Novel cytotoxic bufadienolides derived from bufalin by microbial hydroxylation and their structure–activity relationships

Microbial transformation was used to prepare novel cytotoxic bufadienolides. Twelve products ( 3– 14) were obtained from bufalin ( 1) by the fungus Mucor spinosus. Their structures were elucidated by high-resolution mass spectroscopy (HR-MS) and extensive NMR techniques, including 1 H NMR, 13 C NMR, DEPT, 1 H – 1 H correlation spectroscopy (COSY), two dimensional nuclear Overhauser effect correlation spectroscopy (NOESY), heteronuclear multiple quantum coherence (HMQC), and heteronuclear multiple bond coherence (HMBC). Compounds 3, 4, 9 and 11– 14 are new mono- or dihydroxylated derivatives of bufalin with novel oxyfunctionalities at C-1β, C-7β, C-11β, C-12β and C-16α positions. The in vitro cytotoxic activities against human cancer cell lines of 3– 14, together with 16 biotransformed products derived from cinobufagin ( 15– 30) were determined by the MTT method, and their structure–activity relationships (SAR) were discussed.