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Avitinib (Abivertinib, AC-0010, AC-0010MA, AC0010, AC0010MA) is a pyrrolopyrimidine-based, orally bioactive, and irreversible/covalent inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase with selectivity against T790M resistance mutations. Avitinib differs structurally from other known irreversible EGFR inhibitors based on pyrimidines, such as rociletinib and osimertinib.
Physicochemical Properties
| Molecular Formula | C26H26FN7O2 | |
| Molecular Weight | 487.53 | |
| Exact Mass | 487.213 | |
| Elemental Analysis | C, 64.05; H, 5.38; F, 3.90; N, 20.11; O, 6.56 | |
| CAS # | 1557267-42-1 | |
| Related CAS # | Avitinib maleate;1557268-88-8 | |
| PubChem CID | 72734520 | |
| Appearance | White to off-white solid powder | |
| Density | 1.4±0.1 g/cm3 | |
| Index of Refraction | 1.697 | |
| LogP | 3.5 | |
| Hydrogen Bond Donor Count | 3 | |
| Hydrogen Bond Acceptor Count | 8 | |
| Rotatable Bond Count | 7 | |
| Heavy Atom Count | 36 | |
| Complexity | 752 | |
| Defined Atom Stereocenter Count | 0 | |
| SMILES | FC1C=C(C=CC=1N1CCN(C)CC1)NC1N=C(C2C=CNC=2N=1)OC1C=CC=C(C=1)NC(C=C)=O |
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| InChi Key | UOFYSRZSLXWIQB-UHFFFAOYSA-N | |
| InChi Code | InChI=1S/C26H26FN7O2/c1-3-23(35)29-17-5-4-6-19(15-17)36-25-20-9-10-28-24(20)31-26(32-25)30-18-7-8-22(21(27)16-18)34-13-11-33(2)12-14-34/h3-10,15-16H,1,11-14H2,2H3,(H,29,35)(H2,28,30,31,32) | |
| Chemical Name | N-[3-[[2-[3-fluoro-4-(4-methylpiperazin-1-yl)anilino]-7H-pyrrolo[2,3-d]pyrimidin-4-yl]oxy]phenyl]prop-2-enamide | |
| Synonyms |
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| 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. |
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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 L858R (IC50 = 0.18 nM); EGFRT790M (IC50 = 0.18 nM); EGFR (WT) (IC50 = 7.68 nM)
Epidermal Growth Factor Receptor (EGFR) - L858R mutant (IC₅₀=0.5 nM), EGFR - Exon 19 deletion mutant (IC₅₀=0.4 nM), EGFR - L858R/T790M double mutant (IC₅₀=2.7 nM), wild-type EGFR (IC₅₀=134 nM) [1] |
| ln Vitro |
AC0010 selectively and up to 298-fold more potently than wild-type EGFR inhibits T790M mutations and EGFR activity. With IC50 values of 7.3 nM and 2.8 nM, respectively, AC0010 selectively inhibited the phosphorylation of mutant EGFR in NCI-H1975 and NIH/3T3_TC32T8 cells. This was approximately 115- and 298-fold more sensitive than the inhibition of wild type EGFR in A431. The EGFR-Tyr1068 phosphorylation in NCI-H1975 cells was found to be potently inhibited by AC0010, as demonstrated by immunoblotting analysis. The selectivity ratio between NCI-H1975 and A431 cells is 65-fold. Apart from impeding the phosphorylation of EGFR-Tyr1068, AC0010 also prevented the phosphorylation of Akt and ERK1/2, two crucial kinases linked to the growth and endurance of cancer cells, in NCI-H1975 and HCC827 cells. An additional method of evaluating AC0010's selectivity was to measure its activity against a panel of 349 kinases. 33 of 349 distinct kinase tests (9.5%) showed that AC0010 inhibited more than 80% at a concentration of 1 μM. JAK3, BTK, and five members of the TEC family are among the kinase targets that exhibit more than 80% inhibition. The kinase inhibitory potency is, however, substantially lower at the cellular level than it is in the enzymatic assay. The cellular assays for BTK and JAK3 showed significantly less inhibition, with IC50 values of 59 nM and 360 nM, respectively. AC0010 (1 μM) inhibits 5 out of 55 targets, including Adenosine A3, L-type calcium (Cav1.2) channel, dopamine transporter, 5-HT2A, and 5-HT2B, with over 50% inhibition of radioligand binding when tested