3-amino-1,2,4-benzotriazine 4-oxide: Characterization of a new metabolite arising from bioreductive processing of the antitumor agent 3-amino-1,2,4-benzotriazine 1,4-dioxide (Tirapazamine)

Tirapazamine (1) is a promising antitumor agent that selectively causes DNA damage in hypoxic tumor cells, following one-electron bioreductive activat ion. Surprisingly, after more than 10 years of study, the products arising from bioreductive metabolism of tirapazamine have not, been completely char acterized. The two:previously characterized metabolites are 3-amino-1,2,4-b enzotriazine 1-oxide (3) and 3-amino-1,2,4-benzotriazine (5). In this work, 3-amino-1,2,4-benzotriazine 4-oxide (4) is identified for the first time a s a product resulting from:one-electron activation of the antitumor agent t irapazamine by the enzymes xanthine/xanthine oxidase and NADPH:cytochrome P 450 oxidoreductase. As part of this work, the novel N-oxide (4) was unambig uously synthesized and characterized using NMR spectroscopy, UV-vis spectro scopy, LC/MS, and X-ray crystallography. Under conditions where the parent drug tirapazamine is enzymatically activated, the metabolite 4 is produced but readily undergoes further]reduction to the benzotriazine (5). Thus, und er circumstances where extensive reductive metabolism occurs, the yield of the 4-oxide: (4) decreases. In contrast, the isomeric two-electron; reducti on product 3-amino-1,2,4-benzotriazine 1-oxide (3) does not readily undergo enzymatic reduction and, therefore, is found as a major bioreductive metab olite under all conditions. Finally, the ability of the 4-oxide metabolite (4) to participate in tirapazamine-mediated; DNA damage is considered.