
Brown powder, 77% yield; mp 168-170 °C; IR ʋ / cm-1 3063, 2548, 1699, 1632, 1358, 1255, 1053, 762; 1H NMR (500 MHz, Acetone-d) d 7.34 (t, J = 8.0 Hz, 1H, Ar-H7′), 7.37 (s, 1H, Ar-H3′), 7.53 (t, J = 8.0 Hz, 1H, Ar-H8′), 7.75 (d, J = 8.0 Hz, 1H, Ar-H6′), 7.96 (d, J = 8.0 Hz, 1H, Ar-H4′), 8.34 (s, 1H, Ar-H9′); 13C NMR (125 MHz, Acetone-d) d 111.1, 112.6, 126.3, 127.3, 128.9, 129.1, 130.8, 136.2, 153.1, 160.1, 162.8, 177.8. All data were compared with the reported literature [40].
5-[(1′-H-indol-3′-yl) methyl]-1,3,4-oxadiazole-2-thiol (4j).
Dark brown liquid, 27% yield; IR ʋ / cm-1 3316, 3128, 2983, 2773, 1688, 1309, 1248, 1176, 739; 1H NMR (500 MHz, Acetone-d) d 3.51 (s, 2H, CH2-H10′), 6.99 (t, J = 8.1 Hz, 1H, Ar-H7′), 7.1 (t, J = 8.1 Hz, 1H, Ar-H6′), 7.22 (s, 1H, Ar-H2′), 7.38 (d, J = 8.1 Hz, 1H, Ar-H8′), 7.58 (d, J = 8.1 Hz, 1H, Ar-H5′); 13C NMR (125 MHz, Acetone-d) d 31.4, 106.6, 111.2, 112.8, 118.9, 119.6, 122.2, 123.0, 124.9, 167.6, 173.8. All data were compared with the reported literature [36].
General procedure for the synthesis of 2-alkylbenzysulfanyl-5-substituted-1,3,4-oxadiazoles derivatives (5a-5j and 6a-6j)
A solution of an alkylating agent (1 mmol) and tetrabutylammonium bromide, TBAB (0.05 mmol) in CH2Cl2 (5 mL) was added to a solution of the corresponding 5-substituted-1,3,4-oxadiazole-2-thiols (4a-4j) (1.1 mmol) and sodium hydroxide (1.25 mmol) in H2O (5 mL). The reaction mixture was stirred slowly at room temperature overnight. Upon completion, the organic layer was separated, washed with water (2 × 7 mL), and dried over anhydrous Na2SO4 and concentrated under reduced pressure. The precipitate obtained was recrystallized from EtOH to afford compounds 5a-5j and 6a-6j (40-92% yields). For the known compounds, the spectroscopic data were compared with literatures.
2-(Benzylsulfanyl)-5-phenyl-1,3,4-oxadiazole (5a)
Cream powder, 92% yield; mp 116-118 ºC; IR ʋ / cm-1 3192, 3063, 2917, 1654, 1344, 1260, 1070, 691; 1H NMR (500 MHz, Acetone-d6) δ4.62 (s, 2H, CH2-H7″), 7.31 (t, J 7.5 Hz, 1H, Ar-H4″), 7.38 (t, J 7.5 Hz, 2H, Ar-H3″, H5″), 7.55 (d, J 7.5 Hz, 2H, Ar-H2″, H6″), 7.6 (t, J 8.0 Hz, 2H, Ar-H3′, H5′), 7.62 (t, J 8.0 Hz, 1H, Ar-H4′), 8.20 (d, J 8.0 Hz, 2H, Ar-H2′, H6′); 13C NMR (125 MHz, Acetone-d6) δ 36.2, 123.8, 126.4, 127.9, 128.7, 129.1, 129.3, 131.8, 136.6, 163.6, 165.6; HRMS (TOF-ES+) m/z, calcd. for C15H13N2OS+ [M + H]+: 269.0749, found: 269.0752; All data were compared with the reported literature [41].
