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Icotinib HCl (formerly known as BPI-2009 HCl), the hydrochloride salt of Icotinib and an macrocyclic analog of erlotinib, is an orally bioavailable, potent and specific EGFR inhibitor with IC50 of 5 nM, including the EGFR, EGFR(L858R), EGFR(L861Q), EGFR(T790M) and EGFR(T790M, L858R). Icotinib solely exhibited significant inhibitory activity against EGFR and its mutants when 88 kinases were profiled. In the human epidermoid carcinoma A431 cell line, itotinib inhibits the growth of tumor cells by blocking EGFR-mediated intracellular tyrosine phosphorylation (IC50 = 45 nM). Icotinib showed strong, dose-dependent antitumor effects in nude mice that were bearing different xenografts derived from human tumors, according to in vivo investigations. 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). According to the data, there was no significant difference between Icotinib and Gefitinib in terms of median progression free survival (PFS), and Icotinib was safer than Gefitinib. In 2011, China approved icotinib for the treatment of lung cancer.
Physicochemical Properties
| Molecular Formula | C22H22CLN3O4 |
| Molecular Weight | 427.89 |
| Exact Mass | 427.129 |
| Elemental Analysis | C, 61.76; H, 5.18; Cl, 8.28; N, 9.82; O, 14.96 |
| CAS # | 1204313-51-8 |
| Related CAS # | Icotinib;610798-31-7;Icotinib-d4;1567366-82-8 |
| PubChem CID | 44609731 |
| Appearance | Light yellow solid powder |
| LogP | 4.034 |
| Hydrogen Bond Donor Count | 2 |
| Hydrogen Bond Acceptor Count | 7 |
| Rotatable Bond Count | 3 |
| Heavy Atom Count | 30 |
| Complexity | 553 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | C#CC1=CC(NC2=C(C=C(OCCOCCOCCO3)C3=C4)C4=NC=N2)=CC=C1.[H]Cl |
| InChi Key | PNNGXMJMUUJHAV-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C22H21N3O4.ClH/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);1H |
| 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;hydrochloride |
| Synonyms | BPI-2009; BPI-2009H; BPI2009; BPI 2009; BPI2009H; 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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| 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); EGFRL861Q; EGFRL858R/T790M; EGFRL858R; EGFRT790M
Icotinib Hydrochloride (BPI-2009H) is a selective inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, targeting wild-type EGFR (Ki = 2.6 nM), EGFR Del746-750 mutant (Ki = 0.1 nM), and EGFR L858R mutant (Ki = 0.2 nM); it has an IC50 of 15 nM for inhibiting wild-type EGFR kinase activity, 0.7 nM for EGFR Del746-750, and 1.0 nM for EGFR L858R [1] Icotinib Hydrochloride (BPI-2009H) shows weak inhibitory activity against other tyrosine kinases (e.g., ErbB2, ErbB4) with IC50 values >10 μM [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]. Icotinib Hydrochloride (BPI-2009H) potently inhibited EGFR tyrosine kinase activity in a dose-dependent manner, with significantly higher potency against EGFR mutants (Del746-750, L858R) than wild-type EGFR. In cancer cell lines with EGFR mutations (PC-9, HCC827), it inhibited cell proliferation with IC50 values of 0.025 μM and 0.019 μM, respectively; in wild-type EGFR-expressing cell lines (A549, H460), the IC50 values were 1.4 μM and 1.2 μM, respectively. Treatment with icotinib induced apoptosis in PC-9 cells (detected by Annexin V/PI staining), with the apoptotic rate increasing from 5.2% (control) to 35.7% at 1 μM. It also suppressed EGFR downstream signaling pathways: Western blot showed reduced phosphorylation of EGFR, ERK1/2, and Akt in PC-9 cells after 2-hour treatment with 0.1-1 μM icotinib. In colony formation assays, icotinib (0.01-1 μM) dose-dependently inhibited the clonogenic growth of PC-9 cells, with almost complete inhibition at 1 μM [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). Oral administration of Icotinib Hydrochloride (BPI-2009H) at doses of 50, 100, and 200 mg/kg once daily significantly inhibited tumor growth in PC-9 (EGFR-mutant) xenograft models in nude mice: tumor volume inhibition rates were 42.3%, 68.7%, and 85.1%, respectively (P < 0.01 vs vehicle). In A549 (wild-type EGFR) xenografts, 200 mg/kg icotinib achieved a tumor inhibition rate of 52.6% (P < 0.05 vs vehicle). Immunohistochemical analysis of tumor tissues showed that icotinib reduced the phosphorylation level of EGFR and its downstream protein ERK1/2 in PC-9 xenografts. No significant body weight loss or obvious toxicological changes were observed in treated mice during the experiment [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 μCi32P-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]. 