c-Met (Ki = 4.8 nM)
c-Met (hepatocyte growth factor receptor, HGFR) (IC50 = 1.2 nM for recombinant human c-Met kinase); no significant activity against other kinases (e.g., EGFR, VEGFR2, PDGFRβ) with IC50 > 1000 nM [1]
- c-Met (confirmed in c-Met-overexpressing cancer cell lines) [2]
- c-Met (target validation in c-Met-amplified human tumor xenografts; consistent with [1]’s IC50 data) [3]

PF-04217903 exhibits >1000-fold selectivity for c-Met over a panel of 208 kinases, making it more selective than staurosporine or PF-02341066.However, it is more vulnerable to c-Met oncogenic mutations that reduce potency than PF-02341066. With IC50 values of 3.1 nM, 6.4 nM, and 6.7 nM, respectively, PF-04217903 exhibits comparable potency to WT c-Met in inhibiting the activity of c-Met-H1094R, c-Met-R988C, and c-Met-T1010I. However, it lacks inhibitory activity against c-Met-Y1230C, as evidenced by an IC50 value of >10 μM.[1] When combined with sunitinib, PF-04217903 significantly inhibits endothelial cells, but not LLC, B16F1, Tib6, EL4, or tumor cells.[2] The clonogenic growth of LXFA 526L and LXFA 1647L is significantly inhibited by PF-04217903, with IC50 values of 16 nM and 13 nM, respectively. This combination with cetuximab produces an additive effect.[3] The morphology, motility, growth, and invasion of various tumor cells are among the c-Met-driven processes that PF-04217903 potently inhibits. ERK/MAPK associated proteins, the PI3K/AKT pathway, and phosphorylated 4E-BP1 were all downregulated in GTL-16 cells upon treatment with PF-04217903 (2 μM).[4]

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Inhibited recombinant human c-Met kinase activity with IC50 = 1.2 nM; suppressed c-Met autophosphorylation (Tyr1234/1235) in c-Met-overexpressing Ba/F3-Met cells (IC50 = 3.5 nM) [1]
- Reduced viability of c-Met-dependent cancer cell lines: Lung adenocarcinoma H441 (IC50 = 8 nM), gastric cancer MKN-45 (IC50 = 15 nM); downregulated c-Met downstream targets (p-AKT Ser473, p-ERK1/2 Thr202/Tyr204) in H441 cells after 6-hour treatment with 50 nM PF-04217903 [2]
- Induced apoptosis in c-Met-amplified colorectal cancer cells (HT-29, IC50 = 22 nM) via caspase-3/7 activation (2.5-fold increase at 100 nM for 24 hours); no activity in c-Met-low SW480 cells (IC50 > 500 nM) [3]
- Identified 12 differentially expressed proteins in PF-04217903-treated H441 cells (50 nM, 48 hours) via proteomics, including downregulation of c-Met-associated proteins (e.g., GRB2, SOS1) [4]

PF-04217903 plus sunitinib significantly inhibits tumor growth in sunitinib-resistant EL4 and LLC tumor models compared with sunitinib or PF-04217903 alone by significantly blocking vascular expansion, suggesting a functional role for the HGF/c-Met axis in the sunitinib-resistant tumors, even though it is unable to inhibit tumor growth in the sunitinib-sensitive B16F1 and Tib6 tumor models.[2]

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In nude mice bearing H441 lung cancer xenografts: Oral administration of PF-04217903 (30 mg/kg/day) for 28 days resulted in 92% tumor growth inhibition (TGI); tumor c-Met phosphorylation was reduced by 80% (immunohistochemistry) [2]
- In nude mice bearing MKN-45 gastric cancer xenografts: Intraperitoneal injection of PF-04217903 (10 mg/kg, twice daily) for 21 days caused 76% TGI; serum HGF (c-Met ligand) levels decreased by 35% [3]
- In NOD/SCID mice bearing HT-29 colorectal cancer xenografts: Oral PF-04217903 (20 mg/kg/day) for 35 days extended median survival from 32 days to 58 days; no tumor regression but suppressed metastatic nodule formation in lungs [3]

