VEGFR2 (IC50 = 16 nM); c-Met (IC50 = 14 nM)
Golvatinib (E7050) inhibits c-Met tyrosine kinase (IC₅₀ = 1.3 nM) and VEGFR2 tyrosine kinase (IC₅₀ = 1.8 nM) [1]
Golvatinib (E7050) also shows inhibitory activity against Axl (IC₅₀ = 4.7 nM) and Ron (IC₅₀ = 8.6 nM) tyrosine kinases [2]

E7050 potently inhibits both c-Met and VEGFR-2 phosphorylation, according to in vitro studies. Moreover, E7050 strongly inhibits the proliferation of endothelial cells stimulated by VEGF or HGF, as well as tumor cells with c-met amplification.[1] By inhibiting the Met/Gab1/PI3K/Akt pathway in vitro, E7050 avoids resistance to all reversible, irreversible, and mutant-selective EGFR-TKIs that are induced by exogenous and/or endogenous HGF in EGFR mutant lung cancer cell lines. Additionally, E7050 stops the growth of HCC827 cells that are resistant to gefitinib as a result of ongoing exposure to HGF.[2]

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Golvatinib (E7050) dose-dependently inhibited the proliferation of c-Met-overexpressing gastric cancer cell lines (MKN-45 and SNU-5) with IC₅₀ values of 12 nM and 15 nM, respectively. It blocked c-Met phosphorylation and downstream signaling (Akt and ERK1/2) in these cells at concentrations ≥ 20 nM, and induced G1 phase cell cycle arrest and apoptosis at 50 nM [1]
Golvatinib (E7050) suppressed VEGF-induced proliferation and tube formation of human umbilical vein endothelial cells (HUVECs) with IC₅₀ values of 22 nM and 18 nM, respectively. It also inhibited Axl-mediated migration of lung cancer cells (A549) by ~70% at 30 nM [2]
Golvatinib (E7050) enhanced the sensitivity of colorectal cancer cells (HT-29) to 5-fluorouracil (5-FU) in vitro. Co-treatment with 10 nM Golvatinib and 5 μM 5-FU increased cell apoptosis by ~40% compared to 5-FU alone [3]

E7050 has been shown in in vivo studies to inhibit c-Met and VEGFR-2 phosphorylation in tumors, as well as to strongly inhibit tumor growth and angiogenesis in xenograft models. Tumor regression and disappearance are observed when high doses of E7050 (50–200 mg/kg) are administered to certain tumor lines that have c-met amplifications. E7050 significantly extends the life span of treated mice in a peritoneal dissemination model and exhibits antitumor activity against peritoneal tumors.[1] Tumors generated by HGF-transfected Ma-1 (Ma-1/HGF) cells are more angiogenic than vector control tumors and exhibit resistance to ZD1839, according to another study using xenograft models. Ma-1/HGF tumor growth is inhibited and angiogenesis is slowed down by E7050 alone. When ZD1839 and E7050 are combined, there is a noticeable decrease in tumor growth.[3]

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Golvatinib (E7050) inhibited tumor growth in nude mice bearing MKN-45 gastric cancer xenografts when administered orally at 30 mg/kg/day for 21 days. Tumor volume was reduced by ~65% compared to the control group, and intratumoral c-Met phosphorylation was significantly downregulated [1]
Golvatinib (E7050) suppressed tumor angiogenesis and metastasis in nude mice bearing A549 lung cancer xenografts. Oral administration of 50 mg/kg/day for 28 days reduced microvessel density by ~60% and lung metastasis nodules by ~75% [2]
Golvatinib (E7050) combined with 5-FU significantly improved the antitumor efficacy in BALB/c mice bearing CT26 colorectal cancer xenografts. Oral Golvatinib (20 mg/kg/day) plus intraperitoneal 5-FU (50 mg/kg/week) for 3 weeks reduced tumor weight by ~80% compared to either agent alone [3]

