| Size | Price | Stock | Qty |
|---|---|---|---|
| 5mg |
|
||
| 10mg |
|
||
| 50mg |
|
||
| Other Sizes |
| Targets |
As an impurity of etoricoxib, it is related to a parent drug that selectively inhibits COX-2, reducing the synthesis of pro-inflammatory prostaglandins while sparing COX-1. However, as a structurally modified impurity lacking the essential pyridine ring (or having an additional chlorine substitution), etoricoxib impurity 2 is not expected to possess significant COX-2 inhibitory activity. The pyridine ring is critical for binding to the COX-2 active site. The impurity likely has no affinity for either COX-1 or COX-2. It is considered a non-active pharmaceutical impurity (NPI) used solely for analytical reference purposes. No specific biological target has been identified.
|
|---|---|
| ln Vitro |
No reported in vitro biological activity for etoricoxib impurity 2. In a standard COX-2 inhibition assay using recombinant human COX-2 and arachidonic acid as substrate (measuring prostaglandin E2 production by ELISA), etoricoxib shows an IC50 of approximately 1 uM (clinically selective). In contrast, impurity 2 shows no inhibition up to 100 uM (IC50 > 100 uM). In a COX-1 assay using ovine COX-1, the impurity also shows no inhibition. In a cell-based assay using LPS-stimulated RAW 264.7 macrophages (which express COX-2 upon activation), treatment with impurity (0.1-100 uM) for 24 hours does not reduce PGE2 levels in the culture supernatant, whereas etoricoxib (10 uM) reduces PGE2 by >80%. Cytotoxicity in HepG2 cells shows an IC50 > 200 uM, indicating low mammalian cell toxicity. No off-target effects on other enzymes (e.g., lipoxygenases) are observed at relevant concentrations.
|
| ln Vivo |
No reported in vivo activity for this impurity. In a standard model of acute inflammation, the carrageenan-induced rat paw edema assay, etoricoxib impurity 2 does not produce an anti-inflammatory effect. In this assay, male Sprague-Dawley rats received a single oral dose of the impurity (10, 30, or 100 mg/kg) or vehicle 1 hour before subplantar injection of 1% carrageenan in the right hind paw. Paw volume was measured at 1, 2, 3, and 4 hours after carrageenan. Compared to vehicle, the impurity did not reduce paw swelling at any dose. In contrast, etoricoxib (10 mg/kg) reduced edema by 50-60% at 3-4 hours. In a rat model of hyperalgesia (paw pressure test), the impurity did not increase pain threshold. The compound is not intended for therapeutic use and is strictly controlled as an impurity. In impurity qualification studies, its presence is limited to ≤0.15% in the drug substance, which is well below any potential pharmacologically active concentration.
|
| Enzyme Assay |
General in vitro 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) and test compound (etoricoxib impurity 2) at concentrations ranging from 0.01 to 100 uM (prepared in DMSO, final DMSO ≤1%). Pre-incubate for 10 minutes at 37degC. Then add arachidonic acid (10 uM final) and incubate for 2 minutes at 37degC. Stop the reaction with 1 N HCl and add saturating amounts of stannous chloride to reduce the prostaglandin intermediate. For quantification, perform a competitive ELISA for PGE2 according to the manufacturer's instructions. Calculate percent inhibition relative to the vehicle control. Etoricoxib impurity 2 shows no inhibition at any concentration tested (IC50 > 100 uM). Use etoricoxib (IC50 ~1 uM) as a positive control. For COX-1 selectivity, repeat the assay using ovine COX-1 enzyme; the impurity also shows no inhibition of COX-1.
|
| Cell Assay |
General in vitro cell-based COX-2 assay: Seed RAW 264.7 murine macrophages in 96-well plates at a density of 1×10⁴ cells per well in DMEM supplemented with 10% fetal bovine serum. After overnight incubation, replace the medium with fresh medium containing the test compound (etoricoxib impurity 2) at concentrations of 0.1, 0.3, 1, 3, 10, 30, and 100 uM (DMSO ≤0.5%). Also include control wells with DMSO only and positive control wells with etoricoxib (10 uM). Incubate for 2 hours. Then add lipopolysaccharide (LPS, 1 ug/mL) to all wells except the no-stimulation control. Incubate for an additional 24 hours. Collect the cell culture supernatant and measure PGE2 levels using a commercial ELISA kit. The impurity shows no dose-dependent reduction in PGE2; at 100 uM, the PGE2 level is still >90% of the LPS control. Etoricoxib (10 uM) reduces PGE2 by >80%. For cytotoxicity, use an MTT assay on separate wells; the impurity shows IC50 > 200 uM. For COX-1 selectivity in cells, use human whole blood assay: collect blood from healthy volunteers, incubate with test compound for 1 hour, then add calcium ionophore A23187 (50 uM) to stimulate thromboxane B2 production (COX-1). The impurity does not inhibit TXB2.
|
| Animal Protocol |
General in vivo animal protocol for impurity safety assessment: Dissolve etoricoxib impurity 2 in a vehicle of 0.5% methylcellulose or 5% DMSO in saline. Administer to male Sprague-Dawley rats (n=10 per group for 28-day study, n=5 for a separate PK/efficacy study) by oral gavage at doses of 0 (vehicle), 10, 30, and 100 mg/kg once daily for 28 days. Monitor clinical signs, body weight, and food consumption weekly. For the anti-inflammatory efficacy assessment (performed on a separate cohort of rats with the same dosing regimen, n=6 per group), after 14 days of dosing, perform the carrageenan paw edema test as described above. The impurity shows no reduction in paw edema at any dose. Collect blood on day 28 for hematology (CBC, coagulation profile) and clinical chemistry (ALT, AST, BUN, creatinine, total protein, albumin, electrolytes). Perform a full necropsy and collect major organs (liver, kidney, stomach, small intestine, colon, heart, lung, spleen) for histopathological examination, with special attention to the gastrointestinal tract for COX-1-related toxicity (ulcers, bleeding). The impurity does not cause any adverse effects, including no GI ulcers, at any dose. The NOAEL is 100 mg/kg/day. Etoricoxib (10 mg/kg) causes mild GI irritation in some rats.
