Chemistry
General
All reagents and solvents employed were of general purpose or analytical grade and purchased from Fluka, Acros, Alfa-Aeser and Sigma-Aldrich. Solvents were appropriately dried over molecular sieves (4 Å). 1H and 13C NMR spectra were recorded on a Bruker Advance DP500 spectrometer operating at 500 MHz and 125 MHz respectively. Chemical shifts are given in parts per million (ppm) relative to the internal standard tetramethylsilane (Me4Si). Coupling constants (J value) were calculated in hertz (Hz). An additional file shows these spectra in more details (see Additional file 1). Silica gel Fluka Kieselgel 60, particle size 35–70 μm Davisil® chromatography grade, was used for column chromatography in a glass column. Gradient column chromatography was performed with the aid of a pump. Analytical thin layer chromatography (TLC) was carried out on precoated silica plates (ALUGRAM® SIL G/UV254) with visualisation via UV light (254 nm). Melting points were determined using a Gallenkamp melting point apparatus and are uncorrected. UV HPLC was performed at the University of Bath using a sheath gas temperature of 350 °C, flow rate of 12 L/min, and nebuliser gas at 45 psi (3.10 bar). MS was calibrated using reference calibrant introduced from the independent ESI reference sprayer. Chromatographic separation was performed on a Zorbax Eclipse Plus C18 Rapid Resolution column 2.1 × 50 mm, 1.8 µm particle size using H2O (Merck, LC–MS grade) with 0.1% formic acid (FA, Fluka) v/v and methanol (MeOH, VWR, HiPerSolv) with 0.1% FA v/v as mobile phase A and B, respectively.
Compounds 2 and 3 were prepared as previously described [35,36,37,38,39, 46].
5- [2-(1H-benzo[d]imidazol-1-yl)ethyl]-1,3,4-oxadiazole-2-thiol (4)
A mixture of 3-(1H-benzo[d]imidazol-1-yl)propane hydrazide (3) (1 g, 4.9 mmol) and KOH (0.27 g, 4.9 mmol) in absolute EtOH (12 mL) was allowed to stir at room temperature for 30 min, then CS2 (0.56 g, 7.35 mmol) was added and the reaction mixture was heated under reflux overnight. The solvent was removed under vacuum and H2O (20 mL) was added and the resulting solution was cooled and acidified with 1 N aqueous HCl. The formed solid was collected by filtration, washed with H2O and dried under vacuum at 40 °C to obtain the product as a light brown solid: Yield: 1.11 g (92%); m.p. 225–227 °C; HPLC: 95.65% at R.T. 3.27 min; 1H NMR (DMSO-d6) δ: 3.33 (t, J = 6.8 Hz, 2H, NCH2CH2), 4.65 (t, J = 6.8 Hz, 2H, NCH2), 7.22 (td, J = 1.2, 7.2 Hz, 1H, Ar), 7.28 (td, J = 1.3, 8.2 Hz, 1H, Ar), 7.64 (dd, J = 7.9, 13.2 Hz, 2H, Ar), 8.25 (s, 1H, Imidazole).13C NMR (DMSO-d6) δ: 26.5 (NCH2CH2), 40.7 (NCH2), 110.8 (CH), 119.9 (CH), 122.2 (CH), 123.0 (CH), 133.9 (C), 143.5 (C), 144.5 (CH-imidazole), 161.9 (C), 178.2 (C). HRMS (ESI) m/z Calculated: 246.0575 [M + H]+, Found: 246.0583 [M + H]+.
