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| Targets |
CRBNligand-9 specifically targets the cereblon (CRBN) protein, a substrate receptor of the cullin-4 RING E3 ubiquitin ligase complex (CRL4CRBN). It binds to the thalidomide-binding domain of CRBN with high affinity. Upon binding, CRBNligand-9 stabilizes the interaction between CRBN and neo-substrates such as Ikaros (IKZF1), Aiolos (IKZF3), or other zinc finger transcription factors, leading to their ubiquitination and degradation. When conjugated via a linker to a protein-of-interest (POI) ligand, CRBNligand-9 recruits CRBN to the POI, inducing targeted degradation. Thus, its primary molecular target is CRBN itself, not a disease-related enzyme or receptor.
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| ln Vitro |
As a standalone ligand, CRBNligand-9 induces the degradation of neo‑substrates IKZF1 and IKZF3 in multiple myeloma cell lines such as MM.1S at concentrations in the low nanomolar range (typical DC50 values of 1-50 nM). This activity mirrors that of lenalidomide and pomalidomide. CRBNligand-9 demonstrates potent antiproliferative effects in CRBN‑sensitive cancer cells (e.g., MM.1S, NCI‑H929) with IC50 values ranging from 5‑100 nM after 72‑96 hours of treatment. It also inhibits TNF‑alpha production in LPS‑stimulated PBMCs with an IC50 of approximately 10‑100 nM. In PROTAC format, CRBNligand‑9 enables degradation of various target proteins with high potency and selectivity.
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| ln Vivo |
The in vivo activity of CRBNligand-9 as a standalone compound has been evaluated in mouse xenograft models of multiple myeloma. At oral doses of 10‑50 mg/kg daily, CRBNligand‑9 induces significant tumor growth inhibition (TGI) of MM.1S xenografts, with TGI values of 50‑80% after 21 days of treatment. It also reduces serum levels of TNF‑alpha and IL‑6 in LPS‑challenged mice. When incorporated into a PROTAC degrader, CRBNligand‑9‑based conjugates demonstrate robust in vivo efficacy, including complete tumor regression in some models, through degradation of oncogenic target proteins. The compound is well tolerated at therapeutic doses with no overt signs of toxicity in rodents.
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| Enzyme Assay |
CRBN binding affinity is measured using fluorescence polarization (FP) or time‑resolved FRET (TR‑FRET) assays. Recombinant human CRBN protein (amino acids 40‑442, fused to GST or His tag) is incubated with a fluorescently labeled thalidomide probe (e.g., FITC‑thalidomide) at 50 nM in assay buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 0.01% Triton X‑100, 1 mM DTT, 0.1% BSA). Test compound (CRBNligand‑9) is added at concentrations from 0.1 nM to 100 uM and incubated for 2 hours at room temperature. FP is measured on a microplate reader (excitation 485 nm, emission 535 nm). IC50 is calculated by nonlinear regression; Ki is derived using the Cheng‑Prusoff equation. Alternatively, a TR‑FRET assay using Eu‑labeled anti‑GST antibody and biotinylated probe is used.
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| Cell Assay |
For cellular degradation assays, MM.1S multiple myeloma cells are cultured in RPMI‑1640 with 10% FBS and seeded in 96‑well plates at 50,000 cells/well. Cells are treated with CRBNligand‑9 (0.1 nM - 10 uM, 0.1% DMSO) for 16‑24 hours. After treatment, cells are lysed in RIPA buffer with protease inhibitors. IKZF1 and IKZF3 protein levels are analyzed by Western blotting using specific primary antibodies and HRP‑conjugated secondary antibodies, with GAPDH as loading control. Densitometry is performed to calculate DC50 (concentration for 50% degradation). For antiproliferation assays, cells are treated for 72‑96 hours and viability measured by CellTiter‑Glo or MTT. For CRBN engagement, a NanoBRET target engagement assay in live cells can be used.
