Synthesis, Characterization and Evaluation of Anti- tubercular Activity of Ofloxacin Chalcone Conjugates

Synthesis and characterization of Ofloxacin Chalcone derivatives were carried out and evaluated for their antitubercular activity. Synthesized Ofloxacin Chalcone derivatives (Compound A to F). These molecules are characterized by analytical methods (TLC), melting point, and Spectroscopic methods (FT-IR, Mass, NMR). The evaluated antitubercular activity of molecules of synthesized compounds and Ciprofloxacin Pyrazinamide, and Streptomycin as standard drugs. concentrations of synthesized derivatives were prepared (0.8, 1.6, 3.12, 6.25, 12.5, 25, 50, 100 μg/ml) by using sterile deionized water as a solvent. Antitubercular Assay was Performed by Mycobacterium tuberculosis using the MABA method. Analyzed MIC values. Compound E showed the best results among 6 Compounds at a very low concentration at 3.12 μg/ml.


INTRODUCTION
The fluoroquinolones are very important antimicrobial agents. the present focus on Ofloxacin derivatives. The structure bears a fluorine group at the C-6 position and acid group at C-3 and ketone group at the C-4 position which is crucial for activity. these quinolones can be synthesized by laboratory synthesis method, research studies found that quinolones have appreciable concentration and their MIC (minimum inhibitory concentration) are good in biological tissues. The fluoroquinolones have been analyzed by various methods which have been described in different kinds of works of literature. This research becomes needful because of rapid progress and development in fluoroquinolones. [1,2].
The boost in fluoroquinolones prescribing was attributable to the introduction and use of newer, against S. Pneumoniae (for example, levofloxacin, Gemifloxacin, and moxifloxacin). Fluoroquinolones have a very useful role in many bacterial infections, Ofloxacin is a broadspectrum antibiotic that belongs to the secondgeneration of fluoroquinolones. It inhibits DNA replication repair of bacterial DNA and RNA transcription of gram-negative and gram-positive cocci acting open DNA topoisomerase enzyme which eventually leads to blockage cell growth. Advances in the quinolone field are likely to provide better compounds capable of dealing with resistant strains. [3][4][5].
Chalcones are important biological compounds that form enone (aromatic ketones), which are collectively called chalcones. Which two aromatic rings are conjugated together through a reactive α-β unsaturated carbonyl system. Chalcones are considered as precursors in many flavonoids, a very large group of plant constituents, and have defense strategies such as anti-microbial. Antifungal, anti-oxidants potential healing of diverse diseases. It has anti-bacterial, anti-inflammatory, analgesic, and other activities [6].
A five or six-membered rings are employed as a substitution at the C-7 position example trovafloxacin having amino pyrrolidine substituent C-7. Fluoroquinolones with piperazinyl moiety at the C-7 position have been reported to possess potent antibacterial activity. Mechanism of action and structural activity relationship studies of quinolones reveals that the site near the C-7 substituent regards as the drug enzyme interaction domain. besides, it is also reported that the cell permeability is dominantly by C-7 substituents. These facts motivated our concerns to develop some C-7 substituted analogs of the quinolone. [7][8][9][10] A wide range of drugs used as fluoroquinolones antibacterial agents, namely norfloxacin, ciprofloxacin. ofloxacin levofloxacin. Levofloxacin and ofloxacin are isomers and chiral versions of the drugs. The main targets identified for levofloxacin activity, topoisomerase II (DNAgyrase inhibitor) in Gram-negative bacteria, and topoisomerase IV inhibitor in Gram-positive bacteria [11][12][13].
Ofloxacin is pale yellow or bright yellow crystalline powder, and with a bitter taste forms colourless needles with spirit or ethanol. Ofloxacin should be stored at 4 o C in the dark to minimize photolytically induced. Mycobacterium tuberculosis is the cause of TB infection, it affects the lungs and other body parts, which is spread through inhalation, of aerosolized droplets. Tuberculosis is highly transmittable during the active stage of the disease and can contaminate an individual through a breath of few as mycobacterium tuberculosis (MTB). After a breath, these bacteria are mainly taken by the alveolar macrophages, but they can escape the host resistant system for a long time at which point they can re-energize to an infectious form under immune-compromised situations of the host. [14][15][16][17].
Tuberculosis can be treated efficiently by using the first-line drug (FLD) Rifampicin (RIF), Pyrazinamide (PZA), Isoniazid (INH), and Streptomycin (SM), Ethambutol (EMB) The firstline therapy often fails to cure TB for several reasons. Replacement and the spread of the disease contribute to the emergence of drugresistant bacteria. The emergence of multidrugresistant TB (MDR-TB), requires the use of second-line drugs that are difficult to procure and are much more toxic and expensive than FLDs. [18][19][20].
Our present focus is to synthesize ofloxacin chalcone derivatives. Both have an antimicrobial, anti-bacterial activity which might be useful for the treatment of MDR-TB.

