Optimizing a weakly binding fragment into a potent RORγt inverse

Jul 10, 2018 - ... Eric Vangrevelinghe , Romain M Wolf , Klemens Kaupmann , Johannes Ottl , Janet Dawson King , Nigel G. Cooke , Klemens Hoegenauer ...
0 downloads 0 Views 6MB Size
Download PDF
Article Cite This: J. Med. Chem. 2018, 61, 6724−6735

pubs.acs.org/jmc

Optimizing a Weakly Binding Fragment into a Potent RORγt Inverse Agonist with Efficacy in an in Vivo Inflammation Model David A. Carcache,†,# Anna Vulpetti,*,†,# Joerg Kallen,§,# Henri Mattes,† David Orain,† Rowan Stringer,∥ Eric Vangrevelinghe,† Romain M. Wolf,† Klemens Kaupmann,‡ Johannes Ottl,§ Janet Dawson,‡ Nigel G. Cooke,† Klemens Hoegenauer,† Andreas Billich,‡ Juergen Wagner,† Christine Guntermann,‡ and Samuel Hintermann*,† Global Discovery Chemistry, §Chemical Biology & Therapeutics, ∥PK Sciences, and ‡Autoimmunity, Transplantation and Inflammation, Novartis Institutes for BioMedical Research, 4002 Basel, Switzerland

J. Med. Chem. 2018.61:6724-6735. Downloaded from pubs.acs.org by UNIV OF SOUTH DAKOTA on 08/12/18. For personal use only.

†

S Supporting Information *

ABSTRACT: The transcription factor RORγt is an attractive drug-target due to its role in the differentiation of IL-17 producing Th17 cells that play a critical role in the etiopathology of several autoimmune diseases. Identification of starting points for RORγt inverse agonists with good properties has been a challenge. We report the identification of a fragment hit and its conversion into a potent inverse agonist through fragment optimization, growing and merging efforts. Further analysis of the binding mode revealed that inverse agonism was achieved by an unusual mechanism. In contrast to other reported inverse agonists, there is no direct interaction or displacement of helix 12 observed in the crystal structure. Nevertheless, compound 9 proved to be efficacious in a delayed-type hypersensitivity (DTH) inflammation model in rats.

■

INTRODUCTION

During early hit-finding efforts to identify starting points for RORγt inhibitors, it became apparent that most of the hits had poor physicochemical properties. This may not be surprising considering the rather large and hydrophobic ligand binding pocket (LBP) of RORγt. However, there are also regions of the LBP where polar interactions are possible, such as the “sulfate pocket”.16 Our aim was to screen for fragments targeting these more favorable regions. Here, we report the identification of a weak binder from a biophysical screen and describe how this hit was converted into a potent RORγt inverse agonist with an unusual indirect mode of action. This tool compound was tested in a model of delayed-type hypersensitivity in rats (proof-of-concept study) and demonstrated efficacy, thus confirming the functional activity of this novel binding mode.

The T helper 17 (Th17) cell pathway has been strongly linked to autoimmunity; indeed, therapeutic interventions with inhibitors of different components of the pathway such as IL-17A, IL-17RA, and IL-23 have shown beneficial effects in multiple autoimmune diseases. Because of its role in the differentiation of Th17 cells, the RORγ2 (also called RORγt) isoform of the nuclear hormone receptor retinoic acid receptor-related orphan receptor C (RORC) is an additional attractive drug-target.1−6 Besides thymus- and T-cell-specific RORγt, the widely expressed RORCvar1 (RORγ1) is also known. Because of the structural identity of the ligand-binding domains of RORγ1 and RORγt, low-molecular weight inhibitors will inevitably target both isoforms. Many RORγt inhibitors have been described covering a large diversity of chemical structures.7−11 For some, inhibition of inflammation in in vivo rodent models of autoimmune disease has been reported.2−5,12−14 A number of compounds such as VTP43742,11 JNJ-3534, AZD-0284, and JTE-451 have advanced into clinical studies. For the most advanced candidate VTP43742, Vitae Pharmaceuticals reported the successful outcome of a four-week proof-of-concept trial in psoriasis patients, but recently, clinical development of the compound was stopped.15 © 2018 American Chemical Society

■

RESULTS AND DISCUSSION

Fragment-Based Screening. To complement classical high-throughput biochemical and cellular screening approaches, a series of fragment-based screening (FBS) Received: April 4, 2018 Published: July 10, 2018 6724

DOI: 10.1021/acs.jmedchem.8b00529 J. Med. Chem. 2018, 61, 6724−6735

Journal of Medicinal Chemistry

Article

Figure 1. Crystal structure of RORγt (Y264−K518) in complex with 1 and RIP140 peptide (depicted in violet) (PDB ID code: 6FZU).

Figure 2. Crystal structure of RORγt (Y264−K518) in complex with 2 (PDB ID code: 6G05) and RIP140 peptide (not shown). The binding pocket generated by SiteMap is shown as a gray surface. The “site points”, shown in white, cover a large region comprising various subpockets labeled in red. The hydrophobic and H-bond donor and acceptor maps are shown in yellow (−1.0 kcal/mol iso-contour), blue (−10 kcal/mol), and red (−10 kcal/mol), respectively.

and a RIP140 coactivator peptide1 (receptor-interacting protein 140), solved at 1.80 Å resolution, revealed that 1 sits in the middle of the large LBP well anchored by two hydrogen bonds (H-bonds) (Figure 1). It makes a direct H-bond with the backbone carbonyl of F377 as well as water-mediated Hbonds with the carbonyl backbone of H323 and the side-chain nitrogen of Q286. In addition the phenyl ring stacks nicely below F378 in an edge-to-face fashion. Finally, the chlorine substituent of 1 points toward the side-chain of C320 forming hydrophobic contacts. Based on the analysis of the crystal structure, we concluded that this fragment could provide an

campaigns were carried out to discover novel starting points for medicinal chemistry optimization. The most attractive hits discovered by screening a proprietary fragment library with differential static light scattering (DSLS) were tested in cocrystallization trials with RORγt in order to obtain structural information on their binding modes. Among the 13 hits successfully cocrystallized, phenyl-acetamide 1 with an IC50 value of 216 μM in an MS affinity assay was identified as a preferred starting point for structure-based design, despite its moderate LE (0.26) and LLE (0.23). The X-ray cocrystal structure of 1 bound to the RORγt (Y264−K518) construct 6725

DOI: 10.1021/acs.jmedchem.8b00529 J. Med. Chem. 2018, 61, 6724−6735

Journal of Medicinal Chemistry

Article

Scheme 1. Medicinal Chemistry Strategy Towards the Identification of the Potent RORγt Inverse Agonist 9

moieties filling this region. The side chains of the hydrophobic residues lining the “hydrophobic hot spot” region are F388, I397, I400, and F401. The “business end” region points toward helices 11 and 12. This portion of RORγt is considered more flexible, as revealed by various crystal structures with agonists, antagonists, and inverse agonists. All the fragments solved by crystal structure determination were bound to an agonist conformation of the RORγt (Y264− K518) construct with the RIP140 peptide bound as exemplified for ligand 2. Medicinal Chemistry Strategy. Based on our understanding of the molecular mechanism for RORγt inverse agonism, which we have recently reported,16 it was anticipated that fragment 1, which binds into the central region of the RORγt binding cavity, would be devoid of any functional activity. It was recognized early on that growing the fragment further toward the “business end” from the ethoxy substituent of 1 would be a preferred strategy to achieve RORγt inverse agonism. The aim of our initial medicinal chemistry strategy was to determine the SAR of the ethoxy substituent, explore the feasibility of attaching larger substituents to occupy the “business end”, and demonstrate functional activity (Scheme 1). The cyclopropoxy analog 3 resulted in a moderate 3-fold improvement of the binding affinity for RORγt, whereas the isopropoxy compound 4 displayed a greater than 10-fold improvement in binding affinity compared to 1 (Table 1). The trifluoroisopropoxy substituent in compound 5 was shown to further improve the binding affinity, likely due to an enhanced interaction with the “hydrophobic hot spot” region, but this was achieved at the expense of ligand efficiency and was not pursued further. Encouraged by these initial results, the benzyl ether 6 was designed with the aim of extending to the “business end” and achieving functional activity. Both enantiomers were prepared, and compounds 6 and 7 showed comparable binding affinity for RORγt in the MS reporter assay, while only the (S)-enantiomer 6 showed the first hints of inverse agonism in a FRET assay (Table 2). Having achieved functional activity by growing the fragment toward the “business end”, we decided to use only the functional assay for the further optimization. The affinity MS reporter assay was geared toward a very robust and direct quantification of compound binding to the target pocket in an affinity window of 0.7 μM to 2 mM, whereas the functional

