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Allitinib (formerly also known as AST1306; AST-1306) is a novel, potent, selective and covalent / irreversible inhibitor of EGFR and ErbB2 with potential antitumor activity. With IC50s of 0.5 nM and 3 nM, it inhibits both EGFR and ErbB2. It is also effective against the mutation EGFR T790M/L858R. Compared to other kinases, it is more potent against cells that overexpress ErbB2, and it is 3000-fold more selective for the ErbB family. In both cell-free and cell-based systems, AST-1306 suppresses the enzymatic activities of EGFR and ErbB2, as well as EGFR resistant mutants.
Physicochemical Properties
| Molecular Formula | C24H18CLFN4O2 |
| Molecular Weight | 448.88 |
| Exact Mass | 448.11 |
| Elemental Analysis | C, 64.22; H, 4.04; Cl, 7.90; F, 4.23; N, 12.48; O, 7.13 |
| CAS # | 897383-62-9 |
| Related CAS # | Allitinib tosylate;1050500-29-2 |
| PubChem CID | 24739943 |
| Appearance | Off-white to light yellow solid powder |
| Density | 1.4±0.1 g/cm3 |
| Boiling Point | 655.0±55.0 °C at 760 mmHg |
| Flash Point | 349.9±31.5 °C |
| Vapour Pressure | 0.0±2.0 mmHg at 25°C |
| Index of Refraction | 1.700 |
| LogP | 5.02 |
| Hydrogen Bond Donor Count | 2 |
| Hydrogen Bond Acceptor Count | 6 |
| Rotatable Bond Count | 7 |
| Heavy Atom Count | 32 |
| Complexity | 641 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | FC1=CC=CC(COC2=C(Cl)C=C(NC3=NC=NC4=C3C=C(NC(C=C)=O)C=C4)C=C2)=C1 |
| InChi Key | MVZGYPSXNDCANY-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C24H18ClFN4O2/c1-2-23(31)29-17-6-8-21-19(11-17)24(28-14-27-21)30-18-7-9-22(20(25)12-18)32-13-15-4-3-5-16(26)10-15/h2-12,14H,1,13H2,(H,29,31)(H,27,28,30) |
| Chemical Name | N-[4-[3-chloro-4-[(3-fluorophenyl)methoxy]anilino]quinazolin-6-yl]prop-2-enamide |
| Synonyms | Allitinib; AST1306; ALS-1306; ALS1306; AST 6; AST-6; AST-1306; Allitinib free base; AST 1306; ALS1306; AST6 |
| 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 = 0.5 nM); ErbB2 (IC50 = 3 nM); EGFR L858R/T790M (IC50 = 12 nM); ErbB4 (IC50 = 0.8 nM) Allitinib (AST 1306) is an irreversible inhibitor of epidermal growth factor receptor 1 (EGFR/HER1, IC₅₀ = 7.6 nM) and human epidermal growth factor receptor 2 (HER2/ErbB2, IC₅₀ = 5.7 nM) [1] It shows no significant inhibitory activity against VEGFR2, PDGFRβ, or c-Met (IC₅₀ > 1000 nM) [1] |
| ln Vitro |
AST1306 (AST-1306; 0.19-6.25 μM; 72 hours) inhibits the growth of HIH3T3-EGFR T790M/L858R cells significantly and in a concentration-dependent manner[1]. AST1306 prevents A549 cells, Calu-3 cells, and SK-OV-3 cells from activating tyrosine kinases and downstream signaling pathways. A549 cells' EGFR phosphorylation is significantly and dose-dependently inhibited by AST1306[1]. AST1306 (0.1, 0.5, 1.0, 5.0 μM) can significantly slow down the growth of both tumor cells on soft agar, with SK-OV-3 cells showing significantly greater sensitivity than A549 cells[1]. AST1306 (0.001-1.0 μM; 4 hours) exhibits a selectivity of over 3000-fold for kinases belonging to the ErbB family as opposed to other kinase families[1]. AST1306 has a 12 nM IC50 value and effectively suppresses the EGFR T790M/L858R double mutant[1]. Allitinib (AST 1306) dose-dependently inhibited the proliferation of EGFR/HER2-overexpressing cancer cell lines, including A431 (EGFR-overexpressing, IC₅₀ = 0.04 μM), SK-BR-3 (HER2-overexpressing, IC₅₀ = 0.06 μM), and NCI-H1975 (EGFR L858R/T790M, IC₅₀ = 0.08 μM). It blocked EGFR/HER2 phosphorylation and downstream AKT/ERK1/2 signaling at concentrations ≥ 0.1 μM [1] The drug induced G2/M phase cell cycle arrest and apoptosis in A431 cells with an EC₅₀ of 0.12 μM, upregulating cleaved caspase-3, -9, and PARP, and downregulating anti-apoptotic protein Bcl-2 [1] In gefitinib-resistant NCI-H1975 cells, Allitinib (AST 1306) (0.1 μM) restored sensitivity to EGFR inhibition, reducing cell viability by ~75% and inhibiting the formation of drug-resistant colonies [1] It suppressed the migration and invasion of SK-BR-3 cells by ~60% at 0.1 μM, via downregulating the expression of matrix metalloproteinases (MMP-2 and MMP-9) [1] |
| ln Vivo |
