Resatorvid   中文名称: 瑞沙托维

TLR4; TNF-R (IC50 = 1.9 nM); IL-6 (IC50 = 1.3 nM)

Resatorvid 的 IC50 值范围为 11 至 33 nM，可抑制 LPS 刺激的人外周血单核细胞 (PBMC) 产生的 NO、TNF-α 和 IL-6 [1]。在剂量为 100 nM 时，Resatorvid（1-100 nM；4 小时）会磷酸化并破坏 IκBβ。 Resatorvid（1-100 nM；15 分钟；PBMC 细胞）强烈抑制 LPS 诱导的细胞外信号调节模型 1/2 (Erk1/2)、p38 和 JNK /SAPK [1]。

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促炎介质如细胞因子和NO在各种炎性疾病中起关键作用。为了成功地对抗炎症性疾病，促炎介质的产生将是一个关键的过程。本研究研究了新型小分子细胞因子产生抑制剂(6R)-6-[N-（2-氯-4-氟苯基）磺胺基]环己基-1-烯-1-羧酸乙酯（TAK-242）的体外作用及其机制。在RAW264.7细胞和小鼠腹膜巨噬细胞中，TAK-242抑制脂多糖（LPS）诱导的NO、肿瘤坏死因子- α (tnf - α)和白细胞介素-6的产生，50%的抑制浓度（IC50）为1.1 ~ 11 nM。TAK-242还能抑制lps刺激的人外周血单个核细胞（PBMCs）产生这些细胞因子，IC50值为11 ~ 33 nM。此外，lps对人pbmc、单核细胞和巨噬细胞诱导的IL-6和IL-12产生的抑制作用相似。TAK-242对LPS和干扰素诱导的RAW264.7细胞IL-6和tnf - α mRNA表达有抑制作用。LPS诱导的丝裂原活化蛋白激酶磷酸化也呈浓度依赖性被抑制。然而，TAK-242不拮抗LPS与细胞的结合。值得注意的是，TAK-242抑制toll样受体（TLR） 4配体诱导的细胞因子产生，而不抑制TLR2、-3和-9配体诱导的细胞因子产生。此外，il -1 β诱导的人PBMCs中IL-8的产生不受TAK-242的显著影响。这些数据表明，TAK-242通过选择性抑制TLR4细胞内信号传导抑制多种细胞因子的产生。最后，TAK-242是一种新型的小分子TLR4信号抑制剂，可能成为治疗炎症性疾病的有前景的药物，其发病机制与TLR4有关。[1]

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从SAR研究结果来看，含1位酯基和6位取代苯基磺胺基的环己烯衍生物具有抑制活性。从改变环己烯环大小的结果来看，活性受到分子形状的影响，并且靶分子上的结合位点可能至少存在两个口袋（酯部分和苯基部分），因为上面提到的每个片段似乎都被严格识别。在所有化合物中，5n/TAK -242，具有环己烯环、乙酯基和2-氯-4-氟苯基磺甲酰基的组合，表现出最有效的抑制NO产生的活性，并对(R)-(+)-5n和(S)-(−)-5n两个对映体的活性进行了评估，以研究抑制的立体化学要求。结果表明，(R)-(+)-5n （IC50 = 1.8 nM）比(S)-(−)-5n （IC50 = 640 nM）的活性高350倍。此外，我们还研究了5n及其对映体对lps刺激的RAW264.7细胞产生细胞因子的抑制作用。(R)-(+)-5n强烈抑制TNF-α和IL-6的产生，IC50值分别为1.9 nM和1.3 nM（表4），比先导化合物5a强120倍。［2］

resporvid（TAK-242；3mg/kg；腹膜内注射；持续2天；雄性C57BL/6小鼠）均显着恢复了LPS引起的体重减轻、TA肌肉损失和肌肉力量损失。 TAK-242 增加间隙空间并逆转 LPS 产生的肌纤维的收缩。在给予 LPS 的小鼠中，TAK-242 抑制分解代谢细胞因子释放到系统中以及骨骼肌的蛋白水解作用 [3]。

