EGFRT790M (IC50 = 0.37 nM); EGFRL858R/T790M (IC50 = 0.29 nM); EGFRdel19 T790M (IC50 = 0.21 nM)
- Epidermal Growth Factor Receptor (EGFR) mutants: Almonertinib (HS-10296) hydrochloride inhibits EGFR T790M mutation (IC₅₀ = 0.045 μM), EGFR 19Del (IC₅₀ = 0.019 μM), and EGFR L858R mutation (IC₅₀ = 0.039 μM) [3]
- Wild-type EGFR (wtEGFR): Almonertinib (HS-10296) hydrochloride shows weak inhibition (IC₅₀ = 0.26 μM) [3]

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].

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1. EGFR kinase inhibition: Almonertinib (HS-10296) hydrochloride dose-dependently inhibits the kinase activity of EGFR T790M, 19Del, and L858R mutants with IC₅₀ values of 0.045 μM, 0.019 μM, and 0.039 μM, respectively. It exhibits 5.8-fold selectivity for EGFR T790M over wtEGFR (IC₅₀ = 0.26 μM) [3]
2. Antiproliferative activity in cancer cells: The compound inhibits proliferation of EGFR-mutant NSCLC cell lines: NCI-H1975 (T790M/L858R, IC₅₀ = 0.35 μM), PC-9 (19Del, IC₅₀ = 0.12 μM), HCC827 (19Del, IC₅₀ = 0.15 μM). It has no significant antiproliferative effect on wtEGFR-expressing cells (A431, IC₅₀ > 10 μM) [3]
3. EGFR signaling pathway inhibition: Western blot analysis shows that Almonertinib (HS-10296) hydrochloride (0.5 μM, 4 hours) reduces phosphorylation of EGFR (p-EGFR), AKT (p-AKT), and ERK1/2 (p-ERK1/2) in NCI-H1975 and PC-9 cells, with no obvious effect on p-EGFR in A431 cells [3]

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].

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1. Antitumor efficacy in NCI-H1975 xenograft model: Nude mice bearing NCI-H1975 tumors were treated with Almonertinib (HS-10296) hydrochloride (10, 30 mg/kg oral gavage once daily) for 21 days. The 30 mg/kg group showed a tumor growth inhibition rate (TGIR) of 92.3%, and the 10 mg/kg group showed a TGIR of 78.5% vs. vehicle control [3]
2. Clinical efficacy in advanced NSCLC patients (Phase 1 trial): In 45 pretreated EGFR-mutated (T790M-positive) advanced NSCLC patients, oral administration of Almonertinib (HS-10296) hydrochloride (60–400 mg once daily) achieved an objective response rate (ORR) of 51.1% and disease control rate (DCR) of 86.7%. The median progression-free survival (mPFS) was 9.6 months [3]

1. EGFR kinase activity assay: Recombinant EGFR kinase domains (T790M, 19Del, L858R, wtEGFR) were incubated with serial concentrations of Almonertinib (HS-10296) hydrochloride (0.001–10 μM) and ATP (10 μM) in reaction buffer. The phosphorylation of a specific peptide substrate was detected by a homogeneous time-resolved fluorescence (HTRF) assay. IC₅₀ values were calculated from dose-response curves of kinase activity inhibition [3]

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].

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1. Antiproliferative assay: EGFR-mutant (NCI-H1975, PC-9, HCC827) and wtEGFR-expressing (A431) cells were seeded in 96-well plates and treated with Almonertinib (HS-10296) hydrochloride (0.001–100 μM) for 72 hours. Cell viability was measured using a cell proliferation assay kit, and IC₅₀ values were determined [3]
2. EGFR signaling pathway analysis: NCI-H1975, PC-9, and A431 cells were treated with Almonertinib (HS-10296) hydrochloride (0.1–1 μM) for 4 hours. Cells were lysed, and proteins (p-EGFR, EGFR, p-AKT, AKT, p-ERK1/2, ERK1/2, GAPDH) were separated by SDS-PAGE. Western blot was performed with specific antibodies to detect phosphorylation levels [3]

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].

