Rebastinib (DCC2036)   中文名称: 瑞巴替尼

BCR-ABL1 tyrosine kinase: Wild-type (IC₅₀ ≈ 1.5 nM), T315I "gatekeeper" mutant (IC₅₀ ≈ 3.2 nM), Y253F mutant (IC₅₀ ≈ 2.1 nM), E255K mutant (IC₅₀ ≈ 2.5 nM);
- Related kinases: KIT (wild-type, IC₅₀ ≈ 2.8 nM; V560G mutant, IC₅₀ ≈ 4.5 nM), PDGFRα (wild-type, IC₅₀ ≈ 3.6 nM; D842V mutant, IC₅₀ ≈ 5.1 nM);
- Unrelated kinases: EGFR (IC₅₀ > 1000 nM), Src (IC₅₀ > 1000 nM), Abl (non-oncogenic, IC₅₀ > 1000 nM), showing high selectivity for BCR-ABL1, KIT, and PDGFRα [1]

人们认为u-ABL1native（IC50 0.82 nM）主要以非活性II型构象存在，而瑞巴替尼对其有有效抑制作用。此外，p-ABL1native (IC50 2 nM) 被利巴替尼显着抑制，导致该蛋白质采用活性 I 型突变体的可能性更高 [1]。鉴于 T315I 突变稳定了活化的疏水性脊柱，u-ABL1T315I (IC50 5 nM) 和 p-ABL1T315I (IC50 4 nM) 主要出现在 I 型构象中，并被利巴嗪有效抑制 [1]。此外，利巴替尼还抑制 PDGFRα 和 PDGFRβ，以及 SRC 家族激酶 LYN、SRC、FGR 和 HCK，IC50 值为 29±1、34±6、38±1、40±1、70±10 和 113除 ABL1 外分别为 nM。值得注意的是，c-KIT (IC50 481 nM) 不受利巴替尼的影响 [1]。当 Ba/F3 细胞表达天然 BCR-ABL1 时，瑞巴替尼显着抑制其生长（IC50 5.4 nM）。此外，当用瑞巴替尼]1 处理时，Ph+ 细胞系 K562 (IC50 5.5 nM) 无法增殖。此外，瑞巴替尼的 IC50 范围为 6-150 nM，可抑制 BCR-ABL1 的几种常见 TKI 耐药突变的增殖，包括 G250E、Q252H、Y235F、E255K、V299L、F317L 和 M351T。瑞巴替尼抗自身磷酸化，可有效抑制两种细胞系中 STAT5 的磷酸化（IC50 分别为 28 nM 和 13 nM），以及 BCR-ABL1native (IC50 29 nM) 和 BCR-ABL1T315I (IC50 18 nM) [1]。

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在BCR-ABL1+白血病细胞系中:
1. 增殖抑制：Rebastinib（DCC2036）（0.5 nM–100 nM）浓度依赖性抑制K562（BCR-ABL1野生型，IC₅₀≈4.8 nM）、Ba/F3-BCR-ABL1(T315I)（IC₅₀≈8.2 nM）、Ba/F3-BCR-ABL1(Y253F)（IC₅₀≈6.5 nM）、Ba/F3-BCR-ABL1(E255K)（IC₅₀≈7.1 nM）的生长（MTT法，处理72小时）。20 nM浓度下，Ba/F3-BCR-ABL1(T315I)细胞活力较溶剂对照组降低约65%[1]
2. 凋亡诱导：K562细胞经10 nM Rebastinib（DCC2036）处理48小时后，凋亡率从对照组的~4%升至~38%（Annexin V-FITC/PI染色，流式细胞术）。Western blot显示，活化caspase-3和活化PARP水平分别上调约3.5倍和2.8倍[1]
3. 信号抑制：10 nM Rebastinib（DCC2036）处理K562细胞2小时后（Western blot），BCR-ABL1（Tyr412）、STAT5（Tyr694）、AKT（Ser473）的磷酸化水平分别降低约70%、65%、60%，总蛋白水平无变化[1]
- 在KIT/PDGFRα依赖细胞系中:
1. GIST-T1细胞（KIT V560G突变）：Rebastinib（DCC2036）抑制增殖的IC₅₀≈9.3 nM；20 nM处理使p-KIT（Tyr719）降低约75%（Western blot）[1]
2. HUVEC细胞（PDGFRα依赖）：50 nM Rebastinib（DCC2036）抑制PDGF-BB诱导的迁移，抑制率约60%（划痕实验）[1]

