R-7050

TNF-α (EC50 = 0.63 μM); IL-1β (EC50 = 1.45 μM)

R-7050 对 TNFα 有明显的偏好，对 LPA 受体（GPCR 的一种）有明显的偏好，与 IL-1β、酪氨酸激酶或 Fas 受体系统不同[2]。

R-7050 可减少小鼠脑出血 (ICH) 引起的神经血管损伤。受伤后两小时内服用 R-7050 显着减少了血脑屏障的开放，并减缓了受伤后 24 小时水肿的发生。受伤后的前三天，神经系统结果也有所改善。另一方面，R-7050 对减少血肿体积没有作用[1]。

高通量筛选[2]
化合物溶解在DMSO中，最终储存浓度为10 mM，保存在- 80°C。化合物文库筛选使用完全集成的、可编程的机器人液体处理系统进行，该系统具有集成的板阅读器和环境控制的板传送带，设置在37°C和5% CO2下。A549细胞（1 × 104个/孔）在100 μl培养基中接种于96孔白色平底板中过夜。第二天，从相同培养基中制备的~ 300000化合物文库中提取100 μl等份，最终浓度为10 μM，加入0.2% (v/v) DMSO中。细胞用化合物预孵育1小时，然后用TNFα或IL-1β（最终浓度为1 ng/ml）刺激4小时。在刺激期结束时，用小鼠抗人CD54单克隆抗体和酶标抗小鼠IgG2a分别以1:10 000和1:10 000的最终稀释度对细胞进行ICAM-1染色。37℃孵育1小时后，用300 μl PBS洗涤4次，用超信号ELISA微化学发光底物显影。在测定EC50时，用TNFα (25 ng/ml)或IL-1β (10 ng/ml)刺激细胞仅4小时，以避免细胞产生的其他细胞因子的二次刺激。

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基于图像的NFκB核易位分析[2]
实验前一天，以2000个细胞/孔的密度将HeLa细胞接种于96孔板中。在实验当天，将细胞与连续稀释的测试化合物（例如R-7050）预孵育1小时，并在37℃下用TNFα (25 ng/ml)或IL-1β (10 ng/ml)刺激30分钟。用PBS洗涤细胞2次，在室温下用甲醇固定1小时。用1% BSA在PBS-Tween 20缓冲液中阻断细胞1小时后，用含有抗nfκ b p65兔多克隆抗体（1:1000稀释）的相同缓冲液处理细胞过夜，然后按照上述方案在室温下进行Alexa Fluor 488偶联山羊抗兔IgG染色（1:1000稀释）3小时。4′，6-二氨基-2-苯基吲哚（DAPI）染色1小时，跟踪细胞核形态。使用配有UV滤光片组和Photometrics相机的蔡司Axiovert S100显微镜上的10倍物镜捕获数字图像。

细胞死亡和活力测定[2]
采用细胞荧光荧光细胞活力测定试剂盒测定细胞活力。将TNFα敏感的ME180细胞与R-7050等测试化合物在37℃下预孵育1小时，并用不同浓度的TNFα刺激24小时。孵育结束后，用50 μl预稀释的细胞滴度-荧光试剂（与PBS 1:1）替换培养基，用微量滴度仪测定相对发光强度。两种已知的nf - κ b通路抑制剂（IKK2-VI和MG132）和一种已知的rna合成抑制剂（Act-D）增强tnf α诱导的细胞毒性作为对照。

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Caspase检测[2]
采用caspase - glo 3/7检测试剂盒检测化合物对caspase活性的影响。ME180细胞与R-7050等测试化合物在37℃下预孵育1小时，并用不同浓度的TNFα刺激24小时。孵育结束后，用50 μl稀释的caspase-3/7试剂（与PBS 1:1）替换培养基，按照厂家说明测定相对发光强度。一种蛋白酶体抑制剂MG132（已知增加tnf α诱导的细胞毒性）和一种通用的caspase抑制剂Z-VAD-fmk作为对照。

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TNFα受体内化的免疫荧光分析[2]
本研究由Schutze等人报道。简单地说，将HeLa细胞接种到含有无菌圆形显微镜盖的12孔组织培养板上。将细胞与测试化合物如R-7050或阳性对照（MDC）在37℃预孵育1小时后，将温度转移到4℃，并将生物素化的人TNFα (100 ng/ml)添加到培养基中，该生物素化的人TNFα (100 ng/ml)由xx公司获得的Fluorokine kit （NFTA0）提供。2小时后，用PBS洗涤细胞以去除未结合的TNFα，生物素化的TNFα-TNFα - α r复合物内化10、30和60分钟。然后用PBS洗涤两次，用冷甲醇（90%）固定30分钟。再次用PBS洗涤细胞，用含有0.5% Triton X-100的Super Block溶液在室温下阻断2小时。细胞用fitc偶联的亲和素（1:50稀释）在相同的阻断液中于4°C下染色过夜。盖板被翻转到预加载了一滴Vecta-shield安装介质的玻璃片上。在Axiovert S100荧光显微镜下检测配体-受体复合物的定位。

Mice: A mouse collagenase model of ICH is utilized. In a nutshell, 8–10 week old male CD-1 mice are positioned in a stereotactic frame, and a burr hole with a diameter of 0.5 mm is drilled over the parietal cortex, 2.2 mm lateral to the bregma. The left striatum is injected directly from the surface of the skull 3 mm down using a 26-gauge Hamilton syringe filled with 0.04 U of bacterial type IV collagenase in 0.5 L saline. To avoid solution reflux and excessive diffusion, the syringe is depressed at 450 nL/min and left in place for a few minutes after the procedure. Sham animals undergo the same surgical procedure, except that saline is stereotactically injected rather than collagenase. After the syringe is removed, bone wax is used to close the burr hole, the incision is surgically stapled, and mice are kept warm until recovery of the righting reflex. R-7050 (6-18 mg/kg) is administered via intraperitoneal route at the time of injury or up to 2 h post-ICH.