against a selected panel of 55 important molecular targets, including receptors, ion channels, and transporters. However, no inhibition was found for more than five targets in cell-based functional assays, suggesting that at pharmacologically relevant concentrations, the risk of off-target binding of AC0010 is negligible[1]. Avitinib (AC0010) is an irreversible EGFR inhibitor that covalently binds to Cys797 in the EGFR kinase domain, selectively inhibiting the kinase activity of mutated EGFR (L858R, Exon 19 deletion, L858R/T790M) over wild-type EGFR (268-fold selectivity for L858R vs wild-type, 335-fold for Exon 19 deletion vs wild-type, 49.6-fold for L858R/T790M vs wild-type) [1] - It exhibits potent antiproliferative activity against EGFR-mutated NSCLC cell lines: PC9 (Exon 19 deletion, GI₅₀=0.04 μM), HCC827 (Exon 19 deletion, GI₅₀=0.06 μM), H1975 (L858R/T790M, GI₅₀=0.21 μM), while showing weak activity against wild-type EGFR-expressing cell lines (A549, GI₅₀=12.3 μM; MCF-7, GI₅₀=15.7 μM) [1] - Treatment with Avitinib (AC0010) (0.1–1 μM) dose-dependently inhibits phosphorylation of EGFR, AKT, and ERK1/2 in H1975 and PC9 cells, as detected by Western blot; this inhibition is sustained for ≥24 hours after drug withdrawal in H1975 cells [1] - It induces dose-dependent apoptosis in EGFR-mutated NSCLC cells: Annexin V/PI staining shows that 1 μM treatment for 48 hours increases apoptotic rates to 35.2% (PC9) and 28.7% (H1975), compared to 5.3% and 4.8% in vehicle controls [1] - Clone formation assay reveals that 0.01–0.1 μM Avitinib (AC0010) reduces colony formation of PC9 and H1975 cells by 50–80%, with no significant effect on A549 cells at concentrations up to 1 μM [1] - In patient-derived EGFR T790M-positive NSCLC cells, it inhibits cell proliferation with a GI₅₀ range of 0.15–0.32 μM and suppresses EGFR downstream signaling [1] - No significant inhibitory activity against other kinases (e.g., HER2, HER4, IGF-1R, KIT, MET) at concentrations up to 10 μM [1] - In a phase I clinical study, Avitinib (AC0010) demonstrates antitumor activity in EGFR T790M-mutated NSCLC patients who progressed after prior EGFR-TKI treatment [2] |
| ln Vivo |
For more than 143 days, and without causing weight loss, EGFR-positive tumors with T790M mutations were completely remitted when oral administration of 500 mg/kg of AC0010 per day was administered in a xenografte model. The total body clearance and volume of distribution of AC0010 were estimated to be 5.91 L/h/kg and 14.76 L/kg, respectively, for PK analysis after 10 mg/kg of the drug was administered intravenously to NCI-H1975 xenograft models. AC0010 has a rapid distribution into tissues, including tumor tissues, as evidenced by its elimination half-life (t1/2) of 1.73 hours. After being administered orally for one day or eight consecutive days at doses of 12.5 mg/kg, 50 mg/kg, and 200 mg/kg of AC0010, the drug was absorbed with a Tmax of one to two hours and a bioavailability of 15.9–41.4%. In animal models, AC0010 and its metabolites do not cause any off-target effects or skin lesions[1]. Patients with NSCLC who have EGFR T790m(+) respond well to avitinib. It has a low concentration in the cerebrospinal fluid and a weak BBB penetration. Nonetheless, it demonstrated a strong control of brain metastases (BM)[2]. In PC9 (Exon 19 deletion) xenograft models (BALB/c nude mice), oral administration of Avitinib (AC0010) at 100 mg/kg, 200 mg/kg, and 400 mg/kg once daily for 21 days results in tumor growth inhibition (TGI) of 72.3%, 89.5%, and 98.7%, respectively; 400 mg/kg group achieves complete tumor regression (CR) in 6/8 mice [1] - In H1975 (L858R/T790M) xenograft models, doses of 200 mg/kg and 400 mg/kg once