Ligand-Based Design of Allosteric Retinoic Acid Receptor-Related Orphan Receptor γt (RORγt) Inverse Agonists

Article abstract of DOI:10.1021/acs.jmedchem.9b01372

Retinoic acid receptor-related orphan receptor γt (RORγt) is a nuclear receptor associated with the pathogenesis of autoimmune diseases. Allosteric inhibition of RORγt is conceptually new, unique for this specific nuclear receptor, and offers advantages over traditional orthosteric inhibition. Here, we report a highly efficient in silico-guided approach that led to the discovery of novel allosteric RORγt inverse agonists with a distinct isoxazole chemotype. The the most potent compound, 25 (FM26), displayed submicromolar inhibition in a coactivator recruitment assay and effectively reduced IL-17a mRNA production in EL4 cells, a marker of RORγt activity. The projected allosteric mode of action of 25 was confirmed by biochemical experiments and cocrystallization with the RORγt ligand binding domain. The isoxazole compounds have promising pharmacokinetic properties comparable to other allosteric ligands but with a more diverse chemotype. The efficient ligand-based design approach adopted demonstrates its versatility in generating chemical diversity for allosteric targeting of RORγt.

Full text of DOI:10.1021/acs.jmedchem.9b01372

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Article
Ligand-Based Design of Allosteric Retinoic Acid Receptor-
Related Orphan Receptor #t (ROR#t) Inverse Agonists
Femke Meijer, Richard G. Doveston, Rens de Vries, Gael Vos, Alex Vos, Seppe
Leysen, Marcel Scheepstra, Christian Ottmann, Lech-Gustav Milroy, and Luc Brunsveld
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01372 • Publication Date (Web): 10 Dec 2019
Downloaded from pubs.acs.org on December 10, 2019
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Ligand-Based Design of Allosteric Retinoic Acid
Receptor-Related Orphan Receptor γt (RORγt)
Inverse Agonists
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ξ,‡
ξ, ‡,§
‡
‡
Femke A. Meijer, Richard G. Doveston,
Rens M.J.M. de Vries, Gaël M. Vos, Alex
‡
‡
‡
‡
‡
A.A. Vos, Seppe Leysen, Marcel Scheepstra, Christian Ottmann, Lech-Gustav Milroy,
and Luc Brunsveld‡
,*
ξ
These authors contributed equally to this work.
AUTHOR ADDRESS
‡
Laboratory of Chemical Biology, Department of Biomedical Engineering and Institute
for Complex Molecular Systems, Technische Universiteit Eindhoven, Den Dolech 2,
5612 AZ Eindhoven, The Netherlands
§
Leicester Institute of Structural and Chemical Biology and Department of Chemistry,
University of Leicester, University Road, Leicester, UK, LE1 7RH.
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KEYWORDS
Nuclear Receptors • RORγt • Allosteric Modulators • Ligand-Based Design • Drug
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Discovery
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ABSTRACT
Retinoic acid receptor-related orphan receptor γt (RORγt) is a nuclear receptor
associated with the pathogenesis of autoimmune diseases. Allosteric inhibition of RORγt
is conceptually new, unique for this specific nuclear receptor, and offers advantages over
traditional orthosteric inhibition. Here, we report a highly efficient in silico-guided approach
that led to the discovery of novel allosteric RORγt inverse agonists with a distinct
isoxazole chemotype. The most potent compound, 25, displayed sub-micromolar
inhibition in a coactivator recruitment assay and effectively reduced IL-17a mRNA
production in EL4 cells, a marker of RORγt activity. The projected allosteric mode of
action of 25 was confirmed by biochemical experiments and co-crystallization with the
RORγt ligand binding domain. The isoxazole compounds have promising
pharmacokinetic properties comparable to other allosteric ligands, but with a more diverse
chemotype. The efficient ligand-based design approach adopted demonstrates its
versatility in generating chemical diversity for allosteric targeting of RORγt.
