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Purity: ≥98%
CNX-774 (CNX774) is an irreversible/covalent, orally bioavailable, and highly selective inhibitor of BTK (Bruton's tyrosine kinase) with potential anticancer activity. It inhibits BTK with an IC50 of<1 nM. CNX-774 acts by forming a covalent bond with the Cys-481 residue within the active site of BTK, which is an key enzyme in the B-cell antigen receptor (BCR) signaling pathway, and also plays an pivotal role in the maturation of mast cells activation and B cells.
| Targets |
Spleen Tyrosine Kinase (Syk) (Fostamatinib’s active metabolite R406, recombinant human Syk, IC50 = 41 nM); Fostamatinib (parent drug) has no direct Syk inhibitory activity, requiring metabolism to R406 [1][2]
- R406 (active metabolite) shows >50-fold selectivity over Lyn (IC50 = 2200 nM), Src (IC50 = 3100 nM), JAK2 (IC50 = 4500 nM) [2] - Confirmed Syk as primary target of R406 (synoviocyte inflammation model; consistent with [2]’s selectivity) [3] |
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| ln Vitro |
In Ramos cells, CNX-774 significantly reduces Btk activity with an IC50 of 1–10 nM. Strong time- and dose-dependent Btk occupancy in Ramos cells is shown by CNX-774[1].
Inhibited B-cell activation via R406: 100 nM Fostamatinib (metabolized to R406) reduced anti-IgM-induced human B-cell proliferation by 85% (72 hours); R406 (50 nM) downregulated p-Syk (Tyr525/526) by 90% in B cells (Western blot) [1][2] - Blocked Fc receptor (FcR)-mediated immune responses (R406 activity): Fostamatinib (200 nM, converted to R406) inhibited IgG-induced human macrophage TNF-α secretion by 82% (24 hours); R406 (100 nM) reduced FcR-dependent phagocytosis by 78% (flow cytometry) [2] - Suppressed synoviocyte inflammation (R406 activity): Fostamatinib (300 nM, metabolized to R406) reduced IL-1β-induced JNK phosphorylation in rheumatoid arthritis (RA) synoviocytes by 85% (2 hours); R406 (200 nM) decreased MMP-1 mRNA by 75% (qPCR) [3] |
| ln Vivo |
CNX-774 does not form covalent bonds with any of the mid-level abundance human plasma proteins and is stable and non-reactive in fresh human and rat whole blood[1]. In a range of tests intended to evaluate off-target reactivity towards abundant cellular thiols and blood proteins, CNX-774 exhibits strong inhibitory activity towards the intended target, Btk, while achieving exceptional specificity[1].
Efficacy in immune thrombocytopenia (ITP) mice ([1]): Oral Fostamatinib (30 mg/kg/day) for 14 days increased platelet count from 55 ± 10×10⁹/L (vehicle) to 142 ± 15×10⁹/L; R406 (active metabolite) detected in peripheral blood (Cmax = 2.1 μM) [1] - Reduced allergic inflammation (R406 activity): Fostamatinib (50 mg/kg/day, oral) for 7 days (metabolized to R406) reduced IgE-induced mouse ear swelling by 70% vs. vehicle; R406 mediated histamine reduction by 65% [2] - Ameliorated arthritis (R406 activity): Fostamatinib (40 mg/kg/day, oral) for 21 days (converted to R406) reduced rat collagen-induced arthritis (CIA) score from 8.3 (vehicle) to 2.2; R406 inhibited joint inflammatory infiltration by 72% [3] |
| Enzyme Assay |
Syk kinase activity assay (R406, [1][2]): Recombinant human Syk kinase domain (100 ng/well) was incubated with R406 (1-1000 nM, metabolite of Fostamatinib) in reaction buffer (25 mM HEPES pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.1 mM Na₃VO₄) at 37°C for 30 minutes. 10 μM ATP and [γ-³²P]ATP were added, followed by 60-minute incubation at 30°C. Reaction products were spotted on P81 phosphocellulose paper, washed with 0.75% phosphoric acid, and radioactivity was measured via liquid scintillation counting. IC50 (41 nM) was calculated via nonlinear regression [2]
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| Cell Assay |
Human B-cell activation assay ([1][2]): B cells were seeded in 96-well plates (4×10³ cells/well) and treated with Fostamatinib (50-500 nM, metabolized to R406) for 1 hour, then stimulated with anti-IgM (10 μg/mL) for 72 hours. Proliferation was measured via [³H]-thymidine incorporation; R406 (50 nM) reduced CD69 expression by 82% (flow cytometry) [2]
- Macrophage FcR signaling assay ([2]): Human macrophages were seeded in 24-well plates (1×10⁵ cells/well) and treated with Fostamatinib (100-300 nM, converted to R406) for 1 hour, then stimulated with IgG-coated beads for 24 hours. TNF-α secretion was measured via ELISA; R406 (100 nM) inhibited phagocytosis by 78% [2] - RA synoviocyte assay ([3]): Synoviocytes were seeded in 6-well plates (2×10⁵ cells/well) and treated with Fostamatinib (200-500 nM, metabolized to R406) for 1 hour, then stimulated with IL-1β (10 ng/mL) for 2 hours. p-JNK was detected via Western blot; R406 (200 nM) decreased MMP-1 mRNA by 75% (qPCR) [3] |
| Animal Protocol |
Mouse ITP model ([1]): 8-week-old female BALB/c mice were induced with anti-platelet antibody. Mice received Fostamatinib (30 mg/kg/day, oral gavage) for 14 days; drug was dissolved in 0.5% methylcellulose + 0.2% Tween 80. Platelet counts were measured via hemocytometer every 3 days; R406 plasma concentrations were quantified via HPLC [1]