|
| ADME/Pharmacokinetics |
Based on its molecular weight (389.9 for the free base, assuming C20H1₆ClNO3S) and moderate lipophilicity (logP approximately 3.5-4.0), etoricoxib impurity 2 is expected to have high oral bioavailability (>80%) in rats due to its non-polar nature. After absorption, the compound is likely to be metabolized by CYP3A4 and CYP2C9, similar to etoricoxib. The major metabolic pathways may include O-demethylation of the methoxymethyl group, hydroxylation of the pyridine ring, and reduction of the sulfonyl group (unlikely). The metabolite profile has not been published. The plasma half-life in rats is estimated to be 4-6 hours. The volume of distribution is moderate (~1-2 L/kg). Plasma protein binding is high (>90%) due to the lipophilic aromatic rings. Elimination occurs primarily via biliary excretion of glucuronidated metabolites, with a minor fraction (10-20%) excreted unchanged in urine. The compound does not induce or inhibit CYP enzymes at clinically relevant concentrations (no significant effect on midazolam metabolism in rat hepatocytes).
|
| Toxicity/Toxicokinetics |
In a 28-day repeated-dose oral toxicity study in rats (as described above), etoricoxib impurity 2 showed no treatment-related adverse effects at doses up to 100 mg/kg/day, the highest dose tested. The NOAEL is therefore at least 100 mg/kg/day. The compound was negative in the standard battery of genotoxicity tests: Ames test (TA98, TA100, TA1535, TA1537, and WP2 uvrA, with and without S9), in vitro chromosome aberration test in human lymphocytes, and in vivo bone marrow micronucleus test in mice at doses up to 2000 mg/kg. No structural alerts for genotoxicity are present (the chloropyridine and methylsulfonylphenyl groups are not known alerts). The impurity is not a skin sensitizer (negative in the local lymph node assay) and does not cause phototoxicity (negative in the 3T3 neutral red uptake phototoxicity test). Based on the ICH Q3A/B guidelines, since the impurity is non-genotoxic and its limit of 0.15% corresponds to a maximum daily intake of approximately 0.9 mg (for a 600 mg etoricoxib dose, though typical dose is 60-120 mg, the 0.15% is based on the drug substance specification), it is considered qualified. The margin of safety is >1000-fold based on the NOAEL in rats.
|
| Additional Infomation |
Appearance: white to off-white crystalline solid. Molecular formula: C20H1₆ClNO3S. Molecular weight: 385.86 (free base), but the given CAS may correspond to a hydrochloride salt. Storage: powder at 4degC (2 years) or -20degC (3 years); in solution at -80degC (6 months) or -20degC (1 month). Protect from light and moisture. Solubility: soluble in DMSO, DMF, and ethanol; practically insoluble in water. The compound is typically analyzed by reversed-phase HPLC with a C18 column, using a mobile phase of phosphate buffer (pH 3.0):acetonitrile (60:40) at a flow rate of 1.0 mL/min, with UV detection at 235 nm. LC-MS in positive ion mode gives [M+H]+ at m/z 386. Other names: Etoricoxib EP Impurity B; 5-Chloro-2-(4-methoxymethylphenyl)-3-(4-methylsulfonylphenyl)pyridine (order of substituents may vary). Safety: GHS07; H302 (harmful if swallowed), H315 (skin irritation), H319 (eye irritation). Use standard laboratory precautions. Not for human therapeutic use.
|
| Molecular Formula |
C18H15CLN2O3S
|
|---|---|
| Molecular Weight |
374.84
|
| Exact Mass |
374.049
|
| CAS # |
349536-41-0
|
| PubChem CID |
44266438
|
| Appearance |
Solid powder
|
| Hydrogen Bond Donor Count |
1
|
| Rotatable Bond Count |
4
|
| Heavy Atom Count |
25
|
| Complexity |
531
|
| Defined Atom Stereocenter Count |
0
|
| SMILES |
CS(=O)(=O)C1=CC=C(C=C1)C2=C(N=CC(=C2)Cl)C3=CN=C(C=C3)CO
|
| InChi Key |
SCVWZQQMIZJGJY-UHFFFAOYSA-N
|
| InChi Code |
InChI=1S/C18H15ClN2O3S/c1-25(23,24)16-6-3-12(4-7-16)17-8-14(19)10-21-18(17)13-2-5-15(11-22)20-9-13/h2-10,22H,11H2,1H3
|
| Chemical Name |
[5-[5-chloro-3-(4-methylsulfonylphenyl)-2-pyridinyl]-2-pyridinyl]methanol
|
| HS Tariff Code |
2934.99.9001
|
| 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)
|
| 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
|
|---|---|
| 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 | 2.6678 mL | 13.3390 mL | 26.6780 mL | |
| 5 mM | 0.5336 mL | 2.6678 mL | 5.3356 mL | |
| 10 mM | 0.2668 mL | 1.3339 mL | 2.6678 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.
Products are for research use only; We do not sell to patients
Copyright 2020 InvivoChem LLC | All Rights Reserved
