Identification of potent RORβ modulators: Scaffold variation

Article abstract of DOI:10.1016/j.bmcl.2018.08.017

We sought to develop RORβ-selective probe molecules in order to investigate the function of the receptor in vitro and in vivo and its role in the pathophysiology of disease. To accomplish this, we modified a potent dual RORβ/RORγ inverse agonist from the primary literature with the goal of improving selectivity for RORβ vs RORγ. Truncation of the Western portion of the molecule ablated activity at RORγ and led to a potent series of RORβ modulators. Continued exploration of this series investigated alternate replacement cores for the aminothiazole ring. Numerous suitable replacements were found during the course of our SAR investigations and are reported herein.

Full text of DOI:10.1016/j.bmcl.2018.08.017

Accepted Manuscript
Identification of potent RORβ modulators: Scaffold variation
Christelle Doebelin, Rémi Patouret, Ruben D. Garcia-Ordonez, Mi Ra Chang,
Venkatasubramanian Dharmarajan, Scott Novick, Anthony Ciesla, Sean
Campbell, Laura A. Solt, Patrick R. Griffin, Theodore M. Kamenecka
PII:
S0960-894X(18)30676-0
https://doi.org/10.1016/j.bmcl.2018.08.017
BMCL 25997
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 June 2018
9 August 2018
13 August 2018
Please cite this article as: Doebelin, C., Patouret, R., Garcia-Ordonez, R.D., Ra Chang, M., Dharmarajan, V., Novick,
S., Ciesla, A., Campbell, S., Solt, L.A., Griffin, P.R., Kamenecka, T.M., Identification of potent RORβ modulators:
Scaffold variation, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.
2018.08.017
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Bioorganic and medicinal chemistry letters
Identification of potent RORβ modulators: Scaffold variation
Christelle Doebelina, Rémi Patoureta, Ruben D. Garcia-Ordoneza, Mi Ra Changa, Venkatasubramanian
Dharmarajana, Scott Novicka, Anthony Cieslaa, Sean Campbellb, Laura A. Soltb, Patrick R. Griffina, Theodore
M. Kamenecka*a
aThe Scripps Research Institute, Scripps Florida, Department of Molecular Medicine, 130 Scripps Way #A2A, Jupiter, FL 33458, USA
bThe Scripps Research Institute, Scripps Florida, Department of Immunology and Microbiology, 130 Scripps Way #C2A, Jupiter, FL 33458, USA
ARTICLE INFO
ABSTRACT
We sought to develop RORβ-selective probe molecules in order to investigate the function
of the receptor in vitro and in vivo and its role in the pathophysiology of disease. To
accomplish this, we modified a potent dual RORβ/RORγ inverse agonist from the primary
literature with the goal of improving selectivity for RORβ vs RORγ. Truncation of the
Western portion of the molecule ablated activity at RORγ and led to a potent series of
RORβ modulators. Continued exploration of this series investigated alternate replacement
cores for the aminothiazole ring. Numerous suitable replacements were found during the
course of our SAR investigations and are reported herein.
Article history:
Received
Received in revised form
Accepted
Available online
Keywords:
Nuclear receptor
ROR
2016 Elsevier Ltd. All rights reserved
Selective ligand
Aminothiophene
The retinoic acid receptor-related orphan receptors (RORα,
RORβ and RORγ) are members of the NR1F subfamily of
NRs and they play an important regulatory role in the
maintenance of a variety of physiological and pathological
processes. The RORs have been implicated in the pathology
of diseases associated with dysregulation of the circadian
rhythm including sleep disorders, bipolar disorder and
schizophrenia. Like all NRs, the RORs display the conserved
domain structure with a variable amino-terminal A/B domain,
a central highly conserved two zinc finger DNA-binding
domain (C domain), a hinge (domain D) and a carboxy-
terminal ligand-binding domain (LBD; E domain). The three
RORs display significant sequence similarities but display
distinct patterns of expression. RORα is expressed in the
liver, skeletal muscle, skin, lungs, adipose tissue, kidney,
thymus and brain.1-2 RORγ is most highly expressed in the
thymus, but significant expression is also found in the liver,
skeletal muscle, adipose tissue and kidney.3 The RORγt
isoform, which has garnered much attention lately due to its
role in TH17 cells, is exclusively expressed in key cells within
the immune system.4 RORβ has a more restricted pattern of
expression and is limited to the central nervous system as well
as in bone.5 RORβ is expressed in regions of the CNS that are
involved in processing of sensory information and
components of the mammalian circadian clock, the
suprachiasmatic nuclei, the retina, and the pineal gland.
RORβ-/- mice show defects in circadian rhythmicity,5 exhibit
increased exploration activity and reduced anxiety
behaviour.6 Aberrant circadian rhythms are associated with
numerous ailments in humans including bipolar disorder,
major depressive disorder, and seasonal affective disorder.7-8
Moreover, RORB genes are associated with bipolar disorder,
epilepsy and schizophrenia.6, 9-10
RORβ is also expressed in retinal progenitor cells during
development and genetic deletion of RORβ results in retinal
degeneration implicating its role in vision. RORβ appears to
play a role in the maturation of photoreceptors as RORβ null
mice are born blind.5, 11 Most recently, it was discovered that
RORβ plays a role in osteogenesis by inhibiting Runx2
activity.7 Levels of RORβ inversely correlated with
osteogenic potential suggesting that suppression of RORβ
may drive osteoblast mineralization. Additionally, RORβ and
a subset of RORβ-regulated genes were increased in bone
biopsies from post-menopausal women compared to pre-
menopausal women suggesting a role for RORβ in human
age-related bone loss.12-13
Although much of the work in the field has focused on
development of RORγ inverse agonists for modulation of
immune function including hundreds of citations, little effort
has focused on RORβ, most likely due to the lack of available
chemical starting points for structure-activity relationship
(SAR) studies. Given the receptors specific tissue distribution
and important physiological functions, the identification of
RORβ-selective small molecules would be valuable chemical
probes and pharmacological tools. Prior to 2014, only all trans
retinoic acid (ATRA) and a synthetic analog (ALRT 1550)
had been reported to bind to RORβ and function as inverse
agonists.14-15 Unfortunately, these ligands bind several other
NRs including the RXRs (co-receptors for many other NRs)
and the RARs.

