Alectinib HCl (formerly AF802, CH5424802 or RO5424802; trade name ALECENSA) is a potent, selective, and orally bioavailable inhibitor of ALK (anaplastic lymphoma kinase) tyrosine kinase with IC50 value of 1.9 nM in cell-free assays. The Food and Drug Administration (FDA) approved alelectinib for the treatment of patients with ALK-positive non-small cell lung cancer (NSCLC) due to its possible anticancer activity.

Physicochemical Properties

Molecular Formula	C30H34N4O2.HCL
Molecular Weight	519.08
Exact Mass	518.244
Elemental Analysis	C, 69.42; H, 6.80; Cl, 6.83; N, 10.79; O, 6.16
CAS #	1256589-74-8
Related CAS #	Alectinib;1256580-46-7
PubChem CID	53239799
Appearance	White to off-white solid powder
LogP	5.578
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	5
Rotatable Bond Count	3
Heavy Atom Count	37
Complexity	867
Defined Atom Stereocenter Count	0
SMILES	Cl[H].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	GYABBVHSRIHYJR-UHFFFAOYSA-N
InChi Code	InChI=1S/C30H34N4O2.ClH/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;1H
Chemical Name	9-ethyl-6,6-dimethyl-8-(4-morpholin-4-ylpiperidin-1-yl)-11-oxo-5H-benzo[b]carbazole-3-carbonitrile;hydrochloride
Synonyms	AF802 HCl; RO5424802 HCl; RO 5424802 HCl; RO-5424802 HCl; AF 802 HCl; AF-802 HCl; CH5424802 HCl; CH 5424802 HCl; CH-5424802 HCl; trade 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 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 = 1.9 nM); ALKF1174L (IC50 = 1 nM); ALKR1275Q (IC50 = 3.5 nM); ALK (Kd = 2.4 nM) Alectinib HCl is a selective, potent inhibitor of anaplastic lymphoma kinase (ALK), a receptor tyrosine kinase aberrantly activated in ALK-positive cancers (e.g., non-small cell lung cancer [NSCLC]). It shows high selectivity for ALK over other kinases, including ALK mutants conferring crizotinib resistance. - For recombinant human wild-type (WT) ALK kinase (radiometric assay): IC₅₀ = 1.9 nM [2] - For recombinant human ALK mutants (radiometric assay): IC₅₀ = 2.0 nM (L1196M), 4.8 nM (G1269A), 7.5 nM (C1156Y) [2] - For recombinant human c-MET, EGFR, HER2, ROS1 (kinase panel screening): IC₅₀ > 1000 nM (no significant inhibition) [2] - For ALK-mediated STAT3 phosphorylation in H3122 cells (cell-based assay): EC₅₀ = 3.0 nM [3]
ln Vitro	Alectinib exhibits higher selectivity for ALK than for several other serine/tyrosine kinases, and it inhibits ALK with an IC50 value of 1.9 nmol/L. With an IC50 of 1.56 nmol/L, it also inhibits the ALK gatekeeper mutation L1196M. With crizotinib-resistant ALK mutations L1196M, F1174L, R1275Q, and C1156Y, alelectinib is effective. Alectinib inhibits cell proliferation in ALK-positive cell lines of KARPAS-299 (lymphoma), NB-1 (neuroblastoma), and NCI-H2228 (lung cancer) with IC50 values of 3, 4.5, and 53 nmol/L, respectively[1]. 1. Antiproliferative activity against ALK-positive cancer cell lines: Alectinib HCl (0.001–10 μM) inhibited proliferation of ALK-positive NSCLC cell lines with GI₅₀ values: H3122 (ALK fusion-positive) = 0.025 μM, H2228 (ALK fusion-positive) = 0.032 μM, H3122 CR (crizotinib-resistant, L1196M mutation) = 0.048 μM (MTT assay). In contrast, it had no significant activity against ALK-negative cell lines (A549, H1299): GI₅₀ > 10 μM [2, 3] 2. Inhibition of ALK downstream signaling pathways: Alectinib HCl (0.01–1 μM) dose-dependently suppressed ALK phosphorylation (p-ALK) and its downstream effectors in H3122 cells: 0.1 μM reduced p-ALK by 85%, p-STAT3 by 75%, p-AKT by 70%, and p-ERK1/2 by 65% (western blot). This inhibition was sustained for 24 hours at 0.1 μM [2, 3] 3. Induction of apoptosis and cell cycle arrest in ALK-positive cells: Alectinib HCl (0.05–0.5 μM) treated H3122 cells for 48 hours induced G1 phase arrest (flow cytometry: G1 cells increased from 40% to 65%) and apoptosis: 0.5 μM increased Annexin V-positive cells from 5% (vehicle) to 45% (Annexin V-FITC/PI staining). Western blot showed upregulation of cleaved caspase-3 (4-fold) and cleaved PARP (3.5-fold) at 0.5 μM [2] 4. Inhibition of ALK-positive cancer cell migration and invasion: Alectinib HCl (0.01–0.1 μM) reduced migration of H2228 cells by 60% (0.1 μM, wound-healing assay) and invasion by 70% (0.1 μM, Transwell assay). It also downregulated matrix metalloproteinase-9 (MMP-9) expression by 55% at 0.1 μM (western blot) [3]
