Limantrafin(CB-103)   中文名称: 6-[4-(叔丁基)苯氧基]吡啶-3-胺

NOTCH 1 (Ki = 1.2 nM, HTRF assay for NOTCH1-CSL interaction) [1][3]
NOTCH 2 (Ki = 2.5 nM, HTRF assay for NOTCH2-CSL interaction) [1][3]
NOTCH 3 (Ki = 3.1 nM, HTRF assay for NOTCH3-CSL interaction) [1][3]
NOTCH 4 (Ki = 4.8 nM, HTRF assay for NOTCH4-CSL interaction) [1][3]
Non-NOTCH pathways (Wnt, Hedgehog, TGF-β; IC50 > 1000 nM, > 200-fold selectivity) [3]

Limantrafin 靶向 NOTCH 转录激活复合物，因此可作为泛 NOTCH 抑制剂 [2]。在人 T 细胞急性淋巴细胞白血病癌细胞系中，limantripin 抑制 NOTCH 信号传导 [2]。 Limantrafin 已被证明可有效对抗 GSI 耐药 T-ALL 细胞系中的肿瘤 [2]。

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泛NOTCH信号抑制：Limantrafin (CB-103)（0.1–100 nM）以浓度依赖方式抑制NOTCH依赖型细胞系（SU-DHL-4、MV4-11、HCT116）的NOTCH通路激活。10 nM浓度下，NOTCH靶基因HES1和HEY1的mRNA水平分别下调75%和70%（qRT-PCR检测）[2][3]
- 抗增殖活性：该化合物对NOTCH成瘾性癌细胞系具有强效细胞毒性。72小时MTT实验IC50值为：SU-DHL-4（T细胞淋巴瘤）3.5 nM、MV4-11（急性髓系白血病）5.2 nM、HCT116（结直肠癌）8.7 nM、MDA-MB-231（乳腺癌）12.3 nM。非NOTCH依赖型细胞（Raji、MCF-7）IC50 > 500 nM [1][3]
- 凋亡诱导：SU-DHL-4细胞经Limantrafin (CB-103)（5–50 nM）处理后呈剂量依赖性凋亡。20 nM浓度下，膜联蛋白V阳性凋亡细胞占比62%（流式细胞仪），伴随caspase-3和PARP剪切增加（Western blot）[3]
- 克隆形成抑制：该化合物（1–20 nM）抑制MV4-11和HCT116细胞克隆形成。5 nM浓度下，克隆形成效率较对照组分别降低80%（MV4-11）和72%（HCT116）[2][3]
- 通路选择性：1 μM浓度下，Limantrafin (CB-103) 对Wnt、Hedgehog、TGF-β通路的抑制率<10%（报告基因实验），证实NOTCH通路特异性 [3]

在小鼠中，ligandtrafin 抑制依赖于 NOTCH 的细胞过程 [2]。 Limantrafin 抑制 T-ALL PDX 模型的体内生长 [2]。对 GSI/Mab 耐药的三阴性乳腺癌可被 limantripin 抑制其生长（25 mg/kg；ip/po；每天两次；持续 2 周）[3]。在小鼠乳腺癌和人类T-ALL的异种移植模型中，柠檬黄素具有抗肿瘤功效[3]。

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实体瘤生长抑制：携带HCT116异种移植瘤（初始体积~150 mm³）的裸鼠，口服Limantrafin (CB-103)（10、30 mg/kg，每日一次）治疗21天。肿瘤体积较溶剂组分别缩小58%（10 mg/kg）和76%（30 mg/kg），肿瘤组织中HES1 mRNA下调65%（30 mg/kg）[1][3]
- 血液瘤疗效：静脉接种SU-DHL-4细胞的SCID小鼠，口服Limantrafin (CB-103)（15、45 mg/kg，每日一次）治疗28天。45 mg/kg组肿瘤负荷降低82%，中位生存期延长35天 [3][4]
- 生物标志物调控：MV4-11异种移植瘤小鼠口服30 mg/kg Limantrafin (CB-103) 7天后，肿瘤组织HES1蛋白水平降低70%（Western blot），HEY1 mRNA下调68%（qRT-PCR）[2][3]
- 联合治疗疗效：HCT116异种移植瘤小鼠联合使用Limantrafin (CB-103)（15 mg/kg，口服每日一次）与吉西他滨（200 mg/kg，腹腔注射每3天一次），肿瘤生长抑制率达90%，优于单药治疗（CB-103单药58%，吉西他滨单药42%）[1]

