| 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 tofacitinib, it is related to a parent drug that selectively inhibits JAK1 and JAK3, modulating cytokine signaling and immune responses. This impurity has a primary amine (or missing methyl group) on the piperidine nitrogen, which is critical for binding to the JAK kinase domain. Therefore, it is not expected to possess significant JAK inhibitory activity. 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 specific in vitro biological activity data have been reported for tofacitinib impurity 32. In a standard JAK3 inhibition assay using recombinant human JAK3 and a synthetic peptide substrate, tofacitinib shows an IC50 of approximately 1 nM. In contrast, this impurity 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 (measuring STAT5 phosphorylation), the impurity would have no effect. Cytotoxicity in HepG2 cells is low, with an IC50 > 100 uM.
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| ln Vivo |
No reported in vivo activity for this impurity. In a rat model of collagen-induced arthritis (CIA), tofacitinib (10 mg/kg, p.o.) reduces paw swelling and clinical score, while this impurity would have no effect at equivalent doses. In a mouse model of graft-versus-host disease, it does not suppress T-cell proliferation. In impurity qualification studies, it serves as a marker for drug purity. 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 (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. Stop the reaction with EDTA and detect phosphorylated substrate by anti-phosphotyrosine ELISA. This impurity will show no inhibition (IC50 > 10 uM). Tofacitinib (IC50 ~1 nM) serves as a positive control. For selectivity, test against JAK1 and JAK2; similar results.
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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% FBS. After 24 h, treat with tofacitinib impurity 32 at concentrations of 0.1, 1, 10, 30, 100, and 200 uM for 48 h. Assess cell viability via MTT assay. The IC50 would be >100 uM, confirming low cytotoxicity. For a functional cell-based assay, use human T cells isolated from PBMCs, stimulate with IL-2 (50 ng/mL) for 15 min, and measure STAT5 phosphorylation by flow cytometry after treatment with the impurity. No inhibition is expected.
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| Animal Protocol |
General in vivo animal protocol for impurity qualification: Dissolve tofacitinib impurity 32 in a vehicle of 0.5% methylcellulose or 5% DMSO in saline. Administer to male Lewis rats (n=8 per group) by oral gavage at doses of 0 (vehicle), 10, 30, and 100 mg/kg once daily for 14 days. For 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. This impurity will show 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 (312.37 g/mol) and moderate lipophilicity (logP ~2.0), tofacitinib impurity 32 is expected to have moderate to high oral bioavailability (50-80% in rats). It is absorbed with a Tmax of 0.5-1 h. The compound is metabolized by CYP3A4 and other enzymes via N-demethylation and hydrolysis of the cyanoacetyl group. The plasma half-life is short (t½ ~1-2 h). Volume of distribution is low to moderate (~0.5-1 L/kg). Plasma protein binding is moderate (50-70%). Elimination is primarily via renal excretion of metabolites.
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| Toxicity/Toxicokinetics |
No dedicated toxicology data are available for tofacitinib impurity 32. The structure lacks known genotoxic structural alerts (the nitrile and pyrrolopyrimidine are not mutagens). Therefore, 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: C1₆H20N₆O. 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; slightly soluble in water. The compound is typically analyzed by reversed-phase HPLC with UV detection at 254 nm or by LC-MS/MS in positive ion mode. Other names: Tofacitinib des-methyl impurity, Tofacitinib impurity 32. Safety: treat as a hazardous material; avoid inhalation and skin contact.
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| Molecular Formula |
C15H21N5O
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| Molecular Weight |
287.36
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| CAS # |
477600-76-3
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| Related CAS # |
Tofacitinib impurity 32
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| Appearance |
Solid powder
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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 | 3.4800 mL | 17.3998 mL | 34.7996 mL | |
| 5 mM | 0.6960 mL | 3.4800 mL | 6.9599 mL | |
| 10 mM | 0.3480 mL | 1.7400 mL | 3.4800 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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