Hypoxia in
tumors results in resistance to both
chemotherapy and
radiotherapy treatments but affords an environment in which
hypoxia-activated
prodrugs (HAP) are activated upon bioreduction to release targeted
cytotoxins. The benzotriazine 1,4-di-N-oxide (BTO) HAP,
tirapazamine (TPZ, 1), has undergone extensive clinical evaluation in combination with
radiotherapy to assist in the killing of hypoxic
tumor cells. Although compound 1 did not gain approval for clinical use, it has spurred on the development of other BTOs, such as the 3-alkyl analogue,
SN30000, 2. There is general agreement that the
cytotoxin(s) from BTOs arise from the one-electron reduced form of the compounds. Identifying the cytotoxic radicals, and whether they play a role in the selective killing of hypoxic
tumor cells, is important for continued development of the BTO class of anticancer
prodrugs. In this study, nitrone spin-traps, combined with electron spin resonance, give evidence for the formation of aryl radicals from compounds 1, 2 and 3-phenyl analogues, compounds 3 and 4, which form
carbon C-centered radicals. In addition, high concentrations of DEPMPO (5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide) spin-trap the •OH radical. The combination of spin-traps with high concentrations of
DMSO and
methanol also give evidence for the involvement of strongly oxidizing radicals. The failure to spin-trap methyl radicals with PBN (
N-tert-butylphenylnitrone) on the bioreduction of compound 2, in the presence of
DMSO, implies that free •OH radicals are not released from the protonated radical
anions of compound 2. The spin-trapping of •OH radicals by high concentrations of DEPMPO, and the radical species arising from
DMSO and
methanol give both direct and indirect evidence for the scavenging of •OH radicals that are involved in an intramolecular process.
Hypoxia-selective cytotoxicity is not related to the formation of aryl radicals from the BTO compounds as they are associated with high aerobic cytotoxicity.