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.

The influence of structure on DT-diaphorase substrate activity, topoisomerase II inhibition activity, and DNA reductive alkylation was studied for the 6-aziridinylpyrrolo[1,2-alpha]benzimidazolequinones (PBIs) and the 6-acetamidopyrrolo[1,2-alpha]benzimidazolequinones (APBIs). The PBIs are reductively activated by DT-diaphorase and alkylate the phosphate backbone of DNA via major groove interactions, while the APBIs are reductively inactivated by this enzyme since only the quinone form inhibits topoisomerase II. Bulk at the 7-position (butyl instead of methyl) significantly decreases k(cat)/K(m) for DT-diaphorase reductase activity for both PBIs and APBIs. As a result, a 7-butyl PBI has little cytotoxicity while the 7-butyl APBI has enhanced cytotoxicity. The type of 3-substituent and the configuration of the 3-position of the PBIs and APBIs influence DT-diaphorase substrate activity to a lesser degree. Bulk at the 7-position (butyl instead of methyl) had an adverse effect on APBI inhibition of topoisomerase II while the configuration of the 3-position had either an adverse or positive effect on inhibition of this enzyme. The configuration of the 3-position, when substituted with a hydrogen bond donor, influences the PBI reductive alkylation of DNA homopolymers. The rationale for this observation is that the R or S stereoisomers will determine if the 3-substituent points in the 3' or 5' direction and thereby influence the hydrogen-bonding interactions. The above findings were used to rationalize the relative cytotoxicity of various PBI and APBI derivatives.

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.

A series of quinone substrates were modeled into the active site of human DT-diaphorase and minimized. Correlation of these models with the substrate specificity k(cat)/K(m) provided insights into the structural requirements of quinone substrates. The W105, F106, and H194 residues can influence the position of the quinone substrate in the active site resulting in formation of one of the two possible Michael anions resulting from hydride transfer from FADH(2). Electron withdrawing groups on the substrate can stabilize these anions resulting in excellent substrate specificity. Inspection of models indicated that the W-105 and F-106 residues form parallel walls that will accommodate large polycyclic substrates. Thus excellent polycyclic substrates of DT-diaphorase were designed. However, the placement of tetrahedral centers on these polycyclic substrates interfered with the W-105 and the F-106 residues resulting in their exclusion from the active site. The histidine (H194) residue permits recognition of substrate enantiomers as a result of hydrogen bonding interactions. As a result of this study, it will be possible to design poor to excellent substrates of DT-diaphorase and take advantage of varying levels of this enzyme in histologically different cancers.

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