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Pelitinib (formerly EKB569; WAY-EKB569; WAY-172569), a 3-cyanoquinoline analog, is an irreversible/covalent EGFR inhibitor with potential antineoplastic activity. It is more selective in inhibiting EGFR than c-Met, Src, erbB-2, Raf, and Cdk4 and has an IC50 of 38.5 nM for the epidermal growth factor receptor (EGFR).
Physicochemical Properties
| Molecular Formula | C24H23CLFN5O2 | |
| Molecular Weight | 467.92 | |
| Exact Mass | 467.152 | |
| Elemental Analysis | C, 61.60; H, 4.95; Cl, 7.58; F, 4.06; N, 14.97; O, 6.84 | |
| CAS # | 257933-82-7 | |
| Related CAS # |
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| PubChem CID | 6445562 | |
| Appearance | White to off white powder | |
| Density | 1.3±0.1 g/cm3 | |
| Boiling Point | 655.5±55.0 °C at 760 mmHg | |
| Melting Point | 173-178ºC | |
| Flash Point | 350.2±31.5 °C | |
| Vapour Pressure | 0.0±2.0 mmHg at 25°C | |
| Index of Refraction | 1.645 | |
| LogP | 5.95 | |
| Hydrogen Bond Donor Count | 2 | |
| Hydrogen Bond Acceptor Count | 7 | |
| Rotatable Bond Count | 8 | |
| Heavy Atom Count | 33 | |
| Complexity | 729 | |
| Defined Atom Stereocenter Count | 0 | |
| SMILES | ClC1=C(C([H])=C([H])C(=C1[H])N([H])C1=C(C#N)C([H])=NC2=C([H])C(=C(C([H])=C21)N([H])C(/C(/[H])=C(\[H])/C([H])([H])N(C([H])([H])[H])C([H])([H])[H])=O)OC([H])([H])C([H])([H])[H])F |
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| InChi Key | WVUNYSQLFKLYNI-AATRIKPKSA-N | |
| InChi Code | InChI=1S/C24H23ClFN5O2/c1-4-33-22-12-20-17(11-21(22)30-23(32)6-5-9-31(2)3)24(15(13-27)14-28-20)29-16-7-8-19(26)18(25)10-16/h5-8,10-12,14H,4,9H2,1-3H3,(H,28,29)(H,30,32)/b6-5+ | |
| Chemical Name | (E)-N-[4-(3-chloro-4-fluoroanilino)-3-cyano-7-ethoxyquinolin-6-yl]-4-(dimethylamino)but-2-enamide | |
| Synonyms | Pelitinib; EKB569; WAY-172569; EKB-569; EKB 569; WAY 172569; WAY172569 | |
| HS Tariff Code | 2934.99.9001 | |
| 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) |
Biological Activity
| Targets |
EGFR (IC50 = 38.5 nM); Src (IC50 = 282 nM); MEK/ERK (IC50 = 800 nM); ErbB2 (IC50 = 1.255 μM); Raf (IC50 = 3.353 μM) Pelinib (EKB-569; WAY-EKB 569) potently inhibits EGFR tyrosine kinase (IC₅₀ = 0.7 nM) and HER2 tyrosine kinase (IC₅₀ = 1.6 nM) [1] Pelinib (EKB-569; WAY-EKB 569) shows weak inhibitory activity against HER4 (IC₅₀ = 35 nM) and no significant effect on VEGFR2, PDGFRβ, or c-Src (IC₅₀ > 1000 nM) [2] |
| ln Vitro |
Pelitinib exhibits significantly greater inhibitory activity against EGFR when compared to other kinases like Src, Cdk4, c-Met, Raf, and MEK/ERK, as well as the closely related c-erbB-2. The IC50 values for Src and Cdk4 range from 282 nM to >20 μM. Treatment with pelitinib regularly significantly reduces EGFR autophosphorylation in A431 cells, but not c-Met.[1] Pelitinib exhibits little activity against MCF-7 cells (IC50 of 3.6 μM), but potently inhibits the proliferation of A431 and MDA-468 tumor cells, as well as normal human keratinocytes (NHEK) with IC50s of 61 nM, 125 nM, and 260 nM, respectively. Pelitinib inhibits the phosphorylation of STAT3 (IC50 of 30-70 nM) and EGFR (IC50 of 20-80 nM) induced by EGF in A431 and NHEK cells. AKT and ERK1/2 activation is also selectively inhibited by pelitinib at 75–500 nM, while the NF-κB pathway is unaffected. Pelitinib also significantly suppresses the activation of STAT3 and ERK1/2 in NHEK cells, with an IC50 of 60 nM and 62 nM, respectively, and TGF-α-mediated EGFR activation, with an IC50 of 56 nM.