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采用小鼠内毒素休克模型检测体外实验中最高抑制剂(R)-(+)-5n/TAK-242的体内药效。在致死性LPS攻击前1小时静脉给药，(R)-(+)-5n以剂量依赖的方式拯救小鼠（图3）。在3mg /kg的剂量下，所有小鼠都存活，(R)-(+)-5n保护小鼠免于致死的最小有效剂量（ED50）值为0.3 mg/kg。LPS刺激后血清中TNF-α、IL-6、IL-1β和NO水平显著升高，(R)-(+)-5n （0.1 ~ 3 mg/kg）抑制LPS诱导的所有细胞因子和NO的产生并呈剂量依赖性。在0.1 mg/kg剂量下，(R)-(+)-5n显著抑制所有细胞因子和NO的产生（图4）。[2]

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在这里，研究人员测试了TAK-242，一种toll样受体4 （TLR4）特异性信号抑制剂，对内毒素诱导的体外肌管萎缩和体内肌肉萎缩的影响。LPS处理小鼠C2C12肌管引起炎症反应（核因子-κB活性、白细胞介素-6和肿瘤坏死因子-α表达增加），激活泛素-蛋白酶体和自噬蛋白水解途径（atroggin -1/MAFbx、MuRF1和LC-II表达增加），导致肌管萎缩。在小鼠中，LPS注射增加了骨骼肌中相同的炎症和蛋白水解途径，并诱导萎缩，导致握力降低。值得注意的是，在体外和体内，用TAK-242预处理细胞或小鼠可以减少或逆转LPS的所有有害影响。总之，我们的研究结果表明，TLR4信号的药理抑制可能是内毒素血症诱导的肌肉萎缩的一种新的治疗干预措施。[3]

LPS诱导的丝裂原活化蛋白激酶的磷酸化也以浓度依赖的方式受到抑制。然而，TAK-242不拮抗LPS与细胞的结合。值得注意的是，TAK-242抑制了Toll样受体（TLR）4配体诱导的细胞因子产生，而不是TLR2、-3和-9配体诱导的。此外，TAK-242对IL-1β诱导的人PBMC产生IL-8没有显著影响。这些数据表明，TAK-242通过选择性抑制TLR4细胞内信号传导来抑制多种细胞因子的产生。最后，TAK-242是一种新型的小分子TLR4信号抑制剂，可能是一种很有前途的炎症性疾病的治疗剂，其发病机制涉及TLR4[1]。

蛋白质印迹分析[1]
细胞类型： PBMC 细胞
测试浓度： 1 nM、10 nM、100 nM
孵育时间：15分钟
实验结果：LPS诱导的丝裂原激活蛋白激酶的磷酸化也以浓度依赖性方式被抑制。

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NO生成抑制活性测定。[2]
RAW264.7细胞以1 × 105个/孔的比例在96孔培养板中孵育过夜。去除细胞培养上清后，在37℃、5% CO2的湿化气氛中，用10 ng/mL LPS和不同浓度的测试化合物刺激细胞20 h。刺激介质为无酚红RPMI1640，含1%热灭活FBS和10 μg/mL卡那霉素。将试验化合物以10 mmol/L溶解于DMF中，用RPMI-1640培养基稀释至适当浓度，加入培养液中。
根据使用2,3-二氨基萘的荧光法，通过测量一氧化氮的稳定代谢物亚硝酸盐的量来估计一氧化氮的产生。简单地说，将25 μL的20 μg/mL DAN加入到50 μL的培养上清中，室温孵育10 min。加入25 μL的0.5 N NaOH后，在460 nm处（激发波长：355nm）测量荧光。

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抑制细胞因子产生的活性测定。[2]
根据制造商的说明，使用特异性酶联免疫吸附试验（ELISA）测定培养上清中TNF-α和IL-6的浓度。

Animal/Disease Models: Male C57BL/6 mice (8-12 weeks of age) treated with lipopolysaccharide (LPS)[3]
Doses: 3 mg/kg
Route of Administration: intraperitoneal (ip)injection; for 2 days
Experimental Results: Pretreatment of mice decreased or reversed all the detrimental effects of LPS.