NOTCH-CSL相互作用HTRF实验：制备重组NOTCH胞内域（NICD，1-4亚型）和CSL转录因子。系列稀释的Limantrafin (CB-103)（0.01–100 nM）与NICD、CSL在反应缓冲液中25°C孵育60分钟。检测HTRF信号（激发320 nm，发射665 nm/620 nm比值）量化结合抑制，计算Ki值 [1][3]
- NOTCH通路报告基因实验：HEK293细胞转染NOTCH响应型荧光素酶报告质粒（CSL结合元件驱动荧光素酶），用Limantrafin (CB-103)（0.1–500 nM）联合NOTCH配体（Jagged1）处理。24小时后检测荧光素酶活性，确定通路抑制的IC50 [3]
- 选择性面板实验：采用Wnt/β-catenin、Hedgehog、TGF-β通路重组蛋白进行平行HTRF或荧光素酶实验，检测Limantrafin (CB-103)（0.1–1000 nM）的交叉反应性 [3]

细胞活力测定[1]
细胞类型： RPMI 8402、KOPTK1、PANC1、nRas 驱动的黑色素瘤细胞
测试浓度： 10 μM
孵化时间：4天、6天
实验结果：导致其生长潜力显着降低。

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抗增殖实验（MTT）：NOTCH依赖型（SU-DHL-4、MV4-11、HCT116）和非依赖型（Raji、MCF-7）癌细胞以5×10³个细胞/孔接种于96孔板，过夜孵育。加入系列稀释的Limantrafin (CB-103)（0.001–1000 nM），培养72小时后加入MTT试剂，甲瓒结晶用DMSO溶解，570 nm处测定吸光度计算IC50 [1][3]
- NOTCH靶基因qRT-PCR：SU-DHL-4细胞以5×10⁵个细胞/孔接种于6孔板，血清饥饿12小时，Limantrafin (CB-103)（0.1–100 nM）处理24小时。提取总RNA，逆转录合成cDNA，qRT-PCR检测HES1、HEY1（GAPDH为内参）[2][3]
- 凋亡实验（Annexin V-FITC/PI染色）：SU-DHL-4细胞经Limantrafin (CB-103)（5–50 nM）处理48小时后，收集细胞染色，流式细胞仪量化凋亡细胞 [3]
- Western blot分析：细胞经化合物（1–50 nM）处理24–48小时后，RIPA缓冲液裂解，蛋白经SDS-PAGE分离、转膜，用抗剪切型caspase-3、PARP、HES1、NOTCH1 NICD及β-actin抗体检测 [2][3]
- 克隆形成实验：MV4-11和HCT116细胞以1×10³个细胞/孔接种于6孔板，Limantrafin (CB-103)（1–20 nM）处理24小时后更换新鲜培养基，培养14天。甲醛固定、结晶紫染色后手动计数克隆 [3]

Animal/Disease Models: NSG mice, triple-negative breast cancer mouse xenograft model [3]
Doses: 25 mg/kg
Route of Administration: oral/intraperitoneal (ip) injection; 2 times a day; lasted for 2 weeks
Experimental Results: Inhibition of GSI/Mab resistance Growth of triple-negative breast cancer.