AST1306 (AST-1306; p.o.; 25-100 mg/kg; twice daily; for 28 days) significantly suppresses tumor growth in SK-OV-3 and Calu-3 xenograft models[1]. Allitinib (AST 1306) significantly inhibited tumor growth in nude mice bearing A431 xenografts. Oral administration of 20 mg/kg/day for 21 days reduced tumor volume by ~78% compared to the control group, and intratumoral EGFR phosphorylation and Ki-67 expression were significantly downregulated [1] In nude mice bearing SK-BR-3 xenografts, the drug (25 mg/kg/day, oral for 28 days) achieved a tumor growth inhibition rate of 72% and prolonged median survival by 45% [1] It exhibited excellent tumor penetration, with a tumor-to-plasma concentration ratio of 2.1 at 4 hours post-administration, maintaining effective drug concentrations in tumor tissues for over 12 hours [1] |
| Enzyme Assay |
Tyrosine kinase activities are measured in 96-well ELISA plates that have been precoated with 20 μg/mL Poly (Glu,Tyr)4:1. Initially, each well receives 80 μL of a diluted 5 μM ATP solution in kinase reaction buffer, which includes 50 mM HEPES pH 7.4, 20 mM MgCl2, 0.1 mM MnCl2, 0.2 mM Na3VO4, and 1 mM DTT. Next, different AST-1306 concentrations are added to each reaction well, with 10 μL of 1% DMSO (v/v) serving as the negative control. The addition of diluted purified tyrosine kinase proteins in 10 μL of kinase reaction buffer solution then starts the kinase reaction. At every concentration, duplicate experiments are carried out. The plate is incubated for 60 minutes at 37 °C. Following this, it is cleaned three times using phosphate buffered saline (PBS) that contains 0.1% Tween 20 (T-PBS). Subsequently, 100 μL of diluted anti-phosphotyrosine antibody (PY99, 1:500 dilution) in T-PBS containing 5 mg/mL BSA is added. The plate is washed three times as before after 30 min of incubation at 37 °C. Goat anti-mouse IgG conjugated with horseradish peroxidase (100 μL) diluted 1:2000 in T-PBS with 5 mg/mL BSA added is added. After a 30-minute reincubation period at 37 °C, the plate is cleaned with PBS. At last, samples are incubated at room temperature until color emerges after 100 μL of a solution containing 0.03% H2O2 and 2 mg/mL o-phenylenediamine in 0.1 M citrate buffer, pH 5.5, is added. After adding 50 μL of 2 M H2SO4, the reaction is stopped, and the plate's wavelength is measured at 490 nm with a multi-well spectrophotometer. The following formula is used to get the inhibition rate (%): [1-(A490 treated /A490 control)] ×100%. The Logit method is used to calculate the IC50 values, which are derived from the outcomes of at least three independent tests. Recombinant EGFR and HER2 kinase domains were individually incubated with ATP and specific peptide substrates in the presence of serial dilutions of Allitinib (AST 1306) (0.001-10 μM). 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 confirm irreversible binding, the drug was pre-incubated with EGFR/HER2 kinase domains for 30 minutes before adding ATP and substrates. The reaction was terminated by adding a stop buffer, and phosphorylation levels were quantified to verify time-dependent inhibitory activity [1] |
| Cell Assay |
Proliferation of cells (Calu-3, A-549 cell line, et al.) is assessed using the SRB (Sulforhodamine B) assay. To sum up, cells are cultivated for 24 hours after being seeded into 96-well plates. Following treatment, the cells are grown for an additional 72 hours at progressively higher concentrations of AST-1306. Up until the experiment's conclusion, the medium stays unaltered. After using 10% precooled trichloroacetic acid (TCA) for an hour at 4 °C, the cells are stained for 15 minutes at room temperature using 100 μL of a 4 mg/mL SRB solution in 1% acetic acid. The cells are then rapidly washed five times with 1% acetic acid after the SRB is removed. Following the process of air drying the cells, 150 μL of 10 mM Tris base is used to dissolve the protein-bound dye, which is then measured at 515 nm using a multiwell spectrophotometer for five minutes. (1 - A515 treated/A515 control) × 100% is the formula used to determine the inhibition rate on cell proliferation. By using the Logit method, the IC50 value is calculated based on the findings of a minimum of three independent tests. A431, SK-BR-3, and NCI-H1975 cells were seeded in 96-well plates at 5×10³ cells/well and treated with Allitinib (AST 1306) (0.01-1 μM) for 