Experimental Section
General
All commercially available reagents and solvents were used without further purification unless otherwise specified. Solvents were dried and re-distilled prior to use according to standard methods. Melting points were determined on a Büchi Melting Point B-540 apparatus (Büchi Labortechnik, Flawil, Switzerland) and are uncorrected. 1H-NMR spectra were measured in DMSO-d6 on a Bruker ARX 300 spectrometer (Bruker, Rheinstetten, Germany). Chemical shifts are reported in parts per million (ppm) using tetramethylsilane (TMS) as internal standard if not specifically mentioned (J in Hz). Mass spectra were obtained on Waters Micromass® Quattro MicroTM API mass spectrometer (Waters Corporation, Milford, United States). Column chromatography was performed on silica gel H and analytical TLC on silica gel HF254 plates.
2,2’-di-[[(3,5-Dimethyl-4-methoxy)pyrid-2-yl]methylenesulfinyl]-5,5’-bis-1H,1’H-benzimidazole (3a).
Compound 2a (1.0 g, 1.68 mmol) in dichloromethane (30 mL) was stirred in an ice-salt bath. When the temperature reached -15°C, MCPBA (0.71 g, 3.50 mmol) was added in batches. Afterwards the mixture was stirred for 1 hr. Saturated sodium carbonate solution (10 mL) was added to adjust the pH to about 9. The organic layer was separated and the aqueous was extracted with dichloromethane (20 mL × 3). The organic phases were combined and dried over anhydrous sodium sulfate. An oil was obtained after concentration, which was then purified by silica gel column chromatography (eluent CH2Cl2-CH3OH= 60:1) to give compound 3a. Yield: 72 %; mp. = 158°C dec; 1H-NMR: δ = 13.66 (s, 1H, Bz-NH), 8.20 (s, 1H, Py-6-H), 7.67–8.01 (m, 3H, Bz-4,6,7-H), 4.78 (dd, 2H, J=19.8/13.8, SOCH2), 3.70 (s, 3H, OCH3), 2.20 (s, 6H, Py-3,5-CH3); MS (ESI-): m/e = 629.5 [M + 1].
Compounds 3b – 3j were similarly prepared:
2,2’-di-[[(3,4-Dimethoxy)pyrid-2-yl]methylenesulfinyl]-5,5’-bis-1H,1’H-benzimidazole (3b). Yield: 70 %; mp. = 127 – 130°C; 1H-NMR: δ = 13.68 (s, 1H, Bz-NH), 8.16 (d, 1H, J=5.5, Py-6-H), 7.67–8.00 (m, 3H, Bz-4,6,7-H), 7.11 (d, 1H, J=5.6, Py-5-H), 4.72 (dd, 2H, J=18.1/13.0, SOCH2), 3.89 (s, 3H, OCH3), 3.78 (s, 3H, OCH3); MS (ESI-): m/e = 633.2 [M + 1].
2,2’-di-[[[3-Methyl-4-(2,2,2-trifluoroethoxy)]pyrid-2-yl]methylenesulfinyl]-5,5’-bis-1H,1’H-benz-imidazole (3c). Yield: 77 %; mp. = 172°C dec; 1H-NMR: δ = 13.68 (s, 1H, Bz-NH), 8.31 (d, 1H, J=5.7, Py-6-H), 7.88 (s, 1H, Bz-4-H), 7.73 (d, 1H, J=8.7, Bz-7-H), 7.62 (d, 1H, J=8.7, Bz-6-H), 7.10 (d, 1H, J=5.7, Py-5-H), 4.93 (q, 2H, J=8.7, CF3CH2O), 4.81 (dd, 2H, J=33.5/13.7, SOCH2), 2.21 (s, 3H, Py-3-CH3); MS (ESI-): m/e = 771.5 [M + 35].
2,2’-di-[[(3-Methoxy-4-chloro)pyrid-2-yl]methylenesulfinyl]-5,5’-bis-1H,1’H-benzimidazole (3d). Yield: 68 %; mp. = 130–133°C; 1H-NMR: δ = 13.72 (s, 1H, Bz-NH), 8.29 (d, 1H, J=5.1, Py-6-H), 7.62 – 7.78 (m, 3H, Bz-4,6,7-H), 7.61 (d, 1H, J=5.1, Py-5-H), 4.85 (s, 2H, SOCH2), 3.90 (s, 3H, OCH3); MS (ESI-): m/e = 639.2 [M – 1].