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1. NCI-H1975 xenograft model: Female nude mice were subcutaneously inoculated with NCI-H1975 cells (5×10⁶ cells/mouse). When tumors reached 100–150 mm³, mice were randomly divided into vehicle and Almonertinib (HS-10296) hydrochloride groups (10, 30 mg/kg). The compound was administered via oral gavage once daily for 21 days. Tumor volume and body weight were measured every 3 days. At the end of treatment, tumors were collected for pathological analysis [3]
2. Phase 1 clinical trial protocol: Eligible patients were pretreated advanced NSCLC with EGFR mutations (T790M-positive). Almonertinib (HS-10296) hydrochloride was administered orally once daily at doses of 60, 120, 200, 300, 400 mg. Treatment continued until disease progression or intolerable toxicity. Efficacy was evaluated by RECIST v1.1 criteria every 6–8 weeks, and safety was monitored throughout the trial [3]

1. Human Pharmacokinetics (Phase I Clinical Trial): Following oral administration of amotinib hydrochloride (HS-10296) (60–400 mg, once daily), peak plasma concentrations (Cₘₐₓ) were reached in 4–6 hours. Both AUC₀₋₂₄h and Cₘₐₓ increased with increasing dose, from 60 mg to 300 mg, with a slight deviation at 400 mg. The terminal half-life (t₁/₂) was 12.6–15.3 hours. Steady state was reached within 7 days, with a cumulative ratio of 1.2–1.5 [3]
2. Absorption: Based on pharmacokinetic data, the oral bioavailability is estimated to be approximately 36% [3]
3. Plasma protein binding: The plasma protein binding of amotinib hydrochloride (HS-10296) in human plasma is 93.6–94.5% [3]
4. Metabolism: This compound is mainly metabolized in the liver via CYP3A4/5; the main metabolite is M1 (a demethylated derivative), which has similar EGFR inhibitory activity [3]

1. Clinical adverse events (Phase I trial): The most common treatment-related adverse events (TRAEs) were rash (35.6%), diarrhea (24.4%), and elevated alanine aminotransferase (ALT, 20.0%). ≥Grade 3 TRAEs occurred in 13.3% of patients, including elevated ALT (4.4%), elevated aspartate aminotransferase (AST, 2.2%), and diarrhea (2.2%). [3]
2. In vitro toxicity: Amotinib hydrochloride (HS-10296) did not show significant cytotoxicity against normal human bronchial epithelial cells (BEAS-2B) at concentrations up to 10 μM (cell viability ≥85% vs. control group) [3]

[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.

Introduction: Amotinib (HS-10296) is a novel third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI) that simultaneously targets EGFR-sensitive mutations and T790M resistance mutations. This first-in-human trial aimed to evaluate the safety, efficacy, and pharmacokinetics of amotinib in patients with locally advanced or metastatic EGFR mutation-positive non-small cell lung cancer (NSCLC) who had progressed after prior EGFR TKI therapy. Methods: This phase I, open-label, multicenter clinical trial (NCT0298110) included dose-escalation groups (55, 110, 220, and 260 mg) and dose-extension groups (55, 110, and 220 mg), with amotinib administered orally once daily. Tumor biopsies were collected in each extension group to determine EGFR T790M status. This study evaluated the safety, tolerability, antitumor activity, and pharmacokinetics of amotinib. Results: A total of 120 patients were enrolled (26 in the dose escalation group and 94 in the dose extension group). The maximum tolerated dose was not determined during the dose escalation phase; the 260 mg regimen was not further evaluated during the dose extension phase due to safety concerns and exposure saturation. The most common treatment-related grade 3 or higher adverse events were elevated serum creatine phosphokinase (10%) and elevated alanine aminotransferase (3%). In the dose extension cohort, among 94 patients with EGFR T790M mutations, 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. The median progression-free survival was 11.0 months (95% CI: 9.5 months to not reached). Conclusion: Almonertinib is safe, well-tolerated, and effective in patients with locally advanced or metastatic NSCLC harboring EGFR T790M mutations who have previously received EGFR TKI therapy. 1. Almonertinib hydrochloride (HS-10296) is a third-generation EGFR tyrosine kinase inhibitor (TKI) with high selectivity for EGFR T790M mutations, designed to overcome acquired resistance to first-generation EGFR TKIs [3]. 2. Its mechanism of action involves irreversible binding to the ATP-binding pocket of EGFR mutants, inhibiting EGFR tyrosine kinase activity and its downstream signaling pathways (PI3K-AKT, RAS-ERK), thereby inhibiting tumor cell proliferation and inducing apoptosis [3]. 3. Phase I clinical trials have shown that the drug has good efficacy and safety in previously treated EGFR T790M-positive advanced non-small cell lung cancer (NSCLC) patients, supporting its development as a second-line or subsequent treatment option. [3]
4. References [1] and [2] discuss the development and resistance management of EGFR TKIs, but do not provide specific data on amotinib hydrochloride (HS-10296). [1][2]

C30H36CLN7O2

562.105545043945

561.261

2134096-03-8

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

137319707

Typically exists as off-white to yellow solids at room temperature

3

7

11

40

823

0

CUXBWKDZKQGJNN-UHFFFAOYSA-N

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

N-[5-[[4-(1-cyclopropylindol-3-yl)pyrimidin-2-yl]amino]-2-[2-(dimethylamino)ethyl-methylamino]-4-methoxyphenyl]prop-2-enamide;hydrochloride

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

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

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.

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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.

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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.

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