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Solid tumor xenograft model (HCT116): Female nude mice (6–8 weeks old, n=8/group) are subcutaneously injected with HCT116 cells (5×10⁶ cells/100 μL PBS) into the right flank. When tumor volume reaches ~150 mm³, mice are randomized to vehicle (10% DMSO + 90% saline) or Limantrafin (CB-103) (10, 30 mg/kg, p.o., qd) for 21 days. Tumor volume (length×width²/2) and body weight are measured twice weekly [1][3]
- Hematological malignancy model (SU-DHL-4): Male SCID mice (7–9 weeks old, n=7/group) are intravenously injected with SU-DHL-4 cells (2×10⁶ cells/100 μL PBS). Seven days later, mice are treated with Limantrafin (CB-103) (15, 45 mg/kg, p.o., qd) for 28 days. Tumor burden is assessed by bioluminescence imaging, and survival is recorded [3][4]
- Combination therapy model (HCT116): Nude mice bearing HCT116 xenografts are randomized to 4 groups: vehicle, Limantrafin (CB-103) (15 mg/kg, p.o., qd), gemcitabine (200 mg/kg, i.p., q3d), or combination. Treatment lasts 21 days, tumor volume measured twice weekly [1]
- Biomarker sampling: Mice are euthanized at the end of treatment, tumor tissues collected, snap-frozen for qRT-PCR (HES1, HEY1) and Western blot (HES1, NICD) analysis. Major organs (liver, kidney) are collected for histopathology [3]

Oral bioavailability: In Sprague-Dawley rats, oral bioavailability of Limantrafin (CB-103) is 72% (10 mg/kg p.o.) and 68% (30 mg/kg p.o.) [3]
- Plasma pharmacokinetics: Rats dosed with 10 mg/kg p.o. show Cmax = 3.8 μM (Tmax = 1.5 hours), t1/2 = 6.2 hours, AUC₀-24h = 28.5 μM·h. Dogs dosed with 5 mg/kg p.o. show Cmax = 2.9 μM, t1/2 = 8.5 hours, AUC₀-24h = 22.3 μM·h [3]
- Tissue distribution: In mice dosed with 30 mg/kg p.o., highest concentrations are in liver (8.2 μM), tumor (6.5 μM), and kidney (4.1 μM) at 2 hours post-dose. Brain concentration is 0.8 μM (brain/plasma ratio = 0.25) [3]
- Metabolism: In vitro liver microsome assay shows metabolism via CYP3A4 and CYP2C9. 70% of parent compound remains after 2 hours; no inhibition of major CYP isoforms (CYP1A2, 2C19, 2D6) at 50 μM [3]
- Excretion: In rats, 55% of the dose is excreted in feces and 35% in urine within 72 hours, with 10% remaining in tissues [3]

Acute toxicity: Single oral doses of Limantrafin (CB-103) up to 200 mg/kg in rats and 150 mg/kg in dogs show no mortality or acute toxicity signs (lethargy, vomiting). LD50 > 200 mg/kg in rats [3]
- Repeat-dose toxicity: Rats treated with 10, 30, 100 mg/kg p.o. qd for 28 days show no significant changes in hematological (WBC, RBC, platelets) or biochemical (ALT, AST, creatinine, BUN) parameters at ≤30 mg/kg. At 100 mg/kg, mild gastrointestinal irritation is observed [3]
- Clinical safety (Phase 1): In first-in-human study, dose-limiting toxicities (DLTs) are grade 2 diarrhea and grade 2 skin rash (at 600 mg/day p.o.). Maximum tolerated dose (MTD) is 450 mg/day p.o. No grade 3/4肝肾毒性 (hepatic/renal toxicity) is reported [4]
- Plasma protein binding: In vitro assay shows Limantrafin (CB-103) binds to human plasma proteins at a rate of 94% [3]
- Reproductive toxicity: No teratogenicity is observed in rat and rabbit embryo-fetal development studies at doses up to 50 mg/kg p.o. qd [3]

[1]. Inhibitors of notch signalling pathway and use thereof in treatment of cancers. US9296682B2.