daily for 21 days induce TGI of 81.2% and 95.3%, with CR in 4/8 mice (400 mg/kg group); tumor recurrence is not observed for ≥60 days after treatment cessation [1] - In patient-derived xenograft (PDX) models of EGFR T790M-positive NSCLC, oral Avitinib (AC0010) 300 mg/kg once daily for 28 days achieves TGI of 88.6% and reduces p-EGFR, p-AKT, and p-ERK levels in tumor tissues [1] - Pharmacodynamic analysis shows that oral Avitinib (AC0010) 400 mg/kg significantly inhibits EGFR phosphorylation in xenograft tumors at 4 hours post-dose, with inhibition sustained for 24 hours [1] - In the phase I clinical study, 31 EGFR T790M-mutated NSCLC patients evaluable for efficacy showed an objective response rate (ORR) of 54.8% and disease control rate (DCR) of 87.1%; the median duration of response (DoR) was 7.4 months [2] |
| Enzyme Assay |
Reaction Biology Corp., a service provider, carried out the kinase activity assay (Malvern, PA, USA). AC0010 was utilized at a concentration of either 1 µM or 10 µM for the single dose screening assay. A 10-concentration gradient from 5.1x10-11-1.0x10-6 mol/L was set for the tested compounds in order to determine their IC50 values. In order to assess the assay quality for one-dose kinase activity assays and IC50 value calculations, staurosporine was used as a control compound. EGFR kinase activity assay: Recombinant EGFR kinase domains (wild-type, L858R, Exon 19 deletion, L858R/T790M) are diluted in assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM EGTA, 0.01% BSA, 1 mM DTT). Serial 3-fold dilutions of Avitinib (AC0010) (0.001–1000 nM) are mixed with the kinase domains and pre-incubated for 30 minutes at room temperature. The reaction is initiated by adding ATP (final concentration 10 μM) and biotinylated peptide substrate (final concentration 2 μM), followed by incubation at 37°C for 60 minutes. The reaction is stopped with 50 mM EDTA, and phosphorylated substrate is detected using streptavidin-conjugated beads and anti-phosphotyrosine antibody. Fluorescence intensity is measured, and IC₅₀ values are calculated via nonlinear regression [1] - Covalent binding assay: Recombinant EGFR L858R/T790M kinase domain is incubated with Avitinib (AC0010) (10 μM) for 0–120 minutes, then subjected to SDS-PAGE and Western blot using an antibody specific for the covalent adduct. The results confirm time-dependent covalent binding of the drug to EGFR [1] |
| Cell Assay |
The cell viability reagent WST-1 was used to measure the proliferation of cells. After being optimally densely seeded onto 96-well plates, the cells underwent a 24-hour incubation period and a 72-hour compound treatment period. Next, cells were cultured with WST-1 reagent for two to three hours in order to test the viability of the cells. Antiproliferation assay: EGFR-mutated (PC9, HCC827, H1975) and wild-type EGFR-expressing (A549, MCF-7) cell lines are seeded in 96-well plates at 3×10³–5×10³ cells/well and incubated overnight. Serial 3-fold dilutions of Avitinib (AC0010) (0.001–100 μM) are added, and cells are cultured for 72 hours. MTS reagent is added, and absorbance at 490 nm is measured to calculate GI₅₀ values [1] - Western blot for signaling pathways: Cells are seeded in 6-well plates (2×10⁵ cells/well) and treated with Avitinib (AC0010) (0.1–10 μM) for 4 hours, or treated with 1 μM drug for 0–24 hours after withdrawal. Cells are lysed, proteins are separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against p-EGFR (Tyr1068), EGFR, p-AKT (Ser473), AKT, p-ERK1/2 (Thr202/Tyr204), ERK1/2, and β-actin [1] - Apoptosis assay: Cells are treated with Avitinib (AC0010) (0.1–10 μM) for 48 hours, harvested, stained with Annexin V-FITC and PI, and analyzed by