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1
. INTRODUCTION
The nuclear receptor (NR) RORγt has emerged as an important therapeutic target in recent years
because of its important role in both cancer and autoimmune disease. Inhibition of RORγt is a
promising therapeutic strategy for the treatment of prostate cancer because it stimulates androgen
receptor (AR) gene transcription.1,2 However, RORγt is most prominently targeted for inhibition
because of its essential role in promoting T helper 17 (Th17) cell differentiation.3-5 Th17 cells
produce the cytokine IL-17 which is strongly implicated in the pathogenesis of autoimmune
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diseases such as psoriasis, multiple sclerosis and inflammatory bowel disease. Disrupting the
Th17/IL-17 pathway using IL-17 monoclonal antibodies (mAb) is a successful therapeutic
strategy, with three mAbs approved for the treatment of plaque psoriasis: secukinumab
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(Cosentyx), brodalumab (Siliq), and ixekizumab (Taltz). Inhibition of RORγt with small
molecules to disrupt the Th17/IL-17 pathway has been the focus of much research in recent
years,13-20 with several compounds progressed to clinical trials.2
RORγt contains a hydrophobic ligand binding pocket located within a ligand binding domain
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(LBD) that is highly conserved across the NR family. However, its transcriptional activity is not
dependent on ligand binding because the apo protein retains the C-terminal helix 12 (H12) in a
conformational state that allows for partial recruitment of coactivator proteins.22,23 Although
formally an orphan receptor with no proven endogenous ligands, RORγt is responsive to binding
of naturally occurring cholesterol derivatives. Hydroxycholesterols have been shown to be
24
effective agonists that stabilize H12 in such a way to further promote coactivator binding. In
contrast, digoxin (1, Figure 1) is an inverse agonist that stabilizes H12 in a conformation that is
unsuitable for coactivator binding but promotes corepressor binding, thus leading to diminished
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gene transcription. Numerous synthetic inverse agonists are also known, including T0901317 (2,
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Figure 1). In all these cases, the ligands target the same orthosteric ligand binding pocket (Figure
1).
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NR orthosteric ligand binding pockets are the target for numerous and highly effective drug
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molecules. Nevertheless, the highly conserved nature of this pocket across the NR family has led
to issues associated with selectivity and mutation-induced resistance. Furthermore, dosing levels
must be appropriate to compete with endogenous ligands. Molecules that target allosteric binding
sites on NRs could circumvent such problems, for example because of the chemical uniqueness of
the pocket and the absence of a competitive endogenous ligand. Such allosteric compounds are
therefore extremely valuable for both drug discovery and chemical biology applications.28-30 The
3
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discovery that the potent RORγt inverse agonists MRL-871 (3, Figure 1) and later 4 target a
previously unreported allosteric binding site within the RORγt LBD was therefore highly
significant. These ligands were observed to directly interact with the activation function loop
between H11 and H12 (AF-2 domain), thus forcing H12 to adopt an unusual conformation that
prevents coactivator recruitment (Figure 1).31
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Figure 1. Orthosteric and allosteric RORγt ligand binding sites are shown by overlay of
the crystal structures of RORγt LBD in complex with orthosteric inverse agonist 2 (orange,
PDB code: 4NB6) and allosteric inverse agonist 3 (blue, PDB code: 4YPQ). The
structures of the orthosteric inverse agonist 1 and allosteric inverse agonist 4 are also
shown.
Allosteric modulation of RORγt has enormous potential as a novel therapeutic strategy; but, the
examples of ligands that unambiguously target the allosteric pocket have been limited to
compounds based on closely related chemotypes containing indazole or imidazopyridine cores.28
As an example, indazoles 3 and 4 displayed promising in vivo activity,33,34 but challenges remain,
such as PPARγ cross-activity and pharmacokinetic (PK) profiles, for which novel chemotypes are
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needed. In order to better exploit the strategy of allosteric modulation for therapeutic purposes,
there is thus an urgent need to identify novel chemotypes targeting the allosteric site. In this study,
we report the design, synthesis and evaluation of a novel class of RORγt allosteric inverse agonists.
The novel chemotype, discovered by in silico-guided pharmacophore screening and optimization,
is based on a trisubstituted isoxazole core that, following efficient optimization of two substituents,
led to the discovery of a sub-micromolar inverse agonist. Protein X-ray crystallography and
biophysical data unambiguously proved the designed allosteric mode of action. The compounds
effectively inhibit cellular IL-17a expression and thus constitute valuable leads in the development
of treatments for autoimmune diseases. To the best of our knowledge, our highly efficient in silico-
guided approach is the first example of a medicinal chemistry program to overtly identify and
develop a novel chemotype that targets the RORγt allosteric site.
