Bruton Tyrosine Kinase (BTK) (recombinant human BTK, IC50 = 1.0 nM); >280-fold selectivity over EGFR (IC50 = 280 nM), ITK (IC50 = 320 nM), JAK2 (IC50 = 350 nM); no activity against Src, Abl, VEGFR2 (IC50 > 1000 nM) [1]
- Confirmed BTK as primary target (prostate cancer model; no additional IC50 values; consistent with [1]’s selectivity) [2]

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.

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Inhibited B-cell-mediated inflammation: 20 nM CGI1746 reduced anti-IgM-induced mouse splenic B-cell proliferation by 85% (72 hours); decreased B-cell activation marker CD86 expression by 82% (flow cytometry) [1]
- Suppressed myeloid cell function: 50 nM CGI1746 reduced LPS-induced TNF-α secretion by human monocytes by 75% (24 hours); inhibited osteoclast differentiation from bone marrow macrophages by 80% (14-day culture) [1]
- Inhibited prostate cancer cell growth: Human prostate cancer PC-3 cells (IC50 = 15.2 nM), DU145 cells (IC50 = 18.7 nM); 100 nM CGI1746 reduced PC-3 cell clone formation by 80% (10-day culture); p-BTK (Tyr223) and p-AKT (Ser473) downregulated by >90% (Western blot) [2]

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].

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In collagen-induced arthritis (CIA) mice (C57BL/6, [1]): Oral CGI1746 (30 mg/kg/day) for 21 days reduced arthritis score from 8.5 (vehicle) to 2.3; serum IL-6/TNF-α levels decreased by 70%/68%; splenic B-cell and myeloid cell infiltration in joints reduced by 75%/72% (immunohistochemistry) [1]
- In PC-3 prostate cancer xenograft mice (nude mice, [2]): CGI1746 (25 mg/kg/day, oral) for 28 days achieved 72% tumor growth inhibition (TGI); tumor Ki-67 (proliferation marker) positive cells reduced by 65% vs. vehicle [2]

BTK kinase activity assay (literature 1): Recombinant human BTK kinase domain (50 ng/well) was incubated with CGI1746 (0.01-100 nM) in reaction buffer (25 mM HEPES pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.1 mM Na₃VO₄) at 37°C for 20 minutes. 10 μM ATP and a fluorescently labeled peptide substrate (sequence: biotin-GGEEEEYFELVAKKKK) were added, followed by 60-minute incubation at 30°C. Phosphorylated substrate was captured by streptavidin-coated 96-well plates, detected via anti-phosphotyrosine antibody, and kinase activity was quantified using homogeneous time-resolved fluorescence (HTRF; excitation 340 nm, emission 665 nm). IC50 was calculated via nonlinear regression analysis [1]

Mouse B-cell proliferation assay (literature 1): Splenic B cells were isolated from C57BL/6 mice and seeded in 96-well plates (4×10³ cells/well). Cells were treated with CGI1746 (1-200 nM) for 1 hour, then stimulated with anti-mouse IgM (10 μg/mL) for 72 hours. Proliferation was measured via [³H]-thymidine incorporation assay; CD86 expression was analyzed by flow cytometry with a FITC-conjugated anti-CD86 antibody [1]
- Human monocyte cytokine assay (literature 1): Human peripheral blood monocytes were seeded in 24-well plates (1×10⁵ cells/well) and treated with CGI1746 (5-100 nM) for 2 hours, then stimulated with LPS (1 μg/mL) for 24 hours. Supernatants were collected; TNF-α levels were detected via ELISA [1]
- Prostate cancer cell assay (literature 2): PC-3/DU145 cells were seeded in 96-well plates (5×10³ cells/well) and treated with CGI1746 (0.1 nM-1 μM) for 72 hours. Viability was measured via MTT assay; IC50 was calculated via four-parameter logistic fitting. For clone formation, cells were seeded in 6-well plates (2×10³ cells/well) with CGI1746 (100 nM) for 10 days, then stained with crystal violet and counted [2]

100 mg/kg; s.c. administration
mouse models

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CIA mouse model (C57BL/6 mice, [1]): Arthritis was induced by intradermal injection of bovine type II collagen (200 μg/mouse) emulsified in complete Freund’s adjuvant. 14 days post-induction, mice received CGI1746 (30 mg/kg/day, oral gavage) for 21 days. Drug was dissolved in 0.5% methylcellulose + 0.2% Tween 80. Arthritis score (0-10, based on joint redness/swelling) was recorded every 3 days; serum cytokines and joint histopathology were analyzed at study end [1]
- PC-3 prostate cancer xenograft model (nude mice, [2]): 6-week-old female nude mice were subcutaneously injected with 2×10⁶ PC-3 cells. When tumors reached 100 mm³, mice received CGI1746 (25 mg/kg/day, oral gavage) for 28 days. Drug was dissolved in 0.5% methylcellulose; tumor volume (length × width² / 2) was measured every 3 days. Tumor tissues were collected for Ki-67 immunohistochemistry [2]

In mice (Reference 1): the oral bioavailability of CGI1746 was 55% (30 mg/kg); the plasma half-life (t₁/₂) was 4.0 h; and the peak plasma concentration (Cmax) 1.2 h after oral administration was 4.3 μM [1]. Plasma protein binding: the binding rate to human plasma proteins was 99.3% (determined by ultrafiltration) [1].

In the 21-day CIA study ([1]): no significant weight loss (>8%); serum ALT (25 ± 3 U/L), AST (48 ± 5 U/L), and BUN (17 ± 2 mg/dL) were all within the normal range; 1/10 mice experienced mild diarrhea (which resolved on day 7) [1]
- In the 28-day prostate cancer study ([2]): no treatment-related deaths; no abnormalities were found in liver and kidney histopathological examination; peripheral blood leukocyte count was normal [2]

[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.

CGI1746 is an irreversible covalent Bruton's tyrosine kinase (BTK) inhibitor that binds to the Cys481 residue of BTK, blocking BTK activation and its downstream signaling in B cells and myeloid cells [1]. Its therapeutic potential covers autoimmune diseases (e.g., rheumatoid arthritis) by inhibiting B cell activation and myeloid cell pro-inflammatory function; and solid tumors (e.g., prostate cancer) by inhibiting BTK-dependent cancer cell proliferation [1][2]. Preclinical data have confirmed its efficacy in alleviating joint inflammation (arthritis) and prostate tumor growth, supporting its potential as a dual-target drug for BTK-dependent inflammatory and neoplastic diseases [1][2].

C34H37N5O4

579.69

579.284

910232-84-7

24857323

White to light yellow solid powder

1.2±0.1 g/cm3

1.627

3.42

2

5

7

43

1070

0

JIFCFQDXHMUPGP-UHFFFAOYSA-N

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)

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

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

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.

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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.

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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.

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 (Please use freshly prepared in vivo formulations for optimal results.)