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CB-103 is a novel and potent γ-secretase inhibitor that is currently being investigated in Phase-1 dose escalation in cancer patients. CB-103 produces Notch loss-of-function phenotypes in flies and mice and inhibits the growth of human breast cancer and leukemia xenografts, notably without causing the dose-limiting intestinal toxicity associated with other Notch inhibitors.
Physicochemical Properties
| Molecular Formula | C15H18N2O |
| Molecular Weight | 242.322 |
| Exact Mass | 242.142 |
| Elemental Analysis | C, 74.35; H, 7.49; N, 11.56; O, 6.60 |
| CAS # | 218457-67-1 |
| Related CAS # | CB-103 HCl;218457-67-1; |
| PubChem CID | 2735289 |
| Appearance | White to light brown solid powder |
| Density | 1.09g/cm3 |
| Boiling Point | 394.2ºC at 760mmHg |
| Melting Point | 89-90ºC |
| Flash Point | 192.2ºC |
| Vapour Pressure | 2.01E-06mmHg at 25°C |
| Index of Refraction | 1.574 |
| LogP | 4.334 |
| Hydrogen Bond Donor Count | 1 |
| Hydrogen Bond Acceptor Count | 3 |
| Rotatable Bond Count | 3 |
| Heavy Atom Count | 18 |
| Complexity | 254 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | 0 |
| InChi Key | WHIWGRCYMQLLAO-UHFFFAOYSA-N |
| InChi Code | 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 |
| Chemical Name | 5-Amino-2-(4-tert-butylphenoxy)pyridine |
| Synonyms | CB 103CB-103 CB103 |
| HS Tariff Code | 2934.99.9001 |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
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] |
| ln Vitro |
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]. 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] |
| ln Vivo |
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]. 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] |
| Enzyme Assay |
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 Assay |
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. 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 Protocol |
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. 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] |
| ADME/Pharmacokinetics |
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] |
| Toxicity/Toxicokinetics |
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] |
| References |
[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. |
| Additional Infomation |
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. 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] |
Solubility Data
| Solubility (In Vitro) | DMSO : ~100 mg/mL (~412.68 mM) |
| Solubility (In Vivo) |
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. 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. 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 4.1268 mL | 20.6339 mL | 41.2677 mL | |
| 5 mM | 0.8254 mL | 4.1268 mL | 8.2535 mL | |
| 10 mM | 0.4127 mL | 2.0634 mL | 4.1268 mL |