Met (IC50 = 0.4 nM); KDR (IC50 = 0.86 nM); Tie-2 (IC50 = 1.1 nM); VEGFR3/FLT4 (IC50 = 2.8 nM); RON (IC50 = 3 nM)

XL880 inhibits tyrosine kinases of the HGF receptor family, with IC50 values of 3 nM for Ron and 0.4 nM for Met. Moreover, KDR, Flt-1, and Flt-4 are inhibited by XL880, with IC50 values of 0.9 nM, 6.8 nM, and 2.8 nM, respectively. XL880 has an IC50 of 40 nM, 29 nM, and 165 nM, respectively, which inhibits the growth of B16F10, A549, and HT29 cell colonies.[1] According to a recent study, XL880 has different effects on cell growth in the gastric cancer cell lines MKN-45 and KATO-III. In MKN-45 cells, XL880 inhibits the phosphorylation of MET and downstream signaling molecules; in KATO-III cells, it targets GFGR2.[2]

XL880, administered orally as a single 100 mg/kg dose, significantly inhibits the phosphorylation of B16F10 tumor Met and ligand (e.g., HGFor VEGF)-induced receptor phosphorylation of Met in the liver and Flk-1/KDR in the lung, both of which lasted for a full day. Tumor burden is reduced when XL880 (30–100 mg/kg, once daily, oral gavage) is administered. Treatment with 30 and 100 mg/kg XL880 reduces the lung surface tumor burden by 50% and 58%, respectively. When mice with B16F10 solid tumors are treated with XL880, there is a dose-dependent inhibition of tumor growth of 64% and 87% at 30 and 100 mg/kg, respectively. The administration of XL880 is well tolerated in both studies, and there is no discernible reduction in body weight.[1] XL880 was created to target HGF abnormal signaling through Met and several receptor tyrosine kinases involved in tumor angiogenesis at the same time. In human xenografts, XL880 produced tumor hemorrhage and necrosis in 2 to 4 hours. Maximum tumornecrosis is seen at 96 hours (after five daily doses), leading to total regression.[3]

One of three assay formats—[³³P]phosphoryl transfer, luciferase-coupled chemiluminescence, or AlphaScreen tyrosine kinase technology—is used to study kinase inhibition. XLFit is used in nonlinear regression analysis to calculate IC50s. ³³P - Transfer of Phosphoryl Assay for Kinase 384-well white, clear-bottomed, high-binding microtiter plates (Greiner, Monroe, NC) are used for reactions. In a 50 μL coating buffer containing 40 μg/mL substrate (poly(Glu, Tyr)), 2 μg/well of protein or peptide substrate is applied to the plates. 3 mM NaN₃, 50 mM NaCl, 27.5 mM NaHCO₃, and 4:1 can all be found. After incubating at room temperature for the entire night, coated plates are once again washed with 50 μL of assay buffer (RT). In a total volume of 20 μL, test compounds and enzymes are mixed with ³³P-γ-ATP (3.3 μCi/nmol). After two hours of RT incubation, aspiration is used to end the reaction mixture. After that, the microtiter plates are cleaned six times using a 0.05% Tween-PBS buffer (PBST). Addition and incorporation of scintillation fluid (50 μL/well) A MicroBeta scintillation counter is used in liquid scintillation spectrometry to measure ³³P. Chemiluminescence Assay with Luciferase Coupled Microtiter plates with medium binding (Greiner), measuring 384 wells, are used for conducting reactions. Step one involves mixing the enzyme and compound and letting them sit for 60 minutes. Step two involves adding ATP and peptide substrate (poly(Glu, Tyr) 4:1) in a final volume of 20 μL, then letting them sit for two to four hours at room temperature. After the kinase reaction, a Victor plate reader is used to measure the luminescence signal, and a 20 μL aliquot of Kinase Glo (Promega, Madison, WI) is added. There is a 50% cap on ATP consumption overall. ALPHAScreen Tyrosine Kinase Assay Utilized are acceptor beads coated with PY100 anti-phosphotyrosine antibody and donor beads coated with streptavidin. The substrate is biotinylated poly(Glu,Tyr) 4:1. The addition of donor/acceptor beads and the subsequent formation of a donor-acceptor bead complex are used to measure the substrate phosphorylation. In 384-well white, medium binding microtiter plates (Greiner), kinase and test compounds are mixed and preincubated for 60 minutes. Next, ATP and biotinylated poly(Glu, Tyr) are added in a total volume of 20 μL. The reaction mixtures are allowed to sit at room temperature for one hour. AlphaScreen bead suspension containing 75 mM Hepes, pH 7.4, 300 mM NaCl, 120 mM EDTA, 0.3% BSA, and 0.03% Tween-20 is added to 10 L to quench reactions. Plates are read using an AlphaQuest reader after being incubated for 2–16 hours at room temperature.

