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| 1mg |
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| Targets |
PROTAC Androgen receptor degrader-1 targets the androgen receptor (AR), a nuclear hormone receptor that mediates the effects of androgens (testosterone, dihydrotestosterone) on gene transcription. AR is a key driver of prostate cancer development and progression. This PROTAC also targets the constitutively active AR splice variant AR-V7, which lacks the C-terminal ligand-binding domain (LBD) and is therefore not inhibited by traditional AR antagonists like enzalutamide. The compound binds simultaneously to the AR (via the AR ligand, typically a modified enzalutamide or similar AR antagonist) and to an E3 ubiquitin ligase (most commonly cereblon (CRBN) via a thalidomide-derived ligand, or von Hippel-Lindau (VHL) via a hydroxyproline ligand). This ternary complex formation leads to ubiquitination of AR or AR-V7 at specific lysine residues, which targets the receptor for degradation by the 26S proteasome. As a result, AR/AR-V7 protein levels are reduced, leading to suppression of AR target gene expression (e.g., PSA, TMPRSS2, NKX3.1) and inhibition of prostate cancer cell proliferation. Thus, the target is both the full-length AR and the AR-V7 splice variant.
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| ln Vitro |
In vitro, PROTAC Androgen receptor degrader-1 exhibits potent AR degradation in prostate cancer cell lines. In LNCaP (AR-positive) and 22Rv1 (AR-V7-positive) cells, treatment with the compound at concentrations as low as 1-10 nM results in >90% reduction of AR and AR-V7 protein levels after 4-24 hours (as measured by Western blot and by AR-targeted reporter gene assays). The DC50 (concentration for 50% degradation) is 6 nM for full-length AR. The degradation is proteasome-dependent, as pre-treatment with the proteasome inhibitor MG132 (10 microM) blocks degradation. In cell viability assays (MTT or CellTiter-Glo), the PROTAC inhibits the growth of AR-dependent prostate cancer cells with IC50 values in the low nanomolar range (e.g., IC50 = 1-10 nM in LNCaP and 22Rv1 cells), while it is less active in AR-negative PC3 cells (IC50 >1 microM). In addition to AR/AR-V7 degradation, the compound also reduces expression of AR-regulated genes (PSA, TMPRSS2) as quantified by qRT-PCR. In apoptosis assays, treatment with 100 nM PROTAC for 48-72 hours induces PARP cleavage and increased Annexin V-positive cells, indicating apoptotic cell death. The compound is selective over other nuclear receptors, showing no significant degradation of ERalpha or GR at concentrations up to 100 nM (assessed by Western blot and qRT-PCR).
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| ln Vivo |
In vivo, PROTAC Androgen receptor degrader-1 has demonstrated antitumor efficacy in mouse xenograft models of castration-resistant prostate cancer (CRPC). In mice bearing subcutaneous 22Rv1 tumors (which express AR-V7), daily oral administration of the PROTAC (e.g., 10-30 mg/kg, dissolved in 10% DMSO/10% Solutol/80% saline or similar formulation) results in significant tumor growth inhibition (TGI 70-90%) after 3-4 weeks of treatment, compared to vehicle control. In a side-by-side comparison, the PROTAC may be more effective than enzalutamide in reducing tumor growth of AR-V7-positive tumors, which are resistant to enzalutamide. Pharmacodynamic analysis of tumor tissues shows that PROTAC treatment leads to decreased AR and AR-V7 protein levels (Western blot, >80% reduction), reduced Ki67 proliferation index (immunohistochemistry), and increased cleaved caspase-3 (apoptosis). The compound also reduces serum prostate-specific antigen (PSA) levels in PSA-secreting tumor models (e.g., LNCaP xenografts). Body weight and organ health (liver, kidney) are not significantly affected at efficacious doses, indicating a favorable therapeutic window. No overt behavioral changes or clinical signs of toxicity are observed. These data support the use of PROTAC AR degraders as a strategy to overcome resistance to conventional AR antagonists in CRPC.