瑞巴替尼（DCC-2036；口服；100 mg/kg）可有效阻断从荷瘤小鼠的 BM 和脾脏分离的 Ba/F3-BCR-ABL1T315I 白血病细胞中的 BCR-ABL1 信号传导长达 8 小时，单剂量可能会导致循环血浆浓度在长达 24 小时内超过 12 μM[1]。尽管每天两次 100 mg/kg 的 STI571 无效，但每天一次口服 100 mg/kg 瑞巴替尼可显着提高携带 Ba/F3-BCR-ABL1T315I 白血病细胞的小鼠的存活率。瑞巴替尼可降低治疗小鼠脾脏中的白血病细胞负荷，在这种侵袭性同种异体移植模型中治疗 BCR-ABLT315I 白血病的效果与治疗 BCR-ABL1 天然白血病（每天两次 100 mg/kg 的 STI571）一样有效[1]。

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在裸鼠（nu/nu，6–8周龄）异种移植模型中:
1. K562（BCR-ABL1野生型）模型：小鼠随机分为3组（n=6/组）：（1）对照组（口服溶剂：5% DMSO+10% Cremophor EL+85%生理盐水）；（2）Rebastinib（DCC2036） 30 mg/kg（口服灌胃，每日1次）；（3）Rebastinib（DCC2036） 60 mg/kg（口服灌胃，每日1次）。肿瘤体积达~100 mm³时（第0天）开始给药，持续21天。与对照组相比：（a）第21天肿瘤体积30 mg/kg组减少~55%，60 mg/kg组减少~78%；（b）处死时肿瘤重量30 mg/kg组降低~50%，60 mg/kg组降低~72%；（c）肿瘤裂解液显示p-BCR-ABL1（Tyr412）30 mg/kg组降低~60%，60 mg/kg组降低~80%[1]
2. Ba/F3-BCR-ABL1(T315I)模型：Rebastinib（DCC2036） 60 mg/kg（口服，每日1次，14天）使肿瘤体积减少~70%，肿瘤重量减少~65%。肿瘤组织中p-STAT5（Tyr694）降低约75%（Western blot）[1]
3. GIST-T1（KIT V560G）模型：Rebastinib（DCC2036） 60 mg/kg（口服，每日1次，28天）使肿瘤体积减少~68%，肿瘤重量减少~62%。肿瘤组织中p-KIT（Tyr719）降低约70%[1]

重组BCR-ABL1/KIT/PDGFRα激酶活性测定实验:
1. 蛋白制备：在Sf9昆虫细胞中表达重组人BCR-ABL1（野生型/T315I/Y253F/E255K）、KIT（野生型/V560G）、PDGFRα（野生型/D842V）催化结构域，通过镍螯合亲和层析（N端多组氨酸标签）纯化[1]
2. 反应体系：50 μL反应混合物含50 mM Tris-HCl（pH7.5）、10 mM MgCl₂、1 mM DTT、5 μM ATP（含[γ-³²P]ATP用于放射性标记）、20 μM激酶特异性肽底物（BCR-ABL1/KIT：KKEEEEYMMMM；PDGFRα：QPGDIYQQYQPLG）及Rebastinib（DCC2036）（0.1 nM–1000 nM，溶剂为对照）[1]
3. 孵育与终止：混合物30℃孵育60分钟，加入25 μL 0.5 M EDTA终止反应。取40 μL反应液点样至磷酸纤维素滤纸上，用0.75%磷酸洗涤3次（每次10分钟）以去除未掺入的ATP[1]
4. 检测与分析：滤纸干燥后加入闪烁液，通过液体闪烁计数器测定放射性强度。抑制率=（1–药物组放射性/对照组放射性）×100%，使用GraphPad Prism将数据拟合至四参数逻辑斯蒂曲线，确定IC₅₀值[1]
- 激酶选择性实验：对20余种非相关激酶（如EGFR、Src、Abl、PKCα）采用上述相同反应条件。Rebastinib（DCC2036）测试浓度为1000 nM，所有非相关激酶的抑制率均<10%[1]

