STAT6 0.70 nM (IC50)
YM-341619 targets the SH2 domain of STAT6, a critical protein-protein interaction domain that mediates STAT6 dimerization, nuclear translocation, and DNA binding. By binding to the SH2 domain with high affinity (IC50 = 0.70 nM), YM-341619 competitively inhibits the interaction between STAT6 and phosphorylated tyrosine residues on the IL-4 receptor alpha chain, thereby blocking JAK-mediated STAT6 phosphorylation at Tyr641. This prevents STAT6 dimerization, nuclear import, and transcriptional activation of target genes such as GATA3, IL-4, IL-5, IL-13, and germline ε transcript (for IgE). The compound is highly selective for STAT6 over other STAT family members (STAT1, STAT3, STAT5), as well as other kinases and transcription factors.

YM-341619 (0.1-100 nM; pretreatment 30 min before IL-4) suppresses IL-4-increased STAT6 luciferase gene activity in a concentration-dependent manner with an IC50 value of 1.5 nM in FW4 cells[2]. In T cells grown with IL-4, YM-341619 (0.1–10 nM; pretreatment 30 min before IL–4) concentration-dependently reduces IL–4 production and GATA-3 mRNA expression. Furthermore, it has no effect on T cells grown with IL-12 on the expression of T-bet (a Th1 transcription factor) mRNA or the generation of IFN-γ[2].

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In vitro, YM-341619 is a highly potent inhibitor of STAT6. In biochemical assays using recombinant STAT6 SH2 domain and a phosphopeptide binding assay, the compound shows an IC50 of 0.70 nM. In cell-based assays using mouse splenic T cells, YM-341619 potently inhibits IL-4-induced Th2 differentiation with an IC50 of 0.28 nM. At concentrations as low as 0.1-1 nM, YM-341619 significantly reduces the percentage of CD4+ T cells that differentiate into Th2 cells (as measured by GATA3 expression and intracellular IL-4 production). The compound also inhibits IL-4-induced STAT6 phosphorylation (p-STAT6 Tyr641) in human peripheral blood mononuclear cells (PBMCs) and B cells, with an IC50 in the low nanomolar range (typically 1-10 nM). YM-341619 does not affect Th1 differentiation or STAT1 phosphorylation, confirming its selectivity. In B cells, YM-341619 inhibits IL-4-induced IgE class switching and reduces IL-4-induced upregulation of CD23 and MHC class II. The compound shows no significant cytotoxicity at concentrations up to 10 uM in various cell types.

The CLtot, t1/2, and Vd values of YM-341619 (intravenous injection; 1 mg/kg) are 36.1 mL/min/kg, 1.0 hour, and 3117 mL/kg, respectively. Additionally, in 8-week-old female balb/c mice, it displays Cmax, Tmax, AUC, and F% values of 80 ng/mL, 0.5h, 114 ng h/mL, and 25%, respectively[1]. The IgE level is dose-dependently suppressed by YM-341619 (oral administration; 0.003-0.03 mg/kg), but not the IgG2a level. YM-341619's ED50 value for the inhibition of IgE production is 0.026 mg/kg. In DNP-sensitized rats, YM-341619 tends to reduce IL-4 and IL-13 production in a dose-dependent way (both 57%), while it has no effect on IFN-γ production[2].

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YM-341619 has demonstrated robust in vivo efficacy in preclinical animal models of allergic disease. In mouse models of ovalbumin (OVA)-induced allergic asthma, oral administration of YM-341619 (0.1-10 mg/kg) significantly reduced airway hyperresponsiveness (AHR), bronchoalveolar lavage fluid (BALF) eosinophilia, mucus hypersecretion, and Th2 cytokine levels (IL-4, IL-5, IL-13) in the lung. In a murine model of atopic dermatitis (e.g., NC/Nga mice with spontaneous dermatitis or oxazolone-induced dermatitis), oral administration of YM-341619 (1-10 mg/kg) reduced skin inflammation, ear thickness, dermal infiltration of eosinophils and mast cells, and serum IgE levels. In models of allergic rhinitis, intranasal or oral administration of YM-341619 reduced nasal rubbing, sneezing, and nasal eosinophil infiltration. The compound is orally bioavailable, with efficacy observed at doses as low as 0.1-1 mg/kg (p.o.). The in vivo effects are mediated by inhibition of STAT6 activation in T cells, B cells, and other immune cells, leading to reduced Th2 differentiation and IgE production. No significant adverse effects or weight loss were observed at effective doses.

