A new class of protein tyrosine kinase inhibitors was identified that is based on an oxindole core (indolinones). Two compounds from this class inhibited the kinase activity of fibroblast growth factor receptor 1 (FGFR1) and showed differential specificity toward other receptor tyrosine kinases. Crystal structures of the tyrosine kinase domain of FGFR1 in complex with the two compounds were determined. The oxindole occupies the site in which the adenine of adenosine triphosphate binds, whereas the moieties that extend from the oxindole contact residues in the hinge region between the two kinase lobes. The more specific inhibitor of FGFR1 induces a conformational change in the nucleotide-binding loop. This structural information will facilitate the design of new inhibitors for use in the treatment of cancer and other diseases in which cell signaling by tyrosine kinases plays a crucial role in disease pathogenesis.

3-Substituted indolin-2-ones have been designed and synthesized as a novel class of tyrosine kinase inhibitors which exhibit selectivity toward different receptor tyrosine kinases (RTKs). These compounds have been evaluated for their relative inhibitory properties against a panel of RTKs in intact cells. By modifying the 3-substituted indolin-2-ones, we have identified compounds which showed selective inhibition of the ligand-dependent autophosphorylation of various RTKs at submicromolar levels in cells. Structure-activity analysis for these compounds and their relative potency and selectivity to inhibit particular RTKs has determined that (1) 3-[(five-membered heteroaryl ring)methylidenyl]indolin-2-ones are highly specific against the VEGF (Flk-1) RTK activity, (2) 3-(substituted benzylidenyl)indolin-2-ones containing bulky group(s) in the phenyl ring at the C-3 position of indolin-2-ones showed high selectivity toward the EGF and Her-2 RTKs, and (3) the compound containing an extended side chain at the C-3 position of the indolin-2-one (16) exhibited high potency and selectivity when tested against the PDGF and VEGF (Flk-1) RTKs. Recent published crystallographic data for two of these 3-substituted indolin-2-ones provides a rationale to suggest that these compounds may bind in the ATP binding pocket of RTKs. The structure-activity analysis supports the use of subsets of these compounds as specific chemical leads for the development of RTK-specific drugs with broad application for the treatment of human diseases.

To improve the antitumor properties and optimize the pharmaceutical properties including solubility and protein binding of indolin-2-ones, a number of different basic and weakly basic analogues were designed and synthesized. 5-[5-Fluoro-2-oxo-1,2-dihydroindol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylaminoethyl)amide (12b or SU11248) has been found to show the best overall profile in terms of potency for the VEGF-R2 and PDGF-Rbeta tyrosine kinase at biochemical and cellular levels, solubility, protein binding, and bioavailability. 12b is currently in phase I clinical trials for the treatment of cancers.

Receptor tyrosine kinases (RTKs) have been implicated as therapeutic targets for the treatment of human diseases including cancers, inflammatory diseases, cardiovascular diseases including arterial restenosis, and fibrotic diseases of the lung, liver, and kidney. Three classes of 3-substituted indolin-2-ones containing propionic acid functionality attached to the pyrrole ring at the C-3 position of the core have been identified as catalytic inhibitors of the vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) RTKs. Some of the compounds were found to inhibit the tyrosine kinase activity associated with isolated vascular endothelial growth factor receptor 2 (VEGF-R2) [fetal liver tyrosine kinase 1 (Flk-1)/kinase insert domain-containing receptor (KDR)], fibroblast growth factor receptor (FGF-R), and platelet-derived growth factor receptor (PDGF-R) tyrosine kinase with IC(50) values at nanomolar level. Thus, compound 1 showed inhibition against VEGF-R2 (Flk-1/KDR) and FGF-R1 tyrosine kinase activity with IC(50) values of 20 and 30 nM, respectively, while compound 16f inhibited the PDGF-R tyrosine kinase activity with IC(50) value of 10 nM. Structural models and structure-activity relationship analysis of these compounds for the target receptors are discussed. The cellular activities of these compounds were profiled using cellular proliferation assays as measured by bromodeoxyuridine (BrdU) incorporation. Specific and potent inhibition of cell growth was observed for some of these compounds. These data provide evidence that these compounds can be used to inhibit the function of these target receptors.

