Toward Hypoxia-Selective DNA-Alkylating Agents Built by Grafting Nitrogen Mustards onto the Bioreductively Activated, Hypoxia-Selective DNA-Oxidizing Agent 3‑Amino-1,2,4-benzotriazine 1,4-Dioxide (Tirapazamine)

Tirapazamine (3-amino-1,2,4-benzotriazine 1,4-dioxide) is a heterocyclic di-<i>N</i>-oxide that undergoes enzymatic deoxygenation selectively in the oxygen-poor (hypoxic) cells found in solid tumors to generate a mono-<i>N</i>-oxide metabolite. This work explored the idea that the electronic changes resulting from the metabolic deoxygenation of tirapazamine analogues might be exploited to activate a DNA-alkylating species selectively in hypoxic tissue. Toward this end, tirapazamine analogues bearing nitrogen mustard units were prepared. In the case of the tirapazamine analogue <b>18a</b> bearing a nitrogen mustard unit at the 6-position, it was found that removal of the 4-oxide from the parent di-<i>N</i>-oxide to generate the mono-<i>N</i>-oxide analogue <b>17a</b> did indeed cause a substantial increase in reactivity of the mustard unit, as measured by hydrolysis rates and DNA-alkylation yields. Hammett sigma values were measured to quantitatively assess the magnitude of the electronic changes induced by metabolic deoxygenation of the 3-amino-1,2,4-benzotriazine 1,4-dioxide heterocycle. The results provide evidence that the 1,2,4-benzotiazine 1,4-dioxide unit can serve as an oxygen-sensing prodrug platform for the selective unmasking of bioactive agents in hypoxic cells.