potent and selective RORβ-modulators, including thiazole,
thiophene, and aminothiophene.
Table 1. Core thiazole replacements
Figure 1. Aminothiophenes as potent RORβ modulators
aRORβ
IC50 (μM)
bRORγ
IC50
(μM)
Recently, a modestly potent dual RORβ/γ ligand was
described by Fauber B. et al. with an EC50 of 1.2 μM and an
EC50 of 0.41 μM respectively.16 A more potent modulator was
identified around the same time by a group from Phenex, first
as a RORγ inverse agonist,17 then as a dual RORβ/γ inverse
Cmpd
Ar1
Ar2
0.24±0.05
2.4±1.6
0.57±0.12
30%
>40
15%
5%
2
4
5
6
agonist (Figure 1,
1
).18
In our previous Communication,19 we reported truncation of
the sulfone containing side chain in the dual RORβ/γ ligand
1
led to RORβ-selective compounds (Figure 1, ). Optimization
2
of the aminothiazole scaffold led to a series of more potent
RORβ-selective ligands. Herein, we continue our SAR
investigations of this scaffold and our efforts to improve the
potency while maintaining the excellent selectivity for RORβ
vs RORγ. The binding potency of the analogs was determined
35%
30%
2.6±1.7
50%
40%
25%
0%
7
8
9
3
in a scintillation proximity assay (SPA) using H-T0901317
and recombinant human RORβ and RORγ Ligand Binding
Domains (LBDs). This assay measures the affinity of the
compounds for RORβ vs RORγ and is tabulated in Tables 1-
4.
Our initial efforts to modify the core (Ar1) were based on our
lead molecule
2 (Table 1), wherein we maintained the (3-
chlorophenyl-5-yl)(2-chlorophenyl) methanone substitution.
Attempts to replace the 2-aminothiazole ring with this
substitution pattern were not very successful. Removal of the
0.38±0.06
15%
30%
n.t.
n.t
10
11
12
2-amino group led to a 10-fold loss in affinity (
thiophene ( ) was 2-fold less potent than the aminothiazole.
The aminothiazole isomer ( ) was even less potent indicating
4). The
5
6
the ring sulfur atom is likely more important than the nitrogen
atom towards potency. A 2-pyridyl ring was also not a viable
5%
replacement (
).
8) nor were some more esoteric heterocycles (7,
9
50%
n.t.
13
Given 4-substitution (Ar2) was shown to be important for
potency in the aminothiazole ring,19 we investigated two other
modifications (Table 1). With the benzo[d][1,3]dioxole ring,
furan, oxazole, and benzothiophene ring systems were not
0.61±0.06
0.034±0.017
0.040±0.011
0.23±0.04
10%
>40
10±1
>40
14
15
3
potent (11
,
12
,
13). The thiophene ring, however, was
. The 3-
tolerated (10), with similar activity to compound
5
CF3-phenyl ring was an alternate substitution at the 4-position
on the aminothiazole ring (14). The corresponding thiazole
(
15) and 2-aminothiophene ring substitutions (
3
) showed 6-
fold improvements in potency. Compound
3
was slightly
14±3
n.t
16
17
18
19
more selective for RORβ vs RORγ than 15 (1000-fold vs 300-
fold, respectively). The simple thiophene (16) was about 10-
fold less potent than the corresponding aminothizaole (
subsequently less selective for RORγ as well. Substituting a
methyl group (17) for amino ( ) in the thiophene series also
led to complete loss of activity. Phenyl (18) or aminophenyl
19) ring replacements were modestly more potent on RORβ
3), and
3
0.18±0.03
0.090±0.028
2.6±0.8
3.8±1.1
(
than 14, but also considerably less selective against RORγ.
a Displacement of [3H]-T09 from human RORβ LBD. Values are the mean ±
SEM of at least three replicates. IC50 or displacement of [3H]-T09 at 1 µM);
b Displacement of [3H]-T09 from human RORγ LBD. Values are the mean ±
Based on this first round of core replacements, three viable
substitutions for aminothiazole were found giving the most