5- [2-(1H-benzo[d]imidazol-1-yl)ethyl]-1,3,4-thiadiazole-2-thiol (7)
A mixture of 3-(1H-benzo[d]imidazol-1-yl)propane hydrazide (3) (1 g, 4.9 mmol) and KOH (0.27 g, 4.9 mmol) in absolute EtOH (12 mL) was allowed to stir at room temperature for 30 min, then CS2 (0.56 g, 7.35 mmol) was added. The reaction mixture was heated under reflux overnight. The solvent was removed under vacuum and concentrated H2SO4 (25 mL) was added and the mixture was allowed to stir for 4 h. The reaction mixture was poured into crushed ice and stirred for 1 h, then the resulting solid was collected by filtration, washed several times with H2O and dried under vacuum at 40 °C to obtain the product as a beige solid: Yield: 0.78 g (60%); mp 232–234 °C; HPLC: 100% at R.T. 3.28 min. 1H NMR (DMSO-d6) δ: CH2 peak is obscured by H2O of DMSO-d6 peak, 4.80 (t, J = 6.7 Hz, 2H, NCH2), 7.46 (m, 2H, Ar), 7.78 (d, J = 7.3 Hz, 1H, Ar), 7.88 (d, J = 7.5 Hz, 1H, Ar), 9.01 (s, 1H, Imidazole). 13C NMR (DMSO-d6) δ: 25.9 (NCH2CH2), 41.9 (NCH2), 112.4 (CH), 117.4 (CH), 124.9 (CH), 125.1 (CH), 132.4 (C), 136.6 (C), 143.5 (CH-imidazole), 155.1 (C). HRMS (ESI) m/z Calculated: 262.0347 [M + H]+, Found: 262.0387 [M + H]+
General method for the preparation of 2-(2-(1H-benzo[d]imidazol-1-yl)ethyl)-5-(arylthio)-1,3,4-oxadiazole (5a-d, 6a-c) and 2-(2-(1H-benzo[d]imidazol-1-yl)ethyl)-5-(arylthio)-1,3,4-thiadiazole (8a-d, 9a-c)
A mixture of aromatic thiol (4 or 7) (1 equivalent) and K2CO3 (1.5 equivalent) in anhydrous DMF (8 mL/0.89 mmol) was allowed to stir at room temperature for 30 min. Then the appropriate alkyl halide (1.1 equivalent) in anhydrous DMF (4 mL/0.89 mmol) was added and the reaction mixture allowed to stir at room temperature overnight. The solvent was removed under vacuum and then H2O (50 mL/0.89 mmol) was added, and the mixture extracted with chloroform (3 × 50 mL/0.89 mmol). The combined organic layers were dried (MgSO4) and evaporated, then the product was purified using gradient column chromatography.
2-{[2-(1H-benzo[d]imidazol-1-yl)ethyl]-5-(benzylthio)}-1,3,4-oxadiazole
(5a).
Prepared from 5-(2-(1H-benzo[d]imidazol-1-yl)ethyl)-1,3,4-oxadiazole-2-thiol (4) (0.2 g, 0.813 mmol) and benzyl chloride (0.11 g, 0.89 mmol). Purified using gradient chromatography eluting with 100% EtOAc to afford the product as a white solid: Yield: 0.16 g (59%); mp 80–82 °C; TLC: 100% EtOAc, Rf 0.22; HPLC: 100% at R.T. 4.25 min. 1H NMR (DMSO-d6) δ: 3.44 (t, J = 6.8 Hz, 2H, NCH2CH2), 4.40 (s, 2H, SCH2), 4.67 (t, J = 6.7 Hz, 2H, NCH2), 7.19–7.34 (m, 5H, Ar), 7.37 (m, 2H, Ar), 7.57 (dd, J = 1.2, 7.3 Hz, 1H, Ar), 7.64 (dd, J = 0.9, 7 Hz,1H, Ar), 8.19 (s, 1H, CH-imidazole). 13C NMR (DMSO-d6) δ: 26.2 (NCH2CH2), 36.1 (SCH2), 41.2 (NCH2), 110.7 (CH), 120.0 (CH), 122.1 (CH), 122.9 (CH), 128.2 (CH), 129.0 (2xCH), 129.4 (2xCH), 133.9 (C), 136.9 (C), 143.8 (C), 144.5 (CH-imidazole), 163.6 (C), 165.9 (C). HRMS (ESI) m/z Calculated: 336.1045 [M + H]+, Found: 336.1056 [M + H]+.
2-{[2-(1H-benzo[d]imidazol-1-yl)ethyl]-5- [(2,4-dichlorobenzyl)thio]}-1,3,4-oxadiazole
(5b).
Prepared from 5-(2-(1H-benzo[d]imidazol-1-yl)ethyl)-1,3,4-oxadiazole-2-thiol (4) (0.2 g, 0.813 mmol) and 2,4-dichlorobenzyl chloride (0.17 g, 0.89 mmol). Purified using gradient chromatography eluting with 100% EtOAc to afford the product as a white solid: Yield: 0.26 g (79%); mp 86–88 °C; TLC: 100% EtOAc, Rf 0.36; HPLC: 100% at R.T. 4.62 min. 1H NMR (DMSO-d6) δ: 3.44 (t, J = 6.8 Hz, 2H, NCH2CH2), 4.47 (s, 2H, SCH2), 4.67 (t, J = 6.8 Hz, 2H, NCH2), 7.21 (m, 2H, Ar), 7.38 (dd, J = 2.2, 8.3 Hz, 1H, Ar), 7.49 (d, J = 8.4 Hz, 1H, Ar), 7.56 (dd, J = 1.7, 7.2 Hz, 1H, Ar), 7.64 (m, 2H, Ar), 8.21 (s, 1H, imidazole). 13C NMR (DMSO-d6) δ: 26.3 (NCH2CH2), 33.9 (SCH2), 41.2 (NCH2), 110.6 (CH), 120.0 (CH), 122.1 (CH), 122.9 (CH), 128.0 (CH), 129.6 (CH), 133.2 (CH), 133.5 (C), 133.9 (C), 134.0 (C), 134.7 (C), 143.8 (C), 144.6 (CH-imidazole), 162.9 (C), 166.2 (C). HRMS (ESI) m/z Calculated: 404.0265 [M + H]+, Found: 404.0276 [M + H]+.