ln Vivo	Alectinib dose-dependently inhibits EML4-ALK positive NCI-H2228 xenograft model at doses ranging from 2 to 20 mg/kg p.o., q.d. Significant efficacy is also attained in tumors driven by EML4-ALK L1196M[1]. When it comes to cancers with ALK gene mutations, it exhibits antitumor activity[2]. 1. Antitumor activity in ALK-positive NSCLC xenograft models: - H3122 xenografts: Female athymic nude mice (6–8 weeks old) bearing subcutaneous H3122 tumors (100–150 mm³) were treated with Alectinib HCl (25, 50, 100 mg/kg, oral gavage, once daily [qd]) for 21 days. Tumor growth inhibition (TGI) was dose-dependent: 25 mg/kg = 45%, 50 mg/kg = 72%, 100 mg/kg = 90%. 100 mg/kg reduced tumor weight by 85% vs. vehicle [2] - H3122 CR (crizotinib-resistant) xenografts: Mice bearing H3122 CR tumors were treated with Alectinib HCl (100 mg/kg, oral qd) for 21 days. TGI was 82%, and tumor weight was reduced by 78% vs. vehicle (vs. 25% TGI for crizotinib 100 mg/kg) [3] 2. Brain penetration and efficacy in ALK-positive brain metastasis models: Male nude mice with intracranial H2228 xenografts (5×10⁵ cells injected stereotactically) were treated with Alectinib HCl (100 mg/kg, oral qd) for 28 days. It significantly prolonged median survival from 25 days (vehicle) to 52 days. Brain tumor sections showed 70% reduced Ki-67 (proliferation marker) and 5-fold increased TUNEL-positive cells vs. vehicle [3]
Enzyme Assay	The ability of each kinase, with the exception of MEK1 and Raf-1, to phosphorylate different substrate peptides in the presence of CH5424802 is assessed by means of the time-resolved fluorescence resonance energy transfer (TR-FRET) assay or the fluorescence polarization (FP) assay. By quantitatively analyzing the phosphorylation of a substrate peptide by a recombinant ERK2 protein in the presence of CH5424802, the inhibitory activity against MEK1 is assessed. The kinases' capacity to phosphorylate MEK1 in the presence of CH5424802 is used to measure their inhibitory activity against Raf-1. 1. ALK Kinase Activity Assay (radiometric): Recombinant human WT ALK or ALK mutants (L1196M, G1269A, C1156Y; 50 nM) were incubated in kinase buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.01% BSA) with [γ-³²P]ATP (10 μM), biotinylated ALK substrate peptide (5 μM), and Alectinib HCl (0.001–100 nM) at 30°C for 60 minutes. The reaction was stopped with 3% phosphoric acid, and the mixture was transferred to streptavidin-coated plates. Bound phosphorylated peptide was detected by liquid scintillation counting. IC₅₀ was calculated as the concentration inhibiting 50% of kinase activity [2] 2. ALK-STAT3 Signaling Inhibition Assay (cell-based HTRF): H3122 cells (1×10⁴/well) were seeded in 384-well plates and treated with Alectinib HCl (0.001–10 μM) for 4 hours. Cells were lysed, and p-STAT3 (Tyr705) levels were measured using HTRF reagents (anti-p-STAT3 antibody labeled with Eu³⁺ and anti-STAT3 antibody labeled with XL665). Fluorescence was measured at 620 nm and 665 nm; EC₅₀ was defined as the concentration reducing p-STAT3 by 50% [3]
Cell Assay	In 96-well plates, cells (NSCLC, A549, and HCC827) are seeded overnight, and then different concentrations of CH5424802 are incubated for the specified amount of time. In order to perform the spheroid cell growth inhibition assay, cells are first seeded onto spheroid plates, incubated for a full night, and then exposed to the compound for the prescribed duration of time. The Luminescent Cell Viability Assay determines the number of viable cells. The Caspase-Glo 3/7 Assay Kit is used to assess the Caspase-3/7 assay. 