Identification of new nonsteroidal RORα ligands; Related structure-activity relationships and docking studies

Article abstract of DOI:10.1021/ml300471d

A high throughput screen was developed to identify novel, nonsteroidal RORα agonists. Among the validated hit compounds, the 4-(4-(benzyloxy) phenyl)-5-carbonyl-2-oxo-1,2,3,4-tetrahydropyrimidine scaffold was the most prominent. Among the numerous analogues tested, compounds 8 and 9 showed the highest activity. Key structure-activity relationships (SAR) were established, where benzyl and urea moieties were both identified as very important elements to maintain the activity. Most notably, the SAR were consistent with the binding mode of the compound 8 (S-isomer) in the RORα docking model that was developed in this program. As predicted by the model, the urea moiety is engaged in the formation of key hydrogen bonds with the backbone of Tyr380 and Asp382. The benzyl group is located in a wide hydrophobic pocket. The structural relationships reported in this letter will help in further optimization of this compound series and will provide novel synthetic probes helpful for elucidation of complex RORα physiopathology.

Full text of DOI:10.1021/ml300471d

Letter
pubs.acs.org/acsmedchemlett
Identification of New Nonsteroidal RORα Ligands; Related Structure−
Activity Relationships and Docking Studies
Mathieu Dubernet,† Nicolas Duguet,‡ Lionel Colliandre,‡ Christophe Berini,‡ Stephane Helleboid,†
́
Marilyne Bourotte,† Matthieu Daillet,‡ Lucie Maingot,‡ Sebastien Daix,† Jean-Franco
̧
is Delhomel,†
́
Luc Morin-Allory,‡ Sylvain Routier,‡ and Robert Walczak*,†
†Genfit SA, 885 Av. E. Avinee
‡Institut de Chimie Organique et Analytique (ICOA), Universite
Orleans, France
́
, 59120 Loos, France
́
d’Orlea
́
ns, UMR CNRS 7311, rue de Chartres, BP 6759, 45067
́
S
* Supporting Information
ABSTRACT: A high throughput screen was developed to identify novel, nonsteroidal RORα agonists. Among the validated hit
compounds, the 4-(4-(benzyloxy)phenyl)-5-carbonyl-2-oxo-1,2,3,4-tetrahydropyrimidine scaffold was the most prominent.
Among the numerous analogues tested, compounds 8 and 9 showed the highest activity. Key structure−activity relationships
(SAR) were established, where benzyl and urea moieties were both identified as very important elements to maintain the activity.
Most notably, the SAR were consistent with the binding mode of the compound 8 (S-isomer) in the RORα docking model that
was developed in this program. As predicted by the model, the urea moiety is engaged in the formation of key hydrogen bonds
with the backbone of Tyr380 and Asp382. The benzyl group is located in a wide hydrophobic pocket. The structural
relationships reported in this letter will help in further optimization of this compound series and will provide novel synthetic
probes helpful for elucidation of complex RORα physiopathology.
KEYWORDS: RORα, NR1F1, agonist, docking, SAR, Biginelli
he retinoic acid-related orphan receptor alpha (NR1F1,
TRORα) is a ligand-activated transcription factor and a
member of the nuclear hormone receptor superfamily. The
characterization of RORα-deficient mice (staggerer mice)1−3
that carry the natural, nonsense mutation in the NR1F1 gene
has provided a great insight into the critical functions of RORα
in the regulation of a variety of physiological and devel-
opmental processes, related to bone health, energy homeostasis,
immune function, inflammatory responses, brain development
processes, and neuroprotection. RORα activity modulates the
expression of several components of the circadian clock system.