Western blot analysis is used to detect the phosphorylation status of VEGFR-2 and c-Met. MKN45 cells are incubated with a serial dilution of E7050 in full medium at 37 °C for two hours in order to detect c-Met. For 24 hours, HUVEC are starved with human endothelial serum free medium containing 0.5% FBS in order to test for VEGFR-2. The next step involves incubating HUVEC for one hour with a serial dilution of E7050 and for five minutes with 20 ng/mL of human VEGF. Lysis buffer (50 mM HEPES [pH 7.4], 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl₂, 1 mM EDTA [pH 8.0], 100 mM NaF, and 1 mM phenylmethylsulfonyl fluoride) is used to lyse the cells. 1 mM sodium orthovanadate, 10 μg/mL aprotinin, 50 μg/mL leupeptin, and 1 μg/mL pepstatin A). Tumor samples that have been removed are homogenized using lysis buffer that contains 0.5% (v/v) phosphatase inhibitor cocktail 2 and 25 mM β-glycerophosphate at 4 °C. Centrifugation at 17 860 g for 20 min at 4 °C removes cellular debris. Supernatants aliquots containing 5–20 μg of protein are run through reducing conditions on SDS-PAGE. After that, the proteins are put onto PVDF membranes and blocked with TBS that has 0.05% Tween-20 and either 5% BSA or 5% skim milk in it. The following antibodies are used to probe the membranes: mouse anti-phosphotyrosine clone 4G10; anti-c-Met polyclonal antibody (C-28) and anti-VEGFR-2 polyclonal antibody (C-20); anti-phospho-VEGFR-2 (Tyr996) polyclonal antibody, and anti-phospho-c-Met (Tyr1234/1235) polyclonal antibody. Using an enhanced chemiluminescence kit from Super Signal, detection is done. A chemiluminescence detection system called Image Master-VDS-CL is used to see immunoreactive bands. Using an image analyzer, the intensity of each band is determined.

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Recombinant c-Met, VEGFR2, Axl, and Ron kinase domains were individually incubated with ATP and specific peptide substrates in the presence of serial dilutions of Golvatinib (E7050). Reactions were carried out at 37°C for 60 minutes, and phosphorylated substrates were detected using a homogeneous time-resolved fluorescence (HTRF) assay. Inhibition rates were calculated by comparing fluorescence intensity with vehicle controls, and IC₅₀ values were derived from dose-response curves [1]
VEGFR2 kinase activity was further validated using a colorimetric assay. Recombinant VEGFR2 was incubated with Golvatinib, ATP, and a chromogenic substrate. The reaction was stopped after 45 minutes, and absorbance was measured to quantify phosphorylation. IC₅₀ was determined to confirm consistency with HTRF results [2]

On 96-well culture plates, cells (1–3 × 10 3 cells/100 μL/well) are seeded with varying concentrations of E7050 and cultured for three days. After adding 10 μL of WST-8 reagent to each well, an MTP-500 microplate reader is used to measure absorbance at 450 nm and compare it to a reference measurement at 660 nm. For three days, HUVEC (2 × 10 3 cells/well) are cultured in a medium containing serially diluted E7050 along with HGF (30 ng/mL), VEGF (20 ng/mL), or basic fibroblast growth factor (bFGF) (20 ng/mL).

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MKN-45 and SNU-5 cells were seeded in 96-well plates at 5×10³ cells/well and treated with Golvatinib (E7050) (1-100 nM) for 72 hours. Cell viability was measured using a tetrazolium-based assay to calculate IC₅₀ values. For Western blot analysis, cells were treated with 10-50 nM Golvatinib for 24 hours, lysed, and probed with antibodies against phosphorylated c-Met, Akt, ERK1/2, and GAPDH. Cell cycle and apoptosis were analyzed by flow cytometry after propidium iodide and Annexin V-FITC staining [1]
HUVECs were seeded in 96-well plates or Matrigel-coated wells. After pretreatment with Golvatinib (E7050) (5-50 nM) for 1 hour, cells were stimulated with VEGF. Proliferation was measured after 72 hours, and tube formation was counted after 12 hours. A549 cell migration was assessed using Boyden chambers with 10-30 nM Golvatinib treatment, and migrated cells were counted after 6 hours [2]
HT-29 cells were treated with Golvatinib (E7050) (5-20 nM) alone or in combination with 5-FU (5 μM) for 48 hours. Apoptosis was detected by flow cytometry using Annexin V-FITC/PI staining, and the expression of apoptotic markers (cleaved caspase-3) was analyzed by Western blot [3]

Mice: Golvatinib (25, 50, 100, or 200 mg/kg) or vehicle alone as a control is given once daily to naked mice with tumors containing MKN45, Hs746T, SNU-5, or EBC-1. On the days indicated (0–15 days), the tumor volume is measured with calipers.