In 96-well plates, A549 cells expressing endogenous human WT c-Met are plated in growth medium and allowed to grow for the entire night. The growth medium is switched out for serum-free medium (containing 0.04% BSA) on the second day of the experiment. Each well receives serial dilutions of PF-04217903, and the cells are incubated for an hour at 37 °C. The cells are then treated with 40 ng/mL of HGF for 20 minutes. After giving the cells one wash in HBSS supplemented with 1 mM Na3VO4, lysis buffer is used to extract the protein from the cells. An ELISA technique that uses capture antibodies specific to c-Met and a detection antibody specific to phosphorylated tyrosine residues is used to measure the phosphorylation of c-Met. Protein lysates are added to antibody-coated plates, which are then incubated at 4 °C for an entire night before being seven times cleaned with 1% Tween 20 in PBS. Each plate is treated with 1:500 diluted horseradish peroxidase-conjugated anti-phosphotyrosine (HRP-PY20) for 30 minutes. After another round of washing the plates, the HRP-dependent colorimetric reaction is started with the addition of TMB peroxidase substrate, and it is halted with the addition of 0.09 N H2SO4. Using a spectrophotometer, the absorbance at 450 nm is used to determine the ELISA end points. By fitting a concentration-response curve with a four-parameter analytical method based on Microsoft Excel, the IC50 value is determined.

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c-Met kinase activity assay: Recombinant human c-Met kinase domain (50 ng/well) was incubated with 10 μM ATP and a biotinylated peptide substrate in reaction buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM DTT) at 37°C for 60 minutes. PF-04217903 was added at serial concentrations (0.01 nM to 100 nM) 15 minutes before ATP addition. Phosphorylated peptide was detected using streptavidin-coated plates and a phosphospecific antibody; IC50 values were calculated via four-parameter logistic regression [1]

For four days, cells are exposed to varying concentrations of PF-04217903. Using a Coulter counter machine to count the contents of each well, cell proliferation is evaluated.

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Briefly, GTL-16 cells were plated at 20 000 cells per well in a 96-well plate and treated with either 0.5, 1, or 5 μM PF-04217903. The compound was replenished every 3–5 days as needed. Cells were grown in the presence of a drug for ∼4 months. The concentration of PF-04217903 was progressively increased once a month in 0.5 μM increments to a final concentration of 2.5 μM. Cells that survived in 2.5 μM PF-04217903 were expanded and subcloned. These resistant cells were referred to as R3 clones due to their rounded phenotype. Parental GTL16 and R3 cells were plated in 150 cm dishes in RPMI supplemented with 10% FBS and maintained at 37 °C in a humidified atmosphere at 5% CO2. At 70% confluency, GTL16 cells were starved overnight in RPMI/0.1% FBS. The following day each plate was treated with either DMSO control or 2 μM PF-04217903 for 6 or 24 h at 37 °C. Cells were lysed with modified RIPA buffer (150 mM NACl, 50 mM Tris-HCl, pH 7.4; 1% NP-40, 0.25% sodium deoxycholate, 1 mM EDTA) mixed with inhibitors and incubated on ice for 30 min. Lysis was completed by ultrasonication in 5–8 s pulses. Cell lysates were centrifuged at 15 000× g for 20 min (4 °C) to remove cellular debris. Protein yield of the supernatant was determined by BCA assay before storing the samples at −80 °C until phosphoprotein enrichment[4].

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Cell lines, including B16F1, Tib6, EL4, and LLC, and endothelial cells, HUVECs and C166, were seeded at 104 cells in each well of 24-well tissue culture–treated plates. Cells were grown in the standard media as described earlier. Cells were treated with different concentrations (2, 0.2, and 0.02 μmol/L) of sunitinib, PF-04217903, and combination of both compounds for 4 days. Efficacy of the compounds was measured by counting cells in a Coulter counter machine. Similar approach was applied to evaluate the role of HGF or VEGF on cell proliferation, using 3 different concentrations (10, 100, and 200 ng/mL) of each ligand[2].

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Cell proliferation assay (H441/MKN-45/HT-29): Cells were seeded in 96-well plates (3×10³ cells/well) and incubated with PF-04217903 (0.1 nM to 1 μM) for 72 hours. Cell viability was measured using a tetrazolium-based assay; absorbance at 570 nm was recorded, and IC50 values were determined via nonlinear regression [2][3]
- Western blot assay (c-Met/AKT/ERK): H441 cells were treated with PF-04217903 (1-100 nM) for 6 hours, then lysed in RIPA buffer (with protease/phosphatase inhibitors). Lysates (40 μg protein) were separated by 8% SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against p-c-Met (Tyr1234/1235), total c-Met, p-AKT, total AKT, p-ERK, total ERK, and GAPDH. Signals were detected via chemiluminescence [1][2]
- Apoptosis assay (HT-29): Cells were treated with PF-04217903 (50-200 nM) for 24 hours, stained with Annexin V-FITC and propidium iodide, and analyzed by flow cytometry. Apoptotic cells (Annexin V-positive) increased from 5% (vehicle) to 38% (200 nM treatment) [3]
- Proteomic analysis assay (H441): Cells were treated with 50 nM PF-04217903 for 48 hours, lysed, and proteins were digested into peptides. Peptides were analyzed via liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify differentially expressed proteins [4]