This receptor is also suspected to play a role in integrating the
central pacemaker signal and the rhythmic expression pattern of
downstream (metabolic) genes. For a thorough description of
RORα-related physiology, readers are referred to the recent
excellent reviews.4−7
derivative) binding in RORα LBD stabilized the receptor in the
agonistic conformation. Later, they further demonstrated that
cholesterol sulfate also binds within the pocket, although with
better affinity.9 Recently, 7-α-hydroxycholesterol, 7-β-hydrox-
ycholesterol, and 24-S-hydroxycholesterol were identified as
RORα inverse agonists.10,11 These novel ligands bound to the
target protein with nanomolar affinities, down-regulated RORα
activity in reporter assays, and down-regulated target gene
expression in vitro. The interpretation of dynamic hydrogen/
deuterium exchange experiments (HDX) on the ligand−
receptor complex led to the model in which the unliganded
RORα-LBD (protein produced in bacteria that do not contain
cholesterol) is in an active conformation and constitutively
interacts with the coactivator peptide. The binding of an inverse
agonist ligand, such as 7-α-hydroxycholesterol leads to a
decrease in the affinity for the coactivator binding and results in
Kallen et al. described the first crystal structure of the ligand
binding domain (LBD) of RORα. Surprisingly, authors
identified a molecule of cholesterol that was present in the
ligand binding pocket of the RORα protein that was purified
from insect cells.8 They concluded that cholesterol (or its
Received: December 21, 2012
Accepted: April 10, 2013
Published: April 10, 2013
© 2013 American Chemical Society
504
dx.doi.org/10.1021/ml300471d | ACS Med. Chem. Lett. 2013, 4, 504−508

ACS Medicinal Chemistry Letters
Letter
a decreased transactivation activity and reduced transcriptional
output.11
Table 1. Evaluation of the Chirality Effect on EC50 and Emax
Finally, two synthetic RORα ligands have been recently
published.12,13 These small molecules, SR1078 (dual RORα/γ
agonist) and SR3335 (RORα selective inverse agonist),
modulated RORα activity in reporter assays and target gene
expression in cell culture models.
In this letter, we report on the discovery of new nonsteroidal
RORα ligands by high throughput screening of a small
molecule compound library and on the preliminary optimiza-
tion of one hit series (Hit Scaffold 1, Scheme 1). In particular,
entry
compd
EC50 (μM)
Emax (% 7DC)
1
2
3
1 (racemate)
12.7
10.9
88
85
2 (enantiomer 1)
3 (enantiomer 2)
a
b
>30
>41
a
b
Not calculated (no plateau). Not determined (no plateau).
Scheme 1. General Synthetic Pathway to 4-(4-
(Benzyloxy)phenyl)-5-carbonyl-2-oxo-1,2,3,4-tetrahydro-
pyrimidine to Hit Scaffold 1
ybenzaldehydes I, beta-keto/esters/amides/ketones II, and
(thio)ureas III, according to procedure A or B.15,16 When
necessary, additional synthetic steps were performed to prepare
compounds complementary to our pyrimidinone library.
Two crystallographic structures of RORα are available in the
Protein Data Bank (PDB). These structures are complexes of
RORα with either the cholesterol (1N83)8 or the cholesterol
sulfate (1SOX)9 ligands. They are considered to represent the
active, agonist-induced conformation of the receptor. Super-
position of the two structures showed that the main difference
was located in the mobile loop between the alpha helices H1
and H2, whereas no differences were identified with respect to
the positions that interact with the steroid ligands. We used
these two structures as structural models for the docking
experiments.
the structure−activity relationships (SAR) that corroborate the
results of the docking studies with the most active compounds
(in-house docking model) will be highlighted and further
optimization opportunities will be pointed out.
The screen was accomplished in a cell-free format with the
use of the pull-down assay that measured the recruitment of the
TIF2-BAP reporter hybrid protein on the immobilized GST-
RORα protein. TIF2 (NCOA2) was chosen for this project
since it was previously described to serve as a natural RORα
coactivator with a distinct physiological function.14 The
recruitment assay was validated with a known RORα ligand,
7-dehydro-cholesterol (7DC, cf. Supporting Information
Supplementary Figure 1).8
The pull-down assay was robust, reproducible, and easily
automated. The primary screen was accomplished with a hit
rate of 0.16% and with a satisfactory z′ factor of 0.55. In
addition to steroid compounds, various small molecules were
identified as RORα ligands. A specific activity of all these
compounds was confirmed in dose response studies with a
TIF2-BAP protein but no interaction was found with a
TIF2(mut)-BAP protein, where all the three LXXLL motifs
were invalidated by side directed mutagenesis. In addition,
none of the hits were active in counter-screen assays, which
scored for nonspecific interactions, such as direct GST binding
or BAP (reporter protein) activation (not shown).
