Chemistry
All chemicals were purchased from common commercial suppliers and used without further purification. Melting points (mp) were determined on a Gallenkamp melting point apparatus and were uncorrected. The IR spectra were recorded on Thermo scientific NICOLET IS10 spectrophotometer. All 1H NMR and 13C NMR spectra were recorded on Bruker AM-300 spectrophotometer at 300 and 100 MHz respectively, in DMSO as a solvent and TMS as an internal standard at Quaid-e-Azam University, Islamabad.
General procedure for the preparation of compounds Synthesis of 2-mercapto benzimidazole (1)
Compound 1 was prepared according to the reported procedure [13].
Synthesis of ethyl 2-(benzimidazolylthio) acetate (2)
An equimolar solution of 2-mercapto benzimidazole (1) (1.50 g, 0.01 mol) and ethylchloroacetate (1.22 mL, 0.01 mol) in dry acetone (4 mL) in presence of anhydrous K2CO3 (1 g) was refluxed on a water bath for 6 h. The solvent was removed by vacuum distillation and the residue was recrystallized from chloroform to furnish compound 2 (1.055 g, 70%). m. p.: 60 64 °C; IR (cm−1) 3042 (SP2 CH), 1722 (C=O of ester), 1684 (C=N), 1320 and 1234 (C–O–C), 1H-NMR (300 MHz, DMSO-d
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) ppm: 1.40 (t, 3H, J = 7 Hz, CH3), 4.08 (q, 2H, J = 6.75 Hz, CH2), 4.68 (s, 2H, S–CH2), 6.93–7.78 (m, 4H, Ar–H), 11.2 (s, 1H, NH). Anal. calcd. For C11H12N2O2S: C, 55.93; H, 5.10; N, 11.86. Found: C, 55.83; H, 5.04; N, 11.75.
Synthesis of [(2-benzimidazolylthio)-acetyl]-hydrazine (3)
Compound 2 (2.36 g, 0.01 mol) and hydrazine hydrate (0.9 mL, 0.02 mol) in ethanol (20 mL) were refluxed for about 5 h on oil bath. After cooling, the resulting solid was filtered, dried and recrystallized from ethanol to obtain compound 3 (1.77 g, 75%). m. p.: 190–193 °C; IR (cm−1): 3311, 3369 (NHNH2), 1680 (C=O); 1H-NMR (300 MHz, DMSO-d
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) ppm: 4.02 (s, 2H, NH2), 4.45 (s, 2H, S–CH2), 7.05–7.95 (m, 4H, Ar–H), 10.55 (s, 1H, NH). Anal. calcd. for C9H10N4OS: C, 48.64; H, 4.80; N, 25.22. Found: C, 47.99; H, 4.69; N, 25.20.
General procedure for the synthesis of benzimidazole-pyrazole hybrids
Equimolar quantities of compound 3 (0.5 g, 0.001 mol) and respective chalcones (0.001 mol) were dissolved in ethanol (50 mL) containing 2–3 mL of glacial acetic acid. A few drops of hydrochloric acid were added as catalyst and the reaction mixture was refluxed for 16–17 h until the completion of reaction. After cooling, the resulting solution was added to ice cold water and resultant precipitates were collected by filtration.
2-(1H-benzimidazol-2-ylsulfanyl)-1-[5-(2-hydroxyphenyl)-3-phenyl-1H-pyrazol-1-yl]ethanone (5a)
Yield 65%, m. p. 190 °C, IR (cm−1). 3340 (OH), 1697 (C=O), 1537 (C=C), 1617 (C=N), 1HNMR (300 MHz, DMSO-d
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) ppm: 11.12 (s, 1H, OH), 9.02 (s, 1H, NH), 7.39–8.28 (m, 13H, Ar–H), 6.97 (pyrazole H), 3.34 (s, 2H, S–CH2). 13C NMR (100 MHz, DMSO-d
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) ppm: 157.55, 153.00, 149.35, 149.35, 145.89, 142.35, 139.74, 133.35, 131.85, 128.92, 128.70, 128.70, 125.76, 125.45, 125.45, 122.53, 122.45, 121.82, 119.87, 118.01, 115.45, 107.23, 107.18, 32.10. Anal. calcd. for C24H18N4O2S: C, 67.60; H, 4.22; N, 13.14. Found: C, 67.54; H, 4.20; N, 13.10.
2-(1H-benzimidazol-2-ylsulfanyl)-1-[3,5-bis(2-hydroxyphenyl)-1H-pyrazol-1-yl]ethanone (5b)
Yield 67%, m. p. 185 °C, IR (cm−1). 2738 (OH), 1698 (C=O), 1642 (C=N), 1540 (C=C), 1HNMR (300 MHz, DMSO-d
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) ppm: 11.12 (s, 1H, OH), 9.00 (s, 1H, NH), 6.95–8.29 (m, 12H, Ar–H), 7.76 (pyrazol H), 3.35 (s, 2H, S–CH2). 13C NMR (100 MHz, DMSO-d
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) ppm: 157.45, 156.67, 153.00, 149.30, 147.35, 145.89, 142.35, 139.74, 131.85, 131.85, 128.09, 125.76, 122.53, 122.45, 121.82, 120.51, 119.87, 118.68, 118.01, 117.19, 115.45, 107.23, 107.18, 32.61. Anal. calcd. for C24H18N4O3S: C, 65.15; H, 4.07; N, 12.66. Found: C, 65.13; H, 4.03; N, 12.62.
