AST1306 targets EGFR (IC50 = 0.5 ± 0.2 nmol/L), ErbB2 (IC50 = 3.0 ± 1.5 nmol/L), ErbB4 (IC50 = 0.8 ± 0.3 nmol/L), and EGFR T790M/L858R double mutant (IC50 = 12 ± 2 nmol/L). It shows >3000-fold selectivity over other kinases tested (Flt-1, KDR, PDGFRα, PDGFRβ, c-Src, c-Met, c-Kit, IGF1R, FGFR1, RON, Abl, EphA2, EphB2, Tie2, SYK, PKCb2, p70S6K, JAK2, Pim2, AKT1, AUR A, CDK2, GSK3b; all IC50 > 10000 nmol/L). [1]

On the development of HIH3T3-EGFR T790M/L858R cells, AST1306 tosylate (AST-1306 (TsOH); 0.19-6.25 μM; 72 hours) can have notable, concentration-dependent inhibitory effects [1]. In A549 cells, Calu-3 cells, and SK-OV-3 cells, AST1306 tosylate suppresses the activation of tyrosine kinases and downstream signaling pathways. In A549 cells, AST1306 tosylates in a dose-dependent manner and notably prevents EGF-induced EGFR phosphorylation [1]. Two tumor cells grown on soft agar can be significantly inhibited by AST1306 tosylate (0.1, 0.5, 1.0, 5.0 μM), with SK-OV-3 cells exhibiting far higher sensitivity than A549 cells [1]. Compared to other kinase families, AST1306 tosylate (0.001-1.0 μM; 4 hours) is more than 3000 times more selective for ErbB family kinases [1]. With an IC50 value of 12±2 nmol/L, AST1306 tosylate efficiently suppresses the EGFR T790M/L858R double mutant[1].

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AST1306 potently inhibited the proliferation of NIH3T3 cells engineered to express EGFR T790M/L858R double mutant in a concentration-dependent manner (IC50 values not shown but effective at low concentrations). In contrast, it was much less potent toward parental NIH3T3 cells. AST1306 also inhibited the growth of NCI-H1975 cells (harboring endogenous EGFR T790M/L858R mutation) in a concentration-dependent manner (IC50 not specified) and blocked EGFR phosphorylation and downstream signaling. [1]
In A549 cells (high EGFR expression), AST1306 dose-dependently inhibited EGF-induced EGFR phosphorylation (bands almost disappeared at 0.01 μmol/L), as well as downstream Erk1/2 and AKT phosphorylation. In Calu-3 cells (ErbB2-overexpressing) and SK-OV-3 cells (overexpressing both EGFR and ErbB2), AST1306 similarly inhibited target and downstream signaling, being >10-fold more potent than lapatinib. [1]
AST1306 suppressed proliferation of a panel of human cancer cell lines with varying IC50 values: Calu-3 (IC50 = 0.23 μmol/L), BT474 (IC50 = 0.97 μmol/L), MDA-MB-468 (IC50 = 6.2 μmol/L), A549 (IC50 = 7.5 μmol/L), NCI-H23 (IC50 = 7.5 μmol/L), SK-OV-3 (IC50 = 6.2 μmol/L), and MCF-7 (IC50 = 16.0 μmol/L). ErbB2-overexpressing cells were more sensitive. [1]
In anchorage-independent growth assays (soft agar), AST1306 dramatically inhibited colony formation of SK-OV-3 and A549 cells, with SK-OV-3 cells showing much higher sensitivity. [1]
Knockdown of ErbB2 by siRNA (sequence: 5’ CGUUUGAGUCCCAUGCCCAATT 3’) in SK-OV-3 cells significantly reduced sensitivity to AST1306 at 6.25 μmol/L (p<0.05) and 12.5 μmol/L (p<0.01). Knockdown of EGFR using two independent siRNAs (#1: 5’ GGUGUAGAAUAUGAAACUATT 3’; #2: 5’ GUCCUUUGGGAAUUUUGGAAATT 3’) did not significantly affect antiproliferative activity. [1]

Tumor growth in SK-OV-3 and Calu-3 xenograft models is significantly inhibited by AST1306 tosylate (AST-1306 (TsOH); oral; 25–100 mg/kg; twice daily; for 28 days [1].