In a 96-well plate containing 10% FBS and EXEL-2880, B16F10, A549, and HT29 cells (1.2 x 10³ per well) are combined with soft agar and seeded on top of a base agar layer. The plates are incubated at 37°C for 12 to 14 days in 21% oxygen, 5% CO₂, and 74% nitrogen under normoxic conditions. In contrast, the plates are incubated at 37°C under hypoxic conditions in a hypoxia chamber with 1% oxygen, 5% CO₂, and 94% nitrogen. After adding 50% Alamar Blue and detecting fluorescence, the number of colonies is assessed for each condition.

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The Met receptor tyrosine kinase and its ligand, hepatocyte growth factor (HGF), are overexpressed and/or activated in a wide variety of human malignancies. Vascular endothelial growth factor (VEGF) receptors are expressed on the surface of vascular endothelial cells and cooperate with Met to induce tumor invasion and vascularization. EXEL-2880 (XL880, GSK1363089) is a small-molecule kinase inhibitor that targets members of the HGF and VEGF receptor tyrosine kinase families, with additional inhibitory activity toward KIT, Flt-3, platelet-derived growth factor receptor beta, and Tie-2. Binding of EXEL-2880 to Met and VEGF receptor 2 (KDR) is characterized by a very slow off-rate, consistent with X-ray crystallographic data showing that the inhibitor is deeply bound in the Met kinase active site cleft. EXEL-2880 inhibits cellular HGF-induced Met phosphorylation and VEGF-induced extracellular signal-regulated kinase phosphorylation and prevents both HGF-induced responses of tumor cells and HGF/VEGF-induced responses of endothelial cells. In addition, EXEL-2880 prevents anchorage-independent proliferation of tumor cells under both normoxic and hypoxic conditions. In vivo, these effects produce significant dose-dependent inhibition of tumor burden in an experimental model of lung metastasis. Collectively, these data indicate that EXEL-2880 may prevent tumor growth through a direct effect on tumor cell proliferation and by inhibition of invasion and angiogenesis mediated by HGF and VEGF receptors.[1]

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To explore the mechanism of action of foretinib (GSK1363089), an oral multi-kinase inhibitor known to target MET, RON, AXL, and vascular endothelial growth factor receptors (VEGFRs), in gastric cancer, we evaluated the effects of the agent on cell growth and cell signaling in the following panel of gastric cancer cell lines: KATO-III, MKN-1, MKN-7, MKN-45, and MKN-74. Of these, only MKN-45 and KATO-III, which harbor MET and fibroblast growth factor receptor 2 (FGFR2) amplification, respectively, were highly sensitive to foretinib. In MKN-45, 1 μM of foretinib or PHA665752, another MET kinase inhibitor, inhibited phosphorylation of MET and downstream signaling molecules as expected. In KATO-III, however, PHA665752 inhibited phosphorylation of MET independently of downstream molecules. Further, 1 μM of foretinib or PD173074, a selective FGFR kinase inhibitor, inhibited phosphorylation of FGFR2 and downstream molecules, suggesting that foretinib targets FGFR2 in KATO-III. We confirmed this novel activity of foretinib against FGFR2 in OCUM-2M, another FGFR2-amplified gastric cancer cell line. Using a phospho-receptor tyrosine kinase array, we found that foretinib inhibits phosphorylation of epidermal growth factor receptor (EGFR), HER3 and FGFR3 via MET inhibition in MKN-45, and EGFR, HER3 and MET via FGFR2 inhibition in KATO-III. Knockdown of HER3 and FGFR3 in MKN-45 with siRNA resulted in the partial inhibition of cell signaling and cell growth. In conclusion, foretinib appears effective against gastric cancer cells harboring not only MET but also FGFR2 amplification, and exerts its inhibitory effects by blocking inter-RTK signaling networks with MET or FGFR2 at their core[2].

Mice without tumors or mice carrying B16F10 tumors are used in in vivo target modulation experiments. Oral gavage with 10 mL/kg of foretinib or vehicle (0.9% normal saline) is used. HGF (10 μg/mouse) is given intraperitoneally 10 minutes prior to harvesting in order to assess Met phosphorylation in the liver. Thirty minutes prior to harvest, or half an hour later, mice receive an intravenous injection of VEGF (10 μg/mouse) to assess Flk-1/KDR phosphorylation in the lung. Immunoblot analysis is used to determine receptor phosphorylation.

