Reaction of the hypoxia-selective antitumor agent tirapazamine with a C1 '-radical in single-stranded and double-stranded DNA: The drug and its metabolites can serve as surrogates for molecular oxygen in radical-mediated DNAdamage reactions

The compound 3-amino-1,2,4-benzotriazine 1,4-dioxide (1, tirapazamine; also known as SR4233, WIN 59075, and tirazone) is a clinically promising antica ncer agent that selectively kills the oxygen-poor (hypoxic) cells found in tumors. When activated by one-electron enzymatic reduction, tirapazamine in duces radical-mediated oxidative DNA strand cleavage. Using the ability to generate a single deoxyribose radical at a defined site in an oligonucleoti de, we recently provided direct evidence that, in addition to initiating th e formation of DNA radicals, tirapazamine can react with these radicals and convert them into base-labile lesions [Daniels et al. (1998) Chem. Res. To xicol. 11, 1254-1257]. The rate constant for trapping of a C1'-radical in s ingle-stranded DNA by tirapazamine was shown to be similar to 2 x 10(8) M-1 s(-1), demonstrating that tirapazamine can substitute for molecular oxygen in radical-mediated DNA strand damage reactions. Because reactions of tira pazamine with DNA radicals may play an important role in its ability to dam age DNA, we have further characterized the ability of the drug and its meta bolites to convert a C1'-DNA radical into a base-labile lesion. We find tha t tirapazamine reacts with a C1'-radical in double-stranded DNA with a rate constant of 4.6 x 10(6) M-1 s(-1). The mono-iii-oxide (3) stemming from bi oreductive metabolism of tirapazamine converts the C1'-radical to an alkali ne-labile lesion more effectively than the parent drug. Compound 3 traps a C1'-radical in single-stranded DNA with a rate constant of 4.6 x 10(8) M-1 s(-1) and in double-stranded DNA with a rate constant of 1.4 x 10(7) M-1 s( -1) We have also examined the rate and mechanism of reactions between the C 1'-radical and representatives from two known classes of "oxygen mimetic" a gents: the nitroxyl radical 2,2,6,6-tetramethylpiperidin-N-oxyl (4, TEMPO) and the nitroimidazole misonidazole (5). TEMPO traps the C1'-radical in sin gle-stranded DNA (7.2 x 10(7) M-1 s(-1)) approximately 3 times less effecti vely than tirapazamine, but 2 times as fast in double-stranded DNA (9.1 x 1 0(6) M-1 s(-1)). Misonidazole traps the radical in single- (6.9 x 10(8) M-1 s(-1)) and double-stranded DNA (2.9 x 10(7) M-1 s(-1)) with rate constants that are roughly comparable to those measured for the mono-N-oxide metabol ite of tirapazamine. Finally, information regarding the chemical mechanism by which these compounds oxidize a monomeric C1'-nucleoside radical has bee n provided by product analysis and isotopic labeling studies.