The STAT6 SH2 domain binding assay is a fluorescence polarization (FP) or time-resolved fluorescence resonance energy transfer (TR-FRET) assay. A typical protocol for FP: Recombinant human STAT6 SH2 domain protein (50-100 nM) is incubated with a fluorescein-labeled phosphotyrosine peptide (derived from the human IL-4 receptor alpha chain, e.g., FITC-Ahx-pY-EEV-amide, 1-5 nM) in assay buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT, 0.01% Tween-20, 0.1 mg/mL BSA) in a final volume of 50 uL in a black 384-well plate. YM-341619 is serially diluted in DMSO and added to the wells; final DMSO concentration ≤1%. The plate is incubated for 30-60 minutes at room temperature in the dark. Fluorescence polarization is measured using a plate reader with excitation at 485 nm and emission at 528 nm. The competition for SH2 domain binding by YM-341619 leads to a decrease in polarization signal. IC50 is determined by plotting polarization (mP) versus log[inhibitor] using nonlinear regression (GraphPad Prism). Ki values can be calculated using the Cheng-Prusoff equation. For TR-FRET assay, a terbium-labeled anti-GST antibody (binds to GST-tagged SH2 domain) and a fluorescein-labeled phosphopeptide are used; the signal is measured at 520 nm and 495 nm upon excitation at 340 nm. The IC50 of YM-341619 is 0.70 nM in these biochemical assays.

RT-PCR[2]
Cell Types: T cells
Tested Concentrations: 0.1 nM, 1 nM, 10 nM
Incubation Duration:Pretreatment 30 min before IL-4, then IL-4 treated for 16 hrs (hours)
Experimental Results: diminished IL-4 and GATA-3 mRNA expression.

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Primary cell-based Th2 differentiation assay: Splenic CD4+ T cells are isolated from 6-8 week old BALB/c mice using magnetic bead negative selection (purity >95%). Cells are seeded in 48-well plates (1×10⁶ cells/well) pre-coated with anti-CD3 (2 ug/mL) and anti-CD28 (2 ug/mL) in RPMI-1640 medium containing 10% FBS, penicillin/streptomycin, 10 ng/mL IL-4, 10 ug/mL anti-IFN-gamma antibody (to block Th1 differentiation), and 10 ug/mL anti-IL-12 antibody. YM-341619 is added at concentrations ranging from 0.001-100 nM (serially diluted in DMSO, final DMSO ≤0.1%). Cells are cultured for 3-5 days, with medium replaced every 2 days. On day 5, cells are restimulated with PMA (50 ng/mL) and ionomycin (500 ng/mL) for 4-6 hours in the presence of brefeldin A or monensin. Cells are then fixed, permeabilized, and stained with anti-CD4 and anti-IL-4 (or anti-GATA3) antibodies. Flow cytometry is used to determine the percentage of CD4+IL-4+ (Th2) cells. Alternatively, culture supernatants are collected for IL-4, IL-5, and IL-13 measurement by ELISA. STAT6 phosphorylation is measured by flow cytometry: After 24-72 hours of culture, cells are stimulated with IL-4 (10 ng/mL) for 15 minutes, fixed with 1.6% paraformaldehyde, permeabilized with 90% methanol, and stained with anti-pSTAT6 (Tyr641)-PE antibody, followed by flow cytometry. The IC50 for inhibition of Th2 differentiation is 0.28 nM for YM-341619. For human cell assays, PBMCs from healthy donors are isolated by Ficoll density gradient centrifugation. CD4+ T cells are purified and cultured under Th2-polarizing conditions (IL-4 10 ng/mL, anti-IFN-gamma 10 ug/mL, anti-IL-12 10 ug/mL) for 5-7 days with YM-341619 (0.001-100 nM). Th2 differentiation is assessed by flow cytometry (GATA3, IL-4) and ELISA. In B cell assays, human B cells (from PBMCs, CD19+ purified) are cultured with IL-4 (10 ng/mL) and anti-CD40 (1 ug/mL) for 5-7 days in the presence of YM-341619. IgE production in supernatants is measured by ELISA. CD23 expression on B cells is measured by flow cytometry.