2-(1H-benzimidazol-2-ylsulfanyl)-1-[5-(2-hydroxyphenyl)-3-(3-hydroxy-4-methoxyphenyl) -1H-pyrazol-1-yl]ethanone (5c)
Yield 59%, m. p. 195 °C, IR (cm−1): 3121 (OH), 1695 (C=O), 1632 (C=N), 1535 (C=C), 1HNMR (300 MHz, DMSO-d
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) ppm: 14.18 (s, 1H, OH), 7.33–8.29 (m, 11H, Ar–H), 7.56 (pyrazol H), 4.01 (s, 3H, OCH3), 3.33 (s, 2H, S–CH2). 13C NMR (100 MHz, DMSO-d
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) ppm; 158.43, 156.67, 149.35, 148.16, 147.35, 145.89, 145.85, 142.35, 139.74, 131.85, 131.85, 129.05, 128.09, 122.53, 122.45, 120.51, 119.87, 118.68, 117.19, 112.24, 111.02, 107.23, 107.18, 56.15, 32.73. Anal. calcd. for C25H20N4O4S: C, 63.55; H, 4.23; N, 11.86. Found: C, 63.51; H, 4.22; N, 11.85.
2-(1H-benzimidazol-2-ylsulfanyl)-1-[5-(4-hydroxyphenyaminol)-3-(2-hydroxyphenyl)-1H-pyrazol-1-yl]ethanone (5d)
Yield 61%, m. p. 200 °C, IR (cm−1)0.3319 (OH), 1681 (C=O), 1616 (C=N), 1485 (C=C), 1HNMR (300 MHz, DMSO-d
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) ppm: 11.13 (s, 1H, OH), 9.01 (s, 1H, NH), 6.95–7.71 (m, 12H, Ar–H), 6.96 (pyrazol H), 3.34 (s, 2H, S–CH2). 13C NMR (100 MHz, DMSO-d
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) ppm: 156.29, 154.12, 153.00,149.35, 149.32, 145.89,142.35,139.74, 138.75, 131.85, 125.76,122.53, 122.45,121.82, 119.99, 119.99, 119.87, 118.01, 115.45, 115.40, 115.40, 107.23,94.57, 32.10. Anal. calcd. for C24H19N5O3S: C, 63.01; H, 4.15; N, 15.31. Found: C, 63.02; H, 4.12; N, 15.29.
2-(1H-benzimidazol-2-ylsulfanyl)-1-(3,5-diphenyl-1H-pyrazol-1-yl)ethanone (5e)
Yield 58%, m. p. 170 °C, IR (cm−1): 3056 (NH), 3217 (OH), 1683 (C=O), 1545 (C=N), 1446 (C=C), 1HNMR (300 MHz, DMSO-d
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) ppm: 11.12 (s, 1H, OH), 9.03 (s, 1H, NH), 6.51–7.12 (m, 14H, Ar–H), 6.78 (pyrazol H), 3.32 (s, 2H, S–CH2). 13C NMR (100 MHz, DMSO-d
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) ppm: 157.55, 149.35, 149.35, 145.89, 142.35, 139.74, 133.35, 130.65, 128.92, 128.92, 128.70, 128.70, 128.66, 128.66, 128.35, 128.35, 125.45, 125.45, 122.53, 122.45, 119.87, 107.23, 107.18, 32.10. Anal. calcd. for C24H18N4OS: C, 70.24; H, 4.39; N, 13.65. Found: C, 70.21; H, 4.36; N, 13.62.
2-(1H-benzimidazol-2-ylsulfanyl)-1-[5-(3-hydroxy-4-methoxyphenyl)-3-phenyl-1H-pyrazol-1-yl]ethanone (5f)
Yield 59%, m. p. 195 °C, IR (cm−1): 3121 (OH), 1695 (C=O), 1632 (C=N), 1535 (C=C), 1HNMR (300 MHz, DMSO-d
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) ppm: 14.01 (s, 1H, OH), 7.23–8.09 (m, 13H, Ar–H), 7.06 (pyrazol H), 4.01 (s, 3H, OCH3), 3.36 (s, 2H, S–CH2–CO). 13C NMR (100 MHz, DMSO-d
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) ppm: 157.50, 149.35, 149.30, 148.16, 145.89, 145.85, 142.35, 139.74, 133.30, 131.85, 129.05, 128.92, 128.70, 128.70, 125.40, 125.40, 122.53, 122.45, 119.87, 112.24, 111.02107.23, 107.18, 56.15, 32.10. Anal. calcd. for C25H20N4O3S: C, 65.78; H, 4.38; N, 12.28. Found: C, 65.75; H, 4.37; N, 12.27.