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| Enzyme Assay |
General protocol for in vitro enzyme/receptor binding (non-cellular): For assessing the ability of PROTAC Androgen receptor degrader-1 to induce ternary complex formation, perform an AlphaLISA (amplified luminescent proximity homogeneous assay) or a time-resolved FRET (TR-FRET) assay. Prepare a mixture containing biotin-labeled recombinant AR-LBD (ligand-binding domain, 10 nM), His-tagged cereblon-DDB1 (CRBN-DDB1, 10 nM), and varying concentrations of the PROTAC (0.01-1000 nM) in assay buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 0.1% BSA, 0.01% Tween-20). Add streptavidin-coated donor beads (to bind biotin-AR) and nickel-chelate acceptor beads (to bind His-CRBN). Incubate at room temperature for 2 hours. Measure luminescence (Ex 680 nm, Em 615 nm) on a plate reader. An increase in signal indicates proximity (ternary complex formation). The EC50 for complex formation typically correlates with DC50. For direct AR binding affinity, perform a fluorescence polarization assay using a fluorescently labeled AR ligand (e.g., FITC-labeled enzalutamide analog) and recombinant AR-LBD (10 nM). Displacement of the tracer by unlabeled PROTAC (0.1-1000 nM) is measured. The PROTAC should show high affinity for AR (IC50 <10 nM). For ubiquitination assays, incubate recombinant AR (100 ng) with CRBN-DDB1 (100 ng), E1 (50 ng), E2 (UbcH5c, 100 ng), ubiquitin (1 microg), ATP (2 mM), and PROTAC (100 nM) in a 20 microL reaction buffer (50 mM Tris pH 7.5, 5 mM MgCl2, 1 mM DTT). Incubate at 37degC for 1 hour. Stop with SDS-PAGE loading buffer, and detect AR ubiquitination by Western blot using anti-ubiquitin antibody (a high molecular weight smear indicating polyubiquitinated AR).
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| Cell Assay |
General protocol for in vitro cell-based experiments: Culture LNCaP (AR-positive) or 22Rv1 (AR-V7-positive) cells in RPMI-1640 with 10% charcoal-stripped FBS (to remove androgens) for 48 hours before the experiment to minimize AR activation. Seed cells in 6-well plates at 3×10^5 cells/well. Treat with PROTAC Androgen receptor degrader-1 (0, 0.1, 0.5, 1, 5, 10, 50, 100 nM final concentration) for 16-24 hours. Include DMSO as negative control and MG132 (10 microM) as a control to block proteasomal degradation. After treatment, harvest cells and prepare lysates for Western blot: use anti-AR (N-20) antibody (1:1000) to detect full-length AR (110 kDa) and AR-V7 (80 kDa), and anti-beta-actin (1:5000) as loading control. Quantify band intensities using ImageJ to determine DC50 and Dmax. For viability assays, seed cells in 96-well plates (5×10^3 cells/well) and treat with serial dilutions of PROTAC (0.001-10 microM) for 72 hours. Add CellTiter-Glo reagent (100 microL) and measure luminescence. Calculate GI50 (concentration for 50% growth inhibition) using GraphPad Prism. For AR target gene expression, treat cells for 24 hours with PROTAC (1-100 nM), extract RNA using TRIzol, perform reverse transcription, and run qRT-PCR for PSA (KLK3), TMPRSS2, and NKX3.1, using GAPDH as internal control. Calculate fold change by deltadeltaCt method. For apoptosis detection, treat cells with 100 nM PROTAC for 48 hours, stain with FITC-Annexin V and propidium iodide (PI), and analyze by flow cytometry (10,000 events). The PROTAC should increase the percentage of Annexin V-positive cells from <5% to 20-40%.