2-(Benzylsulfanyl)-5-(3′-nitrophenyl)-1,3,4-oxadiazole (5b)
Light orange powder, 55% yield; mp 90-92 °C; IR ʋ / cm-1 3045, 2978, 1655, 1501, 1385, 1255; 1H NMR (500 MHz, Acetone-d6) δ4.65 (s, 2H, CH2-H7″), 7.32 (t, J 7.5 Hz, 2H, Ar-H3″, H5″), 7.37 (d, J 7.5 Hz, 2H, Ar-H2″, H6″), 7.38 (t, J 7.5 Hz, 1H, Ar-H4″), 7.57 (d, J 7.7 Hz, 1H, Ar-H6′), 7.94 (t, J 7.7 Hz, 1H, Ar-H5′), 8.43 (dd, J 7.7, 1.8 Hz, 1H, Ar-H4′), 8.75 (d, J 1.8 Hz, 1H, Ar-H2′); 13C NMR (125 MHz, Acetone-d6) δ36.9, 121.6, 125.2, 126.1, 128.3, 128.7, 128.9, 129.2, 130.4, 135.3. 148.6, 163.9, 165.2; HRMS (TOF-ES+) m/z, calcd. for C15H12N3O3S+ [M + H]+: 314.0599, found: 314.0602.
2-(Benzylsulfanyl)-5-(2′-Amino-3′-chlorophenyl)-1,3,4-oxadiazole (5c)
Brown liquid, 70% yield; IR ʋ / cm-1 2927, 2859, 1660, 1385, 1255, 1089, 865 ; 1H NMR (500 MHz, DMSO-d6) δ4.91 (s, 2H, CH2-H7″), 6.55 (d, J 8.5 Hz, 1H, Ar-H4′), 6.85 (t, J 8.5 Hz, 1H, Ar-H5′), 7.2 (m, 1H, Ar-H4″), 7.35 (d, J 8.0 Hz, 2H, Ar-H2″, H6″), 7.36 (t, J 8.0 Hz, 2H, Ar-H3″, H5″), 7.70 (d, J 8.5 Hz, 1H, Ar-H6′); 13C NMR (125 MHz, DMSO-d6) δ31.2, 108.4, 115.2, 115.7, 124.3, 124.5, 128.3, 130.5, 133.0, 139.0, 145.5, 152.7, 167.2; HRMS (TOF-ES+) m/z, calcd. for C15H13ClN3OS+ [M + H]+: 318.0468, found: 318.0472.
2-[(Benzylsulfanyl)‐1,3,4-oxadiazol‐2‐yl]‐5′‐bromophenol (5d)
Light brown powder, 67% yield; mp 97-99 ºC; IR ʋ / cm-1 3397, 3060, 2959, 1622, 1453, 1341, 1107, 748; 1H NMR (500 MHz, CDCl3-d1) δ4.58 (s, 2H, CH2-H7″), 7.06 (d, J 8.8 Hz, 1H, Ar-H3′), 7.30 (t, J 8.3 Hz, 1H, Ar-H4″), 7.36 (t, J 8.3 Hz, 2H, Ar-H3″, H5″), 7.49 (d, J 8.3 Hz, 2H, Ar-H2″, H6″), 7.59 (dd, J 8.8, 2.5, Hz, 1H, Ar-H4′), 7.83 (d, J 2.5 Hz, 1H, Ar-H6′), 10.66 (s, 1H, OH); 13C NMR (125 MHz, CDCl3-d1) δ36.4, 110.7, 112.2, 119.9, 128.6, 129.1, 129.5, 131.4, 136.2, 137.1, 155.9, 163.7, 163.8; HRMS (TOF-ES+) m/z, calcd. for C15H12BrN2O2S+ [M + H]+: 362.9803, found: 362.9801.
2-(Benzylsulfanyl)-5-(4′-chlorophenyl)-1,3,4-oxadiazole (5e)
White solid, 81% yield; mp 151-153 °C; IR ʋ / cm-1 3120, 2878, 1619, 1255, 1089, 865; 1H NMR (500 MHz, Acetone-d6) δ4.63 (s, 2H, CH2-H7″), 7.32 (t, J 7.5 Hz, 1H, Ar-H4″), 7.37 (t, J 7.5 Hz, 2H, Ar-H3″, H5″), 7.55 (d, J 7.5 Hz, 2H, Ar-H2″, H6″), 7.64 (d, J 8.7 Hz, 2H, Ar-H3′, H5′), 8.03 (d, J 8.7 Hz, 2H, Ar-H2′, H6′); 13C NMR (125 MHz, Acetone-d6) δ36.2, 122.6, 127.9, 128.1, 128.4, 128.7, 129.5, 136.6, 137.3, 163.9, 164.9; HRMS (TOF-ES+) m/z, calcd. for C15H12ClN2OS+ [M + H]+: 303.0358, found: 303.0355. All data were compared with the reported literature [42].
