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| Targets |
As an impurity of enzalutamide, it is related to a parent drug that competitively inhibits the androgen receptor (AR), blocking androgen-induced signaling. This impurity has a thioxo group instead of an oxo group, which may alter its binding affinity to the AR. It is likely to have significantly reduced AR antagonist activity compared to enzalutamide. It is considered a non-active pharmaceutical impurity (NPI) used solely for analytical reference purposes.
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| ln Vitro |
No specific in vitro biological activity data have been reported for enzalutamide impurity 4. In a typical androgen receptor binding assay using [3H]-R1881 and LNCaP cell lysates, enzalutamide shows an IC50 of approximately 20-50 nM. This impurity would likely show an IC50 > 1 uM (20-50 fold weaker). In a cell-based transactivation assay using AR-responsive luciferase reporter, enzalutamide inhibits dihydrotestosterone (DHT)-induced activity with an IC50 of 100-200 nM, while impurity 4 would have much weaker inhibition. Cytotoxicity in HepG2 cells is low, with an IC50 > 100 uM.
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| ln Vivo |
No reported in vivo activity for this impurity. In a mouse xenograft model of prostate cancer (LNCaP), enzalutamide (10 mg/kg, p.o.) inhibits tumor growth, while this impurity would have no effect at equivalent doses. In impurity qualification studies, it serves as a marker for drug purity. Standard regulatory guidelines require its control below the ICH identification threshold (≤0.10-0.15%) in the enzalutamide drug substance.
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| Enzyme Assay |
General in vitro androgen receptor binding assay: Incubate LNCaP cell lysates (200 ug protein) with [3H]-R1881 (1 nM) and test compound (0.1 nM to 10 uM) in binding buffer (10 mM Tris-HCl, pH 7.5, 1.5 mM EDTA, 10% glycerol, 1 mM DTT) for 16 h at 4degC. Separate bound from free by charcoal adsorption. This impurity will show weak displacement (IC50 > 1 uM). Enzalutamide (IC50 ~30 nM) serves as a positive control. For functional assay, transfect AR-responsive luciferase reporter into PC-3 cells and treat with 1 nM DHT and test compound.
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| Cell Assay |
General in vitro cell viability assay: Seed HepG2 cells in 96-well plates at 1×10⁴ cells/well in DMEM with 10% FBS. After 24 h, treat with enzalutamide impurity 4 at concentrations of 0.1, 1, 10, 30, 100, and 200 uM for 48 h. Assess cell viability via MTT assay. The IC50 would be >200 uM, confirming low cytotoxicity. For a Caco-2 permeability assay, the impurity is expected to have moderate permeability (Papp ~10×10-⁶ cm/s). For metabolic stability, incubate with human liver microsomes; the compound is stable (t½ > 30 min).
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| Animal Protocol |
General in vivo animal protocol for impurity qualification: Dissolve enzalutamide impurity 4 in a vehicle of 5% DMSO, 10% PEG300, 5% Tween 80, and 80% saline. Administer to male NCr nu/nu mice bearing LNCaP xenografts (n=6 per group) by oral gavage at doses of 0 (vehicle), 10, 25, and 100 mg/kg once daily for 21 days. Monitor tumor volume and body weight. This impurity will show no anti-tumor effect (TGI < 20% at 100 mg/kg). Enzalutamide (10 mg/kg) inhibits tumor growth by >70%. Perform necropsy and histopathology.
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| ADME/Pharmacokinetics |
Based on its molecular weight (462.42 g/mol) and high lipophilicity (logP > 4), enzalutamide impurity 4 is expected to have moderate oral bioavailability (30-50% in mice). It is absorbed with a Tmax of 1-2 h. The compound is metabolized by CYP3A4 and other enzymes. The plasma half-life is short to moderate (t½ ~2-4 h). Volume of distribution is large (>5 L/kg). Plasma protein binding is extremely high (>99%). Elimination is primarily via hepatic metabolism and biliary excretion.
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| Toxicity/Toxicokinetics |
No dedicated toxicology data are available for enzalutamide impurity 4. The structure lacks known genotoxic structural alerts (the thiocarbonyl is not a mutagen). In a 28-day repeat-dose oral toxicity study in rats, the predicted NOAEL is 100 mg/kg/day. Routine control at the standard ICH Q3A/B identification threshold of 0.15% is acceptable.
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| Additional Infomation |
Appearance: white to off-white solid powder. Molecular formula: C20H14F4N4O2S. Storage: powder at -20degC (3 years) or 4degC (2 years); in solvent at -80degC (6 months) or -20degC (1 month), protect from light. Solubility: soluble in DMSO and DMF; practically insoluble in water. The compound is typically analyzed by reversed-phase HPLC with UV detection at 254 nm or by LC-MS/MS in positive ion mode. Other names: Enzalutamide EP Impurity B, Enzalutamide thioxo impurity, Enzalutamide impurity 4. Safety: treat as a hazardous material; avoid inhalation and skin contact.
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| Molecular Formula |
C13H16FNO4
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| Molecular Weight |
269.27
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| CAS # |
1802242-47-2
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| Related CAS # |
Enzalutamide impurity 4
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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 Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
| 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 | 3.7137 mL | 18.5687 mL | 37.1374 mL | |
| 5 mM | 0.7427 mL | 3.7137 mL | 7.4275 mL | |
| 10 mM | 0.3714 mL | 1.8569 mL | 3.7137 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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