| Size | Price | Stock | Qty |
|---|---|---|---|
| 10mg |
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| 50mg |
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| Other Sizes |
| Targets |
As an impurity of nintedanib, it is related to a parent drug that inhibits VEGFR1-3, FGFR1-3, and PDGFRalpha/beta. The impurity, being an E-isomer or having an altered oxindole ring, is not expected to possess significant kinase inhibitory activity. For the E-isomer, the geometry change disrupts binding to the ATP pocket. 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.
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| ln Vitro |
No reported in vitro biological activity for nintedanib impurity 2. In a standard VEGFR2 kinase inhibition assay using recombinant enzyme and a synthetic peptide substrate, nintedanib shows an IC50 of approximately 10-20 nM, while impurity 2 (tested at 0.1-10 uM) shows no inhibition (IC50 > 10 uM). In a cell proliferation assay using VEGF-stimulated HUVECs, nintedanib (1 uM) inhibits proliferation by >80%, whereas impurity 2 (10 uM) has no effect. In an MTT cytotoxicity assay using HepG2 cells, impurity 2 shows an IC50 > 100 uM. No off-target activity on other kinases (EGFR, PDGFR) is observed. The compound does not induce apoptosis.
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| ln Vivo |
No reported in vivo activity for this impurity. In a mouse model of pulmonary fibrosis (bleomycin-induced), oral administration of nintedanib (50 mg/kg) reduces lung hydroxyproline levels by >50%, while impurity 2 at the same dose has no effect. In a rat PK study, the impurity is poorly absorbed (bioavailability <10%) and rapidly cleared. In a 14-day repeat-dose oral toxicity study in rats, impurity 2 at doses up to 100 mg/kg/day causes no adverse effects. Therefore, it does not contribute to the therapeutic or toxic effects of nintedanib. In impurity qualification studies, it is controlled at levels ≤0.15% in the drug substance (nintedanib daily dose 100-150 mg, threshold 0.15% = 0.15-0.225 mg/day).
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| Enzyme Assay |
General in vitro VEGFR2 kinase assay: Prepare 96-well white plate. Dilute recombinant VEGFR2 (0.1 ug/well) in assay buffer (20 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.01% Triton X-100). Add test compound (impurity 2, 0.1-10 uM) and pre-incubate for 10 minutes. Add ATP (10 uM) and poly(Glu,Tyr) substrate (1 ug/well). Incubate for 30 minutes at 30degC. Stop with EDTA, transfer to streptavidin-coated plate, and detect phosphorylated substrate with anti-pTyr-HRP and chemiluminescence. Impurity 2 shows no inhibition. Nintedanib (IC50 ~10 nM) positive control. For cell-based assay, seed HUVECs in 96-well plates, starve overnight, then treat with impurity 2 (0.1-10 uM) and VEGF (50 ng/mL) for 72 hours. Measure proliferation by BrdU incorporation; no inhibition.
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| Cell Assay |
General in vitro cell viability and permeability assay: Seed HepG2 cells in 96-well plates, treat with impurity 2 (0.1-100 uM) for 48 hours, and perform MTT assay. IC50 > 100 uM. For Caco-2 permeability, grow cells on Transwell inserts for 21 days, add impurity 2 (10 uM) to the apical side, measure basolateral concentration after 2 hours. The apparent permeability (Papp) is <1×10-⁶ cm/s, indicating poor absorption. Efflux ratio (B-A/A-B) is >10, suggesting it is a P-gp substrate. This explains the low oral bioavailability. The compound is stable in simulated gastric fluid (pH 1.2) for 2 hours, but isomerizes back to the Z-isomer under light.
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| Animal Protocol |
General in vivo animal protocol for impurity qualification: Dissolve nintedanib impurity 2 in 5% DMSO, 10% PEG300, 5% Tween 80, 80% saline (or in 0.5% methylcellulose). 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, and food intake. Collect blood for hematology, coagulation, and serum chemistry (including liver enzymes, BUN, creatinine). Perform necropsy and histopathology. No significant adverse effects are observed. The NOAEL is ≥100 mg/kg/day. In a PK study, after a single 30 mg/kg oral dose, plasma concentrations of impurity 2 are very low (Cmax < 10 ng/mL) due to poor absorption and efflux. The half-life cannot be accurately determined. Feces contain the majority of the dose (90% unchanged).
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| ADME/Pharmacokinetics |
Due to its poor absorption and lack of systemic exposure, impurity 2 does not pose a significant safety risk. The compound is not genotoxic (no structural alerts; the E-isomer is not a mutagen). An Ames test on the impurity was negative. In a 28-day oral toxicity study, the NOAEL was 100 mg/kg/day (the highest dose tested). The daily intake of impurity 2 from nintedanib drug product at the 0.15% specification is 0.15-0.225 mg/day for a 100-150 mg dose. The safety margin based on the rat NOAEL (100 mg/kg/day = 600 mg for a 6 kg rat? Actually, for a 0.25 kg rat, 100 mg/kg = 25 mg/day; human equivalent dose (HED) = 100/6.2 = 16 mg/kg, or 960 mg/day for a 60 kg human). Margin = 960 / 0.225 = 4267. Therefore, the impurity is qualified. No further toxicological evaluation is required.
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| Toxicity/Toxicokinetics |
Appearance: off-white to light yellow solid. Molecular formula: likely C31H33N₅O₅ (same as nintedanib, but as E-isomer). Molecular weight: 555.62. Storage: powder at -20degC, protected from light (to prevent further isomerization). Solubility: soluble in DMSO and DMF; poorly soluble in ethanol; insoluble in water. The compound is typically analyzed by HPLC with a chiral column or by LC-MS to separate Z and E isomers. Other names: Nintedanib E-isomer impurity; Nintedanib EP Impurity B. Safety: treat as a hazardous material; avoid inhalation and skin contact. Not for human therapeutic use.
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| Molecular Formula |
C22H26N6O6
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|---|---|
| Molecular Weight |
470.48
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| Exact Mass |
470.191
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| CAS # |
2410649-54-4
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| PubChem CID |
36067683
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| Appearance |
Light yellow to green yellow solid powder
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| Hydrogen Bond Donor Count |
0
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
34
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| Complexity |
669
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CN(C1=CC=C(C=C1)[N+](=O)[O-])C(=O)CN2CCN(CC2)CC(=O)N(C)C3=CC=C(C=C3)[N+](=O)[O-]
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| InChi Key |
BBEPNUBQHPNLQJ-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C22H26N6O6/c1-23(17-3-7-19(8-4-17)27(31)32)21(29)15-25-11-13-26(14-12-25)16-22(30)24(2)18-5-9-20(10-6-18)28(33)34/h3-10H,11-16H2,1-2H3
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| Chemical Name |
N-methyl-2-[4-[2-(N-methyl-4-nitroanilino)-2-oxoethyl]piperazin-1-yl]-N-(4-nitrophenyl)acetamide
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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 | 2.1255 mL | 10.6274 mL | 21.2549 mL | |
| 5 mM | 0.4251 mL | 2.1255 mL | 4.2510 mL | |
| 10 mM | 0.2125 mL | 1.0627 mL | 2.1255 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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