1. EGFR kinase activity assay: Recombinant human EGFR tyrosine kinase domain (wild-type, Del746-750, L858R) was incubated with serial concentrations of Icotinib Hydrochloride (BPI-2009H) in reaction buffer containing ATP and a peptide substrate. The phosphorylation level of the substrate was detected by a luminescent or colorimetric method, and the inhibition rate of each drug concentration was calculated. Dose-response curves were generated to determine the IC50 values for EGFR kinase inhibition, and Ki values were calculated using the Cheng-Prusoff equation [1] 2. Selectivity assay for other tyrosine kinases: Recombinant ErbB2, ErbB4, VEGFR2, and PDGFRβ kinases were incubated with Icotinib Hydrochloride (BPI-2009H) (0.001-10 μM), and kinase activity was measured using the same method as the EGFR assay to evaluate the selectivity of icotinib [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]. 1. Cell proliferation assay: Human lung cancer cell lines (PC-9, HCC827, A549, H460) were seeded in 96-well plates and treated with serial concentrations of Icotinib Hydrochloride (BPI-2009H) (0.001-10 μM) for 72 hours. Cell viability was determined by the MTT assay, and IC50 values were calculated by non-linear regression analysis using GraphPad Prism software (each concentration was tested in triplicate, N=3 independent experiments) [1] 2. Apoptosis assay: PC-9 cells were treated with Icotinib Hydrochloride (BPI-2009H) (0.1, 0.5, 1 μM) or vehicle for 48 hours. Cells were harvested, stained with Annexin V-FITC and propidium iodide (PI), and analyzed by flow cytometry to quantify the percentage of apoptotic cells (early apoptosis: Annexin V+/PI-, late apoptosis: Annexin V+/PI+) [1] 3. Western blot analysis of EGFR signaling: PC-9 cells were serum-starved for 12 hours, then treated with Icotinib Hydrochloride (BPI-2009H) (0.1, 0.5, 1 μM) for 2 hours, followed by stimulation with EGF (10 ng/mL) for 15 minutes. Cell lysates were prepared, separated by SDS-PAGE, and transferred to PVDF membranes. The membranes were probed with antibodies against phosphorylated EGFR (p-EGFR), total EGFR (t-EGFR), phosphorylated ERK1/2 (p-ERK1/2), total ERK1/2 (t-ERK1/2), phosphorylated Akt (p-Akt), and total Akt (t-Akt); GAPDH was used as a loading control. The band intensity was quantified by densitometry [1] 4. Colony formation assay: PC-9 cells were seeded in 6-well plates at a density of 500 cells per well and treated with Icotinib Hydrochloride (BPI-2009H) (0.01, 0.1, 1 μM) or vehicle. After 14 days of culture, colonies were fixed with methanol, stained with crystal violet, and counted under a microscope. The colony formation rate was calculated as (number of colonies in treated group / number of colonies in control group) × 100% [1] |
| Animal Protocol |
Mice: In mice containing 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. These studies use taxol (30 mg/kg/dose i.p. once a week) as a positive control group[1]. 1. PC-9 xenograft model in nude mice: PC-9 cells (5 × 10⁶) were subcutaneously injected into the right flank of BALB/c nude mice (6-8 weeks old, male). When tumors reached a volume of approximately 100 mm³, mice were randomly divided into four groups (N=8 per group): vehicle group (0.5% carboxymethylcellulose sodium, CMC-Na), and icotinib groups (50, 100, 200 mg/kg). Icotinib Hydrochloride (BPI-2009H) was formulated in 0.5% CMC-Na and administered by oral gavage once daily for 21 consecutive days. Tumor volume was measured every 3 days using calipers (tumor volume = length × width² / 2), and body weight was recorded to assess toxicity [1] 2. A549 xenograft model in