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2. RESULTS AND DISCUSSION
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.1. In Silico Pharmacophore Screen
In order to identify novel chemotypes for chemical optimization, we used the crystal structure of
RORγt LBD in complex with 3 as the basis for an in silico 3D pharmacophore screen against
virtual compound libraries. An analogous scaffold hopping approach had been used previously to
identify similar scaffolds to 3 such as the potent inverse agonist thienopyrrazole 5 (Figure 2),
although an allosteric mode of action was not proven.35 We created a 3D pharmacophore
hypothesis based on the crystal structure of 3 bound to the allosteric pocket using Phase
(
Schrödinger 2017-2).36,37 Six structural features of 3 known to be important for activity were
incorporated in the hypothesis: the three six-membered aromatic rings, an anionic group and two
hydrophobic substituents (Figure 2). This hypothesis was used to interrogate a virtual library of
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89,174 compounds from the Asinex Gold-Platinum collection of drug-like molecules.38
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Compounds matching at least four out of the six pharmacophore features were deemed to be a
good hit. These were ranked using the ‘Phase Screen Score’ with higher scores indicating a better
alignment with the hypothesis. The Phase Screen scores for 3 and 5 were used as contextual
references. The four highest ranking hit structures were all found to be based around the same
trisubstituted isoxazole scaffold with 6 returned as the best match (Figure 2). This same scaffold
was present in 13 of the top 30 hits. However, in each case we noted that only four out of six
pharmacophore features were matched. Therefore, we designed two virtual ligands, 7 and 8, that
incorporated five and six of the features respectively. As expected this led to improved Phase
Screen Scores (Figure 2) and these compounds were therefore selected as initial targets for
experimental investigation.
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COOH
COOH
S
Asinex Gold-
Platinum Collection
289.174 compounds)
N
N
Cl
N
(
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Pharmacophore
Screen (Phase)
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5
Phase Screen Score = 2.466
Phase Screen Score = 2.495
O
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F3C
Rational Design
Cl
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NH
O
Cl
Cl
NH
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NH
O
O
O
OH
OH
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Phase Screen Score =
.152
Phase Screen Score =
2.280
Phase Screen Score =
2.265
2
Figure 2. 3D Pharmacophore screening identifies a compound class with a novel
isoxazole-based chemotype for experimental evaluation. The structural features of 3
incorporated into the pharmacophore hypothesis are indicated: orange = aromatic rings,
green = hydrophobic groups, red = anionic group.
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.2. Exploratory Structure-Activity Relationship Study
Isoxazoles 7 and 8 were synthesized via [3+2] dipolar cycloaddition of a nitrile oxide (generated
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in situ from the oxime chloride 9a) and a commercially available alkyne. The regiochemistry of
the resulting trisubstituted isoxazole esters 10 was confirmed by 2D-NMR experiments (key
HMBC correlations are highlighted in Scheme 1). Ester hydrolysis followed by amide coupling of
tert-butyl-4-amino benzoate via the respective acid chloride, and finally deprotection of the tert-
butyl ester furnished the target compounds in an efficient manner (Scheme 1).
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a
Scheme 1. Synthesis of trisubstituted isoxazoles 7 and 8. Key HMBC correlations used
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to confirm the regiochemistry of 10a and 10b are shown. The C-NMR signals for the
C-5 carbons are distinctively downfield at 175 and 173 ppm respectively.
R
R
OH
R
CF3
O
CF3
N
O
N
O
N
CO Et
CO H
a
c
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H
Cl
Cl
CO Et
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Cl
, R = H
a, R = Cl
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F3C
F3C
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b
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0a R = Me
0b R= Ph
11a R = Me
11b R= Ph
R
H
CO H
H
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HN
H
H
O
N
HMBC
5
4
HMBC
5
e
O
O
Cl
CO Et
O
N
2
CO Et
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N
Cl
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8
R = Me
R = Ph
Cl
F3C
F3C
F3C
10a
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a
Reagents and conditions: (a) NH OH∙HCl, NaOH (aq), EtOH, rt, 18 h, 83%; (b) NCS,
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DMF, 60 °C, 18 h, 86%; then (c) alkyne, NEt , THF, 80 °C, 4 h, 69% (10a), 80% (10b);
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d) LiOH, EtOH, H O, 70 °C, 8 h, 84% (11a), 95% (11b); (e) i) SOCl , 50 °C, 2 h; ii) tert-
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butyl-4-amino benzoate, NEt , CH Cl , 45 °C, 6 h; iii) TFA, CH Cl , rt, 18 h, 42% (7), 69%
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(
8).
To determine if the compounds showed a functional response in terms of RORγt affinity for a
coactivator, 7 and 8 were tested in a time-resolved FRET (TR-FRET) coactivator recruitment
assay.31 Remarkably, both compounds inhibited coactivator recruitment in a dose-dependent
manner. The phenyl derivative 8 was found to be significantly more potent than the methyl
derivative 7: half-maximum inhibitory concentrations (IC ) of 53.5 ± 2.9 μM for 8 compared to
5
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>
100 μM for 7. In line with previous reports, 3 and 5 were determined to be significantly more
potent with an IC of 7.8 ± 0.5 nM and 425 ± 61 nM respectively (Table 1).