2-(Benzylsulfanyl)-5-(4′-nitrophenethyl)-1,3,4-oxadiazole (5f)
Brown powder, 81% yield; mp 128-130 °C; IR ʋ / cm-1 3338, 2928, 1660, 1502, 1385, 1255, 1089, 657; 1H NMR (500 MHz, Acetone-d) δ3.14 (t, J 7.0 Hz, 2H, CH2-H8′), 3.22 (t, J 7.0 Hz, 2H, CH2-H7′), 4.59 (s, 2H, CH2-H7″), 7.33 (m, 1H, Ar-H4″), 7.35 (m, 2H, Ar-H2″, H6″), 7.37 (d, J 8.5 Hz, 2H, Ar-H3″, H5″), 7.41 (d, J 8.7 Hz, 2H, Ar-H5′, H3′), 8.18 (d, J 8.7 Hz, 2H, Ar-H2′, H6′); 13C NMR (125 MHz, Acetone-d) δ26.0, 32.0, 36.7, 123.5, 128.1, 128.8, 129.1, 129.7, 135.5, 138.2, 146.8, 164.2, 166.3; HRMS (TOF-ES+) m/z, calcd. for C17H16N3O3S+ [M + H]+: 342.0912, found: 342.0909.
(E)-2-(benzylsulfanyl)-5-(1-phenylprop-1-en-2′-yl)-1,3,4-oxadiazole (5g)
Light yellow powder, 53 % yield; mp 118-120 °C; IR ʋ / cm-1 2935, 1656, 1181, 1063, 852; 1H NMR (500 MHz, DMSO-d6) δ 2.34 (d, J 1.4 Hz, 3H, CH3-H3′), 4.73 (s, 2H, CH2-H7″), 7.32 (m, 1H, Ar-H4″), 7.38 (t, J 7.5 Hz, 1H, Ar-H7′), 7.44 (s, 1H, Ar-H1′), 7.47 (t, J 7.5 Hz, 2H, Ar-H6′, H8′), 7.53 (d, J 7.5 Hz, 2H, Ar-H5′, H9′), 7.86 (d, J 8.7 Hz, 2H, Ar-H3″, H5″), 8.24 (d, J 8.7 Hz, 2H, Ar-H2″, H6″); 13C NMR (125 MHz, DMSO-d6) δ14.4, 35.5, 120.9, 123.9, 128.5, 128.6, 129.4, 129.6, 130.1, 134.6, 135.4, 138.1, 164.7, 167.4; HRMS (TOF-ES+) m/z, calcd. for C18H17N2OS+ [M + H]+: 309.1061, found: 309.1058.
2-(Benzylsulfanyl)-5-(1′H-indol-2′-yl)-1,3,4-oxadiazole (5h)
Light yellow powder, 60% yield; mp 178-180 °C; IR ʋ / cm-1 3306, 3120, 2983, 1688, 1309, 1248, 1019, 739; 1H NMR (500 MHz, DMSO-d6) δ 4.48 (s, 1H, CH2-H7″), 7.01 (t, J 8.3 Hz, 1H, Ar-H6′), 7.05 (d, J 1.2, 1H, Ar-H9′), 7.15 (m, 1H, Ar-H5′), 7.17 (t, J 8.4 Hz, 1H, Ar-H4″), 7.22 (t, J 8.4 Hz, 2H, Ar-H3″, H5″), 7.39 (d, J 8.4 Hz, 2H, Ar-H2″, H6″), 7.43 (t, J 8.3 Hz, 1H, Ar-H7′), 7.55 (d, J 8.1 Hz, 1H, Ar-H4′), 10.9 (s, 1H, -NH); 13C NMR (125 MHz, DMSO-d6) δ36.5, 105.2, 112.1, 120.6, 121.2, 121.5, 124.5, 127.8, 127.9, 128.6, 129.0, 136.5, 138.1, 160.8, 162.9; HRMS (TOF-ES+) m/z, calcd. for C17H14N3OS+ [M + H]+: 308.0857, found: 308.0855. All data were compared with the reported literature [39].
