Synthesis and Antimicrobial Activity of 1-[ 2-( 10p-Chlorobenzyl ) phenothiazinyl ]-3-( substituted aryl )-2-propen-1-ones

Aiming at the development of antimicrobial agents, we have synthesized nine chlorobenzyl substituted phenothiazinyl chalcones by condensing 2-acetyl phenothiazine with aldehyde derivatives in dilute ethanolic sodium hydroxide solution at room temperature according to Claisen Schmidt condensation and subsequent reaction of products with p-chlorobenzyl bromide. Structures of these compounds were elucidated by their IR, 'H NMR spectroscopic data and microonalyses. The antimicrobial activity of the new products was evaluated by Filter Paper Disc Method.


Introduction
Infectious diseases caused by micro and myco organisms, viz.bacteria, fungi, viruses and parasites are still a major threat to human health, despite tremendous inventions in drug chemistry.The emergence of wide spread drug resistance, particularly multi-drug resistance 1 against Gram-positive bacteria is a major concern.Among large number of organic compounds with varying structural units, chalcones, well known intermediates for synthesizing variety of heterocyclic compounds, have been found to possess diverse biological properties including antituberculosis 2 , antiplatelet and calcium channel blocking 3 , antifungal 4,5 , antiviral 6 , anti-leishmania 7,8 , anti-oxidant 9 , anti-inflammatory 10-12 and antitumoural 11-14 etc. Reactive α, βunsaturated keto moiety of chalcones plays a pivotal role in exhibiting antimicrobial properties.In the present communication we report synthesis and antimicrobial activity of chlorobenzyl substituted phenothiazinyl chalcones obtained by reaction of phenothiazine with different substituted aldehydes and subsequent reaction of the products with p-chlorobenzyl bromide.Structures of the new products have been assigned on the basis of their microanalyses, IR and 1 H NMR data.

Experimental
Melting points determined in open glass capillaries were uncorrected.Microanalyses were performed on Vario-el-III, Element R and were within the ±0.5% of theoretical values.Molecular weights were determined by Rast's method using camphor solvent.Infrared spectra were recorded in KBr on Thermo Nicolet Nexus FT-IR. 1 H NMR spectra were recorded in CDCl 3 on Bruker WM 400 MHz spectrometer.Purity/homogeneity of the compounds was tested by TLC using silica gel-G (Merck) and purification was done by repeated crystallization.
General procedure for the synthesis of p-chlorobenzyl substituted phenothiazinyl chalcones (1-9)   Two steps reactions were carried out to obtain title compounds.To an ice cold stirred solution of 2-acetyl phenothiazine (2.41 g, 0.0l mol) and benzadehyde derivatives (0.01 mol) in dry ethanol (25 mL) 1.0 mL 70% NaOH aqueous solution was added drop wise and the reaction mixture was further strirred for 6-8 h at room temperature.Solid separated was filtered, washed with water and crystallized from acetic acid Scheme 1.In the second step a mixture containing equimolar quantities (0.005 mol) of these products, p-chlorobenzyl bromide and NaH in DMF (25 mL) was stirred for 48 h.Solids precipitated on dilution of reaction mixture with water were filtered, washed with water and crystallized from acetonitrile.

Antimicrobial activity
Antimicrobial studies on newly synthesized chalcones (1-9) were performed using Filter Paper Disc Diffusion method against Enterococcus faecium, Enterococcus faecalis, Escherichia coli and Bacillus subtilis bacteria and Candida albicans and Aspergillus niger fungi.Whatmann filter paper-1 discs (6.5 mm) sterilized by dry heat at 1400 C were saturated with test solution and placed on the surface of a sterilized nutrient agar medium for bactericidal study and sabouraud dextrose agar and potato dextrose agar medium for Candida albicans and Aspergillus niger respectively for fungicidal study in petri dishes which were preinoculated with test organisms.All these petri dishes were incubated for 48h and inhibition zones were measured.Control solvent (tween-80-water, 1:9, v/v) was used as blank.Among the three concentrations used (5.0, 0.5, 0.05 µg µL -1 ) concentration of each test sample giving maximum inhibition is noted with zone dia in Table 3. Table 1.Physical and analyses data of compounds (1-9).
From Table 3 it is evident that although all the chalcones are active against all the four micro-organisms but compunds faecium and E. feacalis respectively.In order to study the effects of different substituted groups on inhibition zone dia of para substituted chalcones bearing minimum steric hindrance have been arranged in increasing order; electron-withdrawing groups form N(CH 3 ) 2 <OCH 3 < Br order whereas electron donor groups fall in F<Cl<NO 2 order aginst all the four microbes tested, opposite to the sequence in electronegativities of substituents of each series.This indicates increase in electronegativity of substituent group has adverse effect on the antimicrobial activity.However increase in number of methoxy substituents enhances antibacterial activity of their molecules.Moreover compound 9 and compounds 3, 8 & 9 which produce higher inhibition zones than reference drugs, chloramphenicol and benzyl penicillin, in comparatively less concentrations for E. Coli and B. subtilis could be of better use than standard drugs.None of the compounds found active against both fungi tested.