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]

体外活性：CH5424802 以 ATP 竞争方式对 ALK 的解离常数 (KD) 值为 2.4 nM。 CH5424802 对天然 ALK 和 L1196M 具有显着的抑制效力，Ki 分别为 0.83 nM 和 1.56 nM。 CH5424802 可防止表达 EML4-ALK 的 NCI-H2228 NSCLC 细胞中 ALK 的自身磷酸化。 CH5424802 还抑制 STAT3 和 AKT 的磷酸化，但不抑制 ERK1/2 的磷酸化。 CH5424802 完全抑制 STAT3 Tyr705 的磷酸化。 CH5424802 优先对表达 EML4-ALK 的 NCI-H2228 细胞有效，但对 ALK 融合阴性 NSCLC 细胞系无效，包括 HCC827 细胞（EGFR 外显子 19 缺失）、A549 细胞（KRAS 突变体）或 NCI-H522 细胞（EGFR 野生型）单层培养中的（KRAS 野生型和 ALK 野生型）。 CH5424802 在 NCI-H2228 球状细胞中引发凋亡标记物 - caspase-3/7 样激活。 CH5424802 使用 NPM-ALK 融合蛋白阻断两种淋巴瘤系 KARPAS-299 和 SR 的生长，但不影响无 ALK 融合的 HDLM-2 淋巴瘤系的生长。 CH5424802对KARPAS-299表现出高靶点选择性和更强的抗增殖活性。 CH5424802 抑制 KAPRAS-299，IC50 为 3 nM，抑制 KDR，IC50 为 1.4 μM。 CH5424802的代谢稳定性非常高。激酶测定：通过使用时间分辨荧光共振能量转移 (TR-FRET) 测定或荧光偏振 (TR-FRET) 测定在 CH5424802 存在下检查其磷酸化各种底物肽的能力，评估对除 MEK1 和 Raf-1 之外的每种激酶的抑制能力。 FP）测定。在 CH5424802 存在的情况下，通过重组 ERK2 蛋白对底物肽的磷酸化进行定量分析来评估针对 MEK1 的抑制活性。通过检查激酶在 CH5424802 存在的情况下磷酸化 MEK1 的能力来评估对 Raf-1 的抑制活性。细胞测定：将细胞（NSCLC、A549 和 HCC827）接种在 96 孔板中过夜，并与不同浓度的 CH5424802 一起孵育指定时间。对于球状细胞生长抑制测定，将细胞接种在球状体板上，孵育过夜，然后用化合物处理指定的时间。通过发光细胞活力测定来测量活细胞。使用 Caspase-Glo 3/7 检测试剂盒评估 Caspase-3/7 检测。

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1. 对ALK阳性癌细胞的抗增殖活性：
盐酸阿来替尼（0.001–10 μM）抑制ALK阳性NSCLC细胞增殖，GI₅₀值如下：H3122（ALK融合阳性）= 0.025 μM、H2228（ALK融合阳性）= 0.032 μM、H3122 CR（克唑替尼耐药，L1196M突变）= 0.048 μM（MTT法）。相反，其对ALK阴性细胞系（A549、H1299）无显著活性：GI₅₀ > 10 μM [2, 3]
2. 抑制ALK下游信号通路：
盐酸阿来替尼（0.01–1 μM）呈剂量依赖性抑制H3122细胞中ALK磷酸化（p-ALK）及其下游效应分子：0.1 μM使p-ALK降低85%、p-STAT3降低75%、p-AKT降低70%、p-ERK1/2降低65%（western blot）。0.1 μM浓度下，这种抑制作用可维持24小时 [2, 3]
3. 诱导ALK阳性细胞凋亡及细胞周期阻滞：
盐酸阿来替尼（0.05–0.5 μM）处理H3122细胞48小时，诱导G1期阻滞（流式细胞术：G1期细胞比例从40%升至65%）和凋亡：0.5 μM使Annexin V阳性细胞从溶媒组的5%增至45%（Annexin V-FITC/PI双染）。Western blot显示，0.5 μM时切割型caspase-3（4倍）和切割型PARP（3.5倍）上调 [2]
4. 抑制ALK阳性癌细胞迁移和侵袭：
盐酸阿来替尼（0.01–0.1 μM）使H2228细胞迁移能力降低60%（0.1 μM，划痕实验），侵袭能力降低70%（0.1 μM，Transwell实验）。0.1 μM时还可使基质金属蛋白酶-9（MMP-9）表达降低55%（western blot）[3]

口服 CH5424802 剂量依赖性地抑制肿瘤生长（ED50 为 0.46 mg/kg）并抑制肿瘤消退。 20 mg/kg CH5424802治疗显示肿瘤快速消退168%，治疗11天（第28天）后任何小鼠的肿瘤体积<30 mm3，保持有效的抗肿瘤作用，并且自始至终不发生肿瘤再生4周的禁药期。 CH5424802在小鼠体内的半衰期和口服生物利用度分别为8.6小时和70.8%。重复剂量为 6 mg/kg 时，给药后 2、7 和 24 小时的平均血浆水平分别达到 1.7、1.5 和 0.3 nM。 CH5424802的施用导致肿瘤生长预防和肿瘤消退。第 20 天，20 mg/kg 剂量时，KARPAS-299 的肿瘤生长抑制率为 119%，NB-1 的肿瘤生长抑制率为 104%。CH5424802 以剂量依赖性方式（2-20 mg/kg）抑制 STAT3 的磷酸化。在 CH5424802 处理的异种移植肿瘤中也观察到 AKT 磷酸化部分降低。

