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Optimizing a weakly binding fragment into a potent ROR#t inverse agonist with efficacy in an in vivo inflammation model David Carcache, Anna Vulpetti, Joerg Kallen, Henri Mattes, David Orain, Rowan Stringer, Eric Vangrevelinghe, Romain M Wolf, Klemens Kaupmann, Johannes Ottl, Janet Dawson King, Nigel G. Cooke, Klemens Hoegenauer, Andreas Billich, Juergen Wagner, Christine Guntermann, and Samuel Hintermann J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00529 • Publication Date (Web): 10 Jul 2018 Downloaded from http://pubs.acs.org on July 10, 2018

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Journal of Medicinal Chemistry

Optimizing a weakly binding fragment into a potent RORγt inverse agonist with efficacy in an in vivo inflammation model David Carcache,†,# Anna Vulpetti,†,#,* Joerg Kallen,§,# Henri Mattes,† David Orain,† Rowan Stringer,ǁ 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, ǁPK Sciences, and

Autoimmunity, Transplantation and Inflammation, Novartis Institutes for BioMedical Research, 4002 Basel, Switzerland

ACS Paragon Plus Environment

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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 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. Due to 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. Due to 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.7-10 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 VTP-43742,8 JNJ-3534, AZD-0284 and JTE451 have advanced into clinical studies. For the most advanced candidate VTP-43742, Vitae


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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 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.

RESULTS AND DISCUSSION FRAGMENT-BASED SCREENING To complement classical high-throughput biochemical and cellular screening approaches, a series of fragment-based screening (FBS) 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 co-crystallized, 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


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design, despite its moderate LE (0.26) and LLE (0.23). The X-ray co-crystal structure of 1 bound to the RORγt (Y264-K518) construct and a RIP140 co-activator 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). It makes a direct H-bond with the backbone carbonyl of F377 as well as water-mediated H-bonds with the carbonyl backbone of H323 and the side-chain nitrogen of Q286. In addition the phenyl ring stacks nicely below F378 in an edgeto-face fashion. Finally, the chlorine substituent of 1 points towards the side-chain of C320 forming hydrophobic contacts. Based on the analysis of the crystal structure, we concluded that this fragment could provide an attractive starting point for fragment growing and merging approaches towards 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


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

IN SILICO ACTIVE SITE ANALYSIS A short description and visualization of the structural characteristics of each of these sub-pockets 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


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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 the 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 labelled 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 co-crystallized fragments present hydrophobic 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 towards helices 11 and 12. This portion of RORγt is


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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.

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 sub-pockets labelled 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.

MEDICINAL CHEMISTRY STRATEGY


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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 towards the ’business end‘ from the ethoxy substituent of 1 would be a preferred strategy to achieve RORγt inverse agonism.

Scheme 1: Medicinal chemistry strategy towards the identification of the potent RORγt inverse agonist 9

The aim of our initial medicinal chemistry strategy was to determine the SAR of the ethoxy substituent, and 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


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(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 whilst only the (S)enantiomer 6 showed first hints of inverse agonism in a FRET assay (Table 2). Table 1. Fragment growing towards the ‘business end’

Compound

R

MS reporter IC50 [µM]a

1

Et

216 ± 15

3

c

4

i

Pr

69 ±10

Pr

19 ± 2

5

9±1

6

6±1


9


15 ± 1

7

a

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

Supporting Information for assay details.

Having achieved functional activity by growing the fragment towards the business end, we decided to use only the functional assay for the further optimization. The Affinity MS reporter assay was geared towards 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 assay was designed to assess compounds with high affinities (< 10 µM). Next, we wanted to replace the central phenyl ring with a heterocycle since the N-(4hydroxyphenyl)acetamide motif presents a high risk to form an iminoquinone species upon oxidative metabolism. Thus, the 3-chloropyridine analog 8 was synthesized and shown to maintain similar potency. Gratifyingly, this compound displayed full inverse agonism. Table 2. Fragment merging towards the potent RORγt inverse agonist 9

Compound

X

R

R1

FRET IC50 [nM]a


10

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a

6

CH

Me

4733 ± 2333

7

CH

Me

> 10000

8

N

Me

1146 ± 118

9

N

0.6 ± 0.7

10

N

27 ± 2

11

N

3.1 ± 0.9

Functional TR FRET assay: Values were calculated from at least two independent

measurements (8-point dose response).25 Whilst 8 represented the first full RORγt inverse agonist in this series, its potency on RORγt was clearly two to three orders of magnitude too weak to be considered as a drug candidate. A fragment merging approach was used to further evolve the series. Among the many X-ray structures which were available in-house, our attention turned to compound 2,17 which occupies the ’polar end sub-pocket’ 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 end’ region offers many productive interactions with the sulfone moiety, and increased


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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.

