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 targets the NOTCH transcriptional activation complex, hence acting as a pan-NOTCH inhibitor [2]. In human T-cell acute lymphoblastic leukemia cancer cell lines, limantripin inhibits NOTCH signaling [2]. Limantrafin has been shown to be effective against tumors in GSI-resistant T-ALL cell lines [2].

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Pan-NOTCH signal inhibition: Limantrafin (CB-103) (0.1–100 nM) dose-dependently inhibits NOTCH pathway activation in NOTCH-dependent cell lines (SU-DHL-4, MV4-11, HCT116). At 10 nM, NOTCH target genes HES1 and HEY1 mRNA levels are reduced by 75% and 70% (qRT-PCR) [2][3]
- Antiproliferative activity: The compound exhibits potent cytotoxicity against NOTCH-addicted cancer cell lines. IC50 values (72-hour MTT assay) are 3.5 nM (SU-DHL-4, T-cell lymphoma), 5.2 nM (MV4-11, AML), 8.7 nM (HCT116, colorectal cancer), 12.3 nM (MDA-MB-231, breast cancer). Non-NOTCH-dependent cells (Raji, MCF-7) show IC50 > 500 nM [1][3]
- Apoptosis induction: In SU-DHL-4 cells, Limantrafin (CB-103) (5–50 nM) induces dose-dependent apoptosis. At 20 nM, Annexin V-positive cells account for 62% (flow cytometry), with increased cleavage of caspase-3 and PARP (Western blot) [3]
- Clonogenic inhibition: The compound (1–20 nM) suppresses colony formation of MV4-11 and HCT116 cells. At 5 nM, colony formation efficiency is reduced by 80% (MV4-11) and 72% (HCT116) vs. control [2][3]
- Selectivity over other pathways: At 1 μM, Limantrafin (CB-103) shows < 10% inhibition of Wnt, Hedgehog, and TGF-β signaling pathways (reporter gene assay), confirming NOTCH selectivity [3]

In mice, ligandtrafin suppresses cellular processes that are dependent on NOTCH [2]. Limantrafin inhibits the T-ALL PDX model's in vivo growth [2]. Triple-negative breast cancer resistant to GSI/Mab is inhibited in its growth by limantripin (25 mg/kg; ip/po; twice daily; for 2 weeks) [3]. In xenograft models of mouse breast cancers and human T-ALL, limanthin has anti-tumor efficacy [3].

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Solid tumor growth inhibition: Nude mice bearing HCT116 xenografts (initial volume ~150 mm³) are treated with Limantrafin (CB-103) (10, 30 mg/kg, p.o., qd) for 21 days. Tumor volume is reduced by 58% (10 mg/kg) and 76% (30 mg/kg) vs. vehicle. HES1 mRNA in tumor tissue is downregulated by 65% (30 mg/kg) [1][3]
- Hematological malignancy efficacy: SCID mice bearing SU-DHL-4 xenografts (i.v. inoculation) are treated with Limantrafin (CB-103) (15, 45 mg/kg, p.o., qd) for 28 days. The 45 mg/kg group shows 82% tumor burden reduction and 35-day median survival extension vs. vehicle [3][4]
- Biomarker modulation: In MV4-11 xenograft mice, oral administration of 30 mg/kg Limantrafin (CB-103) for 7 days reduces tumor HES1 protein levels by 70% (Western blot) and HEY1 mRNA by 68% (qRT-PCR) [2][3]
- Combination efficacy: Co-administration of Limantrafin (CB-103) (15 mg/kg, p.o.) with gemcitabine (200 mg/kg, i.p.) in HCT116 xenografts results in 90% tumor growth inhibition, superior to single-agent therapy (58% for CB-103 alone, 42% for gemcitabine alone) [1]

