Synthesis and biological evaluation of coumarin based isoxazoles, pyrimidinthiones and pyrimidin-2-ones

2.1 Analytical methods and instruments

Chemicals and solvents were obtained from commercial sources and used as received throughout the investigation. Melting points were determined in open capillaries on a Veego electronic apparatus VMP-D (Veego Instrument Corporation, Mumbai, India) and are uncorrected. IR spectra (4000–400 cm⁻¹) of synthesized compounds were recorded on a Shimadzu 8400-S FT-IR spectrophotometer (Shimadzu India Pvt. Ltd., Mumbai, India) using KBr pellets. Thin layer chromatography was performed on object glass slides (2 × 7.5 cm) coated with silica gel-G and spots were visualized under UV irradiation. ¹H NMR and ¹³C NMR spectra were recorded on a Varian 400 MHz model spectrometer (Varian India Pvt. Ltd., Mumbai, India) using DMSO as a solvent and TMS as an internal standard with ¹H resonant frequency of 400 MHz and ¹³C resonant frequency of 100 MHz. The ¹H NMR and ¹³C NMR chemical shifts were reported as parts per million (ppm) downfield from TMS (Me₄Si). The splitting patterns are designated as follows; s, singlet; br s, broad singlet; d, doublet; t, triplet; q, quartet; m, multiplet. Mass spectra were recorded on the JEOL SX-102 (EI) model. All new compounds were subjected to elemental analysis using a Heraeus Carlo Erba 1180 CHN analyzer (Hanau, Germany).

2.2 Synthesis of 4-chloro-chromen-2-one (3)

4-Hydroxycoumarin 1 (30 g, 0.185 mol) and 60 mL POCl₃ were refluxed for 1 h, cooled, and slowly poured into crushed ice (700 g) with vigorous stirring. The solid was collected by filtration and washed successively with ice-water. Azeotropic distillation with n-hexane and hot filtration of the by-product (15 g, 17%) and then followed by evaporation of solvent, and crystallization yielded (21.9 g, 65%) 4-chloro coumarin with m.p. 87–89 °C (Kováč et al., 2001).

2.3 4-Chloro-3,4′,3′,4″-tercoumarin (by-product) (3a)

Crystallization from acetic acid gave yellowish crystals, m.p. 322–327 °C (Kováč et al., 2001).

2.4 Synthesis of 4-(4-acetyl-phenylamino)-chromen-2-one (4)

4-Chloro-chromen-2-one 3 (10.0 g, 0.055 mol) and 1-(4-amino-phenyl)-ethanone (7.48 g, 0.055 mol) were dissolved in acetone (250 mL). The reaction mixture was refluxed for 6–8 h. Sodium carbonate was added to neutralize HCl evolved during the reaction mixture. After completion of the reaction, the mixture was cooled and poured into crushed ice. The solid separated, was filtered, washed with water, dried and recrystallized from ethanol to give compound 4. Yield: 82%. (Patel et al., 2012).

2.5 General procedure for the synthesis of chalcones 5(a–k)

Compound 4 (4.0 g, 0.014 mol) was dissolved in DMF (50 mL) and appropriate aldehyde (for e.g. benzaldehyde – 1.54 g, 0.014 mol) was added with constant stirring at room temperature. Then KOH solution (40 wt%) was added to the reaction mixture, which was stirred for 24 h at room temperature. On completion of the reaction, the mixture was poured into crushed ice and neutralized with HCl. The product was separated out, filtered, washed with water, dried and recrystallized from ethanol to give compound 5a. m.p. 145–147 °C. Similarly other compounds 5(b–k) were synthesized. (Patel et al., 2012).