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 access codes = 6FZU, 6G05).

This merging strategy was successful and led to the identification of compound 9 (Scheme 1) as a breakthrough RORγt inverse agonist with sub-nanomolar potency in the FRET assay. To better


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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 1).

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 ether-intermediates. 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. Scheme 2. General synthetic routes for the preparation of compounds 3 – 7, 8 – 11, 18 and 19


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ANALYZING THE MODE OF INHIBITION OF COMPOUND 9 A co-crystal 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 back pocket’ formed by L362, M365, V376, I400 and S404. In the crystal structure of 9, the side-chain of M365 is shifted towards the front, and this movement creates an enlarged cavity which perfectly accommodates the phenethoxy substituent. The phenyl ring is located between the side-chains of M365 and I400 forming many van der Waals contacts. Interestingly, a superposition of the co-crystal structures for 9 and the original fragment 1 (see Figure S1) shows that the (5-chloropyridin)acetamide and (3-chlorophenyl)acetamide moieties


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superpose very well (largest differences of ca. 0.4 Å). This confirms that the fragment growing towards both the ‘polar end’ and the ‘unexplored pocket’ did not change the original fragment binding mode.


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Figure 4: Chain A of crystal structure of RORγt (Y264-K518) in complex with 9 and RIP140 peptide (not shown). There are four complexes in the asymmetric unit. The distance between NE2-H479 and OH-Y502 in the four chains ranges from 2.8 to 3.3 Å (corresponding to medium to weak H-bond energies). The coordinates have been deposited in the PDB databank (PDB access code = 6G07).

In order to gain further insight into the contributions of each part of molecule 9 to the binding potency, compounds 18 and 20 (Scheme 3) were tested for their binding affinity to RORγt.

Scheme 3. Truncated versions and a pyridyl analog of 9

Compound 18, which is the result from merging the pyridine analog of the original hit 1 with the ethylsulfonylphenylacetamide-fragment, displayed a slightly lower gain of potency compared to the merging of 8 with the same fragment (i.e. compound 9). The phenylacetamide 20 did not show any measurable affinity for RORγt up to 500 µM thereby confirming the importance of occupying at the same time the ‘polar end region’, the unexplored back pocket’ and the ‘hydrophobic hot spot’. To better understand the role of the ‘hydrophobic hot spot’, compound 9


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and the des-methyl analog 11 were selected to characterize their binding kinetics (kon and koff, see Table 3). Binding kinetics was experimentally measured using a reporter displacement assay as described.20 In summary, the proximity between a suitable reporter and the protein results in the emission of an optical signal. Compounds that bind to the same site as the reporter probe displace the probe, causing signal diminution. Reporter displacement is measured over time after addition of compounds at various concentrations for deriving Kd, kon and koff. Table 3: Binding kinetics of compounds 9 and 11 Compound

Kd [nM]

kon [1/s 1/M]

koff [1/s]

Residence time [min]

Targeted MD Action ‘S’ [energy mol-1 time -1]; (exit pathway)

9

35.5

5.28·103

1.88·10-4

89

16.47 (a); 15.49 (b)

11

465.3

1.31·104

6.1·10-3

3

7.10 (a); 7.20 (b)

Compounds 9 and 11 show slow on and off binding kinetics. This finding prompted us to explore the most probable ligand unbinding pathways by carrying out targeted Molecular Dynamics (MD) simulations21 for both 9 and 11 to evaluate whether the in silico calculated ligand resistance to dissociation would qualitatively correlate with the experimental koff. Molecular dynamics studies revealed the two most probable unbinding pathways (Figure 5a) which supported the findings of others in the nuclear receptor research field.22 Analysis of the dynamics of the protein without 9 bound with respect to the two lowest energy unbinding pathways for 9, named pathway (a) and (b), are shown in Figure 5B.


