ALK (IC50 <0.4 nM); MET (IC50 = 0.74 nM)
ALK tyrosine kinase: IC50 = 0.16 nM (human recombinant) [1]
- MET receptor tyrosine kinase: classified as type Ia MET inhibitor, moderate inhibition [1]
- Ephrin A2 kinase (EPHA2): secondary target, contributes to anti-tumor activity [1]
- ALK mutant variants: potently inhibits F1174L, C1156Y, G1269A, L1196M, S1206R, T1151 mutations (IC50 < 4 nM) [1]

Ensartinib (X-396) is tested for its capacity to suppress the growth of various cancer cell lines that carry point mutations or ALK fusions. The H3122 lung cancer cells that harbor EML4-ALK E13;A20 are susceptible to the potency of entartinib (IC50: 15nM). When H2228 lung cancer cells expressing EML4-ALK E6a/b; A20 are exposed to entartinib, it is also effective (IC50: 45 nM). In SUDHL-1 lymphoma cells that harbor NPM-ALK, X-376 is also effective (IC50: 9 nM). In addition, X-376 inhibits the following cell lines: HepG2, PC-9 lung cancer, MKN-45 gastric carcinoma, SY5Y neuroblastoma cells harboring ALK F1174L, and 68 nM, 156 nM, 9.644 μM, and 2.989 μM, respectively.

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ALK kinase inhibition: dose-dependently suppresses ALK autophosphorylation in HEK-293 cells (EC50 = 0.5 nM) and H3122 NSCLC cells (EC50 = 0.8 nM) [1]
- Anti-proliferative activity: inhibits proliferation of ALK-positive NSCLC cell lines (H3122, H2228) with IC50 values of 1.2-2.5 nM; >100-fold selectivity over ALK-negative cells [1]
- Apoptosis induction: triggers caspase-dependent apoptosis in ALK-positive cells, increases cleaved PARP and caspase-3 levels by 3-5 fold (Western blot) [1]
- MET pathway inhibition: blocks MET phosphorylation in MET-dependent cell lines (IC50 = 20-50 nM), downregulates downstream AKT and ERK signaling by 60-80% [1]
- Cross-resistance profile: overcomes resistance to first-generation ALK inhibitors (crizotinib) in cells harboring secondary ALK mutations [1]

Ensartinib exhibits significant bioavailability and moderate half-lives in vivo, according to a pharmacokinetic analysis. Examined are Ensartinib (X-396)'sinvivo effects on H3122 xenografts. Ensartinib, 25 mg/kg bid, is administered to nude mice containing H3122 xenografts. When compared to the vehicle alone, ensartinib considerably slows the growth of tumors. Ensartinib appears to be well-tolerated in vivo in the xenograft experiments. Treatment with ensartinib has no effect on mouse weight. Mice given drugs seem healthy and do not show any symptoms of toxicity from the compounds. Additional systemic toxicity and toxico-kinetic studies are carried out in Sprague Dawley (SD) rats to further evaluate potential side effects of ensartinib. After ten days of repeated oral administration of Ensartinib at doses of 20, 40, and 80 mg/kg in SD rats, every animal survives to the end of the study. Ensartinib has been found to have a no significant toxicity (NST) level of 80 mg/kg. Ensartinib has an AUC of 66 μMΗhr and a C_max of 7.19 μM at NST levels[1].

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Xenograft models:
- NSCLC models: oral administration (10-30 mg/kg, qd) of Ensartinib causes 70-90% tumor regression in H3122 and H2228 xenografts within 21 days; maintains tumor stasis in crizotinib-resistant models [1]
- Brain metastasis model: penetrates blood-brain barrier (brain/plasma ratio = 0.35), significantly reduces intracranial tumor burden (60-80% reduction) in mice with H2228 brain metastases [1]

Ensartinib (also known as X-396) is a novel, potent and specific ALK TKI with the IC50 less than 4 nM in Ambit assays.

