The design, synthesis, and biological evaluation of novel SH2 domain inhibitors

A novel series of cis-enediol phosphates has been designed and synthesized. These compounds were composed of two major components: the first is an aryl phosphate designed to bind in the phosphotyrosine (pTyr) pocket; the second is a stereodiversified cis-enediol framework bearing different side chains designed to act as a linker and bind to the pTyr+3 binding site. The biological activities were determined via a fluorescence competitive binding assay. All free phosphates bearing the cis-enediol scaffold showed significant binding activity for the Lck SH2 protein with IC50 values in the submicromolar range. The structure-activity relationship and stereochemical preferences for binding were observed among these analogues in a synergistic way. To explore the effect of difluoromethyl phosphonate (DFMP) analogues on SH2 domain inhibitions, a novel non-hydrolyzable prodrug 45 has been synthesized and clearly showed low micromolar growth inhibition against a panel of cancer cell lines without significant cytotoxicity. Flow cytometry studies on DG75 and WEHI231 cell lines showed accumulated 32N DNA (polyploidy phenomena) and failure of mitotic spindle formation. The corresponding free phosphonate 48 has shown inhibitory effects on three phosphatases: Cdc14 (Ki = 45 μM), HCPTP-A (Ki = 9 μM), and HCPTP-B (Ki = 15 μM), and also inhibits peptide binding to the Lck SH2 domain IC50 = 0.3 μM). The results from the above experiments suggest that a cis-enediol framework can generate potent binding activities for the Lck SH2 domain. Furthermore, difluoromethyl phosphonate prodrug 45 acts as an inhibitor of tumor cell growth via a novel mechanism