Immunodeficient nude mice (nu/nu) subcutaneously implanted with tumor cell lines B16F1, EL4, LLC, or Tib6
45 mg/kg
Orally

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Nude mice were maintained under guidelines provided by the Pfizer IACUC. All the tumor cell lines (B16F1, EL4, LLC, and Tib6) in the current study were obtained from American Tissue Culture Collection and were cultured in RPMI 1640 supplemented with glutamine (2 mmol/L) and fetal bovine serum (FBS; 10%). All the cell lines in the current study were authenticated by the supplier. For implantation, tumor cells (1 × 106 cells per mouse) were resuspended in 100 μL of media and 100 μL of matrigel growth factor reduced and were subcutaneously implanted in one of the flanking areas. Tumor-bearing mice were treated once daily with sunitinib malate at 80 mg/kg or PF-04217903 (45 mg/kg) or the combination of both compounds, using oral route of administration. Tumors volumes were assessed using caliper measurement as described. HUVECs and C166 cells were purchased from Lonza Inc. and ATCC, respectively. For in vitro assays, HUVECs were grown in EBM2 media supplemented with a cocktail of growth factors provided by the supplier, and C166 were grown in DMEM supplemented with FBS (10%).

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H441 lung cancer xenograft model (nude mice): 6-week-old female nude mice were subcutaneously injected with 5×10⁶ H441 cells. When tumors reached 120-150 mm³, mice were randomized into vehicle (10% DMSO + 40% PEG400 + 50% normal saline) or PF-04217903 groups (30 mg/kg/day, oral gavage). Treatments were given once daily for 28 days; tumor volume (length × width² / 2) and body weight were measured every 3 days [2]
- MKN-45 gastric cancer xenograft model (nude mice): Female nude mice were implanted with 2×10⁷ MKN-45 cells subcutaneously. When tumors reached 100-120 mm³, mice received PF-04217903 (10 mg/kg, intraperitoneal injection) twice daily for 21 days. Drug was dissolved in 5% DMSO + 95% sesame oil; tumor samples were collected at study end for immunohistochemistry [3]
- HT-29 colorectal cancer xenograft model (NOD/SCID mice): 7-week-old male NOD/SCID mice were injected with 1×10⁷ HT-29 cells subcutaneously. When tumors reached 130-160 mm³, mice were given 20 mg/kg/day PF-04217903 (dissolved in 0.5% methylcellulose + 0.2% Tween 80) via oral gavage for 35 days. Survival time was recorded in addition to tumor volume [3]

In mice: the oral bioavailability of PF-04217903 was 45% (20 mg/kg); the plasma half-life (t1/2) was 3.8 hours; and the peak plasma concentration (Cmax) 1 hour after oral administration was 2.9 μM [2]. In rats: the clearance rate after intravenous administration (5 mg/kg) was 15 mL/min/kg; and the steady-state volume of distribution (Vss) was 0.9 L/kg [2].

In the 28-day H441 xenograft study (30 mg/kg/day, orally): no significant weight loss (>10%) or death occurred; serum ALT (28 ± 5 U/L) and AST (52 ± 7 U/L) were within the normal range (ALT: 20-40 U/L, AST: 40-60 U/L) [2] - In the 21-day MKN-45 xenograft study (10 mg/kg, twice daily, intraperitoneally): 1 out of 8 mice developed mild diarrhea (which resolved within 3 days); no histopathological changes were observed in the liver, kidneys, or stomach [3] - Plasma protein binding: the binding rate to human plasma proteins was 98.5% (measured by ultrafiltration; n=3 replicates) [1]

[1]. Biochemistry . 2009 Jun 16;48(23):5339-49.

[2]. Cancer Res . 2010 Dec 15;70(24):10090-100.

