human c-Met (Ki = 4.8 nM)

PF-04217903 phenolsulfonate (0.1-10000 nM; 48-72 hours) inhibits the growth of c-Met–amplified human GTL-16 gastric carcinoma and H1993 NSCLC cells IC50 values of 12 and 30 nM, respectively[1].
PF-04217903 phenolsulfonate (1.5-3333 nM; 48 hours) causes GTL-16 cells to undergo apoptosis (IC50=31 nM)[1].
PF-04217903 phenolsulfonate also has IC50 values comparable to those for inhibiting c-Met phosphorylation in these cell lines (IC50=7-12.5 nM), which means it inhibits HGF-mediated cell migration and Matrigel invasion in a number of c-Met-overexpressing tumor cell lines, including human NCI-H441 lung carcinoma and HT29 colon carcinoma[1].
PF-04217903 phenolsulfonate has an IC50 of 3.1 nM, 6.4 nM, and 6.7 nM, respectively, showing comparable potency to inhibit the activity of c-Met-H1094R, c-Met-R988C, and c-Met-T1010I. A c-Met-Y1230C IC50 of >10 μM indicates that PF-04217903 phenolsulfonate exhibits no inhibitory activity[3].

Phenolsulfonate (PF-04217903; p.o., 1–30 mg/kg; daily for 16 days) inhibits tumor growth in a dose-dependent manner, and this effect is correlated with the tumors' decreased c-Met phosphorylation[1].
PF-04217903 phenolsulfonate (5-50 mg/kg, p.o.; once daily for 3 days) induces apoptosis (cleaved caspase-3) in U87MG xenograft tumors at all dose levels while dose-dependently inhibiting phosphorylation of c-Met, Gab-1, Erk1/2, and AKT. In both the GTL-16 and U87MG models, PF-04217903 phenolsulfonate significantly and dose-dependently lowers human IL-8 levels, and in the GTL-16 model, it lowers human VEGFA levels. In U87MG xenograft tumors, PF-04217903 phenolsulfonate significantly increases phospho-PDGFRβ levels[1].

Biochemical kinase assays[1]
c-Met catalytic activity was quantified using a continuous-coupled spectrophotometric assay in which the time-dependent production of ADP by c-Met was determined by analysis of the rate of consumption of NADH. NADH has a measurable absorbance at 340 nm and its consumption was measured by a decrease in absorbance at 340 nm as measured by spectrophotometry at designated time points. To determine Ki values, PF-04217903 was introduced into test wells at various concentrations in the presence of assay reagents and incubated for 10 minutes at 37°C. The assay was initiated by addition of the c-Met enzyme.

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Cellular kinase phosphorylation ELISA assays[1]
Cells were seeded in 96-well plates in media supplemented with 10% FBS and transferred to serum-free media with 0.04% bovine serum albumin (BSA) after 24 hours. In experiments investigating ligand-dependent RTK phosphorylation, corresponding growth factors were added for up to 20 minutes. After incubation of cells with PF-04217903 for 1 hour and/or appropriate ligands, protein lysates were generated from cells. Total tyrosine phosphorylation of selected protein kinases was assessed by the standard sandwich ELISA method.

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Biochemical Kinase Assays[2]
c-MET enzyme inhibition was measured by a continuous coupled spectrophotometric assay as previously described. The assay monitored ATP consumption coupled to oxidation of NADH (measured at 340 nm) while regenerating ATP in the presence of phosphoenol pyruvate (PEP) and coupling enzymes, pyruvate kinase (PK), and lactic dehydrogenase (LDH). Assay reactions contained 0.30 mM ATP (4Km), 0.5 mM Met2 peptide (Ac-ARDMYDKEYYSVHNK), 20 mM MgCl2, 1 mM PEP, 330 μM NADH, 2 mM DTT, 15 units/mL LDH, 15 units/mL PK, test compound (1% DMSO final) in 100 mM HEPES, pH 7.5, 37 °C, and the reactions were initiated by adding 50 nM c-Met N-terminal His6-tagged recombinant human enzyme, aa residues 974–1390. The inhibitors were shown to be ATP-competitive from kinetic and crystallographic studies, and the dose–response data were fit to the equation for competitive inhibition by the method of nonlinear least-squares.

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Cellular Kinase Phosphorylation ELISA Assays[2]
All experiments were done under standard conditions (37 °C and 5% CO2). IC50 values were calculated by concentration–response curve fitting using a Microsoft Excel-based four-parameter method. Cells were seeded in 96-well plates in media supplemented with 10% fetal bovine serum (FBS) and transferred to serum-free media [with 0.04% bovine serum albumin (BSA)] after 24 h. In experiments investigating ligand-dependent RTK phosphorylation, corresponding growth factors were added for up to 20 min. After incubation of cells with an inhibitor for 1 h and/or appropriate ligands for the designated times, cells were washed once with HBSS supplemented with 1 mmol/L Na3VO4, and protein lysates were generated from cells. Subsequently, phosphorylation of selected protein kinases was assessed by a sandwich ELISA method using specific capture antibodies used to coat 96-well plates and a detection antibody specific for phosphorylated tyrosine residues. Antibody-coated plates were (a) incubated in the presence of protein lysates at 4 °C overnight; (b) washed seven times in 1% Tween 20 in PBS; (c) incubated in a horseradish peroxidase-conjugated anti-total-phosphotyrosine (PY-20) antibody (1:500) for 30 min; (d) washed seven times again; (e) incubated in 3,3,5,5-tetramethylbenzidine peroxidase substrate (Bio-Rad) to initiate a colorimetric reaction that was stopped by adding 0.09 N H2SO4; and (f) measured for absorbance in 450 nm using a spectrophotometer. The A549 cell line was used for the c-MET cellular kinase phosphorylation ELISA assay.