attractive starting point for fragment growing and merging approaches toward other regions of the large RORγt binding cavity. These regions are referred as the “polar end region” (or “sulfate pocket”), the “unexplored back pocket”, the “hydrophobic hot spot”, and the “business end” as reported previously.16 In Silico Active Site Analysis. A short description and visualization of the structural characteristics of each of these subpockets is provided in this section, along with the SiteMap analysis of compound 217 (Figure 2) for which we had solved the X-ray structure earlier. The Schrödinger SiteMap tool18 provides a wealth of information in the form of computed properties and graphical contour maps, distinguishing regions in a binding pocket that are favorable for occupancy by hydrophobic, H-bond donor and H-bond acceptor ligand groups. The hydrophobic (depicted in yellow), donor (blue), and acceptor (red) maps for the structure are shown in Figure 2. The pocket is almost completely enclosed and mainly hydrophobic. This has been recognized as a challenge to balance potency and favorable physicochemical properties in a single ligand. Opportunities for polar contacts are found in the “polar end region”, in the region underneath the β-sheet portion, and at the bottom of the “unexplored back-pocket”. The “polar end region” is lined by the R367 side chain and the backbone-NH of L287. These groups interact favorably with the SO2 motif of ligand 2. The β-sheet portion of the protein closes the pocket from the top and projects the backbone carbonyl of F377 and the backbone NH of E379 into it (not shown in Figure 1). The backbone carbonyl of F377 makes an H-bond with the NH of the amide of 2, while the CO of the amide makes a water-mediated interaction with the side-chain of Q286 and with the backbone-CO of H323. We called the back-pocket shown in Figure 2 “unexplored” because there were no ligand or fragment bound crystal structures available at that time occupying this region. Later, 1,3-dihydro-2,1,3benzothiadiazole 2,2-dioxide inverse agonists were described that occupy parts of the same subpocket close to Ser404 with the side-chain of M365 adopting a different orientation.19 This subpocket is surrounded by the side chains of hydrophobic amino acids: L362, M365, V376, and I400. However, the most buried region of this pocket is polar due to the presence of the S404 side chain. The “hydrophobic hot spot” region was labeled as such due to the yellow hydrophobic iso-contour map (visible at −1.0 kcal/mol) but also based on the observation that most of the cocrystallized fragments present hydrophobic 6726

DOI: 10.1021/acs.jmedchem.8b00529 J. Med. Chem. 2018, 61, 6724−6735

Journal of Medicinal Chemistry

Article

which occupies the “polar end subpocket” with a large substituent. By overlaying the crystal structure of 2 with the crystal structure of fragment 1, both in complex with RORγt, the N-acetyl moiety in both compounds perfectly overlaps. The NH acts as H-bond donor to the carbonyl backbone of F377, while the acetyl CO accepts a water-mediated interaction with H323 and Q286 (Figure 3). The “polar

Table 1. Fragment Growing Towards the “Business End”

a

Binding assay: Values were calculated from two replicates (8-point dose response). See Supporting Information for assay details.

Table 2. Fragment Merging Towards the Potent RORγt Inverse Agonist 9

Figure 3. Overlay of the crystal structure of RORγt (Y264−K518) in complex with 1 (green) and 2 (white) and RIP140 peptide (not shown). The LBP is represented as cavity (atom-based colored). The coordinates for 1 and 2 have been deposited in the PDB databank (PDB ID codes: 6FZU, 6G05).

end” region offers many productive interactions with the sulfone moiety, and increased affinity was expected by growing into this region. The SO2 group of 2 makes H-bond interactions with both the side-chain of R367 and the backbone NH of L287. This merging strategy was successful and led to the identification of compound 9 (Scheme 1) as a breakthrough RORγt inverse agonist with subnanomolar potency in the FRET assay. To better understand the influence of the configuration at the benzylic position on potency, the (R)enantiomer 10 was also prepared and showed a 45-fold lower potency compared to the (S)-enantiomer, suggesting that the absolute configuration of the benzylic substituent is critical for RORγt potency. The corresponding des-methyl analog 11 turned out to be a weaker inverse agonist, confirming the importance of occupying the so-called “hydrophobic hot spot” (Figure 2). Chemistry. A three-step synthetic sequence was developed for the preparation of compounds described here. This sequence relied on a key nucleophilic aromatic substitution (SNAr). As shown in Scheme 2, starting from commercially available 4-halogeno-nitroarenes (12 and 13), the 4-halogeno group was displaced with an alcohol nucleophile, generated with sodium or potassium hydride, to form the etherintermediates. Béchamp reduction of the nitro group with iron provided the corresponding anilines and 3-aminopyridines (14−17). These aromatic amines were then treated with acetyl chloride to obtain the N-acetyl analogs (3 and 5−8) or coupled with the corresponding aryl acetic acids to give the final compounds 9−11, 18, and 19. Analyzing the Mode of Inhibition of Compound 9. A cocrystal structure of 9 bound to the RORγt (Y264−K518) construct, solved at 1.90 Å resolution, revealed a surprising orientation for the benzyl ether side chain (Figure 4). Instead of occupying the “business-end region”, as assumed in our original design, the side chain is located in the “unexplored

a

Functional TR FRET assay: Values were calculated from at least two independent measurements (8-point dose response).25

assay was designed to assess compounds with high affinities (10000 nM IC50 = 4113 nM; 54% inhibition IC50 = 17 nM IC50 = 23 nM IC50 = 110 nM IC50 = 255 nM

related transcription factors RORα,β and against other nuclear hormone receptors, we examined the agonistic and antagonistic effects in cellular Gal4 reporter gene assays or biochemical TR-FRET assays. Compound 9 did not show any activity against FXR, AR, ER, PR, PXR, GR, PPAR, LXRα/ β, RXR (data not shown), and the related nuclear receptor RORα (Table 4). The compound was only weakly active against RORβ, resulting in partial inhibition in the micromolar range (Table 4), demonstrating that 9 is highly selective. Compound 9 is highly lipophilic, poorly soluble at neutral pH, and highly permeable across PAMPA and Caco-2 monolayers (Table 5). Intrinsic clearance values in hepatic microsomes ranged between 31 to 48 μL·min−1·mg−1, the molecule was stable after a 2 h incubation in either rat plasma or rat intestinal and hepatic S9 fractions. Compound 9 is >99% plasma protein bound in rat and mouse plasma. A Table 5. In Vitro Profiling Data for 9 LogD(7.4) equilibrium solubility (pH 6.8) LogPAMPA Caco-2 Papp A-B/Papp B-A (× 10−6 cm/s) liver microsomes (rat/human/mouse) CLint (μL·min−1·mg−1) plasma protein binding (mouse/rat) (%) 6730

>5.7 0.5 mg/L; 1 μM −4.2 cm·s−1 7.5/4.7 48/31/46 >99/>99 DOI: 10.1021/acs.jmedchem.8b00529 J. Med. Chem. 2018, 61, 6724−6735

