Potent c-Met Inhibitors Research Articles

Discovery of a 5H-Benzo[4,5]cyclohepta[1,2-b]pyridin-5-one (MK-2461) Inhibitor of c-Met Kinase for the Treatment of Cancer

c-Met is a transmembrane tyrosine kinase that mediates activation of several signaling pathways implicated in aggressive cancer phenotypes. In recent years, research into this area has highlighted c-Met as an attractive cancer drug target, triggering a number of approaches to disrupt aberrant c-Met signaling. Screening efforts identified a unique class of 5H-benzo[4,5]cyclohepta[1,2-b]pyridin-5-one kinase inhibitors, exemplified by 1. Subsequent SAR studies led to the development of 81 (MK-2461), a potent inhibitor of c-Met that was efficacious in preclinical animal models of tumor suppression. In addition, biochemical studies and X-ray analysis have revealed that this unique class of kinase inhibitors binds preferentially to the activated (phosphorylated) form of the kinase. This report details the development of 81 and provides a description of its unique biochemical properties.

Abstract 3577: TAS-115: a highly potent c-Met + VEGFRs dual inhibitor with prominently safer profile

Abstract c-Met is a multi-functional proto-oncogenic receptor tyrosine kinase (RTK) involving various malignant phenotypes and aberrant expressions of HGF/c-Met axis, which are associated with poor prognosis of diverse cancers. On the other hand, VEGF/VEGFR inhibition has achieved a notable status of standard therapy for several cancers. Moreover, recent studies suggest that both RTKs work in a complementary way on cancer malignant phenotypes including angiogenesis, metastasis, and hypoxia reaction. Therefore, their dual inhibition seemed to be an attractive strategy for cancer therapy, and indeed several compounds inhibiting both targets have been subjected to clinical evaluation. However, clinical efficacy appeared to be limited so far, probably due to their poor selective of mode of action and toxicity resulting from VEGFR inhibition which result in poor tolerability restricting the dosage in the clinical setting. The optimized potent c-Met + VEGFRs dual inhibitor with better safety profile may become a successful treatment modality meeting the medical needs. To address this issue, we have developed TAS-115, a novel small molecule c-Met + VEGFRs dual inhibitor. In biochemical assay, it was identified to be a potent ATP competitive inhibitor of both c-Met and VEGFR2 at IC50 of around 10 nM that is equal or more potent than those of other known inhibitors such as crizotinib, foretinib, or sunitinib. In cellular assays, it also potently inhibited the phosophorylation of both kinases with the same potency as HGF/c-Met or VEGF dependent cellular growth at IC50 range of 10 nM. Importantly, the IC50 values under no HGF/c-Met or VEGF dependent condition were over 20,000 nM. The difference between both conditions were more than 2000-fold, while the other inhibitors showed only 10∼300 hold difference when compared to control, not stimulated cells. This selectivity was also confirmed in a panel of 40 cell lines; the IC50 values were highly correlated with HGF, c-Met mRNA expression level as revealed by coefficient value of R2>0.65.In vivo studies, orally administered TAS-115 exhibited highly potent anti-tumor activity against c-Met positive and negative xenografts including complete tumor eradications accompanying by abolishing of c-Met and VEGFR2 signal cascade. The effective doses (ED50) determined in about 12 xenograft models ranged from 4 to 30 mg/kg. The tolerability study of consecutive dosing for 4 wks revealed no animal death incidence, even at more than 28 times higher exposure over the ED50 values of TAS-115, which was surprising when compared with safety of other VEGFR inhibitors including foretinib, sunitinib and sorafenib, whose therapeutic indices (MTD/ED50) were only 2∼4. Taken together, TAS-115 is a highly potent c-Met + VEGFRs dual inhibitor with prominent selectivity and safer profile indicating its therapeutic potentials in human cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3577. doi:10.1158/1538-7445.AM2011-3577

