Articles Enzyme-Activated, Hypoxia-Selective DNA Damage by 3-Amino-2-quinoxalinecarbonitrile 1,4-Di-N-oxide Goutam Chowdhury, Delshanee Kotandeniya, J. Scott Daniels, † Charles L. Barnes, and Kent S. Gates* Departments of Chemistry and Biochemistry, University of Missouri-Columbia, Columbia, Missouri 65211 Received June 23, 2004 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 O 2 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. Introduction The compound 3-amino-1,2,4-benzotriazine 1,4-N-oxide (1, tirapazamine, TPZ) is a clinically promising antican- cer agent that selectively kills the hypoxic cells (1, 2) found in solid tumors (3, 4). Inside cells, one-electron enzymatic reduction of TPZ (Scheme 1) yields an acti- vated form of the drug, 2 (5-7). In normally oxygenated cells, the radical anion 2a is rapidly destroyed by reaction with O 2 (5, 8). Although this reaction generates super- oxide radical, the toxicity of this species is diminished by cellular enzyme systems such as superoxide dismu- tase, catalase, and glutathione peroxidase (9), and the back-oxidation process represents a detoxification path- way for the activated drug. On the other hand, under the oxygen-poor (hypoxic) conditions found in tumor cells, activated TPZ can partition forward to cause cytotoxic DNA damage (5, 10-13). There is general agreement that one-electron reduction of tirapazamine leads to production of a highly oxidizing DNA-damaging radical; however, the exact identity of this species remains a matter of debate. It has long been proposed that 2b causes DNA strand breaks via direct abstraction of hydrogen atoms from the deoxyribose * To whom correspondence should be addressed. Tel: 573-882-6763. Fax: 573-882-2754. E-mail: gatesk@missouri.edu. † Current address: Millenium Pharmaceuticals, Inc., Cambridge, MA. NOVEMBER 2004 VOLUME 17, NUMBER 11 © Copyright 2004 by the American Chemical Society 10.1021/tx049836w CCC: $27.50 © 2004 American Chemical Society Published on Web 10/02/2004