2-(Benzylsulfanyl)-5-(2′-naphthalen-1′-ol)-1,3,4-oxadiazole (5i)
Cream powder, 89% yield; mp 108-110 °C; IR ʋ / cm-1 2962, 2874, 1601, 1514, 1343, 1176, 1049, 702; 1H NMR (500 MHz, Acetone-d6) δ4.72 (s, 2H, CH2-H7″), 7.33 (m, 1H, Ar-H4″), 7.35 (d, J 8.2 Hz, 1H, Ar-H6′), 7.40 (d, J 8.8 Hz, 2H, Ar-H3″, H5″), 7.46 (d, J 8.2 Hz, 1H, Ar-H8′), 7.52 (d, J 8.8 Hz, 2H, Ar-H2″, H6″), 7.53 (d, J 8.2 Hz, 1H, Ar-H5′), 7.73 (t, J 8.2 Hz, 1H, Ar-H7′), 7.83 (d, J 8.2 Hz, 1H, Ar-H9′), 8.27 (d, J 8.2 Hz, 1H, Ar-H10′); 13C NMR (125 MHz, Acetone-d6) δ35.4, 112.0, 124.6, 126.6, 127.5, 127.8, 128.3, 127.5, 128.7, 128.9, 129.0, 129.2, 135.2, 136.5, 152.8, 163.9, 165.1; HRMS (TOF-ES+) m/z, calcd. for C19H15N2O2S+ [M + H]+: 335.0854, found: 335.0850.
2-(Benzylsulfanyl)-5-((1′H-indol-3′-yl)methyl)-1,3,4-oxadiazole (5j)
Dark brown liquid, 70% yield; IR ʋ / cm-1 3373, 2962, 1699, 1637, 1151, 1038, 743; 1H NMR (500 MHz, Acetone-d6) δ2.89 (s, 2H, CH2-H10′), 4.76 (s, 3H, CH2-H7″), 7.00 (t, J 8.1 Hz, 1H, Ar-H6′), 7.1 (t, J 8.1 Hz, 1H, Ar-H7′), 7.22 (s, 1H, Ar-H2′), 7.33 (dd, J 8.1 Hz, 1H, Ar-H5′), 7.35 (d, J 8.1 Hz, 1H, Ar-H8′), 7.38 (t, J 7.5 Hz, 2H, Ar-H3″, H5″), 7.39 (m, J 7.5 Hz, 1H, Ar-H4″), 7.43 (d, J 7.5 Hz, 2H, Ar-H2″, H6″); 13C NMR (125 MHz, Acetone-d6) δ31.4, 36.3, 106.6, 111.2, 118.9, 119.6, 122.2, 123.0, 128.6, 128.8, 129.0, 129.1, 129.6, 136.1, 167.6, 173.8; HRMS (TOF-ES+) m/z, calcd. for C18H16N3OS+ [M + H]+: 322.1014, found: 322.1009. All data were compared with the reported literature [36].
2-(4″-Nitrobenzyl)sulfanyl)-5-phenyl-1,3,4-oxadiazole (6a)
Cream powder, 90% yield; mp 118-120 ºC; IR ʋ / cm-1 3062, 2917, 1654, 1520, 1341, 1260, 1070, 691; 1H NMR (500 MHz, DMSO-d6) δ4.72 (s, 2H, CH2-H7″), 7.65 (t, J 7.5 Hz, 2H, Ar-H4′), 7.70 (t, J 8.2 Hz, 1H, Ar-H3′, H5′), 7.78 (d, J 8.2 Hz, 2H, Ar-H2′, H6′), 7.95 (d, J 8.8 Hz, 2H, Ar-H3″, H5″), 8.22 (d, J 8.8 Hz, 2H, Ar-H2″, H6″); 13C NMR (125 MHz, DMSO-d6) δ35.4, 123.4, 124.1, 126.9, 129.9, 130.8, 132.6, 145.4, 147.4, 163.4, 165.9; HRMS (TOF-ES+) m/z, calcd. for C15H12N3O3S+ [M + H]+: 314.0599, found: 314.0602.
2-[(4″-Nitrobenzyl)sulfanyl)-5-(3′-nitrophenyl)]-1,3,4-oxadiazole (6b)
Light orange powder, 55% yield; mp 90-92 °C; IR ʋ / cm-1 3003, 2932, 1655, 1501, 1385, 1255; 1H NMR (500 MHz, Acetone-d6) δ4.65 (s, 2H, CH2-H7″), 7.32 (t, J 8.1 Hz, 3H, Ar-H3″, H5″), 7.37 (d, J 8.1 Hz, 2H, Ar-H2″, H6″), 7.57 (d, J 8.1 Hz, 1H, Ar-H6′), 7.94 (t, J 8.1 Hz, 1H, Ar-H5′), 8.43 (dd, J 8.1, 1.8 Hz, 1H, Ar-H4′), 8.75 (d, J 1.8 Hz, 1H, Ar-H2′); 13C NMR (125 MHz, Acetone-d6) δ36.9, 121.6, 125.2, 126.1, 128.3, 128.7, 128.9, 129.2, 130.4, 135.3, 148.6, 163.9, 165.2; HRMS (TOF-ES+) m/z, calcd. for C15H11N4O5S+ [M + H]+: 359.0451, found: 359.0455.
