Synthesis , characterization and biological evaluation of some novel 9-benzooxazol-4-yl-3 , 4-diphenyl-1-oxa-6-thia-2 , 4 , 9-triaza-spiro [ 4 . 4 ] non-2-en-8-ones

A series of various novel 4-benzooxazol-4-yl-9-phenyl-1,6-dithia-4,9-diaza-spiro[4.4]nonane-3,8-diones (5a-e) and 9-benzooxazol-4-yl-3,4-diphenyl-1-oxa-6-thia-2,4,9-triaza-spiro[4.4]non-2-en-8-ones (6a-e) were synthesized in good yields by using 4-nitro-1 H -benzooxazole (1) as starting material and 4-amino-1 H -benzooxazole (2) , N -benzooxazol-4-yl-2-chloro-acetamide (3) and 3-benzooxazol-4-yl-2-phenylimino-thiazolidin-4-ones (4a-e) as intermediates. After structural confirmation, the title compounds were screened for their antimicrobial and anti-inflammatory activity.


Introduction
A literature search revealed that, thiazolidinone derivatives may exhibit antibacterial, 1 antituberculosis, 2 antiviral 3 and anticancer 4 properties. In addition, some thiazolidinones were recently reported as novel inhibitors of mycobacterial rhamnose synthetic enzymes. 5 Small ring heterocycles containing nitrogen, sulfur and oxygen have been under investigation for a long time because of their important medicinal and biological properties. Among these types of heterocyclic molecules, spirothiazolidin based compounds have been shown to have various important biological activities such as antimicrobial, 6 antileukemic, 7 anti-helminthic, anticonvulsant, 8 antibacterial, 9 antifungal, 10 anti-tubercular, 11 anticancer 12 activity. Recent observations suggest that substituted benzoxazoles possess potential activity with lower toxicities in the chemotherapeutic approach in man. 13 Careful literature survey revealed that targets containing benzoxazole moiety have remarkable biological activities like antibacterial, 14 antihistaminic, 15 antiparasitics, 16 3.70 (s, 3H, CH 3 ); 13

Antimicrobial activity
The disc diffusion method 19 was used for the screening of anti microbial activity. The in vitro antibacterial activity of the synthesized compounds 5a-e and 6a-e was tested against three gram-positive bacteria i.e. Staphylococcus aureus, Staphylococcus albus, Streptococcus faecalis and against three gram-negative bacteria such as Escherichia coli, Proteus mirabilis, Salmonella typhi using a nutrient agar medium. The antifungal activity of the compounds was screened against two representative fungal organisms namely Candia albicans and Aspergillus fumigatus using Sabouraded dextrose agar medium. Amicacin (300 μg/ml) and Fluconazole (300 μg/ml) were used as reference compounds for the study of antibacterial and antifungal activity respectively. The lowest concentration (highest dilution) of the compounds at which, there was no visually detectable bacterial growth was taken as minimum inhibitory concentration (MIC). The zone of inhibition in mm caused by the various compounds on the microorganisms was measured.
IJBR (2014) 05 (01) www.ssjournals.com 08 ( The results (Table 1) of the antimicrobial screening of the tested compounds revealed that, all the tested compounds exhibited moderate to significant antimicrobial activity against both bacteria and fungi comparable with that of reference compounds. Compound 5a compare with other molecules was found to be totally inactive against P. mirabilis. Compound 5c was good active only against S. faecalis and almost inactive towards E. coli. This compound exhibited moderate activity against the rest of organisms. Highest antibacterial activity was observed in the product 6d against S. aureus, S. albus and S. faecalis, but shows only moderate activity against E. coli and P. mirabilis. This compound also performed high activity against two fungal organisms with marked activity index. Both compounds 6b and 6c exhibited highest antifungal activity against C. albicans and A. fumigatus. The remaining compounds exhibit moderate to good activity against all organisms employed. It can be concluded that the antimicrobial activity of such compounds may change by introduction or elimination of a specific group.

Anti-inflammatory activity
The anti-inflammatory activity screening for the prepared compounds 5a-e and 6a-e (at a dose of 10 mg/kg body weight) was determined in vivo by the acute carrageenan-induced paw oedema standard method. 20 The anti-inflammatory properties were recorded at successive time intervals 0.5, 1, 2, 3, and 4 h and compared with reference standard, Indomethacin (at a dose of 10 mg/kg body weight). From the obtained results (Table 2), it was noticed that after 1 h, compounds 5b, 5d, 6b and 6d exhibited considerable anti-inflammatory properties with potency 0.72, 0.64, 0.72 and 0.56 respectively. The remaining compounds showed lower to moderate activity.

Conclusion
The outstanding properties of this new class of antibacterial and antifungal substances deserve further investigation in order to clarify the mode of action at molecular level, responsible for the activity observed. More extensive study is also warranted to determine additional physicochemical and biological parameters to have a deeper insight into structure-activity relationship and to optimize the effectiveness of this series of molecules.