nude mice: A549 cells (1 × 10⁷) were subcutaneously injected into nude mice, and the modeling and grouping methods were the same as the PC-9 model. Mice were treated with Icotinib Hydrochloride (BPI-2009H) at 200 mg/kg or vehicle by oral gavage once daily for 21 days. Tumor volume and body weight were measured regularly, and tumor inhibition rate was calculated as [(mean tumor volume of control group - mean tumor volume of treated group) / mean tumor volume of control group] × 100% [1] 3. Immunohistochemical analysis of tumor tissues: At the end of the experiment, tumor tissues were harvested, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into 4 μm slices. The sections were immunostained with antibodies against p-EGFR and p-ERK1/2, and the staining intensity was evaluated by a pathologist in a blinded manner (scored as 0-3 based on the percentage of positive cells and staining intensity) [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 Hydrochloride (BPI-2009H) showed good oral absorption in rats: the oral bioavailability was approximately 60% after a single oral dose of 50 mg/kg. The plasma concentration reached the peak (Cmax) at 1-2 hours post-administration, with a peak concentration (Cmax) of 1.2 μg/mL and an area under the curve (AUC0-24h) of 8.5 μg·h/mL. The terminal half-life (t1/2) was about 6 hours. Icotinib was widely distributed in tissues, with high concentrations in the lung, liver, and kidney, and low concentrations in the brain. It was mainly metabolized in the liver via cytochrome P450 enzymes (CYP3A4 as the major isoform), and the metabolites were excreted primarily through the feces (approximately 70%) and urine (approximately 20%) within 48 hours [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. In acute toxicity studies, the median lethal dose (LD50) of Icotinib Hydrochloride (BPI-2009H) in mice was >2000 mg/kg after oral administration, indicating low acute toxicity. In a 28-day subchronic toxicity study in rats, oral administration of icotinib at doses of 50, 100, and 200 mg/kg/day showed no significant changes in body weight, food intake, or hematological parameters (RBC, WBC, platelet count). Biochemical analysis of serum showed no abnormal levels of alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), or creatinine (Cr), suggesting no obvious hepatotoxicity or nephrotoxicity. Histopathological examination of major organs (liver, kidney, heart, lung) revealed no drug-induced pathological changes. The plasma protein binding rate of icotinib was approximately 90% in human plasma (in vitro) [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 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 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 (BPI-2009H) is a novel, orally available, selective EGFR tyrosine kinase inhibitor (TKI) developed for the treatment of non-small cell lung cancer (NSCLC). Its mechanism of action involves competitive binding to the ATP-binding pocket of the EGFR tyrosine kinase domain, inhibiting EGFR phosphorylation and blocking downstream Ras/Raf/MEK/ERK and PI3K/Akt signaling pathways, thereby suppressing tumor cell proliferation and inducing apoptosis [1] Icotinib Hydrochloride (BPI-2009H) exhibits higher selectivity for EGFR (especially mutant EGFR) than first-generation EGFR-TKIs such as gefitinib and erlotinib, with weaker inhibitory effects on other tyrosine kinases (ErbB2, VEGFR2), which may reduce off-target toxicities. Preclinical studies demonstrated potent anti-tumor efficacy in EGFR-mutant NSCLC models, supporting its clinical development for the treatment of EGFR-mutant NSCLC [1] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.84 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 (5.84 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 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: ≥ 2.5 mg/mL (5.84 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 4: 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 | 2.3370 mL | 11.6852 mL | 23.3705 mL | |
| 5 mM | 0.4674 mL | 2.3370 mL | 4.6741 mL | |
| 10 mM | 0.2337 mL | 1.1685 mL | 2.3370 mL |