The dinitrobenzamide
aziridine CB 1954 (1) and its
nitrogen mustard analogue
SN 23862 (6) are
prodrugs that are activated by enzymatic nitroreduction in
tumors. Bioactivation of 1 is considered to be due to reduction of its 4-nitro group to the
hydroxylamine and subsequent formation of the N-acetoxy derivative; this acts as a reactive center, in concert with the
aziridine moiety, to provide a bifunctional
DNA cross-linking agent (Knox model). It is currently unclear whether bioactivation of 6 occurs by the same mechanism or results from the electronic effects of nitroreduction on reactivity of the
nitrogen mustard moiety. To discriminate between these mechanisms, we have synthesized the
hydroxylamine and
amine derivatives of 1 and 6, plus related compounds, and determined their alkylating reactivities in aqueous
solution, using LC/MS to identify reaction pathways. The relationships between substituent electronic effects, reactivity, and cytotoxicity were determined using the UV4 cell line, which is defective in nucleotide excision repair (thus avoiding differences in repair kinetics). Alkylating reactivity correlated with the electron-donating character of the ortho or para substituent in the case of the mustards, with a less marked electronic effect for the
aziridines. Importantly, there was a highly significant linear relationship between cytotoxic potency and alkylating reactivity in both the
aziridine and the mustard series, with the notable exception of 4, the 4-hydroxylamine of 1, which was 300-fold more toxic than predicted by this relationship. This demonstrates that the high potency of 4 does not result from activation of the
aziridine ring, supporting the Knox model. The single-step bioactivation of 6, to amino or
hydroxylamine metabolites with similar potency to 4, is a potential advantage in the use of dinitrobenzamide mustards as
prodrugs for activation by
nitroreductases.