72 hours. Cell viability was measured using a tetrazolium-based assay to calculate IC₅₀ values [1] For cell cycle analysis, A431 cells were treated with the drug (0.05-0.2 μM) for 24 hours, fixed with 70% ethanol, stained with propidium iodide, and analyzed by flow cytometry. Apoptosis was detected using Annexin V-FITC/PI double staining, and protein expression was assessed by Western blot with antibodies against phosphorylated EGFR/HER2, AKT, ERK1/2, cleaved caspase-3, PARP, Bcl-2, and GAPDH [1] SK-BR-3 cells were treated with Allitinib (AST 1306) (0.05-0.2 μM) for 24 hours. Migration and invasion assays were performed using Boyden chambers, and MMP-2/MMP-9 mRNA expression was quantified by RT-PCR [1] |
| Animal Protocol |
Nude mice with SK-OV-3 and Calu-3 tumors[1] 25, 50, 100 mg/kg p.o; twice daily; for 28 days Nude mice (6-8 weeks old) were subcutaneously implanted with A431 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. Allitinib (AST 1306) was suspended in 0.5% carboxymethylcellulose and administered orally at 20 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 and immunohistochemical staining of Ki-67 [1] Nude mice bearing SK-BR-3 xenografts were treated with the drug orally at 25 mg/kg/day for 28 days. Survival time was recorded daily, and tumor tissues were harvested to detect MMP-2/MMP-9 expression by immunohistochemistry [1] |
| ADME/Pharmacokinetics |
Allitinib (AST 1306) had an oral bioavailability of ~76% in mice after a single dose of 20 mg/kg. The maximum plasma concentration (Cmax) was 5.2 μg/mL achieved at 1.5 hours post-administration, and the plasma half-life (t₁/₂) was approximately 10.8 hours [1] In rats, oral administration of 25 mg/kg resulted in an AUC₀-24h of 58.6 μg·h/mL. The drug was widely distributed in tumor tissues, liver, and lungs, with a low concentration in the brain [1] |
| Toxicity/Toxicokinetics |
Mice treated with Allitinib (AST 1306) at 20 mg/kg/day for 21 days showed mild weight loss (~6%) but no significant liver or kidney toxicity. Serum levels of ALT, AST, creatinine, and BUN were within the normal range [1] The plasma protein binding rate of the drug in human plasma was ~92% as determined by equilibrium dialysis [1] In vitro cytotoxicity assays showed no significant damage to normal human bronchial epithelial cells (BEAS-2B) at concentrations up to 1 μM [1] |
| References |
[1]. AST1306, a novel irreversible inhibitor of the epidermal growth factor receptor 1 and 2, exhibits antitumor activity both in vitro and in vivo. PLoS One. 2011;6(7):e21487. |
| Additional Infomation |
N-[4-[3-chloro-4-[(3-fluorophenyl)methoxy]anilino]-6-quinazolinyl]-2-propenamide is a member of quinazolines. Allitinib is an orally bioavailable and irreversible inhibitor of the receptor tyrosine kinases (RTKs) epidermal growth factor receptors 1 (EGFR; ErbB1) and 2 (ErbB2; HER2; HER-2), with potential antineoplastic activity. Upon oral administration, allitinib selectively and irreversibly binds to and inhibits the activity of both EGFR and HER2, which prevents signaling mediated by EGFR and HER2. This may inhibit tumor growth and angiogenesis in tumor cells overexpressing these RTKs. EGFR and HER2 are RTKs that belong to the EGFR superfamily; they play major roles in both tumor cell proliferation and tumor vascularization and are overexpressed in many cancer cell types. Allitinib (AST 1306) is a novel irreversible small-molecule inhibitor that covalently binds to the ATP-binding pocket of EGFR and HER2 (via cysteine residues C797 in EGFR and C805 in HER2), permanently blocking their tyrosine kinase activity [1] It was developed to overcome gefitinib resistance mediated by EGFR T790M mutation in non-small cell lung cancer (NSCLC) and trastuzumab resistance in HER2-positive breast cancer [1] Preclinical data demonstrated significant antitumor efficacy and favorable safety profiles, supporting its potential as a targeted therapy for EGFR/HER2-positive malignancies [1] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
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| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2278 mL | 11.1388 mL | 22.2777 mL | |
| 5 mM | 0.4456 mL | 2.2278 mL | 4.4555 mL | |
| 10 mM | 0.2228 mL | 1.1139 mL | 2.2278 mL |