Design, synthesis, antibacterial, and antifungal studies of novel 3-substituted coumarinyl-triazine derivatives

Chemistry

Scheme 1 outlines the synthetic pathway used to obtain compounds 6a–m. The starting material 2-oxo-2H-chromene-3-carboxylic acid (1) was prepared from 2-hydroxybenzaldehyde and malononitrile via consecutive Knoevenagel and Pinner reactions in water [31]. Chlorination of compound 1 with thionyl chloride followed by condensation with hydrazine hydrate of the resultant acid chloride yielded the 2-oxo-2H-chromene-3-carbohydrazide (2). The resulting carbohydrazide was cyclized using potassium hydroxide in the presence of carbon disulfide to form the oxadiazole derivative 3 [32]. Compound 4 was obtained in a good yield by the reaction of the oxadiazole intermediate product 3 with 2,4,6-trichloro-1,3,5-triazine in the presence of 10% sodium bicarbonate solution at 0–5°C in acetone. Compound 4 was then allowed to react with morpholine in acetone at 45–50°C to give the precursor 5 to the final desired products 6a–m. The synthesis of compounds 6a–m was confirmed by IR, ¹H NMR, ¹³C NMR, MS, and elemental analysis.

Scheme 1

Antimicrobial activity

The results of the antibacterial and antifungal activities of compounds 6a–m are presented in Table 1. These results reveal that compounds 6b, 6c, 6i, 6j, and 6m display significant activity against Gram +ve bacteria, Staphylococcus aureus and Bacillus subtilis. Compounds 6d, 6e, 6h, 6k, and 6l show moderate to good inhibition, whereas compounds 6a, 6f, and 6g are definitely less active. Compounds 6b, 6h, 6i, 6j, and 6m display good activity against Gram -ve bacteria, Escherichia coli and Salmonella typhi, and compounds 6c, 6e, 6g, 6k, and 6l show moderate to good inhibition.

Compounds 6c, 6h, 6j, and 6m display excellent inhibition against fungi Aspergillus niger. Compounds 6b, 6d, 6i, 6k, and 6l show moderate to good inhibition. Compounds 6a, 6e, 6f, and 6g show weaker activity towards tested fungi. Among the synthesized compounds, 6j and 6m exhibit good activity against fungi Aspergillus clavatus. Compounds 6b, 6c, 6d, and 6i show moderate to good activity against fungi.

Conclusion

New coumarinyl-triazine derivatives were synthesized. In vitro antibacterial and antifungal activities against two Gram -ve (E. coli, S. typhi), two Gram +ve (S. aureus and B. subtilis) bacterial and two fungal strains (A. niger and A. clavatus) were assayed. Compounds 6b and 6j exhibit good activity against both Gram +ve and -ve strains. Compounds 6j and 6m exhibit good activity against fungi.

General

Melting points were determined on an electrothermal apparatus (capillary method) and are uncorrected. The IR spectra (4000–400 cm^-1) were recorded on a Shimadzu 8400-S FT-IR spectrophoto meter with KBr pellets. ¹H NMR spectra were recorded in DMSO-d₆ on a Bruker Avance II 400 MHz and ¹³C NMR spectra were recorded in DMSO-d₆ at 100 MHz. Mass spectra were recorded on a Finnegan Mat spectrometer at 70 eV. Elemental analyses were carried out on ‘Haraeus Rapid Analyzer’. To monitor the reactions, thin layer chromatography (TLC) was performed on silica gel coated (Merck 60 GF-254, 0.2 mm thickness) sheets.

Preparation of 2-oxo-2H-chromene-3-carboxylic acid (1)

A mixture of salicylaldehyde (12.2 g, 0.1 mol), malononitrile (6.6 g, 0.1 mol), and 0.025 M aqueous NaOH solution (pH 12.4, 50.0 mL) was vigorously stirred at room temperature and then heated under reflux for 2 h. 3-Cyano-2-iminocoumarin was separated. Then, the solution was treated with concentrated HCl (1.25–2.0 mL), and the resultant heterogeneous mixture was heated at 90°C with stirring for 1.5 to 2.5 h. After cooling, the mixture was treated with aqueous NaOH solution (1.5 M, 20 mL). After removal of a solid precipitate of 3-cyanocoumarin, the solution was heated at 90°C and stirred for 2 h. The final solution was cooled to room temperature and acidified with concentrated HCl to pH≤2. The resultant product was separated. The progress of the reaction was monitored by TLC using acetone/toluene (8:2) as eluent. The crude product was purified by crystallization from absolute ethanol: yield 85%; mp 184–186°C (lit. mp 189–192°C, [31]).

