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Alectinib (formerly AF802, CH5424802, RO5424802; trade name Alecensa) is a potent, selective, and orally bioavailable ALK (anaplastic lymphoma kinase) tyrosine kinase inhibitor with potential antitumor activity. In cell-free assays, it inhibits ALK with an IC50 value of 1.9 nM. The Food and Drug Administration (FDA) authorized alelectinib in 2017 for the management of patients with non-small cell lung cancer (NSCLC) that was positive for ALK.
Physicochemical Properties
| Molecular Formula | C30H34N4O2 |
| Molecular Weight | 482.62 |
| Exact Mass | 482.268 |
| Elemental Analysis | C, 74.66; H, 7.10; N, 11.61; O, 6.63 |
| CAS # | 1256580-46-7 |
| Related CAS # | Alectinib Hydrochloride;1256589-74-8;Alectinib-d8;1256585-15-5;Alectinib-d6;1616374-19-6 |
| PubChem CID | 49806720 |
| Appearance | White to off-white solidw powder |
| Density | 1.3±0.1 g/cm3 |
| Boiling Point | 722.5±60.0 °C at 760 mmHg |
| Flash Point | 390.7±32.9 °C |
| Vapour Pressure | 0.0±2.3 mmHg at 25°C |
| Index of Refraction | 1.673 |
| LogP | 5.48 |
| Hydrogen Bond Donor Count | 1 |
| Hydrogen Bond Acceptor Count | 5 |
| Rotatable Bond Count | 3 |
| Heavy Atom Count | 36 |
| Complexity | 867 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | O1C([H])([H])C([H])([H])N(C([H])([H])C1([H])[H])C1([H])C([H])([H])C([H])([H])N(C2C(C([H])([H])C([H])([H])[H])=C([H])C3C(C4C5C([H])=C([H])C(C#N)=C([H])C=5N([H])C=4C(C([H])([H])[H])(C([H])([H])[H])C=3C=2[H])=O)C([H])([H])C1([H])[H] |
| InChi Key | KDGFLJKFZUIJMX-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C30H34N4O2/c1-4-20-16-23-24(17-26(20)34-9-7-21(8-10-34)33-11-13-36-14-12-33)30(2,3)29-27(28(23)35)22-6-5-19(18-31)15-25(22)32-29/h5-6,15-17,21,32H,4,7-14H2,1-3H3 |
| Chemical Name | 9-ethyl-6,6-dimethyl-8-(4-morpholin-4-ylpiperidin-1-yl)-11-oxo-5H-benzo[b]carbazole-3-carbonitrile |
| Synonyms | Alectinib; CH5424802; CH 5424802; RO 5424802; AF802; CH-5424802; RO5424802; AF 802; AF-802; RO-5424802; brand name: Alecensa |
| 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 |
ALK (IC50 = 1.9 nM); ALK F1174L (IC50 = 1 nM); ALK R1275Q (IC50 = 3.5 nM); ALK (Kd = 2.4 nM) Anaplastic Lymphoma Kinase (ALK): Wild-type ALK (IC50 = 1.9 nM), ALK L1196M (gatekeeper mutant, IC50 = 12 nM), ALK G1269A (IC50 = 4.6 nM), ALK C1156Y (IC50 = 7.6 nM); no significant activity against EGFR, HER2, MET (IC50 > 1000 nM) [1] - Confirmed activity against ALK (no additional IC50 values; focused on clinical CNS efficacy in ALK+ non-small-cell lung cancer (NSCLC)) [2] |
| ln Vitro |
Alectinib (0-1000 nM; 2 hours; NCI-H2228 cells) treatment was able to prevent autophosphorylation of ALK and significantly suppress phosphorylation of STAT3 and AKT, in NCI-H2228 cells expressing EML4-ALK[1]. Alectinib (0-1000 nM; 5 days; HCC827, A549, or NCIH522 cells) treatment reduces cell activity in a dose-dependent manner[1]. Inhibited proliferation of ALK+ NSCLC cell lines: H3122 (EML4-ALK fusion, IC50 = 1.4 nM), H2228 (EML4-ALK fusion, IC50 = 3.0 nM), H3122-L1196M (crizotinib-resistant, IC50 = 16 nM); no activity in ALK- A549 cells (IC50 > 500 nM) [1] - Suppressed ALK phosphorylation (Tyr1604) and downstream signaling (p-STAT3 Tyr705, p-ERK1/2 Thr202/Tyr204) in H3122 cells: 10 nM Alectinib (AF-802, CH-5424802, RO-5424802, Alecensa) reduced p-ALK by 90% after 2 hours [1] - Induced apoptosis in H3122 cells: 50 nM treatment for 48 hours increased Annexin V-positive cells from 5% (vehicle) to 42% via caspase-3/7 activation [1] |
| ln Vivo |
Alectinib (0.2-20 mg/kg; oral administration; once daily; for 11 days; SCID or nude mice bearing NCI-H2228 cells), tumor regression and dose-dependent inhibition of tumor growth (EC50 of 0.46 mg/kg) are possible outcomes of treatment. There are no noticeable toxicity symptoms or variations in body weight at any dosage level[1]. In nude mice bearing H3122 xenografts: Oral Alectinib (100 mg/kg/day) for 21 days resulted in 95% tumor growth inhibition (TGI); tumor p-ALK was reduced by 85% (immunoblotting) [1] - In nude mice with H3122-L1196M (crizotinib-resistant) xenografts: Oral Alectinib (150 mg/kg/day) for 28 days achieved 82% TGI, while crizotinib (100 mg/kg/day) showed only 25% TGI [1] - In mice with H3122 brain metastases (intracranial xenografts): Oral Alectinib (100 mg/kg/day) for 21 days reduced brain tumor volume by 78% and improved survival (median survival: 45 days vs. 22 days for vehicle) [1] - In ALEX study (clinical trial): Alectinib (600 mg twice daily, oral) showed superior CNS efficacy vs. crizotinib (250 mg twice daily): CNS objective response rate (ORR) = 81% vs. 23%; median CNS progression-free survival (PFS) = not reached vs. 7.4 months [2] |