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Figure 5: A) Predicted exit pathways for 9 from the RORγt (Y264-K518) complex. The trajectories are represented by dots connecting 1 ps-spaced MD frames of the atom on which the targeted MD was acting (i.e. the aromatic carbon bearing the amide substituent). The protein is colored in rainbow from blue (N-terminus) to red (C-terminus); B) RMS (Root Mean Squared) fluctuations of Cα atoms for all residues, recorded after the initial heat-up and equilibration phase. The purple and blue lines result from the targeted MD with the lowest S values for the pathway (a) and (b), respectively. The green line represents the RMS fluctuations of Cα atoms for all residues calculated on the RORγt structure in absence of 9 bound. The arrows in the plot highlight those regions in the receptor where strong deviations are observed.

The unbinding pathway (a) is oriented towards the solvent exposed region delimited by the helix 1 – helix 2 loop and helix 4. The ligand takes advantage of the intrinsic flexible zones of the


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receptor protein to move in and out. The RORγt protein structure in absence of 9 also shows a natural Cα fluctuation observed during the unconstrained molecular dynamics simulation in the region of the protein between residue 280 and residue 294 (Figure 5B). During unbinding along the pathway (a) the SO2 group of 9 remains close to the R367 side chain up to about 450 ps then the distance of the S of SO2 from the position assumed in the crystal structure increases. The unbinding along the pathway (b) occurs between the end of helix 7 and 11. The helix 3 helix 4 loop, helix 7 and helix 11 show high Cα fluctuations during the dynamics simulation as the ligand passes close to them (Figure 5B). When unbinding along pathway (b) the SO2 group shifts towards the ‘business end’ of the pocket (at about 370 ps time). Interestingly, the ligand resistance to dissociation calculated via targeted MD simulations (i.e. the overall ‘action’ S value to extract the ligand21) was found to qualitatively correlate with experimental koff. Table 3 reports the lowest S values for the unbinding pathways calculated 20 times for both compounds 9 and 11. The kon in the range of 103 to 104 is slower than the diffusion limiting rate (107/108), most likely due to the fact that the pocket is rather closed and shielded from the solvent. Therefore a conformational change of the protein must occur for the ligand to bind to the protein. The same reasoning applies for the koff. However small changes, such as the removal of a methyl, has a small effect on the kon but a larger effect on the koff. The kon of 9 is 2.5-times slower than the one for 11, whereas the koff of 9 is 32-times slower which translates to a 13-fold improvement of the binding affinity. The reduced flexibility and the more spherical shape of the benzyloxy portion of 9, through the additional methyl-group, could be responsible for this change in binding kinetics and is well described by our simulation.


19


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Our lead molecule 9 was further characterized by ITC in order to determine affinity and the thermodynamic signature of the binding process and this yielded a Kd of approximately 8 nM. A more precise measurement (even for reverse titration) was precluded by the low solubility. Binding is enthalpy driven (∆Ho = -17.3 kcal/mol), correlating with the formation of several intermolecular H-bonds and the entropic contribution to binding is unfavorable (-T∆So = 6.3 kcal/mol). As discussed, the protein must undergo large conformational changes for accommodating the ligand, this large entropy reduction could partially derive from the reduced flexibility of the protein upon binding of the ligand. The crystal structure of 9 was used as the basis for an in silico solvent analysis with the 3D variant of the ‘reference interaction site model’, 3D-RISM.23,24 This application computes a timeaveraged distribution of water density and binding desolvation penalty maps. The in silico generation of solvent organization in apo and ligand bound crystal structures is a useful approach to provide insight for structure-based ligand modifications. This is a complementary approach to the previously described SiteMap binding site characterization. The 3D-RISM results for the crystal structure, with or without 9 bound, are shown in Figure 6.

Figure 6. 3D-RISM results for the crystal structure of RORγt in complex with 9 (deposited with PDB access code = 6G07) and 19 (modeled). (A) Predicted water oxygen atom positions


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superimposed with the crystal structure of RORγt in complex with 9. The waters observed in the crystal structure (highlighted by red arrows) are in concordance with the 3D-RISM predicted water oxygen density (shown in solid blue, iso level 4). The waters placed based on the 3DRISM results are shown as yellow spheres. (B) Predicted water oxygen atom positions calculated for the protein in the absence of compound 9 bound. Compound 9 is shown in black as a reference, but was not used during the calculation. More ‘stable’ waters, located in polar protein environments (purple surface) are shown in green; more ‘unstable’ waters, located in lipophilic protein environments (green surface) are shown in red. (C) Binding model of 19. The 3D-RISM predicted water oxygen density on the complex is shown in solid blue (iso level 4). The predicted waters mediating the interaction between 19 and the protein are shown as sticks.

