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| Targets |
Piperidine‑GNE‑049‑N‑Boc binds to the CBP/p300 bromodomain of its target protein, acting as the warhead in PROTACs such as dCBP‑1. The primary targets are the histone acetyltransferases p300 (EP300) and CREB‑binding protein (CBP, CREBBP). These proteins are transcriptional co‑activators that play critical roles in gene expression regulation, including the activation of oncogenes such as MYC and androgen receptor (AR) target genes. In PROTAC constructs, Piperidine‑GNE‑049‑N‑Boc engages the target protein p300/CBP, while a separate ligand recruits the E3 ubiquitin ligase cereblon (CRBN), leading to selective ubiquitination and subsequent proteasomal degradation of p300/CBP.
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| ln Vitro |
Piperidine-GNE-049-N-Boc (S17) is suitable for the synthesis of dCBP-1[1].
As a standalone compound, Piperidine‑GNE‑049‑N‑Boc inhibits p300/CBP with biochemical IC50 values of 1.1 nM (CBP) and 2.3 nM (p300) in TR‑FRET assays. It displays excellent selectivity (>3820‑fold) over other bromodomains such as BRD4. In cellular assays, Piperidine‑GNE‑049‑N‑Boc (via its parent GNE‑049) inhibits a BRET cellular assay with an IC50 of 12 nM and suppresses MYC expression in MV‑4‑11 cells with an EC50 of 14 nM. When incorporated into the PROTAC dCBP‑1, it induces potent and selective degradation of both p300 and CBP, achieving near‑complete degradation (<98%) in multiple cancer cell lines, including AR‑positive prostate cancer cells. |
| ln Vivo |
In vivo efficacy has been demonstrated for Piperidine‑GNE‑049‑N‑Boc‑based PROTAC degraders (e.g., dCBP‑1) in preclinical models of multiple myeloma and prostate cancer. In mice bearing castration‑resistant prostate cancer xenografts, treatment with the degraders induced significant tumor growth inhibition and suppressed AR target gene expression. The degraders also abrogate enhancer activity driving MYC oncogene expression, leading to anticancer effects. In multiple myeloma xenograft models, dCBP‑1 degrader demonstrated exceptional potency in killing myeloma cells and eliminating tumor‑promoting enhancer activity. These in vivo activities are attributed to the sustained degradation of p300/CBP, which disrupts oncogenic transcriptional programs, including AR signaling and MYC expression.
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| Enzyme Assay |
The binding affinity of Piperidine‑GNE‑049‑N‑Boc to purified p300/CBP bromodomain is typically measured by time‑resolved fluorescence resonance energy transfer (TR‑FRET) or bioluminescence resonance energy transfer (BRET) assays. Recombinant CBP bromodomain (50 nM) is incubated with a biotinylated acetylated histone peptide and a europium‑labeled anti‑GST antibody in assay buffer (50 mM HEPES, pH 7.4, 0.01% Triton X‑100, 1 mM DTT). Test compound is added at increasing concentrations (0.1 nM - 10 microM) and incubated for 1‑2 hours. TR‑FRET signal is measured at 665 nm and 620 nm after excitation at 337 nm. IC50 values are calculated by nonlinear regression. For BRET assays, a fusion protein of CBP and luciferase is used.
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| Cell Assay |
For cellular assays, cells are seeded in 96‑well plates (5,000‑10,000 cells/well) in RPMI‑1640 with 10% FBS and incubated overnight. Cells are treated with Piperidine‑GNE‑049‑N‑Boc (as a standalone compound or as part of a PROTAC) at concentrations ranging from 0.1 nM to 10 microM (0.1% DMSO) for 6‑72 hours. Target engagement is assessed by BRET assay using a CBP‑luciferase fusion protein and a substrate. For degradation studies, p300/CBP protein levels are quantified by Western blotting after 6‑24 hours of treatment with the PROTAC degrader. Cell viability is measured by MTT or CellTiter‑Glo after 48‑72 hours. MYC expression is assessed by qPCR or Western blotting to confirm downstream pathway modulation.
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| Animal Protocol |
For in vivo studies, Piperidine‑GNE‑049‑N‑Boc is formulated as a PROTAC degrader (e.g., dCBP‑1) in a vehicle such as 10% DMSO, 40% PEG300, 5% Tween‑80, and 45% saline. Male immunodeficient mice bearing subcutaneous xenografts (e.g., VCaP prostate cancer or multiple myeloma cells) are treated when tumors reach ~150‑200 mm3. The degrader is administered via oral gavage or intraperitoneal injection at doses of 10‑50 mg/kg daily or every other day for 2‑4 weeks. Tumor volumes are measured every 2‑3 days using calipers. Endpoints include tumor growth inhibition, body weight monitoring, and analysis of p300/CBP degradation in tumor homogenates by Western blot. Blood samples are collected for pharmacokinetic analysis. For CNS penetration studies, unbound drug concentrations in brain and plasma are measured.