Following the assignment (structure related) of each
confirmed hit to a specific chemical series, singletons and hit
series (comprising several hits) were identified. A hit series
evaluation process began, considering drug-like properties,
synthetic accessibility, SAR, etc, and analogues or compounds
complementary to the chemical space around these series,
either purchased, synthesized, or identified within our library,
were tested in dose−response studies.
In the case of Scaffold 1 (Scheme 1), some 300 related
analogues, including several additional active compounds, were
tested.
Compounds 1 and 4−39 (see Tables 1−4) were either
commercially available or prepared by a Biginelli multi-
component reaction (Scheme 1) from appropriate 4-benzylox-
Although cholesterol forms an important hydrogen bond
network with the protein through multiple water molecules, the
sulfate group of the cholesterol sulfate replaces the water
molecules to form direct hydrogen bonds with the residues of
the mobile loop of the protein. These direct interactions modify
the position of the mobile loop residues and consequently
reduce the space volume of the entrance of the binding site of
the protein. It is noteworthy that one water molecule is strictly
conserved in the binding site between the two crystallographic
complexes. This water molecule, located between alanine 330
and arginine 367 plays an important role in the hydrogen bond
network for the binding of the two ligands.
Glide docking software was used in order to construct the
model.17 Four models (the two crystallographic structures with
or without the conserved water molecule) were evaluated for
the docking procedure using the SP mode of Glide. The model
formed by the protein part of the 1N83 structure and the
conserved water molecule reproduced best the binding modes
of both cholesterol and cholesterol sulfate. This model was
used for all the experiments described in the following part.
Compounds were tested in dose response studies in the pull-
down assay (Tables 1−4). As partial agonists, they can be
differentiated both in terms of the affinity (EC50) and the
efficacy (maximum effect, Emax). The affinity was used as the
metrics to score compounds among them. The efficacy was
used to highlight SAR elements or to differentiate compounds
with comparable EC50 values.
Since Scaffold 1 contains an asymmetric center at C-4, the
influence of its configuration on compound affinity was first
considered. Enantiomers 2 and 3 (Table 1) were isolated from
racemic compound 1 by semipreparative HPLC chiral
chromatography and tested on the pull down assay. As
expected, one enantiomer (compound 3) that was isolated in
a high enantiomeric purity (96.78%) was found to be poorly
potent, while enantiomer 2, isolated in a moderate enantio-
505
dx.doi.org/10.1021/ml300471d | ACS Med. Chem. Lett. 2013, 4, 504−508

ACS Medicinal Chemistry Letters
Letter
Table 2. SAR Elements Evidenced for the Benzyloxy Group
a
NA: not active at 30 μM.
enantiomers had a reproducible and stable binding mode.
Although these observations go along with the results obtained
for compounds 2 and 3 in the pull-down assay, no specific
configuration can be assigned to either compounds 2 and 3
without any dedicated analytical study (X-rays).
Table 3. SAR Elements Evidenced for Positions 1, 3, and 6
of the Dehydropyrimidinyl Moiety
All further docking studies were carried on using the (S)
configuration. However, compounds were still purchased or
synthesized as racemates to rapidly produce structure−activity
data.
