title: 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.
abstract: Synthetic routes have been investigated to prepare a novel C8-epoxide-functionalized pyrrolo[2,1-c][1,4]benzodiazepine 6 as a potential sequence-selective DNA cross-linking agent (Wilson et al. Tetrahedron Lett. 1995, 36, 6333-6336). A successful synthesis was accomplished via a 10-step route involving a pro-N10-Fmoc cleavage method that should have general applicability to other pyrrolobenzodiazepine (PBD) molecules containing acid- or nucleophile-sensitive groups. During the course of this work, a one-pot reductive cyclization procedure for the synthesis of PBD N10-C11 imines from nitro dimethyl acetals was also discovered, although this method results in C11a racemization which can reduce DNA binding affinity and cytotoxicity. The target epoxide 6 was shown by thermal denaturation studies to have a significantly higher DNA-binding affinity than the parent DC-81 (3) or the C8-propenoxy-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 cross-linking of DNA observed for a difunctional agent. This was confirmed by an electrophoretic assay which demonstrated efficient induction of interstrand cross-links in plasmid DNA at concentrations >1 microM. Higher levels of interstrand cross-linking were observed at 24 h compared to 6 h incubation. A Taq polymerase stop assay indicated a preference for binding to guanine-rich 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 rationalized 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) human ovarian carcinoma cell lines and an IC(50) of 0.2 microM in A2780cis(R). The significant activity of 6 in the cisplatin-resistant CH1cis(R) cell line (IC(50) = 47 nM) gave a resistance factor of 0.8 compared to the parent cell line, demonstrating no cross-resistance with the major groove cross-linking agent cisplatin.
authors.affiliation: CRC Gene Targeted Drug Design Research Group, School of Pharmacy and Biomedical Sciences, University of Portsmouth, St. Michael's Building, White Swan Road, Portsmouth, Hants. PO1 2DT, U.K.
authors.forename: S C
authors.initials: SC
authors.lastname: Wilson
authors.forename: P W
authors.initials: PW
authors.lastname: Howard
authors.forename: S M
authors.initials: SM
authors.lastname: Forrow
authors.forename: J A
authors.initials: JA
authors.lastname: Hartley
authors.forename: L J
authors.initials: LJ
authors.lastname: Adams
authors.forename: T C
authors.initials: TC
authors.lastname: Jenkins
authors.forename: L R
authors.initials: LR
authors.lastname: Kelland
authors.forename: D E
authors.initials: DE
authors.lastname: Thurston
mesh_terms: D000970:Antineoplastic Agents / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D001569:Benzodiazepines / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D001570:Benzodiazepinones / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D002945:Cisplatin / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D004247:DNA / Q000737:chemistry*; D019008:Drug Resistance, Neoplasm; D004852:Epoxy Compounds / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D008958:Models, Molecular; D009691:Nucleic Acid Denaturation; D010084:Oxidation-Reduction; D011758:Pyrroles / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D019914:Taq Polymerase; D014407:Tumor Cells, Cultured
chemical_list: C122053:8-(2,3-epoxypropoxy)-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo(2,1-c)(1,4)benzodiazepin-5-one; D000970:Antineoplastic Agents; D001570:Benzodiazepinones; D003432:Cross-Linking Reagents; D004852:Epoxy Compounds; D011758:Pyrroles; D001569:Benzodiazepines; D004247:DNA; D019914:Taq Polymerase; D002945:Cisplatin