Abstract LB-390: Antitumor efficacy of crizotinib (PF-02341066), a potent and selective ALK and c-Met RTK inhibitor, in EML4-ALK driven NSCLC tumors in vitro and in vivo

Abstract EML4-ALK fusion was recently characterized as an “Addicted Oncogene” in a subset of human lung adenocarcinoma, and it plays an essential role in regulation of the tumor cell survival, growth and metastasis. Crizotinib is a potent and selective ATP competitive small molecule inhibitor of ALK and c-Met. It is currently in clinical trials for advanced non-small cell lung cancers positive for ALK fusion. This report summarizes the pre-clinical pharmacology studies for crizotinib in the EML4-Alk positive tumor models to assess the pharmacodynamic inhibition of EML4-ALK, antitumor efficacy, PKPD relationships and antitumor mechanism of action in vitro and in vivo. Crizotinib potently inhibited the catalytic activity of ALK kinase (Ki = 0.5 nM) and the autophosphorylation of cellular EML4-ALK V1, V2, V3a and V3b with IC50 values ranging from 26–74 nM. Crizotinib also inhibited cell proliferation and induced apoptosis in NCI-H3122 human NSCLC cells harboring EML4-ALK V1 fusion with IC50s of 63 nM and 110 nM respectively. In the EML4-ALK V3a/b positive NCI-H2228 human NSCLC cells, crizotinib completely inhibited ALK phosphorylation (IC50 = 74 nM) but only partial inhibited cell proliferation and survival. This observation is consistent with the notion that only a portion of H2228 cells are EML4-ALK positive by Exon Array analysis. Crizotinib demonstrated marked tumor growth inhibition and regression in H3122 xenograft model at well tolerated dose levels. The antitumor efficacy of crizotinib was dose dependent and demonstrated a strong correlation to pharmacodynamic inhibition of ALK phosphorylation in vivo. PKPD modeling was conducted to understand the relationships between crizotinib plasma concentration to ALK target inhibition (EC50 = 19 nM) and antitumor efficacy (EC50 = 23 nM). Collectively, the results from H3122 model indicated that significant inhibition of EML4-ALK during the entire treatment period was necessary to achieve robust antitumor efficacy. Additional in vivo studies with crizotinib demonstrated dose dependent inhibition of EML4-ALK mediated signal transduction (STAT3, AKT, Erk, PLCγ, c-Myc), tumor cell proliferation (Ki67) and induction of apoptosis (caspase-3). Crizotinib also dose dependently inhibited total EML4-ALK levels in H3122 tumors indicating an additional antitumor mechanism of action by crizotinib in these tumors. Furthermore, a dose dependent increase in phospho-EGFR levels was observed after 4 days of drug treatment in H3122 model, indicating a compensation mechanism of “oncogene switching” in tumor cell signaling, and a potential resistance mechanism that may compromise “patient” responses to crizotinib treatment in these tumors. In conclusion, crizotinib was shown to be a potent inhibitor of EML4-ALK. It demonstrated marked antitumor efficacy in EML4-ALK dependent human NSCLC tumors in vitro and in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-390. doi:10.1158/1538-7445.AM2011-LB-390

Structural Basis for Selective Small Molecule Kinase Inhibition of Activated c-Met

The receptor tyrosine kinase c-Met is implicated in oncogenesis and is the target for several small molecule and biologic agents in clinical trials for the treatment of cancer. Binding of the hepatocyte growth factor to the cell surface receptor of c-Met induces activation via autophosphorylation of the kinase domain. Here we describe the structural basis of c-Met activation upon autophosphorylation and the selective small molecule inhibiton of autophosphorylated c-Met. MK-2461 is a potent c-Met inhibitor that is selective for the phosphorylated state of the enzyme. Compound 1 is an MK-2461 analog with a 20-fold enthalpy-driven preference for the autophosphorylated over unphosphorylated c-Met kinase domain. The crystal structure of the unbound kinase domain phosphorylated at Tyr-1234 and Tyr-1235 shows that activation loop phosphorylation leads to the ejection and disorder of the activation loop and rearrangement of helix αC and the G loop to generate a viable active site. Helix αC adopts a orientation different from that seen in activation loop mutants. The crystal structure of the complex formed by the autophosphorylated c-Met kinase domain and compound 1 reveals a significant induced fit conformational change of the G loop and ordering of the activation loop, explaining the selectivity of compound 1 for the autophosphorylated state. The results highlight the role of structural plasticity within the kinase domain in imparting the specificity of ligand binding and provide the framework for structure-guided design of activated c-Met inhibitors.