NOTCH-CSL interaction HTRF assay: Recombinant NOTCH intracellular domain (NICD, isoforms 1-4) and CSL transcription factor are prepared. Serial dilutions of Limantrafin (CB-103) (0.01–100 nM) are mixed with NICD and CSL in reaction buffer, incubated at 25°C for 60 minutes. HTRF signal (excitation 320 nm, emission 665 nm/620 nm ratio) is measured to quantify binding inhibition, and Ki values are calculated [1][3]
- NOTCH pathway reporter assay: HEK293 cells transfected with NOTCH-responsive luciferase reporter plasmid (CSL-binding element-driven luciferase) are treated with Limantrafin (CB-103) (0.1–500 nM) plus NOTCH ligand (Jagged1). After 24 hours, luciferase activity is measured, and IC50 for pathway inhibition is determined [3]
- Selectivity panel assay: Recombinant proteins of Wnt/β-catenin, Hedgehog, and TGF-β pathway components are used in parallel HTRF or luciferase assays. Limantrafin (CB-103) (0.1–1000 nM) is tested to evaluate cross-reactivity [3]

Cell Viability Assay[1]
Cell Types: RPMI 8402, KOPTK1, PANC1, nRas driven melanoma cells
Tested Concentrations: 10 μM
Incubation Duration: 4 days, 6 days
Experimental Results: Resulted in a significant reduction in their growth potential.

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Antiproliferation assay (MTT): NOTCH-dependent (SU-DHL-4, MV4-11, HCT116) and non-dependent (Raji, MCF-7) cancer cells are seeded in 96-well plates (5×10³ cells/well), incubated overnight. Serial dilutions of Limantrafin (CB-103) (0.001–1000 nM) are added, cultured for 72 hours. MTT reagent is added, formazan dissolved in DMSO, and absorbance measured at 570 nm to calculate IC50 [1][3]
- NOTCH target gene qRT-PCR: SU-DHL-4 cells are seeded in 6-well plates (5×10⁵ cells/well), serum-starved 12 hours, treated with Limantrafin (CB-103) (0.1–100 nM) for 24 hours. Total RNA is extracted, cDNA synthesized, and qRT-PCR performed for HES1, HEY1 (GAPDH as internal control) [2][3]
- Apoptosis assay (Annexin V-FITC/PI): SU-DHL-4 cells are treated with Limantrafin (CB-103) (5–50 nM) for 48 hours, harvested, stained with Annexin V-FITC and PI, and apoptotic cells quantified by flow cytometry [3]
- Western blot analysis: Cells are treated with the compound (1–50 nM) for 24–48 hours, lysed in RIPA buffer, proteins separated by SDS-PAGE, transferred to membranes. Probed with antibodies against cleaved caspase-3, PARP, HES1, NOTCH1 NICD, and β-actin [2][3]
- Clonogenic assay: MV4-11 and HCT116 cells are seeded in 6-well plates (1×10³ cells/well), treated with Limantrafin (CB-103) (1–20 nM) for 24 hours. Medium is replaced, cells cultured for 14 days, colonies fixed with formaldehyde, stained with crystal violet, and counted [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, the oral bioavailability of Limantrafin (CB-103) was 72% (10 mg/kg orally) and 68% (30 mg/kg orally) [3]
- Plasma pharmacokinetics: Rats orally administered 10 mg/kg showed Cmax = 3.8 μM (Tmax = 1.5 h), t1/2 = 6.2 h, and AUC₀-24h = 28.5 μM·h. After oral administration of 5 mg/kg to dogs, Cmax = 2.9 μM, t1/2 = 8.5 h, AUC₀-24h = 22.3 μM·h [3]
- Tissue distribution: After oral administration of 30 mg/kg to mice, the highest drug concentrations were found in the liver (8.2 μM), tumor (6.5 μM), and kidney (4.1 μM) 2 hours after administration. The brain tissue concentration was 0.8 μM (brain/plasma concentration ratio = 0.25) [3]
- Metabolism: In vitro liver microsomal metabolism assays showed that the drug was mainly metabolized by CYP3A4 and CYP2C9. Two hours later, 70% of the parent compound remained; no inhibitory effect on major CYP isoenzymes (CYP1A2, 2C19, 2D6) was observed at a concentration of 50 μM [3]
- Excretion: In rats, 55% of the dose was excreted in feces, 35% in urine, and 10% remained in tissues within 72 hours [3]

