Inhibitors of topoisomerase II based on the benzodiimidazole and dipyrroloimidazobenzimidazole ring systems: controlling DT-diaphorase reductive inactivation with steric bulk.

Described herein are the synthesis, cytotoxic properties, and topoisomerase II inhibition assays of benzodiimidazole and dipyrroloimidazobenzimidazole structural variants of the pyrrolo[1, 2-a]benzimidazole or APBI ring system. These ring variants were designed to inhibit topoisomerase II, much as the APBIs are able to do. Since only the quinone form of the APBIs can intercalate DNA, two-electron reduction to the hydroquinone by DT-diaphorase is known to deactivate these compounds. Indeed, the APBIs possess a high inverse correlation with the cellular concentration of DT-diaphorase. Therefore one feature of the ABPI structural variants is the excessive bulk about the quinone ring, which was predicted to diminish DT-diaphorase substrate activity. Another feature is the presence of one or two alkylating centers, which would permit alkylation of DNA and/or topoisomerase II. Inhibition assays for topoisomerase II-mediated relaxation of supercoiled DNA indicate that the benzodiimidazole and dipyrroloimidazobenzimidazole quinone ring systems are catalytic inhibitors of topoisomerase II. Both quinone systems exhibit cytotoxicity perhaps due to the lack of inactivation by DT-diaphorase as well as topoisomerase II inhibition. One quinone displayed the novel feature of cytotoxicity selectively against melanoma cell lines. In conclusion, the benzodiimidazole and dipyrroloimidazobenzimidazole quinone ring systems will be subjected to future analogue development and structure-activity studies.

Evidence for DNA phosphate backbone alkylation and cleavage by pyrrolo[1,2-a]benzimidazoles: small molecules capable of causing base-pair-specific phosphodiester bond hydrolysis.

This report presents evidence that a reduced pyrrolo[1,2-a]benzimidazole (PBI) cleaves DNA as a result of phosphate alkylation followed by hydrolysis of the resulting phosphate triester. The base-pair specificity of the phosphate alkylation results from Hoogsteen-type hydrogen bonding of the reduced PBI in the major groove at only A.T and G.C base pairs. Alkylated phosphates were detected by 31P NMR and the cleavage products were detected by 1H NMR and HPLC. Evidence is also presented that a reduced PBI interacts with DNA in the major groove rather than in the minor groove or by intercalation.

Pyrrolo[1,2-a]benzimidazole-based quinones and iminoquinones. The role of the 3-substituent on cytotoxicity.

The influence of the 3-substituent on the cytotoxicity of the 6-aziridinylpyrrolo[1,2-a]-benzimidazole quinones (PBIs), the 6-acetamidopyrrolo[1,2-alpha]benzimidazole quinones (APBIs), and the 6-acetamidopyrrolo[1,2-alpha]benzimidazole iminoquinones (imino-APBIs) was investigated by comparing LC50 mean graphs consisting of 60 cancer lines. Increasing lipophilicity of the 3-substituent of PBIs and APBIs increased the cytotoxicity specifically in melanoma cell lines. The 3-substituent does not influence DNA cleavage by reduced PBIs, except for the 3-carbamate derivative which shows enhanced cleavage. This property of the 3-carbamate is rationalized in terms of the PBI major groove binding model. The imino-APBIs show enhanced cytotoxicity in melanoma and renal cancer cell lines; the correlation coefficient for log LC50 vs log lipophilicity is 0.8 to 0.9. COMPARE correlations revealed that the PBIs are activated by DT-diaphorase but that the APBIs and imino-APBIs are inactivated by this enzyme. Thus, the latter two agents are cytotoxic only as quinones. It was noted that APBIs possess a similar cytotoxic profile to three anthracycline analogues. This observation suggests mechanistic similarities between both types of cytotoxic agents. Major conclusions of this study pertain to the design of agents displaying cytotoxicity specifically against melanoma and renal cancers and to the use of 60-cell line mean graphs and COMPARE in cancer drug QSAR.

Structure-activity studies of benzimidazole-based DNA-cleaving agents. Comparison of benzimidazole, pyrrolobenzimidazole, and tetrahydropyridobenzimidazole analogues.

The synthesis and cytotoxic properties of benzimidazole-based DNA-cleaving agents are presented herein. These agents include pyrrolo[1,2-a]benzimidazole (PBI), benzimidazole (BI), and tetrahydropyrido[1,2-a]benzimidazole (TPBI) analogues. As a result of these studies, it is concluded that the pyrrolo ring is not necessary for cytotoxicity (PBI is only slightly more cytotoxic than BI) but that homologation of the pyrrolo ring by one carbon results in a system, TPBI, prone to decomposition. Another conclusion is that the 6-aziridinyl derivative of the PBI system is more potent than the 7-aziridinyl derivative. Comparative studies with known antitumor agents revealed that the benzimidazole-based DNA-cleaving agents possess a unique spectrum of activity. Noteworthy observations are the high level of cytotoxicity against melanoma cell lines and the complete absence of activity against leukemia cell lines. The reductive activation and DNA-cleavage properties of the most active analogue (BI-A) are also presented. Reduction of the quinone ring to the hydroquinone results in nucleophile and proton trapping by the aziridinyl group. Documented nucleophiles include water and the oxygen anion of 5'-dAMP. In addition, reduced BI-A reacts with DNA to form a stable adduct, which cleaves at G+A bases upon heating in basic gel-loading solution.

Pyrrolo[1,2-a]benzimidazole-based aziridinyl quinones. A new class of DNA cleaving agent exhibiting G and A base specificity.

Pyrrolo[1,2-a]benzimidazole(PBI)-based aziridinyl quinones cleave DNA under reducing conditions specifically at G + A bases without any significant cleavage at C + T bases. The postulated mechanisms involve phosphate alkylation by the reductively activated aziridine to afford a hydrolytically labile phosphotriester as well as the classic N(7) purine alkylation followed by depurination and backbone cleavage. Evidence is presented that the phosphate alkylation mechanism could contribute. The PBIs possess a unique spectrum of cytotoxicity against cancer cells (inactive against leukemia but active against nonsmall cell lung, colon, CNS, melanoma, ovarian, and renal cancers). Also reported are results of in vivo antitumor activity screens.