Enzyme-activated, hypoxia-selective DNA damage by 3-amino-2-quinoxalinecarbonitrile 1,4-di-N-oxide

The compound 3-amino-2-quinoxalinecarbonitrile 1,4-dioxide (4) displays potent hypoxia-selective cytotoxicity in cell culture. This compound is structurally similar to the known hypoxia-selective DNA-damaging agent tirapazamine (1, TPZ), but the ability of 4 to cause DNA damage under low-oxygen conditions has not previously been characterized. The results presented here provide the first evidence that 4 causes reductively activated DNA damage under hypoxic conditions. The findings indicate that one-electron reduction of 4 by NADPH:cytochrome P450 reductase yields an oxygen-sensitive intermediate (5). This activated intermediate is rapidly destroyed by reaction with O2 under aerobic conditions, but goes forward to cause DNA damage under low-oxygen conditions. Analysis of the DNA damage indicates that reductive activation of 4 leads to production of a highly reactive, freely diffusible oxidizing radical that causes sequence-independent cleavage of the deoxyribose backbone and oxidative damage to the heterocyclic bases in duplex DNA. On the basis of the experiments reported here, the chemical nature of the DNA damage caused by redox-activated 4 is analogous to that reported previously for TPZ.