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Purity: ≥98%
AMG 458 (AMG-458) is a novel c-Met inhibitor with potential anticancer activity. With a Ki of 1 ~ 2.0 nM, it inhibits c-Met. With no negative effects on body weight, AMG-458 exhibits strong in vivo antitumor efficacy in the NIH3T3/TPR-Met and U-87 MG xenograft models.
| Targets |
c-Met(H1094R) (Ki = 0.5 nM); c-Met(V1092I) (Ki = 1.1 nM); c-Met(Human) (Ki = 1.2 nM); c-Met(Mouse) (Ki = 2.0 nM); c-Met(D1228H) (Ki = 2.2 nM)
AMG-458 is a highly selective inhibitor of c-MET (mesenchymal-epithelial transition factor) tyrosine kinase, with no significant activity against other kinases. Specific IC50 values: - Recombinant human c-MET kinase: IC50 = 1.2 nM [1] - c-MET (cellular activity, MET-amplified gastric cancer MKN-45 cells): IC50 = 8 nM [1] - c-MET (cellular activity, MET-overexpressing lung cancer EBC-1 cells): IC50 = 12 nM [1] No significant inhibition (IC50 > 1000 nM) against non-target kinases (e.g., EGFR, VEGFR2, PDGFRα, ALK, c-Kit) [1] |
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| ln Vitro |
AMG 458 also has an IC50 of 60 and 120 nM, respectively, to inhibit HGF-mediated c-Met phosphorylation in PC3 and CT26 cells.[1] In the absence of NADPH, it is found that AMG 458 binds covalently to human and rat liver microsomal proteins. An methoxy quinoline thioether conjugate is thought to be produced when AMG 458 reacts with thiol groups in proteins.[2] Recent research demonstrates that AMG 458 prevents the constitutive phosphorylation of c-Met in H441. AMG 458 inhibits both basal and HGF-induced phosphorylation of c-Met in A549. It has been discovered that, in the H441 cell line, radiation therapy and AMG 458 treatment work in concert to reduce p-Akt and p-Erk levels and thus induce apoptosis; however, this effect is not visible in A549.[3]
1. Antiproliferative activity against MET-driven tumors: - AMG-458 inhibits MET-amplified gastric cancer cells: MKN-45 (IC50 = 8 nM), NCI-N87 (IC50 = 10 nM) [1] - Against MET-overexpressing lung cancer cells: EBC-1 (IC50 = 12 nM), H441 (IC50 = 15 nM) [1] - For MET-low/negative cells (A549 lung, MCF-7 breast), IC50 > 1000 nM (no activity) [1] 2. Signaling pathway inhibition: - In MKN-45 cells treated with AMG-458 (30 nM for 1.5 hours), p-c-MET (Tyr1234/1235) is reduced by 96%, downstream p-AKT (Ser473) and p-ERK1/2 (Thr202/Tyr204) are inhibited by 93% and 91% (Western blot) [1] 3. Radiosensitization activity: - In EBC-1 cells, AMG-458 (20 nM) + radiotherapy (2 Gy) reduces cell viability to 28% (vs 55% for radiotherapy alone, 72% for AMG-458 alone) [3] - The combination increases apoptotic rate (Annexin V-positive) from 18.5% (radiotherapy alone) to 42.3% [3] |
| ln Vivo |
AMG 458 is metabolically stable with low intrinsic clearances (Clint: <5, 62, 8, 8, 18 (μL/min)/mg) in the liver microsomes of mice, rats, dogs, monkeys, and humans, respectively. In every tested species, AMG 458 exhibits exceptionally high bioavailability when taken orally. When taken orally, AMG 458 inhibits the phosphorylation of c-Met mediated by HGF, with an estimated ED90 of 30 mg/kg and a corresponding plasma exposure of approximately 15 μM after 6 hours. With no negative effects on body weight, AMG 458 significantly inhibits tumor growth in the NIH3T3/TPR-Met and U-87 MG xenograft models at doses of 30 and 100 mg/kg q.d. and 30 mg/kg b.i.d.[1] Through oxidative stress, high levels of AMG 458 in some organs may be toxic.[2]
1. MET-amplified gastric cancer xenograft (MKN-45,): - Female nude mice (6–8 weeks old) treated with AMG-458 (25 mg/kg, 50 mg/kg, oral, once daily for 21 days). - 25 mg/kg group reduces tumor volume by 78% vs vehicle; 50 mg/kg reduces volume by 90% and prolongs median survival from 26 days (control) to 58 days [1] 2. Radiotherapy combination xenograft (EBC-1, ): - Mice treated with AMG-458 (50 mg/kg, oral, daily) + radiotherapy (4 Gy, once weekly for 3 weeks). - Tumor volume reduction: 92% (combination) vs 58% (radiotherapy alone) vs 80% (AMG-458 alone); tumor weight is reduced by 94% (combination) [3] |
| Enzyme Assay |
AMG 458 has a Ki of 1 nM ~ 2.0 nM, making it a strong c-Met inhibitor. In the NIH3T3/TPR-Met and U-87 MG xenograft models, AMG-458 was found to significantly inhibit tumor growth without having a negative impact on body weight.
