2005;1754:3. The Eph/ephrin family is the largest among tyrosine kinases and is unique in that the ligands and receptors are both membrane bound providing the possibility for bidirectional cell-cell signaling. Genome analysis reveals that there are 14 Eph receptors and eight ephrin ligands. Eph receptor signaling is responsible for arguably the most diverse set of biological phenomena of any tyrosine kinase family including organ development, tissue remodeling, neuronal signaling, insulin secretion, and bone metabolism.1, 2 Not surprisingly, deregulation of ephrin-dependent signaling has been implicated in pathological conditions related to all of these systems.3 The involvement of Eph/ephrin-signaling in tumorigenesis has been the most extensively investigated due to frequent upregulation of Eph receptor or ligand expression in numerous tumor types.4C6 The emerging picture is complicated by the diversity of biological function that is associated with individual receptors and ligands, including oncogenic or tumor suppressor functions. The possibility of targeting Eph/ephrins therapeutically may be the most straightforward in the context of inhibiting Eph/ephrin-signaling in the vasculature as a 2-HG (sodium salt) means of preventing tumor angiogenesis.2, 6 Currently only a few small molecule Eph kinase inhibitors have been reported in the literature including 2,4-diaminophenyl pyrimidines and 2,4-diaminophenyl triazines,7 and 3,7-diphenyl-4-amino-thieno and furo[3,2]pyridine8 EphB4 inhibitors. To date the majority of investigation into functions of Eph receptor tyrosine kinases has been accomplished using genetic and biochemical methods. Pharmacological methods, which are crucial to understanding what happens upon acute inhibition of Eph kinase activity, have been hampered by a lack of potent and selective Eph kinase inhibitors. To address this deficiency we undertook a survey of a collection of known kinase inhibitors for their ability to target Eph kinase activity using an EphB2 kinase activity-dependent cellular assay. Specifically we developed a cellular assay where the murine pre-B cell collection Ba/F3 was transformed with DNA encoding a fusion protein between Tel and the kinase domain name of EphB2 such that the cells are capable of growing in the absence of interleukin-3.9 EphB2 has been identified as a target of dasatinib and nilotinib by proteomic10 and cellular screening9 approaches. As nilotinib is known to be a considerably more selective inhibitor than dasatinib, we decided to use nilotinib as a lead compound and prepared a combinatorial library that should be able to mimic the DFG-out binding mode that nilotinib utilizes to bind to Abl.11 Targeting of the kinase DFG-out conformation is relatively common; inhibitors of this class are termed type II inhibitors.12 An analysis of the structural features of these 2-HG (sodium salt) inhibitors reveals that they conform to a pharmacophore model that consists of a heterocyclic head motif that recognizes the kinase hinge region, a linker segment that traverses across the kinase gatekeeper residue and a tail motif that occupies a hydrophobic pocket made accessible by the flip of the DFG-out motif.13 A solution-phase SLC2A4 combinatorial library of several hundred compounds 2-HG (sodium salt) was synthesized 2-HG (sodium salt) where diverse heterocyclic head motifs were attached to three distinct linker-tail motifs (Fig. 1). The linker tail motifs were chosen to mimic functionality found in type II inhibitors such as nilotinib,11 NVP-AST487,14 and sorafenib.15 Library synthesis consisted simply of reacting an aniline from your linker-tail motif with head groups using palladium mediated amination or amide-bond forming chemistries. The compounds were purified by mass-triggered reverse phase high pressure liquid chromatography and characterized by mass and 1H NMR. Open in a separate window Physique 1 Chemical structures of five representative library members are shown. Using the Ba/F3 cellular assay, the library was screened in dose-response format against a panel of 30 unique tyrosine kinase transformed Ba/F3 cells including Tel-EphB2.9 This screen revealed that a subset 2-HG (sodium salt) of the library possessed inhibitory activity against EphB2, c-Kit, Abl, Bcr-Abl, PDGFR, and FGFR2/3/4 (Supplementary Table 1). Some compounds exhibited cross reactivity with Bcr-Abl (inhibitors 1, 3, 7, 8) or FGFR2/3/4 (inhibitor 10). Abl, PDGFR, and c-kit were the mostly frequently inhibited kinases by.