Journal of Medicinal Chemistry

Article

pharmacokinetic evaluation of compound 9 was undertaken in male Sprague−Dawley rats (1 mg/kg i.v.; 3 mg/kg by oral gavage) yielding an i.v. half-life of 1.3 h, blood clearance of 76 mL/min/kg, and a volume of distribution of 5.3 L/kg. After oral administration of a crystalline suspension, the maximum concentration in plasma (Cmax = 36 nM) was reached after 2.7 h and oral bioavailability was 10%. Based on this encouraging profile, 9 was selected for an in vivo proof-of-concept study. We wanted to confirm that this novel binding mode and mechanism of action would indeed lead to the desired pharmacodynamic activity in a mechanistic model before embarking on further optimization of physicochemical properties. We decided to test the compound at a rather high dose of 50 mg/kg with b.i.d. dosing. This dose was selected based on past experience with the goal to achieve the maximal possible efficacy. Pharmacology. Based on our experience with RORγt inverse agonist pharmacology, we decided to profile 9 in a DTH model in rats using an immunization/challenge protocol. Lewis rats were immunized with methylated bovine serum albumin (mBSA) as the antigen in the presence of complete Freund’s adjuvant (CFA). Two weeks later, the DTH response was elicited by a single injection of mBSA into the right ear and swelling was monitored for 48 h. Immediately prior to the antigen challenge, compound 9 was dosed by oral gavage at 50 mg/kg twice daily throughout the study. To determine the involvement of IL-17A in this DTH model, we neutralized this cytokine by a single injection of an anti-IL-17A antibody (10 mg/kg) before the mBSA antigen challenge. Blockade of IL17A by the antibody inhibited ear swelling by approximately 50% compared to control animals. Treatment of rats with the RORγt inhibitor 9 resulted in a significant reduction of the ear swelling score and was comparable to the IL-17A antibody treated group. The inhibitory effect was already evident after 6 h and persisted until the end of the study (Figure 7A). To assess the effect of RORγt inhibition on proinflammatory cytokine levels, inhibition of IL-6 cytokine expression in ear punch homogenates was assessed. Compound 9 treatment led to an approximately 50% reduction of IL-6 production at the end of the DTH experiment (Figure 7B), whereas treatment with the anti-IL-17 antibody led to a slightly higher degree of inhibition of IL-6 expression. Compound 9 reduced the frequencies of IL-17A producing cells in ex vivo mBSA recall assays using auricular draining lymph node cells compared to untreated controls (up to 30%); however, statistical significance was not reached most likely due to the high variability in the control and a “nonresponder” in the treatment group, respectively (Figure 7C, p = 0.0625, unpaired t test). Exposures of 9 at the end of the experiment (2 h after last dose) reached 1237 ± 598 nM in blood and 2305 ± 1251 nM in lymph nodes. The rather high interanimal variability might be due to suboptimal physicochemical properties (e.g., the poor solubility), which might influence oral absorption.

Figure 7. (A) Compound 9 attenuates mBSA-induced DTH responses in female Lewis rats. Rats were immunized with mBSA/ CFA, and 2 weeks later, the right ear of the animals was challenged with mBSA in 5% glucose, while the left ear was injected with vehicle (5% glucose). Starting just prior to antigen challenge, 9 was administered twice daily via oral gavage at 50 mg/kg for 2 days, and ear swelling was monitored. As an active comparator, a single bolus injection of an anti-IL-17 antibody (10 mg/kg) prior to antigen challenge was used. The mean ± SEM of the swelling ratios between antigen-challenged and vehicle injected ears are shown (n = 5). (B) Inhibition of IL-6 production in ear homogenates after treatment with compound 9 or with an anti-IL-17 antibody. All groups, n = 5. **P < 0.01 Dunnett’s test. (C) Draining lymph node cells were prepared and stimulated ex vivo with mBSA for 24 h, and frequencies of IL17producing cells were determined by ELISpot. Individual data and mean ± SEM are depicted (n = 4−5). Data is representative from two independent studies.

helix 12 as hypothesized. Instead, the compound binds into the “unexplored back pocket” and maintains the “Tyr-His lock” (hydrogen bond between NE2−H479 and OH−Y502), which typically characterizes the agonist conformation. The hydrogen bond is present in all four chains observed in the crystal structure ranging from 2.8 to 3.3 Å. The crystal structure of 9 and the water solvation analysis are consistent with the hypothesis that the mechanism of RORγt inverse agonism might be due to trapped (“unstable”, “displaceable”) water molecules in a hydrophobic environment at the “business end”, which can be released into bulk solvent when Y502 in helix 12 moves away from its agonist position (Figure S2). Without 9 bound, these water molecules are free to exchange with bulk solvent via the opening between Q286 and H323 (i.e., helix 12 and Y502 do not have to move away, see Figure 2 and pathway (a) in Figure 5). Interestingly, we have observed such trapped “unstable” water molecules for different chemical series of inverse agonists (not necessarily with a moiety in the back pocket), providing an indirect destabilization of helix 12.16 For these “nonsteric clash inverse agonists”, cocrystallization with a coactivator peptide and helix 12 in agonist position is still possible. Different protein conformations in solution with respect to those observed by crystal structure could support the hypothesized destabilization of helix 12, a potential novel functional mechanism of RORγt inverse agonism. Another

■

CONCLUSION In summary, we described the identification of the RORγt inverse agonist 9 derived from a weakly binding fragment hit. Subsequent fragment growing and merging efforts led to a highly potent inverse agonist, efficiently blocking IL-17 production in primary cells and in a human whole blood assay. Surprisingly, when compound 9 was cocrystallized with RORγt, the benzylic substituent did not sterically clash with 6731