2-(4″-Nitrobenzyl)sulfanyl)]-5-[(3′-Chloro-2′-aniline)-1,3,4-oxadiazole (6c)
Brown solid, 70% yield; mp 45-48 °C; IR ʋ / cm-1 2997, 2859, 1660, 1533, 1385, 1255, 1089, 865; 1H NMR (500 MHz, DMSO-d6) δ4.91 (s, 2H, CH2-H7″), 6.55 (d, J 8.2 Hz, 1H, Ar-H4′), 6.85 (t, J 8.2 Hz, 1H, Ar-H5′), 7.66 (d, J 8.2 Hz, 1H, Ar-H6′), 7.73 (d, J 8.5 Hz, 2H, Ar-H2″, H6″), 8.22 (d, J 8.5 Hz, 2H, Ar-H3″, H5″); 13C NMR (125 MHz, DMSO-d6) δ31.2, 108.4, 115.2, 115.7, 124.3, 124.5, 128.3, 130.5, 133.0, 139.0, 145.5, 152.7, 167.2; HRMS (TOF-ES+) m/z, calcd. for C15H12ClN4O3S+ [M + H]+: 363.0319, found: 363.0315.
2-((4″-Nitrobenzyl)sulfanyl)-5-(5′-Bromo-2′-hydroxy)-1,3,4-oxadiazole) (6d)
Light brown crystal powder, 78% yield; mp 129-131 ºC; IR ʋ / cm-1 3395, 2928, 1619, 1520, 1453, 1342, 1107, 748, 696; 1H NMR (500 MHz, CDCl3-d1) δ4.52 (s, 2H, CH2-H7″), 6.94 (d, J 8.5 Hz, 1H, Ar-H3′), 7.44 (d, J 8.5 Hz, 1H, Ar-H4′), 7.60 (d, J 8.5 Hz, 2H, Ar-H3″, H5″), 7.70 (d, J 8.5 Hz, 1H, Ar-H2″, H6″), 8.5 (s, 1H, Ar-H6′); 13C NMR (125 MHz, CDCl3-d1) δ35.7, 110.8, 112.2, 119.8, 124.1, 128.6, 130.1, 136.5, 145.6, 147.4, 156.3, 162.9, 164.5; HRMS (TOF-ES+) m/z, calcd. for C15H11BrN3O4S+ [M + H]+: 407.9653, found: 407.9649.
2-((4″-Nitrobenzyl)sulfanyl)-5-(4′-Chlorophenyl)-1,3,4-oxadiazole (6e)
Yellow powder, 80% yield; mp 110-112 ºC; IR ʋ / cm-1 3120, 2878, 1619, 1525, 1255, 1089, 865; 1H NMR (500 MHz, DMSO-d6) δ4.47 (s, 2H, CH2-H7″), 7.41 (d, J 8.6 Hz, 2H, Ar-H3″, H5″), 7.53 (d, J 8.8 Hz, 2H, Ar-H2′, H6′), 7.70 (d, J 8.8 Hz, 2H, Ar-H3′, H5′), 7.97 (d, J 8.6 Hz, 2H, Ar-H2″, H6″); 13C NMR (125 MHz, DMSO-d6) δC 40.1, 127.0, 128.9, 133.5, 134.8, 135.6, 142.0, 150.2, 152.1, 168.4, 169.9; HRMS (TOF-ES+) m/z, calcd. for C15H11ClN3O3S+ [M + H]+: 348.0209, found: 348.0205.