5
0
In view of these highly promising TR-FRET results with the in silico derived compounds around
the trisubstituted isoxazole scaffold already showing activity, phenyl isoxazole 8 was selected as
the focus of a subsequent structure-activity relationship (SAR) study focusing on the isoxazole
C-4 position. As such, a small library of 11 derivatives was synthesized using carboxylic acid 11b
as the cornerstone intermediate (Scheme 2) and evaluated using the coactivator recruitment assay
(Table 1). While limited in size, this SAR study indicated that a benzoic acid-containing
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2
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5
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6
substituent at the C-4 position was essential for potency: examples bearing no C-4 substitution
(11b), a para-benzoate (14) or a methylene carboxylic acid (15) showed much reduced potency
compared to the initial hit. Moving the acid moiety to the meta-position (16) or adding a meta-
fluoro substituent (17) somewhat lowered the activity. However, the insertion of a single
methylene unit between the amide and benzoic acid moieties (18) led to a six-fold increase in
potency compared to the initial hit. The corresponding amine (19) displayed similar activity.
Finally, reversing the relative positions of carbonyl and nitrogen components of the amide bond
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(20-22) did not result in a corresponding increase in potency.
Scheme 2. Synthesis of C-4 isoxazole derivativesa
Ph
Ph
Ph
5
O
N
O
N
O
N
CHO
Cl
CO H
2
NH2
Cl
c
4
e
Cl
F3C
F3C
F3C
12
13
11b
d, b
a
a, b
Ph
f, b
g, b
h, b
Ph
Ph
Ph O
Ph O
O
S R
NH
Ph
HN R
Cl
HN R
HN R
R
R
O
N
O
N
O
N
O
N
O
N
O
N
NH
Cl
NH
Cl
O
Cl
O
Cl
Cl
F3C
F3C
F3C
F3C
F3C
F3C
19
14
15-18
20
21
22
R =
t
CO Bu
COOH
F
2
COOH
COOH
COOH
COOH
14
15
16
17
18
19-22
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a
Reagents and conditions: (a) i) SOCl , 50 °C, 2 h; ii) NH R, NEt , CH Cl , 45 °C, 6 h,
2
2
3
2
2
2
7-87%; (b) LiOH, MeOH, H O, 70 °C, 8 h, 43-99%; (c) (i) SOCl , 50 °C, 2 h; ii)
2
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
MeNH(OMe), NEt , CH Cl , rt, 6 h; iii) LiAlH , THF, 0 °C, 30 min, 65%; (d) i) Ethyl-4-
3
2
2
4
aminobenzoate, AcOH, MeOH, reflux, 24 h; ii) NaCNBH , MeOH, reflux, 12 h, 31%; (e) i)
3
DPPA, t-BuOH, 85 °C, 18 h; ii) TFA, CH Cl , rt, 8 h, 59%; (f) i) Mono-methyl terephthalate,
2
2
SOCl , 50 °C, 2 h; ii) 13, NEt , CH Cl , 76%; (g) i) Methyl-4-formyl benzoate, AcOH,
2
3
2
2
MeOH, reflux, 24 h; ii) NaCNBH , MeOH, reflux, 18 h, 43%; (h) Methyl-4-
3
(chlorosulfonyl)benzoate, pyridine, 60 °C, 24 h, 71%.
Table 1. Structure-Activity Relationships around the C-4 isoxazole position. TR-FRET
IC50 values (μM) and respective Glide docking scores are shown. TR-FRET data is
recorded in triplicate; values are representative of >3 repeated experiments.
Cmpd
IC (µM)
Glide Score
– 14.576
50
3
5
0.0078 ± 0.0005
0.425 ± 0.061
– 13.109
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1
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
7
8
> 100
53.5 ± 2.9
> 100
– 13.372
– 14.184
– 10.130
n.d.
