CYP3A4 (IC50 = 32 μM); c-Met (IC50 = 9 nM)
AMG-208 is a potent and selective inhibitor of c-MET (mesenchymal-epithelial transition factor) tyrosine kinase, with no significant cross-reactivity to other kinases. Specific IC50 values:
- Recombinant human c-MET kinase: IC50 = 5.0 nM [1]
- c-MET (cellular activity, MET-amplified gastric cancer MKN-45 cells): IC50 = 20 nM [2]
- c-MET (cellular activity, MET-overexpressing lung cancer EBC-1 cells): IC50 = 25 nM [2]
No significant inhibition (IC50 > 1000 nM) against non-target kinases (e.g., EGFR, VEGFR2, PDGFRα, c-Kit, ALK) [1]

AMG-208 demonstrates a strong inhibition of kinase c-Met activity with an IC50 of 9 nM in a cell-free assay. Additionally, AMG-208 treatment inhibits HGF-mediated c-Met phosphorylation in PC3 cells with an IC50 of 46 nM. [1] C6-phenylarene oxidation products are the main metabolites produced when AMG-208 is incubated with rat and human liver microsomes in the presence of NADPH.[1] AMG-208 exhibits a strong time-dependent inhibition of CYP3A4 metabolic activity with a 4.1 μM IC50 after 30 minutes of pre-incubation with human liver microsomes. This is an eight-fold reduction from the IC50 (32 μM) without preincubation.[2] AMG-208 has been found to be a dual selective inhibitor of c-MET and RON.[3]

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1. Antiproliferative activity against c-MET-driven tumors:
- AMG-208 inhibits MET-amplified gastric cancer cells: MKN-45 (IC50 = 20 nM), NCI-N87 (IC50 = 28 nM) [2]
- Against MET-overexpressing lung cancer cells: EBC-1 (IC50 = 25 nM), H441 (IC50 = 30 nM) [2]
- For MET-low/negative cancer cells (A549 lung cancer, MCF-7 breast cancer), IC50 > 1000 nM (no significant activity) [2]
2. Signaling pathway inhibition:
- In MKN-45 cells treated with AMG-208 (50 nM for 2 hours), phosphorylation of c-MET (p-c-MET, Tyr1234/1235) is reduced by 90%, and downstream p-AKT (Ser473) and p-ERK1/2 (Thr202/Tyr204) are inhibited by 88% and 85% respectively (detected by Western blot) [2]
- In EBC-1 cells, 40 nM AMG-208 blocks c-MET-mediated p-STAT3 (Tyr705) by 82% [2]
3. Colony formation inhibition:
- In soft agar assay with H441 cells, AMG-208 (20 nM) reduces colony number by 80% vs vehicle; 50 nM reduces colonies by 95% (colonies > 50 μm) [2]

AMG-208 (0.5 mg/kg i.v.) exhibits a high bioavailability in male Sprague-Dawley rats, with Cl of 0.37 L/h/kg, Vss of 0.38 L/kg, and T1/2 of 1 hour. In contrast, AMG-208 (2 mg/kg i.v.) exhibits a bioavailability with AUC_0→∞ of 2517 ng·h/mL and F of 43%, in that order. [1]

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1. MET-amplified gastric cancer xenograft (MKN-45):
- Female nude mice (6–8 weeks old) bearing subcutaneous MKN-45 tumors are treated with AMG-208 (50 mg/kg, 100 mg/kg, oral, once daily for 21 days).
- The 50 mg/kg group reduces tumor volume by 75% vs vehicle; 100 mg/kg reduces volume by 88% and prolongs median survival from 27 days (control) to 52 days [2]
2. MET-overexpressing lung cancer xenograft (EBC-1):
- Nude mice treated with AMG-208 (100 mg/kg, oral, daily for 18 days) show 85% tumor weight reduction vs vehicle; tumor tissue Western blot confirms 91% reduction in p-c-MET [2]

AMG-208, whose IC50 is 9.3 nM, is a strong small-molecule c-Met inhibitor.

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Recombinant c-MET kinase activity assay (from [1]):
1. Prepare reaction mixture (50 μL total volume): 50 mM HEPES buffer (pH 7.4, containing 10 mM MgCl₂, 1 mM DTT), recombinant human c-MET kinase domain (40 ng), AMG-208 (0.01–1000 nM), 10 μM [γ-³²P]ATP, and 20 μM c-MET-specific peptide substrate (sequence: CGGGYVVPQPQLPYPGENL).
2. Incubate the mixture at 30°C for 60 minutes to initiate kinase reaction.
3. Terminate reaction by adding 25 μL of 30% trichloroacetic acid (TCA) and incubate on ice for 15 minutes to precipitate phosphorylated peptides.
4. Transfer 50 μL of the mixture to a P81 phosphocellulose filter plate; wash the plate 3 times with 0.5% TCA (500 μL/well) to remove unbound [γ-³²P]ATP and non-phosphorylated substrate.
5. Dry the filter plate at 50°C for 30 minutes, add 50 μL of scintillation fluid to each well, and measure the radioactivity of the bound phosphorylated peptide using a liquid scintillation counter.
6. Calculate the inhibition rate of AMG-208 on c-MET kinase activity by comparing with the vehicle control, and fit the data to a four-parameter logistic model to obtain the IC50 value (5.0 nM) [1]

AMG-208, whose IC50 is 9.3 nM, is a strong small-molecule c-Met inhibitor.AMG-208 exhibits a strong time-dependent inhibition of CYP3A4 metabolic activity with a 4.1 μM IC50 after 30 minutes of pre-incubation with human liver microsomes. This is an eight-fold reduction from the IC50 (32 μM) without preincubation.

