DNA crosslinking and biological activity of a hairpin polyamide–chlorambucil conjugate

A prototype of a novel class of DNA alkylating agents, which combines the DNA crosslinking moiety chlorambucil (Chl) with a sequence‐selective hairpin pyrrole–imidazole polyamide ImPy‐β‐ImPy‐γ‐ImPy‐β‐Dp (polyamide 1), was evaluated for its ability to damage DNA and induce biological responses. Polyamide 1‐Chl conjugate (1‐Chl) alkylates and interstrand crosslinks DNA in cell‐free systems. The alkylation occurs predominantly at 5′‐AGCTGCA‐3′ sequence, which represents the polyamide binding site. Conjugate‐induced lesions were first detected on DNA treated for 1 h with 0.1 µM 1‐Chl, indicating that the conjugate is at least 100‐fold more potent than Chl. Prolonged incubation allowed for DNA damage detection even at 0.01 µM concentration. Treatment with 1‐Chl decreased DNA template activity in simian virus 40 (SV40) in vitro replication assays. 1‐Chl inhibited mammalian cell growth, genomic DNA replication and cell cycle progression, and arrested cells in the G2/M phase. Moreover, cellular effects were observed at 1‐Chl concentrations similar to those needed for DNA damage in cell‐free systems. Neither of the parent compounds, unconjugated Chl or polyamide 1, demonstrated any cellular activity in the same concentration range. The conjugate molecule 1‐Chl possesses the sequence‐selectivity of a polyamide and the enhanced DNA reactivity of Chl.