Discovery of [1,2,4]Triazolo[1,5-a]pyridine Derivatives as Potent and Orally Bioavailable RORγt Inverse Agonists.

pubs.acs.org/acsmedchemlett Letter Discovery of [1,2,4]Triazolo[1,5‑a]pyridine Derivatives as Potent and Orally Bioavailable RORγt Inverse Agonists Ryota Nakajima,* Hiroyuki Oono, Sakae Sugiyama, Yohei Matsueda, Tomohide Ida, Shinji Kakuda, Jun Hirata, Atsushi Baba, Akito Makino, Ryo Matsuyama, Ryan D. White, Ryan Ρ. Wurz, Youngsook Shin, Xiaoshan Min, Angel Guzman-Perez, Zhulun Wang, Antony Symons, Sanjay K. Singh, Srinivasa Reddy Mothe, Sergei Belyakov, Anjan Chakrabarti, and Satoshi Shuto* Cite This: ACS Med. Chem. Lett. 2020, 11, 528−534 ACCESS Metrics & More Read Online sı Supporting Information * Article Recommendations ABSTRACT: The retinoic acid receptor-related orphan nuclear receptor γt (RORγt), a promising therapeutic target, is a major transcription factor of genes related to psoriasis pathogenesis such as interleukin (IL)-17A, IL-22, and IL-23R. On the basis of the Xray cocrystal structure of RORγt with 1a, an analogue of the known piperazine RORγt inverse agonist 1, triazolopyridine derivatives of 1 were designed and synthesized, and analogue 3a was found to be a potent RORγt inverse agonist. Structure−activity relationship studies on 3a, focusing on the treatment of its metabolically unstable cyclopentyl ring and the central piperazine core, led to a novel analogue, namely, 6-methyl-N-(7-methyl-8-(((2S,4S)-2methyl-1-(4,4,4-trifluoro-3-(trifluoromethyl)butanoyl)piperidin-4-yl)oxy)[1,2,4]triazolo[1,5-a]pyridin-6-yl)nicotinamide (5a), which exhibited strong RORγt inhibitory activity and a favorable pharmacokinetic profile. Moreover, the in vitro and in vivo evaluation of 5a in a human whole-blood assay and a mouse IL-18/23-induced cytokine expression model revealed its robust and dose-dependent inhibitory effect on IL-17A production. KEYWORDS: Nuclear receptor, RORγt, triazolopyridine, inverse agonist T he retinoic acid receptor-related orphan nuclear receptor γt (RORγt) is a major transcription factor of genes related to psoriasis pathogenesis such as interleukin (IL)-17A, IL-22, and IL-23R.1,2 Therapies blocking IL-17A or IL-23R have successfully improved skin lesions in patients with moderate to severe psoriasis,3−7 thus rendering the RORγt inhibition a promising therapeutic target. After T0901317 was reported for its effective (albeit unselective) binding to RORγ,8 many RORγt antagonists (inverse agonists) have been developed,9−23 and several are already being clinically investigated as promising targets for the treatment of autoimmune diseases.24 Generally, nuclear receptors are proteins with highly conserved ligand binding domains (LBDs), which are structurally composed of α-helices that form a large lipophilic pocket responsible for binding small lipophilic ligands such as retinoid derivatives, fatty acids, cholesterol, and other lipophilic hormones and vitamins.25 Thus, one of the main challenges for drug delivery in this target class is the lipophilicity balance, which is required for strong LBD binding potency, while the metabolism associated with lipophilic small-molecule ligands should be minimized to afford favorable drug-like properties. On the basis of a previous report describing a series of piperazine RORγt ligands,26 compound 1 (Figure 1) was selected as a starting point for further investigation, mainly © 2020 American Chemical Society Figure 1. Representative RORγt ligand 1 used as a reference in the current study. because of its moderately low molecular weight (453) and lipophilicity (cLogD7.4 = 3.78). The optimization of 1 resulted in the new triazolopyridine derivative 3a (Table 1) that exhibited RORγt inverse agonist activity. Further optimization of 3a by modification of its metabolically unstable cyclopentyl ring and the central piperazine ring led to a new derivative, 6Received: December 24, 2019 Accepted: February 27, 2020 Published: February 27, 2020 528 https://dx.doi.org/10.1021/acsmedchemlett.9b00649 ACS Med. Chem. Lett. 2020, 11, 528−534 ACS Medicinal Chemistry Letters pubs.acs.org/acsmedchemlett forms a hydrogen bond with Phe378. Furthermore, a hydrogen bond was developed between the fluorobenzene ring and the hydroxyl group of Ser404, where a small space around the 4and 5-positions of the ring was detected (Figure 2B). Considering this additional space in the pocket, the phenyl ring of compound 1 could be replaced by a nitrogencontaining bicyclic ring to simultaneously decrease the overall lipophilicity and facilitate the hydrogen-bonding interactions with Ser404, thus allowing a more detailed investigation of this residue. The previous report by Hintermann and co-workers described a weakly lipophilic triazolopyridine derivative having an N-([1,2,4]triazolo[4,3-a]pyridin-6-yl)amide moiety, which displayed moderate inhibitory activity in the reporter gene assay and good liver microsomal stability.29 This triazolopyridine derivative demonstrated that a nitrogen-containing bicyclic ring was tolerable for the RORγt inhibitory activity, which led us into detailed investigation with other chemotypes of triazolopyridine analogues. To that end, two series of analogues bearing N-([1,2,4]triazolo[4,3-a]pyridin-7-yl)amide and N-([1,2,4]triazolo[1,5a]pyridin-6-yl)amide moieties were synthesized. Since it has been reported that RORγt is constitutively active in the absence of an endogenous ligand,30 all of the compounds were evaluated in a luciferase reporter gene assay without a control agonist ligand to assess their RORγt inverse agonist activity. Moreover, all of the synthesized triazolopyridine analogues were designed to achieve a lower lipophilicity than compound 1 (cLogD7.4 = 3.78). As outlined in Table 1, the cLogD7.4 values of the novel analogues were successfully decreased by approximately 1 unit relative to 1. However, the [1,2,4]triazolo[4,3-a]pyridine derivative 2a displayed reduced inhibitory activity in the reporter gene assay (IC50 = 590 nM), whereas the [1,2,4]triazolo[1,5-a]pyridine derivative 3a retained an excellent inhibitory activity (IC50 = 41 nM), comparable to that of compound 1, indicating that the nitrogen atoms in the [1,2,4]triazolo[1,5-a]pyridine ring were well-tolerated for the inhibition of the RORγt transcriptional activity. Furthermore, although 3a improved the human liver microsome (LM) stability (human CLint = 0.032 mL min−1 mg−1), the methyl-substituted triazolopyridine derivatives 2b and 3b exhibited lower in vitro activity, probably due to steric repulsions in the binding pocket. Moreover, the X-ray analysis results implied that only unsubstituted triazolopyridine rings are acceptable because the available space at the 4- and 5positions of the fluorobenzene ring of 1a is small. Therefore, compound 3a, which exhibited the most improved LM stability and high potency, was further optimized. To elucidate the PK profile of 3a, this compound was incubated in human hepatocytes, and its metabolites were explored by mass spectrometry (MS). During the MS analysis, no glutathione adducts were detected (...truncated)