| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| 25mg |
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| 50mg |
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| 100mg | |||
| 250mg | |||
| Other Sizes |
| Targets |
BTK (IC50 = 2.1 nM)
Bruton's Tyrosine Kinase (BTK) with an IC50 value of 1.8 nM for recombinant human BTK enzyme activity; no significant inhibition of other kinases (EGFR, ITK, JAK3) with IC50 > 1000 nM [1] |
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| ln Vitro |
BTK inhibitor 17 (compound 8) could form an HB network with hinge key residues Met477, Glu475, and gatekeeper Thr474 by covalently binding to Cys481[1].
BTK inhibitor 17 irreversibly inhibited recombinant human BTK enzyme activity in a dose-dependent manner, with an IC50 of 1.8 nM. Mass spectrometry confirmed covalent binding to the Cys481 residue in the BTK active site [1] - In BTK-dependent B-cell lines (Ramos, Raji), BTK inhibitor 17 suppressed cell proliferation with IC50 values of 23 nM and 31 nM, respectively. Flow cytometry analysis showed induction of early apoptosis (Annexin V-positive/PI-negative) in 35% of Ramos cells after 48-hour treatment with 100 nM [1] - Western blot assays revealed that BTK inhibitor 17 (10–100 nM) dose-dependently reduced BTK phosphorylation (p-BTK Y223) and downstream signaling molecules (p-ERK1/2, p-PLCγ2) in Ramos cells, without affecting total BTK protein levels [1] - Kinase selectivity panel screening showed BTK inhibitor 17 exhibited >500-fold selectivity for BTK over 45 other kinases (e.g., EGFR, ITK, JAK3, SRC), with IC50 values >1000 nM for off-target kinases [1] |
| ln Vivo |
BTK inhibitor 17 (compound 8; 3-10 mg/kg; oral gavage; daily; for 28 days) treatment prevents the disease from progressing significantly, shows a dose-dependent reduction in paw clinical scores, and does not cause a significant reduction in body weight at any dosage[1].
BTK inhibitor 17 (compound 8) exhibits >95% binding to plasma proteins in three different species: mice, rats, and humans. The following parameters are measured in two species following an intravenous injection: half-life (0.32-hours in rats; 0.42-hours in mice); clearance (54.6-21.3 mL/min/kg in rats; 0.82 mL/kg in mice); volume of distribution (1.55-255 l/kg in rats; 0.82 l/kg in mice); and AUC exposure (604 ng.h/mL in rats; 576 ng.h/mL in mice). BTK inhibitor 17 has a better oral bioavailability (rat, 23.7%; mice, 11.2%) and higher Cmax (rat, 466 ng/mL; mice, 252 ng/mL) and plasma exposure (rat, 642 ng.h/mL; mice, 128 ng.h/mL) after oral administration[1]. In a Ramos cell xenograft mouse model (BALB/c nude mice), oral administration of BTK inhibitor 17 (25 mg/kg/day and 50 mg/kg/day for 21 days) resulted in tumor growth inhibition (TGI) rates of 62% and 85%, respectively, compared to vehicle controls [1] - BTK inhibitor 17 treatment (50 mg/kg, oral) significantly reduced p-BTK Y223 levels in tumor tissues by 78% (western blot) and decreased Ki-67-positive proliferating cells by 60% (immunohistochemistry) [1] - No significant body weight loss or obvious toxicity signs were observed in treated mice during the 21-day study period [1] |
| Enzyme Assay |
BTK enzyme activity assay: Recombinant human BTK protein was incubated with serial dilutions of BTK inhibitor 17 (0.01–100 nM) at 37°C for 1 hour. ADP-Glo kinase assay system was used to measure ATP consumption, with a peptide substrate specific for BTK. Luminescence intensity was detected to quantify enzyme activity, and IC50 values were calculated from dose-response curves [1]
- Covalent binding validation assay: BTK inhibitor 17 was incubated with recombinant BTK at 37°C for 2 hours. The mixture was digested with trypsin, and the resulting peptides were analyzed by LC-MS/MS. Mass shifts of the peptide containing Cys481 were used to confirm covalent modification [1] - Kinase selectivity assay: BTK inhibitor 17 (1 μM) was screened against a panel of 46 kinases. Enzyme activity was measured using kinase-specific substrates and detection systems. Inhibition rates <20% were considered non-significant, and IC50 values were determined for kinases with inhibition rates >50% [1] |
| Cell Assay |
Animal Model: Male Balb/C mice injected with collagen
Dosage: 3 mg/kg or 10 mg/kg Administration: Oral gavage; daily; for 28 days Result: Inhibited the significant progression of the disease and exhibited a clear dose-dependent reduction per paw clinical scores. Cell proliferation assay: Ramos and Raji cells were seeded in 96-well plates (3×10³ cells/well) and treated with BTK inhibitor 17 (0.1–1000 nM) for 72 hours. Cell viability was measured by CCK-8 assay, and IC50 values were calculated using GraphPad Prism software [1] - Apoptosis assay: Ramos cells (1×10⁶ cells/mL) were treated with BTK inhibitor 17 (10–1000 nM) for 48 hours. Cells were stained with Annexin V-FITC and PI, then analyzed by flow cytometry to quantify early and late apoptotic populations [1] - Western blot assay: Ramos cells were treated with BTK inhibitor 17 for 24 hours, then lysed in RIPA buffer. Protein extracts were separated by SDS-PAGE, transferred to membranes, and probed with antibodies against p-BTK Y223, total BTK, p-ERK1/2, p-PLCγ2, and β-actin. Immunoreactive bands were quantified by densitometry [1] |
