AST-1306 (TsOH)

Biological Activity

AST-1306 is a novel irreversible epidermal growth factor receptor inhibitor for EGFR and ErbB4 with IC50 of 0.5 and 0.8 nM, respectively. AST-1306 selectively inhibits the tyrosine kinase activities of ErbB2 with an IC50 of 3 nM. AST-1306 also potently prevents the EGFR T790M/L858R double mutant with an IC50 value of 12 nM. For this, AST-1306 is approximately 500-fold more potent than lapatinib. AST-1306 is more than 3000-fold selective for ErbB family kinases over other kinase families including PDGFR, KDR and c-Met. AST-1306 might covalently bind to specific amino acid residues of EGFR and ErbB2, AST-1306 significantly and concentration-dependently prevents the growth of HIH3T3-EGFR T790M/L858R cells. AST-1306 effectively suppresses EGFR phosphorylation in HIH3T3-EGFR T790M/L858R cells. Moreover, AST-1306 prevents the growth of NCI-H1975 cells expressing the EGFR T790M/L858R mutation in a concentration-dependent manner. AST-1306 blocks phosphorylation of EGFR and downstream pathways as well. In addition, AST-1306 dose-dependently and markedly inhibits EGF-induced EGFR phosphorylation in A549 cells. AST-1306 inhibits the phosphorylation of EGFR and ErbB2, and downstream signaling in human cancer cells including A549 cells, Calu-3 cells and SK-OV-3 cells. Twice daily oral administration of AST-1306 gives rise to a dramatic prevention of tumor growth in SK-OV-3 and Calu-3 xenograft models. In SK-OV-3 models, tumors almost completely disappears after treatment with AST-1306 for 7 days. In contrast, AST-1306 only slightly inhibits the growth of tumor in HO-8910 and A549 xenograft models. Therefore, the antitumor efficacy of AST-1306 is greater in ErbB2-overexpressing tumor models than in models expressing low levels of ErbB2. AST-1306 is well tolerate. In addition, oral administration of AST-1306 twice daily for 3 weeks dramatically suppresses the growth of tumor in the FVB-2/Nneu models. After treatment for 11 days, tumors almost completely disappears. The body weights of the mice reduces by less than 20% during treatment. AST-1306 has entered in a phase I clinical trial in the treatment of EGFR-overexpressing tumors.

Protocol (for reference only)

Cell Experiment
Cell lines	Calu-3 and A-549 cell lines
Preparation method	Using the SRB (Sulforhodamine B) assay to evaluate cell (including Calu-3, A-549 cell line et al.) proliferation . Briefly, seeding cells into 96-well plates and grown for 24 hours. Then treating the cells with increasing concentrations of AST-1306 and grown for a further 72 hours. The medium remains unchanged until the completion of the experiment. Then fixing the cells with 10% precooled trichloroacetic acid (TCA) for 1 hour at 4 °C and staining for 15 min at room temperature with 100 μL of 4 mg/mL SRB solution in 1% acetic acid. Then removingthe SRB , and quickly rinsing the cells five times with 1% acetic acid. After cells are air-dried, protein-bound dye is dissolved in 150 μL of 10 mM Tris base for 5 min and measured at 515 nm using a multiwell spectrophotometer.Calculating the inhibition rate on cell proliferation as (1 - A515 treated/A515 control) × 100%. Obtaining the IC50 value by the Logit method and determining from the results of at least 3 independent tests.
Concentrations	0.001-1 μM
Incubation time	72 hours

Animal Experiment
Animal models	Nude mice bearing SK-OV-3 xenograft tumors and SK-OV-3FVB-2/Nneu transgenic mouse
Formulation	0.5% hydroxypropyl methylcellulose (HPMC), grinding with agate morta
Dosages	25 mg/kg, 50 mg/kg and 100 mg/kg
Administration	p.o., twice daily

Chemical Information

Molecular Weight	621.08
Formula	C24H18ClFN4O2
CAS Number	1050500-29-2
Solubility (25°C)	DMSO 50 mg/mL
Storage	Powder          -20°C   3 years ;  4°C   2 years In solvent       -80°C   6 months ;  -20°C   1 month

Conversion of different model animals based on BSA (PMID: 27057123)

Species	Mouse	Rat	Rabbit	Guinea pig	Hamster	Dog
Weight (kg)	0.02	0.15	1.8	0.4	0.08	10
Body Surface Area (m2)	0.007	0.025	0.15	0.05	0.02	0.5
Km factor	3	6	12	8	5	20

Animal A (mg/kg) = Animal B (mg/kg) multiplied by	Animal B Km
	Animal A Km

For example, to modify the dose of Compound A used for a mouse (20 mg/kg) to a dose based on the BSA for a rat, multiply 20 mg/kg by the K_m factor for a mouse and then divide by the K_m factor for a rat. This calculation results in a rat equivalent dose for Compound A of 10 mg/kg.

References

[1] Jin-fang Jiang, et al. Metabolic research of domestically developed small molecule tyrosine kinase inhibitors

[2] Ji Xu, et al. ERBB4 promotes the proliferation of gastric cancer cells via the PI3K/Akt signaling pathway

[3] Guangyuan Song, et al. miR-551b regulates epithelial-mesenchymal transition and metastasis of gastric cancer by inhibiting ERBB4 expression

[4] Olga Martinho, et al. HER Family Receptors are Important Theranostic Biomarkers for Cervical Cancer: Blocking Glucose Metabolism Enhances the Therapeutic Effect of HER Inhibitors

[5] Hui Zhang, et al. AST1306, a potent EGFR inhibitor, antagonizes ATP-binding cassette subfamily G member 2-mediated multidrug resistance