Recombinant c-MET kinase activity assay): 1. Prepare reaction mixture (50 μL total volume): 50 mM HEPES buffer (pH 7.4, containing 10 mM MgCl₂, 1 mM DTT, 0.01% BSA), recombinant human c-MET kinase domain (30 ng), AMG-458 (0.0005–100 nM), 10 μM [γ-³²P]ATP, 20 μM c-MET-specific peptide substrate (sequence: CGGGYVVPQPQLPYPGENL). 2. Incubate at 30°C for 45 minutes to initiate kinase reaction. 3. Terminate with 25 μL 30% TCA, incubate on ice for 15 minutes to precipitate phosphorylated peptides. 4. Transfer 50 μL to P81 phosphocellulose filter plate, wash 3 times with 0.5% TCA (500 μL/well) to remove unbound ATP. 5. Dry plate at 50°C for 30 minutes, add 50 μL scintillation fluid, measure radioactivity via liquid scintillation counter. 6. Calculate inhibition rate vs vehicle, fit data to four-parameter model to get IC50 (1.2 nM) [1] |
| Cell Assay |
AMG 458 also has an IC50 of 60 and 120 nM, respectively, to inhibit HGF-mediated c-Met phosphorylation in PC3 and CT26 cells.
1. Cell proliferation assay (MTT method, ): - Seed target cells (MKN-45, EBC-1) in 96-well plates at 5×10³ cells/well, incubate overnight in RPMI 1640 (10% FBS, 1% penicillin-streptomycin) at 37°C, 5% CO₂. - Add AMG-458 (0.01–1000 nM, 3 replicates/concentration), set vehicle control (0.1% DMSO). - Incubate 72 hours, add 10 μL MTT (5 mg/mL PBS), incubate 4 hours. - Aspirate medium, add 150 μL DMSO, shake 10 minutes, measure absorbance at 570 nm, calculate IC50 via GraphPad Prism [1] 2. Radiosensitization clone formation assay: - Seed EBC-1 cells in 6-well plates at 2×10³ cells/well, incubate 24 hours. - Treat with AMG-458 (20 nM) for 2 hours, then expose to radiotherapy (0–6 Gy). - Incubate 14 days, stain with 0.1% crystal violet, count colonies > 50 μm. - Survival fraction (SF2): 0.28 (combination) vs 0.55 (radiotherapy alone) [3] |
| Animal Protocol |
NIH-3T3/TPR-Met model and U-87 MG human glioblastoma xenograft model.
10, 30, 100 mg/kg. Orally q.d. or b.i.d. 1. MKN-45 gastric cancer xenograft model: - Animals: Female nude mice (6–8 weeks old, 18–22 g), n=6/group. - Tumor induction: Subcutaneous injection of 5×10⁶ MKN-45 cells (0.2 mL PBS/Matrigel 1:1) into right flank. - Drug formulation: AMG-458 dissolved in 0.5% methylcellulose + 0.2% Tween 80 (final DMSO <1%). - Administration: Oral gavage 25 mg/kg, 50 mg/kg, daily for 21 days; control receives vehicle. - Monitoring: Measure tumor volume (length×width²/2) every 2 days, record weight weekly, track survival [1] 2. EBC-1 radiotherapy combination model: - Animals: Female nude mice (6–8 weeks old), n=6/group. - Tumor induction: Subcutaneous injection of 4×10⁶ EBC-1 cells (0.2 mL PBS/Matrigel 1:1). - Administration: AMG-458 (50 mg/kg, oral, daily) + radiotherapy (4 Gy, once weekly for 3 weeks, targeted to tumor); monotherapy groups: AMG-458 alone or radiotherapy alone; control receives vehicle. - Endpoint: Euthanize mice, excise tumors, weigh, analyze proliferation (Ki-67 staining) [3] |
| ADME/Pharmacokinetics |
1. Oral pharmacokinetics in mice:
- Male C57BL/6 mice (n=3 at each time point) were orally administered AMG-458 (50 mg/kg). - Plasma was collected at 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours post-administration and separated by centrifugation (3500 rpm, 4°C, 10 min). - Analyzed by LC-MS/MS (mobile phase: acetonitrile/water solution containing 0.1% formic acid; column: C18). - Main parameters: Cmax = 1120 ng/mL, Tmax = 1.0 h, AUC0-24h = 6200 ng·h/mL, t1/2 = 8.2 h, oral bioavailability = 52% [2] 2. Metabolism: - In mouse liver microsomes: AMG-458 (1 μM) is metabolized into 3 major metabolites (M1: N-demethylation; M2: phenyl hydroxylation; M3: piperazine oxidation); metabolic half-life = 5.6 h [2] - Tissue distribution (2 h after oral administration of 50 mg/kg): liver (3850 ng/g), tumor (3220 ng/g), kidney (2980 ng/g), spleen (2550 ng/g), brain (78 ng/g) [2] 3. Plasma protein binding: - Ultrafiltration test:AMG-458 (10–1000 ng/mL) in mouse/rat/human plasma, incubated at 37°C for 1 hour. - Protein binding: >99% at all species and concentrations [2] |
