Isotopic Labeling Experiments That Elucidate the Mechanism of DNA Strand Cleavage by the Hypoxia-Selective Antitumor Agent 1,2,4-Benzotriazine 1,4-Di-N-oxide

Abstract

The 1,2,4-benzotriazine 1,4-dioxides are an important class of potential anticancer drugs that selectively kill the low-oxygen (hypoxic) cells found in solid tumors. These compounds undergo intracellular one-electron enzymatic reduction to yield an oxygen-sensitive drug radical intermediate that partitions forward, under hypoxic conditions, to generate a highly reactive secondary radical that causes cell killing DNA damage. Here, we characterized bioreductively activated, hypoxia-selective DNA-strand cleavage by 1,2,4-benzotriazine 1,4-dioxide. We found that one-electron enzymatic activation of 1,2,4-benzotriazine 1,4-dioxide under hypoxic conditions in the presence of the deuterium atom donor methanol-d4 produced nondeuterated mono-N-oxide metabolites. This and the results of other isotopic labeling studies provided evidence against the generation of atom-abstracting drug radical intermediates and are consistent with a DNA-damage mechanism involving the release of hydroxyl radical from enzymatically activated 1,2,4-benzotriazine 1,4-dioxides.