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The article presents spectral and kinetic evidence that protonated form of tirapazamine (TPZ) alters dehydration to the benzotriazinyl radical causing oxidative damage to DNA and oxidizing TPZ. It proposes that the form of TPZ goes through a bond homolysis to discharge the oxidizing radical and the 1-oxide of TPZ. Moreover, it reveals that the metabolism of 1,2,4-benzotriazine 1,4-dioxide class of hypoxia-selective prodrugs (HSPs) leads to the formation of carbon- and nitrogen-centered radicals.

The mechanism by which the 1,2,4-benzotriazine 1,4-dioxide (BTO) class of bioreductive hypoxia-selective prodrugs (HSPs) form reactive radicals that kill cancer cells has been investigated by steady-state radiolysis, pulse radiolysis (PR), electron paramagnetic resonance (EPR), and density functional theory (DFT) calculations. Tirapazamine (TPZ, 3-amino BTO, 1) and a series of 3-substituted analogues, -H (2), -methyl (3), -ethyl (4), -methoxy (5), -ethoxymethoxy (6), and -phenyl (7), were reduced in aqueous solution under anaerobic steady-state radiolysis conditions, and their radicals were found to remove the substrates by short chain reactions of different lengths in the presence of formate ions. Multiple carbon-centered radical intermediates, produced upon anaerobic incubation of the compounds with cytochrome P450 reductase enriched microsomes, were trapped by N-tefl-butyl-α-phenylnitrone and observed using EPR. The highly oxidizing oxymethyl radical, from compound 5, was identified, and experimental spectra obtained for compounds 1, 2, 3, and 7 were well simulated after the inclusion of aryl radicals. The identification of a range of oxidizing radicals in the metabolism of the BTO compounds gives a new insight into the mechanism by which these HSPs can cause a wide variety of damage to biological targets such as DNA. [ABSTRACT FROM AUTHOR]