Synthesis , Antimicrobial and Antitubercular Activities of Some Novel Carboxamide Derivatives of 2-quinolones

A series of novel substituted N-(3-acetyl-2-oxoquinolin-1(2H)-yl)benzamide (AJQC1AJQC12) have been synthesized upon refluxing 3-acetyl-1-amino-quinolin-2-one and substituted benzoic acid in the presence of dry redistilled pyridine and silicon tetrachloride as coupling agent. 3-acetyl-1-amino-quinolin-2-one (AJQ1-AJQ12) were synthesized from substituted 3-acetyl coumarin upon refluxing with hydrazine hydrate and ethanol. The structures of the final carboxamide derivatives were confirmed by IR, 1H NMR and mass spectra. The synthesized compounds were screened for their antimicrobial activity by tube dilution method and antitubercular activity by microplate Alamar blue assay. Most of the compounds have exhibited promising antibacterial, antifungal and antitubercular activities.


INTRODUCTION
2-Quinolones (carbostyrils or 1-aza coumarins) are isosteric with coumarins and isomeric to 4-quinolones could become the probable potential candidate for antibacterial activity 1 .2-Quinolone derivatives were found to be associated with various biological activities such as antitumor 2 , anti-inflammatory 3 , antiplatelet, antitubercular 4 , antioxidant 5 and antidepressant activity.Pyrazinamide is a nicotinamide analogue that has been used as a first-line drug to treat tuberculosis 6 .Various compounds possessing the -NHCO-group were found to inhibit photosynthetic electron transport 7 .Amides are usually stable, neutral and have both hydrogen-bond acceptor/ donator properties which are very important for the synthesis of versatile heteroaromatic molecules.The amide functionality is the common backbone of numerous organic molecules and natural products that bear diverse chemical and pharmacological features 8 .A series of quinoline carboxamide derivatives have also been evaluated for positive inotropic activity 9 , as potential radio ligands in the neurodegeneration systems 10 and antiviral against acyclovir resistant herpes simplex virus 11 .By considering the above potent pharmacological properties of these quinoline ring containing compounds, it was contemplated to synthesize some novel 2-quinolone carboxamide derivatives.The final synthesized compounds were evaluated for their in vitro antimicrobial and antitubercular activities and compared with standard drugs.

MATERIAL AND METHODS
All the chemicals were of analytical grade: substituted salicylaldehyde, ethylacetoacetate, absolute ethanol, piperidine, hydrazine hydrate, substituted benzoic acid, silicon tetrachloride and dry redistilled pyridine.Melting points were determined by open capillary method and are uncorrected.The purity of the compounds was monitored by thin layer chromatography (TLC) using silica gel G plates.The spots were visualized under UV light and by the exposure to iodine vapors.The homogeneity of the compounds were checked on silica gel-G coated plate by using Chloroform: Methanol (7:3) as solvent.All IR spectra were recorded in Alpha Bruker using ATR method. 1 H NMR spectra were recorded on Bruker spectrophotometer (400 MHz) in DMSO-d 6 solvent using tetra methyl silane (TMS) as an internal standard.Mass spectra was recorded by LCMS method.

Antimicrobial Activity
All the synthesized compounds were evaluated for their minimum inhibitory concentration by tube dilution method (14).The synthesized test compounds were tested at different concentrations and ciprofloxacin and fluconazole was used as standard.Serial dilutions of the test compound was made in a liquid medium which was inoculated with a standardized number of organisms and incubated for 24 hrs.The lowest concentration of test compound preventing appearance of turbidity is considered to be the minimal inhibitory concentration (MIC).After preparation of different concentrations of the antimicrobial agent in brain heart infusion broth (by using the broth dilution method), we inoculate them with the tested organism.Then after incubation we can determine the MIC by choosing the lowest concentration in which no growth occurs.

Antitubercular activity
The antitubercular activity of test compounds were assessed against Mycobacterium tuberculosis using microplate Alamar blue assay 15 .This methodology is non toxic, uses a thermally stable reagent and shows good correlation with proportional and BACTEC radiometric method.200 µl of sterile deionized water was added to all outer perimeter wells of sterile 96 well plate to minimised evaporation of medium in the test wells during incubation.The 96 well plate received 100 µl of the Middlebrook 7H9 broth and serial dilution of compounds were made directly on plate.The final drug concentrations of the tested compounds were 0.01 to 20.0 µl/ml.The plates were covered and sealed with parafilm and incubated at 37 ¿ C for 5 days.After this, 25 µl of freshly prepared 1:1 mixture of Alamar blue reagent and 10% tween 80 was added to the plate and incubated for 24 hours.A blue color in the well was interpreted as no bacterial growth and pink color was interpreted as growth.The minimum inhibitory concentration was defined as lowest drug concentration which prevented the color change from blue to pink.

Antitubercular Activity
The test compounds (AJQC1-AJQC12) were evaluated for their antitubercular activity against M.tuberculosis using Microplate Alamar Blue assay.A blue colour in the well was interpreted AJPY12 showed promising antitubercular activity compared to the standard drug streptomycin and isoniazid.The presence of 2-quinolone moiety with electron donating groups like methyl, hydroxy, amino and dimethylamino has accounted for their antitubercular activity.The results of the antitubercular activity are summarized in Table 2.

CONCLUSION
This study reports the successful synthesis of carboxamide derivatives of 2-Quinolone moiety with moderate yields and most of the synthesized compounds showed potent antimicrobial and antitubercular activity.