Almonertinib (HS10296) is a novel, potent, orally bioavailable, 3rd-generation irreversible inhibitor of the epidermal growth factor receptor (EGFR) mutant form T790M, with potential antineoplastic activity. HS-10296 attaches itself to the secondarily acquired resistance mutation EGFR T790M, inhibits the tyrosine kinase activity of EGFR T790M, stops EGFR T790M-mediated signaling, and causes tumor cells that express EGFR T790M to die. Tumor cell proliferation and tumor vascularization are significantly influenced by EGFR, a receptor tyrosine kinase that is mutated in a variety of tumor cell types.

Physicochemical Properties

Molecular Formula	C30H35N7O2
Molecular Weight	525.6446
Exact Mass	525.285
Elemental Analysis	C, 68.55 H, 6.71 N, 18.65 O, 6.09
CAS #	1899921-05-1
Related CAS #	Almonertinib mesylate;2134096-06-1;Almonertinib hydrochloride;2134096-03-8; 1899921-05-1; 2134096-04-9 (sulfate); 2134096-07-2 (fumarate); 2134096-08-3
PubChem CID	121280087
Appearance	Off-white to light yellow solid powder
LogP	4.2
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	7
Rotatable Bond Count	11
Heavy Atom Count	39
Complexity	823
Defined Atom Stereocenter Count	0
InChi Key	DOEOECWDNSEFDN-UHFFFAOYSA-N
InChi Code	InChI=1S/C30H35N7O2/c1-6-29(38)32-24-17-25(28(39-5)18-27(24)36(4)16-15-35(2)3)34-30-31-14-13-23(33-30)22-19-37(20-11-12-20)26-10-8-7-9-21(22)26/h6-10,13-14,17-20H,1,11-12,15-16H2,2-5H3,(H,32,38)(H,31,33,34)
Chemical Name	N-[5-[[4-(1-cyclopropylindol-3-yl)pyrimidin-2-yl]amino]-2-[2-(dimethylamino)ethyl-methylamino]-4-methoxyphenyl]prop-2-enamide
Synonyms	Almonertinib; Ameile; HS-10296; HS 10296; HS10296; HS-10296; Aumolertinib; Ameile; HS-10297; Egfr T790M inhibitor HS-10296; Aumolertinib [USAN]; Aumolertinib
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	EGFRT790M (IC50 = 0.37 nM); EGFRL858R/T790M (IC50 = 0.29 nM); EGFRdel19 T790M (IC50 = 0.21 nM)
ln Vitro	HS-10296 is a potentially anti-tumor drug that is administered orally and inhibits the EGFR mutant form T790M, which may be used to treat non-small cell lung cancer[2]. Furthermore, HS-10296 may be able to suppress mutations that are sensitive to EGFR, such as G719X, del19, L858R, and L861Q[3].
ln Vivo	The experimental results of nude mice showed that compared with the control group and the positive control ositinib (AZD9291) group, the tumor growth was significantly inhibited, the weight of nude mice, the tumor volume and the tumor mass were significantly reduced in the almonertinib treatment group (all P<0.05)[4].
Cell Assay	NSCLC cells H1975 and PC-9 were cultured in vitro. The effects of almonertinib on the proliferation, apoptosis, invasion, and migration of H1975 and PC-9 cells were detected by CCK-8 assay, apoptotic assay and Transwell assay. The expression of invasion and migration related proteins was detected by Western blotting[4].
Animal Protocol	Centrifuge H1975 cells, wash with PBS, and suspend in serum-free RPMI 1640 medium. Then inject 3 × 106 cells per mouse subcutaneously into the right shoulder of 4-6 week old nude mice. After the tumor grows to 100 mm3, it will be randomly divided into three groups, with 5 mice in each group. Each group will be given 0.2 mL of dimethyl sulfoxide, 5 mg/kg AZD9291, and 5 mg/kg of amitinib by gavage, once every two days. Monitor the weight and tumor size of nude mice before each injection. The tumor volume is 2/2 of its length and width, and the entire process lasts for 21 days[4].
References	[1]. Safety, Efficacy, and Pharmacokinetics of Almonertinib (HS-10296) in Pretreated Patients With EGFR-Mutated Advanced NSCLC: A Multicenter, Open-label, Phase 1 Trial [published online ahead of print, 2020 Sep 9]. J Thorac Oncol. 2020;S1556-0. [2]. Next-Generation EGFR Tyrosine Kinase Inhibitors for Treating EGFR-Mutant Lung Cancer beyond First Line. Front Med (Lausanne). 2017 Jan 18;3:76. [3]. Management of acquired resistance to EGFR TKI-targeted therapy in advanced non-small cell lung cancer. Mol Cancer. 2018 Feb 19;17(1):38. [4]. Effect of almonertinib on the proliferation, invasion, and migration in non-small cell lung cancer cells. Zhong Nan Da Xue Xue Bao Yi Xue Ban . 2021 Oct 28;46(10):1045-1053. doi: 10.11817/j.issn.1672-7347.2021.201009.
Additional Infomation	Almonertinib is a third-generation EGFR tyrosine kinase inhibitor targeting EGFR-sensitizing and T790M resistance mutations. It is being investigated against advanced or metastatic EGFR-mutated non-small cell lung cancer (NSCLC). Aumolertinib is an orally available inhibitor of the epidermal growth factor receptor (EGFR) mutant form T790M, with potential antineoplastic activity. Upon administration, aumolertinib binds to and inhibits EGFR T790M, a secondarily acquired resistance mutation, inhibits the tyrosine kinase activity of EGFR T790M, prevents EGFR T790M-mediated signaling and leads to cell death in EGFR T790M-expressing tumor cells. EGFR, a receptor tyrosine kinase that is mutated in many tumor cell types, plays a key role in tumor cell proliferation and tumor vascularization. Drug Indication Treatment of lung cancer Mechanism of Action Almonertinib inhibits EGFR tyrosine kinase targeting EGFR-sensitizing and T790M resistance mutations.

Solubility Data

Solubility (In Vitro)

DMSO: ~100 mg/mL (~190.2 mM) Ethanol: ~6 mg/mL (~11.4 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 6.25 mg/mL (11.89 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 62.5 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: ≥ 6.25 mg/mL (11.89 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 62.5 mg/mL clear DMSO stock solution to 900 μL corn oil and mix evenly.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.9024 mL	9.5122 mL	19.0244 mL
	5 mM	0.3805 mL	1.9024 mL	3.8049 mL
	10 mM	0.1902 mL	0.9512 mL	1.9024 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.