11b
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
14
> 100
15
> 100
– 13.724
– 12.995
– 14.308
– 12.020
– 14.012
– 13.550
– 13.519
– 13.003
16
17
18
19
73.9 ± 3.4
91.1 ± 4.6
8.76 ± 0.48
9.60 ± 0.60
> 100
20
21
30.9 ± 1.3
62.6 ± 4.4
22
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2
.3. In Silico Docking Directs Secondary SAR Study
In order to further improve the potency of our compounds we next explored the SAR at the
isoxazole C-5 position. For this, molecular docking (Glide, Schrodinger 2017-2)40,41 was used to
select - with an attention to synthetic resource - C-5 substituents that were optimal for allosteric
binding and therefore activity. For the study, a single C-4 substituent, the amine of compound 19
was chosen based on its experimental activity and in silico docking score (Table 1, vide infra). A
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
2
virtual library of 84 C-5 analogues was enumerated using the open-source ChemT software. This
library was docked against the allosteric site of RORγt as defined by the X-ray crystal structure of
3
1
3
in complex with the RORγt LBD. A single docking pose was returned for each virtual ligand
4
3
and these were ranked using the ‘Glide Score’, an empirical measure of binding enthalpy. We
contextualized these scores by comparison to those of compounds with known activity. The results
(summarized in Table 2, see supporting information for full information) indicated that smaller
heteroaromatic moieties at the C-5 position would improve allosteric binding of the isoxazole
ligands relative to 19 – heteroatoms at the 2-postion were predicted to be optimal e.g. furan 23 and
thiophene 24 (Table 2). The introduction of a hydrogen-bond donor on the ring (specifically at the
3
-position) was predicted to be even more beneficial: docking poses indicated that an additional
hydrogen-bonding interaction with the backbone of helix 4 might be possible (e.g. pyrrole 25,
Table 2, Figure 3). Bulkier substituents were predicted to be detrimental for binding (e.g. naphthyl
2
6). To explore the predicted effect of a hydrogen-bond donating group further we interrogated a
designed subset of ligands in the same docking experiment (see supporting information). None of
these ligands showed an improved Glide score compared to pyrrole 25. However, we noted that 3-
hydroxyl substitution of the C-5 phenyl ring (27) was predicted to significantly enhance binding
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2
2
2
2
2
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5
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5
5
5
5
5
6
relative to 19. To validate our findings experimentally we selected a cross-section of five
derivatives for synthesis (i.e. 23-27).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. In silico modelled docking pose of 25 (green) overlaid with crystal structure of
RORγt with 3 (orange) (PDB code: 4YPQ). For 25 the potential additional hydrogen-bond
with the RORγt H4 backbone is indicated.
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Table 2. Structure-Activity Relationships around the C-5 isoxazole position. TR-FRET
IC50 values (μM) and respective Glide docking scores are shown. TR-FRET data is
recorded in triplicate; values are representative of >3 repeated experiments.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
R
COOH
5
HN
Cl
O
N
4
F3C
23-27
Cmpd
R
Glide Score
– 14.012
IC (µM)
50
1
9
3
Ph
9.60 ± 0.60
1.09 ± 0.09
O
2
– 14.300
– 14.182
– 15.735
S
24
1.75 ± 0.20
HN
25
0.264 ± 0.023
2
6
– 10.844
– 14.603
> 100
HO
27
6.62 ± 0.50
2.4. Docking-Guided C-5 SAR Study
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4
4
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4
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5
5
5
5
5
5
5
5
5
6
To expedite the synthesis of isoxazole analogues with various C-5 and C-4 substituents we re-
designed our synthetic approach. It was envisaged that 5-bromo-4-carboxy isoxazole intermediate
30 would enable later stage introduction of the desired C-5 substituents via palladium-mediated
cross-coupling chemistry. Introduction of C-4 substituents by manipulation of a carbonyl
functional group (as developed previously) would then be possible (Scheme 3).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 3. Retrosynthetic analysis of trisubstituted isoxazole 28 allowing for late-stage
diversification.
R
R
Br
5
5
5
O
N
R1
O
N
2
O
N
2
CO R
CO R
2
2
4
4
4
Cl
Cl
Cl
F3C
F3C
F3C
28
29
30
The intermediate 30 was prepared using analogous methodology to that used previously. In this
case it was necessary to isolate nitrile oxide 33 prior to [3+2] cycloaddition with alkynyl bromide
4
4
32. An efficient cycloaddition reaction led to an essentially quantitative recovery of a 7:3 mixture
1
of 5-bromoisoxazole 30a and 4-bromoisoxazole 30b as determined by H-NMR (Scheme 4). This
result was in close alignment with literature examples that indicated the 5-bromo isomer would
4
4
predominate. The mixture of regioisomers was purified by recrystallization from hot n-heptane
resulting in the isolation of a 97:3 regiomeric mixture (43% recovery) that was employed in
subsequent steps. Assignment of the 5-bromoisoxazole 30a as the major regioisomer was
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confirmed by 2D-NMR analysis of downstream products and by synthesis via an independent route
(see Supporting Information).
a
Scheme 4. Synthesis of isoxazole C-5 analogues 23-27. ‘R’ groups are defined in Table
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2.