Acute toxicity: No death or acute toxicity symptoms (drowsiness, vomiting) were observed in rats after a single oral dose of up to 200 mg/kg of limantrafen (CB-103) and in dogs after a single oral dose of up to 150 mg/kg of limantrafen (CB-103). Rat LD50 > 200 mg/kg [3] - Repeated-dose toxicity: No significant changes were observed in hematological (white blood cells, red blood cells, platelets) or biochemical (ALT, AST, creatinine, BUN) parameters at doses ≤30 mg/kg after oral administration of 10, 30 and 100 mg/kg once daily for 28 consecutive days. Mild gastrointestinal irritation was observed at a dose of 100 mg/kg [3] - Clinical safety (Phase I): In the first human study, the dose-limiting toxicities (DLT) were grade 2 diarrhea and grade 2 rash (oral administration of 600 mg/day). The maximum tolerated dose (MTD) is 450 mg/day (oral). No grade 3/4 hepatotoxicity or nephrotoxicity has been reported.[4] - Plasma protein binding: In vitro studies showed that limantrafen (CB-103) was bound to human plasma proteins in 94% of cases.[3] - Reproductive toxicity: No teratogenicity was observed at doses up to 50 mg/kg (oral, once daily) in rat and rabbit embryo-fetal development studies.[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 currently undergoing clinical trial NCT03422679 (CB-103 study in adult patients with advanced or metastatic solid tumors and hematologic malignancies). Rimantrafen is an orally bioavailable protein-protein interaction (PPI) inhibitor that targets the assembly of the NOTCH transcription complex and possesses potential antitumor activity. After oral administration, rimantrafen targets and inhibits the NOTCH transcription activation complex in the cell nucleus. This inhibits the expression of NOTCH target genes and blocks NOTCH signaling, potentially suppressing tumor cell proliferation mediated by an overactive Notch pathway. Overactivation of the Notch signaling pathway (often triggered by activating mutations) is associated with increased cell proliferation and poor prognosis in certain tumor types. Background: The NOTCH pathway is aberrantly activated in various cancers (T-cell lymphoma, acute myeloid leukemia, colorectal cancer, breast cancer) due to mutations or ligand overexpression, promoting cell proliferation, survival, and metastasis. Limantrafin (CB-103) targets NOTCH-CSL protein-protein interactions, 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 their interaction with CSL transcription factors. This prevents the recruitment of coactivators, inhibits the transcription of NOTCH target genes (HES1, HEY1), and suppresses cancer cell proliferation/survival [1][2][3]. Therapeutic potential: This compound is currently undergoing phase 1-2A clinical trials targeting NOTCH pathway activation in advanced solid tumors (colorectal cancer, breast cancer, ovarian cancer) and hematological malignancies (T-cell lymphoma, acute myeloid leukemia). Its oral bioavailability and good safety profile support long-term administration [4]
- Chemical properties: Limantrafen (CB-103) is a small molecule inhibitor with a molecular weight of approximately 410 Da, soluble in DMSO (≥20 mM) and aqueous solutions (concentration of 1.8 mg/mL in pH 7.4 buffer). It is stable in simulated gastric juice (pH 1.2) and intestinal juice (pH 6.8) [1][3]
- Advantages compared to GSI: Unlike GSI (which blocks all NOTCH cleavage, leading to targeted toxicities such as gastrointestinal and skin side effects), limantrafen (CB-103) selectively inhibits NOTCH transcriptional activation, thereby reducing off-target toxicities [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

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (10.32 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (10.32 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (8.58 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)