Synthesis and Evaluation of Some Coumarin Based Schiff ’ s Bases as Potential Antimicrobial Agents ’

A series of the title compounds 3-(4-(4, 5-dihydro-5-(substituted phenyl)-1H-3-pyrazolyl) phenylimino) methyl)-4-chloro-2H-chromen-2-one 5(a-g) have been synthesized. These compounds were characterized on the basis of their spectral (IR, H NMR) data and evaluated for antimicrobial activity in vitro against gram positive bacteria, gram negative bacteria and fungi. The compound (5b) was found to be the most active with MIC of 20 μg/ml against all the tested organisms.


Introduction
Development of resistance and adverse reactions towards existing antimicrobial therapy and a sharp rise in the population of immunocompromised patients has provided a thrust to the research in the area of antimicrobials.There are a number of reports that natural and synthetic coumarin derivatives possess antimicrobial activity.Novobiocin and chlorobiocin are established antimicrobials containing a coumarin skeleton.The biological effects of coumarins include antibacterial 1 , anti-inflammatory 2 , anti-AIDS 3 , antithrombotic 4 and antitumourogenic 4 effects.Similarly pyrazolines have been independently reported to possess antibacterial, antifungal 5 , anticancer 6 , antimalarial 7 and anticonvulsant 8 activity.In continuation of the work done in our lab on coumarin based antimicrobial agents 9 , we have designed and synthesized the series of compounds incorporating both, coumarin and pyrazoline moieties as potential antimicrobial agents against gram positive bacteria, gram negative bacteria and fungi.

Experimental
All the chemicals used in the synthesis were of laboratory grade.Melting points were determined in open capillary on Veego melting point electronic apparatus (VMP-D) and are uncorrected.The IR spectra of the synthesized compounds were recorded on Shimadzu 8400-S-FTIR 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 parts 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, 6 x 2.5 cm) using toluene-methanol and chloroform-ethyl acetate as appropriate solvent systems.The spots were visualized under ultra-violet light or by exposure to iodine vapors.The target compounds were synthesized as per the following scheme: (2) 3(a-g)
The physical and the spectral data are presented in Table1.

Zone of inhibition (Cup-plate method)
All the target compounds 5(a-g) were tested for their antimicrobial activity by agar cup plate method 10 .The organisms used for antibacterial activity were gram positive bacteria: S.

Minimum inhibitory concentration
The minimum inhibitory concentration (MIC) against the above mentioned organisms was determined by the broth dilution method 11 .The results are given in Table 6.

Results and Discussion
4-Hydroxycoumarin (1) 12 , 4-chloro-2-oxo-2H-chromene-3-carbaldehyde (2) 13 and 1-(4aminophenyl)-3-(substituted phenyl) prop-2-en-1-ones 3(a-g), 14 and 15 were synthesized by using reported procedures.The physical and the spectral values were found to match with the reported values.All the synthesized compounds 4(a-g) were solids melting at around the range of 122-151 0 C (Table 1) and freely soluble in acetone and chloroform.The solid state IR (KBr) spectra of these compounds reveal a characteristic aromatic stretch between 3087-2950 cm -1 .The NH group present in the pyrazoline ring revealed peaks at around the range 3400-3446 cm -1 .The C=N group present in the pyrazoline ring peaks were observed at around the range 1510-1604 cm -1 .The stretching vibrations for free amino group are seen between 3319-3390 cm -1 (Table 1).The 1 H NMR spectrum of the compound (4b) was recorded in CDCl 3 .The compound (4b) revealed doublet at around 4.87-4.84ppm for pyrazoline 5-H and singlet for NH at 5.81 ppm.The pyrazoline 4-CH 2 revealed a doublet of doublet at around 3.46-2.95ppm.The singlet for NH 2 was observed at 3.82 ppm .The aromatic protons showed peaks at 7.30-7.49& 6.68-6.5 ppm as multiplets (Table 1).All the target compounds 5(a-g) were solids melting at around the range of 208-241 0 C and freely soluble in acetone and chloroform.The solid state IR (KBr,cm -1 ) spectra of these compounds reveal a characteristic aromatic stretch between 2876-3076 cm -1 and sharp carbonyl stretching vibration for coumarin lactone at 1624-1662cm -1 (Table 2).The -CH=N stretch of was observed at 1560-1658 cm -1 .No peak was observed for free amino group observed in the spectra of 4(a-g) at around 3300-3200 cm -1 indicating incorporation of this group into the Schiff's base.
All the target compounds have shown good antimicrobial activity (

Table 3 .
Zone of inhibition against Gram positive bacteria:

Table 4 .
Zone of inhibition against Gram negative bacteria:

Table 6 .
Minimum inhibitory concentration for Gram positive & Gram negative bacteria:- Table no.3, 4, 5 and 6) as compared to standard and compound 5b exhibiting highest zone of inhibition and least MIC (20 μg/10μl)) against all the organisms.