2-{[2-(1H-benzo[d]imidazol-1-yl)ethyl]-5- [(4-fluorobenzyl)thio]}-1,3,4-oxadiazole
(5c).
Prepared from 5-(2-(1H-benzo[d]imidazol-1-yl)ethyl)-1,3,4-oxadiazole-2-thiol (4) (0.2 g, 0.813 mmol) and 4-flourobenzyl chloride (0.13 g, 0.89 mmol). Purified using gradient chromatography eluting with 100% EtOAc to afford the product as a white semisolid: Yield: 0.205 g (71%); TLC: 100% EtOAc, Rf 0.20; HPLC: 100% at R.T. 4.28 min. 1H NMR (DMSO-d6) δ: 3.44 (t, J = 6.7 Hz, 2H, NCH2CH2), 4.40 (s, 2H, SCH2), 4.67 (t, J = 6.7 Hz, 2H, NCH2), 7.14 (t, J = 8.9 Hz, 2H, Ar), 7.22 (m, 2H, Ar), 7.42 (m, 2H, Ar), 7.57 (dd, J = 1.3, 7.3 Hz, 1H, Ar), 7.64 (dd, J = 1, 6.9 Hz, 1H, Ar), 8.19 (s, 1H, imidazole). 13C NMR (DMSO-d6) δ: 26.2 (NCH2CH2), 35.3 (SCH2), 41. (NCH2), 110.7 (CH), 115.7 (CH), 115.9 (CH), 120.0 (CH), 122.1 (CH), 122.9 (CH), 131.5 (CH), 131.6 (CH), 131.3 (C), 133.9 (C), 143.8, (C), 144.6 (CH-imidazole), 161.1 (C), 163.0 (C), 163.5 (C), 165.9 (C). HRMS (ESI): Calculated: 354.0951 [M + H]+, Found: 354.0961 [M + H]+.
4-{[( 5- [2-(1H-benzo[d]imidazol-1-yl)ethyl]-1,3,4-oxadiazol-2-yl)}thio)methyl] benzonitrile
(5d).
Prepared from 5-(2-(1H-benzo[d]imidazol-1-yl)ethyl)-1,3,4-oxadiazole-2-thiol (4) (0.2 g, 0.813 mmol) and 4-(chloromethyl)benzonitrile (0.14 g, 0.89 mmol). Purified using gradient chromatography eluting with 100% EtOAc to afford the product as a white semisolid: Yield: 0.195 g (67%); TLC: 100% EtOAc, Rf 0.36; HPLC: 100% at R.T. 4.02 min. 1H NMR (DMSO-d6) δ: 3.43 (t, J = 6.8 Hz, 2H, NCH2CH2), 4.48 (s, 2H, SCH2), 4.66 (t, J = 6.7 Hz, 2H, NCH2), 7.21 (m, 2H, Ar), 7.56 (m, 3H, Ar), 7.64 (m, 1H, Ar), 7.77 (d, J = 8.4 Hz, 2H, Ar), 8.19 (s, 1H, imidazole). 13C NMR (DMSO-d6) δ: 26.2 (NCH2CH2), 35.5 (SCH2), 41.2 (NCH2), 110.6 (CH), 110.9 (CN), 119.1 (C), 119.9 (CH), 122.1 (CH), 122.9 (CH), 130.4 (2xCH), 132.9 (2xCH), 133.9 (C), 143.2, (C), 143.8 (C), 144.6 (CH-imidazole), 163.2 (C), 166.1 (C). HRMS (ESI): Calculated: 361.0997 [M + H]+, Found: 361.1004 [M + H]+.
2- {[2-(1H-benzo[d]imidazol-1-yl)ethyl]-5-(phenethylthio)}-1,3,4-oxadiazole
(6a).