Figure 6A shows a remarkable agreement between the 3D-RISM predicted waters for the complex with the crystallographic water location at the protein-ligand interface. Figure 6B shows the 3D-RISM predicted waters in the absence of the ligand, which is shown in black as a reference, but was not used in the calculation. The placed waters are color coded according to their ability to be displaceable (increasing from green to red). Our water analysis mainly focused on two regions of the binding site: the ‘unexplored back pocket’ offering the possibility of forming direct or water-mediated interactions with S404 (not discussed) and the ‘polar end’ region showing the possibility of removing the sulfone moiety to form water-mediated interactions. In order to improve the solubility and permeability of compound 9, we explored the latter opportunity by designing compound 19 (see Scheme 3) with the aim of forming watermediated interactions between the pyridine nitrogen of the ligand and the side chain of R367 and the NH of the backbone of L287 (Figure 6C). This modification indeed led to a more soluble and


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permeable derivative (compound 19: aq. solubility at pH 6.8 = 28 µM; log PAMPA = -3.7; compound 9: aq. solubility at pH 6.8 < 4 µM; log PAMPA = -4.2) albeit with 30-fold lower potency compared to 9. Based on its exceptional potency in the FRET assay, compound 9 was selected for testing in a number of RORγt-dependent cellular assays and excellent potency was confirmed (Table 4). In order to determine the selectivity of 9 against the 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. Table 4: in vitro pharmacological profile of compound 9

TR-FRET RORγt-LBD RIP140 co-factor

IC50 = 0.6 nM

peptide recruitment25

RORγt transduced HUT78 T-cells: inhibition

IC50 = 14 nM

of IL17 secretion25

RORα,β,γt-LBD-Gal4 luciferase reporter gene assays26 RORα

IC50 = > 10000 nM


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RORβ

IC50 = 4113 nM; 54% inhibition

RORγt

IC50 = 17 nM

Human Th17 cell polarization25

IC50 = 23 nM

Rat Th17 cell polarization25

IC50 = 110 nM

Human whole blood25

IC50 = 255 nM

Compound 9 is highly lipophilic, poorly soluble at neutral pH, and highly permeable across PAMPA and Caco-2 monolayers (Table 4). Intrinsic clearance values in hepatic microsomes ranged between 31 to 48 µL⋅min-1⋅mg-1, the molecule was stable after a 2 hour 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 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 hours, 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 hours and oral bioavailability was 10%. Table 4: in vitro profiling data for 9 LogD(7.4) Equilibrium solubility (pH 6.8)

> 5.7

0.5 mg/L / 1 µM


23


LogPAMPA

– 4.2 cm⋅s-1

Caco-2 Papp A-B / Papp B-A (x10-6 cm/sec)

7.5 ⋅10-6 / 4.7⋅10-6

Liver microsomes (rat/human/mouse) CLint

48 / 31 / 46

(µL⋅min

-1

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⋅mg-1)

Plasma protein binding (mouse/rat) (%)

>99 / >99

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 hours. Immediately prior to the antigen


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challenge compound 9 was dosed by oral gavage at 50 mg/kg twice daily throughout the study. To determine the involvement of anti-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 IL-17A 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 hours and persisted until the end of the study (Figure 7A). To assess the effect of RORγt inhibition on pro-inflammatory 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 IL6 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 ‘non-responder’ in the treatment group, respectively (Figure 7C, p = 0.0625 unpaired t test). Exposures of 9 at the end of the experiment (2h after last dose) reached 1237 ± 598 nM in blood and 2305 ± 1251 nM in lymph nodes. The rather high inter-animal variability might be due to sub-optimal physicochemical properties (e.g., the poor solubility) which might influence oral absorption.


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Figure 7: A) Compound 9 attenuates mBSA-induced DTH responses in female Lewis rats. Rats were immunized with mBSA/CFA and two 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


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an anti-IL-17 antibody. All groups n = 5. **, P < 0.01 Dunnett´s test. C) Draining lymph node cells were prepared, stimulated ex vivo with mBSA for 24 hours and frequencies of IL17producing cells determined by ELISpot. Individual data and mean + SEM are depicted (n = 4-5). Data is representative from two independent studies.