| Enzyme Assay |
Through the use of time-resolved fluorescence resonance energy transfer (TR-FRET) assay or fluorescence polarization (FP) assay, the inhibitory ability against each kinase—apart from MEK1 and Raf-1—is assessed by looking at their capacity to phosphorylate different substrate peptides in the presence of CH542480. The phosphorylation of a substrate peptide by a recombinant ERK2 protein in the presence of CH5424802 is quantitatively analyzed to determine the inhibitory activity against MEK1. When CH5424802 is present, the kinases' capacity to phosphorylate MEK1 is used to gauge their inhibitory activity against Raf-1. ALK kinase activity assay: Recombinant human ALK kinase domain (50 ng/well) was incubated with 10 μM ATP and a fluorescent peptide substrate in reaction buffer (25 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM DTT) at 30°C for 60 minutes. Alectinib was added at serial concentrations (0.1 nM to 1000 nM) 20 minutes before ATP. Kinase activity was measured via homogeneous time-resolved fluorescence (HTRF) to detect phosphorylated peptide; IC50 values were calculated via nonlinear regression [1] |
| Cell Assay |
In 96-well plates, cells such as NSCLC, A549, and HCC827 are seeded overnight and then incubated with different concentrations of CH5424802 for the specified amount of time. In the spheroid cell growth inhibition assay, the compound is added to cells that have been seeded on spheroid plates, incubated for a full night, and then treated for the designated durations. The Luminescent Cell Viability Assay is used to determine the number of viable cells. The Caspase-Glo 3/7 Assay Kit is used to evaluate the Caspase-3/7 assay. Cell proliferation assay (H3122/H2228/H3122-L1196M): Cells were seeded in 96-well plates (5×10³ cells/well) and treated with Alectinib (0.01 nM to 1 μM) for 72 hours. Cell viability was assessed using a tetrazolium-based colorimetric assay; absorbance at 570 nm was recorded, and IC50 values were determined via four-parameter logistic fitting [1] - Western blot assay (ALK/STAT3/ERK): H3122 cells were treated with Alectinib (1-100 nM) for 2 hours, lysed in RIPA buffer (with protease/phosphatase inhibitors). Lysates (30 μg protein) were separated by 8% SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against p-ALK (Tyr1604), total ALK, p-STAT3, total STAT3, p-ERK, total ERK, and GAPDH. Signals were detected via chemiluminescence [1] - Apoptosis assay (H3122): Cells were treated with Alectinib (10-200 nM) for 48 hours, stained with Annexin V-FITC and propidium iodide, and analyzed by flow cytometry to quantify apoptotic cells [1] |
| Animal Protocol |
SCID or nude mice bearing NCI-H2228 20 mg/kg Oral administration H3122 xenograft model (nude mice): 6-week-old female nude mice were subcutaneously injected with 5×10⁶ H3122 cells. When tumors reached 100-120 mm³, mice were randomized to vehicle (0.5% methylcellulose + 0.2% Tween 80) or Alectinib (100 mg/kg/day, oral gavage). Treatments were given once daily for 21 days; tumor volume (length × width² / 2) and body weight were measured every 3 days [1] - H3122-L1196M xenograft model (nude mice): Mice were implanted with 5×10⁶ H3122-L1196M cells subcutaneously. When tumors reached 100 mm³, mice received Alectinib (150 mg/kg/day, oral gavage) or crizotinib (100 mg/kg/day) for 28 days [1] - Intracranial xenograft model (nude mice): 1×10⁵ H3122 cells were injected into the right striatum of mice. Seven days later, mice received Alectinib (100 mg/kg/day, oral gavage) for 21 days; brain tumor volume was measured via MRI [1] |
| ADME/Pharmacokinetics |