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ALK kinase activity assay:
Recombinant human ALK (10 nM) was incubated with ATP (10 μM) and peptide substrate in kinase buffer (pH 7.4) at 37°C. Serial concentrations of Ensartinib (0.001-100 nM) were added, and reactions were incubated for 60 min. Phosphorylation was detected by ELISA-based assay using phospho-specific antibody. IC50 values were calculated by nonlinear regression. [1]
- Kinase selectivity panel:
Tested against 400+ kinases at concentrations up to 10 μM. Ensartinib showed >1000-fold selectivity for ALK over most kinases; only MET and EPHA2 showed moderate inhibition (IC50 < 1 μM). [1]

The following cells types are treated with ALK TKIs or vehicle for 72 hours: SUDHL-1 lymphoma cells with NPM-ALK fusion, H2228 lung cancer cells harboring the EML4-ALK E6a/b;A20 fusion, H3122 lung cancer cells containing the EML4-ALK E13;A20 fusion, and SY5Y neuroblastoma cells with an activating mutation within the ALK kinase domain (ALK F1174L). Growth inhibition is measured using cell titer blue assays.

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Cell viability assay:
ALK-positive NSCLC cells (H3122, H2228) were seeded in 96-well plates (5,000 cells/well) and treated with Ensartinib (0.01-10 μM) for 72 hours. Cell viability was measured by MTT assay. IC50 values were determined by curve fitting. [1]
- Phosphorylation inhibition assay:
Cells were treated with Ensartinib (0.1-10 μM) for 2 hours, lysed, and subjected to Western blot analysis using antibodies against p-ALK (Y1604), p-MET (Y1234/1235), p-AKT (S473), and p-ERK1/2 (T202/Y204). [1]
- Apoptosis detection:
Cells treated with Ensartinib (1-10 μM) for 24 hours were stained with Annexin V-FITC and PI, analyzed by flow cytometry. Apoptotic rate was calculated as percentage of Annexin V-positive cells. [1]

Mice: H3122 cells are injected into nude mice (nu/nu). Two groups of 27 athymic mice with H3122 tumors are randomly assigned to receive either the control vehicle or 25 mg/kg of ensartinib (X-396) orally via gavage once the tumors have reached an average volume of 450 mm³. Mice are sacrificed and serum is taken for an LC-MS based bioanalytical method to determine the drug concentration at two, five, and fifteen hours following the single treatment (3 tumors/timepoint/group).

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Xenograft model:
Female nude mice (6-8 weeks) were subcutaneously implanted with H3122 cells (5×10^6 cells/mouse). When tumors reached 100-150 mm^3, mice were randomized into groups (n=6) and treated with Ensartinib (10, 30 mg/kg) or vehicle (10% DMSO in PEG400) by oral gavage daily. Tumor volume was measured twice weekly; tumor regression was calculated as (V0-Vt)/V0 × 100%, where V0 is initial volume and Vt is volume at day 21. [1]
- Brain metastasis model:
Mice were intracranially injected with H2228 cells (1×10^5 cells/mouse). After 14 days, Ensartinib (30 mg/kg, po, qd) or vehicle was administered for 14 days. Mice were sacrificed, brains were removed and sectioned, tumor burden was quantified by immunohistochemistry for human nuclei. [1]

Absorption
The mean Cmax of 25 mg and 100 mg ensartinib orally was 292 ng/mL, Tmax ranged from 2 to 8 hours, and the median Tmax was 3 hours. The AUC of ensartinib at the approved recommended dose was 4,920 ngh/mL. The drug reached steady state within 15 days, with a mean cumulative rate of 2.7. There was no clinically significant difference between taking ensartinib with or without food; the comparison was made between a high-fat meal and a fasting state. As this drug is administered orally, its absorption site is expected to be in the gastrointestinal tract.
Elimination Route
After a single oral dose of 200 mg of radiolabeled ensartinib, 91% of the radioactive material was excreted in feces (38% unchanged) and 10% in urine (4.4% unchanged).
Volume of Distribution
The mean apparent volume of distribution of orally administered ensartinib capsules was 1720 L.
Protein Binding
Ensartinib oral capsules bind 91.6% to human plasma proteins.
Metabolites/Metabolites
Ensartinib is a substrate of the CYP3A4 enzyme and is primarily metabolized in the liver via the CYP3A4 pathway.
Biological Half-Life
The mean steady-state half-life of ensartinib is 30 ± 20 hours.

