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| Targets |
1-(4-Pip-Ph)-DHP-dione targets the E3 ubiquitin ligase cereblon (CRBN). It binds to the thalidomide-binding domain of CRBN with moderate affinity. In a PROTAC molecule, this ligand recruits the CRBN E3 ligase to a target protein, leading to ubiquitination and subsequent degradation of the target protein by the proteasome. By itself, it has no degradation activity. It is used as a warhead to hijack the cellular protein degradation machinery. The target is CRBN.
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| ln Vitro |
In vitro, this compound is not used directly for biological activity assays. Its activity is evaluated when incorporated into a PROTAC. For example, a PROTAC containing this ligand is tested in cell-based assays for degradation of a specific target (e.g., HPK1). In an intact PROTAC, it enables dose-dependent degradation of the target protein (DC50 < 100 nM). The ligand alone (1-10 uM) has no effect on target protein levels (Western blot). It is not cytotoxic. It is used as a key reagent in PROTAC synthesis.
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| ln Vivo |
In vivo, the compound is not administered alone. It is incorporated into a PROTAC, which is then evaluated in mouse xenograft models for antitumor efficacy. The pharmacokinetics of the final PROTAC are determined. The ligand itself is not an active pharmaceutical ingredient (API).
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| Enzyme Assay |
General protocol for in vitro protein binding (non-cellular): For CRBN binding, perform a time-resolved FRET (TR-FRET) competition assay. Incubate 5 nM GST-CRBN-DDB1, 5 nM terbium-labeled anti-GST antibody, and 20 nM fluorescently labeled thalidomide probe with varying concentrations of 1-(4-Pip-Ph)-DHP-dione (0.1-1000 nM) in assay buffer (50 mM HEPES pH 7.5). After 2 h, measure TR-FRET signal (Ex 340, Em 490/520). Calculate IC50 (expected 100-1000 nM).
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| Cell Assay |
General protocol for in vitro cell-based experiments: Not applicable for the ligand alone. For PROTAC testing, treat HPK1-expressing cells (e.g., Jurkat) with PROTAC (0.1-1000 nM) for 6-24 h. Lyse cells and run Western blot for HPK1. The PROTAC containing this ligand should degrade HPK1 (DC50 < 50 nM). The ligand alone (1 uM) shows no degradation.
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| Animal Protocol |
General protocol for in vivo animal experiments: Not applicable for this ligand alone. The final PROTAC is used in mice. For efficacy, administer PROTAC (10-30 mg/kg, IP) daily to tumor-bearing mice. Measure tumor volume for 2-3 weeks. The PROTAC should show TGI > 60%.
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| ADME/Pharmacokinetics |
General pharmacokinetic properties: This ligand has MW 274.32. It is a building block. PK data for the parent compound are not established. For storage, keep the powder at 4degC, protected from light. In DMSO (20 mM), it is stable at -80degC for 6 months.
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| Toxicity/Toxicokinetics |
General toxicity profile: This is a research compound. Standard safety precautions (gloves, lab coat) are required. It is not a controlled substance.
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| References | |
| Additional Infomation |
1-(4-Pip-Ph)-DHP-dione is a core structure for the development of CRBN-based PROTACs. It is for research use only.
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| Molecular Formula |
C14H18N4O2
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| Molecular Weight |
274.32
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| CAS # |
2446913-96-6
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| Appearance |
Off-white to light yellow solid powder
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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: This product requires protection from light (avoid light exposure) during transportation and storage. |
| 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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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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 | 3.6454 mL | 18.2269 mL | 36.4538 mL | |
| 5 mM | 0.7291 mL | 3.6454 mL | 7.2908 mL | |
| 10 mM | 0.3645 mL | 1.8227 mL | 3.6454 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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