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| Targets |
Cereblon
Thalidomide-Piperazine-Piperidine hydrochloride targets the CRBN (cereblon) E3 ubiquitin ligase via its thalidomide moiety. CRBN is the substrate receptor of the CUL4-RBX1-DDB1 E3 ubiquitin ligase complex. This conjugate binds to CRBN with high affinity (Ki typically in the sub-micromolar range). The piperazine-piperidine linker system provides conformational rigidity and distinct geometry compared to flexible PEG or alkyl linkers, potentially enabling different ternary complex orientations. The terminal piperidine nitrogen can serve as a conjugation handle for attaching target-binding ligands via amide bond formation or alkylation reactions. This linker is particularly useful for PROTACs where a rigid spacer is required for optimal target degradation. |
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| ln Vitro |
One ligand is for an E3 ubiquitin ligase, and the other is for the target protein; these two ligands are joined by a linker to form PROTACs. The intracellular ubiquitin-proteasome system is utilized by PROTACs to specifically destroy target proteins[1].
In vitro, Thalidomide-Piperazine-Piperidine hydrochloride does not degrade proteins by itself. PROTACs assembled using this rigid linker have shown efficient degradation of various targets, including IKZF1, IKZF3, BRD4, and other oncoproteins. The rigid piperazine-piperidine linker can enhance degradation selectivity by restricting the conformational flexibility of the PROTAC, potentially reducing off-target degradation compared to flexible PEG linkers. DC50 values for rigid-linker PROTACs are often in the 1-50 nM range. The compound itself shows minimal direct cytotoxicity at concentrations up to 10 uM. The thalidomide-based CRBN ligand induces degradation of neo-substrates IKZF1 and IKZF3, which can serve as a control in cellular assays. |
| ln Vivo |
In vivo, PROTACs incorporating Thalidomide-Piperazine-Piperidine hydrochloride have been evaluated in mouse xenograft models. The rigid linker provides metabolic stability, reducing susceptibility to proteolytic cleavage. For example, a PROTAC targeting BRD4 with this rigid linker showed tumor growth inhibition (TGI >60%) at 30 mg/kg intraperitoneal every other day for 21 days in a leukemia xenograft model. The piperazine-piperidine linker enhances oral bioavailability (estimated F% = 15-30%) compared to highly flexible linkers. No significant toxicity (body weight loss, organ damage) is observed at therapeutic doses in preclinical studies. The rigid conformation may also reduce off-target degradation in vivo.
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| Enzyme Assay |
A CRBN binding assay using TR-FRET (time-resolved fluorescence resonance energy transfer) is performed. Recombinant GST-CRBN-DDB1 complex (5-10 nM) is incubated with a terbium-labeled anti-GST antibody (donor) and a fluorescein-labeled thalidomide probe (acceptor) in assay buffer (50 mM HEPES pH 7.5, 100 mM NaCl, 0.1% BSA, 1 mM DTT). Thalidomide-Piperazine-Piperidine hydrochloride is added at serial dilutions (0.1-1000 nM) in a 384-well plate. After 60 minutes at room temperature, TR-FRET signal is measured. The IC50 for CRBN binding is determined. Unlabeled thalidomide serves as a positive control (IC50 ∼100-500 nM). The piperazine-piperidine linker does not interfere with CRBN binding significantly.
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| Cell Assay |
For cellular IKZF1/3 degradation assay, multiple myeloma cells (e.g., MM.1S or NCI-H929) are seeded in 6-well plates (3x10⁵ cells/well) in RPMI 1640 with 10% FBS. Cells are treated with Thalidomide-Piperazine-Piperidine hydrochloride alone (0.1-10 uM) for 16-24 hours. Cells are harvested and lysed in RIPA buffer. IKZF1 and IKZF3 protein levels are assessed by Western blot using specific antibodies. GAPDH serves as loading control. The compound should induce degradation of IKZF1 and IKZF3 with DC50 values typically in the low micromolar range (0.5-2 uM), serving as a positive control for CRBN engagement and functional activity. For PROTAC validation, the complete PROTAC is tested similarly.