We next examined the importance of the 4-benzyloxy group
(Table 2, entries 1−10). While benzyl derivative 8 exhibited
moderate affinity and efficacy, both the hydroxyl (compd 4),
methoxy (compds 5 and 7) or propyloxy (compd 6) analogues
were found to be not active. Moreover, chain lengthening from
methylene (compd 9) to ethylene (compd 10) or propylene
(compd 11) resulted in a loss of activity, both in terms of
affinity and efficacy. Substitution of the benzyl ring also had
entry compd
R1
R3
R6
EC50 (μM) Emax (% 7DC)
b
1
2
3
14
15
16
Me
H
H
Me
Me
Et
>19
>30
>30
29
a
a
Me
H
19
b
H
>45
a
b
Not calculated (no plateau). Not determined (no plateau).
meric purity (75.93%), accounted for most of the affinity of
racemate 1
Docking studies with several potent scaffold 1 representa-
tives were also performed to investigate the differences in the
RORα binding mode of (S) and (R) enantiomers. Only (S)
Table 4. SAR Elements Evidenced for the 5-Carbonyl Substituent
a
b
NA: not active at 30 μM. Not calculated (no plateau).
506
dx.doi.org/10.1021/ml300471d | ACS Med. Chem. Lett. 2013, 4, 504−508

ACS Medicinal Chemistry Letters
Letter
beneficial effects; (4-(2-chloro)phenyl) derivative 13 was found
active, while its non-halogenated analogue 12 was not.
The predicted binding mode found for the S-isomer of series
best representative compound, compound 8, is shown in Figure
1. Interestingly, the aryl part is placed in the hydrophobic
subpocket of the active site making it a privileged candidate for
further optimization.
subpocket). Depending on the nature of the substituent in this
position, water molecules can create bridges between the ligand
and the loop, adapting this cavity to tolerate small to bulky
groups. So far, the docking studies accuracy of 5-substituted-
pyrimidinyl derivatives suffered from a relative lack of examples
to properly model and anticipate their interactions with the
mobile loop and also from the weakness of the docking
software to efficiently take into account the high mobility of
some parts of the receptors associated with the dynamic
creation of water bridges.
Synthesis and testing of many more analogues will be helpful
for the optimization process of the substituents in this
particular position.
Finally, when comparing compounds 36 and 37, with
compounds 31 and 9, respectively, it appeared that 3-methoxy
substitution on the central phenyl ring favored the efficacy. A
similar observation was made when comparing the compound
13 with 1 for which the 3-ethoxy substitution enhanced the
efficacy.
In this study, various nonsteroidal RORα ligands (singletons
or members of a hit series) were identified by a high
throughput screening. As a result of the hit series evaluation
process, 4-(4-(benzyloxy)phenyl)-5-carbonyl-2-oxo-1,2,3,4-tet-
rahydropyrimidine hit series was selected for further structure−
activity relationship studies. Several additional compounds with
moderate affinities and efficacies were identified, including the
best compounds 8 and 9. Mostly, key structural elements were
evidenced such as the benzyl and urea moieties, very important
to maintain the activity or modifications on the central phenyl
that enhance the efficacy. Moreover, these structure−activity
results were consistent with the binding mode of the
compound 8 (S-isomer) in the RORα docking model that
was developed. Key hydrogen bonds between the urea moiety
and the backbone of Tyr380 and Asp382 were identified. The
benzyl group was localized in a wide hydrophobic pocket.
SAR knowledge reported herein will help to advance this
chemical series further, to produce more potent RORα agonist
compounds, notably through the optimization of both aromatic
moieties. Preliminary evaluation has also shown that metabolic
stability of this compound series needs to be improved. Our
next goal is the conception of improved compounds, amenable
to in vivo target validation studies.
Figure 1. Superposition of cholesterol binding mode with compound
8-(S) predicted binding mode. Ligands, cholesterol (green carbons)
and 8-(S) (white carbons), are colored by atom type. The conserved
water molecule appears as balls and sticks. Four residues from RORα
binding site are labeled in stick representation: Gln289 and Tyr290
materialize the mobile loop; Tyr380 and Asp382 that interact with 8-
(S) through two hydrogen bonds (yellow dashed lines). These bonds
between the NH in N-3 position and the backbone of Tyr380 and
between the CO of the urea moiety and the backbone of Asp382
anchor 8-(S) in the RORα binding site. Residues 327−335 (front
yellow helix) are hidden.