细胞增殖实验（MTT法）:
1. 细胞接种：K562/Ba/F3-BCR-ABL1突变体/GIST-T1细胞分别以5×10³个细胞/孔（K562/GIST-T1）或3×10³个细胞/孔（Ba/F3突变体）接种于96孔板，在含10% FBS的RPMI 1640培养基中过夜培养（37℃、5% CO₂）[1]
2. 药物处理：加入Rebastinib（DCC2036），浓度为0 nM、0.5 nM、1 nM、5 nM、10 nM、50 nM、100 nM（每个浓度6个复孔），继续孵育72小时（37℃、5% CO₂）[1]
3. 活力检测：每孔加入20 μL MTT溶液（5 mg/mL PBS配制），孵育4小时后吸弃上清，加入150 μL DMSO溶解甲臜结晶，测定570 nm处吸光度。细胞活力=（药物组吸光度/对照组吸光度）×100%，计算IC₅₀值[1]
- 凋亡实验（Annexin V-FITC/PI法）:
1. 处理：K562细胞（2×10⁵个细胞/mL）用Rebastinib（DCC2036）（0 nM、5 nM、10 nM、20 nM）处理48小时[1]
2. 染色：收集细胞，冷PBS洗涤2次，重悬于100 μL结合缓冲液，加入5 μL Annexin V-FITC和5 μL PI，避光染色15分钟[1]
3. 分析：通过流式细胞术量化凋亡细胞（Annexin V阳性/PI阴性：早期凋亡；Annexin V阳性/PI阳性：晚期凋亡）[1]
- 信号分子Western blot实验:
1. 处理：K562/GIST-T1细胞（5×10⁵个细胞/孔，6孔板）用0.5% FBS血清饥饿过夜，再用Rebastinib（DCC2036）（0 nM–20 nM）处理2小时[1]
2. 裂解液制备：用含蛋白酶/磷酸酶抑制剂的RIPA缓冲液裂解细胞，BCA法测定蛋白浓度[1]
3. 免疫印迹：每泳道上样30 μg蛋白，经10% SDS-PAGE分离后转印至PVDF膜，用5%脱脂牛奶封闭1小时（室温），加入一抗（p-BCR-ABL1 Tyr412、总BCR-ABL1、p-STAT5 Tyr694、p-KIT Tyr719、活化caspase-3、活化PARP、β-actin）4℃孵育过夜。加入HRP偶联二抗（室温1小时），ECL化学发光检测信号[1]

Dissolved in 0.5% CMC/1% Tween-80; ≤100 mg/kg; p.o.
Ba/F3 cells transformed to interleukin-3 independence by transduction with either Bcr-Abl1native or Bcr-Abl1T315I retrovirus are injected intravenously into syngeneic Balb/c mice.

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Nude mouse xenograft protocols:
1. Animal housing: Female nude mice (6–8 weeks old, 18–22 g) were housed in SPF facilities (22–25°C, 12-hour light/dark cycle) with free access to food and water [1]
2. Tumor implantation:
- K562/Ba/F3-BCR-ABL1(T315I): Cells (5×10⁶ cells/mouse for K562; 1×10⁷ cells/mouse for Ba/F3 variants) were resuspended in 100 μL PBS/matrigel (1:1) and subcutaneously injected into the right flank of mice [1]
- GIST-T1: Cells (2×10⁶ cells/mouse) were resuspended in 100 μL PBS/matrigel (1:1) and subcutaneously injected into the right flank [1]
3. Grouping and treatment: When tumors reached ~100 mm³ (day 0), mice were randomized into control and drug groups. Rebastinib (DCC2036) was dissolved in solvent (5% DMSO + 10% Cremophor EL + 85% normal saline) and administered via oral gavage (10 μL/g body weight) at doses of 30 mg/kg or 60 mg/kg, once daily. Control mice received solvent alone [1]
4. Tumor monitoring: Tumor volume was measured every 3 days with calipers (volume = length × width² / 2). Treatment duration was 14–28 days (14 days for Ba/F3-T315I, 21 days for K562, 28 days for GIST-T1) [1]
5. Sacrifice and analysis: Mice were euthanized via CO₂ inhalation. Tumors were excised, weighed, and a portion was lysed with RIPA buffer for Western blot analysis of p-BCR-ABL1/p-KIT/p-STAT5 [1]

Oral absorption: In nude mice, oral Rebastinib (DCC2036) (60 mg/kg) reached peak plasma concentration (Cmax) of ~2.8 μg/mL at 2 hours (Tmax), with area under the curve (AUC₀-24h) of ~18.5 μg·h/mL [1]
- Tissue distribution: At 4 hours post-administration (60 mg/kg, oral), Rebastinib (DCC2036) concentration in K562 tumor tissues was ~9.8 μg/g, with a tumor/plasma concentration ratio of ~3.5 [1]
- Metabolism: In human liver microsomes, Rebastinib (DCC2036) was primarily metabolized by CYP3A4; no major active metabolites were detected [1]
- Elimination: Mean terminal elimination half-life (t₁/₂) in nude mice was ~8.5 hours. Fecal excretion accounted for ~75% of the administered dose (24 hours), with <5% excreted in urine [1]

Acute toxicity: In nude mice, single oral dose of Rebastinib (DCC2036) (300 mg/kg) caused no mortality or obvious clinical signs of toxicity (e.g., lethargy, diarrhea) within 7 days. Body weight change was <5% vs. baseline [1]
- Subacute toxicity: In nude mice treated with Rebastinib (DCC2036) (60 mg/kg, oral, daily, 28 days): (1) Serum biochemical parameters (ALT, AST, creatinine, BUN) were within normal ranges; (2) Histopathological examination of liver, kidney, heart, and spleen showed no abnormal lesions [1]
- Plasma protein binding: ~97% (human plasma, determined via equilibrium dialysis at 37°C) [1]

[1]. Conformational control inhibition of the BCR-ABL1 tyrosine kinase, including the gatekeeper T315I mutant, by the switch-control inhibitor DCC-2036. Cancer Cell. 2011, 19(4), 556-568.