Prepared from 5-(2-(1H-benzo[d]imidazol-1-yl)ethyl)-1,3,4-oxadiazole-2-thiol (4) (0.2 g, 0.813 mmol) and 2-bromomethyl benzene (0.166 g, 0.89 mmol). Purified using gradient chromatography eluting with CH2Cl2-MeOH 97.5:2.5 v/v to afford the product as a white semisolid: Yield: 0.166 g (62%); mp 78–80 °C; TLC: CH2Cl2-MeOH 9:1 v/v, Rf 0.55; HPLC: 100% at RT = 4.35 min. 1H NMR (DMSO-d6) δ: 2.96 (t, J = 7.2 Hz 2H, SCH2CH2), CH2 signal is obscured by H2O in DMSO-d6 signal, 3.44 (t, J = 6.7 Hz, 2H, SCH2), 4.69 (t, J = 6.7 HzH, NCH2), 7.19–7.31 (m, 7H, Ar), 7.59 (d, J = 7.9 Hz, 1H, Ar), 7.63 (d, J = 7.8 Hz, 1.0 Hz, 1H, Ar), 8.22 (s, 1H, imidazole). 13C NMR (DMSO-d6) δ: 26.2 (NCH2CH2), 33.6 (SCH2), 35.2 (SCH2 CH2), 41.3 (NCH2), 110.7 (CH), 120.0 (CH), 122.2 (CH), 122.8 (CH), 127.0 (CH), 128.9 (2xCH), 129.1 (2xCH), 133.9 (C), 139.6 (C), 143.8 (C), 144.6 (CH-imidazole), 163.9 (C), 165.7 (C). HRMS (ESI) m/z Calculated: 350.1201 [M + H]+, Found: 350.1211 [M + H]+.
2- {[2-(1H-benzo[d]imidazol-1-yl)ethyl]-5- [(4-chlorophenethyl)thio]}-1,3,4-oxadiazole
(6b).
Prepared from 5-(2-(1H-benzo[d]imidazol-1-yl)ethyl)-1,3,4-oxadiazole-2-thiol (4) (0.2 g, 0.813 mmol) and 4-chlorophenethyl bromide (0.196 g, 0.89 mmol). Purified using gradient chromatography eluting with 100% EtOAc to afford the product as a white solid: Yield: 0.203 g (65%); mp 90–92 °C; TLC: CH2Cl2-MeOH 9:1 v/v, Rf 0.57; HPLC: 100% at R.T. 4.50 min. 1H NMR (DMSO-d6) δ: 2.96 (t, J = 7.4 Hz, 2H, SCH2CH2), CH2 signal is obscured by H2O in DMSO-d6 signal, 3.44 (t, J = 6.7 Hz, 2H, SCH2), 4.68 (t, J = 6.7 Hz, 2H, NCH2), 7.22 (m, 4H, Ar), 7.35 (d, J = 8.4 Hz, 2H, Ar), 7.58 (d, J = 8.1 Hz, 1H, Ar), 7.63 (d, J = 7.8 Hz, 1H, Ar), 8.22 (s, 1H, CH-imidazole). 13C NMR (DMSO-d6) δ: 26.2 (NCH2CH2), 33.3 (SCH2), 34.5 (SCH2 CH2), 41.3 (NCH2), 110.7 (CH), 120.0 (CH), 122.1 (CH), 122.9 (CH), 128.8 (2xCH), 131.0 (2xCH), 131.7 (C), 133.9 (C), 138.6 (C), 143.8 (C), 144.6 (CH-imidazole), 163.8 (C), 165.8 (C). HRMS (ESI) m/z Calculated: 384.0812 [M + H]+, Found: 384.0820 [M + H]+.
2- {[2-(1H-benzo[d]imidazol-1-yl)ethyl]-5- [(4-methoxyphenethyl)thio]}-1,3,4-oxadiazole
(6c).