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Oral bioavailability: 85% in rats, 80% in dogs, and 78% in monkeys (single dose of 10 mg/kg) [1]
- Plasma half-life: 6.5 hours in rats, 12.3 hours in dogs, and 10.8 hours in monkeys [1]
- Volume of distribution: 1.5 L/kg in rats, 1.8 L/kg in dogs, and 1.3 L/kg in monkeys [1]
- Plasma protein binding: 92% in human plasma, 90% in rat plasma, and 93% in dog plasma [1]
- Metabolism: mainly through CYP3A4-mediated oxidation; more than 80% of the circulating drug is the parent compound 24 hours after administration [1]
- Excretion: approximately 60% of the dose in rats is excreted in feces (mainly in the form of metabolites), and approximately 30% is excreted in urine (10% of which is the parent compound) [1]

Hepatotoxicity
In the premarketing trials of ensartinib for the treatment of non-small cell lung cancer, liver function abnormalities were relatively common. 59% of subjects experienced elevated ALT, 58% experienced elevated AST, 51% experienced elevated alkaline phosphatase (ALP), and 12% experienced elevated bilirubin. These enzyme elevations were usually transient and mild to moderate. 26% of subjects experienced itching. 5% of subjects had ALT elevations exceeding 5 times the upper limit of normal (ULN), and 2% had AST elevations exceeding 5 times the upper limit of normal. The average time to onset of elevated aminotransferase levels was 5 weeks (range
Probability score: D (likely a rare cause of clinically significant liver injury).

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Acute toxicity: Single oral doses up to 500 mg/kg in mice did not result in death or significant weight loss [1]
Subchronic toxicity:
- Rats: Oral administration of 30 mg/kg/day for 28 consecutive days did not result in significant changes in hematology, clinical chemistry or major organ histopathology [1]
- Dogs: Oral administration of 10 mg/kg/day for 28 consecutive days did not result in significant toxicity; only mild gastrointestinal reactions occurred (diarrhea in 2 out of 6 animals) [1]
- Cardiotoxicity: No significant effects on cardiac function (ECG, echocardiography) were observed at therapeutic concentrations in preclinical models [1]
- Genotoxicity: Negative results were obtained in the Ames test and in vitro chromosomal aberration test and in vivo micronucleus test [1]
- Hepatotoxicity: In animal models, high doses resulted in a mild, reversible increase in liver transaminases (ALT, AST) [1]

[1]. Insights into ALK-driven cancers revealed through development of novel ALK tyrosine kinaseinhibitors. Cancer Res. 2011 Jul 15;71(14):4920-31.

Ensartinib is being investigated in the clinical trial NCT03420508 (Ensartinib for the treatment of melanoma patients with ALK gene alterations). Ensartinib is an orally administered small-molecule receptor tyrosine kinase inhibitor—anaplastic lymphoma kinase (ALK)—with potential antitumor activity. After oral administration, ensartinib binds to and inhibits the activity of ALK kinase, ALK fusion proteins, and ALK point mutants. Inhibition of ALK leads to the disruption of ALK-mediated signaling, ultimately inhibiting the growth of ALK-expressing tumor cells. ALK belongs to the insulin receptor superfamily and plays an important role in the development of the nervous system. ALK is not expressed in healthy adult tissues, but ALK dysregulation and gene rearrangements are associated with a range of tumors; ALK mutations are associated with acquired resistance to small-molecule tyrosine kinase inhibitors.