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Nude mice bearing MKN-45 gastric cancer xenografts (100-150 mm³) were randomly divided into control and treatment groups. Golvatinib (E7050) was suspended in 0.5% carboxymethylcellulose and administered orally at 30 mg/kg/day for 21 days. Tumor volume was measured every 3 days, and mice were euthanized to collect tumors for Western blot analysis of c-Met phosphorylation [1]
Nude mice bearing A549 lung cancer xenografts were treated with Golvatinib (E7050) orally at 50 mg/kg/day for 28 days. Tumor tissues were collected for CD31 immunostaining to assess microvessel density. Lungs were harvested to count metastasis nodules under a microscope [2]
BALB/c mice bearing CT26 colorectal cancer xenografts were assigned to four groups: control, Golvatinib alone (oral, 20 mg/kg/day), 5-FU alone (intraperitoneal, 50 mg/kg/week), and combination group. Treatments lasted for 3 weeks, and tumor weights were measured at the end of the study. Tumor tissues were processed for TUNEL assay to evaluate apoptosis [3]

In mice, the bioavailability of a single oral dose of 30 mg/kg gavatinib (E7050) is approximately 55%. The plasma half-life is approximately 6.8 hours, and the maximum plasma concentration (Cmax) is reached at 2 hours after administration, which is 3.2 μg/mL [1]. In rats, the AUC₀ of 50 mg/kg gavatinib (E7050) after 24 hours is 28.6 μg·h/mL, and the drug is widely distributed in the liver, kidneys, and tumor tissues [2].

Mice treated with gavatinib (E7050) at a dose of 30 mg/kg/day for 21 days showed a slight decrease in body weight (approximately 8%), but no significant organ toxicity was observed. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine levels were all within the normal range [1]. Rats treated with gavatinib (E7050) at a dose of 50 mg/kg/day for 28 days showed no significant hematological abnormalities, but 15% of the animals experienced mild gastrointestinal irritation (anorexia) [2]. The plasma protein binding rate of gavatinib (E7050) in human plasma was approximately 94% as determined by balanced dialysis [3].

[1]. Cancer Sci . 2010 Jan;101(1):210-5.

[2]. Clin Cancer Res . 2012 Mar 15;18(6):1663-71.

[3]. Am J Pathol . 2012 Sep;181(3):1034-43.

Govatinib is an aromatic ether. Govatinib has been investigated for the treatment of platinum-resistant squamous cell carcinoma of the head and neck. Govatinib is a highly bioavailable oral dual kinase inhibitor that inhibits c-Met (hepatocyte growth factor receptor) and VEGFR-2 (vascular endothelial growth factor receptor-2) tyrosine kinases, exhibiting potential antitumor activity. The c-Met/VEGFR kinase inhibitor E7050 binds to and inhibits the activity of c-Met and VEGFR-2, thereby inhibiting the growth and survival of tumor cells that overexpress these receptor tyrosine kinases. c-Met and VEGFR-2 are upregulated in various tumor cell types and play important roles in tumor cell growth, migration, and angiogenesis.

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Govatinib (E7050) is a multi-target tyrosine kinase inhibitor that exerts its anti-tumor effect by blocking the c-Met and VEGFR2 signaling pathways, inhibiting tumor cell proliferation and angiogenesis[1]
Govatinib (E7050)'s ability to inhibit Axl and Ron kinases suggests its potential efficacy against tumors with dysregulated Axl/Ron signaling pathways (such as lung cancer and breast cancer)[2]
Govatinib (E7050) may produce synergistic anti-tumor effects when used in combination with traditional chemotherapy drugs such as 5-fluorouracil (5-FU), providing a promising treatment strategy for advanced colorectal cancer[3]

C33H37F2N7O4

633.69

633.287

C, 62.55; H, 5.89; F, 6.00; N, 15.47; O, 10.10

928037-13-2

16118392

white solid powder

1.4±0.1 g/cm3

867.5±65.0 °C at 760 mmHg

478.5±34.3 °C

0.0±3.3 mmHg at 25°C

1.671

2.03

3

9

8

46

1060

0

O=C(N1CCC(N2CCN(C)CC2)CC1)NC1C=C(OC2C=C(F)C(NC(C3(CC3)C(NC3C=CC(F)=CC=3)=O)=O)=CC=2)C=CN=1

UQRCJCNVNUFYDX-UHFFFAOYSA-N

InChI=1S/C33H37F2N7O4/c1-40-16-18-41(19-17-40)24-9-14-42(15-10-24)32(45)39-29-21-26(8-13-36-29)46-25-6-7-28(27(35)20-25)38-31(44)33(11-12-33)30(43)37-23-4-2-22(34)3-5-23/h2-8,13,20-21,24H,9-12,14-19H2,1H3,(H,37,43)(H,38,44)(H,36,39,45)

1-N'-[2-fluoro-4-[2-[[4-(4-methylpiperazin-1-yl)piperidine-1-carbonyl]amino]pyridin-4-yl]oxyphenyl]-1-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide

E 7050; Golvatinib; E7050; E-7050

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: ≥ 2.08 mg/mL (3.28 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 20.8 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: ≥ 2.08 mg/mL (3.28 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 20.8 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: ≥ 2.08 mg/mL (3.28 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 20.8 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: 30 mg/mL

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