Design, synthesis, and evaluation of a novel sequence-selective epoxide-containing DNA cross-linking agent based on the pyrrolo[2,1-c][1,4]benzodiazepine system

Synthetic routes have been investigated to prepare a novel C8-epoxide-funct ionalized pyrrolo[2,1-c] [1,4]benzodiazepine 6 as a potential sequence-sele ctive DNA cross-linking agent (Wilson et al. Tetrahedron Lett. 1995, 36, 63 33-6336). A successful synthesis was accomplished via a 10-step route invol ving a pro-N10-Fmoc cleavage method that should have general applicability to other pyrrolobenzodiazepine (PBD) molecules containing acid- or nucleoph ile-sensitive groups. During the course of this work, a one-pot reductive c yclization procedure for the synthesis of PBD N10-C11 imines from nitro dim ethyl acetals was also discovered, although this method results in C11a rac emization which can reduce DNA binding affinity and cytotoxicity. The targe t epoxide 6 was shown by thermal denaturation studies to have a significant ly higher DNA-binding affinity than the parent DC-81 (3) or the C8-propenox y-PBD (15), which is structurally similar but lacks the epoxide moiety. The time course of effects upon thermal denaturation indicated a rapid initial binding phase followed by a slower phase consistent with the stepwise cros s-linking of DNA observed for a difunctional agent. This was confirmed by a n electrophoretic assay which demonstrated efficient induction of interstra nd cross-links in plasmid DNA at concentrations >1 mu M. Higher levels of i nterstrand cross-linking were observed at 24 h compared to 6 h incubation. A Tag polymerase stop assay indicated a preference for binding to guanine-r ich sequences as predicted for bis-alkylation in the minor groove of DNA by epoxide and imine moieties. The pattern of stop sites could be partly rati onalized by molecular modeling studies which suggested low-energy models to account-for the observed binding behavior. The epoxide PBD 6 was shown to have significant cytotoxicity (45-60 nM) in the A2780, CH1, and CH1cis(R) h uman ovarian carcinoma cell lines and an IC50 Of 0.2 mu M in A2780cis(R). T he significant activity of 6 in the cisplatin-resistant CH1cis(R) cell line (IC50 = 47 nM) gave a resistance factor of 0.8 compared to the parent cell line, demonstrating no cross-resistance with the major groove cross-linkin g agent cisplatin.