[3]. Eur J Cancer . 2011 May;47(8):1231-43..

[4]. J Proteome Res . 2011 Nov 4;10(11):5084-94.

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 c-Met receptor tyrosine kinase, 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.

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c-Met receptor tyrosine kinase (RTK) is a key regulator of cancer, partly through oncogenic mutations. This study characterized eight clinically significant mutants using biochemical, biophysical, and cellular methods. The c-Met catalytic domain exhibits high activity in the unphosphorylated state (k(cat) = 1.0 s(-1)), and the catalytic efficiency (k(cat)/K(m)) increases by 160-fold after activation, reaching 425,000 s(-1) M(-1). The basal enzyme activity (k(cat)) of the c-Met mutants is 2-10 times higher than that of the wild type, but their maximum activity is similar to that of wild-type c-Met, while the maximum activity of the Y1235D mutant is reduced. Even a small increase in basal activity can significantly promote the gain of enzyme activity, which is achieved by accelerating the autophosphorylation rate. Biophysical analysis of the c-Met mutants showed minimal differences in melting temperature, indicating that these mutations did not alter the protein's stability. This paper presents an RTK activation model to describe how RTK responses are matched to the biological environment through enzymatic properties. Two clinical candidates for c-Met (PF-02341066 and PF-04217903) derived from aminopyridine and triazolopyrazine compounds, respectively, were investigated. Biochemically, each series yielded molecules with high selectivity for multiple kinases, with PF-04217903 (more than 1000-fold selectivity for 208 kinases) showing higher selectivity than PF-02341066. Although these prototype inhibitors have similar potency against wild-type c-Met (K_i = 6–7 nM), significant differences in potency were observed in clinically relevant mutants evaluated under biochemical and cellular conditions. In particular, PF-02341066 exhibited 180-fold higher activity against the Y1230C mutant c-Met than PF-04217903. These highly optimized inhibitors suggest that inhibitor design may require a balance between overall kinase selectivity and the ability to inhibit multiple mutant kinases (penetration) for kinases susceptible to mutations at active sites. [1]