2.6 General procedure for the synthesis of isoxazoles 6′(a–k)

Compound 5a (4.0 g, 0.010 mol) was dissolved in ethanol (25 ml) and hydroxylamine hydrochloride (0.69 g, 0.010 mol) was added to the solution. Then a solution of 40% KOH was added to the reaction mixture and refluxed for 10 h. The progress of the reaction was monitored on TLC. After completion of the reaction, the mixture was cooled and poured into crushed ice and neutralized with dilute HCl. The product was separated out, filtered, washed with water, dried and recrystallized from alcohol to give 6′a. Similarly other isoxazoles 6′(b–k) were synthesized.

2.7 General procedure for the synthesis of pyrimidinthiones 7′(a–k)

Compound 5a (4.0 g, 0.01 mol) was dissolved in ethanol (25 ml) and then thiourea (0.76 g, 0.01 mol) was added to the solution. Then a solution of KOH (5 ml of 40%) was added to the reaction mixture and refluxed for 8 h. The progress of the reaction was monitored on TLC. After completion of the reaction, the mixture was then cooled and poured into crushed ice and neutralized with dilute HCl. The product was separated out, filtered, washed with water, dried and recrystallized from alcohol to give 7′a. Similarly other pyrimidinthiones 7′(b–k) were synthesized.

2.8 General procedure for the synthesis of pyrimidin-2-ones 8′(a–k)

Compound 5a (4.0 g, 0.01 mol) was dissolved in ethanol (25 ml) and then urea (0.60 g, 0.01 mol) was added to the solution. Then a solution of KOH (5 ml of 40%) was added to the reaction mixture and refluxed for 8 h. The progress of the reaction was monitored on TLC. After completion of the reaction, the mixture was then cooled and poured into crushed ice and neutralized with dilute HCl. The product was separated out, filtered, washed with water, dried and recrystallized from alcohol to give 8′a. Similarly other pyrimidinthiones 8′(b–k) were synthesized.

2.9 Antimicrobial assay

The synthesized compounds 6′a–k, 7′a–k, 8′a–k were examined for antimicrobial activity against several bacterial (Staphylococcus aureus MTCC 96, Bacillus cereus MTCC 430, Escherichia coli MTCC 739, Pseudomonas aeruginosa MTCC 741, Klebsiella pneumoniae MTCC 109, Salmonella typhi MTCC 733, Proteus vulgaris MTCC 1771, Shigella Flexneri MTCC 1457) and fungal (Aspergillus niger MTCC 282, Aspergillus fumigatus MTCC 343, Aspergillus clavatus MTCC 1323, Candida albicans MTCC 183) species using paper disk diffusion technique (Gillespie, 1994). The sterilized (autoclaved at 120 °C for 30 min) medium (40–50 °C) was inoculated (1 ml/100 ml of medium) with the suspension (10⁵ cfu ml⁻¹) of the micro-organism (matched to McFarland barium sulfate standard) and poured into a petridish to give a depth of 3–4 mm. The paper impregnated with the test compounds (μg ml⁻¹) in dimethyl sulfoxide was placed on the solidified medium. The plates were pre-incubated for 1 h at room temperature and incubated at 37 °C for 24 and 48 h for anti-bacterial and anti-fungal activities, respectively. Ciprofloxacin (100 μg/disk) and Ketoconazole (100 μg/disk) were used as standard for antibacterial and anti-fungal activities, respectively. The observed zone of inhibition is presented in Tables 1–3. MIC of the compound was determined by the agar streak dilution method (Hawkey and Lewis, 1994). A stock solution of the synthesized compound (100 μg ml⁻¹) in dimethyl sulfoxide was prepared and graded quantities of the test compounds were incorporated in specified quantity of molten sterile agar (nutrient agar for anti-bacterial activity and sabouraud dextrose agar medium for anti-fungal activity). A specified quantity of the medium (40–50 °C) containing the compound was poured into a petridish to give a depth of 3–4 mm and allowed to solidify. Suspension of the micro-organism was prepared to contain approximately 10⁵ cfu ml⁻¹and applied to plates with serially diluted compounds in dimethyl sulfoxide to be tested and incubated at 37 °C for 24 and 48 h for bacteria and fungi, respectively. The MIC was considered to be the lowest concentration of the test substance exhibiting no visible growth of bacteria or fungi on the plate. The observed MIC is presented in Tables 1 and 2.

