AS1517499 is a potent STAT6 inhibitor with IC50 of 21 nM. The STAT6 (signal transducers and activators of transcription 6) protein is activated by interleukin (IL)-4 and IL-13, and plays an important role in T-helper cell 2 (Th2) differentiation. STAT6 might therefore be an excellent therapeutic target for various allergic conditions, including asthma and atopic diseases. It shows potent STAT6 inhibition with an IC50 value of 21 nM, and also inhibits IL-4-induced Th2 differentiation of mouse spleen T cells with an IC50 value of 2.3 nM and without influencing T-helper cell 1 (Th1) differentiation induced by IL-12. AS1517499 selectively inhibits Th2 differentiation without affecting Th1 differentiation.

Physicochemical Properties

Molecular Formula	C20H20CLN5O2
Molecular Weight	397.86
Exact Mass	397.131
Elemental Analysis	C, 60.38; H, 5.07; Cl, 8.91; N, 17.60; O, 8.04
CAS #	919486-40-1
Related CAS #	1448428-04-3; 919486-40-1;1448428-05-4 (HCl);
PubChem CID	10340781
Appearance	Typically exists as white to light brown solids at room temperature
LogP	2.929
Hydrogen Bond Donor Count	4
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	8
Heavy Atom Count	28
Complexity	491
Defined Atom Stereocenter Count	0
SMILES	O=C(C1C(NCC2C=CC=CC=2)=NC(NCCC2C=C(Cl)C(O)=CC=2)=NC=1)N
InChi Key	OZRMEKAUZBKTTC-UHFFFAOYSA-N
InChi Code	InChI=1S/C20H20ClN5O2/c21-16-10-13(6-7-17(16)27)8-9-23-20-25-12-15(18(22)28)19(26-20)24-11-14-4-2-1-3-5-14/h1-7,10,12,27H,8-9,11H2,(H2,22,28)(H2,23,24,25,26)
Chemical Name	5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-4-methylbenzamide
Synonyms	AS-1517499; AS1517499; AS 1517499
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