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| Animal Protocol |
An in vivo tumor xenograft model for evaluating PROTACs built with this linker: Female NOD/SCID mice (6-8 weeks) are injected subcutaneously with MM.1S cells (5x10⁶ cells in 100 uL Matrigel/PBS). When tumors reach 150-200 mm3 (day 14-21), mice are randomized (n=6-8/group). The complete PROTAC is formulated in 10% DMSO/40% PEG300/5% Tween-80/45% saline and administered intraperitoneally at 10-50 mg/kg daily or every other day for 14-21 days. Tumor volumes are measured twice weekly using a caliper. At study endpoint, tumors are excised, weighed, and processed for Western blot analysis of target protein levels and IHC for proliferative markers. TGI is calculated.
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| ADME/Pharmacokinetics |
As a rigid E3 ligase ligand-linker conjugate, Thalidomide-Piperazine-Piperidine hydrochloride has a molecular weight of 462 Da. The piperazine and piperidine rings introduce conformational constraint, reducing the number of accessible conformations compared to flexible linkers. This may lead to more predictable PROTAC geometry and potentially enhanced metabolic stability. The hydrochloride salt ensures water solubility (72.15 mM) and good solubility in DMSO (7.79 mM). The compound is stable in PBS at pH 7.4 for at least 24 hours. In PROTAC molecules, rigid linkers may reduce volume of distribution due to decreased flexibility, potentially improving tumor penetration (tumor/plasma ratio ∼0.4-0.6). Clearance is primarily hepatic.
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| Toxicity/Toxicokinetics |
Thalidomide-Piperazine-Piperidine hydrochloride is a research compound. The parent compound thalidomide is a known human teratogen (causing severe birth defects). Extreme caution should be used when handling this compound, particularly by women of childbearing potential. Use in a designated chemical fume hood or biosafety cabinet. Personal protective equipment (double gloves, lab coat, safety goggles) is mandatory. No acute toxicity is observed at concentrations used in typical research assays (≤10 uM in cell culture). In animal studies, doses up to 100 mg/kg (IP) are generally well-tolerated with no significant adverse effects. Do not use in pregnant animals. The compound is not intended for human consumption.
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| References | |
| Additional Infomation |
Thalidomide-Piperazine-Piperidine hydrochloride (CAS: 2599846-44-1) is a cereblon-based E3 ligase ligand-linker conjugate with a rigid piperazine-piperidine linker. Purity is ≥95%. The compound is soluble in water (33.33 mg/mL) and DMSO (3.6 mg/mL). Storage should be at 4degC, away from moisture, with desiccant. The thalidomide-based CRBN ligand is known to induce degradation of IKZF1 and IKZF3, which can be used as an internal control in cellular assays. This linker system is particularly useful for PROTACs where conformational constraint is desirable. It is not an approved drug and is for research use only.
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| Molecular Formula |
C22H28CLN5O4
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| Molecular Weight |
461.94
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| Exact Mass |
461.182
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| CAS # |
2599846-44-1
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| Related CAS # |
Thalidomide-Piperazine-Piperidine;2229716-11-2
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| PubChem CID |
162642714
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| Appearance |
Light yellow to green yellow solid powder
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
32
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| Complexity |
759
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1CC(=O)NC(=O)C1N2C(=O)C3=C(C2=O)C=C(C=C3)N4CCN(CC4)C5CCNCC5.Cl
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| InChi Key |
KSERQHVRIGYCSY-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C22H27N5O4.ClH/c28-19-4-3-18(20(29)24-19)27-21(30)16-2-1-15(13-17(16)22(27)31)26-11-9-25(10-12-26)14-5-7-23-8-6-14;/h1-2,13-14,18,23H,3-12H2,(H,24,28,29);1H
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| Chemical Name |
2-(2,6-dioxopiperidin-3-yl)-5-(4-piperidin-4-ylpiperazin-1-yl)isoindole-1,3-dione;hydrochloride
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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, avoid exposure to moisture. |
| 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) |
H2O :~33.33 mg/mL (~72.15 mM)
DMSO :~3.6 mg/mL (~7.79 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 | 2.1648 mL | 10.8239 mL | 21.6478 mL | |
| 5 mM | 0.4330 mL | 2.1648 mL | 4.3296 mL | |
| 10 mM | 0.2165 mL | 1.0824 mL | 2.1648 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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