Synthesis and Biological Evaluation of 4-( 3-Hydroxy-benzofuran-2-yl ) coumarins

Various 4-bromomethylcoumarins (1a-k) were reacted with methyl salicylate to yield 2-(2-oxo-2H-chromen-4-ylmethoxy)benzoic acid methyl esters (2a-k). Formations of (3a-k) were achieved by using DBU under microwave irradiation. Structures of all the compounds were established on the basis of their spectral data. All the compounds were tested in vitro for their antimicrobial activity and cell cytotoxicity. All the tested compounds (2b-k) and (3a-k) were shown to be better activity against Staphylococcus aureus than the standard Ciprofloxacin. The compound (3k) (R = 6-OMe) was found to be more potent cytotoxic than the standard 5-fluorouracil.


Introduction
Benzofuran and its derivatives [1] have attracted considerable interest in recent years for their versatile properties in chemistry and pharmacology.3-Benzofuran-5-aryl-1-pyrazolylcarbonyl-4-oxo-naphthyridin [2] was found to be the most potent antitubercular agent against Mycobacterium tuberculosis, even better than standard drug isoniazid.Benzofuran derivatives [3] were found to exhibit favorable antibacterial activity against Staphylococcus aureus and Bacillus subtilis which were better than the control drugs Cefotaxime and Ketoconazole.2-Phenylbenzofurans [4] exhibited enhanced antiprotozoal activity against Trypanosoma brucei rhodesiense and Plasmodium falciparum.2-Arylbenzofurans [5] were isolated from the roots of Glycyrrhiza uralensis.They showed significant in vitro protein tyrosine phosphate-1B inhibitory activity.Technetium-99m labeled pyridyl benzofuran derivatives [6] was tested as potential probes for imaging -amyloid plaques in Alzheimer's brains using single photon emission computed tomography.
Coumarin moieties are widely featured in a broad range of pharmacological and biologically active compounds [7,8].Phosphorohydrazine derivatives of coumarin displayed high in vivo antitumour activity against P388 leukemia [9].Coumarin pyrazoline hybrids were found to possess the highest cytotoxicity against colorectal cell line HCT-116 with IC 50 value of 0.01 M [10].Thiazolyl coumarin derivatives showed significant inhibition against Haemophilus influenzae with a MIC value of 15 M which is less than that of tetracycline [11].Benzo[d]thiazolyl coumarins [12] demonstrated anti-HIV activity against HIV-1 cell with EC 50 < 7 g/mL.
Based on the survey of recent literature studies on coumarins and benzofurans and in our effort to discover novel antimicrobial [13][14][15][16] and anticancer agents [17,18], the aim of our work is synthesis of 4-(3-hydroxy-benzofuran-2yl)coumarins and the evaluation of them for their therapeutic importance.
The IR spectrum of the compound 4-(3-hydroxybenzofuran-2-yl)-6-methylcoumarin (3a) (R = 6-CH 3 ) displayed a lactone carbonyl stretching frequency at 1729 cm −1 , whereas the -OH stretching frequency appeared at 3450 cm −1 .The 1 H NMR spectrum of the compound (3a) showed a singlet at  2.42 and 6.93 due to CH 3 and C 3 -H protons, respectively.The aromatic protons were resonated as a multiplet at  7.23-8.48.The -OH proton observed as a singlet at  11.42 that was confirmed by D 2 O exchange.The mass spectrum (LC-MS) of the compound (3g) displayed a [M+H] peak at 307.The 13 C NMR spectral data of compound (3h) are given in the experimental section.
Antibacterial activity was carried out against three Grampositive bacteria, namely, Staphylococcus aureus, Enterococcus faecalis, and Streptococcus mutans, and three Gram-negative bacteria, namely, Escherichia coli, Klebsiella pneumonia, and Pseudomonas aeruginosa.Ciprofloxacin was used as a standard.Antifungal activity was carried out against two fungi, namely, Candida albicans and Aspergillus fumigatus.Fluconazole was used as a standard.
In general, uncyclized compounds (2b-k) are more potent than the cyclized compounds (3a-k) against S. mutans and E. coli.The cyclized compounds (3a-k) are more potent than the uncyclized compounds (2b-k) against E. faecalis and C. albicans.It is interesting to found that both cyclized and uncyclized compounds showed better activity against S. aureus than the standard Ciprofloxacin.

In Vitro Cell Cytotoxicity
All the newly synthesized compounds (2b-k) and (3a-k) were evaluated for their cytotoxicity against DAL cell using trypan blue dye exclusion assay.The detail procedure has been described in our earlier publications [26,27].
The investigation of in vitro cell cytotoxicity (Table 2) revealed that most of the tested compounds exhibited good activity.The compounds (2d) (R = 6-Br), (2h , and (3k) (R = 6-OMe) were found to be highly active (>70%) against DAL cell at the concentration of 100 g/mL.The compound (2f) (R = 5,6benzo) was found to be poor active (25%) against DAL cell at the concentration of 100 g/mL.The rest of the compounds were found to be moderately active (>40%).In general, the cyclized compounds (3a-k) were found to be more potent than the uncyclized compounds (2b-k).

Experimental Section
The melting points were measured with an electric melting point apparatus and are uncorrected.The IR spectra were obtained using a Shimadzu-8400S FT-IR spectrophotometer.

General Procedure for the Synthesis of Compounds (2a-k).
Methyl salicylate (0.304 g, 2.0 mmol) and anhydrous K 2 CO 3 (1.38 g, 10 mmol) were stirred in 25 mL of dry acetone for 30 min.4-Bromomethylcoumarin (1a-k) (2.0 mmol) was added and stirring was continued for 24 h.The reaction mixture was concentrated to one-fourth volume and poured onto crushed ice.The solid separated was filtered and washed with 10 mL of 5% HCl.Then, it was washed with 50 mL of cold water.The crude product was dried and recrystallised from ethanol.

General Procedure for the Synthesis of Compounds (3a-k).
A mixture of 2-(2-oxo-2H-chromen-4-ylmethoxy)-benzoic acid methyl ester (2a-k) (2.0 mmol), DBU (0.3 g, 2.0 mmol) and DMF (25 mL) were added to a microwave tube equipped with a magnetic stir bar.The microwave tube was fitted with a reflux condenser and irradiated in a microwave reactor at a temperature of 140 ∘ C for 4 min at a maximum power of 370 W.Then, completion of the reaction mixture was poured onto ice cold water and neutralized with dil HCl solution.The solid separated was filtered and washed with 100 mL of cold water.The crude product was dried and recrystallised from ethanol.

Conclusion
All the tested compounds (2b-k) and (3a-k) were shown to exhibit better activity against Staphylococcus aureus than the standard Ciprofloxacin.The compound (3k) (R = 6-OMe) was found to be more potent cytotoxic than the standard 5fluorouracil.