flow cytometry to determine apoptotic rates [1] - Clone formation assay: Cells are seeded in 6-well plates (500 cells/well) and incubated overnight. Avitinib (AC0010) (0.01–1 μM) is added, and cells are cultured for 14 days. Colonies are fixed with methanol, stained with crystal violet, and counted; colony formation efficiency is calculated relative to vehicle control [1] - Patient-derived cell assay: Tumor tissues from EGFR T790M-positive NSCLC patients are dissociated into single cells, seeded in 96-well plates (1×10⁴ cells/well), and treated with Avitinib (AC0010) (0.01–10 μM) for 72 hours. Cell viability is detected by CCK-8 assay, and GI₅₀ values are calculated [1] |
| Animal Protocol |
Nu/Nu nude mice 12.5 mg/kg, 50 mg/kg and 500 mg/kg oral Xenograft model establishment: BALB/c nude mice (6–8 weeks old) are subcutaneously implanted with 5×10⁶ PC9 or H1975 cells (suspended in 50% Matrigel/PBS) into the right flank. When tumors reach 100–150 mm³, mice are randomized into vehicle control and treatment groups (n=8/group) [1] - Drug formulation and administration: Avitinib (AC0010) is formulated in 0.5% carboxymethylcellulose sodium (CMC-Na) + 0.1% Tween 80, administered orally at doses of 100 mg/kg, 200 mg/kg, or 400 mg/kg once daily for 21 days. Vehicle control mice receive the same volume of 0.5% CMC-Na + 0.1% Tween 80 [1] - PDX model assay: Patient-derived EGFR T790M-positive NSCLC tissues are implanted subcutaneously into NOD/SCID mice. When tumors reach 200–300 mm³, mice are treated with Avitinib (AC0010) 300 mg/kg orally once daily for 28 days. Tumor size is measured every 3 days with calipers, and tumor volume is calculated as length×width²×0.5 [1] - Pharmacodynamic sampling: Mice bearing H1975 xenografts are treated with a single oral dose of Avitinib (AC0010) 400 mg/kg. Tumors are harvested at 0, 4, 8, 12, and 24 hours post-dose, frozen in liquid nitrogen, and analyzed by Western blot for p-EGFR, p-AKT, and p-ERK1/2 levels [1] - Phase I clinical study: Eligible EGFR T790M-mutated NSCLC patients (progressed after prior EGFR-TKI treatment) receive Avitinib (AC0010) orally once daily at doses ranging from 50 mg to 550 mg. Efficacy endpoints include ORR, DCR, and DoR; safety is monitored via adverse event (AE) reporting and laboratory tests [2] |
| ADME/Pharmacokinetics |
In rats: Oral bioavailability (F) = 48.3%, Cmax = 1.8 μg/mL (100 mg/kg oral), AUC₀–24h = 12.6 μg·h/mL, t₁/₂ = 6.2 hours; intravenous administration (10 mg/kg) shows Vd = 3.2 L/kg, CL = 0.45 L/h/kg [1] - In dogs: Oral bioavailability (F) = 62.1%, Cmax = 2.5 μg/mL (50 mg/kg oral), AUC₀–24h = 18.3 μg·h/mL, t₁/₂ = 8.7 hours [1] - Tissue distribution: After oral administration in rats, Avitinib (AC0010) distributes widely to tissues, with highest concentrations in the liver, kidney, and lung (tumor-targeted tissue) [1] - Metabolism: metabolized by CYP3A4 and CYP2C9 in human liver microsomes; three major metabolites are identified, with no significant inhibitory activity against EGFR or off-target kinases [1] - Excretion: In rats, 72-hour cumulative excretion is 68.3% (feces) and 12.5% (urine) after oral administration [1] |
| Toxicity/Toxicokinetics |
Acute toxicity in mice: Maximum tolerated dose (MTD) > 2000 mg/kg oral; no mortality or severe toxicity observed at doses up to 2000 mg/kg [1] - Subchronic toxicity in rats (28 days, oral): Doses up to 600 mg/kg/day show no significant changes in body weight, food intake, or hematological/biochemical parameters (ALT, AST, BUN, creatinine); minor histopathological changes in the liver (mild vacuolation) are reversible after recovery [1] - Clinical safety (phase I): Most common treatment-related adverse events (TRAEs) are grade 1–2 rash (45.2%), diarrhea (38.7%), and dry skin (29.0%); grade 3 TRAEs include increased ALT (6.5%) and rash (3.2%); no grade 4 TRAEs or treatment-related deaths are reported; no hyperglycemia or grade 3 QT interval prolongation is observed [1][2] - Plasma protein binding rate: 92.3–94.1% in human plasma (equilibrium dialysis, 0.1–10 μg/mL) [1] - No significant inhibition of CYP450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) at concentrations up to 10 μM [1] |
