AS1517499

STAT6 (IC50 = 21 nM)

在不影响 IL-12 诱导的 T 辅助 1 (Th1) 细胞的情况下，AS1517499 表现出强烈的 STAT6 抑制作用，IC50 值为 21 nM。它还抑制 IL-4 诱导的小鼠脾 T 细胞的 Th2 分化，IC50 值为 2.3 nM。区别。 AS1517499 特异性抑制 Th2 分化，在不影响 Th1 分化的情况下 [1]。共孵化会抑制事件发生。 IL-13 (100 ng/mL) 磷酸化 STAT6 并上调 RhoA，RhoA 是一种单体 GTP 酶，负责培养的人 BSM 细胞中平滑肌收缩的 Ca2+ 敏感性。这两种效应均与 AS1517499 (100 nM)[2]有关。

在最后一次卵清蛋白抗原攻击后，反复主动对该抗原致敏的 BALB/c 小鼠表现出支气管肺泡灌洗液中 IL-13 水平升高以及支气管组织中 STAT6 磷酸化。为了响应乙酰胆碱，这些小鼠表现出 BSM 收缩性增加，并且支气管组织具有更高的 RhoA 表达。每次腹腔注射 AS1517499 (10 mg/kg) 前暴露一小时的卵清蛋白几乎消除了抗原诱导的 RhoA 上调和 BSM 高反应性 [2]。

STAT6（信号转导子和转录激活子6）蛋白被白细胞介素（IL）-4和IL-13激活，在T辅助细胞2（Th2）分化中起着重要作用。因此，STAT6可能是治疗各种过敏性疾病（包括哮喘和特应性疾病）的极好靶点。我们合成了一系列2-{[2-（4-羟基苯基）乙基]氨基}嘧啶-5-甲酰胺衍生物，并评估了它们的STAT6抑制活性。在这些化合物中，4-（苄氨基）-2-{[2-（3-氯-4-羟基苯基）乙基]氨基}嘧啶-5-甲酰胺（2t，AS1517499）显示出强烈的STAT6抑制作用，IC（50）值为21 nM，还抑制了IL-4诱导的小鼠脾脏T细胞的Th2分化，IC（50中）值为2.3 nM，并且不影响IL-12诱导的T辅助细胞1（Th1）分化[1]。

正常人BSM细胞在添加了5%胎牛血清、0.5 ng/ml人表皮生长因子（hEGF）、5μg/ml胰岛素、2 ng/ml人成纤维细胞生长因子碱性（hFGF-b）、50μg/ml庆大霉素和50 ng/ml两性霉素b的SmBM培养基中维持。细胞在37°C的加湿气氛（5%CO2）中维持，每48至72小时喂食一次，当细胞达到90至95%融合时传代。然后将hBSMC（第7-9代）以3500个细胞/cm2的密度接种在6孔板和8孔室载玻片上，当观察到80%至85%的融合时，在添加重组人IL-13之前，在没有血清的情况下培养细胞24小时。在加入IL-13（100ng/ml）前30分钟处理AS1517499（100nM）或其载体（0.3%DMSO）。在一些实验中，AS1517499在添加IL-13后0（共孵育）、3或12小时进行处理。在另一系列实验中，在施用IL-13前15分钟，还施用了选择性Rho激酶抑制剂Y-27632（1μM）或其载体（0.3%DMSO）。在IL-13处理后的指定时间，用PBS洗涤细胞，立即收集，用1×SDS样品缓冲液（250μl/孔）破碎，并用于蛋白质印迹分析[2]。

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]

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1 or 10 mg/kg/d; dissolved in 20% DMSO in saline or its vehicle; i.p injection

Male BALB/c mice

[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.

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]

C20H20CLN5O2

397.86

397.131

C, 60.38; H, 5.07; Cl, 8.91; N, 17.60; O, 8.04

919486-40-1

10340781

Typically exists as white to light brown solids at room temperature

2.929

120.61

4

6

8

28

491

0

O=C(C1C(NCC2C=CC=CC=2)=NC(NCCC2C=C(Cl)C(O)=CC=2)=NC=1)N

OZRMEKAUZBKTTC-UHFFFAOYSA-N

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)

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

AS-1517499; AS1517499; AS 1517499

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.28 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (6.28 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。