Prepared from 5-(2-(1H-benzo[d]imidazol-1-yl)ethyl)-1,3,4-oxadiazole-2-thiol (4) (0.2 g, 0.813 mmol) and 4-methoxyphenethyl bromide (0.19 g, 0.89 mmol). Purified using gradient chromatography eluting with 100% EtOAc to afford the product as a white solid: Yield: 0.2 g (75%); mp 65–67 °C; TLC: CH2Cl2-MeOH 9:1 v/v, Rf 0.57; HPLC: 100% at R.T. 4.34 min. 1H NMR (DMSO-d6) δ: 2.89 (t, J = 6.8 Hz, 2H, SCH2CH2), CH2 signal is obscured by H2O in DMSO-d6 signal, 3.44 (t, J = 6.8 Hz, 2H, SCH2), 3.73 (s, 3H, OCH3), 4.69 (t, J = 6.7 Hz, 2H, NCH2), 6.85 (d, J = 8.7 Hz, 2H, Ar), 7.13 (d, J = 8.7 Hz, 2H, Ar), 7.20 (td, J = 1.2, 7.3 Hz, 1H, Ar), 7.25 (td, J = 1.2, 8.1 Hz, 1H, Ar), 7.59 (d, J = 7.9 Hz, 1H, Ar), 7.63 (d, J = 7.7 Hz, 1, 1H, Ar), 8.22 (s, 1H, imidazole). 13C NMR (DMSO-d6 δ: 26.2 (NCH2CH2), 33.9 (SCH2), 34.4 (SCH2 CH2), 41.3 (NCH2), 55.9 (OCH3), 110.7 (CH), 114.3 (2xCH), 120.0 (CH), 122.1 (CH), 122.9 (CH), 130.1 (2xCH), 131.5 (C), 140.0 (C), 143.8 (C), 144.6 (CH-imidazole), 158.4 (C), 164.0 (C), 165.7 (C). HRMS (ESI) m/z Calculated: 380.130 [M + H]+, Found: 380.1320 [M + H]+.
2- {[2-(1H-benzo[d]imidazol-1-yl)ethyl]-5-(benzylthio)}-1,3,4-thiadiazole
(8a).
Prepared from 5-(2-(1H-benzo[d]imidazol-1-yl)ethyl)-1,3,4-thiadiazole-2-thiol (7) (0.2 g, 0.76 mmol) and benzyl chloride (0.11 g, 0.84 mmol), and purified using gradient chromatography eluting with CH2Cl2-MeOH 97.5:2.5 v/v to afford the as a white solid: Yield: 0.167 g (62%); mp 88–90 °C; TLC: CH2Cl2-MeOH 9:1 v/v, Rf 0.5; HPLC: 100% at R.T. 4.24 min. 1H NMR (DMSO-d6) δ: 3.44 (t, J = 6.8 Hz, 2H, NCH2CH2), 4.40 (s, 2H, SCH2), 4.67 (t, J = 6.7 Hz, 2H, NCH2), 7.19–7.34 (m, 5H, Ar), 7.37 (m, 2H, Ar), 7.57 (m, 1H, Ar), 7.64 (m, 1H, Ar), 8.19 (s, 1H, imidazole). 13C NMR (DMSO-d6) δ: 26.2 (NCH2CH2), 36.1 (SCH2), 41.3 (NCH2), 110.7 (CH), 120.0 (CH), 122.1 (CH), 122.9 (CH), 128.2 (CH), 129.0 (2xCH), 129.4 (2xCH), 133.9 (C), 136.9 (C), 143.8 (C), 144.5 (CH-imidazole), 163.6 (C), 165.9 (C). HRMS (ESI): Calculated: 352.0816 [M + H]+, Found: 352.0850 [M + H]+.
2- {[2-(1H-benzo[d]imidazol-1-yl)ethyl]-5- [(2,4-dichlorobenzyl)thio]} -1,3,4-thiadiazole
(8b).
Prepared from 5-(2-(1H-benzo[d]imidazol-1-yl)ethyl)-1,3,4-thiadiazole-2-thiol (7) (0.2 g, 0.76 mmol) and 2,4-dichlorobenzyl chloride (0.16 g, 0.84 mmol). Purified using gradient chromatography eluting with CH2Cl2-MeOH 96:4 v/v to afford the product as a white solid: Yield: 0.22 g (69%); mp 84–86 °C; TLC: CH2Cl2-MeOH 9:1 v/v, Rf 0.55; HPLC: 100% at R.T. 4.60 min; 1H NMR (DMSO-d6) δ: 3.44 (t, J = 6.7 Hz, 2H, NCH2CH2), 4.47 (s, 2H, SCH2), 4.67 (t, J = 6.7 Hz, 2H, NCH2), 7.21 (m, 2H, Ar), 7.37 (dd, J = 8.3 Hz, 2.2 Hz, 1H, Ar), 7.49 (d, J = 8.4 Hz, 1H, Ar), 7.56 (m, 1H, Ar), 7.63 (m, 1H, Ar), 7.66 (d, J = 2.2 Hz, 1H, Ar), 8.21 (s, 1H, imidazole). 13C NMR (DMSO-d6) δ: 26.3 (NCH2CH2), 33.9 (SCH2), 41.2 (NCH2), 110.6 (CH), 120.0 (CH), 122.1 (CH), 122.9 (CH), 128.0 (CH), 129.6 (CH), 133.2 (CH), 133.5 (C), 133.9 (C), 134.0 (C), 134.7 (C), 143.8 (C), 144.6 (CH-imidazole), 162.9 (C), 166.2 (C). HRMS (ESI) m/z Calculated: 420.0037 [M + H]+, Found: 420.0035 [M + H]+.