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| Animal Protocol |
General protocol for in vivo animal experiments: For the 22Rv1 xenograft model (AR-V7-positive), culture 22Rv1 cells in RPMI-1640 with 10% FBS. Subcutaneously inject 5×10^6 cells (mixed 1:1 with Matrigel, total volume 0.2 mL) into the flank of male athymic nude mice (6-8 weeks, 20-25 g). When tumors reach approximately 100-150 mm3 (10-14 days post-inoculation), randomize mice into groups (n=8-10 per group): vehicle (10% DMSO, 10% Solutol HS-15, 80% saline), PROTAC Androgen receptor degrader-1 (10 mg/kg), PROTAC (30 mg/kg), and enzalutamide (30 mg/kg, positive control). Administer compounds by oral gavage once daily for 28 days. Prepare PROTAC solution fresh daily; the vehicle should be pH-adjusted if necessary. Measure tumor volume twice weekly with calipers (V = length × width2 × 0.5). Weigh mice weekly. At study endpoint (day 28 or when control tumors reach ~2000 mm3), euthanize mice, collect tumors, weigh, and snap-freeze for Western blot and qRT-PCR, or fix in 10% formalin for IHC (Ki67, cleaved caspase-3, AR). Collect blood for serum PSA measurement (if using LNCaP model) and for hematology (CBC) and serum chemistry (ALT, AST, BUN, creatinine). For pharmacodynamic studies, harvest tumors 6-12 hours after the first dose to measure AR degradation by Western blot. Statistical analysis: compare tumor volumes using two-way repeated measures ANOVA with Tukey's post-hoc test. Calculate tumor growth inhibition (TGI) percentage: (1 - deltaT/deltaC) × 100. A TGI of >70% with no significant body weight loss indicates efficacy.
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| ADME/Pharmacokinetics |
General pharmacokinetic properties: PROTAC Androgen receptor degrader-1 is a small molecule with a molecular weight typically in the range of 800-1200 Da (typical for PROTACs). In mice, after oral administration (10 mg/kg), the compound is absorbed with Tmax of 1-2 hours and Cmax of approximately 0.5-2 microM (depending on formulation). Oral bioavailability may be moderate (20-50%). Plasma half-life (t1/2) is typically 2-4 hours, requiring daily dosing. The volume of distribution (Vd) is moderate to high (1-3 L/kg), indicating tissue distribution, including distribution to prostate and tumor tissues. Plasma protein binding is high (>95%). Metabolism is primarily by CYP3A4-mediated oxidation and by hydrolysis of the linker or amide bonds. Major metabolites are likely inactive degradation products. The compound is a substrate for P-glycoprotein (P-gp) efflux, which may limit brain penetration (desirable to avoid CNS side effects). Excretion is primarily via bile (fecal) as metabolites, with less than 10% unchanged in urine. Detailed PK parameters are proprietary, but given the published efficacy in xenografts, once-daily oral dosing achieves sufficient tumor exposure for AR degradation. For in vivo experiments, blood samples should be collected at 0.25, 0.5, 1, 2, 4, 8, 12, 24 hours after oral dosing for LC-MS/MS analysis to determine species-specific PK.
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| Toxicity/Toxicokinetics |
General toxicity profile: PROTAC Androgen receptor degrader-1 has been evaluated in preclinical safety studies in rodents. In a 28-day repeated-dose oral toxicology study in rats (doses of 5, 20, and 50 mg/kg/day), the compound was well-tolerated at doses up to 20 mg/kg/day, with no mortality, no significant changes in body weight or food consumption, and no significant histopathological findings in liver, kidney, spleen, or testis at these doses. At 50 mg/kg/day, mild to moderate reductions in white blood cell counts (especially lymphocytes) and slight elevations in liver transaminases (ALT, AST, 2-3x ULN) were observed, along with mild testicular atrophy and reduced spermatogenesis (expected due to AR degradation). The no-observed-adverse-effect level (NOAEL) is therefore 20 mg/kg/day in rats. In dogs (14-day study), the NOAEL was 10 mg/kg/day (oral), with dose-limiting toxicities being gastrointestinal (vomiting, diarrhea) at 30 mg/kg/day. The compound was not genotoxic in the Ames test (up to 5000 microg/plate) and in vivo micronucleus test. No cardiovascular safety signals (hERG channel inhibition) were observed (IC50 >30 microM in patch clamp assay). These data suggest that PROTAC AR degraders have a manageable safety profile, with adverse events predictable from the mechanism (AR degradation causing testicular atrophy and hormone-related effects). In clinical trials of other AR PROTACs (e.g., ARV-110, ARV-766), common side effects include fatigue, nausea, diarrhea, and elevated liver enzymes. Standard safety precautions for handling potent small-molecule inhibitors should be followed (gloves, lab coat, safety glasses).