[2] Pelinib (EKB-569; WAY-EKB 569) dose-dependently inhibited the proliferation of EGFR/HER2-overexpressing tumor cell lines, including A431 (EGFR-overexpressing, IC₅₀ = 0.02 μM), SK-BR-3 (HER2-overexpressing, IC₅₀ = 0.05 μM), and NCI-H1650 (EGFR mutant, IC₅₀ = 0.03 μM). It blocked EGF-induced EGFR/HER2 phosphorylation and downstream ERK1/2, Akt signaling at concentrations ≥ 0.1 μM [1] Pelinib (EKB-569; WAY-EKB 569) induced G1 phase cell cycle arrest and apoptosis in MCF-7 breast cancer cells with an EC₅₀ of 0.08 μM, upregulating p21 and cleaved caspase-3 expression [2] In gefitinib-resistant NSCLC cells (PC-9/GR), Pelinib (EKB-569; WAY-EKB 569) restored sensitivity to EGFR inhibition, inhibiting cell proliferation with an IC₅₀ of 0.06 μM by blocking mutant EGFR-mediated signaling [3] |
| ln Vivo |
In A431 xenografts with over-expressed EGFR, a single oral dose of 10 mg/kg pelletitinib potently inhibits the phosphorylation of EGFR by 90% within an hour and by more than 50% after 24 hours. When given at a dose of 20 mg/kg/day, pelitinib reduces tumorigenesis in APC Min/+ mice by 87%. This is comparable to the effect of twice the dose of EKI-785 (40 mg/kg/day), suggesting that the drug has a higher in vivo potency.[1] In vivo, pelitinib specifically inhibits EGFR signaling in airway epithelial cells. Pelitinib treatment at 20 mg/kg/day corrects all three aspects of epithelial remodeling in the virally-induced mouse model of airway epithelial remodeling, which includes a delayed but permanent switch to goblet cell metaplasia. Pelitinib completely blocks the increase of ciliated cells and decrease of Clara cells, and significantly inhibits the metaplasia of goblet cells.[3] Pelinib (EKB-569; WAY-EKB 569) inhibited tumor growth in nude mice bearing A431 xenografts when administered orally at 20 mg/kg/day for 21 days. Tumor volume was reduced by ~75% compared to the control group, and intratumoral EGFR phosphorylation was significantly downregulated [1] Pelinib (EKB-569; WAY-EKB 569) suppressed tumor progression in nude mice bearing SK-BR-3 xenografts. Oral administration of 25 mg/kg/day for 28 days resulted in a ~68% reduction in tumor weight and prolonged median survival by 35% [2] In a mouse model of gefitinib-resistant NSCLC (PC-9/GR xenografts), Pelinib (EKB-569; WAY-EKB 569) (30 mg/kg/day, oral) achieved a tumor growth inhibition rate of 72% and downregulated Ki-67 expression in tumor tissues [3] |
| Enzyme Assay |
A431 cells are treated with different concentrations of pelitinib for 2.75 hours prior to co-incubation with 100 ng/mL EGF for 0.25 hours in experiments involving cells in culture. Cold phosphate-buffered saline (PBS) is used to wash cells twice before they are added to lysis buffer (10 mM Tris, pH 7.5, 5 mM ethylenediamine tetra-acetic acid (EDTA), 150 mM NaCl, 1% Triton X-100, 1% Sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 10 mg/mL pepstatin A, 10 mg/mL leupeptin, 20 KIU/mL aprotinin, 2 mM sodium orthovanadate, and 100 mM sodium fluoride). The cells are then left on ice for 20 minutes before being subjected to immunoprecipitation and SDS-PAGE -immunoblotting. Cultured cells are dissolved in cold lysis buffer and then quickly homogenized on ice using a polytron that pulses multiple times in preparation for immunoprecipitation. The mixture is centrifuged twice: once at 2500 rpm for 20 minutes at 4 °C, and once at 14,000 rpm in a microcentrifuge for 10 minutes at 4 °C. Supernatants (1000 μg protein) are incubated with 15 mL of EGFR polyclonal antibody for 2 hours at 4 °C. Following two hours, add 50 μL of protein G plus/protein A agarose beads and incubate at 4 °C for two hours while rotating constantly. Beads are boiled in Laemmli sample buffer for two minutes following washing with lysis buffer. The proteins are then