Foretinib (GSK-1363089; XL-880; EXEL2880; GSK-089)

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.

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.

[1]. Cancer Res . 2009 Oct 15;69(20):8009-16.

[2]. Invest New Drugs . 2012 Aug;30(4):1352-60.

[3]. Clin Cancer Res . 2010 Jul 1;16(13):3507-16.

C34H34F2N4O6

632.65

632.24

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;

Solid powder

COC1=CC2=C(C=CN=C2C=C1OCCCN3CCOCC3)OC4=C(C=C(C=C4)NC(=O)C5(CC5)C(=O)NC6=CC=C(C=C6)F)F

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)