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In SK-OV-3 xenograft models (ErbB2-overexpressing), oral administration of AST1306 (100, 50, 25 mg/kg, twice daily) caused dramatic tumor growth suppression; tumors almost completely disappeared after 7 days of treatment. In Calu-3 xenografts (ErbB2-overexpressing), similar potent inhibition was observed. In contrast, AST1306 only weakly suppressed tumor growth in HO-8910 and A549 xenografts (low ErbB2 expression). Lapatinib (50 mg/kg, twice daily) also showed antitumor activity in ErbB2-overexpressing models, but AST1306 was more efficacious than lapatinib in the SK-OV-3 model at the same dose and schedule. Body weights of mice showed no obvious differences during treatment, indicating good tolerability. [1]
In a single-dose pharmacodynamic study (100 mg/kg) in SK-OV-3 xenografts, AST1306 inhibited EGFR and ErbB2 phosphorylation in tumors within 2 hours, and the inhibition lasted at least 24 hours. [1]
In FVB-2/Nneu transgenic mouse model (expressing ErbB2/Neu proto-oncogene), oral administration of AST1306 (100, 50, 25 mg/kg, twice daily) for 3 weeks caused dramatic tumor growth suppression; after 11 days, tumors almost completely disappeared. Body weights decreased by less than 20% during treatment. [1]

Tyrosine kinase activities were determined using enzyme-linked immunosorbent assay (ELISA). Serine/threonine kinase activities were detected by Caliper Mobility Shift Assay on EZ Reader according to the instruction. For irreversible binding assessment, rapid dilution experiments were performed: 9 μL of 100-fold normal amount of enzyme (800 nM for EGFR, 1800 nM for ErbB2) was mixed with 1 μL of AST1306 at a final concentration 100-fold IC50 for each enzyme or with vehicle control (DMSO). Lapatinib was used as positive control. After 30 minutes incubation at room temperature, 1 μL of the mix was diluted into 99 μL of solution containing substrate peptide (sequence: 5-FAM-EEPLYWSFPAKKK-CONH2, 1.5 μmol/L) and ATP (2.3 μmol/L for EGFR, 15 μmol/L for ErbB2). The microplate was placed in the EZ Reader and wells were repeatedly sampled for 180 minutes. No EGFR or ErbB2 activity was recovered after incubation with AST1306, demonstrating irreversible inhibition. [1]
Molecular docking simulation: The crystal structure of EGFR kinase domain in complex with lapatinib (PDB entry 1XKK) was used as EGFR target. 3D models of ErbB2 kinase domain were created using the MODELLER program based on homologous EGFR. Structure-based analysis was performed using docking program AUTODOCK4.0 with Lamarckian genetic algorithm (step size 0.2 Å for translation and 5° for orientation/torsion; number of generations 500,000; energy evaluations 2,500,000; docking runs 20). AST1306 was predicted to covalently bind to Cys797 of EGFR (distance 4.4 Å from β-carbon of Michael acceptor to sulfur atom) and Cys805 of ErbB2. [1]

Cell Proliferation Assay [1]
Cell Types: NIH3T3 Parental and NIH3T3 Cell
Tested Concentrations: 0.19, 0.39, 0.78, 1.56, 3.13, 6.25 μM
Incubation Duration: 72 hrs (hours)
Experimental Results: Significant concentration-dependent inhibition of EGFR on the growth of HIH3T3- T790M/L858R cells.

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Western Blot Analysis[1]
Cell Types: A549 cells, Calu-3 cells and SK-OV-3 cells
Tested Concentrations: 0.001, 0.01, 0.1, 1.0 μM
Incubation Duration: 4 hrs (hours)
Experimental Results: Inhibition of tyrosine kinases and downstream signaling pathways activation.

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Cell proliferation was evaluated using Sulforhodamine B (SRB) assay after 72 hours treatment with indicated concentrations of AST1306. [1]
For Western blot analysis of receptor tyrosine kinase phosphorylation and downstream signaling, starved cells were treated with AST1306 for 4 hours, stimulated with EGF (50 ng/mL) for 15 minutes, then collected and processed for immunoblotting. Whole-cell lysates were assayed for total and phosphorylated proteins. [1]
For siRNA transfection, small interfering RNA against EGFR or ErbB2, as well as control siRNA, were designed and synthesized. One pair of ErbB2 siRNA (sequence: 5’ CGUUUGAGUCCCAUGCCCAATT 3’), and two pairs of EGFR siRNA (#1: 5’ GGUGUAGAAUAUGAAACUATT 3’; #2: 5’ GUCCUUUGGGAAUUUUGGAAATT 3’) were used. siRNA (final concentration 50 nmol/L) was transfected into SK-OV-3 cells with oligofectamine reagent. AST1306 treatment was done 48 hours after transfection, and growth-inhibitory effects were determined by SRB assay. [1]
Anchorage-independent growth assay: Cells (8000/mL) were exposed to AST1306 in 1 mL of 0.3% basal medium Eagle’s agar containing 10% FBS. Culture was maintained at 37°C in 5% CO2 atmosphere for two weeks. Average colony number was calculated and colonies photographed. [1]