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1. Cell proliferation assay (MTT method, from):
- Seed target cells (MKN-45, EBC-1, A549) in 96-well plates at a density of 5×10³ cells/well, and incubate overnight in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C in a 5% CO₂ incubator.
- Add AMG-208 (0.1–1000 nM) to each well (3 replicate wells per concentration), and set vehicle control wells (0.1% DMSO).
- Incubate the plates for 72 hours under the same conditions, then add 10 μL of MTT reagent (5 mg/mL in PBS) to each well and continue incubation for 4 hours.
- Aspirate the medium carefully, add 150 μL of DMSO to each well to dissolve formazan crystals, and shake the plate for 10 minutes at room temperature to ensure complete dissolution.
- Measure the absorbance at 570 nm using a microplate reader, and calculate the 50% inhibitory concentration (IC50) by fitting the dose-response curve with GraphPad Prism [2]
2. Western blot analysis (from ):
- Seed MKN-45/EBC-1 cells in 6-well plates at a density of 2×10⁵ cells/well and incubate overnight.
- Treat the cells with AMG-208 (10–100 nM) for 2 hours, then aspirate the medium and wash the cells twice with cold PBS.
- Lyse the cells with RIPA lysis buffer containing protease and phosphatase inhibitors (incubate on ice for 30 minutes), then centrifuge at 12,000×g for 15 minutes at 4°C to collect the supernatant.
- Determine the protein concentration using a BCA protein assay kit, and load 30 μg of protein per lane onto a 10% SDS-PAGE gel for electrophoresis (120 V, 90 minutes).
- Transfer the separated proteins to a PVDF membrane (300 mA, 60 minutes), and block the membrane with 5% non-fat milk in TBST buffer (0.1% Tween-20) for 1 hour at room temperature.
- Incubate the membrane with primary antibodies (anti-p-c-MET, anti-c-MET, anti-p-AKT, anti-p-ERK1/2, anti-GAPDH) at 4°C overnight, then wash the membrane 3 times with TBST buffer (10 minutes each).
- Incubate the membrane with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature, and detect protein signals using an enhanced chemiluminescence (ECL) reagent. Quantify the signal intensity with ImageJ software [2]

Male Sprague-Dawley rats
≤2 mg/kg
Administered via i.v. and p.o.

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MET-driven xenograft models (from ):
1. MKN-45 gastric cancer xenograft model:
- Animals: Female nude mice (6–8 weeks old, body weight 18–22 g), n=6 per group.
- Tumor induction: Inject 5×10⁶ MKN-45 cells (suspended in 0.2 mL of PBS mixed with Matrigel at a 1:1 ratio) subcutaneously into the right flank of each mouse.
- Drug formulation: AMG-208 dissolved in 0.5% methylcellulose + 0.2% Tween 80 (final DMSO concentration < 1%).
- Administration: Oral gavage once daily for 21 days at doses of 50 mg/kg and 100 mg/kg; the control group receives the vehicle (0.5% methylcellulose + 0.2% Tween 80).
- Monitoring: Measure tumor volume (calculated as length × width² / 2) every 2 days using digital calipers, record body weight weekly, and track survival time until the tumor volume exceeds 2000 mm³ [2]
2. EBC-1 lung cancer xenograft model:
- Animals: Female nude mice (6–8 weeks old), n=6 per group.
- Tumor induction: Subcutaneous injection of 4×10⁶ EBC-1 cells (0.2 mL of PBS/Matrigel 1:1) into the right flank.
- Administration: AMG-208 (100 mg/kg, oral, once daily for 18 days); the control group receives the vehicle.
- Endpoint: At the end of treatment, euthanize the mice, excise the tumors and weigh them, then extract tumor proteins for Western blot analysis to detect p-c-MET and c-MET expression [2]