SEM of at least three replicates. IC50 or displacement of [3H]-T09 at 1 µM);
n.t. = not tested.
increased affinity for RORγ, there is still a 300-fold window
of selectivity for RORβ. 3,4-bis-substitution was not tolerated
(
38). Attempts to bis-chlorinate the benzoyl group at the 5-
position of the aminothiophene was less successful leading to
mostly impotent compounds 39 43). few (2-
Hence, a second round of SAR was investigated with each of
these core substitutions (Tables 2-4).
(
-
A
methoxyphenyl)methanone substituted aminothiophenes
were also synthesized. While pairing with the 3-CF3-phenyl
A quick scan of 4-phenylthiazole aryl substitutions did not
identify compounds more potent than 15 (Table 2). Most, in
aryl substitution (44) reduced ROR
selectivity against ROR was also diminished. Additional
aryl group substitutions continued to reduce affinity for
RORβ (45 47) as well as selectivity vs RORγ. An interesting
 affinity by 2-fold,
fact, showed less potency on RORβ with the exception of 22
.

The 2-methoxy benzoyl variant of 15 also showed good
affinity for RORβ with good selectivity vs RORγ (25).
-
comparison is 37 vs 46 wherein the 2-OMe group
significantly reduces RORβ potency and selectivity against
RORγ.
Table 2. SAR of 4-substitution on the thiazole ring
Table 3. SAR of 4-substitution on the thiophene ring
aRORβa
IC50 (μM)
bRORγ
IC50
Cmpd
Ar
R
(μM)
2-Cl
2-Cl
2-Cl
0.034±0.017
2.4±1.6
25%
10 ± 1
n.t.
15
4
Cmpd
Ar
R
aRORβ
IC50 (μM)
bRORγ
IC50 (μM)
2-Cl
2-Cl
2-Cl
2-Cl
0.23 ± 0.04
0.57 ± 0.13
0.36
14 ± 3
5%
16
5
n.t.
20
2-Cl
2-Cl
35%
n.t.
21
50%
26
27
0.087±0.02
14 ± 3
0.026±0.027
6.8±3.8
n.t.
22
23
2-Cl
2-Cl
0.98 ± 0.16
0.09±0.02
20%
28
29
2-Cl
25%
3,4-Cl2
0.80±0.48
20%
20%
24
25
0.33±0.076
2-OMe 0.094±0.009
2-Cl
2-Cl
0.38±0.06
0.47±0.09
30%
30%
30
31
a Displacement of [3H]-T09 from human RORβ LBD. Values are the mean ±
SEM of at least three replicates. IC50 or displacement of [3H]-T09 at 1 µM);
b Displacement of [3H]-T09 from human RORγ LBD. Values are the mean ±
SEM of at least three replicates. IC50 or displacement of [3H]-T09 at 1 µM);
n.t. = not tested.
2-Cl
0.015±0.006
0.46±0.24
32
The starting point for SAR in the thiophene series was 16
(Table 3). Several substituted aryl rings at the 3-position of
the
tolerated. Some showed nice improvements in potency for
RORβ (27 29) though the degree of selectivity for RORγ
varied. Compound 27 was the most RORβ-selective analog
identified in the thiophene series (~160-fold selectivity for
RORβ vs RORγ). The 3-Cl-4-pyridyl motif seemed to be a
more selective substitution pattern conferring reasonable
RORβ-selectivity in two compounds (27, 35). Increasing the
size and hydrophobicity of the aryl substitution had varying
effects on RORβ potency, yet this also seemed to increase
affinity for RORγ (29, 32-34).
2-chlorophenyl(thiophen-2-yl)methanones
were
2-OMe
2-OMe
2-OMe
0.18±0.02
0.22±0.02
0.23±0.04
0.78±0.17
0.79±0.32
10.7
33
34
35
,
a Displacement of [3H]-T09 from human RORβ LBD. Values are the mean ±
SEM of at least three replicates. IC50 or displacement of [3H]-T09 at 1 µM);
b Displacement of [3H]-T09 from human RORγ LBD. Values are the mean ±
SEM of at least three replicates. IC50 or displacement of [3H]-T09 at 1 µM);
n.t. = not tested.
There are clearly some trends in SAR between the different
series investigated. Potent and selective RORβ ligands were
identified in each series, but it was also possible to lose
selectivity in each series, depending on substitution patterns
of the groups incorporated.
Finally, SAR optimization of the 2-aminothiophene core was
investigated (Table 4). While mono-3-CF3 substitution on the
4-phenyl ring was potency enhancing
(3), bis-CF3
substitution led to loss of affinity for RORβ (36). Bis-
substitution with smaller groups (Cl, 37), however, led to a
10-fold boost in binding affinity for RORβ and one of the
most potent analogs identified in this study. Despite an

