Synthesis and Evaluation of Some Coumarin Containing Potential Antimicrobial Agents

A series of the Schiff’s bases incorporating coumarin and chalcone moeities, 3-(4-(4-(substituted phenyl)prop-1-ene-3-one) phenylimino) methyl)4-chloro-2h-chromen-2-one 4(a-g) were synthesized as potential antimicrobial agents. These compounds were characterized on the basis of their spectral (IR, H NMR) data and evaluated for antimicrobial activity in vitro against gram positive and gram negative bacteria and fungi. Compound 4b was found to be most active with an MIC of 20 μg/mL against all the tested organisms.


Introduction
There are a number of reports that natural and synthetic coumarin derivatives posses antimicrobial activity [1][2] .Novobiocin and chlorobiocin are established antimicrobials containing a coumarin skeleton.A number of the amino substituted coumarin derivatives have been reported as the inhibitors of DNA Gyrase as potential antibacterials 3 .Elgamal et al 4 have reported synthesis and antimicrobial properties of hydrazino derivatives of 5chloro-6-formylfuranocoumarins.Similarly chalcones have also been shown to display antimicrobial activity 5. Development of resistance towards existing antimicrobial therapy and a sharp rise in the population of immonocompromised patients have provided a thrust to the research in the area of antimicrobials.In continuation of the work done in our laboratory on coumarin based antimicrobial agents 5 , we have designed a series of compounds incorporating coumarin and chalcone moeities in the structure in hope of achieving a better antimicrobial activity.The compounds have been synthesized via simple steps given in Scheme 1.

Experimental
All the chemicals used in the synthesis were of laboratory grade.Melting points were determined in open capillary on Veego (model: VMP-D) electronic apparatus and are uncorrected.The IR spectra of the synthesized compounds were recorded on shimadzu 8400-S FT-IR spectrophotometer using potassium bromide.The 1 H NMR spectra were recorded in CDCl 3 using NMR varian-mercury 300 MHZ spectrometer and chemical shifts are given in units as per million, downfield from tetramethylsilane (TMS) as an internal standard.To monitor the reactions, as well as, to establish the identity and purity of reactants and products, thin layer chromatography was performed on precoated aluminium sheets (silica gel 60 f 254, 5 x 2.5 cm) using toluene-methanol and chloroform-ethyl acetate as appropriate solvent systems.

General procedure for Synthesis of 3-(4-(4-substituted phenyl) prop-1-ene-3-one) phenylimino) methyl)-4-chloro-2H-chromen-2-one 4(a-g)
4-chloro-3-coumarinaldehyde (0.01mol) was dissolved in 20ml of rectified spirit.To this solution the chalcone 3(a-g) (0.01 mol) was added and refluxed for 7-8 hrs.On completion of the reaction, water was added to this solution until cloudiness persisted.It was then kept aside to cool when the poduct separated out as precipitates.The solid obtained was filtered, washed with water, dried in air and recrystallized from methanol.The physical and spectral data is provided in Table I.

Zone of inhibition
All the target compounds 4(a-g) were tested for their antimicrobial activity by agar cup plate method 7 by using nutrient agar broth media.The organisms used were gram positive bacteria: S. aureus (ATCC 9144), B. subtilis (ATCC 6633) and S. epidermis (ATCC 12228), gram negative bacteria: E. coli (ATCC 25922), S. typhi and P. aeruginosa (ATCC 9027) .The antifungal activity was evaluated against A. Niger (ATCC 10594) and C. Albicans (ATCC 10231) by using potato dextrose agar as media.Amoxycillin and fluconazole were used as standards for antibacterial and antifungal activity respectively.The observations are given in Table II and III for antibacterial activity and in Table IV for antifungal activity.

Minimum inhibitory concentration
The Minimum inhibitory concentration (MIC) against the above organisms was determined by the method of broth dilutions 8 .The results are given in Table V.   I) of these compounds reveal a characteristic aromatic stretch between 2900-3100cm -1 .Sharp α, β-unsaturated carbonyl group stretch at around 1620-1741 cm -1 and sharp carbonyl stretching vibration for coumarin lactone at around 1750 cm -1 were also seen.The stretching vibrations for C=N group are seen at around 1534-1602 cm -1 .The 1 H NMR spectra (Table I) of were recorded in CDCl3.The aromatic protons showed peaks at 7.4-8.6ppm as multiplet.The olefinic protons displayed characteristic doublets at around 8.8-8.5 and 8.4-8.3.Absence of peak by aldehydic proton confirms formation of schiff's base.Absence of singlet by amino protons confirms that the intermediates the chalcone 3(a-g) have been condensened successfully with 4-chloro-2-oxo-2H-chromene-3-carbaldehyde (2).This is further confirmed by appearance of imine peak proton at around 9.4 ppm.All the target compounds 4(a-g) have shown good in-vitro antimicrobial activity (Table III and IV) against all the tested gram +ve and gram -ve bacteria and fungi.Compound 4b exhibited highest zone of inhibition and least MIC (20 μg/mL) against all organisms.

Results and Discussion
Scheme 1

Table I .
Physical and spectral data of synthesized compounds.

Table II .
Zone of inhibition of synthesized compounds against gram positive bacteria.

Table III :
Zone of inhibition of synthesized compounds against gram negative bacteria.

Table IV :
Zone of inhibition of synthesized compounds against fungi.

Table V :
Minimum inhibitory concentration for the synthesized compounds (μg/ml).