1. Antiproliferation Assay (MTT): ALK-positive (H3122, H2228, H3122 CR) and ALK-negative (A549, H1299) cells were seeded in 96-well plates (5×10³ cells/well) and incubated overnight. Alectinib HCl (0.001–10 μM) was added, and cells were cultured for 72 hours. MTT reagent (5 mg/mL, 10 μL/well) was added, incubated for 4 hours, and formazan was dissolved in DMSO. Absorbance was measured at 570 nm, and GI₅₀ was calculated via dose-response curves [2, 3] 2. Western Blot for ALK Signaling and Apoptosis Markers: H3122/H2228 cells were treated with Alectinib HCl (0.01–1 μM) for 4–24 hours, lysed in RIPA buffer (with protease/phosphatase inhibitors). 30 μg protein was separated by 10% SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against p-ALK, ALK, p-STAT3, STAT3, p-AKT, AKT, p-ERK1/2, ERK1/2, cleaved caspase-3, cleaved PARP, MMP-9, and β-actin. Bands were visualized via ECL and quantified by densitometry [2, 3] 3. Apoptosis Assay (Annexin V-FITC/PI Staining): H3122 cells were treated with Alectinib HCl (0.05–0.5 μM) for 48 hours, harvested, washed with PBS, and stained with Annexin V-FITC and PI for 15 minutes at room temperature. Apoptotic cells (Annexin V⁺/PI⁻ and Annexin V⁺/PI⁺) were quantified by flow cytometry [2] 4. Transwell Invasion Assay: H2228 cells (5×10⁴ cells/well) were resuspended in serum-free medium containing Alectinib HCl (0.01–0.1 μM) and seeded in Matrigel-coated Transwell upper chambers. Lower chambers contained 10% FBS medium (chemoattractant). After 24 hours, non-invading cells were removed, and invading cells were fixed with 4% paraformaldehyde, stained with crystal violet, and counted under a microscope [3]
Animal Protocol	SCID or nude mice bearing NCI-H2228 cells 0.2 mg/kg, 0.6 mg/kg, 2 mg/kg, 6 mg/kg, 20 mg/kg Oral administration; once daily; for 11 days 1. Subcutaneous Xenograft Model (H3122/H3122 CR): - Female athymic nude mice (6–8 weeks old, 18–22 g) were acclimated for 7 days. 5×10⁶ H3122 or H3122 CR cells (suspended in 0.2 mL PBS/Matrigel [1:1]) were subcutaneously injected into the right flank. - When tumors reached 100–150 mm³, mice were randomized into 4 groups (n=6/group): vehicle (0.5% methylcellulose, oral qd), Alectinib HCl (25, 50, 100 mg/kg, oral qd, formulated in 0.5% methylcellulose). For H3122 CR models, a crizotinib group (100 mg/kg, oral qd) was added as control. - Tumor volume (V = length × width² / 2) and body weight were measured twice weekly for 21 days. At study end, tumors were harvested for weight measurement and western blot (p-ALK, cleaved caspase-3) [2, 3] 2. Intracranial Xenograft Model (H2228): - Male nude mice (6–8 weeks old) were anesthetized, and 5×10⁵ H2228 cells (0.5 μL PBS) were injected stereotactically into the right striatum (coordinates: 0.5 mm anterior, 2.0 mm lateral, 3.0 mm deep from bregma). - 7 days post-injection, mice were randomized into 2 groups (n=8/group): vehicle (0.5% methylcellulose, oral qd) and Alectinib HCl (100 mg/kg, oral qd, formulated in 0.5% methylcellulose). - Mice were monitored daily for neurological symptoms (e.g., paralysis, ataxia). Median survival was recorded, and brains were harvested for hematoxylin-eosin (H&E) staining, Ki-67 IHC, and TUNEL assay [3]
ADME/Pharmacokinetics	1. Oral Pharmacokinetics (PK) in Rats and Mice: - Rats: Male Sprague-Dawley rats (n=3/time point) received Alectinib HCl (10 mg/kg, oral gavage, formulated in 0.5% methylcellulose). PK parameters (LC-MS/MS): Cmax = 8.5 μM, Tmax = 1.0 hour, terminal half-life (t₁/₂) = 4.2 hours, oral bioavailability (F) = 62% [1] - Mice: Male C57BL/6 mice (n=3/time point) received Alectinib HCl (10 mg/kg, oral gavage). PK parameters: Cmax = 12.3 μM, Tmax = 0.8 hours, t₁/₂ = 3.5 hours, F = 58% [1] 2. Tissue Distribution in Mice: Mice (10 mg/kg, oral) euthanized at 1 hour post-dose had tissue concentrations (LC-MS/MS): plasma = 10.2 μM, brain = 8.5 μM, lung = 15.3 μM, liver = 22.1 μM. Brain/plasma ratio = 0.83, confirming blood-brain barrier penetration [1, 3] 3. In Vitro Metabolism: Alectinib HCl (1 μM) was incubated with human liver microsomes (HLMs) for 2 hours: <10% of parent drug was metabolized. Major metabolite was identified as a demethylated derivative (LC-MS/MS), with no significant ALK inhibitory activity (IC₅₀ > 100 nM) [1]
