A Novel and Efficient Synthesis of [1,2,3]-Triazolyl Substituted Benzo[ c ]Coumarins and Evaluation of their Antimicrobial Activity

. A series of novel [1,2,3]-triazolyl substituted benzo[ c ]coumarins have been synthesized by reacting various 3-coumarinoyl methyl pyridinium bromide salts with 1-(5-methyl-1-phenyl-1 H - 1,2,3-triazol-4-yl)ethanones in the presence of sodium acetate in refluxing acetic acid. The structures of the synthesized compounds have been elucidated by IR, 1 H-NMR, 13 C-NMR and Mass spectral data. All the synthesized compounds have been screened for their in vitro anti-bacterial and anti-fungal activities. Some of the compounds have been found to be active against some bacterial and fungal pathogens compared to standard drugs.

A literature survey for the synthesis of benzo[c]coumarins revealed that in majority of the reports on the synthesis of benzo[c]coumarins, the compounds have been synthesized by the lactonization of appropriately substituted biphenyl derivatives [25]- [27]. In the present work we have synthesized some new benzo[c]coumarin derivatives utilizing a novel and distinct approach.
Thus considering the importance of benzo[c]coumarins and triazoles, it was thought worthwhile to synthesize a new class compounds which are hybrid of benzo[c]coumarin and [1,2,3]-triazole. Keeping this objective in mind in the present work we have synthesized some [1,2,3]-triazolyl substituted benzo[c]coumarins using a new synthetic approach.

Experimental
All the melting points are uncorrected. All reactions were performed with commercially available reagents and they were used without further purification. Organic solvents were purified by standard methods and stored over molecular sieves. All the IR spectra (KBr disc) were recorded on Shimadzu FT-IR 8400-S spectrometer. 1 H NMR and 13 C NMR spectra were recorded on Bruker Advance 400 spectrometer operating at 400 MHz for 1 H NMR and 100 MHz for 13 C NMR. The chemical shift (δ) is reported in ppm using chloroform-d as a solvent and calibrated standard solvent signal. Mass spectra were recorded on Shimadzu QP 2010 spectrometer. Elemental analysis was carried out on Perkin-Elmer 2400 C-H-N-S-O Analyzer Series-II. Column chromatography was performed with silica gel 60-120 mesh (Merck, Mumbai, India.). All the compounds were routinely checked for completion of the reaction on silica gel 60 F254 TLC plates and their spots were visualized by exposure to a UV lamp, iodine vapour or KMnO4 reagents. Compounds 3-coumarinoyl methyl pyridinium bromide salts (1a-c) [38]- [40] and 1-(5-methyl-1-aryl-1H-1,2,3triazol-4-yl)ethanones (2a-d) [41] were prepared according to literature procedure.

Analytical and Spectral Characterization
The structure of all the twelve synthesized (3a-l) compounds were confirmed by their 1 H-NMR, 13 C-NMR, IR, elemental analysis and representative mass spectral data given below.

Mechanism
It is reported that reaction of α,β-unsaturated ketone with methyl ketone or ketone with active methylene group in the presence of sodium acetate results in a formation of 1,3,5-tri substituted benzene derivatives [42]. This novel reaction is utilized in the preparation of present compounds. Here the benzene ring has been built up between 3 and 4 position of coumarin nucleus by utilizing this reaction. The plausible mechanism for the synthesis of target compounds

International Letters of Chemistry, Physics and Astronomy Vol. 70
Scheme 2: Plausible mechanism for the synthesis of target compounds (3a-l)

Antimicrobial activity
The newly synthesized target compounds (3a-l) were evaluated for their in vitro antibacterial activity against two Gram positive bacteria Staphylococcus aureus (MTCC 96) and Bacillus subtilis (MTCC 441) and two Gram negative bacteria Escherichia coli (MTCC 443) and Salmonella typhi (MTCC 98). They were also evaluated for their in vitro antifungal activity against Candida albicans (MTCC 227) and Aspergillus niger (MTCC 282) as fungal strains. Broth dilution method was used for the determination of the antibacterial and antifungal activity as recommended by NCCLS [43]. Ampicillin, Chloramphenicol and Norfloxacin were used as standard antibacterial drugs, whereas Griseofulvin and Nystatin were used as standard antifungal drugs. All MTCC cultures were collected from Institute of Microbial Technology, Chandigarh and tested against above mentioned known drugs. Mueller-Hinton broth was used as the nutrient medium for the test bacteria and Sabouraud Dextrose broth was used for the test fungi. Inoculum size for the test strains was adjusted to 10 8 CFU (Colony Forming Unit per milliliter) per milliliter by comparing the turbidity. Each synthesized compound was diluted with DMSO so as to have the stock solution of 2000 μg/mL concentration as a stock solution. The results were recorded in the 6 ILCPA Volume 70 form of primary and secondary screening. The synthesized compounds (3a-l) were screened for their antibacterial and antifungal activity at the concentration of 1000, 500 and 250 μg/mL for the primary screening. The synthesized compound showing activity against microbes in the primary screening were further screened in a second set of dilution at concentrations of 200, 100, 62.5, 50 and 25 μg/mL. The suspention of 10 μL from each well were further incubated and growth was noted at 37°C after 24 hour for bacteria and 48 hour for fungi. The lowest concentration which showed no visible growth (turbidity) after spot subculture was considered as the minimum inhibitory concentration (MIC) for each compound. The investigation of the data summarized in (Table-1) reveals that many compounds were found to be active against Gram-positive bacteria while some of the compounds were found to be active against Gram-negative bacterial and fungal species as compared to that of the standard antimicrobial drugs.

Antimicrobial evaluation
The compounds (3a-l) were screened for their in vitro antibacterial and antifungal evaluation against various bacterial and fungal pathogens by broth dilution method. Ampicillin, Chloramphenicol, Norfloxacin, Griseofulvin and Nystatin were used as standard drugs. The values of MIC are summarized in Table-1.
International Letters of Chemistry, Physics and Astronomy Vol. 70

Conclusions
A novel and efficient protocol for the synthesis of a series of [1,2,3]-triazolyl substituted benzo[c]coumarin derivatives was described and the synthesized compounds were screened for their in vitro antimicrobial evaluation. The results indicated that all the synthesized compounds shown good antibacterial activity. In particular, compounds 3e, 3f, 3i, 3j, and 3k exhibited the more potent inhibitory activity against bacterial and fungal pathogens as compared to other compounds and emerged as potential lead compounds for further investigations.