2-((4″-Nitrobenzyl)sulfanyl)-5-(4′-nitrophenethyl)-1,3,4-oxadiazole (6f)
Brown powder. 69% yield; mp 20-122 °C; IR ʋ / cm-1 3538, 2928, 1660, 1502, 1385, 1255, 1069, 657; 1H NMR (500 MHz, DMSO-d6) δ3.14 (t, J 7.1 Hz, 2H, CH2-H7′), 3.22 (t, J 7.1 Hz, 2H, CH2-H8′), 4.59 (s, 2H, CH2-H7″), 7.52 (d, J 8.6 Hz, 2H, Ar-H2′, H6′), 7.69 (d, J 8.6 Hz, 2H, Ar-H3″, H5″), 8.13 (d, J 8.6 Hz, 2H, Ar-H3′, H5′), 8.18 (d, J 8.6 Hz, 2H, Ar-H2″, H6″); 13C NMR (125 MHz, DMSO-d6) δ26.0, 31.4, 35.2, 123.9, 124.1, 130.2, 130.7, 145.4, 146.6, 147.3, 148.4, 162.8, 167.7; HRMS (TOF-ES+) m/z, calcd. for C17H15N4O5S+ [M + H]+: 387.0763, found: 387.0759.
(E)-2-((4″-Nitrobenzyl)sulfanyl)- 5-(1-phenylprop-1′-en-2′-yl)-1,3,4-oxadiazole (6g)
Yellow powder, 68% yield; mp 120-122 °C; IR ʋ / cm-1 3103, 2935, 1656, 1508, 1181, 1063, 852; 1H NMR (500 MHz, DMSO-d6) δ2.34 (d, J 1.4 Hz, 3H, CH3-H2′), 4.73 (s, 2H, CH2-H7″), 7.38 (t, J 7.5 Hz, 1H, Ar-H7′), 7.44 (s, 1H, Ar-H3′), 7.47 (t, J 7.5 Hz, 2H, Ar-H6′, H8′), 7.53 (d, J 7.5 Hz, 2H, Ar-H5′, H9′), 7.86 (d, J 8.7 Hz, 2H, Ar-H3″, H5″), 8.24 (d, J 8.7 Hz, 2H, Ar-H2″, H6″). 13C NMR (125 MHz, DMSO-d6) δ14.4, 35.5, 120.9, 123.9, 128.5, 128.6, 129.4, 129.6, 130.1, 134.6, 135.4, 138.1, 164.7, 167.4; HRMS (TOF-ES+) m/z, calcd. for C18H16N3O3S+ [M + H]+: 354.0912, found: 354.0915.
2-((4″-Nitrobenzyl)sulfanyl)-5-(1′H-Indol-2′-yl)-1,3,4-oxadiazole (6h)
Light brown powder, 60% yield; mp 180-182 ºC; IR ʋ / cm-1 3307, 3078, 2963, 1684, 1517, 1340, 1245, 1019, 742; 1H NMR (500 MHz, DMSO-d6) δ4.74 (s, 2H, CH2-H7″), 7.10 (d, J 8.0 Hz, 1H, Ar-H5′), 7.17 (s, 1H, Ar-H9′), 7.26 (t, J 8.0 Hz, 1H, Ar-H6′), 7.49 (t, J 8.0 Hz, 1H, Ar-H7′), 7.67 (d, J 8 Hz, 1H, Ar-H4′), 7.79 (d, J 8.5 Hz, 2H, Ar-H3″, H5″), 8.21 (d, J 8.5 Hz, 2H, Ar-H2″, H6″), 12.22 (s, 1H, -NH); 13C NMR (125 MHz, DMSO-d6) δ35.6, 105.6, 112.8, 120.9, 122.0, 124.1, 124.8, 127.7, 130.8, 131.2, 138.2, 145.4, 147.4, 161.2, 162.6; HRMS (TOF-ES+) m/z, calcd. for C17H13N4O3S+ [M + H]+: 353.0708, found: 353.0710.
2-((4″-Nitrobenzyl)sulfanyl)-5-(2′-naphthalen-1′-ol)-1,3,4-oxadiazole (6i)
Cream powder, 70% yield; mp 120-121 °C; IR ʋ / cm-1 2962, 2874, 1601, 1514, 1343, 1176, 1049, 702; 1H NMR (500 MHz, Acetone-d6) δ4.72 (s, 2H, CH2-H7″), 7.37 (t, J 8.2 Hz, 1H, Ar-H6′), 7.38 (d, J 7.8 Hz, 1H, Ar-H9′), 7.53 (t, J 8.2 Hz, 1H, Ar-H5′), 7.77 (t, J 8.2 Hz, 1H, Ar-H7′), 7.81 (d, J 8.8 Hz, 2H, Ar-H3″, H5″), 7.96 (d, J 8.1 Hz, 1H, Ar-H8′), 8.22 (d, J 8.8 Hz, 2H, Ar-H2″, H6″), 8.38 (d, J 7.8 Hz 1H, Ar-H10′), 10.43 (s, 1H, OH); 13C NMR (125 MHz, Acetone-d6) δ 35.4, 111.1, 112.8, 124.3, 124.3, 124.5, 126.4, 127.3, 129.0, 129.1, 130.8, 136.2, 145.5, 147.4, 152.9, 163.4, 165.1; HRMS (TOF-ES+) m/z, calcd. for C19H14N3O4S+ [M + H]+: 380.0705, found: 380.0703.