Targets	STAT6 (IC50 = 21 nM)
ln Vitro	Without influencing IL-12-induced T helper 1 (Th1) cells, AS1517499 demonstrated strong STAT6 inhibition with an IC50 value of 21 nM. It also inhibited IL-4-induced Th2 differentiation of mouse splenic T cells with an IC50 value of 2.3 nM. distinction. Without impacting Th1 differentiation, AS1517499 specifically inhibits Th2 differentiation [1]. Co-incubation inhibits events. IL-13 (100 ng/mL) phosphorylated STAT6 and upregulated RhoA, a monomeric GTPase responsible for smooth muscle contraction's Ca2+ sensitivity in cultured human BSM cells. Both of these effects were linked to AS1517499 (100 nM)[2].
ln Vivo	Following the final ovalbumin antigen challenge, BALB/c mice that had been repeatedly and actively sensitized to the antigen showed elevated IL-13 levels in bronchoalveolar lavage fluid and STAT6 phosphorylation in bronchial tissue. In response to acetylcholine, these mice exhibit increased BSM contractility, and bronchial tissue has higher RhoA expression. A single ovalbumin exposure hour prior to each injection of AS1517499 (10 mg/kg) intraperitoneally virtually eliminated antigen-induced RhoA upregulation and BSM hyperresponsiveness [2].
Enzyme Assay	The STAT6 (signal transducers and activators of transcription 6) protein is activated by interleukin (IL)-4 and IL-13, and plays an important role in T-helper cell 2 (Th2) differentiation. STAT6 might therefore be an excellent therapeutic target for various allergic conditions, including asthma and atopic diseases. We synthesized a series of 2-{[2-(4-hydroxyphenyl)ethyl]amino}pyrimidine-5-carboxamide derivatives and evaluated their STAT6 inhibitory activities. Among these compounds, 4-(benzylamino)-2-{[2-(3-chloro-4-hydroxyphenyl)ethyl]amino}pyrimidine-5-carboxamide (2t, AS1517499) showed potent STAT6 inhibition with an IC(50) value of 21 nM, and also inhibited IL-4-induced Th2 differentiation of mouse spleen T cells with an IC(50) value of 2.3 nM and without influencing T-helper cell 1 (Th1) differentiation induced by IL-12[1].
Cell Assay	Normal human BSM cells were maintained in SmBM medium supplemented with 5% fetal bovine serum, 0.5 ng/ml human epidermal growth factor (hEGF), 5 μg/ml insulin, 2 ng/ml human fibroblast growth factor-basic (hFGF-b), 50 μg/ml gentamicin, and 50 ng/ml amphotericin B. Cells were maintained at 37°C in a humidified atmosphere (5% CO2), fed every 48 to 72 hours, and passaged when cells reached 90 to 95% confluence. Then the hBSMCs (passages 7–9) were seeded in 6-well plates and 8-well chamber slides at a density of 3,500 cells/cm2 and, when 80 to 85% confluence was observed, cells were cultured without serum for 24 hours before addition of recombinant human IL-13. AS1517499 (100 nM) or its vehicle (0.3% DMSO) was treated 30 minutes before the addition of IL-13 (100 ng/ml). In some experiments, AS1517499 was treated 0 (co-incubation), 3, or 12 hours after the addition of IL-13. In another series of experiments, a selective Rho-kinase inhibitor Y-27632 (1 μM) or its vehicle (0.3% DMSO) was also applied 15 minutes before the IL-13 application. At the indicated time after the IL-13 treatment, cells were washed with PBS, immediately collected, and disrupted with 1× SDS sample buffer (250 μl/well), and used for Western blot analyses[2].
Animal Protocol	Male BALB/c mice were housed in a pathogen-free facility.Preparation of a murine model of allergic bronchial asthma, which has an in vivo AHR, was performed as described previously. In brief, BALB/c mice (8 wk of age) were actively sensitized by intraperitoneal injections of 8 μg ovalbumin with 2 mg Imject Alum on Day 0 and Day 5. The sensitized mice were challenged with aerosolized OVA-saline solution (5 mg/ml) for 30 minutes on Days 12, 16, and 20. A control group of mice received the same immunization procedure but inhaled saline aerosol instead of OVA challenge. The aerosol was generated with an ultrasonic nebulizer and introduced to a Plexiglas chamber box (130 × 200 mm, 100 mm height) in which the mice were placed. Animals also received intraperitoneal injection with AS1517499 (1 or 10 mg/kg/d; dissolved in 20% DMSO in saline) or its vehicle 1 hour before each antigen inhalation (Days 12, 16, and 20). Twenty-four hours after the last OVA challenge, mice were killed by exsanguination from abdominal aorta under urethane (1.6 g/kg, intraperitoneally) anesthesia.[2] 1 or 10 mg/kg/d; dissolved in 20% DMSO in saline or its vehicle; i.p injection Male BALB/c mice
References	[1]. Synthesis and evaluation of 2-{[2-(4-hydroxyphenyl)-ethyl]amino}pyrimidine-5-carboxamide derivatives as novel STAT6 inhibitors. Bioorg Med Chem. 2007 Jan 15;15(2):1044-55. [2]. A novel STAT6 inhibitor AS1517499 ameliorates antigen-induced bronchial hypercontractility in mice. Am J Respir Cell Mol Biol. 2009 Nov;41(5):516-24.
Additional Infomation	Interleukin-13 (IL-13) is one of the central mediators for development of airway hyperresponsiveness in asthma. The signal transducer and activation of transcription 6 (STAT6) is one of the major signal transducers activated by IL-13, and a possible involvement of IL-13/STAT6 pathway in the augmented bronchial smooth muscle (BSM) contraction has been suggested. In the present study, the effect of a novel STAT6 inhibitor, AS1517499, on the development of antigen-induced BSM hyperresponsiveness was investigated. In cultured human BSM cells, IL-13 (100 ng/ml) caused a phosphorylation of STAT6 and an up-regulation of RhoA, a monomeric GTPase responsible for Ca2+ sensitization of smooth muscle contraction: both events were inhibited by co-incubation with AS1517499 (100 nM). In BALB/c mice that were actively sensitized and repeatedly challenged with ovalbumin antigen, an increased IL-13 level in bronchoalveolar lavage fluids and a phosphorylation of STAT6 in bronchial tissues were observed after the last antigen challenge. These mice had an augmented BSM contractility to acetylcholine together with an up-regulation of RhoA in bronchial tissues. Intraperitoneal injections of AS1517499 (10 mg/kg) 1 hour before each ovalbumin exposure inhibited both the antigen-induced up-regulation of RhoA and BSM hyperresponsiveness, almost completely. A partial but significant inhibition of antigen-induced production of IL-13 was also found. These findings suggest that the inhibitory effects of STAT6 inhibitory agents, such as AS1517499, both on RhoA and IL-13 up-regulations might be useful for asthma treatment.[2]

Solubility Data

Solubility (In Vitro)

DMSO:≥ 35 mg/mL Water:<1 mg/mL Ethanol:<1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.28 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 (6.28 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	2.5134 mL	12.5672 mL	25.1345 mL
	5 mM	0.5027 mL	2.5134 mL	5.0269 mL
	10 mM	0.2513 mL	1.2567 mL	2.5134 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.