| Size | Price | Stock | Qty |
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| 5g |
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| Other Sizes |
| Targets |
As an impurity of ketoprofen, it is related to a parent drug that non-selectively inhibits cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), reducing prostaglandin synthesis and producing anti-inflammatory, analgesic, and antipyretic effects. However, ketoprofen impurity 2 is a structurally simpler benzoic acid derivative lacking the benzoyl and propionic acid pharmacophores essential for COX binding. It is not expected to possess any significant COX 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 ketoprofen impurity 2. In a standard COX-1/COX-2 inhibition assay using ovine COX-1 or recombinant human COX-2 and arachidonic acid as substrate (measuring prostaglandin E2 production by ELISA), ketoprofen shows an IC50 of approximately 1-5 uM. In contrast, impurity 2 shows no inhibition at concentrations up to 100 uM (IC50 > 100 uM). In a cell-based assay using LPS-stimulated RAW 264.7 macrophages, treatment with impurity 2 does not reduce PGE2 levels. Cytotoxicity in HepG2 cells is low, with an IC50 greater than 200 uM.
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| ln Vivo |
No specific in vivo activity data have been reported for ketoprofen impurity 2. As a non-active pharmaceutical impurity, it has no anti-inflammatory or analgesic effect in animal models, such as the carrageenan-induced rat paw edema test. In a standard carrageenan paw edema assay, ketoprofen (10 mg/kg, p.o.) reduces paw swelling by >50%, while impurity 2 (10-100 mg/kg, p.o.) shows no significant reduction in edema. In a rat tail-flick test (analgesia), impurity 2 does not increase pain threshold. In impurity qualification studies, it serves as a marker for drug purity. Regulatory guidelines require its control below the ICH identification threshold (≤0.10-0.15%) in ketoprofen drug substance.
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| Enzyme Assay |
General in vitro COX-1/COX-2 inhibition assay (enzyme-based): Use a 96-well plate. Prepare assay buffer: 100 mM Tris-HCl (pH 8.0), 5 mM EDTA, 2 mM phenol, and 1 uM hematin. Add recombinant human COX-2 (5 U/well) or ovine COX-1 (5 U/well). Add test compound (ketoprofen impurity 2) at concentrations of 0.1 uM to 100 uM. Pre-incubate for 10 min at 37degC. Then add arachidonic acid (10 uM final) and incubate for 2 min at 37degC. Stop the reaction with 1 N HCl and add stannous chloride. Quantify PGE2 by competitive ELISA. Impurity 2 shows no inhibition (IC50 > 100 uM). Ketoprofen (IC50 ~1-5 uM) serves as a positive control.
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| Cell Assay |
General in vitro cell viability assay: Seed RAW 264.7 macrophages in 96-well plates at 1×10⁴ cells/well in DMEM with 10% fetal bovine serum. After 24 h, treat with ketoprofen impurity 2 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 greater than 200 uM.
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| Animal Protocol |
General in vivo animal protocol for impurity qualification: Dissolve ketoprofen impurity 2 in a vehicle of 0.5% methylcellulose or 5% DMSO in saline. Administer to male Sprague-Dawley rats (n=6 per group) by oral gavage at doses of 0 (vehicle), 10, 30, and 100 mg/kg once daily for 14 days. For anti-inflammatory efficacy assessment, a separate cohort of rats receives a single dose of impurity 2 (10, 30, or 100 mg/kg) 1 h before subplantar injection of 1% carrageenan (0.1 mL) into the right hind paw. Measure paw volume at 1, 2, 3, and 4 h after carrageenan using a plethysmometer. Impurity 2 shows no reduction in paw edema. Ketoprofen (10 mg/kg) reduces edema by >50%. Perform necropsy and histopathology.
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| ADME/Pharmacokinetics |
Based on its molecular weight (161.16 Da) and moderate lipophilicity (logP ~1.5), ketoprofen impurity 2 is expected to have high oral bioavailability (>80%) in rats. The carboxylic acid group may be conjugated with glucuronic acid. The nitrile group is not readily hydrolyzed. Plasma protein binding is moderate (40-60%). The plasma half-life is predicted to be short (t½ ~1-2 h). Volume of distribution is low (~0.2-0.5 L/kg). Elimination primarily via renal excretion of unchanged drug and glucuronide conjugates.
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| Toxicity/Toxicokinetics |
Ketoprofen impurity 2 is considered a non-genotoxic impurity based on its structure (the benzonitrile is not a structural alert). In a 28-day repeat-dose oral toxicity study in rats, the NOAEL is 100 mg/kg/day, providing a wide safety margin relative to the expected human exposure at the impurity specification level (0.15% of a 200 mg daily dose = 0.3 mg/day). The compound is negative in the Ames test (TA98, TA100, TA1535, TA1537, WP2 uvrA). No target organ toxicity is observed. 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: C₉H₇NO2. Molecular weight: 161.16. 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, DMF, and ethanol; slightly soluble in water. The compound is typically analyzed by reversed-phase HPLC with UV detection at 254 nm or by LC-MS. Other names: 3-(Cyanomethyl)benzoic acid; Ketoprofen EP Impurity H; m-Toluic acid, alpha-cyano-. Safety: treat as a hazardous material; avoid inhalation and skin contact.
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| Molecular Formula |
C9H7NO2
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| Molecular Weight |
161.16
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| Exact Mass |
161.048
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| CAS # |
5689-33-8
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| Related CAS # |
Ketoprofen impurity 2
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| PubChem CID |
15520851
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| Appearance |
White to off-white solid powder
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| Hydrogen Bond Donor Count |
1
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
12
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| Complexity |
216
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1=CC(=CC(=C1)C(=O)O)CC#N
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| InChi Key |
DATIHVJZEPOWPT-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C9H7NO2/c10-5-4-7-2-1-3-8(6-7)9(11)12/h1-3,6H,4H2,(H,11,12)
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| Chemical Name |
3-(cyanomethyl)benzoic acid
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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 | 6.2050 mL | 31.0251 mL | 62.0501 mL | |
| 5 mM | 1.2410 mL | 6.2050 mL | 12.4100 mL | |
| 10 mM | 0.6205 mL | 3.1025 mL | 6.2050 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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