Potent and Orally Bioavailable Inverse Agonists of RORÎ³t Resulting

Article Cite This: J. Med. Chem. 2018, 61, 7796−7813

pubs.acs.org/jmc

Potent and Orally Bioavailable Inverse Agonists of RORγt Resulting from Structure-Based Design Frank Narjes,*,† Yafeng Xue,⊥ Stefan von Berg,† Jesper Malmberg,† Antonio Llinas,‡ Roine I. Olsson,† Johan Jirholt,§ Hanna Grindebacke,§ Agnes Leffler,§ Nafizal Hossain,† Matti Lepistö,† Linda Thunberg,□ Hanna Leek,□ Anna Aagaard,⊥ Jane McPheat,¶ Eva L. Hansson,¶ Elisabeth Bäck,¶ Stefan Tångefjord,⊥ Rongfeng Chen,■ Yao Xiong,■ Ge Hongbin,■ and Thomas G. Hansson†

J. Med. Chem. 2018.61:7796-7813. Downloaded from pubs.acs.org by RMIT UNIV on 10/31/18. For personal use only.

†

Medicinal Chemistry, ‡DMPK, and §Bioscience, Respiratory, Inflammation and Autoimmunity, IMED Biotech Unit, AstraZeneca, Gothenburg, SE-43183 Mölndal, Sweden ⊥ Structure, Biophysics & FBLG and ¶Mechanistic Biology and Profiling, Discovery Sciences, IMED Biotech Unit, AstraZeneca, Gothenburg, SE-43183 Mölndal, Sweden □ Early Product Development, Pharmaceutical Sciences, IMED Biotech Unit, AstraZeneca, Gothenburg, SE-43183 Mölndal, Sweden ■ Pharmaron Beijing Company, Ltd., Taihe Road, BDA, Beijing 100176, PR China S Supporting Information *

ABSTRACT: Retinoic acid receptor related orphan receptor γt (RORγt), has been identified as the master regulator of TH17-cell function and development, making it an attractive target for the treatment of autoimmune diseases by a smallmolecule approach. Herein, we describe our investigations on a series of 4-aryl-thienyl acetamides, which were guided by insights from X-ray cocrystal structures. Efforts in targeting the cofactor-recruitment site from the 4-aryl group on the thiophene led to a series of potent binders with nanomolar activity in a primary human-TH17-cell assay. The observation of a DMSO molecule binding in a subpocket outside the LBD inspired the introduction of an acetamide into the benzylic position of these compounds. Hereby, a hydrogen-bond interaction of the introduced acetamide oxygen with the backbone amide of Glu379 was established. This greatly enhanced the cellular activity of previously weakly cell-active compounds. The best compounds combined potent inhibition of IL-17 release with favorable PK in rodents, with compound 32 representing a promising starting point for future investigations.

■

INTRODUCTION

Nuclear receptors are ligand-inducible transcription factors, whose function can be modulated by the binding of small molecules to the ligand-binding domain (LBD) of the receptor, thereby inducing conformational changes to the cofactor-recruitment binding site and ultimately affecting gene transcription.17−19 RORγt has been shown to be active in the absence of ligands, but sterol derivatives were described to affect receptor function and to control TH17 differentiation.20−23 The crystal structures of two sterol derivatives, agonist 25-hydroxychesterol and inverse agonist digoxin, provided insights into the molecular mechanisms leading to the different functional effects.24,25 Efforts to discover orally bioavailable inverse agonists of RORγt, as an alternative to antibody treatment, have generated a variety of different structural classes.26−29 Compounds affecting the receptor by binding to the LBD are not expected to exhibit isoform selectivity, because the two isoforms of RORγ differ only at their N-termini and share the same DNA-