DOI: 10.1021/acs.jmedchem.8b00529 J. Med. Chem. 2018, 61, 6724−6735

Journal of Medicinal Chemistry

Article

Synthesis. N-(3-Chloro-4-cyclopropoxyphenyl)acetamide (3). Prepared according to the general procedures described for 6. 2-Chloro-1-cyclopropoxy-4-nitrobenzene. This was prepared from 2-chloro-1-fluoro-4-nitrobenzene and cyclopropanol (31% yield). 1H NMR (600 MHz, DMSO-d6) δ 8.33 (d, J = 2.8 Hz, 1H), 8.29 (dd, J = 2.8 Hz, 1H), 7.65 (d, J = 9.1 Hz, 1H), 4.19−4.15 (m, 1H), 0.96−0.89 (m, 2H), 0.82−0.78 (m, 2H). MS (ESI): no ionization peak found. UPLC purity: 100%. 3-Chloro-4-cyclopropoxyaniline. This was prepared from 2chloro-1-cyclopropoxy-4-nitrobenzene (71% yield). 1H NMR (600 MHz, DMSO-d6) δ 7.08 (d, J = 8.7 Hz, 1H), 6.62 (d, J = 2.7 Hz, 1H), 6.51 (dd, J = 8.7, 2.7 Hz, 1H), 4.92 (s, 2H), 3.77 (tt, J = 6.1, 3.0 Hz, 1H), 0.75−0.60 (m, 4H). MS (ESI): [M + H]+ m/z 184.2. UPLC purity: 99%. N-(3-Chloro-4-cyclopropoxyphenyl)acetamide (3). This was prepared from 3-chloro-4-cyclopropoxyaniline and AcCl (54% yield). 1H NMR (600 MHz, DMSO-d6) δ 9.96 (s, 1H), 7.77 (s, 1H), 7.44−7.33 (m, 2H), 3.90 (s, 1H), 2.02 (s, 2H), 0.86−0.76 (m, 2H), 0.74−0.62 (m, 2H). MS (ESI): [M + H]+ m/z 226.3. UPLC purity: 95%. N-(3-Chloro-4-((1,1,1-trifluoropropan-2-yl)oxy)phenyl)acetamide (5). Prepared according to the general procedures described for 6. 2-Chloro-4-nitro-1-((1,1,1-trifluoropropan-2-yl)oxy)benzene. This was prepared from 2-chloro-1-fluoro-4-nitrobenzene and 1,1,1trifluoropropan-2-ol (quantitative yield). 1H NMR (600 MHz, DMSO-d6) δ 8.39 (d, J = 2.7 Hz, 1H), 8.27 (dt, J = 9.3, 2.7 Hz, 1H), 7.64 (dd, J = 9.3, 2.1 Hz, 1H), 5.67−5.59 (m, 1H), 1.54−1.49 (m, 3H). MS (ESI): no ionization peak found. UPLC purity: 95%. 3-Chloro-4-((1,1,1-trifluoropropan-2-yl)oxy)aniline. This was prepared from 2-chloro-4-nitro-1-((1,1,1-trifluoropropan-2-yl)oxy)benzene (76% yield). 1H NMR (600 MHz, DMSO-d6) δ 6.98 (d, J = 8.62 Hz, 1 H) 6.63 (s, 1 H) 6.48 (d, J = 8.80 Hz, 1 H) 5.13 (br. s., 2 H) 4.72−4.86 (m, 1 H) 1.38 (d, J = 6.42 Hz, 3 H). MS (ESI): [M + H]+ m/z 240.1. UPLC purity: > 95%. N-(3-Chloro-4-((1,1,1-trifluoropropan-2-yl)oxy)phenyl)acetamide (5). This was prepared from 3-chloro-4-((1,1,1-trifluoropropan-2-yl)oxy)aniline and AcCl (21% yield).). 1H NMR (600 MHz, DMSO-d6) δ 10.02 (s, 1H), 7.80 (d, J = 2.5 Hz, 1H), 7.41 (dd, J = 9.0, 2.6 Hz, 1H), 7.29 (d, J = 9.0 Hz, 1H), 5.14 (hept, J = 6.5 Hz, 1H), 2.02 (s, 3H), 1.43 (d, J = 6.4 Hz, 3H). MS (ESI): [M + H]+ m/z 282.1. UPLC purity: 99%. Prototypical Procedure 1. (S)-N-(3-Chloro-4-(1-phenylethoxy)phenyl)acetamide (6). (S)-2-Chloro-4-nitro-1-(1-phenylethoxy)benzene. A suspension of KH (30% in oil, 457 mg, 3.42 mmol) in anhydrous THF (15 mL) under nitrogen was treated at 0 °C with (S)-1-phenylethanol (413 μL, 3.42 mmol) and stirred at 0 °C for 10 min, then 2-chloro-1-fluoro-4-nitrobenzene (12, 400 mg, 2.28 mmol) was added in one portion, and the dark mixture was stirred at room temperature for 1 h. Water and DCM were added to the mixture, and the aqueous phase was extracted three times with DCM. The combined organic phases were then dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by flash chromatography (SiO2, heptane/AcOEt) to afford the title compound as a yellow solid (96 mg, 14% yield). 1H NMR (600 MHz, DMSO-d6) δ 8.32 (d, J = 2.8 Hz, 1H), 8.12 (dd, J = 9.2, 2.8 Hz, 1H), 7.45−7.42 (m, 2H), 7.41−7.37 (m, 2H), 7.32 (t, J = 1.4 Hz, 1H), 7.28 (d, J = 9.3 Hz, 1H), 5.88 (q, J = 6.4 Hz, 1H), 1.65 (d, J = 6.3 Hz, 3H). MS (ESI): no ionization peak found. UPLC purity: 100%. (S)-3-Chloro-4-(1-phenylethoxy)aniline. A solution of (S)-2chloro-4-nitro-1-(1-phenylethoxy)benzene (95 mg, 0.34 mmol) in THF (2.0 mL) was treated with Fe (76 mg, 1.37 mmol) and H2O (92 μL, 5.13 mmol) at room temperature. AcOH (196 μL, 3.42 mmol) was then added dropwise, and the mixture was heated to 60 °C for 1 h. The mixture was quenched by adding a 10% aqueous solution of NaHCO3 and then allowed to cool to room temperature and extracted twice with AcOEt. The combined organic phases were dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by flash chromatography (SiO2, heptane/AcOEt) to afford the title compound as a yellow oil (60 mg, 67% yield). 1H

hypothesis to explain the inverse agonism mechanism of 9 relies on a possible cross-talk of helix 7 (lining the “back pocket”) and helix 11 (bearing the H479 residue), which are in direct contact (Figure S3). It might be conceivable to think that the binding of 9, even if far from the “business end”, could impact the dynamics of the protein in that region of the LBP and thereby its function. Thus, the helix 11 flexibility could be influenced by the binding of the ligand in the “unexplored back pocket” and indirectly lead to different protein conformations in solution where the H479/Y502 H-bond is either weakened or not present. In addition to crystal structure elucidation, binding of compound 9 was further characterized by its kinetics and thermodynamic features. The enclosure of the pocket is very likely responsible for the slow on and off kinetics measured for compound 9, suggesting that protein conformational changes have to occur upon ligand association and dissociation. Minimal structural modification (removal of one methylgroup) showed substantial effects on the kinetics, which was reproduced by molecular dynamics simulations. ITC measurement showed that the binding of 9 leads to a rigidified complex characterized by an entropy loss, which is overcompensated by a large enthalpy gain consistent with several hydrogen bond interactions (e.g., in the “polar end” pocket). Finally, we have shown that compound 9 efficiently attenuated the in vivo ear swelling response in an mBSA-induced DTH model in rat. Likewise, IL-6 expression was reduced in ear homogenates, and the frequency of IL-17 producing cells was attenuated in an ex vivo antigen specific recall assay. Overall, the reported data clearly demonstrate that compound 9 is an attractive RORγt inhibitor with an unusual indirect mode of inhibition. Further exploration of how different back pocket substituents influence the physicochemical properties is not completed yet, and results will be reported in due course. Since the environment in the back pocket is more polar in RORγt (S404) compared to RORα and RORβ (where the corresponding amino acids are glycine and alanine, respectively), we expect that adding polarity in this region will be tolerated. This would lower the high lipophilicity of the series and should help to identify analogs with improved solubility and increased metabolic stability.

■

EXPERIMENTAL SECTION

General Experimental Information. Chemicals were purchased from commercial sources and were used without further purification. Reactions were magnetically and mechanically stirred and monitored by thin-layer chromatography (TLC) on Merck silica gel 60 F254 glass plates (visualized by UV fluorescence at l = 254 nm) or analytical Waters Acquity UPLC instrument equipped with PDA detector, Waters Acquity SQD mass spectrometer, and Waters Acquity HSS T3 1.8 mm2.1 V50 mm column. Peak detection is reported at full scan 210−450 nm eluting with a gradient composed of water/0.05% formic acid/3.75 mm ammonium formate and acetonitrile/0.05% formic acid. Mass spectrometry results are reported as the ratio of mass over charge. The purity of new compounds was >95%, as determined by 1H NMR and LC−MS after chromatography. Flash column chromatography was performed with Biotage SNAP silica gel cartridges, eluting with distilled technicalgrade solvents on an Isolera Four apparatus from Biotage. NMR data were recorded on a Bruker spectrometer operating at 400 or 600 MHz. Chemical shifts (d) are reported in ppm. The data are reported as s = singlet, d = doublet, t = triplet, q = quadruplet, m = multiplet, and bs = broad singlet. Compounds 1, 4, and 20 are commercially available. 6732