METHODS
The Blanc chloromethylation also called blanc reaction is the chemical reaction of aromatic rings with HCHO and HCl to form chloromethyl arenes. In 1923 Gustave Louis Blanc discovered the reaction. When the concentration is high, the formation of side products due to the second addition is observed.

PROCEDURE
A compound of Ofloxacin 0.01 M and 0.01 M of compounds (1-Naphthol, 4-Amino Phenol) were transferred to separate beakers in 5ml of Ethanol and 5% Sodium Hydroxide respectively, and both were mixed stirred on a magnetic stirrer at 70 0 c.To the above solution, 35 parts formaldehyde solution and 35% concentrated HCl was added dropwise. The reaction mixture was stirred for 4 hours at 70 0 c using a magnetic stirrer. The resultant mixture was cooled and basified with the addition of NH 3 solution. The solid was separated by filtration and dried. The above Blanc product was treated with equimolar quantities of 0.01 M of Pyridine and Acetyl Chloride to produce an Acetylated product. The acetylated product was collected and dried. The acetylated product was treated with an equimolar quantity of 0.01 M Benzaldehyde and it was stirred in an ice bath for 1 hour. After 1 hour 10% Sodium Hydroxide was added as a catalyst and stirring was further continued for 3 hours. Then the Ofloxacin Chalcone product was collected by filtration, dried, recrystallized by using ethanol.

Experimental Investigation
All the compounds were determined by melting point apparatus and Infrared spectra of the compounds were recorded on FT-IR Spectrophotometer, Thin layer chromatography using silica gel plates (Merck) monitored the progress of each reaction in the present examination. The chemicals used are obtained from Sd fine chemicals limited-Mumbai and are of Analytical reagent grade.

RESULTS
The necessary Ofloxacin Chalcone derivatives are synthesized from commercially available reagents and are mentioned in the scheme. The title compounds of ofloxacin chalcone were formed via Blanc reaction then acetylation with acetyl chloride and chalcone formation in presence of benzaldehyde, the structures of all compounds confirmed by IR, NMR. MASS spectral data. The procedure is promising to yield good title derivatives, the IR spectrum is recorded gives the presence of various functional groups. 1 H NMR shows multiple signals to the resonance of ofloxacin chalcone derivatives, Mass spectrum is characterized by M+1 peak, All synthesized compounds characterized by Physicochemical properties like molecular weight, melting point, Rf values then solubility tested using different solvents, all compounds dissolved in DMSO, Insoluble in chloroform and water, mixed solubility result with ethyl acetate and spirit.

Evaluation of Anti-Tubercular Activity
Anti-TB activity using Alamar Blue Dye

Procedure
The anti -Mycobacterial activity of compounds was evaluated using the microplate Alamar Blue assay (MABA method). It is a non-toxic methodology, uses a thermally stable reagent, and shows good correlation with a comparative and radiometric method. sterile deionized water was added to all outer border wells of sterile 96 wells plate to lessen the evaporation of medium in the test wells during the incubation process. The 96 wells plate received 100 µl of the broth (Middlebrook 7H9) and sequential dilution of compounds were made directly on a plate. The ultimate drug concentrations tested were 100 to 0.2 µg/ml. Plates covered, sealed with parafilm at 37ºC five days incubated. 25 µl of freshly arranged 1:1 mixture of Alamar Blue reagent and 10% tween 80 was added to the plate and incubated for 24 hrs. the well was interpreted as no bacterial growth with blue color, and the well shows growth with red color. The MIC was welldefined as the lowest drug concentration which prevented the color change from blue to pink. [21,22]

CONCLUSION
Ofloxacin Chalcone derivatives were synthesized and investigated for their antitubercular activity by the MABA method. comparing the antitubercular activity of Ofloxacin Chalcone derivatives their MIC values were significant. The beneficial effects of these drugs reveal that the results thus hold a great promise for the use of ofloxacin derivatives as potential future antitubercular drugs. Further synthetic work is extended on semi-synthetic derivatives of ofloxacin.