Tumorigenesis is a multistep process in which oncogenes play a key role in tumor formation, growth, and maintenance. MET was discovered as an oncogene that is activated by its ligand, hepatocyte growth factor. Deregulated signaling in the c-Met pathway has been observed in multiple tumor types. Herein we report the discovery of potent and selective triazolopyridazine small molecules that inhibit c-Met activity.

Inhibition of angiogenesis is a promising and clinically validated approach for limiting tumor growth and survival. The receptor tyrosine kinase Tie-2 is expressed almost exclusively in the vascular endothelium and is required for developmental angiogenesis and vessel maturation. However, the significance of Tie-2 signaling in tumor angiogenesis is not well understood. In order to evaluate the therapeutic utility of inhibiting Tie-2 signaling, we developed a series of potent and orally bioavailable small molecule Tie-2 kinase inhibitors with selectivity over other kinases, especially those that are believed to be important for tumor angiogenesis. Our earlier work provided pyridinyl pyrimidine 6 as a potent, nonselective Tie-2 inhibitor that was designed on the basis of X-ray cocrystal structures of KDR inhibitors 34 (triazine) and 35 (nicotinamide). Lead optimization resulted in pyridinyl triazine 63, which exhibited >30-fold selectivity over a panel of kinases, good oral exposure, and in vivo inhibition of Tie-2 phosphorylation.

Abstract. In interphase cells, a-casein kinase I (et-CKI) is found associated with cytosolic vesicular structures, the centrosome, and within the nucleus. To identify the specific vesicular structures with which a-CKI is associated, established cell lines and primary rat neurons were immunofluorescently labeled with an antibody raised to a-CKI. In nonneuronal cells, a-CKI colocalizes with vesicular structures which align with microtubules and are partially coincident with both Golgi and endoplasmic reticulum markers. In neurons, o~-CKI colocalizes with synaptic vesicle markers. When synaptic vesicles were purified from rat brain, they were highly enriched in a CKI, based on activity and immunoreactivity. The synaptic vesicle-associated CKI is an extrinsic kinase and was eluted from synaptic vesicles and purified. This purified CKI has properties most similar to o~-CKI. When the activities of casein kinase I or II were specifically inhibited on isolated synaptic vesicles, CKI was shown to phosphorylate a specific subset of vesicle proteins, one of which was identified as the synaptic vesicle-specific protein SV2. As with ot-CKI, the synaptic vesicle CKI is inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2). However, synthesis of PIP2 was detected only in plasma membranecontaining fractions. Therefore, PIP2 may spatially regulate CKI. Since PIP2 synthesis is required for secretion, this inhibition of CKI may be important for the regulation of secretion. CELLS transport proteins and lipids throughout their cytoplasm using small vesicles containing specific integral membrane proteins (2-5, 13, 24, 35, 38, 52, 58, 62, 72-74). This transport system is microtubule dependent and requires motor proteins (16,19,31). In this process, vesicles bud from donor membranes and are shunted to specific acceptor membranes where the vesicles fuse and release their contents. These general events can be divided into constituitive and regulated pathways (13,17,24,35,41,65,(72)(73)(74). Although distinct, the two pathways appear to have similar components in common. A typical constitutive pathway would be the shuttling of vesicles within and between the endoplasmic reticulum, the Golgi organelle, and the plasma membrane. The movement and CaE+-dependent fusion of synaptic vesicles provides an example of a regulated secretory pathway.Stidhof and Jahn (74) physin and synaptobrevin, and two proteins integral to the plasma membrane, syntaxin and neurexin are involved in docking (4,5,10,11,26,62,68,75). Synaptotagmin, another vesicle integral membrane protein, is thought to act in both the positioning of vesicles at the plasma membrane and the Ca2+-dependent step of the fusion process (12,22,34,37,55,60,61). Finally, the SNAPs (N-ethylmaleimidesensitive factor-associated proteins) have been linked to vesicle targeting and fusion (11,15,35,71,77).Within each of these stages, the myriad of processes involved are extensively regulated. Evidence exists showing that G-proteins (16-18, 23, 41, 42), phosphoinositide kinases (23, 45-48, 69),...

The recognition that aberrant angiogenesis contributes to the pathology of inflammatory diseases, cancer, and myocardial ischemia has generated considerable interest in the molecular mechanisms that regulate blood vessel growth. The receptor tyrosine kinase Tie-2 is expressed primarily by vascular endothelial cells and is critical for embryonic vasculogenesis. Interference with the Tie-2 pathway by diverse blocking agents such as soluble Tie-2 receptors, anti-Tie-2 intrabodies, anti-Ang-2 antibodies, and peptide-Fc conjugates has been shown to suppress tumor growth in xenograft studies. An alternative strategy for interfering with the Tie-2 signaling pathway involves direct inhibition of the kinase functions of the Tie-2 receptor. Herein we describe the development of alkynylpyrimidine amide derivatives as potent, selective, and orally available ATP-competitive inhibitors of Tie-2 autophosphorylation.