DOI: 10.1021/acs.jmedchem.8b00529 J. Med. Chem. 2018, 61, 6724−6735

Journal of Medicinal Chemistry

Article

NMR (600 MHz, DMSO-d6) δ 7.42−7.37 (m, 2H), 7.36−7.32 (m, 2H), 7.28−7.24 (m, 1H), 6.71 (d, J = 8.8 Hz, 1H), 6.59 (d, J = 2.7 Hz, 1H), 6.34 (dd, J = 8.8, 2.7 Hz, 1H), 5.29 (q, J = 6.4 Hz, 1H), 4.89 (s, 2H), 1.51 (d, J = 6.4 Hz, 3H). MS (ESI): [M + H]+ m/z 248.3. UPLC purity: 98%. (S)-N-(3-Chloro-4-(1-phenylethoxy)phenyl)acetamide (6). A solution of (S)-3-chloro-4-(1-phenylethoxy)aniline (60 mg, 0.24 mmol) and Et3N (101 μL, 0.73 mmol) in anhydrous DCM (3 mL) was treated with AcCl (21 μL, 0.29 mmol) at room temperature. The mixture was stirred for 1 h and then quenched by adding a 10% aqueous solution of NaHCO3. The aqueous phase was extracted three times with DCM, and the combined organic phases were dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by flash chromatography (SiO2, heptane/AcOEt) to afford the title compound as a yellow oil (13 mg, 18% yield). 1H NMR (600 MHz, DMSO-d6) δ 9.89 (s, 1H), 7.74 (d, J = 2.6 Hz, 1H), 7.40 (dd, J = 8.1, 1.5 Hz, 2H), 7.38−7.33 (m, 2H), 7.29−7.25 (m, 1H), 7.22 (dd, J = 9.0, 2.6 Hz, 1H), 6.99 (d, J = 9.0 Hz, 1H), 5.54 (q, J = 6.4 Hz, 1H), 1.99 (s, 3H), 1.56 (d, J = 6.4 Hz, 3H). MS (ESI): [M + H]+ m/ z 290.1. UPLC purity: 100%. [α]29D +30.0 (c 1.0, DMSO). (R)-N-(3-Chloro-4-(1-phenylethoxy)phenyl)acetamide (7). Prepared according to the general procedures described for 6. (R)-2-Chloro-4-nitro-1-(1-phenylethoxy)benzene. This was prepared from 2-chloro-1-fluoro-4-nitrobenzene and (R)-1-phenylethanol (44% yield). 1H NMR (600 MHz, DMSO-d6) δ 8.32 (d, J = 2.8 Hz, 1H), 8.12 (dd, J = 9.2, 2.8 Hz, 1H), 7.47−7.42 (m, 2H), 7.42− 7.37 (m, 2H), 7.33−7.30 (m, 1H), 7.28 (d, J = 9.3 Hz, 1H), 5.88 (q, J = 6.4 Hz, 1H), 1.65 (d, J = 6.4 Hz, 3H). MS (ESI): no ionization peak found. UPLC purity: 96%. (R)-3-Chloro-4-(1-phenylethoxy)aniline. This was prepared from (R)-2-chloro-4-nitro-1-(1-phenylethoxy)benzene (52% yield). 1H NMR (600 MHz, DMSO-d6) δ 7.39 (dd, J = 8.2, 1.1 Hz, 2H), 7.34 (t, J = 7.6 Hz, 2H), 7.28−7.24 (m, 1H), 6.71 (d, J = 8.8 Hz, 1H), 6.59 (d, J = 2.7 Hz, 1H), 6.34 (dd, J = 8.7, 2.7 Hz, 1H), 5.29 (q, J = 6.4 Hz, 1H), 4.89 (s, 2H), 1.51 (d, J = 6.4 Hz, 3H). MS (ESI): [M + H]+ m/z 248.3. UPLC purity: 98%. (R)-N-(3-Chloro-4-(1-phenylethoxy)phenyl)acetamide (7). This was prepared from (R)-3-chloro-4-(1-phenylethoxy)aniline and AcCl (50% yield). 1H NMR (600 MHz, DMSO-d6) δ 9.89 (s, 1H), 7.75 (d, J = 2.6 Hz, 1H), 7.43−7.39 (m, 2H), 7.38−7.33 (m, 2H), 7.29−7.25 (m, 1H), 7.22 (dd, J = 9.0, 2.6 Hz, 1H), 6.99 (d, J = 9.1 Hz, 1H), 5.54 (q, J = 6.4 Hz, 1H), 1.99 (s, 3H), 1.56 (d, J = 6.3 Hz, 3H). MS (ESI): [M + H]+ m/z 290.1. UPLC purity: 100%. [α]28D −30.0 (c 1.0, DMSO). (S)-N-(5-Chloro-6-(1-phenylethoxy)pyridin-3-yl)acetamide (8). This was prepared from (S)-5-chloro-6-(1-phenylethoxy)pyridin-3amine and AcCl according to general procedure for the preparation of 6 (63% yield). 1H NMR (600 MHz, DMSO-d6) δ 10.11 (s, 1H), 8.18−8.14 (m, 2H), 7.44−7.40 (m, 2H), 7.38−7.33 (m, 2H), 7.30− 7.24 (m, 1H), 6.19 (q, J = 6.5 Hz, 1H), 2.03 (s, 3H), 1.58 (d, J = 6.5 Hz, 3H). MS (ESI): [M + H]+ m/z 291.3. UPLC purity: 98%. [α]28D −4.0 (c 1.0, DMSO). Prototypical Procedure 2. (S)-N-(5-Chloro-6-(1-phenylethoxy)pyridin-3-yl)-2-(4-(ethylsulfonyl)phenyl)acetamide (9). (S)-3Chloro-5-nitro-2-(1-phenylethoxy)pyridine. A suspension of NaH (60% in oil, 91 mg, 2.28 mmol) in anhydrous THF (10 mL) under argon was treated at 0 °C with (S)-1-phenylethanol (375 μL, 3.11 mmol) and 15-crown-5 (452 μL, 2.28 mmol). The mixture was stirred at 0 °C for 15 min, and 2,3-dichloro-5-nitropyridine (13, 400 mg, 2.07 mmol), dissolved in THF (1.5 mL), was added dropwise. The mixture was allowed to warm to room temperature and stirred for 2 h, then poured onto H2O, and the aqueous phase was extracted three times with DCM. The combined organic phases were then dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by flash chromatography (SiO2, heptane/AcOEt) to give the title compound (164 mg, 27% yield). 1H NMR (600 MHz, DMSOd6) δ 8.97 (d, J = 2.6 Hz, 1H), 8.72 (d, J = 2.6 Hz, 1H), 7.46−7.44 (m, 2H), 7.39−7.35 (m, 2H), 7.31−7.28 (m, 1H), 6.37 (q, J = 6.5 Hz, 1H), 1.65 (d, J = 6.5 Hz, 3H). MS (ESI): no ionization peak found. UPLC purity: 100%.