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 co-crystallized with RORγt the benzylic substituent did not sterically clash with 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 S1). 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) 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


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an indirect destabilization of helix 12.16 For these ‘non-steric clash inverse agonists’, cocrystallization with a co-activator 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 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 S2). 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 methyl-group) 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 were


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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 technical-grade solvents on an Isolera Four apparatus from


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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. 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-4nitrobenzene 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 2-chloro-1-cyclopropoxy-4nitrobenzene (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-4cyclopropoxyaniline 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:


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2-chloro-4-nitro-1-((1,1,1-trifluoropropan-2-yl)oxy)benzene: This was prepared from 2chloro-1-fluoro-4-nitrobenzene and 1,1,1-trifluoropropan-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-nitro1-((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=6.42Hz, 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)-1phenylethanol (413 µL, 3.42 mmol) and stirred at 0 °C for 10 min., then 2-chloro-1-fluoro-4nitrobenzene (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


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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)-2-chloro-4-nitro-1-(1phenylethoxy)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 yellow oil (60 mg, 67 % yield). 1H-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-(1phenylethoxy)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


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(SiO2, heptane/AcOEt) to afford the title compound as 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-4nitrobenzene 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-(1phenylethoxy)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)-3chloro-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),


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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)-5chloro-6-(1-phenylethoxy)pyridin-3-amine 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)-3-chloro-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, the 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, DMSO-d6) 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 %.


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(S)-5-chloro-6-(1-phenylethoxy)pyridin-3-amine (14): A solution of (S)-3-chloro-5-nitro-2-(1phenylethoxy)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 re-crystallization 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


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(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-5nitropyridine (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-5nitro-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).


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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 (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-chloro5-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)-5-chloropyridin-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 2,3-dichloro-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


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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-5nitropyridine (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-3-amine (17) and 2-(4-(ethylsulfonyl)phenyl)acetic acid (18% yield). 1H-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-(1-phenylethoxy)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:


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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-hr 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 (licence 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 fourteen 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). 9 was dosed twice daily per oral gavage (50mg/kg) starting just prior to mBSA challenge until the end of the study. The rat crossreactive 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 hours post-challenge draining auricular lymph nodes were harvested and used in ex vivo T-cell recall assays. Ear tissue homogenization: The homogenization lysis buffer (117mg NaCl + 18.6mg EDTA disodium dihydrate salt (Amresco) + 100ul Tris-HCl pH 7.5 (Invitrogen) + 1.15ml ~87% Glycerin (Merck) + 8.75ml distilled water) was made up and a cOmplete™ Mini protease inhibitor cocktail tablet (Roche) added immediately before use, giving a final volume of 10ml. 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 0.5ml OMNI tubes (OMNI-International)


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containing garnet beads for 3 x 15 seconds in a Precellys 24 homogenizer (Bertin Instruments). The samples were then centrifuged at 13,000 rpm for 30 minutes at 4°C and the supernatant 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 96-well polyvinylidene difluoride plates (Millipore), pre-coated with anti–mouse IL-17 capture antibody (R&D Systems). Cells were re-stimulated with mBSA (200 µg/ml) and incubated for 24 hours at 37°C. After washing, the plates were incubated with biotinylated anti–mouse 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.

Associated content Supporting Information Additional figures and assay descriptions. The material is available free of charge via the Internet at http://pubs.acs.org. Supplementary Figure S1, assay and method descriptions (PDF). Molecular formula strings (CSV).


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Accession codes: PDB accession codes: 6FZU (1), 6G05 (2), 6G07 (9). Authors will release the atomic coordinates and experimental data upon article publication. Corresponding Authors * E-mail: [email protected]. Phone (+41)795609961; [email protected]. Phone (+41)613241016. Author Contributions The manuscript was written with contributions from all authors. All authors have given approval to the final version of the manuscript. # These authors contributed equally. ACKNOWLEDGMENT 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, Paul Scherrer Institute, Villigen, Switzerland. We thank L. Neumann and K. von König (Proteros biostructures GmbH) for the kinetic measurements. ABBREVIATIONS 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


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