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Crenigacestat (formerly also known as LY3039478) is a novel, potent and orally bioavailable small molecule Notch inhibitor with an IC50 of ~1nM in cell based assays. Also an inhibitor of γ-secretase. LY3039478 potently inhibits mutant Notch receptor activity. In a xenograft tumor model, LY3039478 inhibited N1ICD cleavage and expression of Notch-regulated genes in the tumor microenvironment. LY3039478 is being investigated in Phase I trial.
Physicochemical Properties
| Molecular Formula | C22H23F3N4O4 |
| Molecular Weight | 464.4376 |
| Exact Mass | 464.167 |
| CAS # | 1421438-81-4 |
| Related CAS # | 1421438-81-4;1421439-98-6 (H2O); |
| PubChem CID | 71236992 |
| Appearance | White to off-white solid powder |
| Density | 1.4±0.1 g/cm3 |
| Boiling Point | 818.7±65.0 °C at 760 mmHg |
| Flash Point | 448.9±34.3 °C |
| Vapour Pressure | 0.0±3.1 mmHg at 25°C |
| Index of Refraction | 1.594 |
| LogP | 1.83 |
| Hydrogen Bond Donor Count | 3 |
| Hydrogen Bond Acceptor Count | 8 |
| Rotatable Bond Count | 7 |
| Heavy Atom Count | 33 |
| Complexity | 720 |
| Defined Atom Stereocenter Count | 2 |
| SMILES | C[C@@H](C(=O)N[C@H]1C2=CC=CC=C2C3=C(N=CC=C3)N(C1=O)CCO)NC(=O)CCC(F)(F)F |
| InChi Key | YCBAQKQAINQRFW-UGSOOPFHSA-N |
| InChi Code | InChI=1S/C22H23F3N4O4/c1-13(27-17(31)8-9-22(23,24)25)20(32)28-18-15-6-3-2-5-14(15)16-7-4-10-26-19(16)29(11-12-30)21(18)33/h2-7,10,13,18,30H,8-9,11-12H2,1H3,(H,27,31)(H,28,32)/t13-,18-/m0/s1 |
| Chemical Name | 4,4,4-trifluoro-N-((S)-1-(((S)-5-(2-hydroxyethyl)-6-oxo-6,7-dihydro-5H-benzo[d]pyrido[2,3-b]azepin-7-yl)amino)-1-oxopropan-2-yl)butanamide |
| Synonyms | LY3039478; LY 3039478; LY-3039478 |
| 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 |
γ-secretase inhibitor (leading to inhibition of the NOTCH signaling pathway). |
| ln Vitro |
K07074 cells, a primary mouse liver tumor cell line, show anticancer activity in response to crenigacestat (100 nM) [2]. In mouse and human model systems, crenigacestat (LY3039478) lowers the expression of Myc and cyclin A1, two components of the NOTCH-driven proliferation signature. In CCRCC cells, treatment with crenigacestat (LY3039478) similarly results in G0/G1 cell cycle arrest [3]. Assay for cell viability [2]. Treatment with LY3039478 significantly inhibited the viability of clear cell renal cell carcinoma (ccRCC) cell lines Caki and 769-P in a concentration-dependent manner. Treatment with 1 µM LY3039478 led to decreased expression of Myc and cyclin A1 (CCNA1) as assessed by qRT-PCR. Treatment of Caki cells with LY3039478 resulted in a significant increase in the proportion of cells in the G0/G1 phase and a decrease in cells in S and G2/M phases, as determined by flow cytometry. |
| ln Vivo |
The in vivo efficacy of CCRCC was demonstrated by the significant improvement in survival and delay in tumor growth in an independent cohort of mice given oral Crenigacestat (8 mg/kg) three times a week [3]. In immunodeficient NOD-scid IL2Rγ null mice bearing subcutaneous 769-P ccRCC xenografts, treatment with LY3039478 (8 mg/kg, administered orally three times a week) significantly increased overall survival compared to vehicle control. LY3039478 treatment also led to decreased tumor growth in ccRCC xenografts in an independent cohort of mice. |
| Cell Assay |
Cell viability assay [2]. Cell Types: K07074 cells. Tested Concentrations: 100 nM. Incubation Duration: 24-96 hrs (hours). Experimental Results: Effectively inhibited the growth of K07074 cells. For cell viability assays, ccRCC cell lines (Caki and 769-P) were treated with varying concentrations of LY3039478 and compared to DMSO controls. Cell viability was assessed, and the results demonstrated concentration-dependent growth inhibition. For gene expression analysis, cells were treated with 1 µM LY3039478. RNA was then extracted, and qRT-PCR was performed using gene-specific primers for Myc and CCNA1, with GAPDH as an internal control. For cell cycle analysis, Caki cells were treated with LY3039478. Cells were then harvested, stained with propidium iodide, and analyzed by flow cytometry to determine the distribution of cells in different phases of the cell cycle. |