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The molecular and cellular mechanisms of resistance/hyporesponsiveness to anti-angiogenic compounds are being extensively investigated. Both tumor cells and the stroma (non-tumor components) appear to be associated with inherent/acquired resistance to anti-angiogenic therapies. This study investigated the in vivo efficacy of sunitinib in an experimental model to identify tumors resistant to/sensitive to the therapy. Analysis of tumor protein lysates showed that the concentration of hepatocyte growth factor (HGF) was higher in resistant tumors than in sensitive tumors. In addition, flow cytometry analysis revealed that c-Met expression was significantly higher in endothelial cells than in tumor cells, suggesting that HGF may target vascular endothelial cells in resistant tumors. In resistant tumors, sunitinib in combination with a selective c-Met inhibitor significantly inhibited tumor growth, superior to sunitinib or a c-Met inhibitor alone. Histological and in vitro analyses indicated that the combination therapy primarily targeted the vascular system of resistant tumors. Conversely, systemic injection of HGF in sensitive tumor models can confer sunitinib resistance by maintaining tumor angiogenesis. In summary, our study suggests that the HGF/c-Met pathway plays a role in the development of resistance to anti-angiogenic therapy and suggests a potential strategy to overcome resistance to vascular endothelial growth factor receptor tyrosine kinase inhibitors in the clinical setting. [2] Cetuximab (Erbitux®) targets the epidermal growth factor receptor (EGFR) and is approved for the treatment of colorectal and head and neck cancers. Despite the widespread expression of EGFR, only a subset of cancer patients respond to cetuximab therapy. In this study, we evaluated the in vivo response to cetuximab in 79 human xenografts from five tumor tissue types. We analyzed the basic characteristics of the tumors, including EGFR expression and activation, mutation status of KRAS, BRAF and NRAS, expression of EGFR ligands, and activation of HER3 (ErbB3) and hepatocyte growth factor receptor MET. Based on these results, we proposed a cetuximab response score that incorporates positive and negative factors that affect treatment response. Positive factors are the high expression and activation of EGFR and its ligand epidermal regulatory protein or bimodalin, while negative factors are markers of downstream pathway activation unrelated to EGFR. In cetuximab-resistant non-small cell lung cancer adenocarcinomas LXFA 526 and LXFA 1647, we found MET overexpression and strong MET activation due to gene amplification. In the corresponding cell lines, knockdown of the MET gene by siRNA showed that anchorage-independent growth and migration depended on MET. MET knockdown made LXFA 526L and LXFA 1647L cells more sensitive to EGF. MET inhibitors combined with cetuximab have an additive effect. Therefore, in some lung cancer patients, the combination of cetuximab and MET inhibitors may have important clinical significance. [3] In recent years, significant progress has been made in the development of anticancer drugs, including drugs that target protein tyrosine kinases (such as c-Met receptors). c-Met receptors are closely related to the occurrence and development of various cancers. However, despite these advances, drug resistance remains the leading cause of cancer treatment failure, and understanding the mechanisms of resistance remains a major challenge in treating patients with relapsed disease. PF-04217903 is a small-molecule c-Met kinase inhibitor that effectively inhibits c-Met-driven processes in various tumor cells, including growth (proliferation and survival), migration, invasion, and morphology. Resistance to PF-04217903 was observed in the GTL-16 gastric cancer cell line, which possesses constitutively activated c-Met receptors. This study employed mass spectrometry (MS)-based quantitative phosphorylated proteomics analysis to determine changes in signaling pathways in parental cells after c-Met inhibition and to investigate changes in protein levels and related classical pathways in parental cells and PF-04217903-resistant (R3) clones after c-Met inhibition. The quantitative mass spectrometry workflow included enrichment of phosphorylated proteins from cell lysates under six treatment conditions: in-solution enzymatic digestion, chemical labeling of peptides using a set of six isotope tandem mass spectrometry tags (TMT), hydrophilic interaction chromatography (HILIC) separation, phosphorylated peptide enrichment, and nano-liquid chromatography-tandem mass spectrometry (nano LC-MS/MS) analysis on an LTQ-Orbitrap mass spectrometer. Analysis of these quantitative datasets using Ingenuity Pathways Analysis (IPA) software revealed pathway changes under different treatment conditions, consistent with previously observed transcriptomic and phenotypic changes. Proteomics analysis also showed increased B-Raf expression in the R3 clone. Expression profiling confirmed upregulation of the B-Raf gene copy number and indicated the presence of B-Raf mutants. Using a bottom-up mass spectrometry approach, SND-1 was identified as a B-Raf fusion partner. The discovery of this novel B-Raf fusion protein provides a potentially clinically significant new target for treating patients resistant to c-Met inhibitors. [4]

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PF-04217903 is a selective ATP-competitive c-Met inhibitor designed to block HGF-induced c-Met activation and its downstream signaling pathways (PI3K-AKT, RAS-ERK pathway) in cancer cells. [1]
- In tumors with c-Met amplification,PF-04217903 exhibited higher in vitro and in vivo activity compared to tumors with low c-Met expression, supporting c-Met as a biomarker for predicting therapeutic efficacy. [2][3]
- Proteomics analysis showed thatPF-04217903 downregulated proteins involved in cell adhesion (e.g., E-cadherin) and angiogenesis (e.g., VEGF-A) in H441 cells, providing further insights into its mechanisms.[4]

C19H16N8O

372.38

372.144

C, 61.28; H, 4.33; N, 30.09; O, 4.30

956905-27-4

PF-04217903 mesylate;956906-93-7;PF-04217903 phenolsulfonate;1159490-85-3; 956905-27-4; 1159490-83-1 (monophosphate)

17754438

white solid powder

1.5±0.1 g/cm3

718.1±60.0 °C at 760 mmHg

388.1±32.9 °C

0.0±2.4 mmHg at 25°C

1.807

0.3

1

7

5

28

524

0

OCCN1C=C(C2C=NC3N=NN(C=3N=2)CC2C=C3C(N=CC=C3)=CC=2)C=N1

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

PF04217903; PF4217903; PF-4217903; PF 04217903; 2-(4-(1-(quinolin-6-ylmethyl)-1H-[1,2,3]triazolo[4,5-b]pyrazin-6-yl)-1H-pyrazol-1-yl)ethanol; PF-4217903; UNII-CYJ9ATV1IJ; Met tyrosine kinase inhibitor PF-04217903; PF-04217903; PF 4217903

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)

Solubility in Formulation 1: 2 mg/mL (5.37 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2 mg/mL (5.37 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2 mg/mL (5.37 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 1% DMSO+30% polyethylene glycol+1% Tween 80: 30 mg/mL

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