2.10 Antimycobacterial assay

The antimycobacterial screening for test compounds was performed against M. tuberculosis H37Rv using the L.J. (Lowenstein and Jensen) MIC method (Isenberg, 1992) for the measurement of MIC. Stock solutions of primary 1000, 500, 250, and secondary 200, 100, 62.5, 50, 25, 12.5, 6.25, 3.25 μg/mL dilutions of each test compound in dimethylsulfoxide (DMSO) were added in the liquid L.J. Medium and then media were sterilized by the inspissation method. A culture of M. tuberculosis H37Rv growing on the L.J. medium was harvested in 0.85% saline in bijou bottles. These tubes were then incubated at (37 ± 1) °C for 24 h followed by streaking of M. tuberculosis H37Rv (5 × 10⁴ bacilli per tube). Growth of bacilli was seen after 12 days, 22 days and finally 28 days of incubation. Tubes having the compounds were compared with control tubes where medium alone was incubated with M. tuberculosis H37Rv. The concentration at which no development of colonies or <20 colonies occurred was taken as MIC concentration of the test compound. The standard strain M. tuberculosis H37Rv was tested with known drugs rifampicin, isoniazid, ethambutol and pyrazinamide.

3.1 Chemistry

Synthetic route of isoxazoles, pyrimidinthiones and pyrimidin-2-ones is depicted in Scheme 1. The target compounds were obtained from coumarin based chalcones 5a–k and their cyclization with hydroxyl amine hydrochloride, thiourea and urea led to the formation of corresponding isoxazoles (6′a–k), pyrimidinthiones (7′a–k) and pyrimidin-2-ones (8′a–k). The presence of singlet signal at 7.81 ppm for one proton of –CH in ¹H NMR spectra and band at 1588 cm⁻¹ for C⚌N in IR spectrum confirms the formation of isoxazole compound 6’a. ¹³C NMR data showed peak at 168.23, 164.27 and 97.54 ppm for corresponding carbons of C–O, C⚌N and –CH of isoxazole respectively. The singlet signal observed at 9.83 ppm in ¹H NMR spectra of compound 7′a was attributed to –SH of pyrimidine ring. In addition, the band at 1571 cm⁻¹ (–SH str.) also confirmed the formation of compound 7′a. In ¹³C NMR data, peak at 176.11 ppm was observed for C–SH of 7′a. Compound 8′a showed singlet peak at 9.83 ppm for one proton of –OH of pyrimidine ring and appearance of the –OH group at 3145 cm⁻¹ in IR spectra confirmed the formation of pyrimidin-2-one ring. The presence of carbon of C–OH at pyrimidine ring was confirmed by appearance of peak at 157.23 ppm.

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Scheme 1

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Ar (a–k) – Aromatic aldehydes

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3.2 Antimicrobial activity

Investigation on antimicrobial screening data (Tables 1 and 2) of target compounds resulted in a good deal of activity against all the mentioned bacteria and fungi. The obtained results generally indicate that the majority of these series of compounds are more active against Gram-positive and Gram-negative bacteria than the fungi.

3.3 Antimycobacterial activity

The results of the in vitro antimycobacterial activity of synthesized compounds against M. tuberculosis H37 RV were obtained from the Lowenstein Jensen MIC method and the measurement of potency of the analogs and standard drugs is shown in Table 3. Analogs 6′j, 7′b, 7′c, 7′g, 7′j, and 8′j showed MIC value below 100 μg/mL, in which 7′g and 7′j displayed significant MIC = 25 μg/mL with 99% inhibition, while rest of the compounds showed MIC in the range of 100–1000 μg/mL. None of the compounds were potent as standard drugs.