The nuclear receptor retinoic acid receptor related orphan receptor γ (RORγ, NR1F3, or RORc) exists in two isoforms, with one isoform (RORγ or RORc1) widely expressed in a variety of tissues, and the expression of the second isoform (RORγt or RORc2) restricted to the thymus and cells of the immune system.1 RORγt is the key transcription factor for the function and development of CD4+ T-helper 17 (TH17) cells and related IL-17-producing immune cells, such as innate lymphoid 3 cells and γδ T cells.1−5 TH17 cells are characterized by the production of the cytokines IL-17A, IL-17F, and IL-22 and play a key role in host defense against extracellular pathogens.6,7 Dysregulation of the TH17 pathway has been implicated in the pathology of a variety of autoimmune disorders, such as psoriasis, Crohn’s disease, multiple sclerosis, and rheumatoid arthritis.7−12 Antibodies targeting IL-17; its receptor, IL-17RA; or the cytokines involved in the inflammatory pathway, such as IL-23, have shown clinical efficacy in treating psoriasis and related autoimmune disorders.13−16 © 2018 American Chemical Society

Received: May 16, 2018 Published: August 10, 2018 7796

DOI: 10.1021/acs.jmedchem.8b00783 J. Med. Chem. 2018, 61, 7796−7813

Journal of Medicinal Chemistry

Article

Figure 1. Selection of published inverse agonists of RORγt.

and ligand-binding domains.30−40 Recent clinical candidates from these efforts include GNE-3500, 41 GSK805, 42,43 GSK2981278,30 and VTP-4374244 (Figure 1). GSK2981278, whose structure has not been disclosed, was developed for the topical treatment of psoriasis but failed to show efficacy in a Phase 1 study,45 whereas Vitae Pharmaceuticals recently reported positive results for VTP-43742 from a Phase 2a study for the same indication. Oral administration over 4 weeks was found to be safe and efficacious, resulting in a 24% reduction in the psoriasis-area severity index.46 We recently disclosed a series of benzoxazepine-based modulators, which we eventually abandoned because of the metabolic instability of the compounds.39 The hit-identification approaches we conducted to find new starting points included X-ray-based fragment screening,47 and the characterization of compounds from the literature. Among these were derivatives 1 and 2, previously described by scientists from GSK.48,49 Both compounds were potent binders in a radioligandbinding assay (SPA) and behaved as inverse agonists in a FRET assay, showing functional suppression of the recruitment of a coactivator peptide derived from steroid-receptor coactivator 1 (SRC1) to RORγt LBD. The activity of 2 observed in the FRET assay was about 10-fold lower compared with that in the binding assay, which we ascribed to the conditions of the FRET assay, placing a limit on the lowest IC50 that can be determined at around 0.02 μM. They efficiently suppressed IL-17A production from primary human TH17 cells, with thiophene 2 being 30-fold more potent. Compound 2 is highly lipophilic, and its pharmacokinetic properties in mouse were characterized by high plasma clearance and low oral bioavailability.49 Compound 3 was prepared as part of an early effort to understand the SAR and reduce lipophilicity. It was about 60-fold less active in the SPA assay, but we were intrigued by the observation that despite the lack of the benzoate moiety, 3 behaved as an inverse agonist in the FRET assay. Even though 3 exhibited submicromolar binding potency and efficacy in the FRET assay, it was not able to suppress IL-17 release from primary human TH17 cells. In this paper we describe our SAR efforts around 3, which were guided by insights from X-ray crystal structures on the binding modes of compounds as the series evolved. These resulted in a class of compounds that efficiently suppressed IL17 production in primary human cells and had favorable PK properties in rodent species.

Figure 2. (a) X-ray structure of 1 (green) binding inside RORγt LBD (gray; 2.30 Å, PDB: 5NI5). Compound 1 and key residues of RORγt LBD are shown as sticks. (b) Overlay of 1 (green) and 25hydroxycholesterol (25-HC, gray), showing the interaction of 25-HC with the water molecule (dotted sphere) and the Tyr−His lock.