(S)-5-Chloro-6-(1-phenylethoxy)pyridin-3-amine (14). A solution of (S)-3-chloro-5-nitro-2-(1-phenylethoxy)pyridine (164 mg, 0.59 mmol) in THF (4 mL) was treated with Fe (131 mg, 2.35 mmol) and H2O (159 μL, 8.83 mmol) at room temperature. AcOH (337 μL, 5.88 mmol) was then added dropwise, and the mixture was heated to 60 °C for 1 h. The mixture was quenched by adding a 10% aqueous solution of NaHCO3 and then allowed to cool to room temperature and extracted twice with AcOEt. The combined organic phases were dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by flash chromatography (SiO2, heptane/ AcOEt) to give the title compound as a brown oil (100 mg, 65% yield). 1H NMR (600 MHz, DMSO-d6) δ 7.42−7.37 (m, 3H), 7.36− 7.30 (m, 2H), 7.25 (tt, J = 6.8, 1.3 Hz, 1H), 7.13 (d, J = 2.6 Hz, 1H), 6.04 (q, J = 6.5 Hz, 1H), 5.01 (s, 2H), 1.53 (d, J = 6.5 Hz, 3H). MS (ESI): [M + H]+ m/z 249.2. UPLC purity: 96%. (S)-N-(5-Chloro-6-(1-phenylethoxy)pyridin-3-yl)-2-(4(ethylsulfonyl)phenyl)acetamide (9). A solution of (S)-5-chloro-6(1-phenylethoxy)pyridin-3-amine (14, 5.4 g, 21.7 mmol) in DCM (300 mL) under Ar was treated with Et3N (9.1 mL, 65.1 mmol), TBTU (8.02 g, 25.0 mmol), and 2-(4-(ethylsulfonyl)phenyl)acetic acid (5.70 g, 25.0 mmol). The mixture was stirred for 2 h at room temperature, then poured onto H2O and extracted twice with DCM. The combined organic phases were dried over Na2SO4, filtered, and concentrated in vacuo. Purification by flash chromatography (SiO2, DCM/AcOEt, and heptane/AcOEt), followed by recrystallization in EtOH, gave the title compound (7.0 g, 70%) as a white solid. 1H NMR (600 MHz, DMSO-d6) δ 10.44 (s, 1H), 8.18 (dd, J = 16.0, 2.3 Hz, 2H), 7.84 (d, J = 8.0 Hz, 2H), 7.59 (d, J = 8.0 Hz, 2H), 7.42 (d, J = 7.6 Hz, 2H), 7.35 (t, J = 7.5 Hz, 2H), 7.27 (s, 1H), 6.19 (q, J = 6.5 Hz, 1H), 3.80 (s, 2H), 3.28 (d, J = 7.3 Hz, 2H), 1.59 (d, J = 6.5 Hz, 3H), 1.10 (t, J = 7.3 Hz, 3H). 13C NMR (150 MHz, DMSO-d6) δ 168.53, 153.62, 142.56, 141.69, 136.85, 135.62, 130.54, 130.48, 130.28 (2C), 128.41 (2C), 127.85 (2C), 127.45, 125.62 (2C), 116.51, 73.53, 49.17, 42.47, 22.96, 7.17. MS (ESI): [M + H]+ m/z 459.3. UPLC purity: 100%. [α]28D −2.0 (c 1.0, DMSO). Melting point: 141.7 °C. (R)-N-(5-Chloro-6-(1-phenylethoxy)pyridin-3-yl)-2-(4(ethylsulfonyl)phenyl)acetamide (10). Prepared according to the general procedures described for 9. (R)-3-Chloro-5-nitro-2-(1-phenylethoxy)pyridine. This was prepared from 2,3-dichloro-5-nitropyridine (13) and (R)-1-phenylethanol (41% yield). 1H NMR (600 MHz, DMSO-d6) δ 8.97 (d, J = 2.5 Hz, 1H), 8.72 (d, J = 2.6 Hz, 1H), 7.46−7.44 (m, 2H), 7.37 (dd, J = 8.4, 6.9 Hz, 2H), 7.31−7.28 (m, 1H), 6.36 (q, J = 6.6 Hz, 1H), 1.65 (d, J = 6.5 Hz, 3H). MS (ESI): no ionization peak found. UPLC purity: 100%. (R)-5-Chloro-6-(1-phenylethoxy)pyridin-3-amine (15). This was prepared from (R)-3-chloro-5-nitro-2-(1-phenylethoxy)pyridine (77% yield). 1H NMR (600 MHz, DMSO-d6) δ 7.39 (dd, J = 7.3, 1.6 Hz, 3H), 7.35−7.30 (m, 2H), 7.29−7.21 (m, 1H), 7.13 (d, J = 2.6 Hz, 1H), 6.04 (q, J = 6.5 Hz, 1H), 5.01 (s, 2H), 1.53 (d, J = 6.5 Hz, 3H). MS (ESI): [M + H]+ m/z 249.3. UPLC purity: 97%. (R)-N-(5-chloro-6-(1-phenylethoxy)pyridin-3-yl)-2-(4(ethylsulfonyl)phenyl)acetamide (10). This was prepared from (R)5-chloro-6-(1-phenylethoxy)pyridin-3-amine (15) and 2-(4(ethylsulfonyl)phenyl)acetic acid (79% yield). 1H NMR (600 MHz, DMSO-d6) δ 10.44 (s, 1H), 8.18 (dd, J = 15.2, 2.4 Hz, 2H), 7.87− 7.82 (m, 2H), 7.61−7.57 (m, 2H), 7.44−7.40 (m, 2H), 7.38−7.33 (m, 2H), 7.29−7.24 (m, 1H), 6.19 (q, J = 6.5 Hz, 1H), 3.80 (s, 2H), 3.28 (q, J = 7.3 Hz, 2H), 1.58 (d, J = 6.6 Hz, 3H), 1.10 (t, J = 7.4 Hz, 3H). MS (ESI): [M + H]+ m/z 459.4. UPLC purity: 98%. [α]28D +2.0 (c 1.0, DMSO). N-(6-(Benzyloxy)-5-chloropyridin-3-yl)-2-(4-(ethylsulfonyl)phenyl)acetamide (11). Prepared according to the general procedures described for 9. 2-(Benzyloxy)-3-chloro-5-nitropyridine. This was prepared from 2,3-dichloro-5-nitropyridine (13) and benzyl alcohol (68% yield). 1H NMR (600 MHz, DMSO-d6) δ 9.07 (d, J = 2.5 Hz, 1H), 8.76 (d, J = 2.5 Hz, 1H), 7.50 (d, J = 7.0 Hz, 2H), 7.42 (t, J = 7.5 Hz, 2H), 7.37 6733

DOI: 10.1021/acs.jmedchem.8b00529 J. Med. Chem. 2018, 61, 6724−6735

Journal of Medicinal Chemistry

Article

∼87% glycerin (Merck) + 8.75 mL of distilled water) was made up, and a cOmplete Mini protease inhibitor cocktail tablet (Roche) was added immediately before use, giving a final volume of 10 mL. An 8 mm punch biopsy was removed from the ear, weighed, minced into small pieces, and a 10-fold volume of lysis buffer added (w/v). The samples were homogenized in 0.5 mL OMNI tubes (OMNIInternational) containing garnet beads for 3 × 15 s in a Precellys 24 homogenizer (Bertin Instruments). The samples were then centrifuged at 13,000 rpm for 30 min at 4 °C, and the supernatant was frozen at −20 °C until further analysis. Ex Vivo Antigen Recall Study. The frequencies of mBSA-specific IL-17 producing cells were determined by ELISpot as previously described.25 Briefly, draining lymph node cells were seeded into 96well polyvinylidene difluoride plates (Millipore), precoated with antimouse IL-17 capture antibody (R&D Systems). Cells were restimulated with mBSA (200 μg/mL) and incubated for 24 h at 37 °C. After washing, the plates were incubated with biotinylated antimouse IL-17 detection antibody (R&D Systems) overnight at 4 °C, followed by incubation with streptavidin−alkaline phosphatase concentrate. The spots were developed with an ELISpot blue color module (R&D Systems) and counted with an ELISpot reader (AID). Quadruplicate wells were averaged, and results are presented as number of spots/106 cells.

