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Icotinib (formerly known as BPI-2009; trade name Conmana), a side chain-cyclized form of erlotinib, is an orally available and specific EGFR inhibitor with potent anticancer activity. Icotinib was licensed in China in 2011 to treat non-small cell lung cancer (NSCLC). It shares structural similarities with erlotinib. With an IC50 of 5 nM, it inhibits all forms of EGFR, including EGFR(T790M), EGFR(L858R), EGFR(L861Q), and EGFR(T790M, L858R). Icotinib only demonstrated significant inhibitory activity against EGFR and its mutants when profiled using 88 kinases. Icotinib inhibits the proliferation of tumor cells and blocked EGFR-mediated intracellular tyrosine phosphorylation (IC50 = 45 nM) in the human epidermoid carcinoma A431 cell line. Icotinib showed strong dose-dependent antitumor effects in nude mice bearing different human tumor xenografts, according to in vivo studies. In mice, the medication was well tolerated at dosages up to 120 mg/kg/day without causing death or appreciable body weight loss while the mice were being treated. Gefitinib was used as an active control in a head-to-head randomized, double-blind phase III trial that was recently completed for patients with advanced non-small cell lung cancer (NSCLC) (Trial registration ID: NCT01040780). The information demonstrates that Icotinib outperformed Gefitinib in terms of safety and median progression-free survival (PFS), with Icotinib outperforming Gefitinib in terms of safety.
Physicochemical Properties
| Molecular Formula | C22H21N3O4 |
| Molecular Weight | 391.42 |
| Exact Mass | 391.153 |
| Elemental Analysis | C, 67.51; H, 5.41; N, 10.74; O, 16.35 |
| CAS # | 610798-31-7 |
| Related CAS # | Icotinib Hydrochloride;1204313-51-8;Icotinib-d4;1567366-82-8 |
| PubChem CID | 22024915 |
| Appearance | White to off-white solid powder |
| Density | 1.3±0.1 g/cm3 |
| Boiling Point | 581.3±50.0 °C at 760 mmHg |
| Flash Point | 305.3±30.1 °C |
| Vapour Pressure | 0.0±1.6 mmHg at 25°C |
| Index of Refraction | 1.649 |
| LogP | 1.88 |
| Hydrogen Bond Donor Count | 1 |
| Hydrogen Bond Acceptor Count | 7 |
| Rotatable Bond Count | 3 |
| Heavy Atom Count | 29 |
| Complexity | 553 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | O1C([H])([H])C([H])([H])OC([H])([H])C([H])([H])OC([H])([H])C([H])([H])OC2C([H])=C3C(C(=NC([H])=N3)N([H])C3=C([H])C([H])=C([H])C(C#C[H])=C3[H])=C([H])C1=2 |
| InChi Key | QQLKULDARVNMAL-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C22H21N3O4/c1-2-16-4-3-5-17(12-16)25-22-18-13-20-21(14-19(18)23-15-24-22)29-11-9-27-7-6-26-8-10-28-20/h1,3-5,12-15H,6-11H2,(H,23,24,25) |
| Chemical Name | N-(3-ethynylphenyl)-2,5,8,11-tetraoxa-15,17-diazatricyclo[10.8.0.014,19]icosa-1(12),13,15,17,19-pentaen-18-amine |
| Synonyms | BPI-2009; BPI2009; BPI 2009; BPI2009H; BPI-2009H; 610798-31-7; Conmana; BPI-2009; N-(3-ethynylphenyl)-7,8,10,11,13,14-hexahydro-[1,4,7,10]tetraoxacyclododecino[2,3-g]quinazolin-4-amine; UNII-9G6U5L461Q; 9G6U5L461Q; ICOTINIB [WHO-DD]; BPI 2009H; Icotinib; Trade name: Conmana |
| 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 |
| 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 = 5 nM); EGFR L858R ; EGFR L858R/T790M ; EGFR T790M ; EGFR L861Q Icotinib (BPI2009) is a selective inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, with an IC₅₀ of 5.4 nM for wild-type EGFR, 2.6 nM for EGFR L858R mutant, and 18.5 nM for EGFR del19 mutant [1] It shows weak inhibitory activity against HER2 (IC₅₀ = 400 nM) and no significant activity against VEGFR2, PDGFRβ, or c-Src (IC₅₀ > 1000 nM) [1] |
| ln Vitro |
Iconitib at 0.5 μM inhibits kinase activity in 91%, 99%, 96%, 61%, and 61% of the cases. With IC50s of 1, 4.06, 12.16, 16.08, and 40.71 μM, iconetib inhibits the growth of A431 and BGC-823, A549, H460, and KB cell lines. Icotinib only exhibits significant inhibitory activity against EGFR and its mutants when profiled with 88 kinases. Icotinib inhibits the proliferation of tumor cells by blocking EGFR-mediated intracellular tyrosine phosphorylation (IC50=45 nM) in the human epidermoid carcinoma A431 cell line[1].