Preparation of 2-oxo-2H-chromene-3-carbohydrazide (2)

A mixture of 2-oxo-2H-chromene-3-carboxylic acid (1, 19.0 g, 0.1 mol) and thionyl chloride (17.8 g, 0.15 mol) was heated under reflux for 1–2 h. Excess of thionyl chloride was distilled off and the residue was cooled in an ice-bath. The resulting precipitate was dissolved in methanol and the solution was treated immediately and slowly with hydrazine hydrate (4.8 g, 0.1 mol) with constant stirring. The mixture was refluxed for 4 to 5 h. Excess solvent was removed by distillation under reduced pressure and the residue was poured into ice-cold water. The resultant solid was filtered, dried, and crystallized from absolute ethanol: yield 89%; mp 140–142°C (lit. mp 137–140°C, [25]); ¹H NMR: δ 8.24 (1H, s, H-4, coumarin), 8.12 (m, 1H, CONHNH₂), 7.62–7.39 (4H, m, Ar-H), 4.78 (s, 2H, NH₂, D₂O exchangeable).

Preparation of 3-(5-mercapto-1,3,4-oxadiazole-2-yl)-2H-chromene-2-one (3)

The mixture of 2-oxo-2H-chromene-3-carbohydrazide (2, 20.4 g, 0.1 mol), CS₂ (7.6 mL, 0.1 mol), and KOH solution (10 mL, 0.05 mol) in methanol (82 mL) was heated under reflux for 6 to 8 h. Then, the mixture was poured into crushed ice. The product was filtered, washed with water, and crystallized from ethanol. The progress of the reaction was monitored by TLC using acetone/toluene (8:2) as eluent. Product 3 was obtained in a 73% yield; mp 163–166°C (lit. mp 166–168°C, [25]); IR (cm^-1): 2591(SH), 1726 (CO, coumarin), 1629, 1531 (C=N, oxadiazole), 1043 (oxadiazole); ¹H NMR: δ 14.51 (s, 1H, SH), 8.21 (1H, s, H-4, coumarin), 7.58–7.37 (m, 4H, Ar-H).

Preparation of 3-(5-(4,6-dichloro-1,3,5-triazin-2-ylthio)-1,3,4-oxadiazole-2-yl)-2H-chromene-2-one (4)

To a stirred solution of cyanuric chloride (18.4 g, 0.1 mol) in acetone (92 mL) at 0–5°C, the solution of 3-(5-mercapto-1,3,4-oxadiazole-2-yl)-2H-chromene-2-one (3, 24.6 g, 0.1 mol) in acetone (112 mL) was added and pH was maintained neutral by addition of 10% sodium bicarbonate solution. The stirring was continued at 0–5°C for 4 h. After completion of the reaction, the stirring was stopped and the solution was treated with crushed ice. The solid product obtained was filtered and dried. The progress of the reaction was monitored by TLC using ethyl acetate/hexane (6:4) as eluent. The crude product was purified by crystallization from absolute ethanol. Product 4 was obtained in an 80% yield; mp 179–182°C (lit. mp 178–180°C, [25]); IR (cm^-1): 1725 (C=O), 1624, 1527 (C=N, oxadiazole), 1040 (C-O-C, oxadiazole), 696 (C-S-C), 833 (s-triazine), 752 (C-Cl.); ¹H NMR: δ 8.30 (1H, s, H-4, coumarin), 7.51–7.38 (m, 4H, Ar–H).

