Characterization of radicals formed following enzymatic reduction of 3-substituted analogues of the hypoxia-selective cytotoxin 3-amino-1,2,4- benzotriazine 1,4-dioxide (tirapazamine)

The mechanism by which the 1,2,4-benzotriazine 1,4-dioxide (BTO) class of bioreductive hypoxiaselective prodrugs (HSPs) form reactive radicals that kill cancer cells has been investigated by steadystate 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-tert-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.