Synthesis and Biological Evaluation of Mannich Bases of Isoxazoline Derivatives as Novel Anti-Microbial Agents

A novel series of compounds were synthesized by condensation reaction of substituted acetophenone (1a-b) with substituted aldehyde (2a-b) in presence of alcoholic sodium hydroxide to get intermediate chalcones (3ac), which were further treated with hydroxylamine hydrochloride in presence of sodium hydroxide to get isoxazolines derivatives (4a-c). The latter were refluxed with substituted primary amines and formaldehyde for 6-10 h to afford Mannich bases. The synthesized compounds were characterized on the basis of their spectral (IR, HNMR) data and evaluated for the antimicrobial activity by using Zone of Inhibition by cup plate method and Minimum Inhibitory Concentration by broth dilution method. KeywordsChalcone, Isoxazoline, Mannich bases, Antimicrobial activity. Introduction Heterocyclic compounds have so far been synthesized mainly due to the wide range of biological activities 1 . Isoxazoline derivatives have been reported in the literature to possess antifungal 2 , antibacterial 3 , anticonvulsant 4 , anti-inflammatory 5 , antiviral 6 and analgesic activity 7 . Mannich bases of heterocyclic molecules have been grabbing the attention of the synthetic chemists for their wide range of biological activities ranging from antibacterial, antifungal, anticancer, antiparkinson to anticonvulsant and anti–HIV 8-10 . In the present work, chalcones (3a-c) were synthesized by the reacting of substituted acetophenones (1a-b) with substituted aldehydes (2a-b) in the presence of aqueous solution of sodium hydroxide and ethanol at room temperature by Claisen-Schmidt condensation method. The synthesized chalcones were reacted with hydroxylamine hydrochloride in presence of sodium hydroxide to obtain isoxazolines derivatives (4a-c) 11 . The synthesized isoxazolines were reacted with formaldehyde and primary amines to afford Mannich bases (5a-l). Synthesis and Biological Evaluation of Mannich Bases of Isoxazoline Derivatives 1761


Introduction
Heterocyclic compounds have so far been synthesized mainly due to the wide range of biological activities 1 .Isoxazoline derivatives have been reported in the literature to possess antifungal 2 , antibacterial 3 , anticonvulsant 4 , anti-inflammatory 5 , antiviral 6 and analgesic activity 7 .Mannich bases of heterocyclic molecules have been grabbing the attention of the synthetic chemists for their wide range of biological activities ranging from antibacterial, antifungal, anticancer, antiparkinson to anticonvulsant and anti-HIV [8][9][10] .In the present work, chalcones (3a-c) were synthesized by the reacting of substituted acetophenones (1a-b) with substituted aldehydes (2a-b) in the presence of aqueous solution of sodium hydroxide and ethanol at room temperature by Claisen-Schmidt condensation method.The synthesized chalcones were reacted with hydroxylamine hydrochloride in presence of sodium hydroxide to obtain isoxazolines derivatives (4a-c) 11 .The synthesized isoxazolines were reacted with formaldehyde and primary amines to afford Mannich bases (5a-l).

Experimental
All the chemicals used in the synthesis were of laboratory grade.Melting points were determined in open capillary on Veego (model: VMP-D) electronic apparatus and are uncorrected.The IR spectra of the synthesized compounds were recorded on Shimadzu 8400-S FT-IR Spectrophotometer using potassium bromide or chloroform.The 1 H NMR spectra were recorded in CDCl 3 or DMSO using NMR Varian-Mercury 300 MHz spectrometer and chemical shifts are given in units as per million, downfield from Tetramethylsilane (TMS) as an internal standard.To monitor the reactions, as well as, to establish the identity and purity of reactants and products, thin layer chromatography was performed on microscopic slides (2 x 7.5 cm) coated with silica gel-G, using appropriate solvent systems and the spots were visualized under ultra-violet light or by exposure to iodine vapors.
To this solution appropriately substituted benzaldehyde (0.01mol) was added and stirred for about 12 h.The precipitated product was filtered, dried and recrystalized from ethanol.