2-[(4″-Nitrobenzylsulfanyl)-5-(1′H-indol-3′-yl)methyl)]-1,3,4-oxadiazole (6j)
Dark brown liquid, 40% yield; IR ʋ / cm-1: 3373, 3122, 2962, 1699, 1637, 1509, 1309, 1151, 1038, 743; 1H NMR (500 MHz, Acetone-d6) δ 2.89 (s, 2H, CH2-H10′), 4.76 (s, 3H, CH2-H7″), 7.00 (t, J 8.1 Hz, 1H, Ar-H6′), 7.10 (t, J 8.1 Hz, 1H, Ar-H7′), 7.22 (s, 1H, Ar-H2′), 7.33 (d, J 8.1 Hz, 1H, Ar-H5′), 7.35 (d, J 8.1 Hz, 1H, Ar-H8′), 7.38 (t, J 7.6 Hz, 2H, Ar-H3″, H5″), 7.43 (d, J 7.6 Hz, 2H, Ar-H2″, H6″); 13C NMR (125 MHz, Acetone-d6) δ31.4, 36.3, 106.6, 111.2, 118.9, 119.6, 122.2, 123.0, 128.6, 128.8, 128.9, 129.1, 129.6, 136.1, 167.6, 173.8; HRMS (TOF-ES+) m/z, calcd. for C18H15N4O3S+ [M + H]+: 367.0865, found: 367.0867.
Anti-mycobacterial Activity (MIC and MBC evaluations)
Bacterial culture
M. smegmatis and M. tuberculosis H37Ra, were purchased from American Type Culture Collection (ATCC, USA). Their stock cultures were maintained on Middlebrook 7H10 (M7H10) agar slants supplemented with oleic acid, albumin, dextrose and catalase (OADC) enrichment and in Middlebrook 7H9 (M7H9) broth supplemented with albumin, dextrose and catalase (ADC) enrichment at 4 ºC and -25 ºC respectively. The test cultures were activated from the stock cultures by culturing on the M7H10 agar and incubated for two days for M. smegmatis and 10 days for M. tuberculosis H37Ra at 37 ºC in 4% CO2.
Determination of minimum inhibitory concentration (MIC)
The MIC is defined as the lowest concentration of the assayed antimicrobial agent that inhibits the visible growth of the bacterium. 5-substituted-1,3,4-oxadiazole-2-thiol derivatives (5a-j and 6a-j) were screened for anti-mycobacterial MIC values against M. smegmatis and M. tuberculosis H37Ra. The test was performed by using the TEMA method as previously described in Mohamad et al. [43-44] and Wong et al., [45]. The test concentrations of the compounds were in the range of 68 µM and 2300 µM. The control drug used for M. smegmatis was streptomycin with an initial concentration of 6.25 µg mL-1. Isoniazid was used as the control drug for M. tuberculosis H37Ra with an initial concentration of 1.25 µm mL-1. The MIC was determined after the addition of MTT into the microplate. A colour change from yellow to purple was observed and the MIC value was recorded as the lowest concentration of compounds and drugs that remained yellow colour.
Determination of minimum bactericidal concentration (MBC)
The minimum bactericidal concentration (MBC) of 5-substituted-1,3,4-oxadiazole-2-thiol derivatives (5a-j and 6a-j) were determined after the MIC evaluation. The MBC is defined as the lowest concentration of an individual compound or drug that is lethal towards the mycobacteria. A loopful of the culture broth from all wells ≥ MIC value in microplates were streaked onto M7H10 agar plates in replicates after initial incubation prior the addition of MTT reagent. The streaking was done by using sterilized inoculating loops. The agar plates were sealed and incubated for 3 days for M. smegmatis and 28 days for M. tuberculosis H37Ra at 37 °C in 4% CO2. The growth of mycobacteria was observed. The MBC was interpreted as the lowest concentration, which showed no growth of the mycobacteria.
