Abstract

The radical species produced following one-electron reduction of tirapazamine (3-amino-1,2,4-benzotriazine 1,4-dioxide, TPZ) by cytochrome P(450) reductase-enriched microsomes have been investigated using electron paramagnetic resonance (EPR) spectroscopy. Spin trapping with 5,5'-dimethylpyrroline 1-N-oxide (DMPO) gave a composite spectrum of a carbon-centered radical and the well-known DMPO-OH adduct. Using (17)O-labeled water resulted in a change in the EPR spectrum to that of DMPO-(17)OH, indicating that this radical species is formed with solvent involvement and not from release of a (*)OH radical from one-electron-reduced TPZ. Furthermore, using the closely related spin trap 5-diethoxyphosphoryl-5-methylpyrroline N-oxide (DEPMPO), which is less prone to oxidation than DMPO, gave only a carbon-centered radical spectrum without any involvement of a (*)OH radical. Reduction of a more soluble analogue of TPZ, in redox equilibrium with its 1-oxide derivative, led to spin trapping of both a carbon-centered radical and a nitrogen-centered radical by N-tert-butyl-alpha-phenylnitrone (PBN). The multicentered nature of this nitrogen-centered radical spectrum provides support for the formation of a benzotriazinyl radical following one-electron reduction of this class of bioreductive drug.