[2]. Development of a novel first-in-class oral inhibitor of the NOTCH pathway.

[3]. Non clinical pharmacology, pharmacokinetics and safety profiling of CB-103: A novel first-in-class small molecule inhibitor of the NOTCH pathway.

[4]. First-in-human phase 1-2A study of CB-103, an oral Protein-Protein Interaction Inhibitor targeting pan-NOTCH signalling in advanced solid tumors and blood malignancies.

CB-103 is under investigation in clinical trial NCT03422679 (Study of CB-103 in Adult Patients With Advanced or Metastatic Solid Tumours and Haematological Malignancies).
Limantrafin is an orally bioavailable protein-protein interaction (PPI) inhibitor that targets the assembly of the NOTCH transcription complex, with potential antineoplastic activity. Upon oral administration, limantrafin targets and inhibits the NOTCH transcriptional activation complex in the cell nucleus. This inhibits the expression of NOTCH target genes and prevents NOTCH signaling, which may inhibit the proliferation of tumor cells mediated by an overly-active Notch pathway. Overactivation of the Notch signaling pathway, often triggered by activating mutations, has been correlated with increased cellular proliferation and poor prognosis in certain tumor types.

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Background: The NOTCH pathway is aberrantly activated in multiple cancers (T-cell lymphoma, AML, colorectal cancer, breast cancer) by mutations or ligand overexpression, promoting cell proliferation, survival, and metastasis. Limantrafin (CB-103) targets the NOTCH-CSL protein-protein interaction, a unique mechanism distinct from γ-secretase inhibitors (GSIs) [1][2][4]
- Mechanism of action: Limantrafin (CB-103) binds to the NICD of NOTCH 1-4, blocking interaction with CSL transcription factor. This prevents recruitment of co-activators, inhibiting transcription of NOTCH target genes (HES1, HEY1) and suppressing cancer cell proliferation/survival [1][2][3]
- Therapeutic potential: The compound is being evaluated in Phase 1-2A clinical trials for advanced solid tumors (colorectal, breast, ovarian) and blood malignancies (T-cell lymphoma, AML) with NOTCH pathway activation. Its oral bioavailability and favorable safety profile support long-term administration [4]
- Chemical feature: Limantrafin (CB-103) is a small-molecule inhibitor with a molecular weight of ~410 Da, soluble in DMSO (≥20 mM) and aqueous formulations (1.8 mg/mL in pH 7.4 buffer). It is stable in simulated gastric (pH 1.2) and intestinal (pH 6.8) fluids [1][3]
- Advantage over GSIs: Unlike GSIs (which block all NOTCH cleavage, causing on-target toxicities like gastrointestinal and skin side effects), Limantrafin (CB-103) selectively inhibits NOTCH transcriptional activation, reducing off-target toxicity [2][4]

C15H18N2O

242.322

242.142

C, 74.35; H, 7.49; N, 11.56; O, 6.60

218457-67-1

CB-103 HCl;218457-67-1;

2735289

White to light brown solid powder

1.09g/cm3

394.2ºC at 760mmHg

89-90ºC

192.2ºC

2.01E-06mmHg at 25°C

1.574

4.334

48.14

1

3

3

18

254

0

0

WHIWGRCYMQLLAO-UHFFFAOYSA-N

InChI=1S/C15H18N2O/c1-15(2,3)11-4-7-13(8-5-11)18-14-9-6-12(16)10-17-14/h4-10H,16H2,1-3H3

5-Amino-2-(4-tert-butylphenoxy)pyridine

CB 103CB-103 CB103

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)

DMSO : ~100 mg/mL (~412.68 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (10.32 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 25.0 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 2 中的溶解度: ≥ 2.5 mg/mL (10.32 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (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 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 3 中的溶解度: ≥ 2.08 mg/mL (8.58 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澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入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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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。