1,2-benzisoxazole phosphorodiamidates as novel anticancer prodrugs requiring bioreductive activation

Several 1,2-benzisoxazole phosphorodiamidates have been designed as prodrugs of phosphoramide mustard requiring bioreductive activation. Enzymatic reduction of 1,2-benziosoxazole moiety is expected to result in the formation of imine intermediate due to the cleavage of the N-O bond. The imine should then be spontaneously hydrolyzed to a ketone metabolite, thereby facilitating base-catalyzed beta-elimination of cytotoxic phosphoramide mustard. As expected, the proposed prodrugs 4, 9, and 12 were at least 3-5-fold more potent cytotoxins than control compounds 5 and 15, which lack in the phosphoramide mustard group. Upon incubation with phenobarb-induced rat liver S-9 fraction, compounds 4, 9, and 12 underwent extensive NADPH-dependent metabolism with concomitant generation of alkylating activity under both hypoxic and oxic conditions. Corresponding ketone metabolites were detected for 9 and 15. NADPH-dependent bioreduction of 15 to its ketone metabolite 16 was located in the microsomal fraction and inhibited by SKF-525A and pCMBA. Compared with phenobarb-induced rat liver microsomal fraction, incubation of 15 with rat or human P450 reductase microsomes showed moderate generation of 16. Microsomal cytochrome P450 and/or P450 reductase appear to be involved in the reductive metabolism of 1,2-benzisoxazole moiety under hypoxic as well as oxic conditions.