Almonertinib hydrochloride (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 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.

Physicochemical Properties

Molecular Formula	C30H36CLN7O2
Molecular Weight	562.105545043945
Exact Mass	561.261
CAS #	2134096-03-8
Related CAS #	Almonertinib;1899921-05-1;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	137319707
Appearance	Typically exists as off-white to yellow solids at room temperature
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	7
Rotatable Bond Count	11
Heavy Atom Count	40
Complexity	823
Defined Atom Stereocenter Count	0
InChi Key	CUXBWKDZKQGJNN-UHFFFAOYSA-N
InChi Code	InChI=1S/C30H35N7O2.ClH/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);1H
Chemical Name	N-[5-[[4-(1-cyclopropylindol-3-yl)pyrimidin-2-yl]amino]-2-[2-(dimethylamino)ethyl-methylamino]-4-methoxyphenyl]prop-2-enamide;hydrochloride
Synonyms	Almonertinib; HS-10296 HCl; HS 10296; HS-10296 hydrochloride; 2134096-03-8; Almonertinib (hydrochloride); ALMONERTINIB HYDROCHLORIDE; N-(5-((4-(1-Cyclopropyl-1H-indol-3-yl)pyrimidin-2-yl)amino)-2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxyphenyl)acrylamide hydrochloride; N-[5-[[4-(1-cyclopropylindol-3-yl)pyrimidin-2-yl]amino]-2-[2-(dimethylamino)ethyl-methylamino]-4-methoxyphenyl]prop-2-enamide;hydrochloride; N-(5-{[4-(1-cyclopropylindol-3-yl)pyrimidin-2-yl]amino}-2-{[2-(dimethylamino)ethyl](methyl)amino}-4-methoxyphenyl)prop-2-enamide hydrochloride; MFCD31807629;HS10296
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.
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	NSCLC may be treated with almonertinib (HS-10296), an oral inhibitor of the EGFR mutant T790M that has possible anti-tumor activity [2]. Almonertinib (HS-10296) has the ability to inhibit G719X, del19, L858R, and L861Q, among other EGFR-sensitive mutations[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]. Next-Generation EGFR Tyrosine Kinase Inhibitors for Treating EGFR-Mutant Lung Cancer beyond First Line. Front Med (Lausanne). 2017 Jan 18;3:76. [2]. Management of acquired resistance to EGFR TKI-targeted therapy in advanced non-small cell lung cancer. Mol Cancer. 2018 Feb 19;17(1):38. [3]. 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.
Additional Infomation	Introduction: Almonertinib (HS-10296) is a novel, third-generation EGFR tyrosine kinase inhibitor (EGFR TKI) that targets both EGFR-sensitizing and T790M resistance mutations. This first-in-human trial aimed to evaluate the safety, efficacy, and pharmacokinetics of almonertinib in patients with locally advanced or metastatic EGFR mutation-positive NSCLC that had progressed after pevious EGFR TKI therapy. Methods: This phase 1, open-label, multicenter clinical trial (NCT0298110) included dose-escalation (55, 110, 220, and 260 mg) and dose-expansion cohorts (55, 110, and 220 mg) with once daily oral administration of almonertinib. In each expansion cohort, tumor biopsies were obtained for the determination of EGFR T790M status. The safety, tolerability, antitumor activity, and pharmacokinetics of almonertinib were evaluated. Results: A total of 120 patients (26 patients in the dose-escalation cohort and 94 patients in the dose-expansion cohort) were enrolled. The maximum tolerated dose was not defined in the dose-escalation phase; the 260 mg regimen was not further evaluated in the dose-expansion phase owing to safety concerns and saturation of exposure. The most common treatment-related grade greater than or equal to 3 adverse events were increased blood creatine phosphokinase (10%) and increased alanine aminotransferase (3%). Among 94 patients with the EGFR T790M mutation in the dose-expansion cohort, the investigator-assessed objective response rate and disease control rate were 52% (95% confidence interval [CI]: 42-63) and 92% (95% CI: 84-96), respectively. Median progression-free survival was 11.0 months (95% CI: 9.5-not reached) months. Conclusions: Almonertinib is safe, tolerable and effective for patients with locally advanced or metastatic NSCLC harboring the EGFR T790M mutation who were pretreated with EGFR TKIs.

Solubility Data

Solubility (In Vitro)

DMSO : ~83.33 mg/mL (~148.25 mM) H2O : ~12.5 mg/mL (~22.24 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.70 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 20.8 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: ≥ 2.08 mg/mL (3.70 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. 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. Solubility in Formulation 3: ≥ 2.08 mg/mL (3.70 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 20.8 mg/mL clear DMSO stock solution to 900 μL of 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	1.7790 mL	8.8951 mL	17.7901 mL
	5 mM	0.3558 mL	1.7790 mL	3.5580 mL
	10 mM	0.1779 mL	0.8895 mL	1.7790 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.