Table 4. SAR of 4 and 5 substitutions of the amino thiophene
ring.
To better understand the structural basis of binding of dual
RORβ/RORγ inverse agonist and aminothiophene analog
1
44 to RORβ, we performed differential hydrogen/deuterium
exchange (HDX) mass spectrometry (Figure 2) using purified
RORβ ligand binding domain (LBD). HDX data show a clear
difference in structural perturbations between the apo
receptor and the liganded complexes indicating that both
and 44 bind to the LBD in different manners. Furthermore,
the HDX signature for is reminiscent of compound binding
to the canonical ligand binding pocket (LBP) in RORβ. This
profile is similar to 25-hydroxycholesterol (25-HC) binding
to RORβ LBD with observed deprotection (increased
exchange with solvent D2O) changes within the LBP
indicative of displacement of a fortuitous E. coli ligand such
as stearic acid that co-purifies with the RORβ LBD.20
1
Cmpd
Ar
R
aRORβ
IC50 (μM)
bRORγ
IC50 (μM)
1
2-Cl
0.040±0.011
0.24±0.07
> 40
25%
3
2-Cl
36
On the contrary, HDX profiles for the aminothiophene analog
44 and T09 suggests compound binding to the AF2 cleft that
harbors the co-regulator binding site in NRs. Allosteric site
compounds that bind near the AF2 cleft were recently
described for the nuclear receptor RORγ.21 Differential
2-Cl
2-Cl
<0.010±0.03
45%
> 3.0
n.t.
37
38
binding modes observed between 1 and 44 suggests that these
2,3-Cl
2,4-Cl
2,5-Cl
2,6-Cl
3,4-Cl
55%
45%
15%
40%
45%
n.t.
n.t.
39
40
41
42
43
44
molecules are anchored by a variegated set of interactions that
help stabilize probe binding and could therefore explain the
2-fold difference in their affinities.
n.t.
n.t.
n.t.
2-OMe 0.076±0.024
> 5.0
2-OMe
2-OMe
2-OMe
0.13±0.02
0.20±0.02
0.33±0.04
2.9±0.73
1.0±0.16
> 5.0
45
46
47
Figure 3: Gal4-LBD assay of compounds
1 and 44.
a Displacement of [3H]-T09 from human RORβ LBD. Values are the mean ±
SEM of at least three replicates. IC50 or displacement of [3H]-T09 at 1 µM);
b Displacement of [3H]-T09 from human RORγ LBD. Values are the mean ±
SEM of at least three replicates. IC50 or displacement of [3H]-T09 at 1 µM);
n.t. = not tested.
To further confirm pharmacology of the compounds in vitro,
compounds 22 33 37 44 and 45 were screened in HEK293T
cells in a Gal4-RORβ::UAS-Luc or Gal4-RORγ::UAS-Luc
reporter assay with counter-screening against Gal4-
VP16::UAS-Luc (data not shown). While 22 and 33 did not
,
,
,
A 3-CF3 phenyl ring at the 4-position of the thiazoles (15) and
display any significant cellular activity, 37
exhibited RORβ inverse agonism (37, RORβ IC50= 3.1 µM
44, RORβ IC50=2.5 µM, 45, RORβ IC50=3.3 µM) with no
, 44 and 45
aminothiophenes (
16) appears to be optimal for both potency and selectivity. A
2-Cl or 2-OMe benzoyl group appears to be optimal at the 5-
position of the thiazoles (15 25) and aminothiophenes ( 44),
and 2-position of the thiophenes (16).
3), and the 3-position of the thiophenes
,
(
activity against RORγ or ROR
shown).
 (Figure 3 and data not
,
3,
RORβ ± 1
RORβ ± 25 -HC
RORβ ± 44
RORβ ± T09
Figure 2: Conformational dynamics probed by HDX of RORβ inverse agonist 1, 25-Hydroxycholesterol (25-HC), 44 and T09

