Investigations into the synthesis, DNA-binding and efficacy of novel phenazine-1,6-carboxamides as potential anti-cancer drug candidates

Abstract

Bifunctional nitrogen mustards form DNA inter-strand crosslinks and are clinically important due to their broad-spectrum anti-tumour activity. However, the crosslinks are readily repaired by cancer cells, leading to fatal drug resistance, and their tendency to form monofunctional adducts exacerbates their carcinogenicity and mutagenicity. Equipping planar aromatic chromophores with alkylating groups to direct the alkylation of DNA by intercalation enhances alkylation efficacy, cytotoxicity, and modulates adduct specificity. Therefore, a N1,N6-bis(2-(aziridin-1-yl)ethyl)phenazine-1,6-dicarboxamide molecule named “Phenazir” was designed that bears aziridine groups at positions on the chromophore that predisposes them to nucleophilic attack by a guanine and adenine in the major and minor groove, respectively. It was anticipated that the resultant novel crosslinks would be difficult for cancer cells to repair, and therefore, Phenazir would be less susceptible to the development of resistance in a clinical setting. This thesis investigates the synthesis and biological activity of Phenazir and its analogues, and its binding to DNA through spectroscopic methods. Phenazir and its analogues were successfully synthesised and cytotoxicity measurements revealed that Phenazir had IC50 concentrations in the 5 to 25 nM range, and that it caused extensive DNA double strand breaks. Cytotoxicity measurements of other analogues, with different linker lengths and substitutions of the phenazine core, resulted in up to a 30-fold increase in activity. UV-Vis experiments demonstrated that the phenazine-1,6-carboxamide scaffold intercalated and alkylated DNA under mild conditions. NMR experiments were unsuccessful in obtaining solution structures of a Phenazir-oligomer complex, likely hindered by aggregation or polymerisation of Phenazir at high concentrations. However, molecular dynamics studies suggest that the aziridines in intercalated Phenazir are appropriately located to promote DNA alkylation. MALDI and nanoESI-MS were used to identify the alkylated adducts formed upon complexing a 5’ – TpG – 3’-containing oligomer with Phenazir, demonstrating that Phenazir alkylates efficiently at the TpG step with some evidence of interstrand crosslinking. Overall, the work in this thesis has led to the development of phenazine-1,6-carboxamides as a new class of highly active, DNA-targeting cytotoxic molecules that explore the previously untapped potential of threading bis-alkylation as a drug design approach.