Synthesis and Biological Evaluation of 2-( 3-Methyl-2-oxoquinoxalin-1 ( 2 H )-yl )-N '-( substituted phenyl-methyledene / ethylidene ) acetohydrazides

A series of quinoxaline derivatives was prepared and evaluated for antitubercular, antibacterial and antifungal activities. The title compounds were prepared by condensation of substituted aromatic aldehydes and substituted acetophenones with 2-(3-methyl-2-oxoquinoxalin-1(2H)-yl) acetohydrazide. Structures of all these compounds were confirmed by their spectral studies. Among synthesized compounds (4r, 4t, 4u, 4w and 4x) have shown good anti tubercular activity (25 μg mL) when compared to reference drugs pyrazinamide (10 μg mL) and streptomycin (7.5 μg mL). In this study, few derivatives showed broad spectrum of antimicrobial activity at low concentration. The MICs (Minimum inhibitory concentration) of some compounds are 2-4 μg mL.


Introduction
Tuberculosis (TB) is still the greatest single infectious cause of mortality worldwide.However, powerful new anti-TB drugs with new mechanisms of action have not been developed in the last over thirty years 1,2 .It is expected that development of the new effective anti-TB drug will bring us various outcomes such as shortening the total duration of treatment, improvement of the treatment completion ratio, prevention and treatment of the multiple drug resistant tuberculosis (MDR-TB) and reducing the total medical expenditure.
A new anti-TB drug needs to show the well pharmacokinetic distribution and permeation into lung tissue and cells.Furthermore, it is also desired that the novel candidate exhibits the potent bactericidal activity both against exponential and stable phase of M. tuberculosis in vivo.In addition, it is ideal that the novel agent possesses narrow anti-microbial spectrum specialized only against mycobacterial species 3,4 .Schiff bases are also known for their antibacterial and antitubercular activity [5][6][7] .Quinoxalines constitutes an important class of compounds; some analogs are synthesized and evaluated for antimicrobial activity and many posses diverse biological activity such as insecticidal, fungicidal, herbicidal and anthelmintic 8 .

Experimental
Melting points were determined in open capillaries on a Thermonic melting point apparatus and were uncorrected.Infra-red spectra were recorded on Fourier Transform IR spectrophotometer (Shimadzu FT-IR 8700) using in KBr disc method (ν max in cm -1 ). 1 H NMR spectra were recorded in CDCl 3 or DMSO on Bruker-200 NMR spectrophotometer using TMS as internal reference standard (chemical shifts in δ ppm); and LCMS recorded on Shimadzu 2010A spectrophotometer.

Antimicrobial activity
All the synthesized compounds were tested for antimicrobial activity by tube dilution method [9][10][11] .Four bacteria, two fungal and a Mycobacterium strains were used as the antimicrobial test strains namely: Staphylococcus aureus ATCC 12598, Enterococcus fecalis ATCC 35550, Escherichia coli ATCC 25922, Klebsiella pneumonia ATCC 29665, Candida albicans ATCC 2091, Aspergillus fumigates ATCC 13073 and Mycobacterium tuberculosis H 37 Rv.Ciprofloxacin and fluconazole were used as standard drugs against bacteria and fungi respectively.Pyrazinamide and Streptomycin were used as standard drugs against Mycobacterium.In all determinations tests were performed to find out minimum inhibitory concentration (MIC) at two fold concentration from 1 µg mL -1 -500 µg mL -1 .Results are presented in Table 1.Table 1.Antitubercular, antibacterial and antifungal activities of compounds (4k-4q & 4s-4x) (MIC in µg mL -1 )

General procedure for synthesis of compounds (4k-4q & 4s-4x)
A solution of substituted aromatic aldehydes or acetophenones in DMF was added drop wise to a solution of compound (3) in DMF.The mixture was refluxed for 3-5 h in presence of catalytic amount of glacial acetic acid, cooled and poured onto water containing crushed ice.The precipitate thus obtained was filtered, washed with cold water and recrystalised from alcohol / DMSO-water mixture to obtain (4k-4q & 4s-4x).Results are tabulated in Table 2.

Results and Discussion
It was observed that a strong nucleophile can attack directly, without help from an acid catalyst.For a weak nucleophile such as amine (-NH 2 ) an acid catalyst is needed so that the carbonyl carbon is prepared to share a pair of electrons as a new covalent bond.The experiments proved that the use of catalytic amount of acid not only fasten the reaction but also improves the product yield.

Antimicrobial activity
Antimicrobial activity of all the compounds was shown in Table 2.Among the series of synthesized compounds, 4r, 4t, 4u, 4w and 4x have shown considerable antitubercular activity at 25 µg mL -1 in comparison to pyrazinamide 10 µg mL -1 .The Schiff bases modified at N=CH-, the replacement of H with CH 3 has resulted in better activity.The electron releasing nature of CH 3 may also be accounted for this fact.Compounds 4r-4x have shown significant antifungal activity even at lower concentrations (1-4 µg mL -1 ) than standard drug fluconazole (8 µg mL -1 ).The substitutions on the aromatic ring influenced quantitatively, on biological activities.Compounds 4k (m-NO 2 ) and 4l (p-NO 2 ) contains nitro group which is known for its antibacterial activity.