1a.
1b.
Scheme 1. Reagents and Conditions: (a) R1PhCOCl, AlCl3 or SnCl4, DCE, 40
̊
̊C;
(c) diphenylphosphorylazide, Et3N, tBuOH, 90
̊
̊
̊ .
It is unlikely that there are cell permeability issues with 22 and
33 given the high hydrophobicity of the compounds.
Fortunately, compounds do not appear to be toxic at the doses
tested (10 µM and below). However, it is possible that these
compounds are neutral antagonists, much like analogs
previously described.19 Despite binding to the receptor and
competing out T09, perhaps they are too small or lack specific
interactions required to induce a conformational change
within the receptor which can disrupt the AF2 surface and
alter coregulator interactions. Also, both 22 and 33 lack the
2-amino group substitution on the core heterocycle found in
with arylboronic acids with in situ trifluoroacetyl cleavage
gave final products in good yield (36-38, 45-47). To introduce
variation in the acetyl portion of the molecule, the Suzuki
coupling can be done first (51
54), then protecting group
exchange (54 55). Lewis acid mediated acylation and final

deprotection under mild conditions (K2CO3/methanol)
affords final products (39-44).
In summary, we have identified a series of disubstituted
aminothiophenes as potent RORβ inverse agonists. Starting
from a potent dual RORβ/RORγ inverse agonist 1, removal
37 and 44
-
45 which may be a minimal requirement for
, is a potent
, as previously
of the sulfone-containing side chain ablated affinity for
RORγ.19 Optimization of the 4-phenyl and 5-benzoyl groups
led to improvements in RORβ potency without compromising
selectivity for RORγ. Fine tuning of the core led to cell-active
compounds with good potency. Optimization of drug
metabolism properties to identify in vivo active probes is on-
going and will be reported in due course.
cellular activity in this series. By comparison,
dual inverse agonist of ROR
reported (Figure 3).18
1
 and ROR
Different approaches were used in the synthesis of the analogs
described herein. For the thiazole series described in Table 2,
the corresponding aminothiazoles were fashioned as
described in the previous Communication.19 The 2-amino
group was then oxidatively removed using t-BuONO.22
Acknowledgments
This work was supported by the National Institute of Mental
Health (MH108173 to T.M.K. and P.R.G).
For the thiophenes described in Table 3, a simple 2-step
approach was used (Scheme 1a). Acylation of the thiophene
at the 2-position in presence of Lewis acid (AlCl3) gave 3-
Corresponding Author
bromo-thiophen-2-yl-aryl-methanone derivates
followed by a Suzuki-Miyaura coupling of substituted
arylboronic acids lead to final products ( 16 26-35). The
aminothiophenes were obtained following methods described
in Scheme 1b. A Curtius rearrangement of the 4-
bromothiophen-2-carboxylic acid (50) in the presence of
diphenylphosporylazide (DPPA) and t-BuOH afforded the
BOC-protected 2-aminothiophene (51). The BOC-protecting
group was replaced with an acid stable trifluoroacetate group
(49a-b)
* E-mail: kameneck@scripps.edu
5
,
,
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


Truncation of a dual ROR/ROR inverse
agonist ablated activity at ROR
Substituted 2-aminothiophenes were potent
ROR-selective inverse agonists
Differential HDX-MS analysis of ligands with
ROR highlights receptor perturbations