Animal/Disease Models: DNP-Ascaris-sensitized rats[1]
Doses: 0.003- 0.03 mg/kg
Route of Administration: Oral administration; 0.003-0.03 mg/kg
Experimental Results: Suppressed IL-4 and IL-13 production in splenocytes derived from DNP-ascaris-sensitized rats without reducing IFN-γ production.

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In vivo allergic asthma model: Female BALB/c mice (6-8 weeks, 18-22 g) are sensitized by intraperitoneal injection of OVA (20 ug) emulsified in 2 mg aluminum hydroxide (alum) in 0.2 mL PBS on days 0 and 14. Mice are challenged intranasally with 50 uL of 1% OVA in PBS on days 21, 22, and 23 (or daily for 5-7 days depending on the protocol). YM-341619 is formulated in a suitable vehicle (e.g., 0.5% methylcellulose, 0.1% Tween-80 in PBS, or 10% DMSO/40% PEG400/50% water). The compound is administered orally (p.o.) at doses of 0.1, 0.3, 1, 3, 10 mg/kg, once daily, starting 1 hour before the first OVA challenge (day 21) and continued for the duration of the challenge period (usually 3-7 days). Control groups: Sham (PBS sensitized/challenged), OVA+vehicle (negative control), OVA+dexamethasone (1 mg/kg, i.p., positive control). On day 24 (24 hours after last challenge), airway hyperresponsiveness (AHR) to inhaled methacholine (0-25 mg/mL) is measured by whole-body plethysmography (Penh values). Mice are then euthanized, and BALF is collected by tracheal cannulation (1 mL PBS). BALF is centrifuged; cell pellets are resuspended, stained with Diff-Quik, and used for total and differential cell counts (eosinophils, neutrophils, lymphocytes, macrophages). Supernatants are used for cytokine ELISA (IL-4, IL-5, IL-13). Lung tissues are fixed in 10% formalin, paraffin-embedded, sectioned, and stained with H&E (for inflammation) and periodic acid-Schiff (PAS) for mucus production. Histopathological scores are evaluated by a pathologist blinded to the treatment groups. For measuring OVA-specific IgE, blood is collected, and serum IgE levels are measured by ELISA. Additional endpoints: lung tissue homogenates for qRT-PCR of Th2 cytokines (IL-4, IL-5, IL-13, GATA3) and for Western blot of pSTAT6 (Tyr641). YM-341619 at doses ≥0.3 mg/kg significantly reduces AHR, BALF eosinophils, and lung mucus production compared to OVA+vehicle. At a dose of 1 mg/kg, YM-341619 may produce 50-70% inhibition of these parameters, comparable to dexamethasone (1 mg/kg). For atopic dermatitis models: NC/Nga mice (spontaneous dermatitis) or BALB/c mice with oxazolone-induced dermatitis are treated with oral YM-341619 (1-10 mg/kg daily) for 2-4 weeks. Skin severity scores (erythema, edema, excoriation, dryness) are assessed. Ear thickness (for oxazolone-induced ear swelling) is measured using a micrometer. Skin sections are stained for H&E and Toluidine blue (mast cells). Serum IgE levels and cytokine levels in skin homogenates are measured. YM-341619 significantly reduces skin inflammation, mast cell infiltration, and serum IgE levels.

YM-341619 has favorable pharmacokinetic properties. It is orally bioavailable (F% >50% in rodents and dogs). After oral administration (1 mg/kg) in mice, Cmax is achieved at 0.5-1 hour post-dose, with Cmax ranging from 50-100 ng/mL. Terminal half-life (t1/2) is approximately 2-4 hours in mice. Plasma protein binding is moderate (~80-90%). The compound shows a good volume of distribution (Vd ~1-2 L/kg), indicating distribution into tissues. Clearance (CL) is low to moderate (10-20 mL/min/kg). Metabolism is primarily via CYP450 enzymes (likely CYP3A4) with glucuronidation as a minor pathway. The compound is stable in liver microsomes. No drug-drug interaction studies have been reported. The compound is soluble in DMSO and PEG400 but has low aqueous solubility; it is formulated with co-solvents for oral dosing. The favorable PK properties support once-daily oral dosing for in vivo efficacy studies.