Drug interaction checkerboard assay of compound 5c and 5d
Compounds 5c and 5d were selected for interaction study with four first-line anti-TB drugs using the checkerboard TEMA assay. The assay was performed in three microtitre plates labelled Microplate 1 (drug dilution), Microplate 2 (compounds dilution) and Microplate 3 (interaction study). The outer of the well of the microtitre plates were filled with sterilised distilled water. A volume of 150 µL of M7H9 broth was added into wells in Microplate 1, except for the first row (Row G). The drug solution at a concentration that was 16 times higher than the MIC concentration was added into Row G and Row F in a volume of 150 µL. A two-fold serial dilution of the drug was performed across the y-axis starting from Row F to Row B. The excess 150 µL solution was discarded from Row B. In Microplate 2, the dilution was carried out similar to that in Microplate 1 with a slight difference. The wells were added with 50 µL of M7H9 broth, except for the first column (Column 2). The compound solution of 50 µL with 16 times higher in MIC concentration was added in Column 2 and Column 3. Two-fold serial dilution of the compound was done across the x-axis starting from Column 3 to Column 7. The excess 50 µL solution from column 7 was discarded.
For the combination study, a volume of 50 µL from Microplates 1 and 2 were transferred into Microplate 3 following their position of the wells. Column 8 until 10 were reserved for drug control. Column 11 was used as a positive control where 100 µL of M7H9 broth was added into all wells. A volume of 100 µL of log-phase bacterial inoculum was then added. The microplates were covered with lids and sealed with parafilm and incubated at 37 °C in 4% CO2 for 24 hours. A volume of 50 µL of freshly prepared MTT reagent was added into all test wells after 24 hours of incubation. The microplates were sealed and incubated for another 24 hours. A resulting yellow colour in the well indicated that the growth of the mycobacteria was inhibited, while purple colour indicated growth of mycobacteria. The assay was performed in triplicate.
The total fractional inhibitory concentration index (∑FICI) can be calculated by the following formula:
Where A = compound, and B = first-line anti-TB drug
The FIC index was used to classify drug interactions between compounds towards the
drug [46-47]. The drug interactions were defined as synergy if the ∑FIC index was ≤ 0.5, additive if the ∑FICI was from 0.5 to 4, and antagonistic if the FICI was > 4.
Time-kill assay of compound 5c and 5d.
The cidal effect of the most active compounds 5c and 5d in combination with INH on the growth kinetics of M. smegmatis was evaluated by performing the time-kill assay [48]. Four sets of bottles containing M7H9 broth in duplicates were prepared and labelled. A volume of 0.5 mL of the mycobacterial inoculum at McFarland Standard 2 was added into all bottles. Bottle 1 contained broth and bacterial inoculum mixed with compound 5c or 5d at its MIC value; Bottle 2 contained broth and bacterial inoculum with INH at its MIC value; Bottle 3 contained broth and bacterial inoculum in combination with INH and compound 5c or 5d at their respective MICs; Bottle 4 (positive control) contained only broth and inoculum. The final total volume of each bottle was 5 mL. The cultured bottles were incubated at 37 °C in 4% CO2 for 72 hours. Culture samples were taken every 8-hour interval to do colony counts using the drop plate method. A volume of 20 µL of culture sample was drawn out of the bottles for a serial dilution up to 10-5. Then, 10 µL from each dilution (10-2, 10-3, 10-4 and 10-5) was dropped in triplicate onto two sets of M7H10 agar plates. The droplets were left to dry at room temperature then the plates were sealed and incubated at 37 °C in 4% CO2 for 72 hours. The colonies formed on the agar were counted and colony-forming unit (CFU) per mL was calculated. The results plotted as a percentage of colonies compared to the time 0 versus time.
Molecular docking studies.
Molecular docking studies were performed using AutoDock 4.2 to identify appropriate binding modes and conformation of the ligand molecules [49]. The crystal structure of pantothenate synthetase (PDB code:3IVX, resolution-1.73 Å) was retrieved from the PDB and used for molecular modelling studies [20]. The structures of all the compounds were sketched using ChemDraw Ultra 13.0 and converted into 3D structures using Hyperchem Pro 8.0 software (>www.hyper.com) [50-51]. AutoDock tools (ADT) version 1.5.6 (www.autodock.scrips. edu) was used to prepare molecular docking.The grid box size was set to a dimension of 74×42×64 in x, y, z coordinates to cover the active site of the enzyme while virtual screening was performed by AutoDock 4.2.5.1. The best binding conformation was selected from the docking log (.dlg) file for each ligand and further interaction analysis was done using PyMol and Discovery Studio Visualizer 4.0 [52-53].