Solvent-free microwave-assisted synthesis and biological evaluation of 2,2-dimethylchroman-4-one based benzofurans

Chemistry

Synthesis of benzofurans 6a–j was accomplished in two steps as shown below. The starting materials 6-acetyl-7-hydroxy-2,2-dimethylchroman-4-one (2a) and 2,2,8,8,-tetramethyl-2,3,7,8-tetrahydropyrano[3,2-g]chromene-4,6-dione (2b) were prepared as previously described [23] (Scheme 1). The precursor chalcones 4a–j (Scheme 2) were synthesized by the Claisen-Schmidt condensation of 6-acetyl-7-hydroxy-2,2-dimethylchroman-4-one (2a) and substituted aromatic aldehydes 3a–j in the presence of potassium hydroxide in ethanol. The chalcones were subjected to cyclization with 1-bromo-3,3-dimethylbutan-2-one (5) to give the 2,2-dimethylchroman-4-one based benzofurans 6a–j in good yields. All these compounds were thoroughly characterized by spectroscopic techniques and elemental analysis.

Scheme 1

Synthesis of 7-hydroxy-2,2-dimethylchroman-4-one (2a) and 2,2,8,8,-tetramethyl-2,3,7,8-tetrahydropyrano[3,2-g]chromene-4,6-dione (2b).

Scheme 2

Synthetic route to 7-hydroxy-2, 2-dimethylchroman-4-one derived chalcones 4a–j and benzofurans 6a–j.

Antimicrobial activity

Compounds 6a–j were screened for their in vitro antibacterial activity against two Gram-positive bacterial strains Staphylococcus aureus (ATCC-6538), Bacillus faecalis (ATCC-6633) and two Gram-negative bacterial strains Escherichia coli (ATCC-25922) and Klebsiella pneumonia (ATCC-13883) by the disc diffusion method [24] at concentrations of 20 μg/mL and 40 μg/mL. The zones of inhibition (in mm) were compared with that for the standard drug ciproflaxin. As can be seen from Table 1, among newly synthesized benzofurans, compound 6f (4-fluorophenyl derivative) shows good activity against S. aureus, K. pneumonia and E. coli bacterial strains. The inhibitory efficiencies of compounds 6h and 6i against K. pneumonia are close to that of standard. Compounds with withdrawing groups (4-F, 4-Cl and 4-Br) at the phenyl ring show zones of inhibition that are comparable to that of the standard against all the bacterial strains.

All synthesized compounds were also screened for their antifungal activity against two pathogenic fungi, Fusarium oxysporum and Aspergillus flavus by the poison plate technique [25] (Table 1). The results of the antifungal screening were compared with the activities of the standard antifungal drugs amphotericin-B and hymexazol. All compounds show moderate activity against the tested fungal strains.

Antioxidant activity

In vitro antioxidant activities of the benzofurans 6a–j were determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay [22]. The DPPH radical scavenging activity evaluation is a rapid and convenient screening technique. The results are shown in Table 2. As can be seen, the best compounds are 6d and 6c, the radical scavenging abilities of which compare favorably with that of the standard. The remaining compounds show moderate activities.

General procedure for the synthesis of benzofurans 6a–j

A mixture of substituted 2′-hydroxychalcone 4a–i (10.0 mmol), 1-bromo-3,3-dimethylbutan-2-one (15.0 mmol) and potassium carbonate (25.0 mmol) was placed in a quartz tube and inserted into a Teflon vial that was screw capped and subjected to microwave irradiation at 320 W for 2–4 min. After completion of reaction (as indicated by TLC), the mixture was poured into ice water, extracted with dichloromethane (2×30 mL), dried over Na₂SO₄, and purified by column chromatography eluting with n-hexane/ethyl acetate (9:1).

Antibacterial activity

Compounds 5a–j were screened for their in vitro antibacterial activity against the bacterial cultures S. aureus, B. subtilis (Gram-positive) and E. coli and Klebsiella (Gram-negative) by the disc diffusion method at concentrations of 20 μg/mL and 40 μg/mL. The cultures were grown in nutrient agar media and sub-cultured for log phasic cultures in a liquid nutrient broth medium for zone of inhibition and further sub-cultured onto media in Petri plates. The broth cultures were diluted with a sterilized saline to bring the final size of inoculum approximately to 10⁵–10⁶ CFU/mL. The compounds were dissolved in acetone, DMSO and diethyl ether for biological assays. Diethyl ether was the preferred solvent. The bacterial cultures were placed on the media and incubated at 37°C for 24 h–48 h along with the diluted compounds introduced through discs dipped and placed over the nutrient media. The zones of bacterial growth inhibitions were measured using the diameter of the zone. All the results were expressed as zone of inhibition (ZOI) in mm. The results were compared with the activity of the standard antibiotic ciproflaxin (20 μg/mL and 40 μg/mL). For the disc diffusion method, the diluted test compounds were introduced onto the disc and once the disc was found completely saturated it was immediately transferred on to surface of the medium with bacterial inoculums spread on the plate evenly. The Petri dishes were incubated at 37°C for 24 h. Bioactivity was determined by measuring diameter of the inhibition zone (DIZ) in mm.

Antifungal activity

The antifungal activity of synthesized compounds was tested against two pathogenic fungi, F. oxysporum and A. flavus, by the poison plate technique. Test compounds were dissolved in diethyl ether (10 mL) before mixing with potato dextrose agar medium (PDA, 90 mL). The final concentration of compounds in the medium was maintained at 500 μg/mL. The fungi were incubated in PDA at 25±1°C for 48–72 h to get long mycelium for antifungal assay. The mycelia disks of approximately 0.45 cm in diameter were cut from the PDA medium with a sterilized inoculation needle and inoculated in the center of a PDA plate. The inoculated plates were incubated at 27±1°C for 3 days. Diethyl ether in sterilized distilled water was taken as control, while hymexazole was used as positive control for the treatment. The growth of the fungal colonies was measured on the third day and the data were statistically analyzed. The in vitro inhibition effects of the test compounds on the fungi were calculated by the given formula CV=(A–B)/A, where A represents the diameter of fungi growth on untreated PDA, B represents the diameter of the fungi growth on treated PDA, and CV represents the rate of inhibition.

DPPH radical scavenging

DPPH radical scavenging activity was measured by the method of Cotelle [26] after standardization with some modifications. A mixture containing 0.2 mL of DPPH (100 μm in methanol) and 2.0 mL of the test solution containing the compound (50, 100, 200 μg/mL) was incubated at 37°C for 30 min. Absorbance of the mixture was measured at 517 nm using a Beckman model DU-40 spectrophotometer. The percentage inhibition of DPPH radical was calculated by comparing the result of the test with that of the control (not treated with extract) using the following equation:

Percent inhibition= Absorbance of test/(Absorbance of control×100)