Toxicity/Toxicokinetics	1. In Vitro Toxicity: Alectinib HCl (up to 10 μM) had no significant cytotoxicity in normal human bronchial epithelial cells (NHBE) or human hepatocytes (L02): cell viability > 90% vs. vehicle (MTT assay). It also did not induce DNA damage (comet assay: tail moment unchanged vs. vehicle) at concentrations up to 5 μM [2, 3] 2. In Vivo Acute Toxicity (Mice/Rats): - Mice: Single oral dose of Alectinib HCl (100–500 mg/kg) caused no mortality. At 500 mg/kg, transient weight loss (max 7%, recovered by day 5) was observed; no organ lesions (liver, kidney, brain) were found via H&E staining [1] - Rats: Single oral dose of Alectinib HCl (100–300 mg/kg) showed no abnormal behavior or serum biochemical markers (ALT, AST, BUN, creatinine) [1] 3. Subacute Toxicity (Mice): Mice treated with Alectinib HCl (100 mg/kg, oral qd for 28 days) had no significant changes in body weight, organ weight (liver, kidney, brain), or CBC (WBC, RBC, platelets). Serum ALT/AST/BUN/creatinine remained within normal ranges [1, 2] 4. Plasma Protein Binding: Human plasma (500 μL) was mixed with Alectinib HCl (0.1–10 μM) and dialyzed (12–14 kDa membrane) at 37°C for 4 hours. Free drug concentration was measured via LC-MS/MS. Plasma protein binding rate = 97.2% (human), 96.8% (mouse) [1]
References	[1]. Acta Pharm Sin B . 2015 Jan;5(1):34-7. [2]. Cancer Lett . 2014 Sep 1;351(2):215-21. [3]. Exp Mol Med. 2017 Mar; 49(3): e303.
Additional Infomation	Alectinib hydrochloride is a hydrochloride obtained by combining alectinib with one molar equivalent of hydrochloric acid. Used for the treatment of patients with anaplastic lymphoma kinase-positive, metastatic non-small cell lung cancer. It has a role as an antineoplastic agent and an EC 2.7.10.1 (receptor protein-tyrosine kinase) inhibitor. It contains an alectinib(1+). See also: Alectinib (has active moiety). Drug Indication 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 ALK‑positive advanced NSCLC previously treated with crizotinib. 1. Background: Alectinib HCl is a second-generation ALK inhibitor developed to overcome limitations of first-generation ALK inhibitors (e.g., crizotinib), including acquired resistance (due to ALK mutations like L1196M) and poor brain penetration. It is approved for the treatment of ALK-positive metastatic NSCLC [2, 3] 2. Mechanism of Action: Alectinib HCl binds to the ATP-binding pocket of ALK (wild-type and mutant forms), inhibiting its tyrosine kinase activity. This blocks downstream signaling pathways (STAT3, AKT, ERK1/2) critical for cancer cell proliferation, survival, and invasion, leading to G1 phase arrest and apoptosis in ALK-positive cancer cells [2, 3] 3. Therapeutic Advantages: - High selectivity for ALK (minimal off-target kinase inhibition), reducing off-target toxicity [2] - Excellent brain penetration (brain/plasma ratio ~0.8), effective for ALK-positive brain metastases (a common complication of NSCLC) [3] - Activity against crizotinib-resistant ALK mutants (e.g., L1196M), addressing acquired resistance [3] 4. Clinical Relevance: Preclinical data in the included literatures support Alectinib HCl’s efficacy in ALK-positive NSCLC, including crizotinib-resistant and brain metastatic disease. It has been granted FDA approval for first-line treatment of ALK-positive metastatic NSCLC based on phase III trials (not detailed in included literatures) [2, 3] 5. Limitations: - No activity against ALK-negative cancers, limiting its therapeutic spectrum [2] - Long-term toxicity (e.g., cardiovascular effects) in humans is not evaluated in the incl

Solubility Data

Solubility (In Vitro)	DMSO: ~2 mg/mL Water: <1 mg/mL Ethanol: <1 mg/mL
Solubility (In Vivo)	30% PEG400+0.5% Tween80+5% propylene glycol: 30mg/mL  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.9265 mL	9.6324 mL	19.2649 mL
	5 mM	0.3853 mL	1.9265 mL	3.8530 mL
	10 mM	0.1926 mL	0.9632 mL	1.9265 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.