In vitro: Icotinib inhibits EGFR activity in a dose-dependent manner, with an IC50 value of 5 nM and complete inhibition at 62.5 nM. Icotinib selectively solely inhibits the EGFR members including the wild type and mutants with inhibition efficacies of 61-99%. Icotinib blocks EGFR-mediated intracellular tyrosine phosphorylation in human epidermoid carcinoma A431 cells in a dose-dependent manner. Meanwhile, in the proliferation assay performed on A431, BGC-823, A549, H460, HCT8, KB and Bel-7402 cell lines, it was found that the relative sensitivity of cell lines to Icotinib is A431 > BGC-823 > A549 > H460 > KB > HCT8 and Bel-7402. Icotinib exhibits a broad spectrum of antitumor activity and it is especially effective against tumors expressing higher levels of EGFR. Icotinib (BPI2009) dose-dependently inhibited the proliferation of EGFR-mutant non-small cell lung cancer (NSCLC) cell lines, including HCC827 (EGFR del19, IC₅₀ = 0.02 μM) and PC9 (EGFR del19, IC₅₀ = 0.03 μM). It also inhibited wild-type EGFR-expressing A549 cells with an IC₅₀ of 1.2 μM [1] The drug blocked EGF-induced EGFR phosphorylation and downstream AKT/ERK1/2 signaling at concentrations ≥ 0.1 μM. It induced G1 phase cell cycle arrest and apoptosis in HCC827 cells, with an EC₅₀ of 0.08 μM for apoptosis, upregulating cleaved caspase-3 and PARP expression [1] In gefitinib-sensitive NSCLC cells, Icotinib (BPI2009) (0.1 μM) suppressed colony formation by ~75% and downregulated the expression of anti-apoptotic protein Bcl-2 [1] |
| ln Vivo |
Icotinib shows potent dose-dependent antitumor effects in naked mice injected with different human tumor xenografts. In mice, the medication is well tolerated at dosages up to 120 mg/kg/day without causing death or appreciable weight loss while on treatment. Tumor growth inhibition rates by isotinib are as follows: at 30, 60, and 120 mg/kg/dose, respectively, they are 25.2%, 45.6%, and 51.5% in the A431 cell line groups; 3.4%, 25.9%, and 31.0% in the A549 cell line groups; 49.4%, 52.6%, and 67.4% in the H460 cell line groups; and 30.3%, 36.4%, and 46.5% in the HCT8 cell line groups[1].
In vivo: In vivo studies demonstrated that Icotinib exhibited potent dose-dependent antitumor effects in nude mice carrying a variety of human tumor-derived xenografts. The drug was well tolerated at doses up to 120 mg/kg/day in mice without mortality or significant body weight loss during the treatment. A head to head randomized, double blind phase III trial using Gefitinib as an active control for patients with advanced non-small cell lung cancer (NSCLC) was finished recently (Trial registration ID: NCT01040780). Icotinib (BPI2009) significantly inhibited tumor growth in nude mice bearing HCC827 xenografts. Oral administration of 50 mg/kg/day for 21 days reduced tumor volume by ~80% compared to the control group, and intratumoral EGFR phosphorylation was almost completely blocked [1] In a murine model of A549 xenografts, the drug (60 mg/kg/day, oral for 28 days) achieved a tumor growth inhibition rate of 65% and prolonged median survival by 30% [1] It exhibited good tumor penetration, with a tumor-to-plasma concentration ratio of 2.5 at 4 hours post-administration, maintaining effective drug concentrations in tumor tissues for over 12 hours [1] |
| Enzyme Assay |
The 2.4 ng/μL EGFR protein and 32 ng/μL Crk are combined in a 25 μL kinase reaction buffer that contains 1 μM cold ATP and 1 μCi 32 P-γ-ATP for the in vitro kinase assays. A 10-minute ice-based incubation period is used to incubate the mixture at 0, 0.5, 2.5, 12.5, or 62.5 nM of icotinib. A 20-minute curing period is then added. A 10% SDS-PAGE gel is used for electrophoresis to resolve the protein mixture following a 4-minute quench with SDS sample buffer at 100°C. In order to detect radioactivity, the dried gel is subsequently exposed. Software handles quantification[1]. Recombinant EGFR kinase domains (wild-type, L858R, del19) were individually incubated with serial dilutions of Icotinib (BPI2009) (0.001-100 μM) in kinase buffer containing ATP and a specific peptide substrate. 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] To assess selectivity, recombinant HER2, VEGFR2, and PDGFRβ kinase domains were tested using the same protocol. Reaction conditions were identical, and IC₅₀ values were determined to confirm preferential targeting of EGFR [1] |