(d, J = 7.3 Hz, 1H), 5.58 (s, 2H)). MS (ESI): no ionization peak found. UPLC purity: 98%. 6-(Benzyloxy)-5-chloropyridin-3-amine (16). This was prepared from 2-(benzyloxy)-3-chloro-5-nitropyridine (91% yield). 1H NMR (600 MHz, DMSO-d6) δ 7.48 (s, 1H), 7.43 (d, J = 7.5 Hz, 2H), 7.38 (t, J = 7.5 Hz, 2H), 7.31 (t, J = 7.3 Hz, 1H), 7.17 (d, J = 2.7 Hz, 1H), 5.28 (s, 2H), 5.06 (s, 2H). MS (ESI): [M + H]+ m/z 235.1. UPLC purity: 100%. N-(6-(Benzyloxy)-5-chloropyridin-3-yl)-2-(4-(ethylsulfonyl)phenyl)acetamide (11). This was prepared from 6-(benzyloxy)-5chloropyridin-3-amine (16) and 2-(4-(ethylsulfonyl)phenyl)acetic acid (48% yield). 1H NMR (600 MHz, DMSO-d6) δ 10.50 (s, 1H), 8.29 (d, J = 2.3 Hz, 1H), 8.20 (d, J = 2.3 Hz, 1H), 7.88−7.83 (m, 2H), 7.61 (d, J = 8.1 Hz, 2H), 7.46 (d, J = 7.5 Hz, 2H), 7.40 (t, J = 7.5 Hz, 2H), 7.34 (t, J = 7.3 Hz, 1H), 5.41 (s, 2H), 3.82 (s, 2H), 3.29 (q, J = 7.4 Hz, 2H), 1.10 (t, J = 7.4 Hz, 3H). MS (ESI): [M + H]+ m/z 445.2. UPLC purity: 98%. N-(5-Chloro-6-ethoxypyridin-3-yl)-2-(4-(ethylsulfonyl)phenyl)acetamide (18). Prepared according to the general procedures described for 9. 3-chloro-2-ethoxy-5-nitropyridine. This was prepared from 2,3dichloro-5-nitropyridine and EtOH (83% yield). 1H NMR (600 MHz, DMSO-d6) δ 9.04 (d, J = 2.5 Hz, 1H), 8.72 (d, J = 2.6 Hz, 1H), 4.55 (q, J = 7.0 Hz, 2H), 1.39 (t, J = 7.0 Hz, 3H). MS (ESI): no ionization peak found. UPLC purity: 97%. 5-Chloro-6-ethoxypyridin-3-amine (17). This was prepared from 3-chloro-2-ethoxy-5-nitropyridine (76% yield). 1H NMR (600 MHz, DMSO-d6) δ 7.45 (d, J = 2.5 Hz, 1H), 7.13 (d, J = 2.6 Hz, 1H), 4.98 (s, 2H), 4.21 (q, J = 7.0 Hz, 2H), 1.27 (t, J = 7.0 Hz, 3H). MS (ESI): [M + H]+ m/z 173.1. UPLC purity: 97%. N-(5-Chloro-6-ethoxypyridin-3-yl)-2-(4-(ethylsulfonyl)phenyl)acetamide (18). This was prepared from 5-chloro-6-ethoxypyridin-3amine (17) and 2-(4-(ethylsulfonyl)phenyl)acetic acid (18% yield). 1 H NMR (600 MHz, DMSO-d6) δ 10.44 (s, 1H), 8.24 (d, J = 2.4 Hz, 1H), 8.16 (d, J = 2.4 Hz, 1H), 7.87−7.82 (m, 2H), 7.62−7.57 (m, 2H), 4.34 (q, J = 7.0 Hz, 2H), 3.81 (s, 2H), 3.28 (q, J = 7.4 Hz, 2H), 1.32 (t, J = 7.0 Hz, 3H), 1.10 (t, J = 7.3 Hz, 3H). MS (ESI): [M + H]+ m/z 383.2. UPLC purity: 100%. (S)-N-(5-Chloro-6-(1-phenylethoxy)pyridin-3-yl)-2-(pyridin-4-yl)acetamide (19). This was prepared from (S)-5-chloro-6-(1phenylethoxy)pyridin-3-amine (14) and 2-(pyridin-4-yl)acetic acid according to the general procedure for the preparation of 9 (55% yield). 1H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 8.49 (d, J = 5.3 Hz, 2H), 8.25−8.01 (m, 2H), 7.50−7.14 (m, 7H), 6.17 (d, J = 6.5 Hz, 1H), 3.68 (s, 2H), 1.57 (d, J = 6.5 Hz, 3H). MS (ESI): [M + H]+ m/z 368.1. UPLC purity: 100%. [α]29D −3.0 (c 1.0, DMSO). In Vivo Pharmacology. DTH Response in Rat. Lewis rats (approximately 120 to 150 g at start of dosing) were purchased from Charles River Laboratories. Animals were housed in enriched environments with an approximate 12 h light/dark cycle, with food and water provided ad libitum. Animal housing and studies were performed in accordance with the Swiss Animal Welfare law, issued by the Kantonal Veterinary Office of Basel Stadt, Switzerland (license number BS-1244). Female Lewis rats were sensitized intradermally on the back with 500 μg of methylated BSA (mBSA; Sigma) homogenized 1:1 with complete Freund’s adjuvant (DIFCO). After 14 days, the right ear of each rat was challenged with 10 μL of mBSA (10 mg/mL in 5% glucose), whereas the left ears received 10 μL of the vehicle (5% glucose). Compound 9 was dosed twice daily per oral gavage (50 mg/ kg) starting just prior to mBSA challenge until the end of the study. The rat cross-reactive anti-IL-17 antibody BZN035 was administered subcutaneously at 10 mg/kg on the day before the mBSA challenge. Ear swelling was measured using digital calipers (Mitutoyo) and is expressed as a ratio of right (mBSA-injected) versus left (control) ear thickness. After 48 h postchallenge, draining auricular lymph nodes were harvested and used in ex vivo T-cell recall assays. Ear Tissue Homogenization. The homogenization lysis buffer (117 mg of NaCl + 18.6 mg of EDTA disodium dihydrate salt (Amresco) + 100 μL of Tris-HCl pH 7.5 (Invitrogen) + 1.15 mL of

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.8b00529. Supplementary Figures S1−S3; assay and method descriptions (PDF) Molecular formula strings (CSV) Accession Codes

PDB accession codes: 6FZU (1), 6G05 (2), 6G07 (9).

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: (+41) 795609961. *E-mail: [email protected]. Phone: (+41) 613241016. ORCID

Anna Vulpetti: 0000-0002-3114-8679 Klemens Hoegenauer: 0000-0001-7266-0137 Samuel Hintermann: 0000-0001-6853-6803 Author Contributions #

These authors contributed equally. The manuscript was written with contributions from all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare the following competing financial interest(s): All authors are current or former employees of Novartis and own shares or options of Novartis.

■

ACKNOWLEDGMENTS We thank C. Guibourdenche, M. Gunzenhauser, F. Gruber, R. Felber, R. Bösch, R. Brunner, C. Delmas, S. Kapps, E. Koch, A. Izaac, A. Moenaert, C. Be, J. Weiss, and F. Ecoeur for excellent technical assistance in compound synthesis, protein expression, purification, crystallization, ITC, DSLS, and for performing cellular assays. We are grateful to C. Betschart, U. Hommel, and K. Briner for helpful discussions. X-ray data collection was performed on the X10SA beamline at the Swiss Light Source, 6734

