The
antitumor agent 3-amino-1,2,4-benzotriazine 1,4-dioxide (
tirapazamine, TPZ, 1) gains medicinal activity through its ability to selectively damage
DNA in the hypoxic cells found inside solid
tumors. This occurs via one-electron enzymatic reduction of TPZ to yield an
oxygen-sensitive
drug radical (2) that leads to oxidatively generated DNA damage under hypoxic conditions. Two possible mechanisms have been considered to account for oxidatively generated DNA damage by TPZ. First, homolysis of the N-
OH bond in 2 may yield the well-known
DNA-damaging agent,
hydroxyl radical. Alternatively, it has been suggested that elimination of water from 2 generates a benzotriazinyl radical (4) as the ultimate
DNA-damaging species. In the studies described here, the TPZ analogue 3-methyl-1,2,4-benzotriazine 1,4-dioxide (5) was employed as a tool to probe the mechanism of DNA damage within this new class of
antitumor drugs. Initially, it was demonstrated that 5 causes redox-activated,
hypoxia-selective oxidation of
DNA and small organic substrates in a manner that is completely analogous to TPZ. This suggests that 5 and TPZ damage
DNA by the same chemical mechanism. Importantly, the methyl substituent in 5 provides a means for assessing whether the putative benzotriazinyl intermediate 7 is generated following one-electron reduction. Two complementary isotopic labeling experiments provide evidence against the formation of the benzotriazinyl radical intermediate. Rather, a mechanism involving the release of
hydroxyl radical from the activated
drug radical intermediates can explain the
DNA-cleaving properties of this class of
antitumor drug candidates.