hydrogen-bond interactions of the sulfone oxygens with Arg367 and Gln286 and of the amide moiety with the backbone carbonyl of Phe377. The amide bond is coplanar with the thiazole ring, with a stabilizing 1,5-O···S interaction.53 The meta-chloro substituent of the 4-aryl residue points to Ser404 (3.5 Å), and the side chain of Met365 has moved significantly (about 3.5 Å) with respect to the apo-structure. An overlay with 25-hydroxycholesterol (Figure 2b) shows that the benzoyl group at the 5-position of the thiazole fills the hydrophobic pocket near helix 12 only partially and does not interact with the so-called Tyr−His lock, composed of His479 on helix 11 and Tyr502 on helix 12. These two residues form part of the activation-function-2 domain (AF2), the interaction site for coregulatory proteins. We observed a conserved water near Trp317 and the His−Tyr lock, which, in the structure with the agonist, 25-hydroxycholesterol, interacts with the 25hydroxy group and stabilizes the agonist conformation.24 Stabilization of the AF2 domain by hydrophobic interactions was suggested as an explanation for the agonism of thiazole amides and keto amides.50,52 For the inverse agonism of 1, a water-trapping mechanism was proposed, whereby the release of the water molecule trapped at the AF2 domain in the hydrophobic environment is energetically favorable, leading to destabilization of helix 12.51 The X-ray structures described above were not published when we started our work, but the results in Table 1 pointed to a key role of the lipophilic benzoyl group for potency and activity in the cell assay. Assuming that the binding mode of 3 would closely resemble that of 1, we hypothesized that it should be possible to combine cell potency and druglike properties by using the appropriate substituents appended to the thiophene-linked aryl group of 3. A focused library of compounds around the aryl substituent in the 4-position of the thiophene was prepared (Table 2). Addition of a cyano group in the 5-position of the 4-aryl group of 3 resulted in a 2-fold gain in potency. Moving the chlorine to the 2-position gave equipotent compound 5, which

■

RESULTS AND DISCUSSION We succeeded in soaking compound 1 into the apo-crystals of RORγt LBD tethered to an SRC2-derived peptide, representing the agonist state of the receptor (Figure 2).24,39 Recently, the cocrystal structures of 1 and of a closely related agonist were published.50−52 Despite the different crystallization conditions, the key interactions of the compounds inside the ligand-binding domain are very similar. These include the 7797



Article

Table 1. Profile of Compounds 1−3

compd

IC50 SPAa [μM]

IC50 FRETb [μM] (% eff)

IC50 IL-17 cellc [μM] (% eff)

log Dd

clogP

LLEe

1 2 3

0.009 0.002 0.11

0.022 (−87) 0.023 (−82) 0.18 (−96)

0.18 (−75) 0.006 (−85) >3

>4.2 4.5 >4.1

5.5 6.0 4.0

2.6 2.6 3.0

a Displacement of tritiated ligand from human RORγt LBD (geometric mean of n ≥ 2). bInhibition or activation of the recruitment of the SRC1derived coactivator peptide (NCOA1, 677−700 aa) to human RORγt LBD (geometric mean of n ≥ 2). Negative percent-efficacy (% eff) values denote inverse agonism. cInhibition of IL-17 production from human primary TH17 cells (geometric mean of n ≥ 3). Negative values of percent efficacy signify inhibition of IL-17 production. dDistribution coefficient between 1-octanol and aqueous phosphate buffer at pH 7.4. eLigand lipophilic efficiency defined as SPA pIC50 − clogP.