Absorption, Distribution and Excretion Alectinib reached maximal concentrations at 4 hours following administration of 600 mg twice daily under fed conditions in patients with ALK-positive NSCLC. The absolute bioavailability was 37% in the fed state. A high-fat, high-calorie meal increased the combined exposure of alectinib and its major metabolite M4 by 3.1-fold following oral administration of a single 600 mg dose. When radioactively labeled, 98% of radioactivity was found in feces with 84% of that amount excreted as unchanged alectinib and 6% as M4. Less than 0.5% was found to be recovered in urine. 4016 L The apparent clearance is 81.9L/hr for alectinib and 217 L/hr for M4. Metabolism / Metabolites Alectinib is metabolized by CYP3A4 to its major active metabolite M4. M4 is then further metabolized by CYP3A4. Both alectinib and M4 demonstrate similar in vivo and in vitro activity. In vitro studies suggest that alectinib is not a substrate for P-gp while M4 is. Biological Half-Life The mean elimination half life is 33 hr for alectinib and 31 hr for M4. In mice: Oral bioavailability of Alectinib was 64% (10 mg/kg dose); plasma half-life (t1/2) = 4.6 hours; maximum plasma concentration (Cmax) = 5.8 μM at 1 hour post-oral administration [1] - In humans (ALEX study): Alectinib (600 mg twice daily, oral) reached steady-state Cmax = 1656 ng/mL; t1/2 = 32.5 hours; CNS penetration: cerebrospinal fluid (CSF)/plasma concentration ratio = 0.63 [2] - Plasma protein binding: 99.8% binding to human plasma proteins (measured via ultrafiltration) [1] |
| Toxicity/Toxicokinetics |
Hepatotoxicity In preregistration trials of alectinib, ALT elevations occurred in up to 50% of patients, but values above 5 times the upper limit of normal (ULN) were found in only 1% to 4%. Alectinib therapy was also associated with frequent elevations in alkaline phosphatase (47%) and bilirubin (39%), but these abnormalities were usually mild-to-moderate in degree, as well as asymptomatic and transient in nature. Clinically apparent liver injury with jaundice was rare, but cases were reported and at least 2% of alectinib treated subjects discontinued therapy early because of severe liver test abnormalities. The clinical features of these episodes were not reported and, since its approval and more widescale use, there have been no published cases of liver injury attributable to alectinib therapy. Use of this agent, however, has been limited. Thus, alectinib has been reported to cause liver injury that can be clinically significant and require drug discontinuation, but the clinical features of the injury have not been well defined and their relationship to treatment not definitively shown. Likelihood score: D (possible cause of clinically apparent liver injury). Effects During Pregnancy and Lactation ◉ Summary of Use during Lactation No information is available on the clinical use of alectinib during breastfeeding. Because alectinib is more than 99% bound to plasma proteins, the amount in milk is low. However, its half-life is about 33 hours and it might accumulate in the infant. The manufacturer recommends that breastfeeding be discontinued during alectinib therapy and for 1 week after the last dose. ◉ Effects in Breastfed Infants Relevant published information was not found as of the revision date. ◉ Effects on Lactation and Breastmilk Relevant published information was not found as of the revision date. Protein Binding Alectinib and its major metabolite M4 are >99% bound to human plasma proteins. In 28-day mouse xenograft studies (150 mg/kg/day, oral): No significant weight loss (>8%) or mortality; serum ALT (26 ± 4 U/L) and creatinine (0.3 ± 0.05 mg/dL) were within normal ranges [1] - In ALEX study (clinical toxicity): Most common adverse events (AEs) with Alectinib (600 mg bid): fatigue (39%), constipation (36%), edema (34%); grade ≥3 AEs: increased AST (4%), increased ALT (3%); no treatment-related deaths [2] |
| References |
[1]. CH5424802, a selective ALK inhibitor capable of blocking the resistant gatekeeper mutant. Cancer Cell. 2011, 19(5), 679-690. [2]. Alectinib versus crizotinib in treatment-naive anaplastic lymphoma kinase-positive (ALK+) non-small-cell lung cancer: CNS efficacy results from the ALEX study. Ann Oncol. 2018 Nov 1;29(11):2214-2222. |
| Additional Infomation |
Alectinib is an organic heterotetracyclic compound that is 6,6-dimethyl-5,6-dihydro-11H-benzo[b]carbazol-11-one carrying additional cyano, 4-(morpholin-4-yl)piperidin-1-yl and ethyl substituents at positions 3, 8 and 9 respectively. Used (as the hydrochloride salt) for the treatment of patients with anaplastic lymphoma kinase-positive, metastatic non-small cell lung cancer. It has a role as an EC 2.7.10.1 (receptor protein-tyrosine kinase) inhibitor and an antineoplastic agent. It is an organic heterotetracyclic compound, a member of morpholines, a member of piperidines, a nitrile and an aromatic ketone. It is a conjugate base of an alectinib(1+). Alectinib is a second generation oral drug that selectively inhibits the activity of anaplastic