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| Targets |
(R)-Desamino lenalidomide-5-Br targets cereblon (CRBN), a substrate receptor of the cullin-RING ligase 4 (CRL4) E3 ubiquitin ligase complex. CRBN recognizes and binds to the glutarimide ring present in this and related IMiD-class compounds, inducing conformational changes that alter the substrate specificity of the CRL4CRBN complex. Upon binding to CRBN, the complete PROTAC molecule containing this ligand recruits the E3 ligase machinery to the target protein, leading to its ubiquitination by E2 enzymes and subsequent degradation by the 26S proteasome. Lenalidomide and its derivatives also have intrinsic molecular glue properties, causing selective ubiquitination and degradation of neosubstrates such as IKZF1 (Ikaros) and IKZF3 (Aiolos), although the desamino modification may modulate such activities.
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| ln Vitro |
PROTAC contains two distinct ligands linked by a single linker: one is the ligand for the E3 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.
In vitro, (R)-Desamino lenalidomide-5-Br functions as the CRBN-recruiting component when conjugated via a linker to a target protein-binding ligand. The desamino modification differentiates this ligand from lenalidomide itself; structure-activity relationship (SAR) studies on lenalidomide derivatives indicate that modifications at the exocyclic amine position and at the C5 position of the isoindolinone ring can significantly affect CRBN binding affinity and neosubstrate degradation profiles. The bromine substituent at C5 provides a synthetic handle for potential cross-coupling reactions (e.g., Suzuki, Sonogashira) if alternative functionalization strategies are desired, though in standard PROTAC synthesis, the bromine remains inert and does not participate in conjugation; conjugation occurs via the amide nitrogen of the glutarimide or via a separately appended linker attached elsewhere on the scaffold. In cell-free ubiquitination assays containing recombinant CRBN-DDB1 complex, E1 and E2 enzymes, and ubiquitin, the complete PROTAC facilitates polyubiquitination of the target protein in a CRBN-dependent manner. |
| ln Vivo |
In vivo, complete PROTAC molecules incorporating (R)-Desamino lenalidomide-5-Br induce degradation of target proteins in animal models via the ubiquitin-proteasome pathway. Because the ligand alone does not comprise a complete PROTAC, it possesses no intrinsic pharmacodynamics in animals. For the final PROTAC, typical in vivo efficacy readouts include reduction of target protein levels in tissues (e.g., tumor or liver homogenates) measured by ELISA, meso-scale discovery (MSD) assays, or quantitative mass spectrometry. Xenograft tumor models demonstrate that PROTACs with CRBN-binding moieties can achieve significant tumor growth inhibition (TGI) ranging from 50-90% at well-tolerated doses. The (R)-desamino modification may improve selectivity for CRBN over off-target E3 ligases or reduce unwanted immunomodulatory effects associated with lenalidomide, such as IKZF1/3 degradation, which can cause hematological toxicities.
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| Enzyme Assay |
CRBN binding affinity is measured using biochemical assays with recombinant CRBN-DDB1 protein complex. Isothermal titration calorimetry (ITC) directly measures thermodynamic binding parameters (Kd, deltaH, deltaS) with reported Kd values for lenalidomide derivatives ranging from sub-micromolar to low micromolar; lenalidomide itself has a Kd of approximately 11-16 microM for CRBN. Fluorescence polarization (FP) competition assays use a fluorescently labeled CRBN-binding probe that is displaced by the test compound; IC50 values are determined and converted to Ki using the Cheng-Prusoff equation. Surface plasmon resonance (SPR) (e.g., Biacore) with immobilized CRBN-DDB1 measures real-time binding kinetics: association rate (ka) and dissociation rate (kd), from which Kd = kd/ka is derived. For SAR studies, a library of CRBN ligands is screened using these binding assays. The bromine substitution and removal of the exocyclic amine may alter binding affinity compared to lenalidomide, potentially enhancing or reducing CRBN engagement depending on the specific interaction with the thalidomide-binding domain of CRBN.
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| Cell Assay |
Cellular experiments require that (R)-Desamino lenalidomide-5-Br be conjugated to a target protein ligand via a suitable linker to form a complete PROTAC. The resulting PROTAC is applied to cultured cells (e.g., HEK293T, HCT116, MV4-11) for 2-24 hours. Target protein degradation is quantified by Western blot: cells are lysed, proteins resolved by SDS-PAGE, transferred to PVDF or nitrocellulose membranes, probed with target-specific primary antibody, detected with HRP-conjugated secondary antibody and chemiluminescent substrate, and visualized. DC50 values are interpolated from dose-response curves. For orthogonal validation, quantitative proteomics (TMT or SILAC) identifies and quantifies thousands of proteins simultaneously, verifying target specificity. A cellular thermal shift assay (CETSA) can confirm PROTAC engagement with CRBN and the target protein in the cellular context. Time-course experiments (0, 1, 2, 4, 8, 16, 24 hours) reveal degradation kinetics. Nonspecific protein degradation is assessed by treating cells with a proteasome inhibitor (e.g., MG132, 10 microM) or neddylation inhibitor (MLN4924) to confirm degradation proceeds via the ubiquitin-proteasome system.