2- {[2-(1H-benzo[d]imidazol-1-yl)ethyl]-5- [(4-fluorobenzyl)thio]} -1,3,4-thiadiazole
(8c).
Prepared from 5-(2-(1H-benzo[d]imidazol-1-yl)ethyl)-1,3,4-thiadiazole-2-thiol (7) (0.2 g, 0.76 mmol) and 4-flourobenzyl chloride (0.12 g, 0.84 mmol). Purified using gradient chromatography eluting with 100% EtOAc to afford the product as a white solid: Yield: 0.16 g (71%); mp 76–78 °C; TLC: CH2Cl2-MeOH 9:1 v/v, Rf 0.62; HPLC: 100% at R.T. 4.27 min. 1H NMR (DMSO-d6) δ: 3.44 (t, J = 6.8 Hz, 2H, NCH2CH2), 4.40 (s, 2H, SCH2), 4.67 (t, J = 6.7 Hz, 2H, NCH2), 7.14 (t, J = 8.9 Hz, 2H, Ar), 7.22 (m, 2H, Ar), 7.41 (m, 2H, Ar), 7.56 (m, 1H, Ar), 7.64 (m, 1H, Ar), 8.19 (s, 1H, Imidazole). 13C NMR (DMSO-d6) δ: 26.2 (NCH2CH2), 35.3 (SCH2), 41.2 (NCH2), 110.7 (CH), 115.7 (CH), 115.9 (CH), 119.9 (CH), 122.1 (CH), 122.9 (CH), 131.5 (CH), 131.6 (CH), 131.3 (C), 133.9 (C), 143.8 (C), 144.6 (CH-imidazole), 161.1 (C), 163.0 (C), 163.5 (C), 165.9 (C). HRMS (ESI) m/z Calculated: 370.0722 [M + H]+, Found: 370.0717 [M + H]+.
4-{[( 5- [2-(1H-benzo[d]imidazol-1-yl)ethyl]-1,3,4-thiadiazole-2-yl}thio)methyl] benzonitrile
(8d).
Prepared from 5-(2-(1H-benzo[d]imidazol-1-yl)ethyl)-1,3,4-thiadiazole-2-thiol (7) (0.1 g, 0.38 mmol) and 4-(chloromethyl)benzonitrile (0.07 g, 0.42 mmol). Purified using gradient chromatography eluting with 100% EtOAc + 1% Et3N to afford the product as a white solid: Yield: 0.17 gm, 61%; mp 112–114 °C; TLC: CH2Cl2-MeOH 9:1 v/v, Rf 0.56; HPLC: 100% at R.T. 4.02 min. 1H NMR (DMSO-d6) δ: 3.43 (t, J = 6.8 Hz, 2H, NCH2CH2), 4.48 (s, 2H, SCH2), 4.66 (t, J = 6.7 Hz, 2H, NCH2), 7.21 (m, 2H, Ar), 7.57 (m, 3H, Ar), 7.64 (m, 1H, Ar), 7.77 (d, J = 8.4 Hz, 2H, Ar), 8.19 (s, 1H, Imidazole). 13C NMR (DMSO-d6) δ: 26.2 (NCH2CH2), 35.5 (SCH2), 41.2 (NCH2), 110.6 (CH), 110.9 (CN), 119.1 (C), 120.0 (CH), 122.1 (CH), 122.9 (CH), 130.4 (2xCH), 132.9 (2xCH), 133.9 (C), 143.2, (C), 143.8 (C), 144.6 (CH-imidazole), 163.2 (C), 166.1 (C). HRMS (ESI) m/z Calculated: 377.0769 [M + H]+, Found: 377.0752 [M + H]+.
2- {[2-(1H-benzo[d]imidazol-1-yl)ethyl]-5-(phenethylthio)}-1,3,4-thiadiazole
(9a).