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Endotoxin Shock Model. [2]
Mice were intraperitoneally injected with LPS at a lethal dose of 7 mg/kg. Survival of mice was recorded for 7 days following LPS challenge. Compound (R)-(+)-5n / TAK-242was dissolved in 10% (w/v) Glucuronylglucosyl-β-cyclodextrin sodium salt and administered intravenously 1 h before the LPS injection.

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Evaluation of Cytokine and NO Production In Vivo. [2]
The blood was collected at the indicated times, and allowed to clot at room temperature. The serum was separated by centrifugation and stored at −80 °C until analysis. The serum levels of TNF-α, IL-6, and IL-1β were determined by specific ELISA according to the manufacturer's instructions.
The serum levels of nitrite and nitrate, which are stable end products of NO metabolism were measured by a fluorometric method. Briefly, 10 μL aliquots of sample were mixed with 20 μL of β-NADPH (0.2 mmol/L) and 20 μL of Aspergillus nitrate reductase (0.25 U/mL) and incubated for 20 min at room temperature to convert nitrate to nitrite. To these samples was added 25 μL of DAN (25 μg/mL) in HCl (2 mol/L) and incubated for 10 min at room temperature. NaOH (25 μL) (0.5 mol/L) was added, and the fluorescence (excitation:  355 nm, emission:  460 nm) was measured. The nitrite concentrations were calculated from a standard curve with sodium nitrite. [2]

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Male C57BL/6 mice aged 8–12 weeks were quarantined in quiet humidified rooms on a 12 h light/dark cycle (7 am/7 pm) for at least 1 week before use. Mice were allowed access to standard rodent diet and water ad libitum. Food intake and body weight were measured every 24 h for 48 h. Muscle catabolism was induced by the i.p. injection of 1 mg/kg LPS, or an equal volume of vehicle (PBS) for controls, as previously described19. Mice were pretreated with TAK-242 (3 mg/kg) or vehicle (PBS containing 0.9% DMSO) by i.p. injection 1 h before LPS injection unless otherwise specified. These LPS and TAK-242 doses and administration times were selected based on several relevant articles. Two days later, mice were euthanised and the TA muscles were collected, immediately frozen in liquid nitrogen, and stored at −80 °C until use.

[1]. A novel cyclohexene derivative, ethyl (6R)-6-[N-(2-Chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate (TAK-242), selectively inhibits toll-like receptor 4-mediated cytokine production through suppression of intracellular signaling.

[2]. Discovery of novel and potent small-molecule inhibitors of NO and cytokine production as antisepsis agents: synthesis and biological activity of alkyl 6-(N-substituted sulfamoyl)cyclohex-1-ene-1-carboxylate. J Med Chem. 2005 Nov 17;48(23):7457-67.

[3]. TAK-242, a Specific Inhibitor of Toll-like Receptor 4 Signalling, Prevents Endotoxemia-Induced Skeletal Muscle Wasting in Mice. Sci Rep. 2020 Jan 20;10(1):694.

Resatorvid is an investigational compound designed for the treatment of severe sepsis.

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Drug Indication
Investigated for use/treatment in sepsis and septicemia.

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Mechanism of Action
TAK-242 suppresses production of inflammatory mediators such as cytokine by inhibiting the signal transduction through Toll-like receptor 4 (TLR4) which is one of the receptors recognizing the bacterial components.