maintained functional activity. A bulkier benzyl ether, as in 10, led to a 20-fold gain in binding affinity; however, 10 behaved as an agonist in the FRET assay, efficiently enhancing recruitment of the coactivator peptide. This indicated that our initial hypothesis of being able to modulate receptor function by proper substitution from the aryl group was at least partly correct. Attempts to further increase the polarity of 9, as in pyrimidine 11, resulted in a significant loss in potency. Compound 12 combined the potency-enhancing nitrile moiety with the methoxy group and gave results similar to 8. Concurrent with the optimization of the aryl group, we also explored the possibility of lowering the lipophilicity by introducing changes to the aryl sulfone moiety. We considered groups that could maintain some of the interactions in the arginine-rich region. A sulfoxide and especially a sulfoximine moiety fulfilled these requirements,54 but the corresponding compounds, 13 and 14, lost activity by 40- and 10-fold in the SPA assay, respectively, whereas in the FRET assay, 10- and 4fold losses were observed (Table 3). A methylsulfonamide, as in 15, did not offer any reduction in lipophilicity but maintained potency with respect to 12. Replacing the phenyl sulfone aromatic ring with a sulfonylated piperidine, as in 16, also led to a considerable loss of activity, which we believe to be due to the different geometries of the saturated ring compared with those of the aromatic ring. To better understand the tight SAR around the sulfone moiety, we performed a solvent analysis of the apo and complex structures with 1 using 3D-RISM.55 The results indicated the presence of several water molecules in this arginine subpocket. Four of these were hydrophobic (unstable) and were replaced by the ethyl group, whereas two hydrophilic (stable) water molecules were each replaced by one of the oxygen atoms of the sulfone upon binding of the ligand (Figure 3). Accordingly, the two oxygen atoms retain favorable polar interactions with the protein. As a result, the ethyl sulfone group seemed to provide the best configuration to replace the different solvent positions and the best binding affinity of the ligand. This would also explain the loss of potency for sulfoxide 13 and sulfoximine 14. We tested some of the more active compounds for their ability to inhibit IL-17A production in primary human TH17 cells (Table 4). We found that all compounds were inactive at concentrations of up to 3.3 μM, apart from 7 and 10. In line

Table 2. Initial SAR around the 4-Aryl Group

a Displacement of tritiated ligand from human RORγt LBD (geometric mean of n ≥ 2). bInhibition or activation of the recruitment of the SRC1-derived coactivator peptide (NCOA1, 677−700 aa) to human RORγt LBD (geometric mean of n ≥ 2). Negative percentefficacy (% eff) values denote inverse agonism; positive values denote agonism and were determined at a single concentration. cDistribution coefficient between 1-octanol and aqueous phosphate buffer at pH 7.4. dLigand lipophilic efficiency defined as SPA pIC50 − log D.

exhibited a lower partition coefficient compared with that of 4, whereas compound 6, with a cyano group into the 4-position, was less active. Trisubstituted derivative 7, bearing a methoxy group in place of the chlorine, gained another 2-fold increase in potency. Removal of the fluorine was tolerated, leading to 8, with improved lipophilic ligand efficiency (LLE) compared with that of 3. Another set of interesting data was provided by the orthosubstituted pyridyl analogues, 9 and 10. Methoxy derivative 9 exhibited diminished binding activity with respect to 3 but 7798



Article

Table 3. SAR around the Sulfonyl Head Group

Displacement of tritiated ligand from human RORγt LBD (geometric mean of n ≥ 2). bInhibition or activation of the recruitment of the SRC1derived coactivator peptide (NCOA1, 677−700 aa) to human RORγt LBD (geometric mean of n ≥ 2). Negative percent-efficacy (% eff) values denote inverse agonism; positive values denote agonism and were determined at a single concentration. cDistribution coefficient between 1-octanol and aqueous phosphate buffer at pH 7.4. dLigand lipophilic efficiency defined as SPA pIC50 − log D. a

the suppression of IL-17A production and toxicity. On the basis of good binding potency and functional activity in the FRET assay, we were expecting to see a more profound response on IL-17 production, and investigated if the lack of activity was due to issues related to cell permeability or efflux. Results from the Caco2-assay indicated that compared with 2, all of our inverse agonists showed improved permeability. Compound recovery was only 40−50%, which was due to their relatively high lipophilicities (see Tables 1−3), low solubilities, and high affinities for plasma proteins. This can lead to unspecific binding or precipitation, making data interpretation difficult. Compounds 7, 8, and 12 had similar permeabilities and moderate efflux, whereas sulfonamide 15 demonstrated lower permeability and higher efflux. We also measured the intrinsic permeabilities of these compounds in an assay where Caco2 cells were incubated with a cocktail of compounds inhibiting the most common transporters present in the cell, including PgP.56 The intrinsic permeability values for all inverse agonists, apart from 2, were high, indicating that these compounds should be able to reach the interiors of the cells. On the basis of the data, low permeability and efflux do not explain the lack of cell potency. Our conclusion is that binding and cofactor-recruitment assays using the truncated form of the receptor in a simplified system are not always predictive of the more complex biology of inhibition of IL-17 production in