Preparation of 3-(5-(4-chloro-6-morpholino-1,3,5-triazin-2-ylthio)-1,3,4-oxadiazole-2-yl)-2H-chromene-2-one (5)

The solution of morpholine (8.7 g, 0.1 mol) in acetone was added dropwise to well-stirred suspension of 3-(5-(4,6-dichloro-1,3,5-triazin-2-ylthio)-1,3,4-oxadiazole-2-yl)-2H-chromene-2-one (4, 39.4 g, 0.1 mol) in acetone (90.0 mL) maintaining the temperature below 40–45°C. The pH was kept neutral by the addition of 10% NaHCO₃ solution. The temperature was gradually raised to 50°C during 2 h and further maintained for 2 h. After completion of the reaction, the solution was poured into ice-cold water. The solid product was filtered, dried, and crystallized from absolute ethanol. Compound 5 was obtained in an 81% yield; mp 212–214°C; IR (cm^-1): 1741 (C=O, coumarin), 1619, 1540 (C=N, oxadiazole), 1256 (C-O-C), 1041 (C-O-C, oxadiazole), 829 (s-triazine), 749 (C-Cl), 701 (C-S-C); ¹H NMR: δ 8.20 (s, 1H, NH), 8.38 (s, 1H, Ar-H), 6.6–8.04 (m, 4H, Ar-H), 3.17 (t, 4H, J = 7.1 Hz, -CH₂), 3.33 (t, 4H, J = 7.1 Hz, CH₂); MS: m/z 445 (M⁺). Anal. Calcd for C₁₈H₁₃ClN₆O₄S (444.85): C, 48.60; H, 2.95; N, 18.89. Found: C, 48.72; H, 2.91; N, 18.93.

General procedure for the preparation of compounds 6a–m

The mixture of 3-(5-(4-chloro-6-morpholino-1,3,5-triazin-2-ylthio)-1,3,4-oxadiazole-2-yl)-2H-chromene-2-one (5, 5 mmol) and the respective substituted arylamine (5 mmol) in 1,4-dioxane (50.0 mL) was heated under reflux with stirring at 80–100°C for 4 h. After completion of the reaction, the pH was adjusted to neutral by the addition of 10% NaHCO₃solution and the mixture was poured into ice-cold water. The solid product 6a–m was filtered, dried, and crystallized from absolute ethanol.

In vitro evaluation of antimicrobial activity

The synthesized coumarin derivatives 6a–m were evaluated for antimicrobial activity against several bacteria (S. aureus MTCC 96, Bacillus cereus MTCC 619, Escherichia coli MTCC 739, S. typhi MTCC 733) and fungi (A. niger MTCC 282, A. clavatus MTCC 1323) species using the paper disc diffusion technique [33]. The Mueller-Hinton agar media were sterilized (autoclaved at 120°C for 30 min), poured at uniform depth of 5 mm, and allowed to solidify. The microbial suspension (105 CFU/mL) (0.5 McFarland Nephelometry Standards) was streaked over the surface of the media using a sterile cotton swab to ensure even growth of the organisms. The tested compounds were dissolved in dimethyl sulfoxide to give solutions of 3.12–100 μg/mL. Sterile filter paper discs measuring 6.25 mm in diameter (Whatman no. 1 filter paper), previously soaked in a known concentration of the respective test compound in dimethyl sulfoxide, were placed on the solidified nutrient agar medium that had been inoculated with the respective microorganism and the plates were incubated for 24 h at 37 ± 1°C. A control disc impregnated with an equivalent amount of dimethyl sulfoxide without any sample was also used and did not produce any inhibition. Penicillin and streptomycin (100 μg/disc) were used as control drugs for antibacterial and griseofulvin (100 μg/disc) for antifungal activity, respectively. Minimum inhibitory concentrations (MICs) of the compounds were determined by the agar streak dilution method [34]. A stock solution of the synthesized compound (100 μg/mL) in dimethyl sulfoxide was prepared and graded quantities of the test compounds were incorporated in a specified quantity of molten sterile agar, that is, nutrient agar for evaluation of antibacterial and sabouraud dextrose agar for antifungal activity, respectively. The medium containing the test compound was poured into a Petri dish at a depth of 4–5 mm and was allowed to solidify under aseptic conditions. A suspension of the respective microorganism of approximately 105 CFU/mL was prepared and applied to plates with serially diluted compounds with concentrations in the range of 3.12–100 μg/mL in dimethyl sulfoxide and incubated at 37 ± 1°C for 24 h (bacteria) or 48 h (fungi). The lowest concentration of the substance that prevents the development of visible growth was reported as the MIC value.