Synthesis and Antibacterial Activity of Some New Phenothiazine Derivatives

A series of some new phenothiazine derivatives were synthesized with the objective for evaluation as antimicrobials. The title compounds were prepared by a five step synthesis scheme. 2-Amino-6-substituted benzothiazoles (1) on diazotization afford 6-substituted benzothiazolyl-2-diazonium chlorides (2). Reaction of 2 with cold solution of -naphthol in dilute NaOH furnishes -(2diazo-6-substituted benzothiazolyl)-sodionaphthoxides (3) which on acidification with concentrated HCl gives -(2-diazo-6-substituted benzothiazolyl)-naphthols (4). Reaction of 4 with p-substituted anilines gives (2-diazo-6-substituted benzothiazolyl)-(p-substituted anilino) naphthalenes (5). This synthesis besides by using conventional methods was also attempted using microwave. Fusion of 5 with sulphur in presence of iodine results in -(2-diazo-6substituted benzothiazolyl)-6substituted [2, 3-b] benzophenothiazines(6). The structures of all these compounds have been supported by elemental analysis and their spectral studies. All synthesized compounds were tested for their antibacterial activity using standard drugs.


Introduction
Phenothiazine derivatives constitutes an important class of thiazines hetrocyclic ring system and possess potent biological activities as neuroleptics 1 , tranquilizers 2 , analgesic 3 , antimalarian 4 , anticancer 5 , CNS-activity 6 , antiviral 7 and antibacrerials 8 .Furthermore a wide spectrums of biological activities including antibacterial 9 , antitumor 10 , antituberculotic 11 and insecticides 12 have been reported in different benzothiazole derivatives.Benzothiazoles have also shown significant effects against cancer 13 .Similarly diazo compounds also shows a biological activity such as such as antibacterial 14 , antiviral 15 , antifungal 16 , In view of the activities exhibit by benzothiazoles, diazo compounds and phenothiazines, we have taken up the environmentally benign and economic synthesis of some new phenothiazine derivatives.Microwave mediated reaction have emerged as a powerful technique to promote a variety of chemical reactions.The growing number of publications in micro-wave-assisted synthesis includes virtually all type of chemical reactions such as additions, cycloadditions, substitutions, eliminations and fragmentations etc 17,18 .Applications of microwave methodology in Heterocyclic chemistry in terms of enhancements in the rate of reaction and in yields are striking 19,20 .

Experimental
All the melting points are uncorrected.The purity of synthesized compounds has been checked by thin layer chromatography.IR spectra are recorded on FT-IR Perkin-Elmer (Spectrum RX1) spectrophotometer (ν max in cm -1 ) using KBr disc. 1 H NMR spectra are recorded in CDCl 3 on a Bruker DRX-300 MHz using TMS as internal standard.The chemical shifts are reported as parts per million (ppm).Microwave synthesis was carried out in a domestic microwave oven model L.G. MS-194W, 230-50 Hz., 800W.

Synthesis of 6-substituted benzothiazolyl-2-diazonium chlorides (2)
A solution of compound 1 (0.001mole) in 5N HCl (20ml) was cooled to 0 0 C. To this solution was added a cold solution of sodium nitrite (1.0gm) drop wise with constant stirring.When the addition was complete, the resultant reaction mixture was left in ice-chest for 1hr.It was used as such for further reaction.

Synthesis of -(2-diazo-6-substituted benzothiazolyl)--sodionaphthoxides (3)
To the ice cold solution of compound 2, a cold solution of -naphthol(0.05mole) in dilute NaOH was added drop wise with constant shaking.A dark red dye resulted which darkened on adding more alkaline solution of -naphthol.When the addition was complete, the resultant reaction mixture was vigorously stirred and filtered off.It was dried and used for further reaction as such.

Synthesis of -(2-diazo-6-substituted benzothiazolyl)--naphtholes (4)
A saturated solution of compound 3 (0.002mole) in water was neutralized with concentrate HCl.A solid separated out which was allowed to stand at room temperature for 30min.It was filtered off and washed with water.The compounds thus prepared, were recrystallised from redistilled ethanol.

Synthesis of -(2-diazo-6-substituted benzothiazolyl)--(p-substituted anilino) naphthalenes (5)
A mixture of compound 4 and p-substituted aniline (equimoler amount) containing anhydrous ZnCl 2 (1gm) in absolute ethanol (50ml) was heated under reflux for 5hr on a steam bath.The solvent was distilled off and the residual solid was washed with water.It was dried in vacuo and recrystallised from methanol.

Microwave synthesis of -(2-diazo-6-substituted benzothiazolyl)--(p-substituted anilino) naphthalenes (5)
A mixture of compound 4 and p-substituted aniline (equimolar amount) in minimum quantity of anhydrous ethanol were taken in RB flask, which was placed in microwave oven and a reflux condenser was attached.The contents were subjected to microwave irradiation.The reaction was completed in 1.5 -2 minutes (monitored with T.L.C.).The solid obtained washed with distill water, dried in vaccuo and recrystallised from ethanol.

Synthesis of -(2-diazo-6-substituted benzothiazolyl)-6-substituted [2, 3-b] benzophenothiazines(6)
Heating of mixture of compound 5 (0.001mole), sulphur (0.002mole) and iodine (1% weight of reaction mixture) in an oil-bath for 2hr afforded a dark green solid which was cooled and washed repeatedly with water.It was dried and recrystallised from benzene as light green crystalline mass.Characterization data of all the synthesized compounds are summarized in Table 1.

Antibacterial activity
All the synthesized compounds (5a-6d) were tested against gram positive bacteria S. aureus and gram negative bacteria E.coli, Pseudomonas aeruginosa and Klebsiella species using paper disc method.The zone of inhibition was measured in mm.The standard drugs used were streptomycine and ceftazidime.The compounds were tested at 200 g/ml concentration.The observations show that compound 6a, 6c 2 , 6c 1 were found more effective against E.coli, Klebsiella species and S. aureus respectively as compared to streptomycine and the compound 6c 2 , 6c 2, 5c 2 more effective against E.coli, Klebsiella species and Pseudomonas aeruginosa respectively as compared to ceftazidime.The results of activity summarized in Table 2.

Conclusions
During our synthesis, we have used microwave methodology for the synthesis of -(2-diazo-6-substituted benzothiazolyl)--(p-substituted anilino) naphthalenes.Microwave-assisted organic synthesis have fascinated the chemist due to its useful ness with reduction of reaction time, environmental friendly methodology etc. Compound (6c 2 ) was effective against E.Coli, Klebsiella, compound (5c 2 ) was effective against Pseudomonas aeruginosa and compound (6c 1 ) was effective against S. aureus.

Table 2 .
The zone of inhibition in mm of the compound as well as standard drugs tested for antibacterial activity.