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1. ALK阳性NSCLC异种移植模型的抗肿瘤活性：
- H3122异种移植瘤：雌性裸鼠（6–8周龄）皮下接种H3122细胞（肿瘤体积100–150 mm³），给予盐酸阿来替尼（25、50、100 mg/kg，口服灌胃，每日1次）处理21天。肿瘤生长抑制率（TGI）呈剂量依赖性：25 mg/kg = 45%、50 mg/kg = 72%、100 mg/kg = 90%。100 mg/kg使肿瘤重量较溶媒组降低85% [2]
- H3122 CR（克唑替尼耐药）异种移植瘤：携带H3122 CR肿瘤的小鼠给予盐酸阿来替尼（100 mg/kg，口服每日1次）处理21天，TGI为82%，肿瘤重量较溶媒组降低78%（对比克唑替尼100 mg/kg组25%的TGI）[3]
2. 脑穿透性及ALK阳性脑转移模型的疗效：
雄性裸鼠立体定向注射5×10⁵ H2228细胞建立颅内异种移植模型，给予盐酸阿来替尼（100 mg/kg，口服每日1次）处理28天。其显著延长中位生存期，从溶媒组的25天增至52天。脑肿瘤切片显示，Ki-67（增殖标志物）降低70%，TUNEL阳性细胞（凋亡）较溶媒组增加5倍 [3]

除 MEK1 和 Raf-1 外，每种激酶在 CH5424802 存在的情况下磷酸化不同底物肽的能力通过时间分辨荧光共振能量转移 (TR-FRET) 测定或荧光偏振 ( FP）测定。通过在 CH5424802 存在的情况下定量分析重组 ERK2 蛋白对底物肽的磷酸化，评估对 MEK1 的抑制活性。在 CH5424802 存在的情况下，激酶磷酸化 MEK1 的能力用于测量其对 Raf-1 的抑制活性。

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1. ALK激酶活性实验（放射法）：
将重组人WT ALK或ALK突变体（L1196M、G1269A、C1156Y；50 nM）与[γ-³²P]ATP（10 μM）、生物素化ALK底物肽（5 μM）及盐酸阿来替尼（0.001–100 nM）共同加入激酶缓冲液（50 mM Tris-HCl pH 7.5、10 mM MgCl₂、1 mM DTT、0.01% BSA），30°C孵育60分钟。3%磷酸终止反应后，将混合物转移至链霉亲和素包被板，液体闪烁计数法检测结合的磷酸化肽段，IC₅₀定义为抑制50%激酶活性的浓度 [2]
2. ALK-STAT3信号抑制实验（细胞水平HTRF法）：
H3122细胞（1×10⁴/孔）接种于384孔板，用盐酸阿来替尼（0.001–10 μM）处理4小时。裂解细胞后，用HTRF试剂（Eu³⁺标记的抗p-STAT3抗体和XL665标记的抗STAT3抗体）检测p-STAT3（Tyr705）水平，检测荧光波长620 nm和665 nm，EC₅₀定义为降低50% p-STAT3的浓度 [3]

在 96 孔板中，将细胞（NSCLC、A549 和 HCC827）接种过夜，然后将不同浓度的 CH5424802 孵育指定的时间。为了进行球状细胞生长抑制测定，首先将细胞接种到球状平板上，孵育一整晚，然后暴露于化合物规定的持续时间。发光细胞活力测定可测定活细胞的数量。 Caspase-Glo 3/7 检测试剂盒用于评估 Caspase-3/7 检测。

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1. 抗增殖实验（MTT法）：
ALK阳性（H3122、H2228、H3122 CR）和ALK阴性（A549、H1299）细胞接种于96孔板（5×10³细胞/孔），过夜孵育。加入盐酸阿来替尼（0.001–10 μM），培养72小时后，每孔加入MTT试剂（5 mg/mL，10 μL），孵育4小时，DMSO溶解甲臜结晶。570 nm处测定吸光度，通过剂量-反应曲线计算GI₅₀ [2, 3]
2. ALK信号及凋亡标志物western blot实验：
H3122/H2228细胞用盐酸阿来替尼（0.01–1 μM）处理4–24小时，用含蛋白酶/磷酸酶抑制剂的RIPA缓冲液裂解。30 μg蛋白经10% SDS-PAGE分离后转至PVDF膜，用抗p-ALK、ALK、p-STAT3、STAT3、p-AKT、AKT、p-ERK1/2、ERK1/2、切割型caspase-3、切割型PARP、MMP-9及β-actin抗体孵育，ECL发光显示条带，光密度法定量 [2, 3]
3. 凋亡实验（Annexin V-FITC/PI双染）：
H3122细胞用盐酸阿来替尼（0.05–0.5 μM）处理48小时后收集，PBS洗涤，室温下用Annexin V-FITC和PI染色15分钟，流式细胞术定量凋亡细胞（Annexin V⁺/PI⁻和Annexin V⁺/PI⁺）[2]
4. Transwell侵袭实验：
H2228细胞（5×10⁴细胞/孔）重悬于含盐酸阿来替尼（0.01–0.1 μM）的无血清培养基，接种于Matrigel包被的Transwell上室，下室含10% FBS培养基（趋化因子）。24小时后去除未侵袭细胞，侵袭细胞用4%多聚甲醛固定、结晶紫染色，显微镜下计数 [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/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]

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]

[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 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).

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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.

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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

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

72.36

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

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

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