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Ensartinib (formerly known as X-396) is a novel, highly potent, selective, and orally available small molecule inhibitor of ALK (anaplastic lymphoma kinase) with an IC50 less than 4 nM in Ambit assays. It is an additional activity MET, ABL, Axl, EPHA2, LTK, ROS1, and SLK tyrosine kinase inhibitor (TKI). Although higher concentrations are needed to prevent autophosphorylation of the wild-type fusion, ensartinib significantly reduces ALK autophosphorylation. When ALK-expressing tumor cells are inhibited, ALK-mediated signaling is disrupted, which ultimately stops tumor cell growth. Consequently, ensartinib may have anticancer effects.
On December 18, 2024, the Food and Drug Administration approved ensartinib (Ensacove, Xcovery Holdings, Inc.) for adult patients with anaplastic lymphoma kinase (ALK)-positive locally advanced or metastatic non-small cell lung cancer (NSCLC) who have not previously received an ALK-inhibitor.Physicochemical Properties
| Molecular Formula | C26H29CL4FN6O3 |
| Molecular Weight | 634.357265233994 |
| Exact Mass | 634.1 |
| Elemental Analysis | C, 49.23; H, 4.61; Cl, 22.35; F, 2.99; N, 13.25; O, 7.57 |
| CAS # | 2137030-98-7 |
| Related CAS # | Ensartinib;1370651-20-9 |
| PubChem CID | 138320013 |
| Appearance | White to light yellow solid powder |
| Hydrogen Bond Donor Count | 5 |
| Hydrogen Bond Acceptor Count | 8 |
| Rotatable Bond Count | 6 |
| Heavy Atom Count | 40 |
| Complexity | 812 |
| Defined Atom Stereocenter Count | 3 |
| SMILES | C[C@@H]1CN(C[C@@H](N1)C)C(=O)C2=CC=C(C=C2)NC(=O)C3=NN=C(C(=C3)O[C@H](C)C4=C(C=CC(=C4Cl)F)Cl)N.Cl.Cl |
| InChi Key | IERUINQRGJAECT-ISUJJMBGSA-N |
| InChi Code | InChI=1S/C26H27Cl2FN6O3.2ClH/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);2*1H/t13-,14+,15-;;/m1../s1 |
| Chemical Name | 6-amino-5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-N-[4-[(3R,5S)-3,5-dimethylpiperazine-1-carbonyl]phenyl]pyridazine-3-carboxamide;dihydrochloride |
| Synonyms | X-396 hydrochloride; X396 hydrochloride; X 396 hydrochloride; Ensartinib dihydrochloride; Ensartinib HCl; Ensartinib hydrochloride |
| 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 | ALK (IC50 <0.4 nM); MET (IC50 = 0.74 nM) |
| ln Vitro | Ensartinib (X-396) dihydrochloride has dual ALK/MET inhibitory activity and a potent IC50 of less than 0.4 nM and 0.74 nM, respectively. In H3122 lung cancer cells expressing EML4-ALK E13;A20, ensetinib dihydrochloride exhibits potency (IC50: 15 nM). In H2228 lung cancer cells expressing EML4-ALK E6a/b; A20, esartinib dihydrochloride is likewise effective (IC50: 45 nM). Moreover, X-376 has a high potency (IC50: 9 nM) in SUDHL-1 lymphoma cells that harbor NPM-ALK. |
| ln Vivo | 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 Cmax of 7.19 μM at NST levels[1]. |
| Cell Assay | In 96-well plates, cells are seeded at 25%–33% confluency and exposed to medications for viability experiments. Ensartinib (10, 30, 100, 300, and 1000 nM) is used to treat the human lung adenocarcinoma cell lines H3122 and H2228. Ensartinib (5, 10, 30, 100, and 300 nM) is used to treat SUDHL-1 lymphoma cells. Ensartinib (30, 100, 300, and 1000 nM) is used to treat SY5Y neuroblastoma cells. After adding Ensartinib for 72 hours, Cell Titer Blue Reagent is added, and the Spectramax spectrophotometer is used to measure the fluorescence. Every experimental point is set up in duplicate three times, and it is run at least twice independently. GraphPad Prism version 5 for Windows is used to calculate IC50s. With a log (inhibitor) vs. response formula, a nonlinear regression model is used to fit the curves[1]. |
| Animal Protocol | Mice: H3122 cells are injected into naked mice (nu/nu). Following the tumors' average volume of 450 mm3, 27 athymic mice with H3122 tumors are randomly assigned to receive either the control vehicle or 25 mg/kg of ensartinib orally via gavage. Mice are killed two, five, and fifteen hours after the single treatment (3 tumors/timepoint/group), and serum is taken for an LC-MS based bioanalytical method to determine the drug concentration[1]. |
| ADME/Pharmacokinetics |
