Synthesis and Antimicrobial Study of New 8-bromo-1 , 3-diaryl-2 , 3-dihydro-1 H-naphtho [ 1 , 2 e ] [ 1 , 3 ] oxazines

A series of new 8-bromo-1,3-diaryl-2,3-dihydro-1H-naphth[1,2e][1,3]oxazines 2a-n are synthesized. 6-Bromonaphthol undergoes ring closure reaction with substituted aryl and heteroarylaldehydes to give naphthoxazine derivatives. Some of these are hydrolyzed to obtain aminobenzylnaphthols 3c,l which are further condensed with different aryl/heteroaldehydes to yield 4a-e. The structures of newly synthesized compounds are confirmed through elemental analysis, spectral studies and single crystal X-ray study. Compounds are screened for their antibacterial and antifungal activity. Some of them exhibited promising activity.


Experimental
TLC was run on a Merck silica gel 60 F254 coated aluminum plates and melting points were taken in open capillary tubes and are uncorrected.Elemental analysis was carried out using Flash EA 1112 Series, CHNSO Analyzer (Thermo).IR spectra in KBr pellets were recorded on Jasco FT/IR-4100 FTIR spectrophotometer. 1 H NMR spectra were recorded in CDCl 3 and in DMSO-d6 on a Bruker DRX-300 (300 MHz) spectrometer using TMS as internal standard and Mass spectra were recorded on a Jeol SX 102/Da-600 mass spectrometer/data system using Argon/Xenon (6kv,10mA) as FAB gas.

General procedure for the synthesis of 1-(aminosubstituted methyl)-2-naphthols 3c and 3l
2c or 2l (1 mmol) were suspended in 20 % HCl (20 mL) and the mixture was stirred and refluxed for 6 h, whereby the crystalline hydrochloride of 3c, 3l separated out.The product was filtered off and washed with EtOAc.The solid was suspended in H 2 O and the mixture was treated with conc.NH 4 OH (3 mL) and extracted with EtOAc.After drying (over Na 2 SO 4 ) and evaporation of the EtOAc phase, crude crystalline compounds were obtained, which were further purified by recrystallization.

General procedure for the synthesis of 8-bromo
To a solution of the appropriate aminonaphthol 3c or 3l (1 mmol) in absolute MeOH (20 mL), an equivalent amount of aryl-or heteroarylaldehyde was added, and the mixture was left to stand at ambient temperature for 48h.The crystalline product separated were filtered off and then recrystallized in methanol.

Antibacterial studies
The newly synthesized compounds were screened for their antibacterial activity against Escherichia coli (ATTC-25922), Staphylococcus aureus (ATTC-25923), Pseudomonas aeruginosa (ATCC-27853) and Klebsiella pneumoniae (recultured) bacterial strains by serial plate dilution method (Barry, 1991;James et al., 1991).Serial dilutions of the drug in Mueller Hinton broth were taken in tubes and their pH was adjusted to 5.0 using phosphate buffer.A standardized suspension of the test bacterium was inoculated and incubated for 16-18 h at 37°C.
A number of antimicrobial discs are placed on the agar for the sole purpose of producing zones of inhibition in the bacterial lawn.Twenty milliliters of agar media was poured into each Petri dish.Excess of suspension was decanted and plates were dried by placing in an incubator at 37°C for an hour.Using a punch, wells were made on these seeded agar plates and minimum inhibitory concentrations of the test compounds in dimethylsulfoxide (DMSO) were added into each labeled well.A control was also prepared for the plates in the same way using solvent DMSO.The Petri dishes were prepared in triplicate and maintained at 37°C for 3-4 days.Antibacterial activity was determined by measuring the diameter of inhibition zone.Activity of each compound was compared with ciprofloxacin as standard (Fenlon, & Cynamon, 1986).Zone of inhibition was determined for newly synthesized compounds at 10 μg/ml concentration and the results are presented in Table 4.

Antifungal studies
Newly prepared compounds were also screened for their antifungal activity against Aspergilus flavus (NCIM No.524), Aspergilus fumigates (NCIM No. 902), Penicillium (S.aurus) (recultured) and Trichophyton mentagrophytes (recultured) in DMSO by serial plate dilution method (Arthington-Skaggs et al., 2000;Verma et al., 1998).Sabourands agar media was prepared by dissolving peptone (1 g), D-glucose (4 g) and agar (2 g) in distilled water (100 ml) and adjusting the pH to 5.7.Normal saline was used to make a suspension of spore of fungal strains for lawning.A loopful of particular fungal strain was transferred to 3 ml saline to get a suspension of corresponding species.Twenty milliliters of agar media was poured into each Petri dish.Excess of suspension was decanted and plates were dried by placing in incubator at 37°C for 1 h.Using a punch, wells were made on these seeded agar plates minimum inhibitory concentrations of the test compounds in DMSO were added into each labeled well.A control was also prepared for the plates in the same way using solvent DMSO.The Petri dishes were prepared in triplicate and maintained at 37°C for 3-4 days.Antifungal activity was determined by measuring the diameter of inhibition zone at 10 μg/ml concentration.Activity of each compound was compared with Fluconazole as standard.Zone of inhibitions were determined and the results are given in Table 5.

Results and discussion
The reaction sequence employed for synthesis of the title compounds are shown in scheme 1 and scheme 2.
The naphthoxazine 2c and 2l were subjected to acid hydrolysis to get Betti bases (Betti, 1941).
Compounds were also screened for their antibacterial and antifungal activity.Almost all the compounds tested showed moderate to good activity against the bacterial and fungal strains.Compounds 2h, 2j, 2l and 4e showed promising results.The good activity could be attributed to the presence of phenyl ring substituted with fluoro, chloro and methyl groups attached to the oxazine ring.Replacing one of the aryl substituent with a different aryl/heteroaryl group showed increase in the activity of 4a, 4b, 4c, 4d and 4e.
In the antifungal activity study, compound 4e emerged with good activity against fungal strains, particularly against Aspergilus flavus.This may be due to the presence of 3-methylphenyl and 4-chlorophenyl groups attached to the naphthoxazine ring.

Conclusions
The present study reports the synthesis of series of new naphthoxazine derivative.Structure of compound 2h was elucidated through single crystal X-ray diffraction.The newly synthesized compounds were screened for their biological activity.Some of the new compounds found to exhibit good activity against tested bacterial and fungal strains.Compounds having fluoro, chloro and methyl substituted phenyl group attached to naphthoxazine showed promising activity.However it is a preliminary study and these newly synthesized compounds should be subjected to detailed pharmacological and toxicological evaluation for their application in clinical use.human immunodeficiency virus type 1 reverse transcriptase.Antimicrobial and Agents Chemotherapy, 39:2602-2605. Zhang, P., Terefenko, E. A., Fensome, A., Wrobel, J., Winneker, R., Zhang, Z. (2003).

Table 2 .
Substituents Ar and Ar' of compounds 4a-e

Table 3 .
Characterization data of compounds 4a-e

Table 4 .
Antibacterial activity of the compounds 2a-n and 4a-e at 10 µg/ml concentration.(Diameter of zone of inhibition in mm)

Table 5 .
Antifungal activity of the compounds 2a-n and 4a-e at 10 µg/ml concentration.(Diameter of zone of inhibition in mm)