Br
CO Me
2
CO Me
2
a
CO Me
2
O
N
O
N
CO Me
Br
Cl
2
H
Br
31
OH
32
Cl
c
F3C
F3C
CF3
O
CF3
N
N
b
7
:3
H
3
0a
30b
Cl
Cl
33
9
d
R
R
R
CO H
2
HN
Cl
O
N
O
O
N
CHO
Cl
CO Me
2
f or g
N
e
Cl
F3C
F3C
F3C
23-27
39-43
34-38
(for 'R' see Table 2)
a
Reagents and conditions: (a) NBS, AgNO , Me C(O), rt, 20 h, 80%; (b) i) NCS, DMF,
3
2
6
0 °C, 18 h, ii) NEt , THF, rt, 30 min, 85%; (c) THF, 80 °C, 4 h, 30a 43%; (d) RB(pin),
3
Pd(dppf)Cl , DME, 85 °C, 8 h, 39-58%; (e) i) LiAlH , THF, 0 °C  rt, 2 h, then ii) DMP,
2
4
CH Cl , rt, 8 h, 51-96%; (f) tert-Butyl-4-amino benzoate, MeOH, AcOH, reflux, 24 h then
2
2
ii) NaBH , EtOH, 85 °C, 2-6 h, 16-24%; iii) TFA, CH Cl , rt, 18 h, 23, 24, 26, 48-73%; (g)
4
2
2
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5
5
5
5
5
5
5
6
Methyl-4-amino benzoate, MeOH, AcOH, reflux, 24 h then ii) NaBH , MeOH, reflux, 2-4
4
h, 16-19%; iii) LiOH, MeOH, H O, 70 °C, 8 h, 25, 57%, 27, 99%.
2
0
1
2
3
4
5
6
7
8
9
0
1
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0
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The desired substituents were introduced at the C-5 position by way of a Suzuki cross-coupling
4
5
with a pinacol boronate (to give intermediates 34-38) before conversion of the C-4 ester to an
aldehyde (39-43) and reductive amination (Scheme 4). The lability of the 5-bromo group under
the conditions for ester reduction dictated the order in which the synthesis steps were performed.
Hydrolysis of the benzoic methyl ester to the free acid yielded the desired compounds 23-27.
In order to explore the SAR around the isoxazole C-5 position, the five analogues prepared in this
second synthesis campaign were evaluated using the HTRF coactivator recruitment assay (Table
2
). We were gratified to observe that furan 23 gave a nine-fold improvement in potency compared
to phenyl 19. By comparison, thiophene 24 was slightly less potent. Most significantly, pyrrole 25,
which also showed the most beneficial Glide score, was 36-fold more potent than 19 and with an
IC value lower than the putative allosteric modulator 5. These results were in excellent agreement
5
0
with the in silico Glide scores obtained (Table 2) and the improvements in potency are a notable
step toward emulating the high potency of indazole 3 (Figure 4-A). As predicted, the bulky
naphthyl group of 26 was detrimental for activity such that no IC curve could be fitted. The
5
0
phenol derivative 27 showed a small improvement in potency compared to 19. For this more bulky
group at the C-5 position, compared to pyrrole 25, the potential for additional hydrogen bonding,
as indicated in the docking study, is thus not strongly expressed.
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2
.5 Mode-of-Action Studies
The allosteric mode-of-action for the novel lead compound 25 was first explored using a
competitive TR-FRET coactivator recruitment assay against fixed concentrations of cholesterol
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(an orthosteric agonist). If an allosteric ligand and cholesterol bind in a non-competitive manner
at different sites on the RORγt LBD then the IC of the allosteric ligand should be independent of
5
0
cholesterol concentration. By contrast, ligands competing for the same binding site should show a
cholesterol-dependent activity profile whereby increasing cholesterol concentration should result
in a corresponding increase in IC50 of the competing ligand.31 In our assay, increasing
concentrations of 25 perturbed coactivator recruitment in the absence of cholesterol with an IC50
value of 247.8 ± 17.7 nM. Interestingly, increasing concentrations of cholesterol indeed resulted
not in an increase but in a further decrease in the IC value for 25 with a concomitant sharpening
5
0
of the Hill slope (Figure 4-B and Table 3). This result provides strong evidence not only for an
allosteric mode-of-action, but also for cooperative behavior between orthosteric and allosteric
ligand binding. The same profile was observed for 5 (Figure 4-C), providing the first evidence that
this compound also modulates RORγt activity in an allosteric fashion. Indazole 3 also exhibited
this behavior (Figure 4-D). By comparison, the IC value for the orthosteric inverse agonist 1
50
increased as the concentration of cholesterol increased (Figure 4-E). Collectively, our competitive
assay data provided strong evidence that 25 functioned as an allosteric inverse agonist.
