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

Resatorvid has IC50 values ranging from 11 to 33 nM, which suppresses NO, TNF-α, and IL-6 produced by LPS-stimulated human peripheral blood mononuclear cells (PBMC) [1]. At a dose of 100 nM, Resatorvid (1-100 nM; 4 hours) phosphorylates and destroys IκBβ. Resatorvid (1-100 nM; 15 minutes; PBMCs cells) strongly suppresses LPS-induced extracellular signal regulation model 1/2 (Erk1/2), p38, and JNK /SAPK [1].

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Proinflammatory mediators such as cytokines and NO play pivotal roles in various inflammatory diseases. To combat inflammatory diseases successfully, regulation of proinflammatory mediator production would be a critical process. In the present study, we investigated the in vitro effects of ethyl (6R)-6-[N-(2-chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate (TAK-242), a novel small molecule cytokine production inhibitor, and its mechanism of action. In RAW264.7 cells and mouse peritoneal macrophages, TAK-242 suppressed lipopolysaccharide (LPS)-induced production of NO, tumor necrosis factor-alpha (TNF-alpha), and interleukin (IL)-6, with 50% inhibitory concentration (IC50) of 1.1 to 11 nM. TAK-242 also suppressed the production of these cytokines from LPS-stimulated human peripheral blood mononuclear cells (PBMCs) at IC50 values from 11 to 33 nM. In addition, the inhibitory effects on the LPS-induced IL-6 and IL-12 production were similar in human PBMCs, monocytes, and macrophages. TAK-242 inhibited mRNA expression of IL-6 and TNF-alpha induced by LPS and interferon-gamma in RAW264.7 cells. The phosphorylation of mitogen-activated protein kinases induced by LPS was also inhibited in a concentration-dependent manner. However, TAK-242 did not antagonize the binding of LPS to the cells. It is noteworthy that TAK-242 suppressed the cytokine production induced by Toll-like receptor (TLR) 4 ligands, but not by ligands for TLR2, -3, and -9. In addition, IL-1beta-induced IL-8 production from human PBMCs was not markedly affected by TAK-242. These data suggest that TAK-242 suppresses the production of multiple cytokines by selectively inhibiting TLR4 intracellular signaling. Finally, TAK-242 is a novel small molecule TLR4 signaling inhibitor and could be a promising therapeutic agent for inflammatory diseases, whose pathogenesis involves TLR4. [1]

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From the results of the SAR study, the cyclohexene derivatives bearing the ester group at 1-position and substituted phenylsufamoyl group at 6-position had the inhibitory activity. It was suggested that the activity was influenced by the shape of molecule from the results of changing the size of cyclohexene ring, and that there might be at least two pockets in the binding site on target molecule (for the ester moiety and the phenyl moiety), since it seemed that each moiety was strictly recognized as mentioned above. Among all the compounds prepared, 5n/TAK-242, having the combination of cyclohexene ring, ethyl ester group, and 2-chloro-4-fluorophenylsufamoyl group, exhibited the most potent suppressive activity for NO production, and the activities of the two enantiomers, (R)-(+)-5n and (S)-(−)-5n, were evaluated in order to investigate the stereochemical requirement for inhibition. As expected, a significant difference was observed between the enantiomers, and (R)-(+)-5n (IC50 = 1.8 nM) exhibited 350-fold more activity than (S)-(−)-5n (IC50 = 640 nM). In addition, the suppressive effects of 5n and both enantiomers on the production of cytokines from LPS-stimulated RAW264.7 cells were examined. (R)-(+)-5n strongly suppressed the production of TNF-α and IL-6 with IC50 values of 1.9 and 1.3 nM, respectively (Table 4), which was 120-fold stronger than those of lead compound 5a. [2]

LPS-induced weight loss, TA muscle loss, and muscular strength loss were all dramatically and significantly restored by resporvid (TAK-242; 3 mg/kg; i.p.; for 2 days; male C57BL/6 mice). TAK-242 increases interstitial space and reverses the contraction of muscle fibers produced by LPS. In mice given LPS, TAK-242 inhibits the release of catabolic cytokines into the system and the proteolysis of skeletal muscle [3].

