Interactions of a Series of Minor Groove Targeted Polybenzamide-Linked Nitrogen Mustards with DNA

Abstract

Simple bifunctional nitrogen mustards are an important class of anti-cancer drugs. They are believed to exert their lethal action by covalently cross-linking two guanine residues in opposite strands of the DNA double helix. However, cross-linking events are rare compared with mono-alkylations (which are considered to be genotoxic), due to the high reactivity of the mustard moieties. Additionally, because the two mustard units are in the same vicinity, alkylation events are restricted to sequences in which a guanine residue is within reach of the covalently mono-bound mustard. Interest in targeting nitrogen mustard units to DNA by tethering them to minor groove binding ligands resulted from the finding that the minor groove binder CC-1065 is a potent cytotoxin, even though it only mono-alkylates DNA. In this investigation we sought to study different aspects of the interactions of a group of experimental anti-cancer drugs. The relationships between structure, chemical reactivity and cytotoxicity of a series of minor groove targeted polybenzamide-linked nitrogen mustards have been evaluated. The reactivity of the mustards varied according to the presence or absence of additional electron withdrawing groups and the overall topology of the mustards was also varied. This investigation showed that although almost all the members of the series have comparable reactivities in the presence of calf thymus DNA and the bis-mustards cross-linked DNA to the same extent, they showed widely varying cytotoxicities against P388 leukemia cells. All the compounds, including the mono-mustards, were significantly more potent than the untargeted nitrogen mustard chlorambucil, despite the higher chemical reactivity of the latter, with the bis-mustard (37) which has a radius of curvature similar to that of DNA being the most potent compound investigated. The nature of the covalent modification of DNA by selected members of the series was identified as being at the N3 of adenine in the minor groove. Adenine-adenine and adenine-guanine cross-link adducts were isolated from reaction of the bis-mustard (39) with calf thymus DNA. The sequence specificity of DNA alkylation was identified as being alkylation of an adenine residue in runs of adenines. Each compound showed a unique pattern of adducts when reacted with calf thymus DNA and analyzed using the sensitive 32P-postlabelling method. Selected members were also studied by the same method for their ability to form adducts in cells in culture. The mono-mustard (42) showed identical adduct pattern in both naked and cellular DNA while no adducts were detected in cells from the reaction of the bis-analogue (37), even at super toxic doses. The mutagenic potential of selected members was evaluated by employing the gpt gene using different molecular techniques. The studies have demonstrated the mutagenicity of the mono-mustard (42) which induced mainly A→T transversion mutations. However, the bis-analogue (37) was cytotoxic rather than mutagenic.