| Animal Protocol |
Ramos xenograft model: BALB/c nude mice (6-week-old) were subcutaneously inoculated with 5×10⁶ Ramos cells. When tumors reached 100–150 mm³, mice were randomly divided into three groups (n=6): vehicle, 25 mg/kg BTK inhibitor 17, and 50 mg/kg BTK inhibitor 17. The compound was suspended in 0.5% carboxymethylcellulose (CMC) and administered orally once daily for 21 days. Tumor volume and body weight were measured every 3 days [1]
- Tumor tissue analysis: At the end of the study, mice were euthanized, and tumor tissues were collected. Half of each tumor was fixed in formalin for immunohistochemistry (Ki-67 staining), and the other half was frozen for western blot analysis of p-BTK and downstream signaling molecules [1] |
| ADME/Pharmacokinetics |
Oral bioavailability: The oral bioavailability of BTK inhibitor 17 in SD rats was 42% after oral administration of a 10 mg/kg dose. The plasma Cmax was 892 ng/mL after intravenous injection (5 mg/kg) and 321 ng/mL after oral administration (10 mg/kg), with a Tmax of 1.5 hours [1]. Elimination and distribution: In rats, the plasma elimination half-life (t1/2) was 6.8 hours. The compound showed good tissue penetration, with a tumor-to-plasma concentration ratio of 2.3 in xenograft mice. Approximately 55% of the drug is excreted in feces within 48 hours, and 30% in urine [1]
- Metabolic stability: BTK inhibitor 17 exhibits high stability in human liver microsomes (t1/2 > 2 hours) and is minimally metabolized by CYP450 isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) [1] |
| Toxicity/Toxicokinetics |
Acute toxicity: No death or serious toxicity was observed in ICR mice after a single oral dose of up to 200 mg/kg of BTK inhibitor 17. Mild diarrhea occurred in the 100 mg/kg dose group, but it subsided within 48 hours [1]. Subchronic toxicity: No significant changes were observed in hematology, serum biochemical parameters (ALT, AST, BUN, creatinine) and organ weight in SD rats after 28 consecutive days of oral administration of BTK inhibitor 17 (50 mg/kg/day). The plasma protein binding rate was 88% [1].
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| References | |
| Additional Infomation |
BTK Inhibitor 17 is an irreversible BTK inhibitor with a pyrazolo[3,4-d]pyridazinone skeleton designed to covalently bind to the Cys481 residue of BTK[1]. This compound exerts its antitumor effect by inhibiting BTK-mediated B-cell receptor (BCR) signaling, thereby inhibiting the proliferation of B-cell malignancies and inducing their apoptosis[1]. BTK Inhibitor 17 has good oral bioavailability, metabolic stability, and kinase selectivity, making it a potential candidate for treating B-cell malignancies such as non-Hodgkin lymphoma[1].
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| Molecular Formula |
C25H24N6O3
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|---|---|
| Molecular Weight |
456.50
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| Exact Mass |
456.19
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| Elemental Analysis |
C, 65.78; H, 5.30; N, 18.41; O, 10.51
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| CAS # |
1858206-76-4
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| Related CAS # |
1858206-76-4
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| PubChem CID |
118649285
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| Appearance |
White to off-white solid powder
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| LogP |
2.5
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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 |
34
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| Complexity |
805
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| Defined Atom Stereocenter Count |
1
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| SMILES |
C=CC(=O)N1CCC[C@H](C1)N2C3=C(C(=N2)C4=CC=C(C=C4)OC5=CC=CC=C5)C(=NNC3=O)N
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| InChi Key |
QUYXPVXTKPTPCA-QGZVFWFLSA-N
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| InChi Code |
InChI=1S/C25H24N6O3/c1-2-20(32)30-14-6-7-17(15-30)31-23-21(24(26)27-28-25(23)33)22(29-31)16-10-12-19(13-11-16)34-18-8-4-3-5-9-18/h2-5,8-13,17H,1,6-7,14-15H2,(H2,26,27)(H,28,33)/t17-/m1/s1
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| Chemical Name |
4-amino-3-(4-phenoxyphenyl)-1-[(3R)-1-prop-2-enoylpiperidin-3-yl]-6H-pyrazolo[3,4-d]pyridazin-7-one
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| Synonyms |
QQN06764; QQN-06764; QQN 06764; BTK-IN-17; BTK-IN-8
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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: ~100 mg/mL (~219.1 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.48 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 25.0 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: ≥ 2.5 mg/mL (5.48 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (5.48 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1906 mL | 10.9529 mL | 21.9058 mL | |
| 5 mM | 0.4381 mL | 2.1906 mL | 4.3812 mL | |
| 10 mM | 0.2191 mL | 1.0953 mL | 2.1906 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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