ALK (IC50 = 1.9 nM); ALK^F1174L (IC50 = 1 nM); ALK^R1275Q (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]

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].

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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]

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].

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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]

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.

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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]

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.

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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]

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

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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]

1. Oral pharmacokinetics (PK) in rats and mice: - Rats: Male Sprague-Dawley rats (n=3 per time point) received alectinib hydrochloride (10 mg/kg, administered by gavage, dissolved in 0.5% methylcellulose). PK parameters (LC-MS/MS): Cmax = 8.5 μM, Tmax = 1.0 h, terminal half-life (t₁/₂) = 4.2 h, oral bioavailability (F) = 62% [1] - Mice: Male C57BL/6 mice (n=3 per time point) received alectinib hydrochloride (10 mg/kg, administered by gavage). Pharmacokinetic parameters: Cmax = 12.3 μM, Tmax = 0.8 h, t₁/₂ = 3.5 h, F = 58% [1]
2. Tissue distribution in mice:
Mice were sacrificed 1 hour after administration of 10 mg/kg orally. The tissue concentrations (LC-MS/MS) were as follows: plasma = 10.2 μM, brain = 8.5 μM, lung = 15.3 μM, liver = 22.1 μM. The brain/plasma ratio was 0.83, confirming that the drug could penetrate the blood-brain barrier [1, 3]
3. In vitro metabolism:
Alectinib hydrochloride (1 μM) was incubated with human liver microsomes (HLM) for 2 hours: <10% of the parent drug was metabolized. The main metabolite was identified as a demethylated derivative (LC-MS/MS), which showed no significant ALK inhibitory activity (IC₅₀ > 100 nM) [1]

1. In vitro toxicity: Alectinib hydrochloride (at concentrations up to 10 μM) showed no significant cytotoxicity to normal human bronchial epithelial cells (NHBE) or human hepatocytes (L02): cell viability > 90% (MTT assay), compared to the solvent control group. At concentrations up to 5 μM, it also did not induce DNA damage (comet assay: comet tail moment showed no change compared to the solvent control group) [2, 3]. 2. In vivo acute toxicity (mice/rat): - Mice: A single oral dose of alectinib hydrochloride (100–500 mg/kg) did not result in death. At a dose of 500 mg/kg, transient weight loss was observed (maximum 7%, recovered by day 5); no organ lesions (liver, kidney, brain) were found by H&E staining [1] - Rats: No abnormal behavior or serum biochemical markers (ALT, AST, BUN, creatinine) were observed after a single oral dose of alectinib hydrochloride (100–300 mg/kg) [1] 3. Subacute toxicity (mice): Mice treated with alectinib hydrochloride (100 mg/kg, once daily for 28 days) showed no significant changes in body weight, organ weight (liver, kidney, brain) or blood cell count (white blood cells, red blood cells, platelets). Serum ALT/AST/BUN/creatinine levels were maintained within the normal range [1, 2]
4. Plasma protein binding rate:
500 μL of human plasma was mixed with alectinib hydrochloride (0.1–10 μM) and dialyzed at 37°C with a 12–14 kDa dialysis membrane for 4 hours. Free drug concentration was determined by LC-MS/MS. Plasma protein binding rate = 97.2% (human), 96.8% (mouse) [1]

[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.

Alectinib hydrochloride is a hydrochloride salt composed of alectinib and an equimolar amount of hydrochloric acid. It is used to treat patients with anaplastic lymphoma kinase-positive metastatic non-small cell lung cancer. It is an antitumor drug and an EC 2.7.10.1 (receptor protein tyrosine kinase) inhibitor. It contains alectinib (1+).
See also: Alectinib (with active moiety).

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Drug Indications Alecensa monotherapy is indicated for first-line treatment of adult patients with anaplastic lymphoma kinase (ALK)-positive advanced non-small cell lung cancer (NSCLC). Alectinib monotherapy is indicated for adult patients with ALK-positive advanced non-small cell lung cancer (NSCLC) who have previously received crizotinib treatment.

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1. Background:
Alectinib hydrochloride is a second-generation ALK inhibitor designed to overcome the limitations of first-generation ALK inhibitors (such as crizotinib), including acquired resistance (due to ALK mutations, such as L1196M) and poor brain penetration. It has been approved for the treatment of ALK-positive metastatic NSCLC [2, 3].
2. Mechanism of Action:
Alectinib hydrochloride binds to the ATP-binding pocket of ALK (wild-type and mutant), inhibiting its tyrosine kinase activity. The drug blocks downstream signaling pathways (STAT3, AKT, ERK1/2), which are essential for the proliferation, survival, and invasion of cancer cells, 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), thus reducing off-target toxicity [2].
- It has excellent brain permeability (brain/plasma ratio of approximately 0.8) and is effective against ALK-positive brain metastases (a common complication of non-small cell lung cancer) [3].
- It is effective against crizotinib-resistant ALK mutants (e.g., L1196M), overcoming acquired resistance [3].
4. Clinical significance:
Preclinical data in the included literature support the efficacy of alectinib hydrochloride in ALK-positive non-small cell lung cancer (including crizotinib-resistant and brain metastases). Metastatic disease. Based on a phase III clinical trial (not detailed in the included literature), the drug has been approved by the FDA for first-line treatment of ALK-positive metastatic non-small cell lung cancer [2, 3].
5. Limitations:
- It is ineffective against ALK-negative cancers, limiting its therapeutic scope [2].
- Its long-term toxicity (e.g., cardiovascular effects) in humans has not been evaluated.

C30H34N4O2.HCL

519.08

518.244

C, 69.42; H, 6.80; Cl, 6.83; N, 10.79; O, 6.16

1256589-74-8

Alectinib;1256580-46-7

53239799

White to off-white solid powder

5.578

2

5

3

37

867

0

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]

GYABBVHSRIHYJR-UHFFFAOYSA-N

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

9-ethyl-6,6-dimethyl-8-(4-morpholin-4-ylpiperidin-1-yl)-11-oxo-5H-benzo[b]carbazole-3-carbonitrile;hydrochloride

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

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)