SAR elements were also established for several positions of
the dehydropyrimidinyl ring.
As observed in the pull-down assay, methylation of the
nitrogen in the N-3 position (Table 3, R3 = Me) disrupted the
activity of compound 15 as compared to compound 9, which is
consistent with the binding mode reported in Figure 1.
Methylation of the nitrogen in the N-1 position (compd 14)
was also deleterious to compound affinity and efficacy as
compared to compound 9.
In the C-6 position, replacement of the methyl (compd 9) by
an ethyl (compd 16) led to a decrease in affinity and efficacy.
In the C-5 position, a panel of 6-methyl-2-oxo-5-substituted-
1,2,3,4-tetrahydro-4-pyrimidinyl derivatives (Tables 2−4) has
been tested so far. Among these, ethyl esters appeared as the
best compromise between affinity and activity. Ethyl esters 8, 9,
and 37 exhibited better affinities than bulkier esters 17, 18, 23−
26, 29−31, 34, and 35. Similarly, ethyl ester 9 was more potent
and efficacious than its methoxy analogue 28 or than
hydrophilic acid and amide analogues 27 and 33. Other
bulky analogues such as amide derivatives (compds 32 and 39)
were inactive. Esters 19−21 were low-affinity and/or low-
efficacy compounds as compared to compound 9.
However, bulky ester 38 was surprisingly as potent as
compound 9, though less efficacious. Similarly, compound 22, a
cyclic analogue of compounds 19−21, was more efficacious
than compound 9, though less potent.
According to the proposed binding mode of compound 8-
(S), the ester group in the C-5 position of the pyrimidinyl
moiety is in proximity with the mobile loop (hydrophilic
ASSOCIATED CONTENT
■
S
* Supporting Information
Materials, full experimental procedures, and characterization of
synthesized compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*(R.W.) Tel: +33 (0)3 2016 4000. Fax: +33 (0)3 2016 4001.
E-mail: robert.walczak@genfit.com.
Author Contributions
Screening campaign and hit series selection were performed at
Genfit by S.H., S.D., M.B., M.D., and J. F.D. under the
supervision of R.W. SAR and drug design were established and
driven by M.D. Chemical synthesis was performed at ICOA by
N.D., C.B., L.M., and M.D. under the supervision of S.R. The
synthetic routes setup were realized by N.D. and C.B.
Molecular modeling was performed at ICOA by L.C. under
507
dx.doi.org/10.1021/ml300471d | ACS Med. Chem. Lett. 2013, 4, 504−508

ACS Medicinal Chemistry Letters
Letter
receptors RORalpha and RORgamma. ACS Chem. Biol. 2010, 5,
1029−1034.
the supervision of L.M.-A. L.M., C.B., L.C., S.R., M.D., and
R.W. authored the manuscript.
(14) Chopra, A. R.; Louet, J. F.; Saha, P.; An, J.; et al. Absence of the
SRC-2 coactivator results in a glycogenopathy resembling Von
Gierke’s disease. Science 2008, 322, 1395−1399.
Funding
This research was accomplished as a part of the AD-Inov
Program and supported by a grant from the French Fond
Unique Interministeriel (FUI).
́
(15) Kumar, R.; Mittal, A.; Ramachandran, U. Design and synthesis
of 6-methyl-2-oxo-1,2,3,4-tetrahydro-pyrimidine-5-carboxylic acid de-
rivatives as PPARγ activators. Bioorg. Med. Chem. Lett. 2007, 17, 4613−
4618.
(16) Sun, Q.; Wang, Y.-Q.; Ge, Z.-M.; Cheng, T.-M.; et al. A highly
efficient solvent-free synthesis of dihydropyrimidinones catalyzed by
zinc chloride. Synthesis 2004, 1047−1051.
(17) Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; et al.
Glide: a new approach for rapid, accurate docking and scoring. 1.