Synthesis and c-Met Kinase Inhibition of 3,5-Disubstituted and 3,5,7-Trisubstituted Quinolines: Identification of 3-(4-Acetylpiperazin-1-yl)-5-(3-nitrobenzylamino)-7- (trifluoromethyl)quinoline as a Novel Anticancer Agent

By use of an improved synthetic strategy, a series of 3,5-disubstituted and 3,5,7-trisubstituted quinolines were readily prepared. 3,5,7-Trisubstituted quinolines 21a-c, 21l, and 27a-c were identified as the most potent c-Met inhibitors with IC(50) of less than 1.0 nM. Compound 21b showed the most promising overall PK profile and has high potency and extraordinary selectivity to c-Met against c-Met family member Ron and 12 other tyrosine kinases. It produced constitutive inhibition of c-Met phosphorylation in c-Met dependent cell lines. At doses of 100 mg/kg, compound 21b showed statistically significant tumor growth inhibition (68-69%) in both NIH-3T3-TPR-Met and U-87 MG human gliobastoma xenograft models. These results clearly indicated that compound 21b is a potent and highly selective c-Met inhibitor. Its favorable in vitro and in vivo profiles warrant further investigation.

Expeditious one-pot synthesis of C3-piperazinyl-substituted quinolines: key precursors to potent c-Met inhibitors

An effective one-pot synthesis of quinolines bearing diverse C3-piperazinyl functions was developed by using a modified Friedländer's protocol. The method not only enables the synthesis of our early reported c-Met inhibitor on a large scale, but also provides a way to generate novel multi-substituted quinolines for further structure-activity relationship (SAR) study.

Fused bicyclic derivatives of 2,4-diaminopyrimidine as c-Met inhibitors

The HGF-c-Met signaling axis is an important paracrine mediator of epithelial–mesenchymal cell interactions involving the regulation of multiple cellular activities including cell motility, mitogenesis, morphogenesis, and angiogenesis. Dysregulation of c-Met signaling (e.g., overexpression or increased activation) is associated with the development of a wide range of tumor types; thus, inhibiting the HGF-c-Met pathway is predicted to lead to anti-tumor effects in many cancers. Elaboration of a 2-arylaminopyrimidine scaffold led to a series of potent c-Met inhibitors bearing a C4-2-amino-N-methylbenzamide group. Specifically, a series of C2-benzazepinone analogs demonstrated potent inhibition of c-Met in enzymatic and cellular assays. Kinase selectivity could be tuned by varying the nature of the alkyl group on the benzazepinone nitrogen.

Abstract B233: c-Met antagonist Avimer™ polypeptide that exhibits potent in vivo and in vitro inhibitor activity

