Discovery of 6,7-dihydro-3H-pyrano[4,3-c]isoxazol-3-ones as a new class of pathogen specific anti-leptospiral agents

Abstract A simple and efficient method for the synthesis of a series of 6,7-dihydro-3 H -pyrano[4,3- c ]isoxazol-3-one derivatives starting from 5-carboalkoxy-2,3-dihydropyranone (5-CDHPs) has been developed. Pyranoisoxazolones 10a-j , dihydronaphthopyran-4-one (DHNPs) class of natural product 12b and 12c and its analogues 12a and 13a-c were preliminarily screened against pathogenic leptospiral serovar Autumnalis strain N2 at various concentrations. Six pyranoisoxazolones, 10b , 10d , 10f , 10g , 10i and 10j which displayed very good anti-leptospiral activity was taken for secondary screening against twelve strains of pathogenic and one non-pathogenic leptospiral serovars. While all the compounds displayed significant anti-leptospiral activity against the pathogenic serovars at MIC of 62.5–500 μg/mL. Compounds 10d , 10g and 10j did not show any significant effect on non-pathogenic serovar. Inhibition of leptospires at a significant level by pyranoisoxazolone 10g was confirmed using RT-qPCR assay. In vivo treatment of BALB/c mice with compound 10g revealed that, it has 95% survivability against the pathogenic strain Canicola and also showed inhibition of renal colonization of leptospires. Compound 10g was found to show cytotoxicity against THP-1 cells only at higher concentration (≥75 μg/mL). Effective binding of compound 10g with leptospiral outer membrane protein LipL32 observed via in silico molecular docking provided a suitable explanation for pathogen specificity of compound 10g . Antibiotics acting against leptospirosis in human are very few. The results obtained from in vitro , in vivo and in silico study reveals that 6,7-dihydro-3 H -pyrano[4,3- c ]isoxazol-3-ones class of compounds are lead molecules for further development as pathogen specific anti-leptospiral agents.