2,2’-di-[[(3-Methyl-4-methoxy)pyrid-2-yl]methylenesulfinyl]-5,5’-bis-1H,1’H-benzimidazole (3e). Yield: 78 %; mp. = 203°C dec; 1H-NMR: δ = 13.61 (s, 1H, Bz-NH), 8.26 (d, 1H, J=1.5, Py-6-H), 7.60 –8.10 (m, 3H, Bz-4,6,7-H), 6.98 (d, 1H, J=1.5, Py-5-H), 4.83 (dd, 2H, J=22.5/9.3, SOCH2), 3.87 (s, 3H, OCH3), 2.16 (s, 3H, Py-3-CH3); MS (ESI-): m/e = 599.4 [M – 1].
2,2’-di-[[(3-Methyl-4-ethoxy)pyrid-2-yl]methylenesulfinyl]-5,5’-bis-1H,1’H-benzimidazole (3f). Yield: 71 %; mp. = 95–98°C; 1H-NMR: δ = 13.65 (s, 1H, Bz-NH), 8.22 (d, 1H, J=5.4, Py-6-H), 7.60–7.80 (m, 3H, Bz-4,6,7-H), 6.95 (d, 1H, J=5.4, Py-5-H), 4.78 (dd, 2H, J=24.6/13.8, SOCH2), 4.12 (q, 2H, J=6.9, CH3CH2O), 2.15 (s, 3H, Py-3-CH3), 1.35 (t, 3H, J=6.9, CH3CH2O); MS (ESI-): m/e = 627.5 [M – 1].
2,2’-di-[(Pyrid-2-yl)methylenesulfinyl]-5,5’-bis-1H,1’H-benzimidazole (3g). Yield: 81 %; mp. = 198°C dec; 1H-NMR: δ = 13.63 (s, 1H, Bz-NH), 8.54 (d, 1H, J=4.2, Py-6-H), 7.60–7.85 (m, 4H, Bz-4,6,7-H, Py-4-H), 7.30–7.40 (m, 2H, Py-3,5-H), 4.77 (dd, 2H, J=26.4/12.9, SOCH2); MS (ESI-): m/e = 511.2 [M – 1].
2,2’-di-[(Pyrid-3-yl)methylenesulfinyl]-5,5’-bis-1H,1’H-benzimidazole (3h). Yield: 76 %; mp. = 211°C dec; 1H-NMR: δ = 13.42 (s, 1H, Bz-NH), 8.47 (d, 1H, J=3.6, Py-6-H), 8.26 (s, 1H, Py-2-H), 7.58–8.08 (m, 3H, Bz-4,6,7-H), 7.52 (d, 1H, J=7.8, Py-4-H), 7.30 (dd, 1H, J=7.8/4.8, Py-5-H), 4.68 (dd, 2H, J=71.4/13.2, SOCH2); MS (ESI-): m/e = 513.1 [M + 1].
2,2’-di-[(Pyrid-4-yl)methylenesulfinyl]-5,5’-bis-1H,1’H-benzimidazole (3i). Yield: 83 %; mp. = 172°C dec; 1H-NMR: δ = 13.48 (s, 1H, Bz-NH), 8.46 (d, 2H, J=4.8, Py-2,6-H), 7.60 – 8.10 (m, 3H, Bz-4,6,7-H), 7.14 (d, 2H, J=5.1, Py-3,5-H), 4.67 (dd, 2H, J=68.7/12.9, SOCH2); MS (ESI-): m/e = 511.3 [M – 1].
2,2’-di-[[(2-Chloro)pyrid-5-yl]methylenesulfinyl]-5,5’-bis-1H,1’H-benzimidazole (3j). Yield: 64 %; mp. = 166 – 169°C; 1H-NMR: δ = 13.50 (s, 1H, Bz-NH), 8.06 (d, 1H, J=2.0, Py-6-H), 7.60–8.00 (m, 3H, Bz-4,6,7-H), 7.53 (dd, 1H, J=8.3/2.2, Py-4-H), 7.45 (d, 1H, J=8.2, Py-3-H), 4.70 (dd, 2H, J=77.6/13.2, SOCH2); MS (ESI-): m/e = 579.3 [M – 1].