Abstract c-Met is a receptor tyrosine kinase (RTK) that is activated upon binding to its only known ligand, hepatocyte growth factor (HGF). Although essential in development, aberrant c-Met signaling can lead to oncogenesis by promoting proliferation, survival, metastasis, and angiogenesis. Deregulated expression/activation of cMet is found in many neoplasms, and the linkage to disease severity and poor prognosis make this RTK an attractive candidate for targeted anticancer therapy. Challenges of inhibiting c-Met/HGF signaling with a classic antibody therapeutic approach have included: (1) the inhibition of receptor activation required more than one antibody, and (2) bivalent antibodies caused receptor activation. Using a novel non-antibody protein technology platform, we have identified potent polypeptide inhibitors of HGF-mediated c-Met activation. This non-antibody class of proteins, termed Avimer (avidity multimer) polypeptides, are based on naturally occurring domain (e.g., A-domain) containing proteins and can be generated using a phage display library and expressed in E. coli. c-Met antagonist Avimer polypeptides potently inhibit HGF-mediated receptor activation, migration, and proliferation in in vitro assays. Improvement in pharmacokinetic profile gained through attaching polyethylene glycol molecules to avimers enabled in vivo studies. In multiple preclinical tumor models (U87MG glioblastoma, KP-4 pancreatic, U118 glioblastoma), Avimer-treated animals exhibited reduced cMet activation in xenografts and significantly decreased tumor growth. These data indicate that the c-Met antagonist Avimer polypeptides are potent c-Met inhibitors and offer an alternative to small molecules or antibodies in targeting cMet/HGF pathway for anticancer therapy. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B233.

Abstract C190: BMS-777607, a potent small molecule c-MET inhibitor, inhibits prostate cancer progression in vitro

Abstract Overexpression of c-MET is highly associated with prostate cancer progression and therapy resistance. BMS-777607 is a selective and potent c-MET inhibitor that was advanced to clinical evaluation. Herein we report the effects of BMS-777607 in androgen-independent prostate cancer cells (PC-3, DU145). When assessed using an MTT assay, BMS-777607 treatment was found to inhibit cell growth in the absence of hepatocyte growth factor (HGF), with an IC50 >5.0 uM for both cell lines. In a wound-healing assay, BMS-777607 suppressed PC-3 and HGF-induced DU145 cell motility in a dose-dependent fashion (IC50 values of 3.0 and 0.5 uM, respectively). Moreover, BMS-777607 efficiently blocked HGF-triggered DU145 cell scattering in a cell scatter assay; with 40% inhibition at 0.2 uM. Using a Matrigel-based Transwell assay, BMS-777607 also inhibited PC-3 cell invasion in a dose-dependent manner, with 60% inhibition occurring at 1 uM. Western blot analysis demonstrated that BMS-777607 blocked c-MET autophosphorylation, and downregulated multiple downstream kinases involved in c-MET signaling pathway, including Src, Fak and Akt. These data indicate that BMS-777607 inhibited prostate cancer progression in vitro including proliferation, motility, scattering and invasion. Furthermore, the finding of more potent effects on cell migration and invasion than cell proliferation suggests that this small molecule c-MET inhibitor could have utility as an anti-metastatic agent for the treatment of advanced prostate cancer. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C190.

E7050: A dual c‐Met and VEGFR‐2 tyrosine kinase inhibitor promotes tumor regression and prolongs survival in mouse xenograft models

c-Met is the cellular receptor for hepatocyte growth factor (HGF) and is known to be dysregulated in various types of human cancers. Activation of the HGF/c-Met pathway causes tumor progression, invasion, and metastasis. Vascular endothelial growth factor (VEGF) is also known as a key molecule in tumor progression through the induction of tumor angiogenesis. Because of their key roles in tumor progression, these pathways provide attractive targets for therapeutic intervention. We have generated a novel, orally active, small molecule compound, E7050, which inhibits both c-Met and vascular endothelial growth factor receptor (VEGFR)-2. In vitro studies indicate that E7050 potently inhibits phosphorylation of both c-Met and VEGFR-2. E7050 also potently represses the growth of both c-met amplified tumor cells and endothelial cells stimulated with either HGF or VEGF. In vivo studies using E7050 showed inhibition of the phosphorylation of c-Met and VEGFR-2 in tumors, and strong inhibition of tumor growth and tumor angiogenesis in xenograft models. Treatment of some tumor lines containing c-met amplifications with high doses of E7050 (50-200 mg/kg) induced tumor regression and disappearance. In a peritoneal dissemination model, E7050 showed an antitumor effect against peritoneal tumors as well as a significant prolongation of lifespan in treated mice. Our results indicate that E7050 is a potent inhibitor of c-Met and VEGFR-2 and has therapeutic potential for the treatment of cancer.

