Identification of an aminothiazole series of RORβ modulators

Article abstract of DOI:10.1016/j.bmcl.2018.03.001

Crystallography has identified stearic acid, ALRT 1550 and ATRA as ligands that bind RORβ however, none of these molecules represent good starting points to develop optimized small molecule modulators. Recently, Compound 1 was identified as a potent dual RORβ and RORγ inverse agonist with no activity towards RORα (Fig. 1). To our knowledge, this is one of only two small molecule RORβ inverse agonists identified in the primary literature from a tractable chemical series and represents an ideal starting point from which to design RORβ-selective modulators. Herein we describe our SAR optimization efforts that led to a series of potent neutral antagonists of RORβ.

Full text of DOI:10.1016/j.bmcl.2018.03.001

Accepted Manuscript
Identification of an aminothiazole series of RORβ modulators
Rémi Patouret, Christelle Doebelin, Ruben D. Garcia-Ordonez, Mi Ra Chang,
Claudia Ruiz, Michael D. Cameron, Patrick R. Griffin, TheodoreM. Kamenecka
PII:
S0960-894X(18)30176-8
https://doi.org/10.1016/j.bmcl.2018.03.001
BMCL 25655
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 December 2017
20 February 2018
1 March 2018
Please cite this article as: Patouret, R., Doebelin, C., Garcia-Ordonez, R.D., Ra Chang, M., Ruiz, C., Cameron, M.D.,
Griffin, P.R., Kamenecka, T.M., Identification of an aminothiazole series of RORβ modulators, Bioorganic &
Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.03.001
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Bioorganic and medicinal chemistry letters
Identification of an aminothiazole series of ROR modulators
Rémi Patouret, Christelle Doebelin, Ruben D. Garcia-Ordonez, Mi Ra Chang, Claudia Ruiz, Michael D.
Cameron, Patrick R. Griffin, Theodore M. Kamenecka*1
The Scripps Research Institute, Scripps Florida, Department of Molecular Medicine, 130 Scripps Way #A2A, Jupiter, FL 33458, USA
ARTICLE INFO
ABSTRACT
Crystallography has identified stearic acid, ALRT 1550 and ATRA as ligands that bind RORβ,
however, none of these molecules represent good starting points to develop optimized small
molecule modulators. Recently, Compound 1 was identified as a potent dual RORβ and RORγ
inverse agonist with no activity towards RORα (Figure 1). To our knowledge, this is one of only
two small molecule RORβ inverse agonists identified in the primary literature from a tractable
chemical series and represents an ideal starting point from which to design RORβ-selective
modulators. Herein we describe our SAR optimization efforts that led to a series of potent
neutral antagonists of ROR.
Article history:
Received
Received in revised form
Accepted
Available online
Keywords:
Nuclear receptor
ROR
Selective ligand
Aminothiazole
2016 Elsevier Ltd. All rights reserved.
born blind.13 Most recently, it was discovered that ROR

The nuclear receptor (NR) superfamily of ligand-regulated
transcription factors has proven to be a rich source of targets
for the development of therapeutics for a wide range of
human diseases. Retinoic acid receptor-related orphan
receptors (RORs) are transcription factors that belong to the
steroid hormone nuclear receptor super family. Several
nuclear receptors are characterized as orphan receptors
because the ligands for these receptors are still unknown or
controversial. The NR1F subfamily, known as the retinoic
plays
a
role in osteogenesis by impacting Runx2
expression.14
Levels of ROR

inversely correlate with osteogenic
potential, suggesting that suppression of ROR
osteoblast mineralization. While ROR -/- mice displayed
bone abnormalities, the bone phenotype in ROR -/- mice
has not been characterized. ROR and a subset of ROR
 may drive



-
acid receptor-related orphan receptors (ROR
ROR [NR1F2] and ROR [NR1F3]), regulate several
physiological processes, including the circadian rhythm,
glucose and lipid metabolism, and immune functions. ROR
is expressed in the liver, skeletal muscle, skin lungs, adipose
tissue, kidney, thymus and brain.1,2 ROR
is most highly
 [NR1F1],
regulated genes were found to be overexpressed in bone
biopsies from post-menopausal women, suggesting a role for


ROR
in human age-related bone loss.15


expressed in the thymus, but significant expression is also
found in the liver, skeletal muscle, adipose tissue and
kidney.3 ROR
 has a more restricted expression pattern and
is found in regions of the central nervous system (CNS) that
are involved in processing of sensory information and
components of the mammalian timing system (circadian
clock), including the suprachiasmatic nuclei (SCN), retina,
pineal gland and bone.4,5 ROR
-/- mice show defects in
circadian rhythmicity.4 Aberrant circadian rhythms are
associated with numerous ailments in humans including
bipolar disorder, schizophrenia, major depressive disorder
Figure 1. Lead dual ROR/ROR antagonist
and seasonal affective disorder.6-12 ROR

is expressed in the
While the in vivo functions of ROR
widely explored over the past decade, the exact roles for
ROR still remain elusive, in part due to lack of specific
 and ROR have been
retina and genetic deletion of ROR
 results in retina
degeneration, implicating its role in vision development.

