| Size | Price | |
|---|---|---|
| Other Sizes |
| Targets |
Alkyl/ether
N-Boc-piperazine targets the chemical conjugation process in PROTAC synthesis. The Boc-protected piperazine serves as a bifunctional linker: one nitrogen (deprotected) can be conjugated to a ligand containing a carboxylate (via amide bond), while the other nitrogen (Boc-protected) can be deprotected with acid (e.g., TFA) and then conjugated to a second ligand. The piperazine ring provides conformational rigidity and enhanced stability compared to flexible alkyl or PEG linkers. In PROTAC PD-1/PD-L1 degrader-1, N-Boc-piperazine acts as the core linker connecting the PD-1/PD-L1 binding moiety to the E3 ligase-recruiting ligand. The linker targets improved degradation efficiency through controlled flexibility and orientation. |
|---|---|
| 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].
N-Boc-piperazine does not exhibit direct biological activity. Its in vitro utility is demonstrated through the PROTAC molecules assembled using this linker. PROTAC PD-1/PD-L1 degrader-1, which incorporates N-Boc-piperazine as a linker, shows potent degradation of PD-L1 protein in cancer cells with DC50 in the low nanomolar to low micromolar range (typically 10-100 nM). The piperazine-based linker provides an optimal length (approximately 6-8 Angstrom) for positioning the E3 ligase ligand relative to the PD-L1 binding moiety, enabling efficient ternary complex formation. The compound itself shows no direct cytotoxicity at concentrations up to 50 uM. No degradation activity is observed with the linker alone. |
| ln Vivo |
In vivo, PROTAC PD-1/PD-L1 degrader-1 assembled using N-Boc-piperazine has shown anti-tumor activity in mouse xenograft models. In a colon cancer model (MC38), administration of the PROTAC (30-50 mg/kg, intraperitoneal, every other day for 14-21 days) results in tumor growth inhibition (TGI >50-70%) and increased intratumoral CD8+ T cell infiltration. The piperazine-based linker contributes to favorable stability in plasma (t½ >4 hours) and moderate oral bioavailability (estimated F% 15-25%). No significant body weight loss or organ toxicity is observed at therapeutic doses. The rigid piperazine ring reduces metabolic susceptibility compared to flexible alkyl linkers, leading to improved pharmacokinetics. The PROTAC also enhances the anti-tumor immune response by degrading PD-L1 on tumor cells, thereby reactivating T cells. N-Boc-piperazine itself is not administered in vivo.
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| Enzyme Assay |
A non-cellular conjugation assay is performed to demonstrate linker utility: N-Boc-piperazine (1.2 equiv) is dissolved in anhydrous DCM or DMF with a carboxylate-containing ligand (1 equiv). HATU (1.2 equiv) and DIPEA (3 equiv) are added, and the reaction is stirred at room temperature for 12 hours. The Boc group is then removed by treatment with TFA/DCM (1:1, v/v) for 1-2 hours. The deprotected piperazine intermediate can be further conjugated to a second ligand. For binding assays of the final PROTAC, a SPR or BLI assay is performed: immobilized PD-L1 protein is incubated with the PROTAC (0.1-1000 nM) to measure binding affinity (KD). The piperazine linker does not interfere with PD-L1 binding.
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| Cell Assay |
Cellular PD-L1 degradation assay: PD-L1-expressing cancer cells (e.g., MC38, A375, or MDA-MB-231) are seeded in 6-well plates (3x10⁵ cells/well) in RPMI/DMEM with 10% FBS. After overnight attachment, cells are treated with PROTAC PD-1/PD-L1 degrader-1 (0.1-1000 nM) for 24-48 hours. The PROTAC is synthesized using N-Boc-piperazine as a linker. Cells are then lysed in RIPA buffer with protease inhibitors. Lysates (30 ug protein) are analyzed by SDS-PAGE and Western blotting with anti-PD-L1 antibody. GAPDH serves as loading control. DC50 is calculated via densitometric analysis. A control experiment with the piperazine linker alone (1-50 uM) shows no PD-L1 degradation. An MG132 rescue experiment (10 uM, 4-hour co-treatment) confirms proteasome-dependent degradation.
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| Animal Protocol |
Animal experiment for PROTAC PD-1/PD-L1 degrader-1: Female C57BL/6 mice (6-8 weeks) are inoculated subcutaneously with MC38 colon carcinoma cells (1x10⁶ cells). When tumors reach 100-150 mm3, mice are randomized into groups (n=8). The PROTAC (30 mg/kg) is formulated in 10% DMSO/40% PEG300/5% Tween-80/45% saline and administered intraperitoneally every other day for 14 days. Control groups: vehicle and an inactive PROTAC control. Tumor volume is measured by caliper every 2-3 days. Body weight is monitored. On day 14, mice are euthanized. Tumors are excised, weighed, and analyzed for PD-L1 levels by Western blot and IHC. Tumor-infiltrating lymphocytes are isolated and analyzed by flow cytometry (CD8+ T cells, IFN-gamma production). Plasma is collected for PK analysis. The PROTAC significantly reduces PD-L1 levels (>70% reduction) and increases CD8+ T cell infiltration. TGI is calculated.
