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-␣ (TNF-␣), and interleukin (IL)-6, with 50% inhibitory concentration (IC 50 ) of 1.1 to 11 nM. TAK-242 also suppressed the production of these cytokines from LPSstimulated human peripheral blood mononuclear cells (PBMCs) at IC 50 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-␣ induced by LPS and interferon-␥ 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-1␤-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.Cytokines and NO are involved in a variety of inflammatory diseases, including sepsis, rheumatoid arthritis (RA), atherosclerosis, inflammatory bowel disease (IBD), asthma, and chronic obstructive pulmonary disease. In RA, for example, interleukin (IL)-1 is considered to be a mediator. Plasma concentrations of IL-1 in patients with RA are elevated, and they correlate with disease activity (Eastgate et al., 1988). The expression of IL-1 in the bronchial epithelium of patients Article, publication date, and citation information can be found at

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To develop a new therapeutic agent for sepsis, screening of the Takeda chemical library was carried out using mouse macrophages stimulated with lipopolysaccharide (LPS) to identify a new class of small-molecule inhibitors of inflammatory mediator production. The lead compound 5a was discovered, from which a series of novel cyclohexene derivatives I bearing a sulfamoyl and ester group were designed, synthesized and tested for their inhibitory activity against nitric oxide (NO) production. Derivatives I were synthesized by the coupling of sulfonyl chlorides and anilines with concomitant double bond migration in the presence of triethylamine, and phenyl ring substitution and modification of the ester and cyclohexene moieties were carried out. Among the compounds synthesized, ethyl (6R)-6-[N-(2-chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate [(R)-(+)-5n, TAK-242] was found to exhibit the most potent suppressive activity for the production of not only NO but also inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) induced by LPS-stimulated mouse macrophages with IC50 values of 1.8, 1.9 and 1.3 nM, respectively. It shows marked beneficial effects in vivo also. Intravenous administration of (R)-(+)-5n at doses of 0.1 mg/kg or more suppressed the production of NO and various cytokines [TNF-alpha, IL-6 and IL-1beta] in the mouse endotoxin shock model. Furthermore, it protected mice from death dose-dependently and all mice survived at a dose of 3 mg/kg. The minimum effective dose to protect mice from lethality in this model was 0.3 mg/kg, which was consistent with those for inhibitory effects on the production of NO and cytokines. Compound (R)-(+)-5n is currently undergoing clinical trials for the treatment of sepsis.

We previously discovered an orally active human gonadotropin-releasing hormone (GnRH) receptor antagonist, thieno[2,3-d]pyrimidine-2,4-dione derivative 1 (sufugolix). To reduce the cytochrome P450 (CYP) inhibitory activity and improve in vivo GnRH antagonistic activity, further optimization of this scaffold was carried out. We focused our synthetic efforts on chemical modification at the 5 and 3 positions of the thieno[2,3-d]pyrimidine-2,4-dione ring based on computational modeling, which resulted in the discovery of 1-{4-[1-(2,6-difluorobenzyl)-5-[(dimethylamino)methyl]-3-(6-methoxypyridazin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl]phenyl}-3-methoxyurea (16b) as a highly potent and orally active GnRH antagonist. Compound 16b showed potent in vitro GnRH antagonistic activity in the presence of fetal bovine serum (FBS) without CYP inhibition. Oral administration of 16b maintained the suppressive effect of the plasma luteinizing hormone levels in castrated cynomolgus monkeys at a 3 mg/kg dose for more than 24 h. Compound 16b is currently under clinical development with the code name of TAK-385.