2,2’-di-[[(3,5-dimethyl-4-methoxy)pyrid-2-yl]methylenesulfonyl]-5,5’-bis-1H,1’H-benzimidazole (4a).
To a solution of 2a (1.0 g, 1.70 mmol) in THF (30 mL), sodium tungstate (0.15 g, 0.45 mmol), distilled water (10 mL) and hydrogen peroxide (30 %, 5 mL, 44.1 mmol) were added. The mixture was stirred at 50°C for 5 hrs. After the solvent was evaporated under vacuum, water (50 mL) was added to the resultant oil. The white solid obtained after filtration was dried. Purification by gel column chromatography (eluent CH2Cl2-CH3OH= 80:1) gave compound 4a. Yield: 82 %; mp. =167 – 170°C; 1H-NMR: δ = 13.89 (s, 1H, Bz-NH), 8.05 (s, 1H, Py-6-H), 7.79 – 8.04 (m, 3H, Bz-4,6,7-H), 5.11 (s, 2H, SO2CH2), 3.71 (s, 3H, OCH3), 2.26 (s, 3H, Py-3-CH3), 2.19 (s, 3H, Py-5-CH3); MS (ESI-): m/e = 661.5 [M + 1].
Compounds 4b – 4j were similarly prepared:
2,2’-di-[[(3,4-Dimethoxy)pyrid-2-yl]methylenesulfonyl]-5,5’-bis-1H,1’H-benzimidazole (4b). Yield: 85 %; mp. = 173 – 175°C; 1H-NMR: δ = 13.88 (s, 1H, Bz-NH), 7.99 (d, 1H, J=5.5, Py-6-H), 7.77–8.01 (m, 3H, Bz-4,6,7-H), 7.09 (d, 1H, J=5.5, Py-5-H), 5.01 (s, 2H, SO2CH2), 3.88(s, 3H, OCH3), 3.78 (s, 3H, OCH3); MS (ESI-): m/e = 665.5 [M + 1].
2,2’-di-[[[3-Methyl-4-(2,2,2-trifluoroethoxy)]pyrid-2-yl]methylenesulfonyl]-5,5’-bis-1H,1’H-benz-imidazole (4c). Yield: 88 %; mp. = 259–262°C; 1H-NMR: δ = 13.88 (s, 1H, Bz-NH), 8.13 (d, 1H, J=5.7, Py-6-H), 7.76–8.00 (m, 3H, Bz-4,6,7-H), 7.08 (d, 1H, J=5.7, Py-5-H), 5.15 (s, 2H, SO2CH2), 4.91 (q, 2H, J=8.7, CF3CH2O), 2.25 (s, 3H, Py-3-CH3); MS (ESI-): m/e = 769.5 [M + 1].
2,2’-di-[[(3-Methoxy-4-chloro)pyrid-2-yl]methylenesulfonyl]-5,5’-bis-1H,1’H-benzimidazole (4d). Yield: 73 %; mp. = 160–163°C; 1H-NMR: δ = 13.93 (s, 1H, Bz-NH), 8.12 (d, 1H, J=5.1, Py-6-H), 7.60–8.00 (m, 3H, Bz-4,6,7-H), 7.61 (d, 1H, J=5.1, Py-5-H), 5.14 (s, 2H, SO2CH2), 3.91 (s, 3H, OCH3); MS (ESI-): m/e = 671.4 [M – 1].
2,2’-di-[[(3-Methyl-4-methoxy)pyrid-2-yl]methylenesulfonyl]-5,5’-bis-1H,1’H-benzimidazole (4e). Yield: 81 %; mp. = 240 – 243°C; 1H-NMR: δ = 13.89 (s, 1H, Bz-NH), 8.26 (d, 1H, J=5.7, Py-6-H), 7.35–7.80 (m, 3H, Bz-4,6,7-H), 6.99 (d, 1H, J=5.7, Py-5-H), 4.94 (s, 2H, SO2CH2), 3.86 (s, 3H, OCH3), 2.16 (s, 3H, Py-3-CH3); MS (ESI-): m/e = 633.4 [M + 1].
