Synthesis and in vitro effects of BMS-753493, an epothilone-folate conjugate designed to selectively target FR expressing tumor cells

4155 The human folate receptor alpha (FRα) is a tumor marker that is selectively overexpressed in certain tumor types of epithelial origin. Folic acid (FA or folate) is a high affinity ligand for the FR and is internalized by FR-mediated endocytosis; therefore, conjugation of a therapeutic to FA is a promising strategy for targeting agents to tumors while limiting exposure to normal tissues having little or no FR. The ability of the high affinity FRα to deliver a diverse range of cargo for successful endocytosis in FR-positive cell lines is well-documented. The epothilones (e.g. ixabepilone) are a class of new microtubule stabilization agents with demonstrated clinical activity. The potent cell-killing effects of the epothilones make them ideal candidates for tumor targeting strategies. An array of epothilone-folate conjugates were prepared as candidates for targeting to FR-expressing tumor cells. Synthesis of the folate receptor recognition fragment began with commercially available FA which was enzymatically converted to pteroic acid followed by conversion to the corresponding N10-trifluoroacetyl derivative. The pteroic acid intermediate was incorporated into a five-step solid phase synthesis of Folate-Asp-Arg-Asp-Cys-OH, which served as the folate-containing precursor to the conjugate. Various epothilones were modified for conjugation to the folate-containing fragment. A cleavable linker comprised of a reductively labile disulfide bond proximal to a carbonate was engineered to release the epothilone following endocytosis. Relative binding affinity (RA) of the epothilone-folate conjugate compared to FA was measured in a radioligand binding assay utilizing FR-expressing cells and tritiated FA. Clonogenic assays were used to assess the cytotoxicity of conjugates against FR-positive tumor cells in vitro. Naturally occurring epothilone A was converted in seven chemical steps to a 12,13-aziridinyl epothilone intermediate. The aziridine moiety was further functionalized to provide a site for linkage to a folate receptor recognition fragment, Folate-Asp-Arg-Asp-Cys-OH. The resulting epothilone-folate conjugate, BMS-753493, was determined to bind competently to the folate receptor with RA=0.77 (compared to FA, RA=1.0). In cell culture, the parent aziridinyl epothilone, BMS-748285, which is released from the conjugate has an IC90 = 4.3 nM when tested against KB tumor cells in a clonogenic assay. Additional cell culture studies demonstrated that the conjugate BMS-753493 is selectively cytotoxic to tumor cells expressing FR. These studies demonstrate the successful synthesis of an epothilone-folate conjugate that effectively binds to FRα in a manner comparable to folic acid. BMS-753493 exhibits potent cytotoxicity towards FR-positive tumor cells. Collectively, the in vitro data suggests that BMS-753493 is a strong candidate for further development.