Synthesis and characterization of substituted bisbenzoxazine as potent antimicrobial agent

Bisbenoxazines derivatives were synthesized by refluxing substituted bisphenol-C, substituted aniline and formaldehyde in presence of 1,4-dioxane with good yields. All the synthesized compounds were characterized by mass, NMR and IR and also evaluated for antimicrobial activity against four different bacterial and two fungal strains. The compounds 1c, 1h, 1j and 1l has found comparatively good active against all the bacterial strains


INTRODUCTION
Benzoxazines are six-membered heterocyclic compounds synthesized via Mannich condensation reaction from phenols, amines, and formaldehyde. The first benzoxazine compound was synthesized by Holly and Cope in 1944 [1]. Later, the study on benzoxazines and their oligomers was further conducted from the 1950s to 1970s [2][3][4]. These benzoxazines were synthesized from monophenols, monoamines, and formaldehyde, but they could not form polymers with high molecular weight.
Therefore, benzoxazines can be used as high-performance matrices in electronics and aerospace industries. The work on microbial screening has not been found on bisbenzoxazine derivatives. The aim of this study is to investigate the biological activity of substituted bisbenzoxazine derivatives.

EXPERIMENTAL
All research chemicals were purchased from Allied Chemicals (Vadodara) and used as received. Thin-layer chromatography was accomplished on 0.2 mm percolated plates of silica gel G60 F254 (Merck). Visualization was made with UV light (254 and 365 nm). IR spectra were recorded on a FT-IR-8400 spectrophotometer using DRS prob. 1 H (400 MHz) and 13 C (400 MHz) NMR spectra were recorded on a Bruker AVANCE II spectrometer in CDCl 3 . Chemical shifts are expressed in δ ppm downfield from TMS as an internal standard. Mass spectra were determined using direct inlet probe on a GCMS-QP 2010 mass spectrometer (Shimadzu). Melting points were measured in open capillaries and are uncorrected.

1. General procedure for the synthesis of substituted bisbenzoxazine derivatives (2a-t)
0.1 mol substituted aniline in 20 ml 1,4-dioxane was slowly added to 0.2 mol formaldehyde in 80 ml, keeping temperature below 10 °C in an ice bath. The mixture was stirred for 10 min and 0.05 mol bisphenol in 50 ml 1,4-dioxane was added. Temperature of the mixture was raised and refluxed for 15h, cooled, poured in the crushed ice, separated yellowish solid filtered, washed well with water and dried at room temperature. Crude product was treated with 3N NaOH solution till unreacted bisphenol was removed completely by inteminent checking with a dilute HCl solution washed well with water and dried at 50 °C. Compounds were recrystallized repeatedly from appropriate solvent system to get pure product 1(a-l).

Biological testing
Bisphenol-C derivatives were screened for their invitro antibacterial and antifungal activities following micro broth dilution method [20][21][22]. Antibacterial activity was screened against gram-negative (Bacillussubtilis, Staphylococcus aureus) and gram-positive (Escherichia coli, Salmonella typhi) microorganisms. Antifungal activity was screened againstAspergillusniger, Aspergillusclavatusmicroorganisms. The standard drugs used for this study were gentamycin, ampicillin, chloramphenicol, ciprofloxacin and norfloxacin were used for antibacterial screening.
Nystatin and griseofulvin were used for antifungal screening. The standard strains used for screening of antibacterial and antifungal activities were procured from Institute of Microbial Technology (IMTECH), Chandigarh, India. Mueller Hinton Broth was used as a nutrient medium for bacteria and Sabouraud Dextrose Broth for fungal growth. Inoculums size for test strain was adjusted to 10 8 CFU/mL by comparing the turbidity. The results were recorded in the form of primary and secondary screening. The stock solution (2000 µg/mL) of the compounds under investigation and standard drugs were prepared by successive dilution. In primary screening, 1000, 500 and 250 µg/mL concentrations of the compounds were used. The compounds which found active in this primary screening were further screened. In secondary screening, 200, 100, 50, 25, 12.5 and 6.25 µg/mL concentrations were used. The inoculated wells were incubated overnight at 37 ºC in a humid atmosphere overnight. The highest dilution showing at least 99 % inhibition was considered as minimum inhibition concentration (MIC).
The investigation of antibacterial and antifungal screening data revealed that all the tested compounds 4a-t showed moderate to potent activity. The compounds 4i, 4k and 4l showed comparatively good activity against all the bacterial strains.

CONCLUSION
In summary, we have described the synthesis of substituted bisbenzoxazine derivatives in excellent yields by simple refluxing. The spectral data are incorporate with the structure of compounds 1a-1. The antimicrobial data reported here which may be the better reference for the future research in the class of pyrimidine derivatives.