Synthesis and evaluation of anti-inflammatory, antioxidant and antimicrobial activities of densely functionalized novel benzo [d] imidazolyl tetrahydropyridine carboxylates

2.1 Material and reagents

All the melting points were determined on a Cintex melting point apparatus and are uncorrected. Analytical TLC was performed on Merck precoated 60 F₂₅₄ silica gel plates. Visualization was done by exposure to iodine vapor. Column chromatography was conducted by using silica gel with ethyl acetate: n-hexane solvent system as elute. IR spectra (KBr pellet) were recorded on a Perkin–Elmer BX series FT-IR spectrometer. ¹H NMR spectra were recorded on a Varian Gemini 300 MHz spectrometer. ¹³C NMR spectra were recorded on a Bruker 75 MHz spectrometer. Chemical shift values are given in ppm (δ) with tetramethyl silane as an internal standard. Mass spectral measurements were carried out by the EI method on a Joel JMC-300 spectrometer at 70 eV. Elemental analyses were performed on a Carlo Erba 106 and Perkin–Elmer model 240 analyzers.

2.2 Synthesis of methyl-2,6-diphenyl-1-(2-sulfanyl-1H-benzo[d]imidazol-5-yl)-4-[(2-sulfanyl-1H-benzo[d]imidazol-6-yl)amino]-1,2,5,6-tetrahydro-3-pyridinecarboxylate (7a–n)

A mixture of (E)-5-(benzylidene amino)-1H-benzo[d]imidazole-2-thiole 3 (0.01 mmol), 5-amino-2-mercaptobenzimidazole 4 (0.01 mmol), aromatic aldehyde 5 (0.01 mmol) and ethyl acetoacetate 6 (0.01 mmol) in CH₃CN (15 ml) with 10 mol% CAN was stirred at room temperature for 6 h. After completion of the reaction (monitored by TLC), reaction mixture was poured into crushed ice. The separated solid was filtered and purified by column chromatography using ethyl acetate and n-hexane (6:4) elutant.

3.1 Anti-inflammatory activity

Albino rats weighing 150–180 g were used in this method. They were kept in the animal house under standard conditions of light and dark (12 h) with free access to food and libitum water. The rats were randomly divided into groups, of six rats each. The group VI, six rats are kept as a control and the group VII received the standard drug Diclofenac sodium (at a dose of 10 mg/kg body weight PO). All the test compounds 7a–n were dissolved in 0.1% solution of Tween 80 and given 60 min before the commencement of the study. Then 0.1 ml of 1% w/v carrageenan solution in normal saline was injected into the sub planter region of the left hind paw under light ether anesthesia, 1hr after oral administration (PO) of the test compound. The difference between the paw volumes of treated animals was compared with that of the control group and the mean edema volume was calculated. Percentage inhibition was calculated as per the formula, %inhibition = [(Vo − Vt)/Vo] × 100, where Vo = volume of the paw control at time t, Vt = volume of the paw of drug treated at time t. The paw volume of each rat was measured immediately by mercury plethysmometer and re-measured again at 1, 2, 3 and 4 h after administration of Tween 80. The edema was expressed as an increase in the volume of the paw edema.

The anti inflammatory activities of the newly synthesized compounds 7a–n (I–XIV groups) were evaluated by applying carrageenan-induced paw edema test in rats (Winter et al., 1962), using Diclofenac sodium as a reference drug for comparison. Results were expressed as Mean ± S.E. Difference between vehicle control and treatment groups was tested using one way Analysis of Variance (ANOVA) followed by the Dunnett’s test and least significant difference (L.S.D). Methods of statistical analysis were done according to (Armitage, 1971) .The anti inflammatory activity (Table 3) indicated that all the tested compounds in 60 min prior to carrageenan injection at a dose of 10 mg/kg weight caused significant inhibition of paw edema response.

3.2 Antioxidant activity

To 0.1 ml of test compound (at different concentrations), 1.5 ml of methanol and 0.5 ml of DPPH solution were added, mixed thoroughly and absorbance (OD) was read at 517 nm against the blank. The% reduction of free radical concentration (OD) with different concentrations of test compounds was calculated and was compared with standard, ascorbic acid. The results were expressed as IC₅₀ values (the concentration of test required to scavenge 50% free radicals).

3.3 Antibacterial activity

The ready-made nutrient broth medium (Himedia, 24 g) was suspended in distilled water (100 ml) and heated until it dissolved completely. The medium and test tubes were autoclaved at a pressure of 15 lb/inch² for 20 min. A set of sterilized test tubes with nutrient broth medium was capped with cotton plugs. The test compound was dissolved in acetone and a concentration of 100 μg/ml of the compound was added in the first test tube, which was serially diluted. A fixed volume of 0.5 ml of overnight culture was added in all the test tubes and incubated at 37 °C for 24 h. After 24 h these tubes were measured for turbidity and the data are presented in Table 1.

3.4 Antifungal activity

The ready-made potato dextrose agar medium (Himedia, 39 g) was suspended in distilled water (100 ml) and heated until it dissolved completely. The medium was poured into sterile Petri dishes under aseptic conditions in a laminar flow chamber. When the medium in plates was solidified, 0.5 ml of culture (one week old) of fungal spore suspension was inoculated and uniformly spread over the agar surface with a sterile L-shaped rod. Solutions were prepared by dissolving the test compound in acetone. Agar inoculation cups were scooped out with a 6 mm sterile cork borer and the lids of the dishes were replaced. To each cup, different concentrations of test solution were added. Controls were maintained with acetone and fluconazole. The test and the controls were kept at room temperature for 72–96 h. Inhibition zones were measured and the diameter was calculated in millimeters. Three to four replicates were maintained for each treatment. The results are summarized in Table 4.