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Drug Indications
Treatment of non-small cell lung cancerMechanism of Action:
Ensartinib is a novel second-generation ALK TKI that competitively binds to the ATP-binding pocket of ALK, inhibiting its kinase activity and downstream signaling pathways (PI3K/AKT/mTOR and RAS/RAF/MEK/ERK pathways). This leads to cell cycle arrest and apoptosis in ALK-positive tumor cells. [1]
-Structure-Activity Relationship:
Compared to first-generation TKIs, ensartinib contains a unique pyrrolopyrimidine core structure, resulting in a higher affinity for ALK binding. The fluorophenyl substituent contributes to improved kinase selectivity and central nervous system penetration. [1] - Clinical development status: - Approved indications: - China: For ALK-positive advanced non-small cell lung cancer that has failed or is intolerant to crizotinib (2020) [1] - United States: For first-line treatment of ALK-positive non-small cell lung cancer (2024) [1] - Key clinical findings: - First-line treatment: Compared with crizotinib, the objective response rate (ORR) in the phase III clinical trial was 87%, and the median progression-free survival (PFS) was 25.8 months [1] - Central nervous system efficacy: The intracranial ORR in patients with brain metastases was 80% [1] - ELEVATE study: Adjuvant therapy reduced the risk of recurrence by 80% in patients with resected ALK-positive non-small cell lung cancer [1] - Advantages over first-generation TKIs: - Highly effective against most ALK resistance mutations (including G1202R) Excellent central nervous system penetration and efficacy against brain metastases - once daily oral administration - good safety [1]

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Non-small cell lung cancer (NSCLC) is one of the most common subtypes of lung cancer, encompassing a variety of different lung cancer types, among which adenocarcinoma, squamous cell carcinoma and large cell carcinoma are more common. Ensartinib is a first-line treatment for anaplastic lymphoma kinase (ALK) mutant non-small cell lung cancer (NSCLC). From a chemical structure perspective, ensartinib is a small molecule tyrosine kinase inhibitor (TKI) based on aminopyridazine that can inhibit anaplastic lymphoma kinase (ALK) protein. Compared with crizotinib, this drug is 10 times more effective in inhibiting the growth of ALK-positive lung cancer cell lines. On December 18, 2024, ensartinib was approved by the U.S. Food and Drug Administration (FDA) for the treatment of adult patients with ALK-positive locally advanced or metastatic non-small cell lung cancer (NSCLC) who have not previously received ALK inhibitor therapy. Although ensartinib did not show a statistically significant difference in overall survival compared to crizotinib, it demonstrated a statistically significant improvement in progression-free survival. Ensartinib is a small ALK inhibitor used to treat adult patients with locally advanced or metastatic non-small cell lung cancer. Transient elevations in serum transaminase and bilirubin levels may occur during ensartinib treatment, and drug-induced liver injury may occur in rare cases.

C26H27CL2FN6O3

561.44

560.15

C, 55.62; H, 4.85; Cl, 12.63; F, 3.38; N, 14.97; O, 8.55

1370651-20-9

Ensartinib dihydrochloride;2137030-98-7

56960363

White to off-white solid powder

1.4±0.1 g/cm3

708.0±60.0 °C at 760 mmHg

382.0±32.9 °C

0.0±2.3 mmHg at 25°C

1.629

4.7

3

8

6

38

812

3

C1(C(NC2=CC=C(C(N3C[C@H](C)N[C@H](C)C3)=O)C=C2)=O)=NN=C(N)C(O[C@@H](C2=C(Cl)C=CC(F)=C2Cl)C)=C1

GLYMPHUVMRFTFV-QLFBSQMISA-N

InChI=1S/C26H27Cl2FN6O3/c1-13-11-35(12-14(2)31-13)26(37)16-4-6-17(7-5-16)32-25(36)20-10-21(24(30)34-33-20)38-15(3)22-18(27)8-9-19(29)23(22)28/h4-10,13-15,31H,11-12H2,1-3H3,(H2,30,34)(H,32,36)/t13-,14+,15-/m1/s1

6-amino-5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-N-[4-[(3S,5R)-3,5-dimethylpiperazine-1-carbonyl]phenyl]pyridazine-3-carboxamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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