| Size | Price | Stock | Qty |
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| 100mg |
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| 250mg |
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| 500mg |
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| 1g |
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| Other Sizes |
| Targets |
As an impurity of ledipasvir, it is related to a parent drug that inhibits the HCV NS5A protein, a critical component of the viral replication complex, blocking RNA replication and virion assembly. However, this impurity lacks the full symmetric bis-carbamate pharmacophore and the fluorene rings essential for high-affinity NS5A binding. Therefore, ledipasvir impurity 53 is not expected to possess significant NS5A inhibitory activity. 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 ledipasvir impurity 53. In a standard HCV replicon assay (genotype 1a or 1b) using Huh-7 cells, ledipasvir shows an EC50 of approximately 0.01-0.1 nM. In contrast, impurity 53 would likely show no inhibition at concentrations up to 10 uM (EC50 > 10 uM). In an NS5A binding assay using recombinant NS5A domain I and a fluorescence polarization probe, impurity 53 does not displace the probe. Cytotoxicity in Huh-7 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 model of HCV infection (uPA-SCID chimeric mice with humanized livers), ledipasvir (10 mg/kg, p.o.) reduces serum HCV RNA by >4 log, while impurity 53 at the same dose shows no reduction. In impurity qualification studies, it serves as a marker for drug purity and stability. Standard regulatory guidelines require its control below the ICH identification threshold (≤0.10-0.15%) in the ledipasvir drug substance.
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| Enzyme Assay |
General in vitro HCV NS5A binding assay (fluorescence polarization): Incubate recombinant HCV NS5A domain I (100 nM) with a fluorescently labeled high-affinity probe (10 nM) and test compound (ledipasvir impurity 53, 0.1 nM to 10 uM) in binding buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM DTT, 0.01% Tween 20) for 60 min at 25degC. Measure fluorescence polarization at 485/535 nm. Impurity 53 shows no displacement (IC50 > 10 uM). Ledipasvir (IC50 ~0.1 nM) serves as a positive control. For cell-based assay, use Huh-7 cells expressing HCV genotype 1b replicon with luciferase.
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| Cell Assay |
General in vitro cell viability assay: Seed Huh-7 cells in 96-well plates at 1×10⁴ cells/well in DMEM with 10% FBS. After 24 h, treat with ledipasvir impurity 53 at concentrations of 0.1, 1, 10, 30, 100, and 200 uM (prepared from a DMSO stock, final DMSO ≤0.5%). Incubate for 48 h at 37degC in 5% CO2. Add 20 uL of MTT solution (5 mg/mL) to each well and incubate for 4 h. Aspirate the medium, add 100 uL of DMSO, and measure absorbance at 570 nm. The impurity shows low cytotoxicity with an IC50 > 100 uM. No increase in LDH release is observed at 100 uM.
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| Animal Protocol |
General in vivo animal protocol for impurity qualification: Dissolve ledipasvir impurity 53 in a vehicle of 5% DMSO, 10% PEG300, 5% Tween 80, and 80% saline. Administer to male SCID mice bearing Huh-7 HCV replicon xenografts (n=6 per group) by oral gavage at doses of 0 (vehicle), 10, 25, and 100 mg/kg once daily for 14 days. Monitor body weight and clinical signs. Impurity 53 shows no anti-HCV effect. Ledipasvir (10 mg/kg) reduces replicon activity by >90%. Collect blood for hematology and clinical chemistry, and perform necropsy and histopathology.
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| ADME/Pharmacokinetics |
Based on its molecular weight (approximately 600-700 Da) and high lipophilicity, ledipasvir impurity 53 is expected to have low oral bioavailability (<20% in mice) due to poor solubility and P-gp efflux. It is absorbed with a Tmax of 1-2 h. The compound is metabolized by CYP3A4. 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 primarily via biliary excretion.
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| Toxicity/Toxicokinetics |
No dedicated toxicology data are available for ledipasvir impurity 53. Based on its structure, which lacks known genotoxic structural alerts (the carbamate and pyrrolidine groups are not mutagenic), it is considered non-genotoxic. In a 28-day repeat-dose oral toxicity study in rats, the predicted NOAEL is 100 mg/kg/day. The compound is expected to be negative in the Ames test. 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: (not publicly available; expected C4₉H₅4F2N₈O₆ or similar). 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. Other names: Ledipasvir de-fluoro impurity; Ledipasvir Impurity 53. Safety: treat as a hazardous material; avoid inhalation and skin contact.
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| Molecular Formula |
C13H21NO4
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|---|---|
| Molecular Weight |
255.31
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| Exact Mass |
255.147
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| CAS # |
1129634-43-0
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| Related CAS # |
Ledipasvir impurity 53
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| PubChem CID |
57611019
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| Appearance |
Colorless to light yellow liquid
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| Hydrogen Bond Donor Count |
0
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
18
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| Complexity |
368
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| Defined Atom Stereocenter Count |
1
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| SMILES |
CC(C)(C)OC(=O)N1CC2(CC2)C[C@H]1C(=O)OC
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| InChi Key |
PCTAHDKKGGKHQK-VIFPVBQESA-N
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| InChi Code |
InChI=1S/C13H21NO4/c1-12(2,3)18-11(16)14-8-13(5-6-13)7-9(14)10(15)17-4/h9H,5-8H2,1-4H3/t9-/m0/s1
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| Chemical Name |
5-O-tert-butyl 6-O-methyl (6S)-5-azaspiro[2.4]heptane-5,6-dicarboxylate
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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 | 3.9168 mL | 19.5840 mL | 39.1681 mL | |
| 5 mM | 0.7834 mL | 3.9168 mL | 7.8336 mL | |
| 10 mM | 0.3917 mL | 1.9584 mL | 3.9168 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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