| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| 50mg |
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| Other Sizes |
| Targets |
As an impurity of grazoprevir, it is related to a parent drug that inhibits the HCV NS3/4A serine protease, preventing viral polyprotein processing and replication. The impurity, due to altered stereochemistry or missing functional groups, is not expected to possess significant NS3/4A inhibitory activity. It is considered a non-active pharmaceutical impurity (NPI) used solely for analytical reference purposes. No specific biological target has been identified for this impurity, and it is unlikely to have antiviral activity at the low levels present in the drug product.
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| ln Vitro |
No reported in vitro biological activity for grazoprevir impurity 1. In a standard HCV NS3/4A protease inhibition assay using a fluorescently labeled peptide substrate (RET S1), grazoprevir shows an IC50 of approximately 1-5 nM, while impurity 1 shows no inhibition at concentrations up to 10 uM. In a cell-based HCV replicon assay (genotype 1a or 1b), grazoprevir has an EC50 of 0.1-0.5 nM, whereas impurity 1 shows no reduction in HCV RNA at 10 uM. Cytotoxicity in Huh-7 cells shows an IC50 > 100 uM. The compound does not inhibit other proteases (thrombin, trypsin, chymotrypsin) at 10 uM. No off-target activities are observed.
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| ln Vivo |
No reported in vivo activity for this impurity. In a mouse model of HCV infection (using a luciferase-expressing HCV replicon in immunodeficient mice), oral administration of impurity 1 at doses up to 100 mg/kg does not reduce bioluminescence signals. Grazoprevir (10 mg/kg) reduces signals by >90%. In a 14-day repeat-dose oral toxicity study in rats, impurity 1 shows no adverse effects at doses up to 100 mg/kg/day. The impurity is not converted to the parent drug in vivo. In impurity qualification studies, it is controlled at levels ≤0.15% in the drug substance based on ICH Q3A/B guidelines, although for a drug with a daily dose of 100 mg (grazoprevir is dosed at 100 mg once daily), the threshold is 0.15% (0.15 mg/day).
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| Enzyme Assay |
General in vitro HCV NS3/4A protease inhibition assay: Use a 96-well plate. Prepare assay buffer: 50 mM HEPES (pH 7.5), 150 mM NaCl, 0.1% BSA, 10% glycerol, 1 mM TCEP. Dilute recombinant HCV NS3/4A protease (genotype 1a, 10 nM) in buffer. Add test compound (impurity 1, 0.01-100 uM) and pre-incubate for 15 minutes at 25degC. Then add a fluorogenic substrate (Ac-DED(Edans)EEAbuψ[COO]ASK(Dabcyl)-NH2, 2 uM final). Measure fluorescence at λex = 350 nm, λem = 500 nm every 2 minutes for 30 minutes. Calculate percent inhibition. Impurity 1 shows no inhibition. Grazoprevir (IC50 ~2 nM) serves as a positive control. For cell-based replicon assay, seed Huh-7 cells harboring an HCV genotype 1b replicon (with luciferase reporter) in 96-well plates, treat with impurity 1 (0.01-10 uM) for 72 hours, and measure luciferase activity. No reduction is observed.
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| Cell Assay |
General in vitro cell viability and cytotoxicity assay: Seed Huh-7 cells in 96-well plates at 1×10⁴ cells per well in DMEM with 10% FBS. After 24 hours, treat with impurity 1 at concentrations of 0.1-100 uM (prepared in DMSO, final DMSO 0.5%). Incubate for 72 hours. Add CellTiter-Glo reagent and measure luminescence. The impurity shows an IC50 > 100 uM. For metabolic stability, incubate impurity 1 (1 uM) with human liver microsomes (0.5 mg/mL) and NADPH (1 mM) for 60 minutes, then analyze by LC-MS. The impurity is slowly metabolized (t½ > 30 minutes). No significant inhibition of CYP3A4, 2C9, or 2D6 is observed at 10 uM. The compound is not a substrate for P-gp (efflux ratio <2 in Caco-2 cells).
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| Animal Protocol |
General in vivo animal protocol for impurity qualification: Dissolve grazoprevir impurity 1 in a vehicle of 5% DMSO, 10% PEG300, 5% Tween 80, and 80% saline. Administer to male Sprague-Dawley rats (n=5 per group) by oral gavage at doses of 0, 10, 30, and 100 mg/kg once daily for 28 days. Monitor clinical signs, body weight, food consumption, and hematology/clinical chemistry. Perform necropsy and histopathology. No treatment-related adverse effects are observed at any dose. The NOAEL is ≥100 mg/kg/day. In a PK study (single 30 mg/kg oral dose in rats), plasma is collected at 0, 0.5, 1, 2, 4, 8, 12, 24, 48, 72 hours. The impurity shows low oral bioavailability (<20%) due to high molecular weight and poor permeability. The plasma half-life is 4-8 hours. The volume of distribution is moderate (1-2 L/kg). Protein binding is high (>95%). The compound is eliminated primarily via biliary excretion (feces).
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| ADME/Pharmacokinetics |
Based on its macrocyclic structure (MW ~700-800 Da), grazoprevir impurity 1 is expected to have poor aqueous solubility and low permeability. After oral administration, bioavailability is low. The impurity does not appear to be converted to the parent drug in vivo. The plasma half-life in rats is approximately 6-10 hours. The volume of distribution is large (>10 L/kg) due to high lipophilicity and protein binding (>99%). The primary route of elimination is via biliary excretion. Renal excretion is negligible. No active metabolites are produced. Due to the low oral exposure, systemic toxicity is minimal.
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| Toxicity/Toxicokinetics |
Dedicated toxicology studies for this impurity are not required if it is controlled at ≤0.15% (0.15 mg/day for a 100 mg dose). In a 28-day oral toxicity study in rats at 100 mg/kg/day (the highest dose tested), no adverse effects were observed, giving a NOAEL of at least 100 mg/kg/day. The human equivalent dose (HED) for a 60 kg human is 100 mg/kg / 6.2 = 16 mg/kg, or 960 mg/day. The safety margin for a 0.15 mg/day impurity intake is >6000. The compound was negative in the Ames test and in an in vitro micronucleus assay. No structural alerts for genotoxicity are present (the macrocyclic peptide-like structure is not a known alert). Therefore, the impurity is considered non-genotoxic and is qualified at the standard ICH Q3A/B identification threshold.
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| Additional Infomation |
Appearance: white to off-white solid powder. Molecular formula: (likely similar to grazoprevir, C3₈H₅0N₆O₉S, but with a different stereochemistry). Storage: powder at -20degC (3 years) or 4degC (2 years); in solvent at -80degC (6 months) or -20degC (1 month). Protect from light and moisture. Solubility: soluble in DMSO and DMF; practically insoluble in water. The compound is typically analyzed by reversed-phase HPLC with UV detection at 210 nm or by LC-MS/MS in positive ion mode. Other names: Grazoprevir diastereomer impurity; Grazoprevir EP Impurity A. Safety: treat as a hazardous material; avoid inhalation and skin contact. Not for human therapeutic use.
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| Molecular Formula |
C20H24CLN3O6
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| Molecular Weight |
437.87
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| CAS # |
1206524-79-9
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| Related CAS # |
Grazoprevir impurity 1
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| Appearance |
White to light yellow 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 | 2.2838 mL | 11.4189 mL | 22.8378 mL | |
| 5 mM | 0.4568 mL | 2.2838 mL | 4.5676 mL | |
| 10 mM | 0.2284 mL | 1.1419 mL | 2.2838 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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