Figure 3. Solvent analysis of RORγt LBD in complex with 1 using 3D-RISM. The arylsulfone group of 1 and key residues of RORγt LBD are shown as sticks. Solvent positions in the apo-pocket are shown as spheres (hydrophobic, unstable waters are in orange, and hydrophilic, stable ones are in cyan).

with their behavior in the functional FRET assay, inverse agonist 7 inhibited IL-17A production with an IC50 of about 0.7 μM, whereas agonist 10 enhanced it to 38% over the baseline of the cell assay at a concentration of 3.3 μM. We noticed some effects on cell viability in the Cell-Titer-Glo readouts for both compounds at 3 μM. This made the interpretation of the cell data problematic, as there was less than a 10-fold difference between the IC50 values observed for

Table 4. Cell-Assay Activity and Physicochemical Data for Selected Analogues compd

IC50 IL-17 cella [μM] (% eff)

CTGa [μM] (% eff)

Caco2b Papp AtB/BtA/efflux [1 × 10−6 cm/s]

Caco2c intrinsic P0 [1 × 10−6 cm/s]

hPPBd [% free]

solubilitye [μM]

2 7 8 10 12 15

0.006 ± 0.0003 (−85) 0.7 ± 0.5 (−57) >3.3 +38% at 3.3 μM >3.3 >3.3

>3 (40%) >3 (60%)  >3 (30%)  

324 52% rec 1.8/7.1/3.9 41% rec 1.6/12/7.6 41% rec  2.6/17.1/5.8 44% rec 0.65/26/40 55% rec

1 (50) 0.3 (50) >10 3 (40%) >10 >10 >10

13 128 28 80 196 125 104 45 >300 286 281 28 40 20 17 7.5

Displacement of tritiated ligand from human RORγt LBD (geometric mean of n ≥ 2). bInhibition or activation of the recruitment of the SRC1derived coactivator peptide (NCOA1, 677−700 aa) to human RORγ LBD (geometric mean of n ≥ 2). Negative percent-efficacy (% eff) values denote inverse agonism; positive values denote agonism and were determined at a single concentration. cInhibition of IL-17 production from human primary TH17 cells (geometric mean of n ≥ 3). Negative values of percent efficacy signify inhibition of IL-17 production; positive values signify potentiation under the assay conditions. Effects of the compounds on cell viability and proliferation were assessed in the same assay with Cell-Titer-Glo measuring total ATP levels; na, not active at 3.3 μM; nt, not tested. dRat-hepatocyte metabolic intrinsic clearance (μL min−1 (million cells)−1). a

group in agonist 10 filled the pocket around the His−Tyr lock, replacing the conserved water observed in the structures of 1 and 8. This hydrophobic interaction is thought to stabilize the agonist conformation of the AF2 domain, and similar observations were published recently for other RORγ agonists.39,50,52,57,58 On the basis of the X-ray structure of 10, we expected that increasing the size of the alkoxy group would eventually lead to a clash with the His−Tyr lock and therefore to inverse agonism. Increasing the size of the alkoxy substituent from methoxy, as in 9, to ethoxy, as in 17, and subsequently to phenoxy, as in 19, resulted in an increase in binding affinity and a gradual switch in function from inverse agonism to agonism in the FRET assay (Table 5), with 19 behaving like benzyl ether 10. Pushing the phenyl group out further by chain

TH17 cells, where RORγ is in its native state and interacting with its endogenous ligands and cofactors. We could obtain X-ray cocrystal structures of inverse agonist 8 and agonist 10 by soaking the compounds into the apocrystals of RORγt LBD (Figure 4), proving that the compounds bind to the receptor and are genuine modulators of RORγt. Both compounds showed an almost perfect overlap for the sulfone headgroup and core heterocycle. The same key interactions described above for 1 were established. For compound 8, the nitrile on the aryl moiety pointed toward the side-chain oxygen of Ser404, indicative of a rather long hydrogen-bond interaction of 3.2 Å, which is similar to the meta-Cl of the 4-arylygroup of 1. The 2-methoxy group is too short to interact with the AF2 domain, whereas the benzyl 7800



