| Size | Price | Stock | Qty |
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| 1mg |
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| 5mg |
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| 10mg |
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| Other Sizes |
| Targets |
HPK1 0.25 nM (IC50)
HPK1-IN-3 specifically targets HPK1 (MAP4K1). It is a potent ATP-competitive inhibitor with an IC50 of 0.25 nM against the enzyme. HPK1 is a serine/threonine kinase that acts as a negative regulator of T-cell receptor (TCR) signaling. By blocking HPK1 activity, the compound is designed to relieve this brake on T-cell activation, promoting a more robust immune response. It also has reported cellular potency in human PBMCs. |
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| ln Vitro |
In human monocyte-derived dendritic cells, treatment with HPK1-IN-3 (Compound 27; 0.25–4 μM; 24 hours) significantly increased the concentration-dependent levels of pro-inflammatory cytokines TNF-α and IL-6 [1].
In cell-free enzymatic assays, HPK1-IN-3 demonstrates potent inhibitory activity against HPK1 (MAP4K1) with an IC50 of 0.25 nM. This data confirms it as a potent ATP-competitive inhibitor. In cellular assays using human peripheral blood mononuclear cells (PBMCs), HPK1-IN-3 exhibits an EC50 of 108 nM for enhancing IL-2 production, demonstrating its functional activity and ability to modulate T-cell responses in a relevant cellular system. |
| ln Vivo |
Specific in vivo activity data for HPK1-IN-3 is not published. However, given its potent in vitro activity and cellular EC50 of 108 nM, it is hypothesized to enhance T-cell responses in vivo. This could be demonstrated in a syngeneic mouse tumor model (e.g., MC38 or CT26), where oral administration of HPK1-IN-3 might slow tumor growth by enhancing CD8+ T-cell infiltration and effector function. It could also be used in combination with an anti-PD-1 antibody to potentially overcome resistance.
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| Enzyme Assay |
The specific in vitro protocol for HPK1-IN-3 uses an ADP-Glo™ Kinase Assay. The assay is performed in white 384-well plates. Recombinant full-length human HPK1 (MAP4K1) enzyme is incubated with a serial dilution of HPK1-IN-3 (e.g., 1 pM to 10 uM) in kinase reaction buffer containing 10 uM ATP and MBP substrate. After 60 minutes at room temperature, ADP-Glo Reagent is added to terminate the reaction and deplete remaining ATP. After 40 minutes, Kinase Detection Reagent is added to convert ADP to ATP and to allow measurement of luminescence using a plate reader. IC50 values are calculated using a four-parameter logistic curve.
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| Cell Assay |
For in vitro cellular assays, human peripheral blood mononuclear cells (PBMCs) are isolated from whole blood using Ficoll-Paque density gradient centrifugation. PBMCs are seeded in 96-well plates pre-coated with anti-CD3 antibody (1 ug/mL). HPK1-IN-3 is added at concentrations ranging from 0.1 nM to 10 uM, along with soluble anti-CD28 antibody (1 ug/mL). After 72 hours of culture, cell supernatants are harvested. IL-2 levels are quantified using an electrochemiluminescence (ECL) or ELISA-based assay. The EC50 for IL-2 production is calculated from the concentration-response curve.
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| Animal Protocol |
An in vivo pharmacodynamic (PD) protocol for HPK1-IN-3 would assess its ability to enhance T-cell activation. C57BL/6 mice are administered HPK1-IN-3 via oral gavage at doses of 1-30 mg/kg. At predetermined time points (e.g., 2, 6, 12, 24 hours post-dose), mice are euthanized and spleens are harvested. Splenocytes are isolated and stimulated ex vivo with anti-CD3/CD28 beads for 4-6 hours in the presence of GolgiPlug (brefeldin A). Cells are then surface-stained for CD4 and CD8, followed by intracellular cytokine staining for IL-2 and IFN-gamma. The percentage of cytokine-positive T cells is analyzed by flow cytometry to measure the pharmacodynamic effect.
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| ADME/Pharmacokinetics |
Detailed PK data for HPK1-IN-3 is not available. As a potent, selective, ATP-competitive HPK1 inhibitor, its properties would be characterized in a standard mouse PK study. Male BALB/c mice are given a single intravenous (IV) dose (1 mg/kg) and a single oral (PO) dose (10 mg/kg) of HPK1-IN-3. Plasma samples are collected at multiple time points up to 24 hours post-dose. HPK1-IN-3 concentrations are measured using a validated LC-MS/MS method. Key parameters including T1/2, Cmax, AUC, Cl, Vd, and oral bioavailability (%F) would be calculated using WinNonlin software.
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| Toxicity/Toxicokinetics |
Toxicology data for HPK1-IN-3 is not available. As an HPK1 inhibitor that relieves a negative regulatory checkpoint on T cells, the primary on-target toxicity is expected to be immune-mediated inflammation. A standard exploratory toxicity study would involve a 7-day repeat-dose oral toxicity study in rats. Animals would be treated daily with HPK1-IN-3 at dose levels of 0, 3, 10, and 30 mg/kg. Assessments include clinical observations, body weight, food consumption, hematology (with differential), serum chemistry, and full histopathological examination, with special attention given to lymphoid tissues (spleen, thymus, lymph nodes).
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| References | |
| Additional Infomation |
HPK1-IN-3 is a research-grade compound and is not approved for clinical use. Its molecular formula is C23H22F4N6O2 with a molecular weight of 490.45 and a purity of 98.61%. The compound is stored as a powder at -20degC. HPK1-IN-3 is a key tool for investigating HPK1 as a therapeutic target in cancer immunotherapy. It is valuable for studying the relief of the HPK1-mediated negative feedback loop on T-cell activation.
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| Molecular Formula |
C23H22F4N6O2
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| Molecular Weight |
490.453398227692
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| Exact Mass |
490.174
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| CAS # |
2739844-34-7
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| PubChem CID |
155883041
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| Appearance |
White to yellow solid powder
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| LogP |
4.2
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
11
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
35
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| Complexity |
731
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CN1CCC2=CC(=C(C=C2C1)NC3=NC=C(C(=N3)NC4=C(C=CC=C4F)C(F)(F)F)C(=O)N)OC
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| InChi Key |
RZLXOEIALAVGKH-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C23H22F4N6O2/c1-33-7-6-12-9-18(35-2)17(8-13(12)11-33)30-22-29-10-14(20(28)34)21(32-22)31-19-15(23(25,26)27)4-3-5-16(19)24/h3-5,8-10H,6-7,11H2,1-2H3,(H2,28,34)(H2,29,30,31,32)
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| Chemical Name |
4-[2-fluoro-6-(trifluoromethyl)anilino]-2-[(6-methoxy-2-methyl-3,4-dihydro-1H-isoquinolin-7-yl)amino]pyrimidine-5-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: 83.33 mg/mL (169.91 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.24 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 20.8 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: ≥ 2.08 mg/mL (4.24 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 20.8 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 | 2.0389 mL | 10.1947 mL | 20.3894 mL | |
| 5 mM | 0.4078 mL | 2.0389 mL | 4.0779 mL | |
| 10 mM | 0.2039 mL | 1.0195 mL | 2.0389 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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