The c-Met receptor tyrosine kinase inhibitor MP470 radiosensitizes glioblastoma cells

PurposeGlioblastoma multiforme (GBM) is resistant to current cytotoxic therapies, in part because of enhanced DNA repair. Activation of the receptor tyrosine kinase c-Met has been shown to protect cancer cells from DNA damage. We hypothesized that inhibiting c-Met would decrease this protection and thus sensitize resistant tumor cells to the effects of radiation therapy.Materials and methodsEight human GBM cell lines were screened for radiosensitivity to the small-molecule c-Met inhibitor MP470 with colony-count assays. Double-strand (ds) DNA breaks was quantified by using antibodies to gamma H2AX. Western blotting demonstrate expression of RAD51, glycogen synthase kinase (GSK)-3β, and other proteins. A murine xenograft tumor flank model was used for in vivo radiosensitization studies.ResultsMP470 reduced c-Met phosphorylation and enhanced radiation-induced cell kill by 0.4 logs in SF767 cells. Cells pretreated with MP470 had more ds DNA damage than cells treated with radiation alone. Mechanistically, MP470 was shown to inhibit dsDNA break repair and increase apoptosis. MP470 influences various survival and DNA repair related proteins such as pAKT, RAD51 and GSK3β. In vivo, the addition of MP470 to radiation resulted in a tumor-growth-delay enhancement ratio of 2.9 over radiation alone and extended survival time.ConclusionsGBM is a disease site where radiation is often used to address both macroscopic and microscopic disease. Despite attempts at dose escalation outcomes remain poor. MP470, a potent small-molecule tyrosine kinase inhibitor of c-Met, radiosensitized several GBM cell lines both in vitro and in vivo, and may help to improve outcomes for patients with GBM.

Design, Synthesis, and Biological Evaluation of Potent c-Met Inhibitors

c-Met is a receptor tyrosine kinase that plays a key role in several cellular processes but has also been found to be overexpressed and mutated in different human cancers. Consequently, targeting this enzyme has become an area of intense research in drug discovery. Our studies began with the design and synthesis of novel pyrimidone 7, which was found to be a potent c-Met inhibitor. Subsequent SAR studies identified 22 as a more potent analog, whereas an X-ray crystal structure of 7 bound to c-Met revealed an unexpected binding conformation. This latter finding led to the development of a new series that featured compounds that were more potent both in vitro and in vivo than 22 and also exhibited different binding conformations to c-Met. Novel c-Met inhibitors have been designed, developed, and found to be potent in vitro and in vivo.

Discovery of novel inhibitors for c-Met by virtual screening and pharmacophore analysis

The overexpression of human c-Met tyrosine kinase induces the tumor proliferation and migration. Commercial drug is still not available for inhibiting this target. The structure of human c-Met was simulated and validated by molecular modeling. Compounds from our laboratory database, including natural products and anticancer agents, were employed for the docking analysis. De Novo drug design was further performed for three compounds with highest DockScore value to discover the novel inhibitors. Through the adsorption, distribution, metabolism, excretion and toxicity (ADMET) descriptor, only compounds A 2, B 3, C 2 and C 5 were selected. Through the pharmacophore analysis, ARG1086, HIS1088, ARG1208, ASN1209, ALA1226, ARG1227, ASP1228, TYR1230, and GLU1233 were suggested as the key residues because of strong pharmacophore features. In addition, the pharmacophore features of candidates consisted with the active site properties. The added fragments produced the strong interaction with the surrounding residues and yielded hydrogen bonds (HBs). Thus, the interaction energy between the ligand and the receptor was enhanced. Besides, the values of several scoring functions (PLP1, PLP2, and DockScore) of candidates were comparatively higher than compounds A, B, C, and Kirin. According to the aforementioned analyses, compounds A, A 2, B, B 3, C, C 2 and C 5 were suggested as the potent c-Met inhibitors. Besides, the scaffolds of compounds A, B, and C provided the direction for further drug design.