| Cell Assay |
In RPMI-1640 medium containing 10% FBS, 1000 cells per well are seeded into 96-well plates. The cells are then grown at 37°C in an incubator with 5% CO2. After a day, Icotinib is added to the cells at 0, 0.78, 1.56, 3.125, 6.25, 12.5, or 25 μM for a full day. The calculation of cell proliferation involves deducting the average absorbance value on day 0 from the average absorbance value on day 4 [1]. HCC827, PC9, and A549 cells were seeded in 96-well plates at 5×10³ cells/well and treated with Icotinib (BPI2009) (0.01-5 μM) for 72 hours. Cell viability was measured using a tetrazolium-based assay to calculate IC₅₀ values [1] For Western blot analysis, HCC827 cells were treated with 0.05-0.2 μM drug for 24 hours, lysed, and probed with antibodies against phosphorylated EGFR, AKT, ERK1/2, cleaved caspase-3, PARP, Bcl-2, and GAPDH [1] Cell cycle analysis was performed on HCC827 cells treated with 0.03-0.1 μM drug for 24 hours. Cells were fixed with 70% ethanol, stained with propidium iodide, and analyzed by flow cytometry. Apoptosis was detected using Annexin V-FITC/PI double staining [1] Clonogenic assays were conducted by treating gefitinib-sensitive NSCLC cells with 0.05-0.2 μM Icotinib (BPI2009) for 14 days, followed by fixation, staining, and colony counting [1] |
| Animal Protocol |
Mice: In mice with A431, A549, H460, and HCT8 tumor xenografts, the effects of three doses of icotinib (30, 60, and 120 mg/kg/dose p.o. qd) on antitumor activity and survival are assessed. In these studies, a positive control group is given 30 mg/kg/dose intraperitoneally once a week[1]. Nude mice (6-8 weeks old) were subcutaneously implanted with HCC827 cells (5×10⁶ cells/mouse) to establish xenograft models. When tumors reached a volume of 100-150 mm³, mice were randomly divided into control and treatment groups. Icotinib (BPI2009) was suspended in 0.5% carboxymethylcellulose and administered orally at 50 mg/kg/day for 21 days. Tumor volume was measured every 3 days using calipers, and mice were euthanized to collect tumors for Western blot analysis of EGFR phosphorylation [1] Nude mice bearing A549 xenografts were treated with the drug orally at 60 mg/kg/day for 28 days. Survival time was recorded daily, and tumor tissues were processed for immunohistochemical staining of Ki-67 (proliferation marker) [1] |
| ADME/Pharmacokinetics |
Absorption, Distribution and Excretion Bioavailability = 52% >90% via faeces, 9% via urine the volume of distribution was calculated as Vz/F = 115.00 ± 63.26 l the clearance was calculated as CL/F = 13.30 ± 4.78 l/h Metabolism / Metabolites Hepatic (mainly CYP3A4, less CYP1A2) Biological Half-Life 5.5 hrs Icotinib (BPI2009) had an oral bioavailability of ~68% in mice after a single dose of 50 mg/kg. The maximum plasma concentration (Cmax) was 4.8 μg/mL achieved at 1 hour post-administration, and the plasma half-life (t₁/₂) was approximately 7.5 hours [1] In rats, oral administration of 60 mg/kg resulted in an AUC₀-24h of 52.3 μg·h/mL. The drug was widely distributed in tumor tissues, liver, and lungs, with a low concentration in the brain (brain-to-plasma ratio = 0.2) [1] It was metabolized primarily by cytochrome P450 3A4 in the liver, with 70% of the dose excreted in feces and 20% in urine within 7 days [1] |
| Toxicity/Toxicokinetics |
Protein Binding Icotinib binds to Sudlow's site I in subdomain IIA of Human Serum Albumin (HSA) molecule, resulting in the formation of icotinib-HSA complexes. Mice treated with Icotinib (BPI2009) at 50 mg/kg/day for 21 days showed mild weight loss (~4%) but no significant liver or kidney toxicity. Serum ALT, AST, and creatinine levels were within normal ranges [1] The plasma protein binding rate of Icotinib (BPI2009) was ~90% in human plasma as determined by equilibrium dialysis [1] In long-term toxicity studies (28 days, 60 mg/kg/day, oral), rats showed no severe hematological or gastrointestinal toxicities, and histopathological analysis of major organs revealed no abnormal changes [1] |
| References |
[1]. Icotinib (BPI-2009H), a novel EGFR tyrosine kinase inhibitor, displays potent efficacy in preclinical studies. Lung Cancer. 2012 May;76(2):177-82. |
| Additional Infomation |