DOI: 10.1021/acs.jmedchem.8b00529 J. Med. Chem. 2018, 61, 6724−6735

Journal of Medicinal Chemistry

Article

H.; Nakayama, M.; Uga, K.; Shibata, A.; Koga, K.; Okui, T.; Shirasaki, M.; Skene, R.; Sang, B.-C.; Hoffman, I.; Lane, W.; Fujitani, Y.; Yamasaki, M.; Yamamoto, S. Discovery of orally efficacious RORγt inverse agonists. Part 1: identification of novel phenylglycinamides as lead scaffolds. Bioorg. Med. Chem. 2018, 26, 483−500. (14) Kono, M.; Oda, T.; Tawada, M.; Imada, T.; Banno, Y.; Taya, N.; Kawamoto, T.; Tokuhara, H.; Tomata, Y.; Ishii, N.; Ochida, A.; Fukase, Y.; Yukawa, T.; Fukumoto, S.; Watanabe, H.; Uga, K.; Shibata, A.; Nakagawa, H.; Shirasaki, M.; Fujitani, Y.; Yamasaki, M.; Shirai, J.; Yamamoto, S. Discovery of orally efficacious RORγt inverse agonists. Part 2: Design, synthesis, and biological evaluation of novel tetrahydroisoquinoline derivatives. Bioorg. Med. Chem. 2018, 26, 470− 482. (15) https://www.allergan.com/News/News/Thomson-Reuters/ Allergan-Reports-3-Increase-in-First-Quarter-2018 (last accessed 2018.05.31). (16) Kallen, J.; Izaac, A.; Be, C.; Arista, L.; Orain, D.; Kaupmann, K.; Guntermann, C.; Hoegenauer, K.; Hintermann, S. Structural states of RORγt: X-ray elucidation of molecular mechanisms and binding interactions for natural and synthetic compounds. ChemMedChem 2017, 12, 1014−1021. (17) Wang, Y.; Cai, W.; Zhang, G.; Yang, T.; Liu, Q.; Chen, Y.; Zhou, L.; Ma, Y.; Cheng, Z.; Lu, S.; Zhao, Y. G.; Zhang, W.; Xiang, Z.; Wang, S.; Yang, L.; Wu, Q.; Orband-Miller, L. A.; Xu, Y.; Zhang, J.; Gao, R.; Huxdorf, M.; Xiang, J. X.; Zhong, Z.; Elliott, J. D.; Lin, X. Discovery of novel N-(5-(arylcarbonyl)thiazol-2-yl)amides and N-(5(arylcarbonyl)thiophen-2-yl)amides as potent RORγt inhibitors. Bioorg. Med. Chem. 2014, 22, 692−702. (18) Halgren, T. A. Identifying and characterizing binding sites and assessing druggability. J. Chem. Inf. Model. 2009, 49, 377−389. (19) Muegge, I.; Collin, D.; Cook, B.; Hill-Drzewi, M.; Horan, J.; Kugler, S.; Labadia, M.; Li, X.; Smith, L.; Zhang, Y. Discovery of 1,3dihydro-2,1,3-benzothiadiazole 2,2-dioxide analogs as new RORC modulators. Bioorg. Med. Chem. Lett. 2015, 25, 1892−1895. (20) Kinetics measurement were done at Proteros biostructures GmbH according to methods described in Neumann, L.; von König, K.; Ullmann, D. HTS reporter displacement assay for fragment screening and fragment evolution toward leads with optimized binding kinetics, binding selectivity, and thermodynamic signature. Methods in Enzymology; Academic Press: Burlington, 2011; Vol. 493, pp 299−320. (21) Wolf, R. M. Extracting ligands from receptors by reversed targeted molecular dynamics. J. Comput.-Aided Mol. Des. 2015, 29, 1025−1034. (22) Genest, D.; Garnier, N.; Arrault, A.; Marot, C.; Morin-Allory, L.; Genest, M. Ligand-escape pathways from the ligand-binding domain of PPARγ receptor as probed by molecular dynamics simulations. Eur. Biophys. J. 2008, 37, 369−379. (23) Beglov, D.; Roux, B. Solvation of complex molecules in a polar liquid: An integral equation theory. J. Chem. Phys. 1996, 104, 8678− 8689. (24) Kovalenko, A.; Hirata, F. Three-dimensional density profiles of water in contact with a solute of arbitrary shape: a RISM approach. Chem. Phys. Lett. 1998, 290, 237. (25) Guendisch, U.; Weiss, J.; Ecoeur, F.; Riker, J. C.; Kaupmann, K.; Kallen, J.; Hintermann, S.; Orain, D.; Dawson, J.; Billich, A.; Guntermann, C. Pharmacological inhibition of RORγt suppresses the Th17 pathway and alleviates arthritis in vivo. PLoS One 2017, 12, e0188391. (26) Guntermann, C.; Piaia, A.; Hamel, M.-L.; Theil, D.; RubicSchneider, T.; del Rio-Espinola, A.; Dong, L.; Billich, A.; Kaupmann, K.; Dawson, J.; Hoegenauer, K.; Orain, D.; Hintermann, S.; Stringer, R.; Patel, D. D.; Doelemeyer, A.; Deurinck, M.; Schümann, J. Retinoic-acid-orphan-receptor-C inhibition suppresses Th17 cells and induces thymic aberrations. JCI insight 2017, 2, e91127.

Paul Scherrer Institute, Villigen, Switzerland. We thank L. Neumann and K. von König (Proteros biostructures GmbH) for the kinetic measurements.

■

ABBREVIATIONS USED DTH, delayed type hypersensitivity; MS, mass spectrometry; NMR, nuclear magnetic resonance; LC, liquid chromatography; LE, ligand efficiency; LLE, ligand-lipophilicity efficiency; TLC, thin layer chromatography

■

REFERENCES

(1) Jetten, A. M. Retinoid-related orphan receptors (RORs): critical roles in development, immunity, circadian rhythm, and cellular metabolism. Nucl. Recept. Signaling 2009, 7, 1−32. (2) Xiao, S.; Yosef, N.; Yang, J.; Wang, Y.; Zhou, L.; Zhu, C.; Wu, C.; Baloglu, E.; Schmidt, D.; Ramesh, R.; Lobera, M.; Sundrud, M. S.; Tsai, P.-Y.; Xiang, Z.; Wang, J.; Xu, Y.; Lin, X.; Kretschmer, K.; Rahl, P. B.; Young, R. A.; Zhong, Z.; Hafler, D. A.; Regev, A.; Ghosh, S.; Marson, A.; Kuchroo, V. K. Small-molecule RORγt antagonists inhibit T helper 17 cell transcriptional network by divergent mechanisms. Immunity 2014, 40, 477−489. (3) Skepner, J.; Ramesh, R.; Trocha, M.; Schmidt, D.; Baloglu, E.; Lobera, M.; Carlson, T.; Hill, J.; Orband-Miller, L. A.; Barnes, A.; Boudjelal, M.; Sundrud, M.; Ghosh, S.; Yang, J. Pharmacologic inhibition of RORγt regulates Th17 signature gene expression and suppresses cutaneous inflammation in vivo. J. Immunol. 2014, 192, 2564−2575. (4) Chang, M. R.; Lyda, B.; Kamenecka, T. M.; Griffin, P. R. Pharmacologic repression of retinoic acid receptor-related orphan nuclear receptor γ is therapeutic in the collagen-induced arthritis experimental model. Arthritis Rheumatol. 2014, 66, 579−588. (5) Banerjee, D.; Zhao, L.; Wu, L.; Palanichamy, A.; Ergun, A.; Peng, L.; Quigley, C.; Hamann, S.; Dunstan, R.; Cullen, P.; Allaire, N.; Guertin, K.; Wang, T.; Chao, J.; Loh, C.; Fontenot, J. D. Small molecule mediated inhibition of RORγ-dependent gene expression and autoimmune disease pathology in vivo. Immunology 2016, 147, 399−413. (6) Kojetin, D. J.; Burris, T. P. REV-ERB and ROR nuclear receptors as drug targets. Nat. Rev. Drug Discovery 2014, 13, 197−216. (7) Cyr, P.; Bronner, S. M.; Crawford, J. J. Recent progress on nuclear receptor RORγ modulators. Bioorg. Med. Chem. Lett. 2016, 26, 4387−4393. (8) Qiu, R.; Wang, Y. Retinoic acid receptor-related orphan receptor γt (RORγt) agonists as potential small molecule therapeutics for cancer immunotherapy. J. Med. Chem. 2018, DOI: 10.1021/ acs.jmedchem.7b01314. (9) Zhang, Y.; Luo, X-y.; Wu, D-h.; Xu, Y. ROR nuclear receptors: structures, related diseases, and drug discovery. Acta Pharmacol. Sin. 2015, 36, 71−87. (10) Fauber, B. P.; Magnuson, S. Modulators of the nuclear receptor retinoic acid receptor-related orphan receptor-γ (RORγ or RORc). J. Med. Chem. 2014, 57, 5871−5892. (11) Gege, C. Retinoid-related orphan receptor gamma t (RORγt) inhibitors from Vitae Pharmaceuticals (WO2015116904) and structure proposal for their Phase I candidate VTP-43742. Expert Opin. Ther. Pat. 2016, 26, 737−744. (12) Kono, M.; Ochida, A.; Oda, T.; Imada, T.; Banno, Y.; Taya, N.; Masada, S.; Kawamoto, T.; Yonemori, K.; Nara, Y.; Fukase, Y.; Yukawa, T.; Tokuhara, H.; Skene, R.; Sang, B.-C.; Hoffman, I. D.; Snell, G. P.; Uga, K.; Shibata, A.; Igaki, K.; Nakamura, Y.; Nakagawa, H.; Tsuchimori, N.; Yamasaki, M.; Shirai, J.; Yamamoto, S. Discovery of [cis-3-({(5R)-5-[(7-Fluoro-1,1-dimethyl-2,3-dihydro-1H-inden-5yl)carbamoyl]-2-methoxy-7,8-dihydro-1,6-naphthyridin-6(5H)-yl}carbonyl)cyclobutyl]acetic acid (TAK-828F) as a potent, selective, and orally available novel retinoic acid receptor-related orphan receptor γt unverse agonist. J. Med. Chem. 2018, 61, 2973−2988. (13) Shirai, J.; Tomata, Y.; Kono, M.; Ochida, A.; Fukase, Y.; Sato, A.; Masada, S.; Kawamoto, T.; Yonemori, K.; Koyama, R.; Nakagawa, 6735

DOI: 10.1021/acs.jmedchem.8b00529 J. Med. Chem. 2018, 61, 6724−6735