ROR null mice suffer from retinal degeneration, and are

molecular probes that would enable investigation of its
function. Previously it was shown that all trans retinoic acid
———

(ATRA) and a synthetic analog (ALRT 1550) bind to ROR

ROR modulator identified devoid of ROR activity, and
served as the basis for our SAR campaign described herein.
and are functional inverse agonists, but these ligands lack
potency and bind other nuclear receptors including the
RXRs and RARs.16,17 Stearic acid was also shown to bind
ROR
 during expression and purification, but does not
activate the receptor.18,19
Our goal was to identify, characterize and develop potent
ROR
properties to facilitate their use as in vivo probes. Recently,
Phenex Pharmaceuticals identified a potent dual ROR and
ROR inverse agonist with no activity toward ROR
(Compound
, Figure 1).20 To our knowledge, this is one of
only two small molecule ROR inverse agonists identified

selective modulators with sufficient ADME



1

in the primary literature from a tractable chemical series and
Scheme 1. Synthesis of aminothiazole analogs
represents an ideal starting point from which to design
ROR
selective modulators.21
We decided to continue our SAR exploration of 2b and
begin with modifications to the East part of the structure
(Table 2). Synthesis of these aminothiazole analogs was
straightforward following chemistry as described in the
primary literature and is shown in Scheme 1.24
Table 2. East part SAR – Substituted aryl rings
Cmpd
2b
3a
3b
3c
R
3-Cl
aROR

IC50

M
bROR
 IC50 M
Figure 2. Truncations of compound 1
0.24±0.05
(50%)
>40
n.t.
n.t.
2-Cl
Table 1. Binding affinity of truncated analogs
4-Cl
(30%)
Compound
aROR

aROR

bROR
IC50
0.059±0.02 0.013±0.001

M
cROR
2-Br
(15%)
(20%)
(15%)
n.t.
%inhib
80
%inhib
85

IC50
M
3d
3e
3-Br
0.41±0.13
(25%)
1
2a
2b
2c
30
50
35
n.t.
2
n.t.
4-Br
0.24±0.05
>40
3f
3-OMe
3-CF3
3-NO2
3-CN
3-SO2Me
(10%)
n.t.
Percent displacement of [3H]-T09 at 1 M; Displacement of [3H]-T09 from
a
b
3g
3h
3i
0.61±0.06
(15%)
>40
n.t.
c
human RORβ LBD. Values are the mean ± SEM of at least three replicates;
Displacement of [3H]-T09 from human RORγ LBD. Values are the mean ± SEM
of at least three replicates; n.t: not tested.
(5%)
n.t.
Compound 1 can be dissected into three fragments of equal size
and complexity (Figure 2). We were curious to see which
portions of the molecule were absolutely required for activity, so
we synthesized three compounds each lacking one portion of the
parent molecule (Figure 2, 2a-c). The binding potency of the
analogs was determined in a scintillation proximity assay (SPA)
(30%)
n.t.
3j
3-Ph
1.4±0.55
nt
3k
3l
3-Cl, 5-CF3
0.14±0.018
(10%)
3m
3n
3o
3p
3q
3r
3-Br, 5-CF3
3,5-CF3
0.063±0.01
0.31±0.074
(60%)
>40
13.4
n.t.
3
using H T0901317 and recombinant human ROR and ROR
LBDs.22 This assay measures the affinity of the compounds for
3,5-Br
ROR vs ROR and is tabulated in Table 1.
3,5-Dimethyl
3-CF3, 4-Br
3-CF3, 4-Cl
(55%)
n.t.
Removal of the benzoyl portion led to 2a and a significant
reduction in activity against ROR as did removal of the C-
4 aryl group (2c, Table 1). Much to our surprise, truncation
of the west part afforded aminothiazole 2b, which was
0.29±0.076
0.12±0.048
(10%)
>40
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
M); 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 M); n.t. = not tested.
a
selective for ROR
ligand binding pocket in ROR
ROR
(7663Å versus 7053Å, respectfully).23 Despite a ten-
fold drop in potency vs compound , this was the first

vs ROR


. This was unexpected as the
is actually larger than it is in



1

The chlorine substitution was best tolerated at the 3-position
in the phenyl ring, but was not tolerated at the 2- and 4-
positions (2b, 3a, 3b, respectively, Table 2). A scan of the
ortho-(3c), meta-(3d), and para-positions (3e) of the phenyl
ring with a bromine atom showed a similar trend, with the 3-
position optimal for potency. Incorporation of additional
substituents at the 3-position revealed a striking preference
significant effect. None of the analogs tested exhibited any
affinity for ROR . One of the most selective compounds 4c
shows >400-fold selectivity for ROR vs ROR


.
Table 4. Combined SAR of both regions
for hydrophobic groups (3g
to loss of affinity (3f 3h
substitution, we synthesized several 3,5-disubstituted
analogs which led to our most potent compounds to date (3l
3m). Interestingly, while 4-substitution wasn’t well tolerated
alone (3b 3e), inclusion of a 3-CF3 group afforded potent
analogs (3q 3r). None of the compounds had much affinity
for ROR
,
3k), whereas polar residues led
,
,
,
3i 3j). Given the bias for meta
,
,
,
Cmpd.
R1
R2
aROR
IC50
bROR
IC50
n.t.

