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| 1mg |
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| Other Sizes |
| Targets |
JAK-IN-1 targets the Janus kinase family of non-receptor tyrosine kinases, specifically JAK1, JAK2, and JAK3. These kinases are essential components of the JAK-STAT signaling pathway, which mediates signal transduction from cytokine and growth factor receptors to the nucleus. JAK3 is primarily expressed in hematopoietic cells and is involved in signaling through the common gamma chain (gammac) of cytokine receptors, including IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. JAK1 and JAK2 are more ubiquitously expressed and are involved in signaling through multiple cytokine receptors. By inhibiting JAK kinases, JAK-IN-1 prevents the phosphorylation and activation of STAT transcription factors, thereby blocking cytokine-induced gene expression. The compound's improved selectivity for JAK3 over JAK1 may offer advantages in reducing off-target effects associated with broader JAK inhibition.
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| ln Vitro |
JAK-IN-1 suppresses the growth of human CD4 and CD8 T cells in a dose-dependent manner following stimulation with anti-CD3/anti-CD28 antibody-coated beads, emulating in part the activation signals that antigen-presenting cells give to T cells. T cells, one of the main cell types for which JAK3 may be important, are used in both cell-based mechanistic and functional experiments where JAK-IN-1 is active [1].
JAK-IN-1 demonstrates potent in vitro activity against JAK kinases and in cell-based assays. The compound inhibits JAK1, JAK2, and JAK3 with IC50 values of 0.26, 0.8, and 3.2 nM, respectively. In cellular assays using human CD4 and CD8 T cells stimulated with anti-CD3/anti-CD28 antibody-coated beads, JAK-IN-1 inhibits T cell proliferation in a dose-dependent manner. This assay partially mimics the activation signals that antigen-presenting cells deliver to T cells. T cells are one of the primary cell types where JAK3 plays an important role, and JAK-IN-1 is useful for cell-based mechanistic and functional experiments. The compound's sub-nanomolar potency against JAK1 and JAK2 and nanomolar potency against JAK3 make it one of the most potent JAK inhibitors described. Its activity has been characterized in both biochemical and cellular assays. |
| ln Vivo |
Greater inhibitory potency for JAK1/JAK3-driven signaling in whole blood assays as opposed to JAK2- or JAK1/JAK2/TYK2-driven signaling indicates that JAK-IN-1 is specific for JAK3 in vivo. JAK-IN-1 dose-dependent inhibition of IL-2-stimulated JAK-IN-1 plasma concentrations was observed. In a dosage- and concentration-dependent manner, JAK-IN-1 inhibits IL-2-driven STAT5 phosphorylation; at a dose of 10 mg/kg (plasma concentration ~480 nM), roughly 50% inhibition is seen [1].
In vivo, JAK-IN-1 demonstrates JAK3-specific activity in whole blood assays. In whole blood assays, JAK1/JAK3-driven signaling is inhibited with greater potency compared to JAK2- or JAK1/JAK2/TYK2-driven signaling, indicating that JAK-IN-1 exhibits JAK3 specificity in vivo. Dose-dependent inhibition of IL-2-stimulated JAK-IN-1 plasma concentrations has been observed. JAK-IN-1 inhibits IL-2-driven STAT5 phosphorylation in a dose- and concentration-dependent manner. At a dose of 10 mg/kg (corresponding to a plasma concentration of approximately 480 nM), approximately 50% inhibition is observed. These in vivo data confirm that JAK-IN-1 is active in physiologically relevant settings and can modulate JAK-STAT signaling in vivo. The compound's ability to inhibit T cell proliferation and JAK-STAT signaling in vivo supports its utility for studying immune-mediated diseases. |
| Enzyme Assay |
In vitro enzyme/receptor binding assays for JAK-IN-1 involve measuring the inhibition of JAK kinase activity using a radiometric or luminescent assay. Recombinant JAK1, JAK2, and JAK3 kinases are incubated with varying concentrations of the test compound, ATP, and a peptide substrate. The transfer of the phosphate group from ATP to the substrate is quantified using radioactive [33P]-ATP or by detecting phosphorylated peptide using a luminescent method such as the ADP-Glo assay. IC50 values are calculated by plotting percent inhibition against compound concentration using non-linear regression analysis. Selectivity assays compare the compound's activity against a panel of kinases to determine its specificity profile. The assay is typically performed in 96-well or 384-well plate format with appropriate positive controls (known JAK inhibitors such as tofacitinib) and vehicle controls. Each concentration is tested in duplicate or triplicate.
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| Cell Assay |
In vitro cellular assays for JAK-IN-1 are performed using human primary T cells or T cell lines. Human CD4 and CD8 T cells are isolated from peripheral blood and stimulated with anti-CD3/anti-CD28 antibody-coated beads to activate T cell receptor signaling. Cells are treated with varying concentrations of JAK-IN-1 for 48-72 hours, and proliferation is measured using [3H]-thymidine incorporation, CFSE dilution, or CellTiter-Glo assays. STAT5 phosphorylation is measured by flow cytometry or Western blot following IL-2 stimulation to assess JAK-STAT signaling inhibition. Cytokine production (IL-2, IFN-gamma, TNF-alpha) is measured by ELISA or multiplex bead-based assays. Cytotoxicity is assessed in parallel using standard viability assays to ensure that observed inhibition is not due to cell death. IC50 values for inhibition of T cell proliferation are calculated from dose-response curves.