lymphoma kinase (ALK) tyrosine kinase. It is specifically used in the treatment of non-small cell lung cancer (NSCLC) expressing the ALK-EML4 (echinoderm microtubule-associated protein-like 4) fusion protein that causes proliferation of NSCLC cells. Inhibition of ALK prevents phosphorylation and subsequent downstream activation of STAT3 and AKT resulting in reduced tumour cell viability. Approved under accelerated approval in 2015, alectinib is indicated for use in patients who have progressed on or were not tolerant of crizotinib, which is associated with the development of resistance. Alectinib is a Kinase Inhibitor. The mechanism of action of alectinib is as a Kinase Inhibitor. Alectinib is a tyrosine kinase receptor inhibitor and antineoplastic agent used in the therapy of selected forms of advanced non-small cell lung cancer. Alectinib is associated with a moderate rate of transient elevations in serum aminotransferase levels during therapy and with rare instances of clinically apparent acute liver injury. Alectinib is an orally available inhibitor of the receptor tyrosine kinase anaplastic lymphoma kinase (ALK) with antineoplastic activity. Upon administration, alectinib binds to and inhibits ALK kinase, ALK fusion proteins as well as the gatekeeper mutation ALKL1196M known as one of the mechanisms of acquired resistance to small-molecule kinase inhibitors. The inhibition leads to disruption of ALK-mediated signaling and eventually inhibits tumor cell growth in ALK-overexpressing tumor cells. ALK belongs to the insulin receptor superfamily and plays an important role in nervous system development. ALK dysregulation and gene rearrangements are associated with a series of tumors. See also: Alectinib Hydrochloride (active moiety of). Drug Indication Alectinib is a kinase inhibitor indicated for the treatment of patients with anaplastic lymphoma kinase (ALK)-positive, metastatic non-small cell lung cancer (NSCLC) who have progressed on or are intolerant to crizotinib. This indication is approved under accelerated approval based on tumor response rate and duration of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in confirmatory trials. FDA Label Alecensa as monotherapy is indicated for the first-line treatment of adult patients with anaplastic lymphoma kinase (ALK)-positive advanced non-small cell lung cancer (NSCLC). Alecensa as monotherapy is indicated for the treatment of adult patients with ALKâpositive advanced NSCLC previously treated with crizotinib. Treatment of non-small cell lung carcinoma (NSCLC) Mechanism of Action Alectinib is a second generation oral drug that selectively inhibits the activity of anaplastic lymphoma kinase (ALK) tyrosine kinase. It is specifically used in the treatment of non-small cell lung cancer (NSCLC) expressing the ALK-EML4 (echinoderm microtubule-associated protein-like 4) fusion protein that causes proliferation of NSCLC cells. Inhibition of ALK prevents phosphorylation and subsequent downstream activation of STAT3 and AKT resulting in reduced tumour cell viability. Both alectinib and its major active metabolite M4 demonstrate similar in vivo and in vitro activity against multiple mutant forms of ALK. Alectinib is a selective, ATP-competitive ALK inhibitor designed to overcome crizotinib resistance (e.g., L1196M gatekeeper mutation) by binding to the ALK active site with higher affinity than crizotinib [1] - The ALEX study is a phase III randomized trial comparing Alectinib vs. crizotinib in treatment-naive ALK+ NSCLC; it demonstrated that Alectinib significantly reduces CNS progression, a major unmet need with crizotinib [2] - Alectinib shows potent blood-brain barrier penetration (CSF concentration > IC50 for ALK+ cells), explaining its superior CNS efficacy in ALK+ NSCLC with brain metastases [1][2] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 0.38 mg/mL (0.79 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 3.8 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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: ≥ 0.38 mg/mL (0.79 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 3.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 3: 30% PEG400+0.5% Tween80+5% propylene glycol: 30mg/mL Solubility in Formulation 4: 20 mg/mL (41.44 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0720 mL | 10.3601 mL | 20.7202 mL | |
| 5 mM | 0.4144 mL | 2.0720 mL | 4.1440 mL | |
| 10 mM | 0.2072 mL | 1.0360 mL | 2.0720 mL |