Furin plays a crucial role in embryogenesis and homeostasis and in diseases such as Alzheimer's disease, cancer, and viral and bacterial infections. Thus, inhibition of furin may provide a feasible and promising approach for therapeutic intervention of furin-mediated disease mechanisms. Here, we report on a class of small molecule furin inhibitors based on 2,5-dideoxystreptamine. Derivatization of 2,5-dideoxystreptamine by the addition of guanidinylated aryl groups yielded a set of furin inhibitors with nanomolar range potency against furin when assayed in a biochemical cleavage assay. Moreover, a subset of these furin inhibitors protected RAW 264.7 macrophage cells from toxicity caused by furin-dependent processing of anthrax protective antigen. These inhibitors were found to behave as competitive inhibitors of furin and to be relatively specific for furin. Molecular modeling revealed that these inhibitors may target the active site of furin as they showed site occupancy similar to the alkylating inhibitor decanoyl-Arg-Val-LysArg-CH 2Cl. The compounds presented here are bona fide synthetic small molecule furin inhibitors that exhibit potency in the nanomolar range, suggesting that they may serve as valuable tools for studying furin action and potential therapeutics agents for furindependent diseases.serine endoprotease ͉ prohormone ͉ proprotein convertase F urin is a membrane-anchored, calcium-dependent serine endoprotease and is expressed in all tissues and cell lines examined (1-3). It is the first and, so far, the best-characterized member of the mammalian subtilisin-like family of prohormone/proprotein convertases (PCs), which convert precursors of many secreted proteins and peptide hormones into their biologically active forms (1-3). Furin is predominantly localized in the trans-Golgi network and cycles between this compartment, the cell surface, and the endosomes (2, 4). Hence, furin is able to access and efficiently process a diverse spectrum of substrates including growth factors, receptors, serum proteins, matrix metalloproteinases, viral envelope glycoproteins, and bacterial toxins, typically at sites with the consensus sequence Arg-Xaa-Lys/Arg-Arg 2 (2).Although furin plays an essential role in embryogenesis and homeostasis, this endoprotease has also been implicated in the neurodegeneration associated with Alzheimer's disease, and tumor metastasis and the activation and virulence of many bacterial and viral pathogens (2). It has been demonstrated that furin inhibitors modulate tumor growth (5) and attenuate the toxicity of anthrax (6) and Pseudomonas aeruginosa (7) toxins and cytomegalovirus (8) in cell culture and animal models. Therefore, furin inhibitors hold great promise as potential therapeutic agents for treating furinmediated diseases and viral and bacterial infections, particularly for short-term therapy.To date, most furin inhibitors reported in the literature have been proteins (9-19) or peptides (20-24), which show excellent potency against furin, and largely mimic the substrate in bindi...

A series of new 3-substituted indolin-2-ones containing a tetrahydroindole moiety was developed as specific inhibitors of receptor tyrosine kinases associated with VEGF-R, FGF-R, and PDGF-R growth factor receptors. These compounds were evaluated for their inhibitory properties toward VEGF-R2 (Flk-1/KDR), FGF-R1, PDGF-Rbeta, p60(c)()(-)()(Src)(), and EGF-R tyrosine kinases and their ability to inhibit growth factor-dependent cell proliferation. Structure-activity relationships of this new pharmacophore have been determined at the level of kinase inhibition. Compounds containing a propionic acid moiety at the C-3' position of the tetrahydroindole ring represented the most potent indolin-2-ones to inactivate the VEGF, FGF, and PDGF receptor kinases. The inhibitory activities of 9d against VEGF-R2 (Flk-1), 9h against FGF-R1, and 9b against PDGF-Rbeta were 4, 80, and 4 nM, respectively. However, all of these compounds were inactive when tested against the EGF-R tyrosine kinase. Compounds 9a and 9b represented the most potent inhibitors of these classes to inhibit both biochemical kinase and growth factor-dependent cell proliferation for these three targets. In addition, compound 9a was cocrystallized with the catalytic domain of FGF-R1 providing evidence to explain the structure-activity relationship results. This study has provided evidence to support the potential of these new tyrosine kinase inhibitors for the treatment of angiogenesis and other growth factor-related diseases including human cancers.

Abstract-The platelet-derived growth factor (PDGF) ligands and their receptors have been implicated as critical regulators of the formation of arterial lesions after tissue injury. SU9518 (3[5-{5-bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl}-2,4-dimethyl-1H-pyrrol-3-yl]propionic acid) is a novel synthetic indolinone that potently and selectively inhibits the cellular PDGF receptor kinase and PDGF receptor-induced cell proliferation. Inhibition of PDGF receptor phosphorylation in cell-based assays occurs within 5 minutes after drug exposure and persists for Ͼ6 hours after drug removal. The pharmacokinetics indicate plasma levels that exceeded the effective concentration required to inhibit the PDGF receptor in cells for up to 8 hours or 7 days after a single oral or subcutaneous administration, respectively. In the rat balloon arterial injury-induced stenosis model, once-daily oral or once-weekly subcutaneous administration of SU9518 reduced intimal thickening of the carotid artery (ratio of neointimal to medial area,

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