The camptothecins are being widely used in the treatment of solid tumors. Two camptothecin analogs are already on the market and a number of others are in various stages of research and development. However, for all their promise, all known camptothecin analogs contain a fundamental structural liability: the unstable E-ring lactone. Until recently it has been dogma in this field that the presence of the E-ring lactone was essential to biological activity. However, there are recent signs that the functionality in the E-ring can be changed without detriment and, indeed, with benefit. It is the hypothesis of this grant that significantly improved derivatives of camptothecin are still needed and that the next generation of camptothecin derivatives should either contain a lactone displaying markedly improved stability or agents that do not contain the E-ring lactone. It is the aim of this grant to discover these derivatives through the chemical synthesis of focused libraries of modified-lactone or non-lactone analogs of camptothecin followed by the rapid assaying of these libraries for stability/chemical properties, ability to inhibit the enzyme toposisomerase 1, and anti-tumor activity. In this grant we propose to conduct the following experiments: 1) Synthesize A,B,E-ring modified alpha-hydroxy lactone campothecins, as well as exploratory and focused libraries concerning non-lactone camptothecins. The focused libraries will be based on promising candidates that have been identified during the early in vitro analyses. 2) Characterize the in vitro potency of the exploratory and focused library of analogs against 9 distinct human cancer cell lines utilizing a rapid and high-throughput MTS assay. This assay will be performed in the absence and presence of physiologically relevant levels of human serum albumin in order to determine any effect this highly abundant blood protein may exert on analog activity. Analogs displaying superior IC50 values (within the 10 nM range or below) in the presence of human serum albumin will be selected for synthetic scale-up and a more thorough in vitro characterization. 3) Perform a thorough analysis of the physical properties of each analog to ensure the net stability prior to more extensive in vivo characterization. 4) Compare the lead, blood-stable camptothecin derivatives with FDA-approved camptothecins for activity in mice bearing human tumor xenografts. Pharmacokinetic and drug metabolism parameters for each of the lead agents will be determined.