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Forty patients were treated in eight dose cohorts. The maximum tolerated dose was defined as 3.6 mg/kg, with a maximum administered dose of 4.5 mg/kg. Dose-limiting toxicities included grade 3 elevations in aspartate aminotransferase and lipase. Additional non-dose-limiting adverse events included hypertension, fatigue, diarrhea, vomiting, proteinuria, and hematuria. Responses were observed in two patients with papillary renal cell cancer and one patient with medullary thyroid cancer. Stable disease was identified in 22 patients. Foretinib pharmacokinetics increased linearly with dose. Pharmacodynamic evaluation indicated inhibition of MET phosphorylation and decreased proliferation in select tumor biopsies at submaximal doses. Conclusions: The recommended dose of foretinib was determined to be 240 mg, given on the first 5 days of a 14-day cycle. This dose and schedule were identified as having acceptable safety and pharmacokinetics, and will be the dose used in subsequent phase II trials.[3]

[1]. Inhibition of tumor cell growth, invasion, and metastasis by EXEL-2880 (XL880, GSK1363089), a novel inhibitor of HGF and VEGF receptor tyrosine kinases. Cancer Res, 2009, 69(20), 8009-8016.

[2]. Foretinib (GSK1363089), a multi-kinase inhibitor of MET and VEGFRs, inhibits growth of gastric cancer cell lines by blocking inter-receptor tyrosine kinase networks. Invest New Drugs, 2011.

[3]. A phase I study of foretinib, a multi-targeted inhibitor of c-Met and vascular endothelial growth factor receptor 2. Clin Cancer Res, 2010, 16(13), 3507-3516.

N1'-[3-fluoro-4-[[6-methoxy-7-[3-(4-morpholinyl)propoxy]-4-quinolinyl]oxy]phenyl]-N1-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide is an aromatic ether.
Foretinib has been used in trials studying the treatment of Cancer, Breast Cancer, Carcinoma, Renal Cell, Recurrent Breast Cancer, and Neoplasms, Head and Neck, among others. Foretinib is an orally available small molecule compound designed to target multiple RTKs implicated in the development, progression and spread of cancer. It inhibits the activation of MET, RON, ERK and AKT, decreased proliferation and increased apoptosis.
Foretinib is an orally bioavailable small molecule with potential antineoplastic activity. Foretinib binds to and selectively inhibits hepatocyte growth factor (HGF) receptor c-MET and vascular endothelial growth factor receptor 2 (VEGFR2), which may result in the inhibition of tumor angiogenesis, tumor cell proliferation and metastasis. The proto-oncogene c-MET has been found to be over-expressed in a variety of cancers. VEGFR2 is found on endothelial and hematopoietic cells and mediates the development of the vasculature and hematopoietic cells through VEGF signaling.

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Mechanism of Action
Activation of MET by mutation is the causative factor in an inherited kidney cancer syndrome, hereditary papilliary renal cell carcinaoma. Mutational activation of MET has also been found in sporadic kidney cancer, lung carcinomas and head and neck carcinomas. MET is a key driver of tumor cell growth, motility, invasion, metastasis and angiogenesis. Foretinib has attractive pharmaceutical properties with high solubility and oral bioavailability and demonstrates nanomolar potency against its targets, VEGFR, MET, which translates to potent activity in cellular assays. In preclinical studies, Foretinib, developed as a balanced inhibitor of these receptor tyrosine kinases, potently inhibited both MET and VEGFR, including mutant activated forms of MET found in hereditary papillary renal carcinomas. The compound also demonstrated dose-dependent growth inhibition in tumor models of breast, colorectal, non-small cell lung cancer and glioblastoma and has been shown to cause substantial tumor regression in all models tested.

C34H34F2N4O6

632.65

632.244

C, 64.55; H, 5.42; F, 6.01; N, 8.86; O, 15.17

849217-64-7

1226999-07-0 (phosphate);849217-64-7;

42642645

White to light yellow solid powder

1.4±0.1 g/cm3

828.5±65.0 °C at 760 mmHg

454.8±34.3 °C

0.0±3.0 mmHg at 25°C

1.649

5.12

2

10

12

46

1010

0

O=C(C1(CC1)C(NC1C=C(F)C(OC2C3C(=CC(=C(C=3)OC)OCCCN3CCOCC3)N=CC=2)=CC=1)=O)NC1C=CC(F)=CC=1

CXQHYVUVSFXTMY-UHFFFAOYSA-N

InChI=1S/C34H34F2N4O6/c1-43-30-20-25-27(21-31(30)45-16-2-13-40-14-17-44-18-15-40)37-12-9-28(25)46-29-8-7-24(19-26(29)36)39-33(42)34(10-11-34)32(41)38-23-5-3-22(35)4-6-23/h3-9,12,19-21H,2,10-11,13-18H2,1H3,(H,38,41)(H,39,42)

1-N'-[3-fluoro-4-[6-methoxy-7-(3-morpholin-4-ylpropoxy)quinolin-4-yl]oxyphenyl]-1-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide

EXEL 2880, XL-880; GSK1363089; GSK 1363089; GSK1363089, EXEL-2880,XL-880; XL880; XL 880; GSK-1363089; GSK089; EXEL2880

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.95 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (3.95 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (3.95 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 30% propylene glycol, 5% Tween 80, 65% D5W: 30mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)