Article

elongation of 10 did not lead to inverse agonism, but derivatives 20 and 21 behaved as agonists. We hypothesized that the flexibility of these substituents allowed them to be accommodated in the binding pocket, avoiding the steric clash with the AF2 domain. Rigidifying the propyl linker of 21 with a double bond supported this hypothesis, as cinnamyl ether 22 was a potent inverse agonist in the FRET assay. Introduction of a substituent to the para-position of the benzyl group of 10 led to the expected change in the mode of action. Ester 23 as well as benzonitrile 24 behaved as inverse agonists. Moving the cyano residue to the meta-position of the phenyl ring as in 25 led to a switch to agonistic behavior in the FRET assay. Functional behavior in the FRET assay was mirrored in the cell assay, but the observed activity on IL-17A secretion was again obstructed by the effects on cell viability for several compounds. Exceptions were cinnamyl ether 22 and ester 23, for which at least 100-fold windows between cell activity and toxicity were observed. Added lipophilicity, as in compounds 17−25, which had measured log D7.4 values of around 4 or greater, came at the price of decreased metabolic stability, as the data from rat hepatocytes demonstrated. Removal of the metabolically labile methylene in benzyl ether 24 led to phenylether 26 with a lower log D7.4 value (3.6) and improved stability compared with that of either 19 or 24. Compound 26 behaved as an inverse agonist in the cell assay, where it exhibited an IC50 of 190 nM and no sign of cytotoxicity. Further SAR around the 4phenyl substituent eventually identified the trifluoromethoxy group as an optimal group in the para-position. Benzylether 27 combined potent inhibition of IL-17A production with a 100fold window over cytotoxicity and reasonable stability in rat hepatocytes. The corresponding phenylether, 28, showed further improvement in metabolic stability. It behaved like a weak partial inverse agonist in the FRET assay, like 26, and suppressed IL-17A secretion with an IC50 of 144 nM in the cell assay with no effect on cell viability. We were also able to identify an inverse agonist with a short alkoxy side-chain by converting the ethoxy group of 17 to a bulkier trifluoroethoxy group. Compound 29 was a potent inhibitor of IL-17 production with no sign of cytotoxicity. Adding the nitrile group to the 5-position resulted in 30, showing a 10-fold increase in binding potency, an improvement in metabolic stability, and a slight increase in cell potency. The maximum achievable inhibition in the cell assay reached a plateau at 66%, and most compounds in this series, apart from ester 23, did not completely abolish IL-17A production. We succeeded in soaking agonist 25 into the apo-crystal (Figure 5), and the only difference with respect to 10 concerned the orientation of the benzyl moiety. In compound 25, the phenyl ring was tilted in comparison with 10, filling a space between helices 11 and 7 and stabilizing the agonist form. Structurally related inverse agonists, such as 23, 24, or 27 would be expected to push against the AF2-domain in either conformation, which was confirmed by the cocrystal structure of 23, described below. The side chain of 30 is too short to interact directly with the helix 11−helix 12 interface, but it seemed to disturb the conserved water when we modeled it into the structure. Release of this water might destabilize the AF2 domain, as recently described for 1.51 The cocrystal structure of 23 with the native RORγt construct in the absence of coactivator peptide39 showed that

Figure 5. Ligand interaction near the His−Tyr lock based on the structure of 25 (1.82 Å, PDB: 5NIB). Compounds 10 (magenta), 25 (yellow), and 30 (gray, modeled on the basis of 8) and key residues of the His−Tyr lock of RORγt LBD are shown as sticks. The ligands are only shown partially for clarity. The conserved water position is shown as a dotted sphere in red, and the position of the CF3 in 30 is shown as dotted spheres in cyan.

the major interactions of 23 were the same as those illustrated for 10 and 25, except for the benzoate ester group. In the agonist form, this group would clash with His479, as hypothesized above and indicated in Figure 6a. Consequently, part of helix 11 and the entirety of helix 12 are disordered and are missing from the electron density in the structure.

