CGI1746 (CGI-1746) is a reversible/non-covalent and highly selective small-molecule inhibitor of the Bruton's tyrosine kinase-Btk with potential anti-inflammatory activity. It inhibits BTK with an IC50 of 1.9 nM. CGI-1746 shows high in vivo antiinflammatory efficacy in an anti-collagen II antibody–induced arthritis (CAIA) model in mice.

Physicochemical Properties

Molecular Formula	C34H37N5O4
Molecular Weight	579.69
Exact Mass	579.284
CAS #	910232-84-7
Related CAS #	910232-84-7
PubChem CID	24857323
Appearance	White to light yellow solid powder
Density	1.2±0.1 g/cm3
Index of Refraction	1.627
LogP	3.42
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	5
Rotatable Bond Count	7
Heavy Atom Count	43
Complexity	1070
Defined Atom Stereocenter Count	0
InChi Key	JIFCFQDXHMUPGP-UHFFFAOYSA-N
InChi Code	InChI=1S/C34H37N5O4/c1-22-27(7-6-8-28(22)37-31(40)23-9-13-25(14-10-23)34(2,3)4)29-21-38(5)33(42)30(36-29)35-26-15-11-24(12-16-26)32(41)39-17-19-43-20-18-39/h6-16,21H,17-20H2,1-5H3,(H,35,36)(H,37,40)
Chemical Name	4-(tert-butyl)-N-(2-methyl-3-(4-methyl-6-((4-(morpholine-4-carbonyl)phenyl)amino)-5-oxo-4,5-dihydropyrazin-2-yl)phenyl)benzamide
Synonyms	CGI-1746; CGI 1746; CGI1746;
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	CGI1746 is selective for Btk, with approximately 1,000-fold selectivity over Tec and Src family kinases. In an ATP-free competition binding test, Btk's dissociation constant is 1.5 nM. CGI1746 suppresses Btk activity by a novel binding mechanism that stabilizes an inactive, nonphosphorylated enzyme structure. CGI1746 inhibits both the auto- and transphosphorylation processes required for enzyme activation. CGI1746 completely reduces anti-IgM-induced murine and human B cell proliferation at IC50s of 134 nM and 42 nM, respectively, but has no effect on anti-CD3- or anti-CD28-induced T cell proliferation. CGI1746 effectively inhibits the proliferation of CD27+IgG+ B cells isolated from the tonsils of four human donors, with an average IC50 of 112 nM. CGI1746 prevents macrophages from producing TNFα, IL-1β, and IL-6 triggered by FcγRIII. CGI1746 suppresses TNFα, IL-1β, and IL-6 production (with three- to eight-fold greater IC50) in human monocytes challenged with immobilized or soluble immune complexes [1]. CGI-1746 does not kill cells as effectively as irreversible BTK inhibitors at the same dose. CGI-1746 inhibits BTK phosphorylation at tyrosine 233 in the SH3 domain, but it does not kill LNCaP or DU145 prostate cancer cells at the same concentrations as Ibrutinib or AVL-292 [2]. However, it significantly reduces phosphorylation of both the BTK-A and BTK-C proteins, indicating that auto-phosphorylation of the BTK-C isoform is inhibited in a manner similar to that of BTK-A.
ln Vivo	CGI1746 inhibits B cell-dependent arthritis. CGI1746 treatment (100 mg/kg, sc, twice-daily dosing) results in a 97% inhibition of overall clinical arthritis scores. CGI1746 treatment significantly reduces TNFα, IL-1β, and IL-6, as well as MCP1 and MIP-1α, on both the mRNA and protein levels in the passive anti-collagen II antibody-induced arthritis (CAIA) model. CGI1746 effectively reduces clinical scores and joint inflammation in mice and rats with established arthritis, similar to TNFα blockade [1].
Animal Protocol	100 mg/kg; s.c. administration mouse models
References	[1]. Specific Btk inhibition suppresses B cell- and myeloid cell-mediated arthritis. Nature Chemical Biology (2011), 7(1), 41-50. [2]. Bruton's tyrosine kinase is a potential therapeutic target in prostate cancer. Cancer Biol Ther. 2015;16(11):1604-15.

Solubility Data

Solubility (In Vitro)

DMSO: 100 mg/mL (172.5 mM) Water:<1 mg/mL Ethanol: 33 mg/mL warmed (56.9 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.31 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 (4.31 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. 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 (4.31 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), suspension 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	1.7251 mL	8.6253 mL	17.2506 mL
	5 mM	0.3450 mL	1.7251 mL	3.4501 mL
	10 mM	0.1725 mL	0.8625 mL	1.7251 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.