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| Animal Protocol |
In vivo animal studies for JAK-IN-1 are conducted using mouse or rat models. Pharmacodynamic studies involve administering the compound via oral gavage or intraperitoneal injection to animals, followed by ex vivo stimulation of whole blood or spleen cells with IL-2 or other cytokines. STAT5 phosphorylation is measured by flow cytometry or phospho-specific ELISA to assess target engagement. Pharmacokinetic studies assess drug concentrations in plasma to correlate with pharmacodynamic effects. Efficacy studies may be conducted in animal models of autoimmune disease, such as collagen-induced arthritis (CIA) or experimental autoimmune encephalomyelitis (EAE), where JAK inhibition has demonstrated therapeutic benefit. Animals are monitored for clinical signs of disease, body weight, and survival. Tissues are collected for histopathological examination and biomarker analysis. Dosing regimens are optimized based on pharmacokinetic-pharmacodynamic relationships.
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| ADME/Pharmacokinetics |
JAK-IN-1 exhibits favorable pharmacokinetic properties for an orally administered small molecule. The compound is orally available, making it suitable for convenient dosing regimens in research settings. Following oral administration, JAK-IN-1 achieves plasma concentrations that are sufficient to inhibit JAK-STAT signaling, as demonstrated by the inhibition of IL-2-driven STAT5 phosphorylation at a dose of 10 mg/kg (plasma concentration of approximately 480 nM). The compound has a molecular formula of C20H24N6O2 and a molecular weight of 380.44 g/mol. It is soluble in DMSO and other organic solvents. The compound's pharmacokinetic profile supports its use in preclinical studies of JAK-STAT signaling and immune-mediated diseases. Comprehensive pharmacokinetic parameters including half-life, volume of distribution, clearance, and oral bioavailability have been characterized in animal models.
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| Toxicity/Toxicokinetics |
JAK-IN-1 is intended for laboratory research use only and has not undergone comprehensive clinical toxicology testing. As a potent JAK inhibitor, the compound would be expected to have immunosuppressive effects, which could increase the risk of infections and malignancies with chronic exposure. Standard in vitro cytotoxicity assays in cell lines are typically performed alongside efficacy studies to rule out nonspecific toxicity. In vivo, animals are monitored for signs of toxicity including body weight changes, behavioral abnormalities, and clinical observations. The compound's selectivity for JAK3 over JAK1 may offer a more favorable safety profile compared to pan-JAK inhibitors, as JAK3 inhibition is more selectively immunosuppressive. However, comprehensive toxicological characterization including genotoxicity, cardiotoxicity, and repeated-dose toxicity studies has not been reported in the public domain. The compound is not approved for human use and is strictly intended for research purposes.
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| References | |
| Additional Infomation |
JAK-IN-1 is a potent, orally available inhibitor of JAK1, JAK2, and JAK3 with IC50 values of 0.26, 0.8, and 3.2 nM, respectively. It shows improved selectivity for JAK3 over JAK1. The compound has a molecular formula of C20H24N6O2 and a molecular weight of 380.44 g/mol. Its chemical name is N-[(2R)-1-(3-cyanoazetidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl]-2-cyclopropyl-5H-pyrrolo[2,3-b]pyrazine-7-carboxamide. JAK-IN-1 inhibits the proliferation of human CD4 and CD8 T cells in a dose-dependent manner. In whole blood assays, the compound demonstrates JAK3-specific activity, inhibiting JAK1/JAK3-driven signaling with greater potency than JAK2- or JAK1/JAK2/TYK2-driven signaling. The compound is not in clinical trials and has not received regulatory approval. It is available from research chemical suppliers for non-clinical research purposes only. JAK-IN-1 is a valuable research tool for studying JAK-STAT signaling and evaluating the therapeutic potential of JAK3-selective inhibition.
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| Molecular Formula |
C20H24N6O2
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| Molecular Weight |
380.443563461304
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| Exact Mass |
380.196
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| CAS # |
1334673-53-8
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| PubChem CID |
54589404
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| Appearance |
White to off-white solid powder
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| LogP |
1
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
28
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| Complexity |
685
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| Defined Atom Stereocenter Count |
1
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| SMILES |
O=C([C@@H](C(C)(C)C)NC(C1=CNC2=C1N=C(C=N2)C1CC1)=O)N1CC(C#N)C1
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| InChi Key |
SIEMFXMJGAMFQR-INIZCTEOSA-N
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| InChi Code |
InChI=1S/C20H24N6O2/c1-20(2,3)16(19(28)26-9-11(6-21)10-26)25-18(27)13-7-22-17-15(13)24-14(8-23-17)12-4-5-12/h7-8,11-12,16H,4-5,9-10H2,1-3H3,(H,22,23)(H,25,27)/t16-/m0/s1
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| Chemical Name |
N-[(2R)-1-(3-cyanoazetidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl]-2-cyclopropyl-5H-pyrrolo[2,3-b]pyrazine-7-carboxamide
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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) |
DMSO : ~66.67 mg/mL (~175.24 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 1.43 mg/mL (3.76 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 14.3 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 1.43 mg/mL (3.76 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 14.3 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.6285 mL | 13.1427 mL | 26.2854 mL | |
| 5 mM | 0.5257 mL | 2.6285 mL | 5.2571 mL | |
| 10 mM | 0.2629 mL | 1.3143 mL | 2.6285 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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