DNA minor groove targeted alkylating agents based on bisbenzimidazole carriers: synthesis, cytotoxicity and sequence-specificity of DNA alkylation

A series of bisbenzimidazoles bearing a variety of alkylating agents [ortho - and meta-mustards, imidazolebis(hydroxymethyl), imidazolebis(methylcarbam ate) and pyrrolebis(hydroxymethyl)], appended by a propyl linker chain, wer e prepared and investigated for sequence-specificity of DNA alkylation and their cytotoxicity. Previous work has shown that, for para-aniline mustards , a propyl linker is optimal for cytotoxicity. Alkaline cleavage assays usi ng a variety of different labelled oligonucleotides showed that the preferr ed sequences for adenine alkylation were 5'-TTTANANAANN and 5'-ATTANANAANN (underlined bases show the drug alkylation sites), with AT-rich sequences r equired on both the 5' and 3' sides of the alkylated adenine. The different aniline mustards showed little variation in alkylation pattern and similar efficiencies of DNA cross-link formation despite the changes in orientatio n and positioning of the mustard, suggesting that the propyl linker has som e flexibility. The imidazole- and pyrrolebis(hydroxymethyl) alkylators show ed no DNA strand cleavage following base treatment, indicating that no guan ine or adenine N3 or N7 adducts were formed. Using the PCR-based polymerase stop assay, these alkylators showed PCR blocks at 5'-C*G sites (the * nucl eotide indicates the blocked site), particularly at 5'-TAC*GA 5'-AGC*GGA, a nd 5'-AGCC*GGT sequences, caused by guanine 2-NH2 lesions on the opposite s trand. Only the (more reactive) imidazolebis(methylcarbamoyl) and pyrrolebi s(hydroxymethyl) alkylators demonstrated interstrand cross-linking ability. All of the bifunctional mustards showed large (similar to 100-fold) increa ses in cytotoxicity over chlorambucil, with the corresponding monofunctiona l mustards being 20- to 60-fold less cytotoxic. These results suggest that in the mustards the propyl linker provides sufficient flexibility to achiev e delivery of the alkylator to favoured (adenine N3) sites in the minor gro ove, regardless of its exact geometry with respect to the bisbenzimidazole carrier. The 'targeted' bisbenzimidazole bis(hydroxymethyl)pyrrole- and imi dazole analogues showed very similar patterns of alkylation to the correspo nding 'untargeted' compounds, with little evidence of additional selectivit y imposed by this AT-preferring carrier.