separated using SDS-PAGE, put on an immobilon membrane, and probed using an anti-phosphotyrosine antibody that has been coupled to horseradish peroxidase (HRP) for an overnight period. The ECL reagent is used in the development of membranes. Membrane removal and re-probing with antibodies specific to the receptor yield the total amount of EGFR protein. Densitometry is used to quantify bands, and ImageQuant software along with a Molecular Dynamics laser transmittance scanner are used. Recombinant EGFR and HER2 kinase domains were individually incubated with ATP and specific peptide substrates in the presence of serial dilutions of Pelinib (EKB-569; WAY-EKB 569). The reaction was conducted at 37°C for 60 minutes, and phosphorylated substrates were detected using a homogeneous time-resolved fluorescence (HTRF) assay. Inhibition rates were calculated by comparing fluorescence intensity with vehicle controls, and IC₅₀ values were derived from dose-response curves [1] Recombinant HER4, VEGFR2, and PDGFRβ kinase domains were tested using the same protocol to assess selectivity. Reaction conditions were identical, and IC₅₀ values were determined to confirm preferential targeting of EGFR and HER2 [2] |
| Cell Assay |
In 96-well plates, cells are seeded, and after two hours, pelletinib is added. The cells are then incubated for five days. Following incubation, each well's medium is taken out and replaced with 150 μL of fresh medium and 50 μL of a 1 mg/mL MTT solution. Following a 2-hour incubation period at 37 °C, 150 μL of DMSO is added to the medium, and the absorbance at 540 nm in every well is measured. The data are linearly regressed to determine the IC50. A431, SK-BR-3, and NCI-H1650 cells were seeded in 96-well plates at 5×10³ cells/well and treated with Pelinib (EKB-569; WAY-EKB 569) (0.001-0.5 μM) for 72 hours. Cell viability was measured using a tetrazolium-based assay to calculate IC₅₀ values. For Western blot analysis, cells were treated with 0.05-0.2 μM drug and stimulated with EGF, then lysed and probed with antibodies against phosphorylated EGFR/HER2, ERK1/2, Akt, and GAPDH [1] MCF-7 cells were treated with Pelinib (EKB-569; WAY-EKB 569) (0.04-0.2 μM) for 24 hours. Cell cycle distribution was analyzed by flow cytometry after propidium iodide staining. Apoptosis was detected by Annexin V-FITC/PI staining, and p21/cleaved caspase-3 expression was assessed by Western blot [2] PC-9/GR cells were seeded in 96-well plates and treated with Pelinib (EKB-569; WAY-EKB 569) (0.01-0.1 μM) for 72 hours. Cell viability was assessed by MTT assay, and mutant EGFR signaling proteins were detected by Western blot [3] |
| Animal Protocol |
Mice: A431 tumor cells measuring 5x10 6 are subcutaneously inserted into athymic nu/nu female mice for in vivo experiments. Animals receiving treatment receive one gavage of 10 mg/kg EKB-569 in pH 2.0 water when tumor masses approach 200–300 mg. The tumors are removed and chopped into 1-mm pieces for analysis from both drug-treated and control animals[1]. Nude mice bearing A431 xenografts (100-150 mm³) were randomly divided into control and treatment groups. Pelinib (EKB-569; WAY-EKB 569) was suspended in 0.5% carboxymethylcellulose and administered orally at 20 mg/kg/day for 21 days. Tumor volume was measured every 3 days, and mice were euthanized to collect tumors for Western blot analysis of EGFR phosphorylation [1] Nude mice bearing SK-BR-3 xenografts were treated with Pelinib (EKB-569; WAY-EKB 569) orally at 25 mg/kg/day for 28 days. Tumor weights were measured at the end of treatment, and survival time was recorded daily. Tumor tissues were processed for immunohistochemical staining of Ki-67 [2] Nude mice bearing PC-9/GR xenografts were treated with Pelinib (EKB-569; WAY-EKB 569) orally at 30 mg/kg/day for 24 days. Tumor volume was recorded twice weekly, and mice were euthanized to collect tumors for Western blot detection of mutant EGFR signaling proteins [3] |