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The in vivo efficacy of (R)-(+)-5n/TAK-242 which was the highest inhibitor in the in vitro assay was examined using mouse endotoxin shock model. When administrated intraveniously 1 h before lethal LPS challenge, (R)-(+)-5n rescued mice in a dose-dependent manner (Figure 3). At a dose of 3 mg/kg, all mice survived and the minimum effective dose (ED50) value of (R)-(+)-5n to protect mice from lethality was 0.3 mg/kg. In this model, TNF-α, IL-6, IL-1β, and NO levels in serum increased significantly after LPS challenge, and (R)-(+)-5n (0.1−3 mg/kg) suppressed the production of all cytokines and NO induced by LPS in a dose-dependent manner. At a dose of 0.1 mg/kg, (R)-(+)-5n significantly inhibited the production of all cytokines and NO (Figure 4). [2]

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Here, researchers tested the effects of TAK-242, a Toll-like receptor 4 (TLR4)-specific signalling inhibitor, on myotube atrophy in vitro and muscle wasting in vivo induced by endotoxin. LPS treatment of murine C2C12 myotubes induced an inflammatory response (increased nuclear factor-κB activity and interleukin-6 and tumour necrosis factor-α expression) and activated the ubiquitin-proteasome and autophagy proteolytic pathways (increased atrogin-1/MAFbx, MuRF1, and LC-II expression), resulting in myotube atrophy. In mice, LPS injection increased the same inflammatory and proteolytic pathways in skeletal muscle and induced atrophy, resulting in reduced grip strength. Notably, pretreatment of cells or mice with TAK-242 reduced or reversed all the detrimental effects of LPS in vitro and in vivo. Collectively, our results indicate that pharmacological inhibition of TLR4 signalling may be a novel therapeutic intervention for endotoxemia-induced muscle wasting. [3]

The phosphorylation of mitogen-activated protein kinases induced by LPS was also inhibited in a concentration-dependent manner. However, TAK-242 did not antagonize the binding of LPS to the cells. It is noteworthy that TAK-242 suppressed the cytokine production induced by Toll-like receptor (TLR) 4 ligands, but not by ligands for TLR2, -3, and -9. In addition, IL-1β-induced IL-8 production from human PBMCs was not markedly affected by TAK-242. These data suggest that TAK-242 suppresses the production of multiple cytokines by selectively inhibiting TLR4 intracellular signaling. Finally, TAK-242 is a novel small molecule TLR4 signaling inhibitor and could be a promising therapeutic agent for inflammatory diseases, whose pathogenesis involves TLR4 [1].

Western Blot Analysis[1]
Cell Types: PBMC Cell
Tested Concentrations: 1 nM, 10 nM, 100 nM
Incubation Duration: 15 min
Experimental Results: LPS-induced phosphorylation of mitogen-activated protein kinase was also inhibited in a concentration-dependent manner.

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Assay for Inhibitory Activity against NO Production. [2]
RAW264.7 cells were plated at 1 × 105 cells/well in 96-well culture plates and incubated overnight. After removing cell culture supernatants, cells were stimulated with 10 ng/mL LPS in the presence of various concentrations of test compounds for 20 h in stimulation medium (phenol red-free RPMI1640 containing 1% heat-inactivated FBS and 10 μg/mL kanamycin) at 37 °C in a humidified atmosphere of 5% CO2 in air. The test compounds were dissolved at 10 mmol/L in DMF, diluted with an RPMI-1640 medium to the appropriate concentrations, and added to the culture.
Production of NO was estimated by measuring the amount of nitrite, a stable metabolite of NO, according to a fluorometric method using 2,3-diaminonaphthalene. Briefly, 25 μL of 20 μg/mL DAN was added to 50 μL of the culture supernatant and incubated at room temperature for 10 min. After adding 25 μL of 0.5 N NaOH, a fluorescence at 460 nm (excitation wavelength:  355 nm) was measured.

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Assay for Inhibitory Activity against Cytokines Production. [2]
Concentrations of TNF-α, and IL-6 in the culture supernatants were determined using specific enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions.

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

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)

Solubility in Formulation 1: 5.5 mg/mL (15.20 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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: 5.5 mg/mL (15.20 mM) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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: 5 mg/mL (13.82 mM) in 10% DMSO + 90% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 4: ≥ 2.75 mg/mL (7.60 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 27.5 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 5: 2.75 mg/mL (7.60 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 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 6: ≥ 2.75 mg/mL (7.60 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 27.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 7: 2.5 mg/mL (6.91 mM) in 5% DMSO + 95% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 8: 0.5 mg/mL (1.38 mM) in 1% DMSO 99% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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