Animal/Disease Models: SK-OV-3 and Calu-3 tumor nude mice [1]
Doses: 25, 50, 100 mg/kg
Route of Administration: po (po (oral gavage)) twice a day; lasted for 28 days.
Experimental Results: Dramatically inhibited tumor growth.

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Human tumor xenograft models: Under sterile conditions, well-developed tumors were cut into 1-mm³ fragments and transplanted subcutaneously into the right flank of nude mice using a trocar. When tumor volume reached 100 to 200 mm³, mice were randomly assigned into control and treatment groups. Control groups received vehicle alone; treatment groups received AST1306 (100, 50, 25 mg/kg) or lapatinib (50 mg/kg) orally, twice daily. Tumor volume (V) was calculated as V = (length × width²)/2. Body weights were measured simultaneously. [1]
Pharmacodynamic study in tumors: Mice bearing established (~200 mm³) SK-OV-3 xenograft tumors were treated with a single dose of AST1306 at 100 mg/kg. Tumors were harvested at indicated times (n=3 per time point) after treatment, immediately frozen in liquid nitrogen, then homogenized in protein extraction solution. Tumor extracts were subjected to SDS-PAGE followed by immunoblotting. [1]
FVB-2/Nneu transgenic mouse model: 38- to 44-week-old female FVB/Nneu mice were obtained. AST1306 was suspended in 0.5% hydroxypropyl methylcellulose (HPMC), grinding with agate mortar. Mice were administered AST1306 at dosage of 100, 50, and 25 mg/kg twice daily, with lapatinib (50 mg/kg) as comparison. Tumors were measured twice a week in two dimensions using a caliper, and tumor volume calculated as L × W × W / 2. [1]

The half-life of AST1306 after a single oral dose (90 mg/kg) is less than 3.6 hours in rats and less than 2.3 hours in dogs (unpublished data). Despite this short half-life, a single dose of AST1306 (100 mg/kg) in SK-OV-3 xenograft models produced sustained inhibition of EGFR and ErbB2 phosphorylation for more than 24 hours, consistent with an irreversible inhibition mechanism. [1]

In xenograft studies, body weights of nude mice measured concurrently showed no obvious differences during AST1306 treatment, demonstrating that AST1306 was well tolerated. [1]
In FVB-2/Nneu transgenic mouse model, body weights of the mice decreased by less than 20% during treatment with AST1306. [1]

[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.

AST1306 is designed as an irreversible ErbB family inhibitor that overcomes acquired resistance to reversible EGFR inhibitors (e.g., gefitinib, erlotinib) caused by mutations such as T790M. It covalently binds to Cys797 in EGFR and Cys805 in ErbB2. Unlike other irreversible inhibitors (e.g., HKI-272, BIBW2992, CI1033, PF-00299804) that are active in both EGFR- and ErbB2-dependent tumor models, AST1306 shows more potent efficacy in ErbB2-dependent models than in EGFR-dependent models, suggesting a distinct profile. The prolonged pharmacodynamic effect (≥24 h target inhibition) allows once- or twice-daily oral dosing. AST1306 is currently in phase I clinical trial in China. [1]

C₃₁H₂₆CLFN₄O₅S

621.08

620.129

1050500-29-2

Allitinib;897383-62-9

25027665

Light yellow to yellow solid powder

8.914

3

9

8

43

848

0

ZMUKJEHWLJBODV-UHFFFAOYSA-N

InChI=1S/C24H18ClFN4O2.C7H8O3S/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;1-6-2-4-7(5-3-6)11(8,9)10/h2-12,14H,1,13H2,(H,29,31)(H,27,28,30);2-5H,1H3,(H,8,9,10)

N-[4-[3-chloro-4-[(3-fluorophenyl)methoxy]anilino]quinazolin-6-yl]prop-2-enamide;4-methylbenzenesulfonic acid

AST-1306 AST 1306AST1306

2934.99.9001

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.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~80.50 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.03 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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.

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 (Please use freshly prepared in vivo formulations for optimal results.)