Topoisomerase I is an important target for the development of new chemotherapeutic agents for the treatment of cancer in humans. Although some members of the camptothecin class of topoisomerase I inhibitors are presently in clinical use as anticancer agents, the camptothecins suffer from a number of inherent limitations, including chemical and metabolic instability due to lactone ring opening and rapid reversibility of topoisomerase I inhibition upon drug removal. Because of these limitations, effective chemotherapy with the camptothecins requires long I.V. infusions and prolonged and continuous exposure. Preliminary studies in our research group, in collaboration with others, have resulted in the synthesis of a new class of topoisomerase I inhibitors, the indenoisoquinolines. A combination of enzyme inhibition studies, in vitro cytotoxicity results in human cancer cell cultures, and in vivo animal studies have provided compelling evidence that the indenoisoquinolines will overcome some of the limitations of the camptothecins. One of the main goals of the presently proposed research program will be to design and synthesize more effective indenoisoquinolines as topoisomerase I inhibitors with potential clinical application in the treatment of cancer in humans. The new compounds will be synthesized using a novel condensation reaction that was developed in the P1's research group. Condensation of Schiff bases with homophthalic anliydrides will provide 3-aryl-4-carboxyisoquinolines, which will be modified by a variety of methods to afford new indenoisoquinolines with enhanced biological activities. One of the strategies to be employed to enhance the anticancer activities of the indenoisoquinolines will be to attach aminoalkyl and polyaminoalkyl side chains to the indenoisoquinolines. Recent results have shown a dramatic increase in both topoisomerase I inhibitory activity and in vitro anticancer activity when a 3'-aminoalkyl substituent is attached the nitrogen atom of the indenoisoquinolines. This boost in activity has been rationalized as being due to: 1) facilitated cellular uptake; 2) ionic bonding of the positively charged, protonated amino group of the side chain with the negatively charged phosphodiesters of the DNA backbone before intercalation; and 3) stabilization of the intercalation complex by ionic bonding. Some of the compounds proposed in the present application are designed to explored the effects of: I) incorporating multiple amino groups in the side chain that will target more than one phosphodiester linkage; 2) altering the distances separating the amino groups and the indenoisoquinolines nucleus; 3) attaching the aminoalkyl side chains to different regions of the indenoisoquinoline system; and 4) modifying the indenoisoquinoline framework so that it more closely resembles camptothecin. Although several crystal structures of human topoisomerase I in covalent and non-covalent complex with DNA have recently been published, there are no crystal structures available of ternary complexes consisting of topoisomerase I, DNA, and an inhibitor. We will attempt to synthesize a conformationally restricted ternary complex in which the inhibitor is covalently bound to the oligonucleotide after DNA cleavage. This should facilitate crystallization of the complex for X-ray structure determination.
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