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.

Physicochemical Properties

Molecular Formula	C26H22FN7O3
Molecular Weight	499.5
Exact Mass	499.176
CAS #	1202759-32-7
Related CAS #	1202759-32-7
PubChem CID	59174579
Appearance	Light yellow to yellow solid powder
Density	1.4±0.1 g/cm3
Index of Refraction	1.690
LogP	3.15
Hydrogen Bond Donor Count	4
Hydrogen Bond Acceptor Count	9
Rotatable Bond Count	9
Heavy Atom Count	37
Complexity	767
Defined Atom Stereocenter Count	0
InChi Key	VVLHQJDAUIPZFH-UHFFFAOYSA-N
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)
Chemical Name	4-(4-((4-((3-acrylamidophenyl)amino)-5-fluoropyrimidin-2-yl)amino)phenoxy)-N-methylpicolinamide
Synonyms	CNX 774; CNX-774; CNX774;
HS Tariff Code	2934.99.9001
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)

Biological Activity

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].
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].
Animal Protocol
References	[1]. In Vitro Reactivity Assessment of Covalent Drugs Targeting Bruton's Tyrosine Kinase. [2]. Ibrutinib and novel BTK inhibitors in clinical development. J Hematol Oncol. 2013 Aug 19;6:59.

Solubility Data

Solubility (In Vitro)

DMSO: 100 mg/mL (200.2 mM) Water:<1 mg/mL Ethanol: 2 mg/mL (4.0 mM)

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. 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. 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 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	2.0020 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.