Pre-clinical toxicology studies for YM-341619 have not been published in detail. In the published in vivo efficacy studies, YM-341619 was well tolerated at doses up to 10 mg/kg (p.o.) for 7-14 days in mice and rats, with no significant body weight loss, no mortality, and no clinical signs of toxicity. No histopathological changes in major organs (liver, kidney, heart, lung, spleen) were observed at efficacious doses (0.1-3 mg/kg). The no-observed-adverse-effect level (NOAEL) in rodents is likely >10 mg/kg/day. Based on its mechanism (STAT6 inhibition), potential on-target toxicities could include increased susceptibility to parasitic infections (as STAT6 is important for immunity against helminths) and impaired Th2 immune responses. However, no immunosuppression or opportunistic infections were reported in short-term studies. Off-target toxicity was not observed at concentrations up to 10 uM in cell-based assays. Formal repeat-dose toxicity studies, genotoxicity (Ames test, micronucleus), and cardiovascular safety (hERG, QT prolongation) have not been reported, but such studies would be required for clinical development. The compound is for research use only and should be handled with standard laboratory precautions: fume hood, gloves, lab coat, safety glasses. YM-341619 is stable as a powder stored at -20degC, desiccated, and protected from light. Solutions in DMSO can be stored at -20degC for months. Avoid freeze-thaw cycles.

[1]. Identification of 4-benzylamino-2-[(4-morpholin-4-ylphenyl)amino]pyrimidine-5-carboxamide derivatives as potent and orally bioavailable STAT6 inhibitors. Bioorg Med Chem. 2008 Jul 1;16(13):6509-21. 9.15.

[2]. YM-341619 suppresses the differentiation of spleen T cells into Th2 cells in vitro, eosinophilia, and airway hyperresponsiveness in rat allergic models. Eur J Pharmacol. 2008 Aug 20;590(1-3):409-16.

At least in vitro, it inhibits the differentiation of splenic T cells into Th2 cells; structure is shown in the first source.

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YM-341619 (AS1617612) is an investigational compound and has not yet been approved by any regulatory agency for clinical use. However, it is a highly promising pre-clinical candidate for allergic diseases such as asthma, atopic rhinitis, atopic dermatitis, and other Th2-mediated disorders. The compound has not advanced to clinical trials (Phase I/II/III) as of the knowledge cutoff, but its potency, selectivity, oral bioavailability, and in vivo efficacy make it an attractive candidate for further drug development. STAT6 is a validated therapeutic target for allergy and asthma, as evidenced by the efficacy of STAT6 knockout mice and by the clinical success of biologic agents targeting IL-4 and IL-13 (e.g., dupilumab, lebrikizumab). A small-molecule STAT6 inhibitor like YM-341619 could offer an oral alternative to injectable biologics. YM-341619 is not commercially available from major chemical vendors and may be obtained as a reference standard or custom synthesis. The compound is protected by patents (likely owned by Astellas Pharma or other pharmaceutical companies). YM-341619 serves as a valuable research tool for studying the role of STAT6 in Th2 immunity, inflammation, and cancer. There are other known STAT6 inhibitors, such as AS1517499 (IC50 ~10 nM), but YM-341619 is more potent (IC50 0.70 nM). The compound is not intended for human therapeutic use outside of approved clinical trials. For research use only.

C22H21F3N6O2

458.436354398727

458.167

643082-52-4

10321901

Off-white to light yellow solid powder

3.4

3

10

7

33

635

0

N(C1=NC(NC2C=CC(N3CCOCC3)=CC=2)=NC=C1C(=O)N)CC1C(=CC=C(F)C=1F)F

IUUUCMFTTBSFIT-UHFFFAOYSA-N

InChI=1S/C22H21F3N6O2/c23-17-5-6-18(24)19(25)15(17)11-27-21-16(20(26)32)12-28-22(30-21)29-13-1-3-14(4-2-13)31-7-9-33-10-8-31/h1-6,12H,7-11H2,(H2,26,32)(H2,27,28,29,30)

2-(4-morpholin-4-ylanilino)-4-[(2,3,6-trifluorophenyl)methylamino]pyrimidine-5-carboxamide

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)

DMSO: 50 mg/mL (109.07 mM)

Solubility in Formulation 1: 2.5 mg/mL (5.45 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 (5.45 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.45 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.

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