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| ADME/Pharmacokinetics |
As a warhead in PROTACs, Piperidine‑GNE‑049‑N‑Boc contributes to favorable drug‑like properties of its PROTAC derivatives. The parent inhibitor GNE‑049 has acceptable pharmacokinetics in mouse, rat, dog, and monkey, with good oral bioavailability, moderate clearance, and a suitable half‑life for once‑daily dosing. The calculated LogP is 4.0 (XLogP3‑AA), indicating moderate lipophilicity. It is soluble in DMSO (>80 mg/mL) and in acidic conditions (e.g., 1 M HCl, 32.5 mg/mL). In rodents, the brain‑to‑plasma unbound concentration ratio (Kp,uu) of GNE‑049 is 0.43, indicating moderate CNS penetration. Storage: as a powder at -20degC for up to 3 years; in solution at -80degC for 6 months or -20degC for 1 month.
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| Toxicity/Toxicokinetics |
Piperidine‑GNE‑049‑N‑Boc is classified as non‑hazardous under normal laboratory handling conditions. The Safety Data Sheet states that the product is not a hazardous substance or mixture. However, under fire conditions, it may decompose and emit toxic fumes. Acute toxicity data specific to Piperidine‑GNE‑049‑N‑Boc are limited. The parent inhibitor GNE‑049 has been evaluated in a rat toxicokinetic study (30‑250 mg/kg daily). At the highest dose (250 mg/kg), adverse CNS‑related signs (e.g., marked hyperactivity, vocalization) were observed, likely due to brain penetration (Kp,uu = 0.43). No genotoxicity or carcinogenicity data are available for Piperidine‑GNE‑049‑N‑Boc. Standard laboratory safety precautions include handling in a well‑ventilated hood, wearing gloves and goggles, and avoiding release into the environment.
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| References | |
| Additional Infomation |
Piperidine‑GNE‑049‑N‑Boc (CAS 1936431‑36‑5) has the molecular formula C30H39F2N7O2 and a molecular weight of 567.67 g/mol. It is a solid powder with a purity of ≥98%. The compound is also known as tert‑butyl 3‑(7‑(difluoromethyl)‑6‑(1‑methyl‑1H‑pyrazol‑4‑yl)‑3,4‑dihydroquinolin‑1(2H)‑yl)‑1‑(piperidin‑4‑yl)‑6,7‑dihydro‑1H‑pyrazolo[4,3‑c]pyridine‑5(4H)‑carboxylate. It is not approved by the FDA, EMA, or any other regulatory body for clinical or therapeutic use. It is exclusively a research chemical for chemical biology and drug discovery. The compound is commercially available for research purposes from multiple chemical suppliers. It is most commonly used as a ligand for the target protein in PROTACs, such as dCBP‑1, for the selective degradation of p300/CBP. It was first described in a patent (e.g., WO2016118666A1) and in the literature by Vannam et al. (Cell Chem Biol. 2020).
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| Molecular Formula |
C30H39F2N7O2
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| Molecular Weight |
567.673173189163
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| Exact Mass |
567.313
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| CAS # |
1936431-36-5
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| PubChem CID |
121365479
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| Appearance |
White to off-white solid powder
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| LogP |
4
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
41
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| Complexity |
916
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CC(C)(C)OC(=O)N1CCC2=C(C1)C(=NN2C3CCNCC3)N4CCCC5=CC(=C(C=C54)C(F)F)C6=CN(N=C6)C
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| InChi Key |
NNCJWLPQMSOHBI-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C30H39F2N7O2/c1-30(2,3)41-29(40)37-13-9-25-24(18-37)28(35-39(25)21-7-10-33-11-8-21)38-12-5-6-19-14-22(20-16-34-36(4)17-20)23(27(31)32)15-26(19)38/h14-17,21,27,33H,5-13,18H2,1-4H3
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| Chemical Name |
tert-butyl 3-[7-(difluoromethyl)-6-(1-methylpyrazol-4-yl)-3,4-dihydro-2H-quinolin-1-yl]-1-piperidin-4-yl-6,7-dihydro-4H-pyrazolo[4,3-c]pyridine-5-carboxylate
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
1M HCl :~32.5 mg/mL (~57.25 mM)
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.7616 mL | 8.8079 mL | 17.6159 mL | |
| 5 mM | 0.3523 mL | 1.7616 mL | 3.5232 mL | |
| 10 mM | 0.1762 mL | 0.8808 mL | 1.7616 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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