Antibacterial Activity
Various Mannich bases derivatives were synthesized and evaluated for the antimicrobial activity by the cup plate method, against three strains of gram positive bacteria and gram negative bacteria and at three concentrations using DMSO as solvent in a nutrient broth as nutrient medium.Antibacterial activity of test compound was evaluated against gram positive bacteria S.aureus (ATCC 9144), B. subtilis (ATCC 6633) and S.epidermis (ATCC12228) and gram negative bacteria E. coli (ATCC 25922), P. aeruginosa (ATCC 9027), S. typhi (ATCC 13044), using Amoxicillin as standard by cup plate method.The bacteria were subcultured in the nutrient agar medium containing peptone (0.6%), yeast extract (0.3%), beef extract (0.13%) and agar (2.1%) in distilled water.The solution was sterilized for 20 min at 15 psi pressure in an autoclave at 121 0 C. The basal medium 15-20 ml was poured in the sterile petridishes.After the solidification of medium, holes of 6 mm diameter were bored to form cups with the help of a sterile cork borer.To this cup 50 μg/mL, 100 μg/mL, 150 μg/mL of the test compounds were added by the micropipette.The petridishes were kept in a cold room to facilitate the diffusion of the solvent for about 2 h.The extent of inhibition was measured by the width of the inhibition zone in mm.

Antifungal Activity
Fungicidal activity of the test compound was evaluated against A. Niger (ATCC 16404) and A.flavas (ATCC 9643) by cup plate method.The fungi were subcultured in the medium containing potato infusion from 200 gm (4 gm), dextrose (20 gm) and agar (15 gm) in distilled water.The pH of the medium is adjusted to 5.6 ± 0.2 and sterilized for 20 min at 121 0 C. The method of testing the antifungal activity is same as that adopted for evaluating antibacterial activity.Fluconazole was used as the standard; DMSO was used as solvent control and potao-dextose-agar as nutrient medium.

Minimum Inhibitory Concentration
The Minimum inhibitory concentration study is done by broth dilution method as follows.

Broth Dilution Method
Prepare the nutrient broth test tubes.In the first tube, inoculum is not added which is used for checking the sterility of medium and as a negative control.In other all test tubes inoculum is added.In all test tubes, test antimicrobial compound is added ranging from 15 to 50μg/10μL except uninoculated (negative control) and control (positive tube).The positive control tube is used to check the suitability of the medium for the growth of test microorganism and the viability of the inoculum.Adjust the final volume in all the test tube by using sterile water.All test tubes are properly shaken and then inoculated at 37 0 C for 24 h.

Conclusion
The approach of the present study was to synthesize various mannich bases and evaluate the antibacterial and antifungal activities.From result generated it can be concluded that test compounds 5b, 5h, 5i, 5j and 5l were found to possess moderate antibacterial activity against gram positive bacteria and gram negative bacteria compared with Amoxicillin.The observed antimicrobial and antifungal activities are attributed to the substitution of 4-chloro, 4-methyl group at 4 position of benzenamine ring of synthesized compounds.These results suggest that the Mannich bases of appropriately substituted isoxazolines have good potential for further development as antimicrobial agents.Further study is required to predict mechanism of action.The data reported in this article may be helpful guide for the medicinal chemist who is working in this area.

Table 5 .
Data of mean of Zone of inhibition of Gram positive Bacteria.

Table 6 .
Data of mean of Zone of inhibition of Gram negative Bacteria.

Table 7 .
Data of mean of Zone of inhibition of fungi.

Table 8 .
Data of minimum inhibitory concentration (μg/mL) of Gram positive bacteria

Table 9 .
Data of minimum inhibitory concentration (μg/ml) of Gram negative bacteria.