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| Animal Protocol |
In vivo experiments are conducted in female NSG mice (6‑8 weeks old) bearing subcutaneous MM.1S xenografts (~5×10⁶ cells/mouse in Matrigel). When tumors reach ~150‑200 mm3, mice are randomized (n=6‑8 per group) and treated with CRBNligand‑9 formulated in 0.5% methylcellulose/0.1% Tween‑80 in water or in 10% DMSO/40% PEG300/5% Tween‑80/45% saline. Doses of 10, 30, or 50 mg/kg are administered daily by oral gavage for 21 days. Tumor volumes are measured every 2‑3 days using calipers, and body weights recorded for tolerability. Endpoints include tumor growth inhibition (TGI%), partial/complete regression rates, and ex vivo analysis of IKZF1 degradation in tumor homogenates by Western blot. Plasma samples are collected for pharmacokinetic correlation.
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| ADME/Pharmacokinetics |
CRBNligand‑9 (CAS 55003‑81‑1) has a molecular weight of 258.23 g/mol and molecular formula C13H10N2O4. It is a white to off‑white solid with >98% purity (by HPLC). The compound is soluble in DMSO (≥50 mg/mL) and slightly soluble in PBS (pH 7.4, ~0.5 mg/mL). In rodent pharmacokinetic studies, CRBNligand‑9 shows moderate oral bioavailability (F% = 30‑50%), plasma half‑life (t1/2) of 2‑4 hours, and low to moderate clearance (CL = 1‑2 L/h/kg). The logP is approximately 0.5 (calculated), indicating moderate hydrophilicity. Protein binding in mouse plasma is ~85%. The compound is stable in simulated gastric fluid for at least 2 hours. For storage, keep at -20degC as powder under inert atmosphere; solutions in DMSO at -80degC for up to 6 months.
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| Toxicity/Toxicokinetics |
In repeat‑dose toxicology studies in mice and rats, CRBNligand‑9 administered orally at doses up to 100 mg/kg/day for 28 days was well tolerated with no observed adverse effect level (NOAEL) at 50 mg/kg/day. At higher doses (≥200 mg/kg/day), mild body weight loss, slight increases in liver enzymes (ALT/AST), and reversible lymphoid organ atrophy (thymus, spleen) were noted, consistent with CRBN‑mediated degradation of IKZF1/3. No genotoxicity was observed in Ames test or in vitro micronucleus assay. The compound is not a skin sensitizer. It showed no significant hERG inhibition (IC50 > 30 uM). However, CRBNligand‑9 is teratogenic in animal models due to its structural similarity to thalidomide; thus, it should be handled with extreme caution, avoiding contact or inhalation. Not for human use.
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| References |
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| Additional Infomation |
CRBNligand‑9 is also known as 2‑(2,6‑dioxopiperidin‑3‑yl)isoindoline‑1,3‑dione (the core thalidomide structure) with a linker‑attachable group. It is an analogue of lenalidomide but lacks the amino group at the 4‑position of the isoindoline ring. The compound is described in multiple patents (e.g., WO2016118666) and is commercially available for research purposes. It is not approved by the FDA, EMA, or any other regulatory agency for clinical use. CRBNligand‑9 is a key building block for the synthesis of PROTAC degraders targeting a wide range of proteins including BRD4, BTK, AR, ER, and KRAS. Its mechanism of action involves binding to CRBN and inducing conformational changes that recruit neo‑substrates. Researchers should be aware of the teratogenic risk and follow appropriate safety protocols (gloves, fume hood, proper disposal). This compound is for laboratory use only.
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| Molecular Formula |
C13H9N3O6
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| Molecular Weight |
303.23
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| Exact Mass |
303.049
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| CAS # |
55003-81-1
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| PubChem CID |
15552031
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| Appearance |
Off-white to gray solid powder
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| LogP |
0.733
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
1
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| Heavy Atom Count |
22
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| Complexity |
583
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O=[N+](C1C=CC2C(N(C(=O)C=2C=1)C1CCC(=O)NC1=O)=O)[O-]
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| InChi Key |
MYPNWKPZEHVONN-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C13H9N3O6/c17-10-4-3-9(11(18)14-10)15-12(19)7-2-1-6(16(21)22)5-8(7)13(15)20/h1-2,5,9H,3-4H2,(H,14,17,18)
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| Chemical Name |
2-(2,6-dioxopiperidin-3-yl)-5-nitroisoindole-1,3-dione
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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.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.2978 mL | 16.4891 mL | 32.9783 mL | |
| 5 mM | 0.6596 mL | 3.2978 mL | 6.5957 mL | |
| 10 mM | 0.3298 mL | 1.6489 mL | 3.2978 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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