Evaluation of functional Analogs of CC-1065 and the Duocarmycins incorporating the cross-linking 9a-chloromethyl-1,2,9,9a-tetrahydrocyclopropa[ c]benz[ e]indol-4-one (C 2BI) alkylation subunit

Abstract

The DNA alkylation properties and in vitro cytotoxic activity of a series of analogs of CC-1065 and the duocarmycins incorporating the 9a-chloromethyl-1,2,9,9a-tetrahydrocyclopropa[ c]benz[ e]inol-4-one (C 2BI) alkylation subunit are detailed. The C 2BI-based agents have been shown to alkylate DNA within the minor groove in a fashion analogous to CC-1065 or duocarmycin. The stereoelectronically-controlled adenine N3 addition to the least substituted cyclopropane carbon occurs with a selectivity that represents a composite of the two enantiomers of the corresponding CBI-based agents. Additional high affinity alkylation sites were detected which were not prominent alkylation sites for either enantiomer of the CBI-based agents. Such sites may represent induced high affinity alkylation sites resulting from DNA cross-linking following complementary strand alkylation at a high affinity alkylation site and each such site detected proved consistent with predicted models of an adenine-adenine cross-linking event. Further, consistent with this interpretation, the C 2BI agents were shown to constitute efficient cross-linking agents with DNA cross-linking being observed at the same concentrations as DNA alkylation. In comparison to the parent CBI-based agents, the C 2BI-based agents proved to be approximately 100–10.000x less effective at DNA alkylation and 100–10,000x less potent in cytotoxic assays. This is suggested to be the consequence of a significant steric deceleration of the adenine N3 alkylation reaction attributable to the additional 9a-chloromethyl substituent. Consistent with this interpretation, the noncovalent binding constant of C 2BICDPI 2 for poly[dA]-poly[dT] proved nearly identical to that of CDPI 3 under kinetic binding conditions, and prolonged incubation of C 2BICDPI 2 with poly[dA]-poly[dT] (72 h, 25°C) provided covalent complexes with a helix stabilization comparable to that observed with (+)- or (−)-CPI-CDPI 2 indicating that the size of the C 2BI subunit inhibits but does not preclude productive DNA alkylation.