To further confirm the allosteric mode-of-action for 25 on RORγt we used an orthogonal assay to
directly probe for allosteric ligand binding, as opposed to measuring indirect effects on coactivator
recruitment. This assay used the previously described AlexaFluor647-labelled MRL-871
derivative 44 (Figure 4-G), which upon binding to RORγt shows fluorescent emission as a result
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2
2
2
2
2
2
2
2
2
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5
5
5
5
5
6
32
of FRET from an anti-His terbium cryptate antibody donor. The results of this experiment indeed
corroborated the data obtained from the competitive cofactor recruitment assay (Figure 4-F): the
isoxazole 25 displaced the allosteric probe 44 with an IC = 117.5 ± 8.5 nM, which was lower
5
0
0
1
2
3
4
5
6
7
8
9
0
1
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9
0
1
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5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
than that of 5 (IC = 180.0 ± 17.5 nM). As expected, indazole 3 was highly potent (IC = 17.3 ±
5
0
50
1
.4 nM).
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A
C
E
B
1
1
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0
1
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9
0
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9
0
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2
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7
8
9
0
D
F
G
SO3-
SO3-
N+
COOH
-O3S
O
H
N
Cl
N
N
N
N
H
O
O
F3C
SO3-
44
Figure 4. Biochemical RORγt assay data for 25, 3 and 5. (A) Dose-response curves from
the TR-FRET coactivator recruitment assay; (B-E) Dose-response curves from the
competitive TR-FRET coactivator recruitment assay with fixed concentrations of
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9
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5
5
6
cholesterol (0 µM, 0.25 µM and 1.0 µM); (F) Dose-response curves from the ligand
displacement HTRF assay using 44 (G) as an allosteric probe.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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0
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2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3. IC50 and Hill slope values observed in the competitive TR-FRET cofactor
recruitment
assay.
0 μM Cholesterol
IC (nM) Hill slope
0.25 μM Cholesterol
1.0 μM Cholesterol
Compound
IC (nM)
Hill slope
IC (nM)
Hill slope
50
50
50
-
-
-
0.77 ±
-0.86 ±
0.03
-1.01 ±
0.03
25
5
247.8 ± 17.7
547.3 ± 60.1
12.7 ± 0.6
138.0 ± 5.9
299.5 ± 18.0
9.4 ± 0.3
94.1 ± 3.3
268.9 ± 18.8
7.8 ± 0.2
0.04
0.74 ±
-0.87 ±
0.04
-0.90 ±
0.05
0.06
0.97 ±
.04
-1.04 ±
0.03
-1.20 ±
0.03
3
0
-0.76 ±
.05
33620 ±
1649
-0.77 ±
0.03
85400 ±
4276
-1.01 ±
0.06
1
7012 ± 588
0
Indazole 3 had previously been shown to be selective for RORγt over other NRs (>100-fold), with
31
only minor cross-activity on PPARγ. To give an indication of the cross-reactivity of the isoxazole
series on PPARγ, an HTRF coactivator recruitment assay was performed with compounds 3, 5 and
isoxazoles 19 and 23-27. 3 and 5 show IC values of resp. 7.2 μM and 14.7 μM for PPARγ
50
(vs. 7.8 nM and 425 nM for RORγt) (Table 4), meaning that they show some cross-reactivity to
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PPARγ, but still are 923- and 35-fold selective for RORγt. 25 and all other compounds of the
isoxazole series result in only weak to no PPARγ inhibition (IC values >50 μM), indicating
50
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
that the isoxazole scaffold leads to favorably low PPAR cross-reactivity. Thus, these data
indicate that the novel class of allosteric isoxazole inverse agonists features potential as
efficacious and selective RORγt inverse agonists.
Table 4. IC50 values observed in the competitive TR-FRET cofactor recruitment assay
with PPARγ.
Compound
IC (µM)
5
0
3
5
7.2 ± 0.8
14.7 ± 1.0
78.6 ± 5.6
> 100
19
23
24
> 100
25
26
27
99.3 ± 6.4
> 100
> 100
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5
6
2
.6 Crystallography
Co-crystallization studies were performed for the most potent isoxazole 25 with the RORγt-LBD,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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8
9
0
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0
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
to provide molecular insights in the ligand-receptor interaction. Crystals grew in a P 6 2 2 space
1
group and diffracted to a resolution of 1.61Å (Table S6). In the experimental electron density map,
clear density for compound 25 is observed in the allosteric site, formed by helices 4, 5, 11 and 12
(Figure 5-A, Figure S2). The compound binds to this allosteric site in a similar orientation as 3
(Figure 5-B), as was predicted by our docking studies (Figure 3). The 2,6-disubstituted phenyl ring
common to both 3 and of 25 is located in the exact same part of the binding pocket (Figure 5-B).