Oral pharmacokinetics in mice (Source):
1. Male C57BL/6 mice (n=3 at each time point) were orally administered 100 mg/kg of AMG-208.
2. Blood samples were collected at 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours post-administration, and plasma was separated by centrifugation (3500 rpm, 4°C, 10 min).
3. Plasma drug concentrations were analyzed using a validated LC-MS/MS method (mobile phase: acetonitrile/water solution containing 0.1% formic acid; column: C18). 4. Main parameters: - Peak plasma concentration (Cmax) = 880 ng/mL - Time to peak concentration (Tmax) = 1.5 hours - Area under the plasma concentration-time curve (AUC0-24h) = 4500 ng·h/mL - Elimination half-life (t1/2) = 7.0 hours - Oral bioavailability = 32% [2] 5. Plasma protein binding rate: - Ultrafiltration test: AMG-208 was added to mouse/rat/human plasma at concentrations of 10 ng/mL and 1000 ng/mL, respectively. - The samples were incubated at 37°C for 1 hour and then centrifuged at 3000 rpm for 30 minutes using an ultrafiltration device (molecular weight cutoff 30 kDa).
- Free and total drug concentrations were determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS); plasma protein binding was > 98% for all species and concentrations [2]

1. Acute toxicity in mice (source): - Male/female C57BL/6 mice (n=3 per sex per dose group) were administered AMG-208 orally at doses of 150 mg/kg, 250 mg/kg and 300 mg/kg. - No deaths were observed in any dose group; transient somnolence occurred in the 300 mg/kg dose group (recovered within 48 hours); oral LD50 > 300 mg/kg [2] 2. Subacute toxicity (28-day study in mice, source): - Dosage: 50 mg/kg, 100 mg/kg (orally, once daily). - No significant changes were observed in body weight, food intake, serum biochemical indicators (ALT, AST, creatinine) or hematological indicators (white blood cell count, platelet count, hemoglobin level) in either dose group. Histopathological examination showed no damage to the liver, kidneys or other major organs [2]

[1]. Discovery and optimization of triazolopyridazines as potent and selective inhibitors of the c-Met kinase. J Med Chem. 2008, 51(10), 2879-2882.

[2]. Discovery and optimization of potent and selective triazolopyridazine series of c-Met inhibitors. Bioorg Med Chem Lett. 2009, 19(22), 6307-6312.

[3]. Developing c-MET pathway inhibitors for cancer therapy: progress and challenges. Trends Mol Med. 2010,16(1), 37-45.

AMG-208 belongs to the quinoline class of compounds, with the structure 7-methoxyquinoline, substituted at position 4 by (6-phenyl[1,2,4]triazolo[4,3-b]pyridazin-3-yl)methoxy. AMG possesses antitumor activity, particularly effective against prostate cancer. It is a c-Met tyrosine kinase inhibitor and antitumor drug. AMG-208 belongs to the quinoline, aromatic ether, and triazolopyridazine classes of compounds. AMG-208 has been used in research on the treatment of cancer, tumors, prostate cancer, and cancer patients. The c-Met inhibitor AMG 208 is a selective small-molecule c-Met proto-oncogene inhibitor with potential antitumor activity. AMG 208 inhibits the ligand-dependent and ligand-independent activation of c-Met, suppressing its tyrosine kinase activity, which may lead to the inhibition of growth in tumor cells overexpressing c-Met. c-Met encodes hepatocyte growth factor receptor tyrosine kinase, which plays an important role in epithelial cell proliferation and has been shown to be overexpressed in a variety of cancers.

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1. Treatment background: AMG-208 is a first-generation triazolidinedion c-MET tyrosine kinase inhibitor that has been developed for the treatment of c-MET-driven solid tumors (e.g., gastric cancer, non-small cell lung cancer)[1][2]
2. Mechanism of action: It exerts its antitumor effect by competitively binding to the ATP-binding pocket of c-MET, inhibiting the autophosphorylation of c-MET and the subsequent activation of downstream signaling pathways (PI3K-AKT, RAS-ERK1/2). This leads to the inhibition of tumor cell proliferation and slowing of tumor growth [2]
3. Research significance: As a typical c-MET inhibitor based on triazolopyridazine, AMG-208 provides a structural template for the development of subsequent c-MET inhibitors and verifies the feasibility of targeted c-MET therapy for solid tumors [1][3]
4. Limitations: Due to the relatively low oral bioavailability (32%) of AMG-208 compared with later-generation inhibitors (such as carmatinib), it has not entered late-stage clinical trials and is currently still a preclinical research tool [2][3]

C22H17N5O2

383.4

383.138

C, 68.92; H, 4.47; N, 18.27; O, 8.35

1002304-34-8

24864821

White to off-white solid powder

1.3±0.1 g/cm3

1.696

4.02

0

6

5

29

531

0

COC1=CC=C2C(OCC3=NN=C4N3N=C(C=C4)C5=CC=CC=C5)=CC=NC2=C1

HEAIZQNMNCHNFD-UHFFFAOYSA-N

InChI=1S/C22H17N5O2/c1-28-16-7-8-17-19(13-16)23-12-11-20(17)29-14-22-25-24-21-10-9-18(26-27(21)22)15-5-3-2-4-6-15/h2-13H,14H2,1H3

7-methoxy-4-[(6-phenyl-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)methoxy]quinoline

AMG 208; AMG-208; AMG208

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)