DNA-DNA interstrand cross-linking by FR66979: Intermediates in the activation cascade

The antitumor antibiotics FR66979 (1), FR900482 (2), and FK973 (3) are similar in structure and biological activity to the DNA cross-linking antitumor antibiotic mitomycin C (4). The cytotoxic effects of 1-3 have been proposed to result from sequential bioreductive cleavage of the N-O bond and condensation of the thus-exposed amine and ketone functions to yield an indole (e.g., 9) which is structurally analogous to the mitosene nucleus of reductively activated mitomycins. We report herein evidence substantiating this proposal based upon study of the reductive activation chemistry of 1 and 2 using thiols and iron(II) in the absence and presence of DNA. Prolonged exposure of reductively activated 1 to sodium borohydride afforded the dihydroindole 11, presumably through trapping of the iminium ion precursor (16). Kinetics measurements strongly implicate a relatively long-lived precursor to the iminium ion, which accumulates following iron(II)-catalyzed thiol-promoted reduction of 1, proposed herein to be one or both of the isomeric aminals 12. Under appropriate conditions, some step or steps between this intermediate and the iminium ion are shown to be rate limiting in DNA cross-linking, in production of the dihydroindole by borohydride trapping, and in the decay of the intermediate(s) competent to produce those same products. These studies clearly demonstrate the strong similarities in the cascade of reactions which follow reductive activation of FR66979 (1) [and presumably by extension FR900482 (2) and FK973 (3)] and the mitomycins.