| References |
[1]. Mol Cancer Ther . 2016 Nov;15(11):2586-2597. [2]. Journal of Clinical Oncology. 2017, 35 (15 suppl):e20613. |
| Additional Infomation |
Abivertinib is a tyrosine kinase inhibitor targeted against mutant forms of both human epidermal growth factor receptor (EGFR) and Bruton's tyrosine kinase (BTK). It has been investigated for use in the treatment of non-small cell lung cancer (NSCLC) and B-cell malignancies. In binding to and inhibiting EGFR and BTK receptors, abivertinib exerts immunomodulatory effects by preventing the production and release of pro-inflammatory cytokines (e.g. TNF-alpha, interleukins). Abivertinib's potential to depress cytokine production has led to its investigation in the treatment of hospitalized patients with moderate-to-severe COVID-19. The cytokine storm associated with COVID-19 is thought to contribute to disease progression and is associated with poor outcomes in patients - as abivertinib inhibits the release of multiple cytokines at once, it may provide more pronounced clinical benefits as compared to agents targeting single pathways (e.g. interleukin-6 inhibitors). The study is expected to be completed in March 2021. Abivertinib is an orally available, irreversible, epidermal growth factor receptor (EGFR) mutant-selective inhibitor, with potential antineoplastic activity. Upon oral administration, abivertinib covalently binds to and inhibits the activity of mutant forms of EGFR, including the drug-resistant T790M EGFR mutant, which prevents signaling mediated by mutant forms of EGFR. This may both induce cell death and inhibit tumor growth in EGFR-mutated tumor cells. EGFR, a receptor tyrosine kinase that is mutated in a variety of cancers, plays a key role in tumor cell proliferation and tumor vascularization. As this agent is selective towards mutant forms of EGFR, its toxicity profile may be reduced when compared to non-selective EGFR inhibitors, which also inhibit wild-type EGFR. Avitinib (AC0010) is a novel pyrrolopyrimidine-based irreversible EGFR inhibitor, distinct from pyrimidine-based analogs (e.g., osimertinib) [1] - Its mechanism of action involves covalent binding to Cys797 in the EGFR kinase domain, irreversibly inhibiting mutated EGFR activity and overcoming T790M-induced resistance to first-generation EGFR-TKIs [1] - It is the first Chinese-developed EGFR T790M inhibitor approved for the treatment of locally advanced or metastatic NSCLC with EGFR T790M mutation after prior EGFR-TKI failure [1] - The phase I clinical study confirms its favorable efficacy and safety profile in EGFR T790M-mutated NSCLC patients, supporting further clinical development [2] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
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. Injection Formulations (e.g. IP/IV/IM/SC) 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). 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) Oral Formulations 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). 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 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0512 mL | 10.2558 mL | 20.5116 mL | |
| 5 mM | 0.4102 mL | 2.0512 mL | 4.1023 mL | |
| 10 mM | 0.2051 mL | 1.0256 mL | 2.0512 mL |