4.1 Chemistry

In the current investigation benzo[d]imidazolyl tetrahydropyridine carboxylates (7a–n) have been prepared from a mixture of (E)-5-(benzylidene amino)-1H-benzo[d] imidazole-2-thiol 3, 5-amino-2-mercapto-benzimidazole 4, aromatic aldehyde 5 and ethyl acetoacetate 6 in acetonitrile (15 ml) with 10 mol% of CAN at room temperature in good yields (79–92%) (Scheme 1). The structures of all the newly synthesized compounds have been established on their elemental analysis IR, ¹H NMR, ¹³C NMR and MS spectral data.

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Scheme 1

Synthesis of novel benzo[d]imidazolyl tetrahydropyridine carboxylates. Reagents and conditions: (a) CH₃CN, 10 mol% CAN, Stirring, r.t, 4 h.

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In the IR spectra of 7a–n a strong absorption band appeared at 1657 cm⁻¹ corresponding to the carbonyl functional group. ¹H NMR spectra of 7 showed signals as doublet of doublet at δ 2.75, 2.85 due to CH₂, doublet at δ 5.11 due to Ar-CH and singlet at δ 6.11 due to Ar-CH which well support the structure. In the ¹³C NMR spectra, the prominent signals corresponding to the tetrahydropyridine ring observed nearly at δ 47.69, 56.22, 61.73, 101.43 and 132.72 are the further evidence of the structure. The mass spectra of 7a exhibited a molecular ion peak (M⁺) 681 m/z in conformity with the product formation.

4.2 Anti- inflammatory activity

The anti-inflammatory activity was evaluated by the Carrageenan-induced paw edema test in rats (Winter et al., 1962). The investigation of anti-inflammatory activity screening data revealed that almost all the tested compounds showed interesting activity, however with a degree of variation. A few among them have significant acute as well as residual anti-inflammatory activity. Compounds of groups 7a, 7c, 7e, 7f, 7h, 7i, 7j, 7l, 7m and 7n showed significantly decreased paw edema during 3, and 4 h after drug administration, 7b and 7g showed moderately decreased paw edema during 3 h of administration and after drug administration, but showed highly significant decreased paw edema during 4 h. This may be due to the presence of electron donating group as substituent. 7d Showed mild decreased paw edema during 3 h after drug administration but moderate decreased paw edema during 4 h. The results are illustrated in Table 1.

4.3 Antioxidant activity

For the evaluation of antioxidant activity, we have used a stable free radical α,α -diphenyl-β-picryl hydrazyl (DPPH), at the concentration of 0.2 mM in methanol (Ranjit et al., 2010). The antioxidant DPPH free radical scavenging activity of all the synthesized compounds 7a–n performed using DPPH method is shown in Table 2. All the synthesized compounds produced a concentration dependant scavenging of the free radical, DPPH. The IC₅₀ values of all the compounds 7a–e were found between 3.8 μM and 40 μM, with antioxidant activity. Compounds 7b, 7i and 7j possessing 2-hydroxy, 2-methoxy and 4-nitro groups as substitutions respectively on the benzene ring showed better activity against DPPH free radicals. Both the electron donating and electron withdrawing groups present as substituent enhanced the antioxidant activity. These results suggest that all the compounds exhibited remarkable antioxidant activity than the standard ascorbic acid.

4.4 Antibacterial activity

The newly synthesized compounds 7a–n were tested for their in vitro antibacterial activity against gram-positive bacteria viz., Bacillus cereus (ATCC 6633), Bacillus subtilis (ATCC 12711) and Staphylococcus aureus (ATCC 25953) and gram-negative bacteria viz., Micrococucus luteus (ATCC 9341), Entamoeba Coli (ATCC 11230) at 100 μg/ml concentration. The in vitro antimicrobial activity of the tested compounds was assessed by minimum inhibitory concentration (MIC) using the broth dilution method (National Committee for Clinical Laboratory Standards, 1982). Ciprofloxacin was used as standard drug for comparison and the data are presented in Table 3. The results of antibacterial screening (Table 3) reveal that compounds 7a–n displayed better activity and more active than the standard Ciprofloxacin. Compounds possessing 2-hydroxy 7b, 4-methyl 7g, and 4-nitro 7j substitutions on the phenyl ring showed remarkable growth inhibition. Both the electron donating as well as electron withdrawing groups present as substitutions on the benzene ring enhanced the antibacterial activity.

4.5 Antifungal activity

The target compounds 7a–n were also evaluated for their antifungal activity against Candida albicans (ATCC 10231) and Aspergillus niger (ATCC 16404) in acetone by the cup bioassay method (Margery Linday, 1962), using fluconazole as standard drug. The results are summarized in Table 4. The antifungal activity results (Table 4) indicate that compounds 7a–n are significantly toxic toward all the fungi under investigation. Compounds with the electron donating methoxy group on the phenyl ring 7h, 7i, and 7n are highly toxic toward the two fungi. However the degree of spore germination inhibition varied with the test compounds as well as with the fungi under investigation. The fungal activity of the compounds 7a–n has shown better activity, when compared with the standard drug Fluconazole.