Absorption The mean Cmax of orally administered 25 mg and 100 mg ensartnib is 292 ng/mL, with the Tmax being between 2 hours and 8 hours and a median Tmax of 3 hours. Ensartinib at approved recommended dosages has an AUC of 4,920 ng\*h/ml. The steady state of the drug is reached within 15 days with a mean accumulation ratio of 2.7. Ensartinib has no clinically significant differences when administered with or without food, with the comparison being between a high-fat meal versus fasted conditions. As the drug is administered orally, the location of the absorption is predicted to be in the gastrointestinal tract. Route of Elimination When a single oral 200 mg dose of radiolabeled ensartinib was administered, 91% of the radioactivity was recovered in feces (38% as unchanged) and 10% in urine (4.4% as unchanged). Volume of Distribution Ensartinib capsules administered orally have a mean apparent volume of distribution of 1,720 L. Protein Binding Ensartinib oral capsules have been found to be 91.6% bound to human plasma protein. Metabolism / Metabolites A substrate for the enzyme CYP3A4, ensartinib is predominantly metabolized in the liver mainly through the CYP3A4 pathway. Biological Half-Life Ensartinib has a mean steady-state half-life of 30 ± 20 hours. |
| Toxicity/Toxicokinetics |
Hepatotoxicity In the prelicensure trials of ensartinib as therapy of NSCLC, liver test abnormalities were frequent, with elevations in ALT levels in 59%, AST in 58%, Alk P in 51%, and bilirubin in 12% of treated participants. The enzyme elevations were usually transient, mild-to-moderate in severity. Pruritus was seen in 26%. ALT elevations above 5 times the upper limit of normal (ULN) arose in 5% and AST in 2% of subjects. The average time to onset of increased aminotransferase levels was 5 weeks (range Likelihood score: D* (possible rare cause of clinically apparent liver injury). |
| References |
[1]. Insights into ALK-driven cancers revealed through development of novel ALK tyrosine kinaseinhibitors. Cancer Res. 2011 Jul 15;71(14):4920-31. |
| Additional Infomation |
Non-small cell lung cancer (NSCLC) is one of the most frequent subtypes of lung cancers and encompasses a variety of different lung cancers, with notable ones being adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. Ensartinib is a first-line treatment for anaplastic lymphoma kinase (ALK)-mutated non-small cell lung cancer (NSCLC). Chemically, ensartinib is an aminopyridazine-based, small molecule tyrosine kinase inhibitor (TKI) which inhibits the anaplastic lymphoma kinase (ALK) protein. The drug is 10-fold more potent in the inhibition of the growth of ALK-positive lung cancer cell lines when compared to [crizotinib]. Ensartinib was approved by the FDA for adult patients with anaplastic lymphoma kinase (ALK)-positive locally advanced or metastatic non-small cell lung cancer (NSCLC) who have not previously received an ALK-inhibitor on December 18, 2024. Although there was no statistically significant difference in overall survival when compared to [crizotinib], ensartinib demonstrated a statistically significant progression-free survival improvement when compared to [crizotinib].
Ensartinib is a small molecule inhibitor of anaplastic lymphoma kinase (ALK) and is used to treat adults with locally advanced or metastatic non-small cell lung cancer. Ensartinib is associated with transient elevations in serum aminotransferase levels and bilirubin during therapy and rare instances of drug induced liver injury. Ensartinib is an orally available small molecule inhibitor of the receptor tyrosine kinase anaplastic lymphoma kinase (ALK), with potential antineoplastic activity. Upon oral administration, ensartinib binds to and inhibits ALK kinase, ALK fusion proteins and ALK point mutation variants. Inhibition of ALK leads to the disruption of ALK-mediated signaling and eventually inhibits tumor cell growth in ALK-expressing tumor cells. In addition, ensartinib inhibits various other kinases, including the receptor tyrosine kinase c-Met (hepatocyte growth factor receptor; HGFR; MET) and the receptor tyrosine kinase C-ros oncogene 1 (ROS1). ALK belongs to the insulin receptor superfamily and plays an important role in nervous system development. ALK is not expressed in healthy adult human tissue but ALK dysregulation and gene rearrangements are associated with a series of tumors; ALK mutations are associated with acquired resistance to small molecule tyrosine kinase inhibitors. ENSARTINIB is a small molecule drug with a maximum clinical trial phase of IV (across all indications) that was first approved in 2024 and has 3 investigational indications. anaplastic lymphoma kinase (ALK) inhibitor for the treatment of ALK-positive non-small-cell lung cancer |
Solubility Data
| Solubility (In Vitro) |
DMSO : ~35.71 mg/mL (~56.29 mM) H2O : ~33.33 mg/mL (~52.54 mM) |
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (3.28 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 (3.28 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 (3.28 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. Solubility in Formulation 4: 14.29 mg/mL (22.53 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.5764 mL | 7.8820 mL | 15.7639 mL | |
| 5 mM | 0.3153 mL | 1.5764 mL | 3.1528 mL | |
| 10 mM | 0.1576 mL | 0.7882 mL | 1.5764 mL |