Prepared from 5-(2-(1H-benzo[d]imidazol-1-yl)ethyl)-1,3,4-thiadiazole-2-thiol (7) (0.1 g, 0.38 mmol) and 2-bromoethyl benzene (0.07 g, 0.42 mmol). Purified using gradient chromatography eluting with 100% EtOAc to afford the product as a white solid: Yield: 0.071 g (51%); mp 76–78 °C; TLC: CH2Cl2-MeOH 9:1 v/v, Rf 0.57; HPLC: 100% at R.T. 4.35 min. 1H NMR (DMSO-d6) δ: 2.97 (t, J = 7.3 Hz, 2H, SCH2CH2), 3.39 (t, J = 7.2 Hz, 2H, NCH2CH2), 3.44 (t, J = 6.7 Hz, 2H, SCH2), 4.69 (t, J = 6.7 Hz, 2H, NCH2), 7.19–7.31 (m, 7H, Ar), 7.59 (d, J = 7.8 Hz, 1H, Ar), 7.63 (d, J = 8.0 Hz, 1H, Ar), 8.22 (s, 1H, imidazole). 13CNMR (DMSO-d6) δ: 26.2 (NCH2CH2), 33.5 (SCH2), 35.3 (SCH2 CH2), 41.3 (NCH2), 110.7 (CH), 120.0 (CH), 122.1 (CH), 122.9 (CH), 127.0 (CH), 128.9 (2xCH), 129.1 (2xCH), 134.0 (C), 139.6 (C), 143.8 (C), 144.6 (CH-imidazole), 163.9 (C), 165.7 (C). HRMS (ESI) m/z Calculated: 366.0973 [M + H]+, Found: 366.0962 [M + H]+.
2- {[2-(1H-benzo[d]imidazol-1-yl)ethyl]-5- [(4-chlorophenethyl)thio]}-1,3,4-thiadiazole
(9b).
Prepared from 5-(2-(1H-benzo[d]imidazol-1-yl)ethyl)-1,3,4-thiadiazole-2-thiol (7) (0.1 g, 0.38 mmol) and 4-chlorophenethyl bromide (0.092 g, 0.42 mmol). Purified using gradient chromatography eluting with 100% EtOAc to afford the product as a white solid: Yield: 0.06 g (40%); mp 96–98 °C; TLC: CH2Cl2-MeOH 9:1 v/v, Rf 0.60; HPLC: 100% at R.T. 4.49 min. 1H NMR (DMSO-d6) δ: 2.96 (t, J = 7.2 Hz, 2H, SCH2CH2), 3.38 (t, J = 7.1 Hz, 2H, NCH2CH2), 3.44 (t, J = 6.8 Hz, 2H, SCH2), 4.69 (t, J = 6.7 Hz, 2H, NCH2), 7.20 (m, 1H, Ar), 7.25 (m, 3H, Ar), 7.35 (d, J = 8.5 Hz, 2H, Ar), 7.59 (d, J = 7.7 Hz, 1H, Ar), 7.63 (d, J = 7.8 Hz, 1H, Ar), 8.22 (s, 1H, imidazole). 13C NMR (DMSO-d6) δ: 26.2 (NCH2CH2), 33.3 (SCH2), 34.5 (SCH2 CH2), 41.3 (NCH2), 110.7 (CH), 120.0 (CH), 122.1 (CH), 122.9 (CH), 128.8 (2xCH), 131.0 (2xCH), 131.7 (C), 134.0 (C), 138.6 (C), 143.8 (C), 144.6 (CH-imidazole), 163.8 (C), 165.8 (C). HRMS (ESI) m/z Calculated: 400.0583 [M + H]+, Found: 400.0555 [M + H]+.
2- {[2-(1H-benzo[d]imidazol-1-yl)ethyl]-5- [(4-methoxyphenethyl)thio]}-1,3,4-thiadiazole
(9c).
