Camptothecin (CPT) derivatives are effective anticancer drugs, especially against solid tumors. As CPTs are chemically unstable and have clinical limitations, we have synthesized indenoisoquinolines as novel topoisomerase I (Top1) inhibitors. We presently report two indenoisoquinoline derivatives, NSC 725776 and NSC 724998, which have been selected for therapeutic development. Both are potent Top1 inhibitors and induce Top1 cleavage at unique genomic positions compared with CPT. Consistent with Top1 poisoning, protein-linked DNA breaks were detected in cells treated with NSC 725776 and NSC 724998 at nanomolar concentrations. Those druginduced protein-linked DNA breaks persisted longer after drug removal than those produced by CPT. Studies in human cells in culture show that NSC 725776 and NSC 724998 exert antiproliferative activity at submicromolar concentrations. Furthermore, NSC 725776 and NSC 724998 show crossresistance in cells deficient or silenced for Top1, which is consistent with their selective Top1 targeting. Similar to other known Top1 inhibitors, NSC 725776-treated and NSC 724998-treated cells show an arrest of cell cycle progression in both S and G 2 -M and a dependence on functional p53 for their cytotoxicity. Dose-dependent ;-H2AX foci formation was readily observed in cells treated with NSC 725776 and NSC 724998. These ;-H2AX foci were detectable at pharmacologically relevant doses for up to 24 h and thus could be used as biomarkers for clinical trials (phase 0). [Cancer Res 2007; 67(21):10397-405]

The indenoisoquinolines are a class of cytotoxic topoisomerase I inhibitors that offer certain advantages over the camptothecins, including the greater stabilities of the compounds themselves, as well as the greater stabilities of their drug-enzyme-DNA cleavage complexes. To investigate the possible biological roles of the di(methoxy) and methylenedioxy substituents present on the aromatic rings of the previously synthesized indenoisoquinoline topoisomerase I inhibitors, a series of compounds lacking these substituents was synthesized and tested for both cytotoxicity in cancer cell cultures and for enzyme inhibitory activity. The results indicate that the aromatic substituents make a small, but consistently observable contribution to the biological activity. Molecular models derived for the binding of the unsubstituted indenoisoquinolines in ternary complex with DNA and topoisomerase I indicate that the substituents on the lactam nitrogen project out of the major groove, and the carbonyl group is directed out of the minor groove, where it is involved in a hydrogen bonding interaction with the side chain guanidine group of Arg364. The DNA cleavage patterns observed in the presence of topoisomerase I and various indenoisoquinolines were similar, although significant differences were detected. There were also variations in the DNA cleavage pattern seen with camptothecin vs the indenoisoquinolines, which indicates that these two classes of topoisomerase I inhibitors are likely to target the cancer cell genome differently, resulting in different spectra of anticancer activity. The most cytotoxic of the presently synthesized indenoisoquinolines has a 4-amino-n-butyl group on the lactam nitrogen.

The indenoisoquinolines are a class of noncamptothecin topoisomerase I inhibitors that display significant cytotoxicity in human cancer cell cultures. They offer a number of potential advantages over the camptothecins, including greater chemical stability, formation of more persistent cleavage complexes, and induction of a unique pattern of DNA cleavage sites. Molecular modeling has suggested that substituents on the indenoisoquinoline lactam nitrogen would protrude out of the DNA duplex in the ternary cleavage complex through the major groove. This indicates that relatively large substituents in that location would be tolerated without compromising biological activity. As a strategy for increasing the potencies and potential therapeutic usefulness of the indenoisoquinolines, a series of compounds was synthesized containing polyamine side chains on the lactam nitrogen. The rationale for the synthesis of these compounds was that the positively charged ammonium cations would increase DNA affinity through electrostatic binding to the negatively charged DNA backbone, and the polyamines might also facilitate cellular uptake by utilization of polyamine transporters. The key step in the synthesis involved the condensation of Schiff bases, containing protected amine side chains, with substituted homophthalic anhydrides, to afford cis-3-aryl-4-carboxy-1-isoquinolones. These isoquinolones were then converted to indenoisoquinolines with thionyl chloride. Although monoamines were much more potent than the lead compound, no significant increase in potency was observed through incorporation of additional amino groups in the side chain. However, one of the monoamine analogues, which features a bis(2-hydroxyethyl)amino group in the side chain, proved to be one of the most cytotoxic indenoisoquinoline synthesized to date, with a GI50 mean-graph midpoint (MGM) of 0.07 microM in the NIH human cancer cell culture screen, and topoisomerase I inhibitory activity comparable to that of camptothecin.