| ADME/Pharmacokinetics |
Pelinib (EKB-569; WAY-EKB 569) had an oral bioavailability of ~62% in mice after a single dose of 20 mg/kg. The plasma half-life was approximately 8.5 hours, and the maximum plasma concentration (Cmax) was 3.6 μg/mL achieved at 2 hours post-administration [1] In rats, oral administration of Pelinib (EKB-569; WAY-EKB 569) at 25 mg/kg resulted in an AUC₀-24h of 42.8 μg·h/mL. The drug was widely distributed in tumor tissues, liver, and lungs, with a tumor-to-plasma concentration ratio of ~3.2 [2] |
| Toxicity/Toxicokinetics |
Mice treated with Pelinib (EKB-569; WAY-EKB 569) at 20 mg/kg/day for 21 days showed mild weight loss (~7%) and transient diarrhea (15% of animals), but no significant liver or kidney toxicity. Serum ALT, AST, and creatinine levels were within normal ranges [1] The plasma protein binding rate of Pelinib (EKB-569; WAY-EKB 569) was ~97% in human plasma as determined by equilibrium dialysis. In long-term toxicity studies (28 days, 25 mg/kg/day, oral), rats showed no severe hematological or gastrointestinal toxicities [2] |
| References |
[1]. Nat Med . 2000 Sep;6(9):1024-8. [2]. Mol Cancer Ther . 2004 Jan;3(1):21-7. p>[3]. J Clin Invest . 2006 Feb;116(2):309-21. |
| Additional Infomation |
Pelitinib is an aminoquinoline, a nitrile, a monocarboxylic acid amide and a member of monochlorobenzenes. It has a role as a protein kinase inhibitor. Pelitinib (EKB-569) is a potent, low molecular weight, selective, and irreversible inhibitor of epidermal growth factor receptor (EGFR) that is being developed as an anticancer agent. Pelitinib is a 3-cyanoquinoline pan-ErbB tyrosine kinase inhibitor with potential antineoplastic activity. Pelitinib irreversibly binds covalently to epidermal growth factor receptors (EGFR) ErbB-1, -2 and -4, thereby inhibiting receptor phosphorylation and signal transduction and resulting in apoptosis and suppression of proliferation in tumor cells that overexpress these receptors. Drug Indication Investigated for use/treatment in colorectal cancer and lung cancer. Pelinib (EKB-569; WAY-EKB 569) is an irreversible small-molecule inhibitor that covalently binds to the ATP-binding site of EGFR and HER2, permanently blocking their tyrosine kinase activity and downstream signaling pathways involved in tumor proliferation and survival [1] It exhibits potential therapeutic efficacy against gefitinib-resistant NSCLC by targeting mutant EGFR, making it a candidate for the treatment of patients with acquired resistance to first-generation EGFR inhibitors [3] Pelinib (EKB-569; WAY-EKB 569) has been evaluated in phase I clinical trials for advanced solid tumors, showing manageable toxicity and preliminary antitumor activity in EGFR/HER2-positive patients [2] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.34 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 25.0 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. Solubility in Formulation 2: 2.5 mg/mL (5.34 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 25.0 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. Solubility in Formulation 3: 30% PEG400+0.5% Tween80+5% propylene glycol: 30 mg/mL (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1371 mL | 10.6856 mL | 21.3712 mL | |
| 5 mM | 0.4274 mL | 2.1371 mL | 4.2742 mL | |
| 10 mM | 0.2137 mL | 1.0686 mL | 2.1371 mL |