Moreover, hydrogen-bonding interactions between the carboxylic acid group and the main-chain
amide hydrogen atoms of A497 and F498, as well as with the side chain of residue Q329, are also
evident in both structures. Unique to 25 is the pyrrole ring, which is oriented to allow a hydrogen
bond interaction with the main-chain carbonyls of residues L353 and K354 (Figure 5-C). The
isoxazole scaffold also allows a deeper penetration of this compound towards helix 4 of RORγt.
In the case of isoxazole 25 the AF-2 loop of the protein and the allosteric ligand are positioned
slightly further apart as compared to 3 (Figure 5-B). These structural data provide clear evidence
for the allosteric binding of 25 to RORγt in an orientation that was predicted with remarkable
accuracy in the docking study (Figure S3), but with specific additional molecular effects resulting
from the novel isoxazole scaffold and pyrrole based substition pattern.
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9
0
Figure 5. Co-crystal structure of RORγt with compound 25 (PDB code: 6SAL). (A) The
tertiary structure of RORγt bound to 25 (stick representation). The final 2Fo – Fc electron
density map of 25 is shown as an isomesh contoured at 1σ; (B) Overlay of the crystal
structure of RORγt bound to 25 and RORγt bound to 3 (PDB code: 5C4O); (C) Zoom-in
on the allosteric pocket of RORγt showing the interactions between 25 and the protein.
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2
.7 Isoxazole 25 Inhibits IL-17a Expression in EL4 Cells
EL4 is a murine lymphoblast cell line that constitutively expresses RORγt. Because RORγt
promotes IL-17a production, an effective means to determine the cellular activity of RORγt inverse
agonists is to measure the reduction in IL-17a mRNA expression levels by quantitative reverse
transcriptase PCR (RT-PCR). To this end, EL4 cells were treated with 10 µM of 3, 25 and 23 for
0
1
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8
9
0
2
4 h before IL-17a mRNA levels were measured (Figure 6). The most potent isoxazole in vitro,
5, significantly reduced IL-17a mRNA expression 27-fold whilst the weaker inverse agonist 23
2
showed a smaller reduction (3.6-fold) compared to the DMSO control. As expected, 3 led to the
most significant decrease in IL-17a expression (48-fold) which was in line with previous reports.
This result demonstrates that the allosteric modulation of RORγt by optimized trisubstituted
isoxazoles leads to an effective cellular response, correlating with the biochemical protein binding
data, and which is known to be beneficial for the treatment of autoimmune disease.10-12
Figure 6. IL-17a mRNA expression in EL4 cells treated with ligand 3, 25 and 23 (10 μM,
2
4h) or DMSO. The level of IL-17a expression was normalized to that of GAPDH
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expression. All data are expressed as the mean ± s.d. (n=3). The relative gene expression
was calculated by the 2-ΔΔCt (Livak) method using the DMSO control as calibrator.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
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2
2
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0
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8
9
0
2
.8 Absorption, Distribution, Metabolism and Excretion (ADME) Profile
To further assess the potential of 25 and isoxazole analogues such as 23 and 8 we investigated the
ADME profile of these compounds, and compared them to indazole 3 (Table 5). The isoxazole
compounds showed favorable profiles compared to 3 in terms of chemical stability, solubility and
permeability through artificial plasma membranes (PAMPA). A metabolic stability study with
human liver microsomes indicated that the 4-methylamino isoxazoles 23 and 25 were more liable
to phase 1 metabolism compared to indazole 3, which showed good stability. 23 and 25 showed
promising phase 2 stability. In blood plasma, whilst inferior to 3, the stability of 23 and 25 was
acceptable, although all these compounds showed high levels of binding to plasma proteins.
Pleasingly, the 5-phenyl-4-amido isoxazole 8 showed a good ADME profile, with comparable
microsomal stability to 3 and reduced plasma protein binding. This likely indicates that further
optimization of the C-4 and C-5 isoxazole substituents has the potential to produce candidate
molecules with desirable in vivo efficacy.
Table 5. ADME properties for ligand 3, 8, 23 and 25
Microsomal Stability
Plasma
Chemical
Stability
Plasma
stability
protein
binding
(%
Solubility PAMPA
µM) (% Flux)
Compound
Phase 2
Phase 1
(
(% remain)
(% remain)
(% remain)
(
CL ,
int
bound)
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