Pharmacological assay
Animals
Albino rats (weighing 180–220 g) were housed at the animal house of the Riphah Institute of Pharmaceutical Sciences under controlled environment (23–25 °C). Animals were kept in plastic cages with sawdust (changed at every 48 h) and were fasted for 24 h before starting the experiment. Animals were provided with tap water ad libitum and standard pellet diet. Experiments performed complied with rules of Institute of Laboratory Animal Resources, Commission on Life Sciences University, National Research Council (1996) and were approved by Ethical Committee of Riphah Institute of Pharmaceutical Sciences, Riphah International University.
Anti-ulcerogenic activity
Albino rats (180–220 g) of either sex were divided into different groups (n = 5). Animals were fasted for 24 h before the study, but had free access to water. Animals in the control group received only normal saline (10 mL/kg). Compound 5a at doses of 100 and 500 µg/kg, (p. o.) was given to the animals in the treatment group. Same procedure was repeated for Compund 5b, 5c, 5d, 5e and 5f. Omeprazole (30 mg/kg) was used as a standard. The rats were sacrificed 1 h later and the stomach removed and observed for ulcers in the glandular region [3]. The surface area of each lesion was measured and scored by method with described by Tan et al. [14] with some modifications. The ulcer index for each rat was taken as the mean ulcer score (0: no ulcer; 1: US ≤ 0.5 mm2; 2: 0.5 < US ≤ 2.5 mm2; 3: 2.5 mm2 < US ≤ 5 mm2; 4: 5 mm2 < US ≤ 10 mm2; 5: 10 mm2 < US ≤ 15 mm2; 6: 15 mm2 < US ≤ 20 mm2; 7: 20 mm2 < US ≤ 25 mm2; 8: 25 mm2 < US ≤ 30 mm2; 9: 30 mm2 < US ≤ 35 mm2; 10: US > 35 mm2). The sum of the length (mm) of all the lesions for each stomach was used as the ulcer index (UI). The percentage of inhibition (% I) was calculated using the following formula:
$$\% {\text{I}} = \, \left( {{\text{USc}}\,{-}\,{\text{USt}}} \right) \, \times \, 100/{\text{USc}}$$
where USc = ulcer surface area of control and USt = ulcer surface area of test animal [14].
Docking studies
Drug likeliness evaluation
Molinspiration server was used to predict number of rotatable bonds, hydrogen bond acceptors and hydrogen bond donors. These parameters help in evaluation of drug likeliness in light of Lipinski’s rule of five [15]. According to this rule, for any compound to be a good drug candidate, it should have Molecular weight (MW) less than 500 Da, H-bond donors (HBD) less than 5, H-bond acceptors (HBA) less than 10, LogP value less or equal to 5 and total rotatable bonds less than 10.
Molecular docking
The 3D structures of SCH28080, ligands (5a–5f), omeprazole, it’s sulfenic acid and sulfenamide derivatives were drawn using DS Visualizer v16.1.0.15350 and saved in protein data bank (PDB) format. Polar hydrogens and charges were added by Autodock tools-1.5.6.
The three dimensional (3D) structure of Pig Gastric H+/K+ ATPase (PDB code 2XZB) was retrieved from protein data bank [16]. The protein structure downloaded from protein data bank was used without any modification.
SCH28080 was used to validate the docking results because the PDB structure (2XZB) used in the docking analysis was obtained from enzyme crystallized along with it and its binding site was evaluated according to bound SCH28080 in the literature [16]. SCH28080 is a well known competitive inhibitor of gastric H+/K+ ATPase having comparable activity as of omeprazole [17]. Available literature indicate that omeprazole gets converted to its sulfenic acid and sulfenamide derivative in acidic environment and these forms bind to the Cys 813 sulfhydral group by making a covalent disulfide linkage [18, 19]. Due to this reason sulfenic acid and sulfenamide derivatives of omeprazole were also docked along with synthesized compounds.
Molecular docking was carried out by help of Pyrx 0.8 and selecting Autodock vina as docking software [20]. Vina search space coordinates were set as x = 29.161, y = 34.533 and z = −70.686. Dimensions of search space were set as x = 29.279, y = 20.040 and z = 27.678. Exhaustiveness was set at 100. All docked poses were saved in PDB format for further analysis on PyMOL Version 1.7.4.5 Edu and DS Visualizer v16.1.0.15350 [21].
Acute toxicity test
The test was performed using increasing doses (10, 30 and 100 mg/kg) of the test compounds, given orally in 10 mL/kg volume to rats. The animals were allowed food ad libitum and kept under observation for mortality in 24 h [22].
Statistical analysis
Data expressed are mean ± standard error of mean (SEM, n = number of experiment). The statistical parameter applied is one-way analysis of variance with post hoc Tukey test, P < 0.05 noted as significantly different.