| Targets |
As a synthetic building block, this compound has no direct biological target. It is a heterobifunctional linker. The piperazine ring is a common motif in medicinal chemistry that can enhance the aqueous solubility of the final PROTAC. The carboxylic acid group can be activated (e.g., with HATU or EDCI) to form an amide bond with a target protein ligand or E3 ligase ligand. The Boc group protects the secondary piperazine nitrogen.
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|---|---|
| ln Vitro |
PROTAC contains two distinct ligands linked by a single linker: one is the ligand for the E142 ubiquitin ligase, and the other is the ligand for the target protein. PROTAC utilizes the intracellular ubiquitin-proteasome system to selectively degrade the target protein.
No direct in vitro activity is reported for the isolated linker. In the context of the final PROTAC, the piperazine linker can improve degradation activity by reducing aggregation and increasing the effective concentration of the two binding domains. The DC50 (nM) is determined in a cellular degradation assay; the piperazine-based linker can reduce non-specific protein binding, leading to a more accurate measurement of degradation potency. |
| ln Vivo |
No direct in vivo activity is reported for the linker alone. For the final PROTAC, in vivo efficacy is evaluated in an animal model. The piperazine ring can significantly improve the water solubility and oral absorption of the final PROTAC compared to a purely lipophilic linker. Efficacy is measured by a reduction in a pharmacodynamic biomarker (e.g., p-ERK for growth factor receptor pathways).
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| Enzyme Assay |
Not applicable for the isolated linker. For the final PROTAC, the in vitro metabolic stability is determined by incubating the compound (1 uM) with liver microsomes (0.5 mg/mL) and an NADPH-regenerating system at 37degC. Aliquots are taken at 0, 5, 10, 20, 30, and 60 minutes. The half-life (t1/2) in the microsomal assay is calculated. The piperazine linker may be susceptible to N-dealkylation, producing a major metabolite.
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| Cell Assay |
For in vitro cell assays, the final PROTAC containing this linker is dissolved in DMSO. Dilutions of the compound are added to 96-well plates containing 2,000 target-expressing cells/well. After 48 hours, the cells are lysed in the wells, and an acceptor bead-conjugated antibody is added. The target protein concentration is measured using a homogeneous AlphaLISA assay (PerkinElmer), which requires no wash steps. The AlphaLISA signal is inversely proportional to the amount of target protein.
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| Animal Protocol |
For in vivo animal experiments, formulate the PROTAC in a 0.9% saline solution containing 0.5% HPMC and 0.2% Tween-80. Administer 100 mg/kg via oral gavage to male Beagle dogs (n=4). Collect blood samples (1 mL) from the jugular vein at 0, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours post-dose. Place blood in K2EDTA tubes and centrifuge to separate plasma. Plasma is analyzed by LC-MS/MS.
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| ADME/Pharmacokinetics |
No PK data is available for the isolated linker. For the final PROTAC, a full PK profile in dogs is generated. The piperazine-based linker can lead to an improved oral bioavailability (F% > 30%) and a longer half-life (t1/2 > 3-5 h) compared to more lipophilic linkers. Key parameters include AUC, Cmax, Tmax, t1/2, CL, Vss, and F% (calculated from IV and PO dosing data).
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| Toxicity/Toxicokinetics |
No toxicity data is available for the isolated linker. For the final PROTAC, a key safety concern is the potential for phospholipidosis, which can be caused by cationic amphiphilic drugs (including those with piperazine rings). This is assessed by measuring the accumulation of the compound in lysosomes in cultured cells (U-2 OS) using the dye LipidTOX. Positive controls (e.g., amiodarone) are used for comparison.
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| Additional Infomation |
See other relationships...
This piperazine-based linker has the molecular formula C17H24N2O4 and a molecular weight of 320.38 g/mol. Its CAS number is 479353-63-4. It is supplied as a white crystalline powder with a minimum purity of 95%. Storage is recommended in a cool, dry place. It is also known as 4-(4-tert-Butoxycarbonyl)piperazinomethylbenzoic acid. |
| Exact Mass |
320.174
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|---|---|
| CAS # |
479353-63-4
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| PubChem CID |
2795516
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| Appearance |
Powder
|
| Hydrogen Bond Donor Count |
1
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| Rotatable Bond Count |
5
|
| Heavy Atom Count |
23
|
| Complexity |
417
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| Defined Atom Stereocenter Count |
0
|
| InChi Key |
JGOFKAHLQQITIG-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C17H24N2O4/c1-17(2,3)23-16(22)19-10-8-18(9-11-19)12-13-4-6-14(7-5-13)15(20)21/h4-7H,8-12H2,1-3H3,(H,20,21)
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| Chemical Name |
4-[[4-[(2-methylpropan-2-yl)oxycarbonyl]piperazin-1-yl]methyl]benzoic acid
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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 |
| 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.) |
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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