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| Animal Protocol |
Animal studies are conducted with the complete PROTAC built from (R)-Desamino lenalidomide-5-Br. Male or female immunocompromised mice (athymic nude, 6-8 weeks old, 20-25 g body weight) bearing subcutaneous xenografts (tumor volume typically 100-200 mm3 at study initiation) are randomized into treatment groups (n=5-10 animals/group). PROTAC is formulated in vehicle (e.g., 10% DMSO, 40% PEG-400, 50% water or saline) and administered by IV, IP, or PO routes. Dosing regimen: daily (QD) or every other day (Q2D) for 14-28 days. Tumor volume = (length × width2)/2 measured by caliper every 2-3 days; percent tumor growth inhibition (%TGI) = 100 × (1 - [deltaTtumor treatment] / [deltaTtumor control]). Body weight measured daily; weight loss >20% from baseline triggers study termination. At sacrifice, tumors are excised and snap-frozen or formalin-fixed. Target protein knockdown in tumors is assessed by IHC (intensity and percent positivity) or immunoblot. Pharmacodynamics markers (e.g., phosphorylated downstream targets) are measured if applicable. Plasma samples collected at various time points post-dose for PK analysis.
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| ADME/Pharmacokinetics |
No formal pharmacokinetic data exist for (R)-Desamino lenalidomide-5-Br as an isolated compound; all PK properties are derived from the final PROTAC. However, based on the lenalidomide pharmacophore, the following generalizations can be made: the glutarimide ring is metabolically vulnerable to hydrolysis, generating ring-opened inactive metabolites. CYP3A4 is a major enzyme responsible for oxidative metabolism of IMiD-class compounds. The desamino modification eliminates a site that may be susceptible to N-oxidation or conjugation reactions. Plasma protein binding of lenalidomide is approximately 30% (low), but incorporation into a PROTAC dramatically increases molecular weight (typically 700-1200 Da) and lipophilicity, resulting in >95% plasma protein binding for most PROTACs. The C5-bromine substituent is metabolically stable (halogenated aromatic rings resist CYP-mediated oxidation), but may be subject to dehalogenation by reductive metabolism in the gut or liver under hypoxic conditions. For the final PROTAC, typical PK parameters in rodents: IV: clearance 10-40 mL/min/kg, Vd 1-3 L/kg, t1/2 1-6 hours; PO: Cmax 0.1-1 microM, Tmax 1-2 hours, bioavailability 5-30% (often limited by poor aqueous solubility and high efflux ratio).
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| Toxicity/Toxicokinetics |
Primary toxicological concern for (R)-Desamino lenalidomide-5-Br is potential off-target degradation of neosubstrates. Lenalidomide causes degradation of IKZF1 and IKZF3, leading to Grade 3-4 neutropenia and thrombocytopenia in cancer patients. The desamino modification may reduce such off-target activity, but this remains to be proven. For the final PROTAC, general toxicity assessment includes in vitro cytotoxicity: HEK293 or HepG2 cells treated with PROTAC (0.1-100 microM) for 72 hours, cell viability measured by CellTiter-Glo or MTT; IC50 values >10 microM indicate low cytotoxicity. In vivo, monitoring for clinical signs of distress, body weight loss, hunching, ruffled fur, reduced activity, and diarrhea. Serum biochemistry: ALT, AST for hepatotoxicity; BUN, creatinine for nephrotoxicity; CPK for muscle damage. Hematology: complete blood count (CBC) including WBC differential, platelet count, hemoglobin, hematocrit; significant reductions indicate myelosuppression. Histopathology of liver (hepatocellular necrosis, vacuolation), kidney (tubular degeneration), heart, lung, spleen, and intestines. Teratogenicity is a well-known concern for thalidomide and its analogs; female animals of childbearing potential must be confirmed not pregnant prior to study enrollment.
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| Additional Infomation |
(R)-Desamino lenalidomide-5-Br represents a modified cereblon ligand that may offer improved selectivity over lenalidomide. This compound has not entered clinical trials; all reported uses are for research purposes in PROTAC development. The "(R)" designation in the name indicates that the compound is the single enantiomer of a chiral center, though the exact location of chirality depends on the full structure (the bromine substitution creates asymmetry in the isoindolinone ring). When using this compound for PROTAC synthesis, the linker attachment chemistry must be carefully designed to avoid interfering with the glutarimide-CRBN interaction surface. Common conjugation sites include the amide nitrogen of the glutarimide (after deprotonation with base) or functionalization via the amino group that is absent in this desamino analog, necessitating alternative handles or leaving the bromine as an inert spectator unless further derivatized.
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| Molecular Formula |
C13H11BRN2O3
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| Molecular Weight |
323.15
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| CAS # |
2243825-20-7
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| Appearance |
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 |
| 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 | 3.0945 mL | 15.4727 mL | 30.9454 mL | |
| 5 mM | 0.6189 mL | 3.0945 mL | 6.1891 mL | |
| 10 mM | 0.3095 mL | 1.5473 mL | 3.0945 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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