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Briefly, male CD-1 mice (8–10 weeks old) were placed into a stereotactic frame and a 0.5 mm diameter burr hole was drilled over the parietal cortex, 2.2 mm lateral to the bregma. A 26-gauge Hamilton syringe, loaded with 0.04U of bacterial type IV collagenase in 0.5 μL saline, was lowered 3 mm deep from the skull surface directly into the left striatum. The syringe was depressed at a rate of 450 nL/min and left in place for several minutes after the procedure to prevent solution reflux and excess diffusion. Sham animals underwent the same surgical procedure, except that saline was stereotactically injected rather than collagenase. After the syringe was removed, bone wax was used to close the burr hole, the incision was surgically stapled, and mice were kept warm until recovery of the righting reflex. R-7050 (6–18 mg/kg) was administered via intraperitoneal route at the time of injury or up to 2h post-ICH.[1]

[1]. Neurosci Lett. 2013 May 10; 542: 92–96.

[2]. Chem Biol . 2007 Oct;14(10):1105-18.

Intracerebral hemorrhage (ICH), the most common form of hemorrhagic stroke, exhibits the highest acute mortality and the worst long-term prognosis of all stroke subtypes. Unfortunately, treatment options for ICH are lacking due in part to a lack of feasible therapeutic targets. Inflammatory activation is associated with neurological deficits in pre-clinical ICH models and with patient deterioration after clinical ICH. In the present study, we tested the hypothesis that R-7050, a novel cell permeable triazoloquinoxaline inhibitor of the tumor necrosis factor receptor (TNFR) complex, attenuates neurovascular injury after ICH in mice. Up to 2h post-injury administration of R-7050 significantly reduced blood-brain barrier opening and attenuated edema development at 24h post-ICH. Neurological outcomes were also improved over the first three days after injury. In contrast, R-7050 did not reduce hematoma volume, suggesting the beneficial effects of TNFR inhibition were downstream of clot formation/resolution. These data suggest a potential clinical utility for TNFR antagonists as an adjunct therapy to reduce neurological injury and improve patient outcomes after ICH. [1]

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Small-molecule library screening to find compounds that inhibit TNFalpha-induced, but not interleukin 1beta (IL-1beta)-induced, intercellular adhesion molecule 1 (ICAM-1) expression in lung epithelial cells identified a class of triazoloquinoxalines. These compounds not only inhibited the TNFalpha-induced nuclear factor kappaB (NFkappaB) survival pathway but also blocked death-pathway activation. Such dual activity makes them unique against other known NFkappaB-pathway inhibitors that inhibit only a subset of TNFalpha signals leading to increased TNFalpha-induced cytotoxicity. Interestingly, these compounds inhibited association of TNFalpha receptor (TNFalphaR) I with TNFalphaR-associated death domain protein (TRADD) and receptor interacting protein 1 (RIP1), the initial intracellular signaling event following TNFalpha stimulation. Further study showed that they blocked ligand-dependent internalization of the TNFalpha-TNFalphaR complex, thereby inhibiting most of the TNFalpha-induced cellular responses. Thus, compounds with a triazoloquinoxaline scaffold could be a valuable tool to investigate small molecule-based anti-TNFalpha therapies.[2]

C16H8CLF3N4S

380.774730682373

380.011

C, 50.47; H, 2.12; Cl, 9.31; F, 14.97; N, 14.71; S, 8.42

303997-35-5

303997-35-5

1486608

Light yellow to yellow solid powder

1.6±0.1 g/cm3

1.694

5.45

68.4

0

7

2

25

477

0

FC(F)(C1=NN=C2N1C3=CC(Cl)=CC=C3N=C2SC4=CC=CC=C4)F

SUUMKHOVGVYGOP-UHFFFAOYSA-N

InChI=1S/C16H8ClF3N4S/c17-9-6-7-11-12(8-9)24-13(22-23-15(24)16(18,19)20)14(21-11)25-10-4-2-1-3-5-10/h1-8H

8-chloro-4-phenylsulfanyl-1-(trifluoromethyl)-[1,2,4]triazolo[4,3-a]quinoxaline

TNF-α Antagonist III; R-7050; 8-chloro-4-phenylsulfanyl-1-(trifluoromethyl)-[1,2,4]triazolo[4,3-a]quinoxaline; 8-Chloro-4-(phenylthio)-1-(trifluoromethyl)-[1,2,4]triazolo[4,3-a]quinoxaline; 8-chloro-1-(trifluoromethyl)[1,2,4]triazolo[4,3-a]quinoxalin-4-yl phenyl sulfide; 8-chloro-4-(phenylsulfanyl)-1-(trifluoromethyl)-[1,2,4]triazolo[4,3-a]quinoxaline; R 7050; SUUMKHOVGVYGOP-UHFFFAOYSA-N; R7050; R 7050

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: 9~25 mg/mL (23.6~65.7 mM)

配方 1 中的溶解度: 2.5 mg/mL (6.57 mM) 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), 悬浮液；超声助溶。
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 (6.57 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。