human c-Met (Ki = 4.8 nM)

PF-04217903 苯酚磺酸盐（0.1-10000 nM；48-72 小时）抑制 c-Met 扩增的人 GTL-16 胃癌和 H1993 NSCLC 细胞的生长，IC50 值分别为 12 和 30 nM[1]。
PF-04217903 苯酚磺酸盐（1.5-3333 nM；48 小时）导致 GTL-16 细胞凋亡 (IC50=31 nM)[1]。
PF-04217903 苯酚磺酸盐的 IC50 值与抑制 c-Met 的值相当。这些细胞系中的磷酸化 (IC50=7-12.5 nM)，这意味着它在许多 c-Met 过表达肿瘤细胞系（包括人 NCI-H441 肺癌和 HT29 结肠癌）中抑制 HGF 介导的细胞迁移和基质胶侵袭[1].
PF-04217903 phenosulfonate 的 IC50 分别为 3.1 nM、6.4 nM 和 6.7 nM，显示出与 c-Met-H1094R、c-Met-R988C 和 c- 活性相当的抑制效力。梅特-T1010I。 c-Met-Y1230C IC50 > 10 μM 表明 PF-04217903 苯酚磺酸盐没有抑制活性[3]。

苯酚磺酸盐（PF-04217903；口服，1–30 mg/kg；每天服用，持续 16 天）以剂量依赖性方式抑制肿瘤生长，这种作用与肿瘤 c-Met 磷酸化降低相关[1]。
PF-04217903 苯酚磺酸盐（5-50 mg/kg，口服；每天一次，持续 3 天）在所有剂量水平下诱导 U87MG 异种移植肿瘤细胞凋亡（裂解的 caspase-3），同时剂量依赖性抑制 c-Met、Gab- 的磷酸化1、Erk1/2 和 AKT。在 GTL-16 和 U87MG 模型中，PF-04217903 苯酚磺酸盐显着且剂量依赖性地降低人 IL-8 水平，并且在 GTL-16 模型中，它降低人 VEGFA 水平。在 U87MG 异种移植肿瘤中，PF-04217903 苯酚磺酸盐显着增加磷酸-PDGFRβ 水平[1]。

生化激酶测定[1]
使用连续耦合分光光度法定量c-Met催化活性，其中通过分析NADH的消耗速率来确定c-Met产生ADP的时间依赖性。NADH在340nm处具有可测量的吸光度，其消耗量是通过在指定时间点通过分光光度法测量的340nm处吸光度的降低来测量的。为了测定Ki值，在检测试剂存在的情况下，将不同浓度的PF-04217903引入测试孔中，并在37°C下孵育10分钟。通过添加c-Met酶启动该测定。

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细胞激酶磷酸化ELISA检测[1]
将细胞接种在补充有10%FBS的培养基中的96孔板中，24小时后转移到含有0.04%牛血清白蛋白（BSA）的无血清培养基中。在研究配体依赖性RTK磷酸化的实验中，加入相应的生长因子长达20分钟。用PF-04217903和/或适当的配体孵育细胞1小时后，细胞产生蛋白质裂解物。通过标准夹心ELISA方法评估所选蛋白激酶的总酪氨酸磷酸化。

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生化激酶测定[2]
如前所述，通过连续偶联分光光度法测量c-MET酶抑制。该测定监测了在磷酸烯醇丙酮酸盐（PEP）和偶联酶、丙酮酸激酶（PK）和乳酸脱氢酶（LDH）存在下再生ATP时，与NADH氧化相关的ATP消耗（在340nm处测量）。检测反应在100 mM HEPES、pH 7.5、37°C中含有0.30 mM ATP（4Km）、0.5 mM Met2肽（Ac-ARDMYKEYYSVHNK）、20 mM MgCl2、1 mM PEP、330μM NADH、2 mM DTT、15单位/mL LDH、15单位/mL PK、测试化合物（1%DMSO终末），反应通过加入50 nM C-Met N-末端His6标记的重组人酶（aa残基974-1390）启动。动力学和晶体学研究表明，抑制剂具有ATP竞争性，剂量反应数据通过非线性最小二乘法拟合到竞争性抑制方程中。