First in human phase I study of MK-2461, a small molecule inhibitor of c-Met, for patients with advanced solid tumors

14657 Background: MK-2461 is a potent small molecule inhibitor of c-Met, a receptor tyrosine kinase involved in tumor cell proliferation and motility. Over-expression of c-Met or Met gene amplification has been associated with poorer prognosis in several tumor types. Methods: Multicenter, open-label, phase I dose escalation study in patients with advanced solid tumors refractory to standard therapy. Drug was administered by mouth daily or twice daily (BID) for 28 days followed by a 14 day rest period during cycle 1. Patients received continuous dosing subsequently for 28-day cycles. Maximum tolerated dose (MTD) was determined using a standard 3+3 dosing design. PK analyses were performed on days 1 and 28 of cycle 1. Results: Fourteen patients (10 M/ 4 F), mean age 54 (range 19–76), have received 31 cycles (range 1–6). Dose levels tested include 60mg daily, 60mg BID, 120mg BID, and 180 mg BID. Toxicity data are available for 11 patients treated at the 60mg daily-120mg BID dosing cohorts. Ten patients (91%) have not experienced > Grade 1 drug-related toxicity. Dose limiting toxicity has not been reached and no objective antitumor responses have been observed. One patient with mucinous carcinoma of the appendix had stable disease for 6 cycles. Common drug related toxicities are outlined in the table below. Four patients experienced serious adverse experiences that were considered not related to MK-2461. PK analysis revealed a rapid Tmax (1–3 hr) across all dosing cohorts with a terminal half life of 6.3 hr following the final day of dosing for the QD dosing cohort. Conclusions: Twice daily administration of MK-2461 at the doses tested is well tolerated. Terminal t1/2 suggests acceptable drug plasma concentrations expected at BID dosing. Dose escalation continues. Toxicity Grade 1 (%) Grade 2 (%) Anorexia 2(18%) 0 Fatigue 1(9%) 1(9%) Nausea 1(9%) 0 Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Merck Merck Merck Exelixis, Merck

Crystal structure of the tyrosine kinase domain of the hepatocyte growth factor receptor c-Met and its complex with the microbial alkaloid K-252a.

The protooncogene c-met codes for the hepatocyte growth factor receptor tyrosine kinase. Binding of its ligand, hepatocyte growth factor/scatter factor, stimulates receptor autophosphorylation, which leads to pleiotropic downstream signaling events in epithelial cells, including cell growth, motility, and invasion. These events are mediated by interaction of cytoplasmic effectors, generally through Src homology 2 (SH2) domains, with two phosphotyrosine-containing sequence motifs in the unique C-terminal tail of c-Met (supersite). There is a strong link between aberrant c-Met activity and oncogenesis, which makes this kinase an important cancer drug target. The furanosylated indolocarbazole K-252a belongs to a family of microbial alkaloids that also includes staurosporine. It was recently shown to be a potent inhibitor of c-Met. Here we report the crystal structures of an unphosphorylated c-Met kinase domain harboring a human cancer mutation and its complex with K-252a at 1.8-A resolution. The structure follows the well established architecture of protein kinases. It adopts a unique, inhibitory conformation of the activation loop, a catalytically noncompetent orientation of helix alphaC, and reveals the complete C-terminal docking site. The first SH2-binding motif (1349YVHV) adopts an extended conformation, whereas the second motif (1356YVNV), a binding site for Grb2-SH2, folds as a type II Beta-turn. The intermediate portion of the supersite (1353NATY) assumes a type I Beta-turn conformation as in an Shc-phosphotyrosine binding domain peptide complex. K-252a is bound in the adenosine pocket with an analogous binding mode to those observed in previously reported structures of protein kinases in complex with staurosporine.