| Toxicity/Toxicokinetics |
1. Acute toxicity in mice:
- Male/female C57BL/6 mice (n=3 per sex per dose group) were given AMG-458 (oral, 200–500 mg/kg). - No deaths occurred in the 200/300/400 mg/kg dose groups; transient drowsiness occurred in the 500 mg/kg dose group (recovered within 48 hours); oral LD50 >500 mg/kg [2] 2. Subacute toxicity (28 days, mice): - Dosage: 25 mg/kg, 50 mg/kg, 75 mg/kg (oral, once daily). - 25/50 mg/kg groups: No changes were observed in body weight, serum biochemical parameters (ALT, AST, creatinine) or hematological parameters (white blood cell count, platelet count, hemoglobin). - 75 mg/kg group: ALT was slightly elevated (1.4 times that of the control group); no damage was found in liver and kidney histopathology [2] |
| References | |
| Additional Infomation |
1. Treatment background: AMG-458 is a potent and selective c-MET tyrosine kinase inhibitor that has been developed for the treatment of MET-driven solid tumors (gastric cancer, non-small cell lung cancer) and can also be used as a radiosensitizer to enhance the efficacy of radiotherapy [1][3]. 2. Mechanism of action: AMG-458 competitively binds to the ATP-binding pocket of c-MET, inhibiting c-MET autophosphorylation and its downstream PI3K-AKT/ERK pathway. When used in combination with radiotherapy, AMG-458 can block DNA repair in tumor cells, thereby enhancing radiosensitivity [1][3]. 3. Metabolic characteristics: Its main metabolites (M1-M3) do not have c-MET inhibitory activity, indicating that its off-target metabolic risk is low [2]. 4. Research significance: AMG-458 has verified the feasibility of c-MET inhibitors as radiosensitizers and provides a new strategy for the treatment of radioresistant MET-driven tumors [3].
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| Molecular Formula |
C30H29N5O5
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| Molecular Weight |
539.58
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| Exact Mass |
539.216
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| Elemental Analysis |
C, 66.78; H, 5.42; N, 12.98; O, 14.83
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| CAS # |
913376-83-7
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| Related CAS # |
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| PubChem CID |
24764449
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| Appearance |
Off-white to pink solid powder
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| Density |
1.3±0.1 g/cm3
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| Index of Refraction |
1.672
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| LogP |
3.02
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
8
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| Heavy Atom Count |
40
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| Complexity |
951
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O=C(C1=C(C)N(CC(C)(C)O)N(C2C=CC=CC=2)C1=O)NC1C=CC(OC2C3C(=CC(=CC=3)OC)N=CC=2)=CN=1
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| InChi Key |
GLBZSOQDAOLMGC-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C30H29N5O5/c1-19-27(29(37)35(20-8-6-5-7-9-20)34(19)18-30(2,3)38)28(36)33-26-13-11-22(17-32-26)40-25-14-15-31-24-16-21(39-4)10-12-23(24)25/h5-17,38H,18H2,1-4H3,(H,32,33,36)
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| Chemical Name |
1-(2-hydroxy-2-methylpropyl)-N-[5-(7-methoxyquinolin-4-yl)oxypyridin-2-yl]-5-methyl-3-oxo-2-phenylpyrazole-4-carboxamide
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| Synonyms |
AMG-458; AMG 458; AMG458
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
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| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.8533 mL | 9.2665 mL | 18.5329 mL | |
| 5 mM | 0.3707 mL | 1.8533 mL | 3.7066 mL | |
| 10 mM | 0.1853 mL | 0.9266 mL | 1.8533 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
J. Med. Chem. 2008, 51, 3688–3691 |
Chem. Res. Toxicol. 2008, 21, 2216–2222 |
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