AMG-208

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 在无细胞测定中显示出对激酶 c-Met 活性的有效抑制，IC50 为 9 nM。此外，AMG-208处理还可抑制PC3细胞中HGF介导的c-Met磷酸化，IC50为46 nM。在 NADPH 存在下，将 AMG-208 与大鼠和人肝微粒体一起孵育，定性地产生 C6-苯芳烃氧化产物作为主要代谢物。 AMG-208 与人肝微粒体预孵育 30 分钟显示出对 CYP3A4 代谢活性的有效时间依赖性抑制，IC50 为 4.1 μM，相对于未预孵育的 IC50 (32 μM) 降低了八倍。 AMG-208 被鉴定为 c-MET 和 RON 双重选择性抑制剂。激酶测定：AMG-208 是一种有效的小分子 c-Met 抑制剂，IC50 为 9.3 nM。细胞测定：AMG-208 与人肝微粒体预孵育 30 分钟显示出对 CYP3A4 代谢活性的有效时间依赖性抑制，IC50 为 4.1 μM，相对于未预孵育的 IC50 (32 μM) 降低了八倍。

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1. 对c-MET驱动肿瘤的抗增殖活性:
- AMG-208抑制MET扩增胃癌细胞：MKN-45（IC50 = 20 nM）、NCI-N87（IC50 = 28 nM）[2]
- 对MET过表达肺癌细胞：EBC-1（IC50 = 25 nM）、H441（IC50 = 30 nM）[2]
- 对MET低表达/阴性癌细胞（A549肺癌、MCF-7乳腺癌），IC50 > 1000 nM（无显著活性）[2]
2. 信号通路抑制:
- 用AMG-208（50 nM，处理2小时）处理MKN-45细胞后，c-MET磷酸化水平（p-c-MET，Tyr1234/1235）降低90%，下游p-AKT（Ser473）和p-ERK1/2（Thr202/Tyr204）的抑制率分别为88%和85%（Western blot检测）[2]
- 在EBC-1细胞中，40 nM AMG-208阻断c-MET介导的p-STAT3（Tyr705）达82% [2]
3. 抑制集落形成:
- 在H441细胞软琼脂集落形成实验中，AMG-208（20 nM）使集落数量较对照组减少80%；50 nM浓度下集落减少95%（集落直径>50 μm）[2]

在雄性 Sprague-Dawley 大鼠中，AMG-208 (0.5 mg/kg iv) 显示出高生物利用度，Cl 为 0.37 L/h/kg，Vss 为 0.38 L/kg，T1/2 为 1 小时，而 AMG-208 ( 2 mg/kg iv) 显示生物利用度，AUC0→∞ 分别为 2517 ng·h/mL，F 为 43%。

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1. MET扩增胃癌异种移植模型（MKN-45）:
- 6~8周龄雌性裸鼠携带皮下MKN-45肿瘤，口服AMG-208（50 mg/kg、100 mg/kg，每日1次，连续21天）。
- 50 mg/kg组肿瘤体积较溶媒组减少75%；100 mg/kg组减少88%，中位生存期从对照组27天延长至52天 [2]
2. MET过表达肺癌异种移植模型（EBC-1）:
- 裸鼠口服AMG-208（100 mg/kg，每日1次，连续18天），肿瘤重量较对照组减少85%；肿瘤组织Western blot证实p-c-MET降低91% [2]

AMG-208的IC50为9.3 nM，是一种强效小分子c-Met抑制剂。

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重组c-MET激酶活性实验（来自文献[1]）:
1. 制备总体积50 μL的反应体系：50 mM HEPES缓冲液（pH 7.4，含10 mM MgCl₂、1 mM DTT）、重组人c-MET激酶结构域（40 ng）、AMG-208（0.01~1000 nM）、10 μM [γ-³²P]ATP、20 μM c-MET特异性肽底物（序列：CGGGYVVPQPQLPYPGENL）。
2. 30°C孵育60分钟，启动激酶反应。
3. 加入25 μL 30%三氯乙酸（TCA）终止反应，冰上孵育15分钟以沉淀磷酸化肽。
4. 取50 μL反应液转移至P81磷酸纤维素滤板，用0.5% TCA（每孔500 μL）洗涤滤板3次，去除未结合的[γ-³²P]ATP和非磷酸化底物。
5. 50°C烘干滤板30分钟，每孔加入50 μL闪烁液，通过液体闪烁计数器测定结合的磷酸化肽的放射性强度。
6. 与溶媒对照组比较，计算AMG-208对c-MET激酶活性的抑制率，将数据拟合四参数逻辑模型获得IC50值（5.0 nM）[1]

AMG-208 的 IC50 为 9.3 nM，是一种强效小分子 c-Met 抑制剂。AMG-208 与人肝脏预孵育 30 分钟后，对 CYP3A4 代谢活性表现出强烈的时间依赖性抑制，IC50 为 4.1 μM微粒体。这比没有预孵育的 IC50 (32 μM) 降低了八倍。