| Animal Protocol |
Animal/Disease Models: CCRCC xenografts were established using 769-P cell line subcutaneously (sc) (sc) implanted in NOD-scid IL2R-deficient mice [3]. Doses: 8 mg/kg. Route of Administration: po (oral gavage), 3 times per week. Experimental Results: Overall survival of CCRCC xenografts was improved compared to vehicle controls. Immunodeficient NOD-scid IL2Rγ null mice were used. Subcutaneous xenografts were established by implanting 769-P ccRCC cells. LY3039478 was formulated in 1% sodium carboxymethyl cellulose (Na-CMC), 0.25% Tween 80, and 0.05% antifoam. The drug was administered via oral gavage at a dose of 8 mg/kg, on a schedule of three times per week (e.g., Monday, Wednesday, Friday). Treatment was initiated after tumors were established. Tumor dimensions were measured at specified time points in triplicate. Vehicle solution was administered to the control group. |
| References |
[1]. Evaluation of the effects of an oral notch inhibitor, crenigacestat (LY3039478), on QT interval, and bioavailability studies conducted in healthy subjects. Cancer Chemother Pharmacol. 2019 Mar;83(3):483-492. [2]. The inhibition of Akt-Pdpk1 interaction efficiently suppresses the growth of murine primary liver tumor cells. Biochem Biophys Res Commun. 2016 May 20;474(1):118-125. [3]. Notch Pathway Is Activated via Genetic and Epigenetic Alterations and Is a Therapeutic Target in Clear Cell Renal Cancer. J Biol Chem. 2017 Jan 20;292(3):837-846. [4]. Abstract 1131: Novel inhibitor of Notch signaling for the treatment of cancer. Experimental and Molecular Therapeutics. 2013. |
| Additional Infomation |
LY3039478 has been used in trials studying the treatment and basic science of LYMPHOMA, Neoplasms, Solid Tumor, COLON CANCER, and BREAST CANCER, among others. Crenigacestat is an orally available inhibitor of the integral membrane protein gamma-secretase (GS), with potential antineoplastic activity. Upon administration, crenigacestat binds to the GS protease complex, thereby blocking the proteolytic cleavage and release of the Notch intracellular domain (NICD), which would normally follow ligand binding to the extracellular domain of the Notch receptor. This prevents both the subsequent translocation of NICD to the nucleus to form a transcription factor complex and the expression of Notch-regulated genes. This results in the induction of apoptosis and the inhibition of growth in tumor cells that overexpress Notch. Overexpression of the Notch signaling pathway plays an important role in tumor cell proliferation and survival. LY3039478 is a clinical-stage γ-secretase inhibitor that targets the NOTCH signaling pathway. In this study, LY3039478 demonstrated both in vitro and in vivo efficacy against clear cell renal cell carcinoma (ccRCC), providing a preclinical rationale for its further investigation in this malignancy. The NOTCH pathway is activated via genetic and epigenetic alterations in ccRCC and represents a potential therapeutic target. |
Solubility Data
| Solubility (In Vitro) | DMSO : ≥ 34 mg/mL (~73.21 mM) |
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.38 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 25.0 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (5.38 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 3: ≥ 2.5 mg/mL (5.38 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1531 mL | 10.7657 mL | 21.5313 mL | |
| 5 mM | 0.4306 mL | 2.1531 mL | 4.3063 mL | |
| 10 mM | 0.2153 mL | 1.0766 mL | 2.1531 mL |