2,2’-di-[[(3-Methyl-4-ethoxy)pyrid-2-yl]methylenesulfonyl]-5,5’-bis-1H,1’H-benzimidazole (4f). Yield: 82%; mp. = 179 – 182°C; 1H-NMR: δ = 13.86 (s, 1H, Bz-NH), 8.05 (d, 1H, J=5.7, Py-6-H), 7.70–8.00 (m, 3H, Bz-4,6,7-H), 6.94 (d, 1H, J=5.7, Py-5-H), 5.10 (s, 2H, SO2CH2), 4.12(q, 2H, J=6.9, CH3CH2O), 2.21 (s, 3H, Py-3-CH3), 1.36 (t, 3H, J=6.9, CH3CH2O); MS (ESI-): m/e = 661.4 [M + 1].
2,2’-di-[(Pyrid-2-yl)methylenesulfonyl]-5,5’-bis-1H,1’H-benzimidazole (4g). Yield: 86 %; mp. = 203–206°C; 1H-NMR: δ = 13.90 (s, 1H, Bz-NH), 8.41 (d, 1H, J=4.5, Py-6-H), 7.75–8.00 (m, 4H, Bz-4,6,7-H, Py-4-H), 7.42 (d, 1H, J=7.8, Py-3-H), 7.34 (dd, 1H, J=4.5/4.5, Py-5-H), 5.11 (s, 2H, SO2CH2); MS (ESI-): m/e = 543.4 [M – 1].
2,2’-di-[(Pyrid-3-yl)methylenesulfonyl]-5,5’-bis-1H,1’H-benzimidazole (4h). Yield: 80 %; mp. = 254– 258°C; 1H-NMR: δ = 13.91 (s, 1H, Bz-NH), 8.49 (d, 1H, J=4.6, Py-6-H), 8.42 (s, 1H, Py-2-H), 7.60–7.83 (m, 3H, Bz-4,6,7-H), 7.55 (d, 1H, J=8.5, Py-4-H), 7.30–7.50 (m, 1H, Py-5-H), 4.95 (s, 2H, SO2CH2); MS (ESI-): m/e = 543.4 [M – 1].
2,2’-di-[(Pyrid-4-yl)methylenesulfonyl]-5,5’-bis-1H,1’H-benzimidazole (4i). Yield: 81 %; mp. = 231– 234°C; 1H-NMR: δ = 13.90 (s, 1H, Bz-NH), 8.52 (d, 2H, J=4.5, Py-2,6-H), 7.70–8.10 (m, 3H, Bz-4,6,7-H), 7.25 (d, 2H, J=4.6, Py-3,5-H), 5.11 (s, 2H, SO2CH2); MS (ESI-): m/e = 543.4 [M – 1].
2,2’-di-[[(2-Chloro)pyrid-5-yl]methylenesulfonyl]-5,5’-bis-1H,1’H-benzimidazole (4j). Yield: 77 %; mp. = 241–244°C; 1H-NMR: δ = 14.01 (s, 1H, Bz-NH), 8.24 (d, 1H, J=1.5, Py-6-H), 7.70–8.20 (m, 3H, Bz-4,6,7-H), 7.74 (dd, 1H, J=8.3/2.0, Py-4-H), 7.54 (d, 1H, J=8.3, Py-3-H), 5.16 (s, 2H, SO2CH2); MS (ESI-): m/e = 611.4 [M – 1].
Molecular modeling
The molecular modeling studies were performed with the Sybyl 6.9.1 package (Tripos Inc., St. Louis, MO) on a SGI Fuel Workstation with the Irix 6.5 platform. All molecular structures were constructed and geometry optimized in vacuo (ε = 1) with the implemented Tripos force field using the Powell method until the convergence criterion of 0.05 kcal/mol change in energy between successive iterations was reached. The charges were calculated by the Gasteiger-Hückel method. Docking studies were performed with FlexX 1.6. During the docking process, the binding site atoms and the ligand atoms were set to be flexible. An incremental construction algorithm was applied to generate and minimize the possible pose. The models were visualized with DS Visualizer v1.5 (Accelrys Inc., San Diego, CA).
Biological assays
The anti-proliferational effects of HeLa cells and BGC-823 cells were tested by the same methods. Tumor cells in RPMI1640 medium with 10 % fetal bovine serum were plated in 96-well microtiter plates (4.0 × 104 cells per well), and allowed to adhere at 37°C with 5 % CO
2 for 4 h. The test compound was then added, and the cells were incubated at 37°C with 5 % CO
2 for 72 h. The cell viability was assessed using a standard MTT assay [
9].