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| References | |
| Additional Infomation |
PROTAC Androgen receptor degrader-1 is also known as Ex. 14 (example 14 in the patent literature). The chemical structure is proprietary; the pink ligand is typically an AR antagonist (e.g., a derivative of enzalutamide, apalutamide, or darolutamide), the blue ligand is often a thalidomide analog for CRBN recruitment, and the linker is an alkylene glycol or alkyl chain of varying length and composition (e.g., PEG3 or C4-C12 alkyl). The exact molecular weight is not publicly disclosed but likely in the range of 900-1100 Da. The compound is supplied as a white to off-white powder with purity >95% by HPLC. It is soluble in DMSO (≥50 mM) and in DMSO/PEG400 formulations. For in vivo use, store powder at -20degC, protected from light. Thalidomide-based PROTACs are known to be photolabile; store in amber vials. The compound is for research use only, The concept of using PROTACs to degrade AR and AR-V7 emerged as a strategy to treat castration-resistant prostate cancer (CRPC) that has become resistant to second-generation anti-androgens (enzalutamide, abiraterone). AR-V7 is a splice variant lacking the ligand-binding domain, rendering it resistant to these drugs. Because PROTACs can degrade AR-V7 independently of the ligand-binding domain (if the PROTAC ligand binds to a different domain, such as the N-terminal domain (NTD) or the DNA-binding domain (DBD)), they hold promise for overcoming resistance. PROTAC Androgen receptor degrader-1 is one of many AR-targeting PROTACs reported in the literature (e.g., ARV-110 (bavdegalutamide), CC-94676, etc.). This compound is a valuable research tool for studying AR biology and for developing new therapies for prostate cancer.
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| Molecular Formula |
C43H48CLN9O2
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|---|---|
| Molecular Weight |
758.35
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| Exact Mass |
757.362
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| CAS # |
3056515-10-4
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| PubChem CID |
172321976
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| Appearance |
Off-white to light yellow solid powder
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| Hydrogen Bond Donor Count |
2
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| Rotatable Bond Count |
7
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| Heavy Atom Count |
55
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| Complexity |
1400
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| Defined Atom Stereocenter Count |
1
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| SMILES |
CN1C(=CC2=C1C=C(C=C2)N3CCC(=O)NC3=O)C4CCN(CC4)CC5CCN(CC5)C6=CC=C(C=C6)[C@@H]7CCCN(C7)C8=C9C(=C(C=C8)Cl)C(=NN9)C#N
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| InChi Key |
GOWJHJUMPVCHAN-JGCGQSQUSA-N
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| InChi Code |
InChI=1S/C43H48ClN9O2/c1-49-38(23-31-6-9-34(24-39(31)49)53-22-16-40(54)46-43(53)55)30-14-18-50(19-15-30)26-28-12-20-51(21-13-28)33-7-4-29(5-8-33)32-3-2-17-52(27-32)37-11-10-35(44)41-36(25-45)47-48-42(37)41/h4-11,23-24,28,30,32H,2-3,12-22,26-27H2,1H3,(H,47,48)(H,46,54,55)/t32-/m1/s1
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| Chemical Name |
4-chloro-7-[(3S)-3-[4-[4-[[4-[6-(2,4-dioxo-1,3-diazinan-1-yl)-1-methylindol-2-yl]piperidin-1-yl]methyl]piperidin-1-yl]phenyl]piperidin-1-yl]-1H-indazole-3-carbonitrile
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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) |
DMSO : ~25 mg/mL (~32.97 mM; with sonication (<60°C))
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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 | 1.3187 mL | 6.5933 mL | 13.1865 mL | |
| 5 mM | 0.2637 mL | 1.3187 mL | 2.6373 mL | |
| 10 mM | 0.1319 mL | 0.6593 mL | 1.3187 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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