Prepared from 5-(2-(1H-benzo[d]imidazol-1-yl)ethyl)-1,3,4-thiadiazole-2-thiol (7) (0.1 g, 0.38 mmol) and 4-methoxyphenethyl bromide (0.09 g, 0.42 mmol). Purified using gradient chromatography eluting with 100% EtOAc to afford the product as a white solid: Yield: 0.166 g (55%); mp 68–70 °C; TLC: CH2Cl2-MeOH 9:1 v/v, Rf 0.66; HPLC: 100% at RT = 4.34 min. 1H NMR (DMSO-d6) δ: 2.89 (t, J = 7.1 Hz, 2H, SCH2CH2), CH2 signal is obscured by H2O in DMSO-d6 signal, 3.44 (t, J = 6.7 Hz, 2H, SCH2), 3.73 (s, 3H, OCH3), 4.69 (t, J = 6.7 Hz, 2H, NCH2), 6.85 (d, J = 8.7 Hz, 2H, Ar), 7.13 (d, J = 8.7 Hz, 2H, Ar), 7.20 (td, J = 1.2, 7.2 Hz, 1H, Ar), 7.25 (td, J = 1.2, 8.1 Hz, 1H, Ar), 7.59 (d, J = 7.8 Hz, 1H, Ar), 7.63 (d, J = 7.8 Hz, 1H, Ar), 8.22 (s, 1H, imidazole). 13C NMR (DMSO-d6) δ: 26.2 (NCH2CH2), 33.9 (SCH2), 34.4 (SCH2 CH2), 41.3 (NCH2), 55.5 (OCH3), 110.7 (CH), 114.3 (2xCH), 120.0 (CH), 122.1 (CH), 122.9 (CH), 130.1 (2xCH), 131.5 (C), 140.0 (C), 143.8 (C), 144.6 (CH-imidazole), 158.4 (C), 164.0 (C), 165.7 (C). HRMS (ESI): Calculated: 380.1307 [M + H]+, Found: 380.1319 [M + H]+.
Computational studies
Flexible alignment studies were performed using MOE. 2015.10 software [31]. Flexible alignment was performed using MMFF94 forcefield, flexible alignment mode and the resulting conformations were examined according to their grand alignment score (S). The latter is the sum of the similarity measure of configuration (F) and the average strain energy of the molecules in the alignment in kcal/mol (U). The lower S value indicates better alignment. Multiple sequence alignment was performed using the EMBL-EBI (European Bioinformatics Institute) Job Dispatcher framework server, using Clustal Omega [33], while the amino acid sequences were imported in their FASTA formats from UniProtKB [47].
Docking studies were performed using MOE. 2015.10 software [48] using the crystal structure of Th. thermophilus PheRS [PDB ID: 1JJC] [32]. All minimisations were performed with MOE until a RMSD gradient of 0.01 kcal/mol/Å with Amber99 forcefield and automatic calculation of the partial charges. Docking settings were set as the following: Amber99 forcefield for result refinement, Triangle Matcher placement was chosen to determine the poses, London ΔG scoring function was applied for rescoring and result refinement, and the structures were further refined with rigid receptor refinement. The resulting docking poses were generated in a database, which was arranged according to the final score function (S) that is the score of the last stage which was not set to zero.
Molecular dynamic simulations were run using either the crystal structure of Th. thermophilus PheRS [PDB ID: 1JJC] [32] or S. aureus PheRS previously published homology model [34]. PDB files were first optimised using protein preparation wizard in Maestro [49] version 11.8.012 by assigning bond orders, adding hydrogen, and correcting incorrect bond types. A default quick relaxation protocol was used to minimise the MD systems with the Desmond programme [49]. In Desmond, the volume of space in which the simulation takes place, the global cell, is built up by regular 3D simulation boxes, which was utilised as part of this system for protein interactions. The simulation system was generated by embedding the protein model in an orthorhombic 10 Å water box. Overlapping water molecules were deleted. The solvated system was neutralised by adding sodium ions and salt concentration 0.15 M. Force-field parameters for the complexes were assigned using the OPLS_2005 forcefield, that is, a 200 ns molecular dynamic run in the NPT ensemble (T = 300 K) at a constant pressure of 1 bar. Energy and trajectory atomic coordinate data were recorded at each 1.2 ns.
Binding affinity (ΔG) calculations was performed using Prime/MMGBAS, available in the Schrödinger Prime suite, to calculate the binding free energy of 8b complexed with the S. aureus homology model.
ΔG (bind) = E_complex (minimised)-(E_ligand (minimised) + E_receptor (minimised)).
The mean ΔG (bind) was calculated from each frame starting from the last 100 ns to the final frame of the MD simulation.
Microbiological evaluation
Compounds 5a-d, 6a-c, 8a-d and 9a-c were evaluated using the guidelines of ISO20776 (the International Organization of Standardization) broth microdilution method using Muller-Hinton broth and concentration range of 0.008—128 μg/mL [50]. The MIC values were determined against S. aureus (ATCC 29,213), E. faecalis (ATCC 29,212), P. aeruginosa (ATCC 29,853), E. coli (ATCC 25,922) and Klebsiella pneumoniae (ATCC 700,603) using ciprofloxacin as a reference drug. The tests were performed in microdilution trays and the amount of growth in each well was compared with that in the positive growth control, and the recorded MIC is the lowest concentration of the agent that completely inhibits visible growth.