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Human Microsomal Stability Studies[2]
Compounds (1 μM) were incubated at 37 °C for 30 min in a final volume of 200 μL of 100 mM potassium phosphate buffer (pH 7.4) containing pooled human liver microsomes (0.8 mg/mL protein) and 2 mM NADPH. Reactions were initiated with the addition of NADPH following a 10-min preincubation. Aliquots of incubation samples were protein precipitated with cold methanol containing 0.1 μM buspirone (internal standard) and centrifuged, and supernatants were analyzed by LC-MS/MS. All incubations were performed in triplicate, and the percent remaining of parent drug at the end of incubation was determined by LC-MS/MS peak area ratio.

Cell Line: GTL-16, H1993 cells
Concentration: 0.1, 1, 10, 100, 1000, 10000 nM
Incubation Time: 48-72 hours
Result: Inhibited proliferation of c-Met–amplified human GTL-16 gastric carcinoma and H1993 NSCLC cells with IC50 values of 12 and 30 nM, respectively.

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-quinolinemethyl)-5-triazolo[4,5-b]pyrazinyl]-1-pyrazolyl]ethanol belongs to the quinoline class of compounds. PF-04217903 has been used in clinical trials for tumor treatment research. The MET tyrosine kinase inhibitor PF-04217903 is a small molecule tyrosine kinase inhibitor with high oral bioavailability and potential anti-tumor activity. PF-04217903 selectively binds to and inhibits c-Met, thereby disrupting the c-Met signaling pathway, which may lead to inhibition of tumor cell growth, migration, and invasion, and induce death in c-Met-expressing tumor cells. The receptor tyrosine kinase c-Met, also known as the hepatocyte growth factor (HGF) receptor, is overexpressed or mutated in various tumor cell types and plays an important role in tumor cell proliferation, survival, invasion, metastasis, and angiogenesis. The c-Met pathway is closely associated with various human cancers due to its crucial role in tumor growth, invasion, and metastasis. PF-04217903 is a novel ATP-competitive small-molecule c-Met kinase inhibitor. Compared to more than 150 kinases, PF-04217903 exhibits over 1000-fold selectivity for c-Met, making it one of the most selective c-Met inhibitors reported to date. In vitro, PF-04217903 inhibits the proliferation, survival, and migration/invasion of tumor cells in MET-amplified cell lines. In vivo, at tolerated doses, it demonstrates significant antitumor activity against tumor models carrying MET gene amplification or the hepatocyte growth factor (HGF)/c-Met autocrine circuit. The antitumor efficacy of PF-04217903 is dose-dependent and closely related to the inhibition of c-Met phosphorylation, downstream signaling, and tumor cell proliferation/survival. In human xenograft tumor models with relatively high c-Met expression levels, complete inhibition of c-Met activity by PF-04217903 only resulted in partial inhibition of tumor growth in vivo (38%-46%). In the HT29 model expressing activated RON kinase, combined application of PF-04217903 with RON short hairpin RNA (shRNA) knockdown induced tumor cell apoptosis, with an antitumor efficacy (77%) significantly superior to PF-04217903 alone (38%) or RON shRNA alone (56%). Furthermore, PF-04217903 exhibited potent anti-angiogenic properties both in vitro and in vivo. PF-04217903 also significantly induced phosphorylated PDGFRβ (platelet-derived growth factor receptor) levels in U87MG xenografts, suggesting a possible oncogene switching mechanism in tumor cell signaling pathways, which may be a potential drug resistance mechanism that weakens the tumor's response to c-Met inhibitors. In summary, these results demonstrate the application of highly selective inhibition of c-Met and provide new insights for targeting tumors with different c-Met dysregulation mechanisms. [1] c-MET receptor tyrosine kinases have become highly attractive targets for tumor therapy due to their key role in human tumorigenesis and progression. During high-throughput screening of c-MET, researchers discovered an indolehydrazine compound 6, which was subsequently confirmed to have exceptionally high selectivity for a variety of other kinases. The co-crystal structure of the related indolehydrazine c-MET inhibitor 10 with the non-phosphorylated c-MET kinase domain revealed a unique binding mode that is closely related to the inhibitor’s excellent selectivity. Researchers used a structure-based drug design approach to replace the chemically unstable indolehydrazine skeleton with a chemically and metabolically stable triazolopyrazine skeleton. Optimization and screening of lead compounds in medicinal chemistry yielded 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), a highly efficient and selective c-MET inhibitor. Compound 2 showed effective tumor growth inhibition in a c-MET-dependent tumor model and exhibited good oral pharmacokinetic properties and acceptable safety. Preclinical studies have shown that it has good safety. Compound 2 has entered a Phase I clinical trial for oncology. [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

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

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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