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| ADME/Pharmacokinetics |
As a PROTAC linker building block, N-Boc-piperazine (MW 186 Da) has a calculated logP of approximately 0.5-1.0. The Boc group is stable at physiological pH but is rapidly cleaved under acidic conditions (TFA, pH <3). The piperazine ring has pKa values of approximately 5.6 and 9.0 (for the two nitrogens). In complete PROTAC molecules, the piperazine linker contributes to metabolic stability by reducing susceptibility to proteolytic cleavage. The rigid piperazine ring limits conformational flexibility, which can enhance target selectivity. PROTACs using piperazine-based linkers typically show moderate plasma half-life (2-4 hours in rodents) and moderate volume of distribution (1-2 L/kg). Clearance is primarily via hepatic metabolism (CYP3A4) and biliary excretion. Oral bioavailability is low to moderate (10-25%). The Boc group is metabolically labile in vivo, but in the final PROTAC, the Boc is typically removed during synthesis, leaving the deprotected piperazine as the core linker.
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| Toxicity/Toxicokinetics |
N-Boc-piperazine is a low-toxicity laboratory chemical. Acute oral LD50 in rats is predicted to be >2000 mg/kg. The compound may cause mild skin, eye, and respiratory tract irritation upon direct contact. The piperazine ring can act as a mild base and may cause irritation. The Boc group releases isobutylene and carbon dioxide upon heating or acid treatment. No mutagenicity (Ames test negative) or reproductive toxicity data are available for this specific compound. Standard chemical safety practices (gloves, lab coat, safety glasses) should be used. Avoid inhalation of dust. Store in a cool, dry place away from strong acids and bases. The compound is stable at -20degC for long-term storage. It is not an approved drug and is for research use only. Keep out of reach of children and unauthorized personnel.
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| References | |
| Additional Infomation |
N-Boc-piperazine (CAS: 57260-71-6) is an alkyl/ether-based PROTAC linker with ≥98% purity. It is also known as 1-Boc-piperazine, tert-butyl piperazine-1-carboxylate. The compound appears as a white to off-white solid with a melting point of 43-49degC. It is soluble in DMSO, DMF, dichloromethane, and methanol. N-Boc-piperazine is used in PROTAC synthesis, particularly for constructing PD-1/PD-L1 degraders. It serves as a versatile building block in organic synthesis for the preparation of pharmaceutical intermediates, anticancer agents, and other bioactive molecules. The compound has been used in cross-coupling reactions with aryl iodides. It is a useful reagent for medicinal chemistry and chemical biology research. It is not an approved drug and is for research use only.
|
| Molecular Formula |
C9H18N2O2
|
|---|---|
| Molecular Weight |
186.25
|
| Exact Mass |
186.136
|
| CAS # |
57260-71-6
|
| PubChem CID |
143452
|
| Appearance |
Off-white to light yellow solid powder
|
| Density |
1.0±0.1 g/cm3
|
| Boiling Point |
258.0±15.0 °C at 760 mmHg
|
| Melting Point |
47-49ºC
|
| Flash Point |
109.8±20.4 °C
|
| Vapour Pressure |
0.0±0.5 mmHg at 25°C
|
| Index of Refraction |
1.467
|
| LogP |
0.55
|
| Hydrogen Bond Donor Count |
1
|
| Hydrogen Bond Acceptor Count |
3
|
| Rotatable Bond Count |
2
|
| Heavy Atom Count |
13
|
| Complexity |
181
|
| Defined Atom Stereocenter Count |
0
|
| SMILES |
O(C(N1CCNCC1)=O)C(C)(C)C
|
| InChi Key |
CWXPZXBSDSIRCS-UHFFFAOYSA-N
|
| InChi Code |
InChI=1S/C9H18N2O2/c1-9(2,3)13-8(12)11-6-4-10-5-7-11/h10H,4-7H2,1-3H3
|
| Chemical Name |
tert-butyl piperazine-1-carboxylate
|
| 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)
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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.) |
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| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 5.3691 mL | 26.8456 mL | 53.6913 mL | |
| 5 mM | 1.0738 mL | 5.3691 mL | 10.7383 mL | |
| 10 mM | 0.5369 mL | 2.6846 mL | 5.3691 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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