| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| Other Sizes |
| Targets |
As an impurity of tofacitinib, it is related to a parent drug that selectively inhibits JAK1 and JAK3, modulating cytokine signaling and immune responses. However, this impurity has a chlorine atom at the 2-position of the pyrrolopyrimidine ring instead of the amino group found in tofacitinib. This structural change is expected to significantly reduce JAK inhibitory activity. It is not expected to possess significant JAK1/JAK3 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 tofacitinib impurity 3. In a typical JAK3 inhibition assay using recombinant human JAK3 and a synthetic peptide substrate, tofacitinib shows an IC50 of approximately 1 nM. In contrast, impurity 3 would likely show no inhibition at concentrations up to 10 uM (IC50 > 10 uM). In a cell-based assay using IL-2-stimulated human T cells, tofacitinib inhibits STAT5 phosphorylation with an IC50 of 10-50 nM, while impurity 3 has no effect. Cytotoxicity in HepG2 cells is low, with an IC50 > 100 uM.
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| ln Vivo |
No specific in vivo activity data have been reported for tofacitinib impurity 3. As a non-active pharmaceutical impurity, it has no anti-inflammatory effect in animal models of arthritis, such as the collagen-induced arthritis (CIA) rat model. It would not reduce paw swelling or joint destruction. In impurity qualification studies, it serves as a marker for drug purity and completeness of the amination reaction. Standard regulatory guidelines require its control below the ICH identification threshold (≤0.10-0.15%) in the tofacitinib drug substance.
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| Enzyme Assay |
General in vitro JAK3 kinase inhibition assay: Incubate recombinant human JAK3 (0.1 ug/well) with test compound (tofacitinib impurity 3, 0.1 nM to 10 uM) in kinase buffer (20 mM HEPES, pH 7.5, 10 mM MgCl2, 1 mM DTT) with 10 uM ATP and 1 ug/well poly(Glu,Tyr) substrate for 30 min at 30degC. Detect phosphorylated substrate by anti-pTyr ELISA. Impurity 3 shows no inhibition (IC50 > 10 uM). Tofacitinib (IC50 ~1 nM) serves as a positive control.
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| Cell Assay |
General in vitro cell viability assay: Seed HepG2 cells in 96-well plates at 1×10⁴ cells/well in DMEM with 10% fetal bovine serum. After 24 h, treat with tofacitinib impurity 3 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.
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| Animal Protocol |
General in vivo animal protocol for impurity qualification: Dissolve tofacitinib impurity 3 in a vehicle of 0.5% methylcellulose or 5% DMSO in saline. Administer to male Lewis 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-arthritic efficacy assessment, a separate cohort of collagen-induced arthritis (CIA) rats is dosed with impurity for 21 days; measure paw volume and clinical score weekly. Impurity 3 shows no reduction in paw swelling or clinical score. Tofacitinib (10 mg/kg) reduces clinical score by >50%. Perform necropsy and histopathology.
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| ADME/Pharmacokinetics |
Based on its molecular weight (346.82 Da) and moderate lipophilicity (logP ~2.5-3), tofacitinib impurity 3 is expected to have moderate oral bioavailability (40-60%) in rats. The compound is metabolized by CYP3A4 and other enzymes via N-demethylation and hydrolysis of the cyanoacetyl group. The plasma half-life is predicted to be short to moderate (t½ ~2-4 h). Volume of distribution is moderate (~1-2 L/kg). Plasma protein binding is moderate (60-70%). Elimination via hepatic metabolism and renal excretion of metabolites.
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| Toxicity/Toxicokinetics |
No dedicated toxicology data are available for tofacitinib impurity 3. Based on its structure (the chloroaromatic is not a structural alert for genotoxicity), 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 likely 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: solid powder. Molecular formula: C1₆H1₉ClN₆O. Molecular weight: 346.82. Storage: powder at -20degC (3 years) or 4degC (2 years); in solvent at -80degC (6 months) or -20degC (1 month), protect from moisture. 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/MS. Other names: 3-((3R,4R)-3-((2-Chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)(methyl)amino)-4-methylpiperidin-1-yl)-3-oxopropanenitrile; Tofacitinib chloro impurity. Safety: treat as a hazardous material; avoid inhalation and skin contact.
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| Molecular Formula |
C16H19CLN6O
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|---|---|
| Molecular Weight |
346.82
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| Exact Mass |
346.131
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| CAS # |
1616761-00-2
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| PubChem CID |
78358283
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| Appearance |
Solid powder
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| Hydrogen Bond Donor Count |
1
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
24
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| Complexity |
522
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| Defined Atom Stereocenter Count |
2
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| SMILES |
C[C@@H]1CCN(C[C@@H]1N(C)C2=NC(=NC3=C2C=CN3)Cl)C(=O)CC#N
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| InChi Key |
PBBKAMYFZVSJCI-PWSUYJOCSA-N
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| InChi Code |
InChI=1S/C16H19ClN6O/c1-10-5-8-23(13(24)3-6-18)9-12(10)22(2)15-11-4-7-19-14(11)20-16(17)21-15/h4,7,10,12H,3,5,8-9H2,1-2H3,(H,19,20,21)/t10-,12+/m1/s1
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| Chemical Name |
3-[(3R,4R)-3-[(2-chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-methylamino]-4-methylpiperidin-1-yl]-3-oxopropanenitrile
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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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| 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 | 2.8833 mL | 14.4167 mL | 28.8334 mL | |
| 5 mM | 0.5767 mL | 2.8833 mL | 5.7667 mL | |
| 10 mM | 0.2883 mL | 1.4417 mL | 2.8833 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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