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1. 细胞增殖实验（MTT法，来自文献[2]）:
- 将靶细胞（MKN-45、EBC-1、A549）以5×10³细胞/孔的密度接种于96孔板，在含10%胎牛血清（FBS）和1%青霉素-链霉素的RPMI 1640培养基中，37°C、5% CO₂培养箱过夜孵育。
- 向每孔加入AMG-208（0.1~1000 nM），每个浓度设3个复孔；设溶媒对照孔（0.1% DMSO）。
- 相同条件下孵育72小时后，每孔加入10 μL MTT试剂（5 mg/mL PBS溶液），继续孵育4小时。
- 小心吸弃培养基，每孔加入150 μL DMSO溶解甲臜结晶，室温振荡10分钟确保完全溶解。
- 酶标仪在570 nm处测定吸光度，通过GraphPad Prism拟合剂量-反应曲线计算50%抑制浓度（IC50）[2]
2. Western blot实验（来自文献[2]）:
- MKN-45/EBC-1细胞以2×10⁵细胞/孔接种于6孔板，过夜孵育。
- AMG-208（10~100 nM）处理细胞2小时，吸弃培养基后用冷PBS洗涤细胞2次。
- 含蛋白酶和磷酸酶抑制剂的RIPA裂解液冰上裂解细胞30分钟，4°C下12,000×g离心15分钟收集上清液。
- BCA蛋白定量试剂盒测定蛋白浓度，每泳道上样30 μg蛋白进行10% SDS-PAGE电泳（120 V，90分钟）。
- 转印蛋白至PVDF膜（300 mA，60分钟），用含5%脱脂牛奶的TBST缓冲液（0.1% Tween-20）室温封闭1小时。
- 4°C下用一抗（抗p-c-MET、抗c-MET、抗p-AKT、抗p-ERK1/2、抗GAPDH）孵育膜过夜，TBST缓冲液洗涤3次（每次10分钟）。
- 室温下用辣根过氧化物酶（HRP）标记二抗孵育1小时，增强化学发光（ECL）试剂检测蛋白信号，ImageJ软件定量信号强度 [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 (from):
1. Male C57BL/6 mice (n=3 per time point) receive AMG-208 via oral gavage at 100 mg/kg.
2. Collect blood samples at 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours post-dosing, and separate plasma by centrifugation (3500 rpm, 4°C, 10 minutes).
3. Analyze plasma drug concentration using a validated LC-MS/MS method (mobile phase: acetonitrile/water with 0.1% formic acid; column: C18).
4. Key parameters:
- Peak plasma concentration (Cmax) = 880 ng/mL
- Time to reach Cmax (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:
- Ultrafiltration assay: Spike AMG-208 into mouse/rat/human plasma at concentrations of 10 ng/mL and 1000 ng/mL.
- Incubate the samples at 37°C for 1 hour, then centrifuge with ultrafiltration devices (30 kDa cutoff) at 3000 rpm for 30 minutes.
- Measure the concentrations of unbound and total drug using LC-MS/MS; the plasma protein binding rate is > 98% across all species and concentrations [2]

1. Acute toxicity in mice (from ):
- Male/female C57BL/6 mice (n=3/sex/dose) receive AMG-208 via oral gavage at doses of 150 mg/kg, 250 mg/kg, and 300 mg/kg.
- No mortality is observed at any dose; 300 mg/kg causes transient lethargy (recovers within 48 hours); oral LD50 > 300 mg/kg [2]
2. Subacute toxicity (28-day study in mice, from):
- Doses: 50 mg/kg, 100 mg/kg (oral, once daily).
- Both dose groups show no significant changes in body weight, food intake, serum biochemical parameters (ALT, AST, creatinine), or hematological indices (white blood cell count, platelet count, hemoglobin level).
- Histopathological examination reveals 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 is a member of the class of quinolines that is 7-methoxyquinoline substituted at position 4 by a (6-phenyl[1,2,4]triazolo[4,3-b]pyridazin-3-yl)methoxy group. AMG exhibits antitumour activity, particularly in prostate cancer. It has a role as a c-Met tyrosine kinase inhibitor and an antineoplastic agent. It is a member of quinolines, an aromatic ether and a triazolopyridazine.
AMG-208 has been used in trials studying the treatment of Cancer, Tumors, Oncology, Prostate Cancer, and Oncology Patients, among others.
c-Met Inhibitor AMG 208 is a selective small-molecule inhibitor of the proto-oncogene c-Met with potential antineoplastic activity. c-Met inhibitor AMG 208 inhibits the ligand-dependent and ligand-independent activation of c-Met, inhibiting its tyrosine kinase activity, which may result in cell growth inhibition in tumors that overexpress c-Met. C-Met encodes the hepatocyte growth factor receptor tyrosine kinase, 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. Therapeutic background: AMG-208 is a first-generation triazolopyridazine-derived c-MET tyrosine kinase inhibitor, 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 anti-tumor effects by competitively binding to the ATP-binding pocket of c-MET, inhibiting c-MET autophosphorylation and subsequent activation of downstream signaling pathways (PI3K-AKT, RAS-ERK1/2). This leads to suppressed tumor cell proliferation and reduced tumor growth [2]
3. Research significance: As a representative triazolopyridazine-based c-MET inhibitor, AMG-208 provided a structural template for the development of subsequent c-MET inhibitors and validated the feasibility of targeting c-MET in solid tumors [1][3]
4. Limitation: Due to relatively low oral bioavailability (32%) compared to later-generation inhibitors (e.g., capmatinib), AMG-208 was not advanced to late-phase clinical trials and remains 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

74.43

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)