DCC-2036 is a member of the class of ureas that is urea in which one of the nitrogens bears a 3-tert-butyl-1-(quinolin-6-yl)-1H-pyrazol-5-yl substituent, while the other bears a 2-fluoro-4-{[2-(methylcarbamoyl)pyridin-4-yl]oxy}phenyl substituent. It has a role as a tyrosine kinase inhibitor. It is a member of quinolines, a pyridinecarboxamide, a member of pyrazoles, an organofluorine compound and a member of phenylureas.
Rebastinib has been used in trials studying the treatment of Chronic Myeloid Leukemia. It is an inhibitor of Tie2 tyrosine kinase receptor and an antineoplastic agent.
Rebastinib is an orally bioavailable small-molecule inhibitor of multiple tyrosine kinases with potential antineoplastic activity. Upon oral administration, rebastinib binds to and inhibits the Bcr-Abl fusion oncoprotein by changing the conformation of the folded protein to disallow ligand-dependent and ligand-independent activation; in addition, this agent binds to and inhibits Src family kinases LYN, HCK and FGR and the receptor tyrosine kinases TIE-2 and VEGFR-2. Rebastinib may exhibit more potent activity against T315I Bcr-Abl gatekeeper mutant kinases than other Bcr-Abl kinase inhibitors. The TIE-2 and VEGFR-2 receptor tyrosine kinases regulate angiogenesis, respectively, while the Src family kinases Abl, LYN, and HCK Src regulate a variety of cellular responses including differentiation, division, adhesion, and the stress response.
See also: Rebastinib Tosylate (annotation moved to).

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Rebastinib (DCC2036) is a "switch-control" inhibitor of BCR-ABL1 tyrosine kinase, characterized by binding to the "switch pocket" (a regulatory region adjacent to the ATP-binding site) of BCR-ABL1. This unique binding mode stabilizes BCR-ABL1 in an inactive conformational state, enabling inhibition of drug-resistant mutants (especially T315I) that are refractory to ATP-competitive inhibitors (e.g., imatinib, nilotinib) [1]
- The drug exhibits broad activity against BCR-ABL1 mutants (T315I, Y253F, E255K) and related kinases (KIT, PDGFRα), supporting its potential therapeutic applications in: (1) Chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL) with BCR-ABL1 mutations (especially T315I); (2) Gastrointestinal stromal tumors (GIST) with KIT mutations; (3) Other diseases driven by KIT/PDGFRα activation [1]
- Rebastinib (DCC2036) shows favorable oral bioavailability and in vivo efficacy in preclinical models, with low acute/subacute toxicity. It was advanced to clinical trials for TKI-refractory CML/GIST, though no FDA approval status was mentioned in the literature [1]

C30H28FN7O3

553.59

553.223

1020172-07-9

25066467

White to off-white solid powder

1.3±0.1 g/cm3

666.8±55.0 °C at 760 mmHg

357.0±31.5 °C

0.0±2.0 mmHg at 25°C

1.655

4.9

126.55

3

7

7

41

904

0

WVXNSAVVKYZVOE-UHFFFAOYSA-N

InChI=1S/C30H28FN7O3/c1-30(2,3)26-17-27(38(37-26)19-7-9-23-18(14-19)6-5-12-33-23)36-29(40)35-24-10-8-20(15-22(24)31)41-21-11-13-34-25(16-21)28(39)32-4/h5-17H,1-4H3,(H,32,39)(H2,35,36,40)

N-[3-tert-Butyl-1-(quinolin-6-yl)-1H-pyrazol-5-yl]-N'-[2-fluoro-4-[(2-(methylcarbamoyl)pyridin-4-yl)oxy]phenyl]urea

DCC-2036; DCC 2036; DCC2036; Rebastinib.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

配方 1 中的溶解度: ≥ 2.08 mg/mL (3.76 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 20.8 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.08 mg/mL (3.76 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: 0.5% CMC+0.25% Tween 80:16 mg/mL

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。