专利-合成路线.pdf

Bioconjugate Chem. 2002, 13, 855−869 855 Cathepsin B-Labile Dipeptide Linkers for Lysosomal Release of Doxorubicin from Internalizing Immunoconjugates: Model Studies of Enzymatic Drug Release and Antigen-Specific In Vitro Anticancer Activity Gene M. Dubowchik,* Raymond A. Firestone,*,† Linda Padilla, David Willner, Sandra J. Hofstead, Kathleen Mosure, Jay O. Knipe, Shirley J. Lasch,§ and Pamela A. Trail§ Bristol-Myers Squibb Pharmaceutical Research Institute, P.O. Box 5100, Wallingford, Connecticut 06492. Received April 2, 2002; Revised Manuscript Received April 30, 2002 The anticancer drug doxorubicin (DOX) has been linked to chimeric BR96, an internalizing monoclonal antibody that binds to a Lewisy-related, tumor-associated antigen, through two lysosomally cleavable dipeptides, Phe-Lys and Val-Cit, giving immunoconjugates 72 and 73. A self-immolative p-aminoben- zyloxycarbonyl (PABC) spacer between the dipeptides and the DOX was required for rapid and quantitative generation of free drug. DOX release from model substrate Z-Phe-Lys-PABC-DOX 49 was 30-fold faster than from Z-Val-Cit-PABC-DOX 42 with the cysteine protease cathepsin B alone, but rates were identical in a rat liver lysosomal preparation suggesting the participation of more than one enzyme. Conjugates 72 and 73 showed rapid and near quantitative drug release with cathepsin B and in a lysosomal preparation, while demonstrating excellent stability in human plasma. Against tumor cell lines with varying levels of BR96 expression, both conjugates showed potent, antigen-specific cytotoxic activity, suggesting that they will be effective in delivering DOX s