Novel pyrimidine and its triazole fused derivatives: Synthesis and investigation of antioxidant and anti-inflammatory activity

2.2.1 General procedure for the synthesis of 6-oxo-4-substituted aryl-2-sulfanyl-1,6-dihydropyrimidine-5-carbonitrile (1a–c)

Mixture equimolar quantities of ethyl cyanoacetate (5.7 g, 50 mmol), thiourea (3.8 g, 50 mmol), appropriate aromatic aldehyde (50 mmol) and potassium carbonate (6.9 g, 50 mmol) in absolute ethanol (50 ml) was gently refluxed till the completion of reaction. The reaction mixture was neutralized with glacial acetic acid to precipitate out the product. The product was isolated and recrystallized from ethanol as yellow crystals.

2.2.2 General procedure for the synthesis of 1-methyl-2-(methylsulfanyl)-6-oxo-4-substituted aryl-1,6-dihydropyrimidine-5-carbonitrile (2a–c)

To a solution of 6-oxo-4-substituted aryl-2-sulfanyl-1,6-dihydropyrimidine-5-carbonitrile (1a–c, 20 mmol) in N,N-dimethyl formamide (DMF, 30 ml), potassium carbonate (5.52 g, 40 mmol) and methyl iodide (5.68 g, 40 mmol) were added and stirred till the completion of reaction at room temperature. Then the reaction mixture was diluted with cold water and neutralized by glacial acetic acid. The product was filtered off and recrystallized from ethanol as creamy crystals.

2.2.3 General procedure for the synthesis of 2-hydrazinyl-1-methyl-6-oxo-4-substituted aryl-1,6-dihydropyrimidine-5-carbonitrile (3a–c)

A mixture of compound 1-methyl-2-(methylsulfanyl)-6-oxo-4-substituted aryl-1,6-dihydropyrimidine-5-carbonitrile (2a–c, 10 mmol) and hydrazine hydrate (80%, 1.90 g, 30 mmol) in absolute alcohol was refluxed till the completion of reaction. The reaction mixture was poured into crushed ice. Then the product was isolated and recrystallized from ethanol/DMF mixture as yellow crystals.

2.2.4 General procedure for the synthesis of 8-methyl-7-oxo-5-substituted aryl-7,8-dihydro[1,2,4]triazolo[4,3-α]pyrimidine-6-carbonitrile (4a–c)

A mixture of compound 2-hydrazinyl-1-methyl-6-oxo-4-substituted aryl-1,6-dihydropyrimidine-5-carbonitrile (3a–c, 5 mmol) in 20 ml formic acid was refluxed till the completion of reaction. The excess of formic acid was distilled. The reaction mixture after cooling was poured into crushed ice. Then the product was isolated and recrystallized from DMF as yellow crystals.

2.2.5 General procedure for the synthesis of 3,8-dimethyl-7-oxo-5-substituted aryl-7,8-dihydro[1,2,4]triazolo[4,3-α]pyrimidine-6-carbonitrile (5a–c)

A mixture of compound 2-hydrazinyl-1-methyl-6-oxo-4-substituted aryl-1,6-dihydropyrimidine-5-carbonitrile (3a–c, 5 mmol) in 20 ml acetic anhydride was refluxed till the completion of reaction. The excess of acetic anhydride was distilled. The reaction mixture after cooling was poured into crushed ice. Then the product was isolated and recrystallized from DMF as yellow crystals.

2.2.6 General procedure for the synthesis of 8-methyl-7-oxo-3-phenyl-5-substituted aryl-7,8-dihydro[1,2,4]triazolo[4,3-α]pyrimidine-6-carbonitrile (6a–c)

A mixture of compound 2-hydrazinyl-1-methyl-6-oxo-4-substituted aryl-1,6-dihydropyrimidine-5-carbonitrile (3a–c, 5 mmol) in 20 ml benzoyl chloride was refluxed till the completion of reaction. The excess of benzoyl chloride was distilled. The reaction mixture after cooling was poured into crushed ice. Then the product was obtained as semisolid.