M

.

M

5a
5b
5c
5d
5e
5f
3,5-(CF3)2
3,5-(CF3)2
3-CF3, 4-Cl
2-OMe
2-F, 6-CF3
2-OMe
0.29±0.13
Table 3. South part SAR – Substituted benzoyl rings
(55%)
n.t.
>40
n.t.
0.16±0.06
(55%)
3-CF3, 4-Cl 2-F, 6-CF3
3-Br, 5-CF3 2-OMe
3-Br, 5-CF3 2-F, 6-CF3
0.16±0.03
(60%)
16.1
n.t.
Cmpd.
R
aROR
IC50

M
bROR
IC50 M


3g
4a
4b
4c
4d
4e
4f
2-Cl
3-Cl
0.61±0.06
>40
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
M); 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 M); n.t. = not tested.
(60)
n.t.

4-Cl
(40)
n.t.

Combining some of the improvements identified in the East
part SAR (R1) with that found in the South part SAR (R2)
were investigated (Table 4). Additivity was not necessarily
observed (5d
less potent binders of ROR
modifications to each ring induce slight shifts in the pocket
to accommodate substitutions. Hence, once optimal
substituents are found for one phenyl ring, the SAR of the
other ring would have to be reinvestigated de novo.
2-CF3
0.09±0.05
(60)
>40
n.t.
3-CF3
,
5f) wherein some compounds were actually
4-CF3
(45)
n.t.

. This might suggest separate
2-Br
0.11±0.012
0.37±0.11
(60)
>40
>40
n.t.
4g
4h
4i
2-OMe
2-Me
To further confirm pharmacology of the compounds in vitro,
2-OCF3
2-F, 6-CF3
2-F, 6-Cl
(45)
n.t.
compounds
HEK293T cells in
ROR ::UAS-Luc or Gal4-ROR
with counterscreening against Gal4-VP16::UAS-Luc (data
not shown). While clearly displayed inverse agonism of all
three receptors (ROR IC50: 0.11 M; ROR IC50: 0.86 M;
ROR IC50: 0.066 M), none of the new analogs displayed
any significant activity. It is unlikely that there are cell
permeability issues given the high hydrophobicity of the
compounds and the compounds do not appear to be toxic at
1,
2b
,
3m
,
5c and 5e were screened in
Gal4-ROR ::UAS-Luc, Gal4-
::UAS-Luc reporter assay
a

4j
0.13±0.028
0.41±0.12
>40
>40


4k
1
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
M); 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 M); n.t. = not tested.
a








the doses tested (10 M and below). However it is possible
With this east-part SAR information in hand, we turned our
attention toward the south part of the structure (Table 3). A
scan of the 2-(3g), 3-(4a), and 4-positions (4b) of the south
part benzoyl ring with a chlorine atom revealed a distinct
preference for substitution at the 2-position (3g). A
that the compounds are neutral antagonists. Despite binding
to the receptor and competeing out T09, they do not induce a
conformational change which can disrupt the AF2 surface
and alter coregulator interactions. Additional studies will
need to be completed to fully understand the phamracology.
complimentary scan with a trifluoromethyl group (4c, 4d,
4e) confirmed this. Inclusion of additional substitutents at
the 2-position showed a range of potencies depending on the
substituent. The 2-Br group was the most potent, but
increasing the size of the group led to reduced binding
Several analogs were investigated for stability in the
presence of mouse liver microsomes (Table 5).
Unfortunately, in vitro stability decreased significantly by
removing the sulfonamide linker (12b). All other analogs
affinity (4g
essentially equipotent to their mono-substituted counterparts
4j vs 4c, and 4k vs 3g). The fluorine substitution had no
, 4h, 4i). A pair of 2,6-disubstituted analogs were
tested also had poor stability as compared to the lead. This
(

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34
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2.9
6.1
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2.5
2.7
5.2
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1
3n
3q
4k
5a
5e
2b
3g
3l
5.2
3m
In summary, we have identified a series of disubstituted
aminothiazoles which bind ROR with excellent potency.
Starting from a potent dual ROR /ROR inverse agonist
removal of the sulfone-containing side chain ablated affinity
for ROR . Optimization of the 4-phenyl and 5-benzoyl
groups led to improvements in ROR potency without
compromising selectivity for ROR . Analogs 3m and 4c


12
13
14
15

1,



were the most potent analogs identified. Current compounds
are not robustly stable in the presence of liver microsomes,
and will require additional manipulation before in vivo
probes are identified. These studies are on-going and will be
reported in due course.
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Corresponding Author
* E-mail: kameneck@scripps.edu
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