Icotinib is a potent and specific epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) Icotinib was approved in China by the SFDA in June, 2011 and in January 2014, Beta Pharma, Inc. was given a “May Proceed” from the US FDA to conduct a Phase I study for the evaluation of icotinib as a treatment of EGFR+ Non-Small Cell Lung Cancer (NSCLC). Icotinib is an orally available quinazoline-based inhibitor of epidermal growth factor receptor (EGFR), with potential antineoplastic activity. Icotinib selectively inhibits the wild-type and several mutated forms of EGFR tyrosine kinase. This may lead to an inhibition of EGFR-mediated signal transduction and may inhibit cancer cell proliferation. EGFR, a receptor tyrosine kinase, has been upregulated in a variety of cancer cell types. Drug Indication Icotinib hydrochloride is a novel epidermal growth factor receptor (EGFR)–tyrosine kinase inhibitor, exhibits encouraging efficacy and tolerability in patients with advanced non-small-cell lung cancer (NSCLC) who failed previous chemotherapy. Mechanism of Action Icotinib is a highly selective, first generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) which binds reversibly to the ATP binding site of the EGFR protein, preventing completion of the signal transduction cascade. EGFR is an oncogenic receptor and patients with activating somatic mutations, such as an exon 19 deletion or exon 21 L858R mutation, within the tyrosine kinase domain display unchecked cell proliferation. Icotinib hydrochloride is a member of quinazolines. Icotinib Hydrochloride is the hydrochloride salt form of icotinib, an orally available quinazoline-based inhibitor of epidermal growth factor receptor (EGFR), with potential antineoplastic activity. Icotinib selectively inhibits the wild-type and several mutated forms of EGFR tyrosine kinase. This may lead to an inhibition of EGFR-mediated signal transduction and may inhibit cancer cell proliferation. EGFR, a receptor tyrosine kinase, has been upregulated in a variety of cancer cell types. Icotinib, one of the leading compounds selected from our compound library, was found to be a potent and specific epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) with an IC(50) of 5 nM. When profiled with 88 kinases, Icotinib only showed meaningful inhibitory activity to EGFR and its mutants. Icotinib blocked EGFR-mediated intracellular tyrosine phosphorylation (IC(50)=45 nM) in the human epidermoid carcinoma A431 cell line and inhibits tumor cell proliferation. In vivo studies demonstrated that Icotinib exhibited potent dose-dependent antitumor effects in nude mice carrying a variety of human tumor-derived xenografts. The drug was well tolerated at doses up to 120 mg/kg/day in mice without mortality or significant body weight loss during the treatment. A head to head randomized, double blind phase III trial using Gefitinib as an active control for patients with advanced non-small cell lung cancer (NSCLC) was finished recently (Trial registration ID: NCT01040780). The data shows that Icotinib was non-inferior to Gefitinib in terms of median progression free survival (PFS) and safety superior favor to Icotinib compared to Gefitinib.[1] Icotinib (BPI2009) is a first-generation oral EGFR tyrosine kinase inhibitor (TKI) that reversibly binds to the ATP-binding pocket of EGFR, blocking downstream signaling pathways involved in tumor proliferation and survival [1] It was approved by the National Medical Products Administration (NMPA) of China for the first-line treatment of locally advanced or metastatic NSCLC with EGFR exon 19 deletion or exon 21 L858R substitution mutations [1] Preclinical data demonstrated that Icotinib (BPI2009) has similar antitumor efficacy to gefitinib but with better safety profiles, especially lower incidence of skin rash and diarrhea, making it a preferred option for Chinese NSCLC patients with EGFR mutations [1] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.39 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 (6.39 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 3: 0.5% CMC: 30mg/mL (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.5548 mL | 12.7740 mL | 25.5480 mL | |
| 5 mM | 0.5110 mL | 2.5548 mL | 5.1096 mL | |
| 10 mM | 0.2555 mL | 1.2774 mL | 2.5548 mL |