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细胞激酶磷酸化ELISA检测[2]
所有实验均在标准条件下（37°C和5%CO2）进行。IC50值通过使用基于Microsoft Excel的四参数方法进行浓度-反应曲线拟合来计算。将细胞接种在补充有10%胎牛血清（FBS）的培养基中的96孔板中，24小时后转移到无血清培养基[含0.04%牛血清白蛋白（BSA）]中。在研究配体依赖性RTK磷酸化的实验中，加入相应的生长因子长达20分钟。将细胞与抑制剂和/或适当的配体孵育1小时和/或指定时间后，用补充有1 mmol/L Na3VO4的HBSS洗涤细胞一次，细胞产生蛋白质裂解物。随后，通过夹心ELISA法评估所选蛋白激酶的磷酸化，该方法使用用于涂覆96孔板的特异性捕获抗体和对磷酸化酪氨酸残基特异性的检测抗体。抗体包被板（a）在蛋白质裂解物存在下在4°C下孵育过夜；（b） 在1%吐温20的PBS溶液中洗涤7次；（c） 在辣根过氧化物酶偶联的抗总磷酸酪氨酸（PY-20）抗体（1:500）中孵育30分钟；（d） 再洗七次；（e） 在3,3,5,5-四甲基联苯胺过氧化物酶底物（Bio-Rad）中孵育以引发比色反应，通过加入0.09N H2SO4停止比色反应；以及（f）使用分光光度计在450nm处测量吸光度。A549细胞系用于c-MET细胞激酶磷酸化ELISA测定。

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人体微粒体稳定性研究[2]
化合物（1μM）在37°C下在最终体积为200μL的100 mM磷酸钾缓冲液（pH 7.4）中孵育30分钟，该缓冲液含有混合的人肝微粒体（0.8 mg/mL蛋白质）和2 mM NADPH。预孵育10分钟后，加入NADPH引发反应。孵育样品的等分试样用含有0.1μM丁螺环酮（内标）的冷甲醇进行蛋白质沉淀并离心，上清液通过LC-MS/MS进行分析。所有孵育均进行三次，孵育结束时母体药物的剩余百分比通过LC-MS/MS峰面积比确定。

细胞系：GTL-16、H1993 细胞
浓度：0.1、1、10、100、1000、10000 nM
孵育时间：48-72 小时
结果：c-Met 扩增的人细胞增殖受到抑制GTL-16 胃癌和 H1993 NSCLC 细胞的 IC50 值分别为 12 和 30 nM。

Female nu/nu mice (GTL-16 xenograft model) 1, 3, 10, 30 mg/kg Oral; daily for 16 days

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Subcutaneous xenograft models in athymic mice.[1] Tumor cells were implanted subcutaneously into the right flank region of each mouse and allowed to grow to the designated size. The athymic mice bearing established tumors were administered PF-04217903 either by oral gavage in 0.5% methylcellulose suspension or by implanting a mini Alzet-pump carrying the drug solution. Tumor volume was measured using electronic digital calipers. Percent (%) inhibition values were calculated as: 100 × {1 − [(treatedfinal day − treatedday 1)/(controlfinal day − controlday 1)]}. Tumor volumes were analyzed using one-way ANOVA. At the end of study, mice were humanely euthanized and tumors were resected. Proteins were extracted from the tumor samples and protein concentrations were determined using a BSA assay (Pierce). The level of proteins of interest in the tumor sample was determined using a capture ELISA method or immunoblotting.

[1]. Sensitivity of selected human tumor models to PF-04217903, a novel selective c-Met kinase inhibitor. Mol Cancer Ther. 2012 Apr;11(4):1036-47.

[2]. Discovery of a novel class of exquisitely selective mesenchymal-epithelial transition factor (c-MET) protein kinase inhibitors and identification of the clinical candidate 2-(4-(1-(quinolin-6-ylmethyl)-1H-[1,2,3]triazolo[4,5-b]pyrazin-6-yl). 2012 Sep 27;55(18):8091-109.

[3]. Enzymatic characterization of c-Met receptor tyrosine kinase oncogenic mutants and kinetic studies with aminopyridine and triazolopyrazine inhibitors. Biochemistry. 2009 Jun 16;48(23):5339-49.

2-[4-[3-(6-quinolinylmethyl)-5-triazolo[4,5-b]pyrazinyl]-1-pyrazolyl]ethanol is a member of quinolines.
PF-04217903 has been used in trials studying the treatment of Neoplasms.
MET Tyrosine Kinase Inhibitor PF-04217903 is an orally bioavailabe, small-molecule tyrosine kinase inhibitor with potential antineoplastic activity. MET tyrosine kinase inhibitor PF-04217903 selectively binds to and inhibits c-Met, disrupting the c-Met signaling pathway, which may result in the inhibition of tumor cell growth, migration and invasion of tumor cells, and the induction of death in tumor cells expressing c-Met. The receptor tyrosine kinase c-Met, also known as hepatocyte growth factor (HGF) receptor, is overexpressed or mutated in many tumor cell types, playing an important role in tumor cell proliferation, survival, invasion, and metastasis and angiogenesis.