The physical constants and spectral (IR, ¹H NMR, mass) characterization and elemental analysis supported the structure of various synthesized compounds (Tables 1 and 2).

3.1.1 Hydrogen peroxide scavenging activity

A solution of hydrogen peroxide (20 mM) was prepared in phosphate buffer saline (pH 7.4). Various concentrations (12.5, 25, 50, 100 μg/ml) of 1 ml of the test samples or standard, ascorbic acid (Ismaili et al., 2008; Rang et al., 2003) in methanol were added to 2 ml of hydrogen peroxide solution in phosphate buffer saline. The absorbance was measured at 230 nm after 10 min (Jayaprakasha et al., 2004).

3.1.2 Nitric oxide scavenging activity

The reaction mixture (6 ml) containing sodium nitroprusside (10 mM, 4 ml), phosphate buffer saline (pH 7.4, 1 ml) and test samples or standard, ascorbic acid solution in dimethyl sulphoxide (1 ml) at various concentrations (12.5, 25, 50, 100 μg/ml) was incubated at 25 °C for 150 min. After incubation, 0.5 ml of reaction mixture containing nitrite ion was removed, 1 ml of sulphanilic acid reagent was added to this, mixed well and allowed to stand for 5 min for completion of diazotization. Then, 1 ml of naphthyl ethylene diamine dihydrochloride was added, mixed and allowed to stand for 30 min in diffused light. A pink colored chromophore was formed. The absorbance was measured at 640 nm (Marcocci et al., 1994).

3.1.3 Lipid peroxidation inhibitory activity

Egg lecithin (3 mg/ml phosphate buffer, pH 7.4) was sonicated in an ultrasonic sonicator for 10 min to ensure proper liposome formation. Test samples (100 μl) of different concentrations (12.5, 25, 50, 100 μg/ml) were added to liposome mixture (1 ml); the control was without test sample. Lipid peroxidation was induced by adding ferric chloride (10 μl, 400 mM) and l-ascorbic acid (10 μl, 200 mM). After incubation for 1 h at 37 °C the reaction was stopped by adding hydrochloric acid (2 ml, 0.25 N) containing trichloroacetic acid (150 mg/ml), thiobarbituric acid (3.75 mg/ml) and butylated hydroxy anisole (0.50 mg/ml). The reaction mixture was subsequently boiled for 15 min, cooled, centrifuged at 1000 rpm for 15 min and the absorbance of the supernatant was measured at 532 nm and compared with that of ascorbic acid (Duh and Yen, 1997).

For all the above antioxidant methods, experiments were done in triplicate and average is taken, the % inhibition at different concentration was calculated by the following formula $$\%\text{Inhibition}=[1-(V_{\text{t}}/V_{\text{c}})]\times 100$$ where, V_t = mean absorption of test compound, V_c = mean absorption of control.

The IC-50 value was derived from the% inhibition at different concentration.

4.2.1 Antioxidant activity

All the synthesized compounds were screened for in vitro antioxidant activity by various methods as scavenging of hydrogen peroxide, scavenging of nitric oxide radical, lipid peroxidation inhibitory activity. In vitro antioxidant activity of synthesized compound is summarized in Table 3.

The investigation of antioxidant screening revealed that some of the tested compounds showed moderate to good antioxidant activity. Particularly, compounds 3a, 3c, 1a, 1c, 3b and 3c have shown more promising antioxidant activity as compare to standard, ascorbic acid, while other derivatives are moderately active. 1c has shown good antioxidant activity as compare to standard by scavenging of nitric oxide radical and scavenging of hydrogen peroxide method, while 3b showed most potent antioxidant activity by scavenging of nitric oxide radical method.

4.2.2 Anti-inflammatory activity

Synthesized compounds were also tested for anti-inflammatory activity. Compared to the standard Diclofenac sodium, they have shown acceptable anti-inflammatory activity. In vitro anti-inflammatory activity of synthesized compounds is summarized in Table 4 and Fig. 6. Among the tested compounds, 3b, 2b, 1b and 1c showed better anti-inflammatory activity. Amongst all 3b found with most potent activity.