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In this study, screening for new small-molecule inhibitors of NO and inflammatory cytokines production led to discover of the lead compound 5a. Cyclohexene derivatives I bearing a 6-(substituted phenyl)sulfamoyl and 1-(alkyl ester) group were designed and synthesized by coupling of sulfonyl chlorides with a variety of anilines in the presence of Et3N with concomitant migration of the double bond. Chemical modification of the substituents on the phenyl ring, ethyl ester moiety, and cyclohexene ring was carried out and an identified novel series of cyclohexene derivatives exhibited strong suppressive effects on NO production from mouse macropharges stimulated with LPS. Among all the compounds prepared, (R)-(+)-5n exhibited the most potent inhibitory effect on the production of inflammatory cytokines such as TNF-α and IL-6 as well as NO. These IC50 values were approximately 100-fold stronger than those of lead compound 5a. Furthermore, (R)-(+)-5n showed significant in vivo efficacies in mouse endotoxin shock model. The ED50 value to protect mice from lethality was 0.3 mg/kg, and at a dose of 3 mg/kg all mice survived. In addition, (R)-(+)-5n suppressed the increase in serum levels of inflammatory mediators such as TNF-α, IL-6, IL-1β, and NO. Compound (R)-(+)-5n (TAK-242) was selected as a candidate for clinical investigation and is a promising new class of therapeutic agent for the treatment of sepsis. Although the molecular target of (R)-(+)-5n has not yet been identified, it is presumed that (R)-(+)-5n selectively inhibits the Toll-like receptor (TLR) 4-dependent signaling pathway. [2]

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Using C2C12 myotubes in vitro and a mouse model of endotoxemia in vivo, we found that TAK-242, a selective inhibitor of TLR4-mediated signalling, blocked systemic and skeletal muscle inflammatory reactions, inhibited the activation of various proteolytic pathways, and ameliorated skeletal muscle wasting induced by LPS. These findings may prove helpful for the development of new therapeutic strategies to attenuate endotoxemia-induced muscle wasting. [3]

C15H17CLFNO4S

361.82

361.055

C, 49.80; H, 4.74; Cl, 9.80; F, 5.25; N, 3.87; O, 17.69; S, 8.86

243984-11-4

11703255

Off-white to yellow solid

1.4±0.1 g/cm3

462.0±55.0 °C at 760 mmHg

68-69ºC

233.2±31.5 °C

0.0±1.1 mmHg at 25°C

1.574

3.58

80.85

1

6

6

23

560

1

C1(C(OCC)=O)=CCCC[C@H]1S(NC1=C(Cl)C=C(F)C=C1)(=O)=O

LEEIJTHMHDMWLJ-CQSZACIVSA-N

InChI=1S/C15H17ClFNO4S/c1-2-22-15(19)11-5-3-4-6-14(11)23(20,21)18-13-8-7-10(17)9-12(13)16/h5,7-9,14,18H,2-4,6H2,1H3/t14-/m1/s1

ethyl (R)-6-(N-(2-chloro-4-fluorophenyl)sulfamoyl)cyclohex-1-ene-1-carboxylate

TAK242; TAK 242; Resatorvid [INN]; CLI-095; CHEMBL225157; H2MZ648C31; TAK-242

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 : ~100 mg/mL (~276.38 mM)
Ethanol : ~20 mg/mL (~55.28 mM)

配方 1 中的溶解度: 5.5 mg/mL (15.20 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% 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。
*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: 5.5 mg/mL (15.20 mM) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: 5 mg/mL (13.82 mM) in 10% DMSO + 90% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*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 4 中的溶解度: ≥ 2.75 mg/mL (7.60 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 27.5 mg/mL澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入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 5 中的溶解度: 2.75 mg/mL (7.60 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将100μL 27.5mg/mL澄清的DMSO储备液加入到900μL 20％SBE-β-CD生理盐水中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 6 中的溶解度: ≥ 2.75 mg/mL (7.60 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 27.5 mg/mL 澄清 DMSO 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 7 中的溶解度: 2.5 mg/mL (6.91 mM) in 5% DMSO + 95% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*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 8 中的溶解度: 0.5 mg/mL (1.38 mM) in 1% DMSO 99% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*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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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。