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The c-Met pathway has been implicated in a variety of human cancers for its critical role in tumor growth, invasion, and metastasis. PF-04217903 is a novel ATP-competitive small-molecule inhibitor of c-Met kinase. PF-04217903 showed more than 1,000-fold selectivity for c-Met compared with more than 150 kinases, making it one of the most selective c-Met inhibitors described to date. PF-04217903 inhibited tumor cell proliferation, survival, migration/invasion in MET-amplified cell lines in vitro, and showed marked antitumor activity in tumor models harboring either MET gene amplification or a hepatocyte growth factor (HGF)/c-Met autocrine loop at well-tolerated dose levels in vivo. Antitumor efficacy of PF-04217903 was dose-dependent and showed a strong correlation with inhibition of c-Met phosphorylation, downstream signaling, and tumor cell proliferation/survival. In human xenograft models that express relatively high levels of c-Met, complete inhibition of c-Met activity by PF-04217903 only led to partial tumor growth inhibition (38%-46%) in vivo. The combination of PF-04217903 with Recepteur d'origine nantais (RON) short hairpin RNA (shRNA) knockdown in the HT29 model that also expresses activated RON kinase-induced tumor cell apoptosis and resulted in enhanced antitumor efficacy (77%) compared with either PF-04217903 (38%) or RON shRNA alone (56%). PF-04217903 also showed potent antiangiogenic properties in vitro and in vivo. Furthermore, PF-04217903 strongly induced phospho-PDGFRβ (platelet-derived growth factor receptor) levels in U87MG xenograft tumors, indicating a possible oncogene switching mechanism in tumor cell signaling as a potential resistance mechanism that might compromise tumor responses to c-Met inhibitors. Collectively, these results show the use of highly selective inhibition of c-Met and provide insight toward targeting tumors exhibiting different mechanisms of c-Met dysregulation.[1]

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The c-MET receptor tyrosine kinase is an attractive oncology target because of its critical role in human oncogenesis and tumor progression. An oxindole hydrazide hit 6 was identified during a c-MET HTS campaign and subsequently demonstrated to have an unusual degree of selectivity against a broad array of other kinases. The cocrystal structure of the related oxindole hydrazide c-MET inhibitor 10 with a nonphosphorylated c-MET kinase domain revealed a unique binding mode associated with the exquisite selectivity profile. The chemically labile oxindole hydrazide scaffold was replaced with a chemically and metabolically stable triazolopyrazine scaffold using structure based drug design. Medicinal chemistry lead optimization produced 2-(4-(1-(quinolin-6-ylmethyl)-1H-[1,2,3]triazolo[4,5-b]pyrazin-6-yl)-1H-pyrazol-1-yl)ethanol (2, PF-04217903), an extremely potent and exquisitely selective c-MET inhibitor. 2 demonstrated effective tumor growth inhibition in c-MET dependent tumor models with good oral PK properties and an acceptable safety profile in preclinical studies. 2 progressed to clinical evaluation in a Phase I oncology setting.[2]

C19H16N8O

372.383341789246

372.145

C, 54.94; H, 4.06; N, 20.50; O, 14.64; S, 5.87

1159490-85-3

PF-04217903;956905-27-4;PF-04217903 mesylate;956906-93-7

17754438

Solid powder

1.673

107.43

1

7

5

28

524

0

C1=CC2=C(C=CC(=C2)CN3C4=NC(=CN=C4N=N3)C5=CN(N=C5)CCO)N=C1

PDMUGYOXRHVNMO-UHFFFAOYSA-N

InChI=1S/C19H16N8O/c28-7-6-26-12-15(9-22-26)17-10-21-18-19(23-17)27(25-24-18)11-13-3-4-16-14(8-13)2-1-5-20-16/h1-5,8-10,12,28H,6-7,11H2

2-[4-[3-(quinolin-6-ylmethyl)triazolo[4,5-b]pyrazin-5-yl]pyrazol-1-yl]ethanol

PF-04217903 phenolsulfonate; PF 04217903 phenolsulfonate; PF04217903 phenolsulfonate

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
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