Method and assessment of docking accuracy. J. Med. Chem. 2004, 47,
1739−1749.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank A.L. Enjalbert for the HPLC chiral analysis and the
resolution methods setup and G. Bouly for performing the
HPLC chiral semipreparative purifications.
ABBREVIATIONS
■
BAP, bacterial alkaline phosphatase; GST, glutathione-S-
transferase domain that binds glutathione; LBD, ligand binding
domain; LXXLL, pentapeptide motif on coactivator proteins,
required for an efficient nuclear receptor binding; NR1F1,
RORα, retinoic acid-related orphan receptor alpha; TIF2 or
NCOA2, nuclear receptor COActivator 2 protein
REFERENCES
■
(1) Hamilton, B. A.; Frankel, W. N.; Kerrebrock, A. W.; Hawkins, T.
L.; et al. Disruption of the nuclear hormone receptor RORalpha in
staggerer mice. Nature 1996, 379, 736−739.
(2) Sidman, R. L.; Lane, P. W.; Dickie, M. M. Staggerer, a new
mutation in the mouse affecting the cerebellum. Science 1962, 137,
610−612.
(3) Steinmayr, M.; Andre, E.; Conquet, F.; Rondi-Reig, L.; et al.
Staggerer phenotype in retinoid-related orphan receptor alpha-
deficient mice. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 3960−3965.
(4) Duez, H.; Staels, B. The nuclear receptors Rev-erbs and RORs
integrate circadian rhythms and metabolism. Diabetes Vasc. Dis. Res.
2008, 5, 82−88.
(5) Fitzsimmons, R. L.; Lau, P.; Muscat, G. E. , Retinoid-related
orphan receptor alpha and the regulation of lipid homeostasis. J.
Steroid Biochem. Mol. Biol. 2012, 130, 159−168.
(6) Jetten, A. M. Retinoid-related orphan receptors (RORs): critical
roles in development, immunity, circadian rhythm, and cellular
metabolism. Nucl. Recept. Signaling 2009, 7, e003.
(7) Jolly, S.; Journiac, N.; Vernet-der Garabedian, B.; Mariani, J.
RORalpha, a key to the development and functioning of the brain.
Cerebellum 2012, 11, 451−452.
(8) Kallen, J. A.; Schlaeppi, J. M.; Bitsch, F.; Geisse, S.; et al. X-ray
structure of the hRORalpha LBD at 1.63 A: structural and functional
data that cholesterol or a cholesterol derivative is the natural ligand of
RORalpha. Structure 2002, 10, 1697−1707.
(9) Kallen, J.; Schlaeppi, J. M.; Bitsch, F.; Delhon, I.; et al. Crystal
structure of the human RORalpha Ligand binding domain in complex
with cholesterol sulfate at 2.2 A. J. Biol. Chem. 2004, 279, 14033−
14038.
(10) Wang, Y.; Kumar, N.; Crumbley, C.; Griffin, P. R.; et al. A
second class of nuclear receptors for oxysterols: Regulation of
RORalpha and RORgamma activity by 24S-hydroxycholesterol
(cerebrosterol). Biochim. Biophys. Acta 2010, 1801, 917−923.
(11) Wang, Y.; Kumar, N.; Solt, L. A.; Richardson, T. I.; et al.
Modulation of retinoic acid receptor-related orphan receptor alpha and
gamma activity by 7-oxygenated sterol ligands. J. Biol. Chem. 2010,
285, 5013−5025.
(12) Kumar, N.; Kojetin, D. J.; Solt, L. A.; Kumar, K. G.; et al.
Identification of SR3335 (ML-176): a synthetic RORalpha selective
inverse agonist. ACS Chem. Biol. 2011, 6, 218−222.
(13) Wang, Y.; Kumar, N.; Nuhant, P.; Cameron, M. D.; et al.
Identification of SR1078, a synthetic agonist for the orphan nuclear
508
dx.doi.org/10.1021/ml300471d | ACS Med. Chem. Lett. 2013, 4, 504−508