Heterocyclic N-oxides are an interesting class of antitumor agents that selectively kill the hypoxic cells found in solid tumors. The hypoxia-selective activity of the lead compound in this class, tirapazamine, stems from its ability to undergo intracellular one-electron reduction to an oxygen-sensitive drug radical intermediate. In the presence of molecular oxygen, the radical intermediate is back-oxidized to the parent molecule. Under hypoxic conditions, the extended lifetime of the drug radical intermediate enables its conversion to a highly cytotoxic DNA-damaging intermediate via a "deoxygenative" mechanism involving the loss of oxygen from one of its N-oxide groups. The natural product myxin is a phenazine di-N-oxide that displays potent antibiotic activity against a variety of organisms under aerobic conditions. In light of the current view of heterocyclic N-oxides as agents that selectively operate under hypoxic conditions, it is striking that myxin was identified from Sorangium extracts based upon its antibiotic properties under aerobic conditions. Therefore, we set out to examine the molecular mechanisms underlying the biological activity of myxin. We find that myxin causes bioreductively activated, radical-mediated DNA strand cleavage under both aerobic and anaerobic conditions. Our evidence indicates that strand cleavage occurs via a deoxygenative metabolism. We show that myxin displays potent cytotoxicity against the human colorectal cancer cell line HCT-116 under both aerobic and anaerobic conditions that is comparable to the cell-killing properties of tirapazamine under anaerobic conditions. This work sheds light on the processes by which the naturally occurring aromatic N-oxide myxin gains its potent antibiotic properties under aerobic conditions. Furthermore, these studies highlight the general potential for aromatic N-oxides to undergo highly cytotoxic deoxygenative metabolism following enzymatic one-electron reduction under aerobic conditions.

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 non-deuterated 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 release of hydroxyl radical from enzymatically-activated 1,2,4-benzotriazine 1,4-dioxides.

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Hypoxia-selective cytotoxins (HSCs) seek to exploit the oxygenpoor nature of tumor tissue for therapeutic gain. Typically, HSCs require activation by one-electron bioreductive enzymes such as NADPH:cytochrome P450 reductase (CYPOR). Thus, successful clinical deployment of HSCs may be facilitated by the development and implementation of diagnostic probes that detect the presence of relevant bioreductive enzymes in tumor tissue. The work described here develops analogues of the well-studied HSC tirapazamine (3amino-1,2,4-benzotriazine 1,4-di-N-oxide, TPZ) as profluorescent substrates of the one-electron reductases involved in bioactivation of HSCs. Hypoxic metabolism of TPZ or 7-fluoro-TPZ by one-electron reductases releases inherently fluorescent mono-N-oxide metabolites that may serve as indicators, probes, markers, or stains for the detection of the enzymes involved in the bioactivation of HSCs. In particular, profluorescent compounds of this type can provide a foundation for fluorescence-based bioassays that help identify tumors responsive to HSCs.

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