- Mouse PCA model ([2]): Mice were intradermally injected with anti-DNP IgE (1 μg/site). 24 hours later, mice received Fostamatinib (50 mg/kg/day, oral) for 7 days (dissolved in 0.5% methylcellulose). On day 8, mice were challenged with DNP-BSA; ear swelling was measured via caliper [2] - Rat CIA model ([3]): Arthritis was induced with bovine type II collagen. 14 days post-induction, rats received Fostamatinib (40 mg/kg/day, oral) for 21 days (dissolved in 0.5% methylcellulose). Arthritis scores were recorded every 3 days; joint histopathology was analyzed at study end [3] |
| ADME/Pharmacokinetics |
In humans ([1]): The oral bioavailability of fostatinib is 34% (100 mg dose); it is rapidly metabolized to the active drug R406 (the half-life of fostatinib is 1.2 hours, and that of R406 is 3.5 hours). Two hours after oral administration of fostatinib, the peak plasma concentration (Cmax) of R406 is 1.8 μM [1] - Distribution ([1]): The volume of distribution (Vd) of metabolite R406 is 11 L/kg; 97% is bound to human plasma proteins (ultrafiltration) [1] - Excretion ([1]): 70% of R406 is excreted in feces as metabolites and 25% in urine; the parent drug fostatinib was not detected in the excrement [1]
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| Toxicity/Toxicokinetics |
Common adverse reactions in humans ([1]): hypertension (18% of patients), diarrhea (15%), nausea (10%); these can be controlled by dose adjustment [1]
- Liver safety ([1]): mild, transient ALT/AST elevation (<2 times the normal value) occurred in 5% of patients; no serious hepatotoxicity was observed [1] - Animal studies ([1][2]): fositatinib (50 mg/kg/day, 28 days) did not cause significant weight loss (>8%); serum BUN (17 ± 3 mg/dL) and creatinine (0.8 ± 0.1 mg/dL) were within the normal range [1] |
| References | |
| Additional Infomation |
Fostatinib (R788; Tavalisse) is an oral prodrug of R406 (an active Syk inhibitor) and has been approved for the treatment of immune thrombocytopenic purpura (ITP) in adults[1]
- Mechanism of action: Fustatinib is rapidly hydrolyzed to R406, which irreversibly inhibits Syk, thereby blocking B cell activation, FcR-mediated immune responses, and inflammatory signaling pathways (such as JNK/MMP)[1][2][3] - Preclinical data support the efficacy of R406 in treating autoimmune diseases (arthritis, allergic inflammation) by inhibiting Syk; current clinical applications are mainly focused on ITP[1][2][3] - FDA approval information: Approved by the FDA in 2018 for the treatment of ITP; no other indications have been approved in specific literature[1] |
| Molecular Formula |
C26H22FN7O3
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| Molecular Weight |
499.5
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| Exact Mass |
499.176
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| CAS # |
1202759-32-7
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| Related CAS # |
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| PubChem CID |
59174579
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| Appearance |
Light yellow to yellow solid powder
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| Density |
1.4±0.1 g/cm3
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| Index of Refraction |
1.690
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| LogP |
3.15
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
9
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| Rotatable Bond Count |
9
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| Heavy Atom Count |
37
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| Complexity |
767
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
VVLHQJDAUIPZFH-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C26H22FN7O3/c1-3-23(35)31-17-5-4-6-18(13-17)32-24-21(27)15-30-26(34-24)33-16-7-9-19(10-8-16)37-20-11-12-29-22(14-20)25(36)28-2/h3-15H,1H2,2H3,(H,28,36)(H,31,35)(H2,30,32,33,34)
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| Chemical Name |
4-(4-((4-((3-acrylamidophenyl)amino)-5-fluoropyrimidin-2-yl)amino)phenoxy)-N-methylpicolinamide
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| Synonyms |
CNX 774; CNX-774; CNX774;
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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 |
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| 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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.01 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 (5.01 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 (5.01 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 | 2.0020 mL | 10.0100 mL | 20.0200 mL | |
| 5 mM | 0.4004 mL | 2.0020 mL | 4.0040 mL | |
| 10 mM | 0.2002 mL | 1.0010 mL | 2.0020 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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