Figure 6. Ligand interaction of compound 23 in RORγt LBD (2.37 Å, PDB: 6FGQ). (a) Overlay of agonists 10 (magenta) and 25 (yellow) with inverse agonist 23 (orange). Key residues of the His−Tyr lock of RORγt LBD are shown as sticks. The ligands are only shown partially for clarity. The conserved water position is shown as a dotted sphere in red. Helix 12 and part of helix 11 are disordered (indicated in the dashed circle) when compound 23 is bound. The putative steric clash between 23 and the side chain of His479 (based on 10 and 25) is shown as a red star (∼1.4 Å in between). (b) Overlay of RORγt complexes with digoxin (PDB 3B0W, blue), 10 (magenta), and 23 (orange).

An overlay with the inverse-agonist structure of digoxin showed a steric clash of ester 23 with His479, which in the digoxin structure is pulled in by the ligand, establishing a hydrogen-bond to one of its sugar hydroxy groups.25 This in turn “pushes” away part of helix 11 and all downstream residues. Overall, 23 seems to achieve the inverse-agonist effect in a similar way but without pulling in His479. Although we were not able to obtain a structure, we believe that 7801



Article

too short and does not interact with the helix 11−helix 12 interface. There is also no conformational change of the receptor apparent upon binding of the acetamide with respect to 8, and on the basis of this structure, we cannot explain the improvements in cell potency of 31 and 32. Hydrogen bonding to Glu379 was also described recently for 6-substituted quinolines and bisaryl carboxamides.35,60 Unlike 31 and 32, which bear simple aryl groups in the 4positions of the thiophenes, acetamides 34−36, with more elaborate benzyl or aryl ether linkages, did not show gains in cell potency with respect to that of their parents. Cell potency remained unaltered for 35 and 36, whereas for 34, a slight 3fold loss in potency, compared with that of 26, was observed. The acetamide substitution also did not seem to affect the function of the compounds, because benzyl ether 37 still behaved as an agonist. Compound 35 was separated into its enantiomers 38 and 39, with the former being about 100-fold more active in the binding and FRET assays. On the basis of an X-ray structure of 32, 38 was assigned the (R)-configuration, and 39 was assigned the (S). Surprisingly, the latter inhibited IL-17 release in the cell assay with the same potency as 38. We hypothesized that the stereocenter might racemize under the assay conditions and investigated this for 38 and 39. The compounds were stable at pH 5, 7.4, and 8 at 37 °C over 2 days, but racemized in the medium of the TH17 assay, explaining the activity observed for 39 (Table 7). In view of these results, it cannot be excluded that some racemization occurs also under the conditions of other in vitro or in vivo assays, and this should be considered when using these compounds. On the basis of their favorable cell potencies and metabolic stabilities in rat hepatocytes, we further profiled compound 28 and three matched pairs with and without the acetamide residue (Table 8). The introduction of the acetamide residue raised the polar surface area by about 30 Å2, lowered the log P, and increased the ligand efficiency (see also Table 6). The compounds tested were not active on RORα in a timeresolved FRET assay conducted in agonist mode. When tested in inverse-agonist mode, compounds 30, 32, and 38 showed micromolar activity, with 36 being the most active with an IC50 of 0.6 μM. No activity could be detected for 27, 32, 36, and 38 in a RORβ-binding assay. Solubility was low for all analogues, except for 32, in which the acetamide improved solubility significantly with respect to that of 7. All compounds showed very high affinities for plasma proteins, and moderate stability in human hepatocytes. Relatively high activity on the hERG channel was noticed for 7 and 30 when tested at a fixed concentration of 11 μM, and it was decreased to acceptable levels in the corresponding acetamides 32 and 36. All compounds, apart from 36, were profiled for their pharmacokinetic behavior in Han Wistar rats (Table 8). Plasma clearances for the non-acetamide-containing compounds, 7, 27, 28, and 30, decreased from high for the benzyl ether, 27 (46.5 mL/min/kg), to very low for 7 and 30 (

Potent and Orally Bioavailable Inverse Agonists of RORÎ³t Resulting

Recommend Documents