DNA ALKYLATION PROPERTIES OF ENHANCED FUNCTIONAL ANALOGS OF CC-1065 INCORPORATING THE 1,2,9,9A-TETRAHYDROCYCLOPROPA[1,2-C]BENZ[1,2-E]INDOL-4-ONE (CBI) ALKYLATION SUBUNIT

Full details of a study of the DNA alkylation properties of analogs of (+)-CC-1065 possessing the 1,2,9,9a-tetrahydrocyclopropa[1,2-c]benz[1,2-e]indol-4-one (CBI)alkylation subunit are described. Both enantiomers of N-BOC-CBI, a simple derivative of the alkylation subunit, exhibit an identical profile of DNA alkylation that is not distinguishable from that of (+)-N-BOC-CPI, the authentic alkylation subunit of (+)-CC-1065. In addition, the intensity of DNA alkylation as judged by the concentration of agent required for SV40 DNA fragment alkylation was determined to follow the following order: (+)-N-BOC-CBI > (-)-N-BOC-CBI (ca. 5-10X) and (+)-N-BOC-CBI > (+)-N-BOC-CPI (ca. 3-6x). A total of 40-45% of the adenines in the double-stranded DNA examined were found to be alkylated over a 10-fold agent concentration range. With three exceptions, the alkylation was found to occur at adenine flanked by a single 5' A or T base with a preference for 5'-d(AA)-3' > 5'-d(TA)-3'. In contrast, (+)-CBI-CDPI2 was found to alkylate DNA in a more selective fashion, and a total of 20-25% of the adenines were alkylated over a 1000-10000-fold agent concentration range. The DNA alkylation was found to proceed with greater intensity (ca. 10000X) and with a readily distinguishable profile of selectivity in which the adenine N-3 alkylation site proved to be flanked by two 5' A or T bases: 5'-d(AAA)-3' > 5'-d(TTA)-3' > 5-d(TAA)-3' greater-than-or-equal-to 5'-d(ATA)-3'. With (+)-CBI-CDPI2 there proved to be a strong preference for the fourth 5' base to be A or T, 5'-d(A/TXXA)-3' > 5'-d(G/CXXA)-3', a weaker preference for the fifth 5' base to be A or T, 5'-d(A/TXXXA)-3' > 5'-d(G/CXXXA)-3', and a weak preference for the 3' base preceding the alkylation site to be a purine base, 5'-d(NNXXAPu)-3' greater-than-or-equal-to 5'-(NNXXAPy)-3', where X = A or T. Potentially contributing to their enhanced cytotoxic potency, the CBI-based agents were found to alkylate DNA with a greater intensity and a faster relative rate than the corresponding CPI-based agent (e.g. k((+)-CBI-indole2)/k((+)-CPI-indole2) = 14). A discussion of the potential origin of the alkylation selectivity and the origin of the distinctions in the CBI and CPI-based agents is provided.