Identification of potent and selective diphenylpropanamide RORγ inhibitors

Article abstract of DOI:10.1021/ml300286h

Retinoic acid-related orphan receptor RORγt plays a pivotal role in the differentiation of T_H17 cells. Antagonizing RORγt transcriptional activity is a potential means to treat T_H17-related autoimmune diseases. Herein, we describe the identification of a series of diphenylpropanamides as novel and selective RORγ antagonists. Diphenylpropanamide 4n inhibited the transcriptional activity of RORγt, but not RORα, in cells. In addition, it suppressed human T_H17 cell differentiation at submicromolar concentrations.

Full text of DOI:10.1021/ml300286h

Letter
pubs.acs.org/acsmedchemlett
Identification of Potent and Selective Diphenylpropanamide RORγ
Inhibitors
Jun R. Huh,‡ Erika E. Englund,† Hang Wang,† Ruili Huang,† Pengxiang Huang,∥ Fraydoon Rastinejad,∥
James Inglese,† Christopher P. Austin,† Ronald L. Johnson,†,⊥ Wenwei Huang,*,† and Dan R. Littman*,‡,§
†NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical
Center Drive, Bethesda, Maryland 20892-3370, United States
‡Molecular Pathogenesis Program, The Kimmel Center for Biology and Medicine of the Skirball Institute, New York University
School of Medicine, New York, New York 10016, United States
§Howard Hughes Medical Institute, 4000 Jones Bridge Road, Chevy Chase, Maryland 20815, United States
∥Sanford-Burnham Medical Research Institute at Lake Nona, 6400 Sanger Road, Orlando, Florida 32827, United States
S
* Supporting Information
ABSTRACT: Retinoic acid-related orphan receptor RORγt
plays a pivotal role in the differentiation of TH17 cells.
Antagonizing RORγt transcriptional activity is a potential
means to treat TH17-related autoimmune diseases. Herein, we
describe the identification of a series of diphenylpropanamides
as novel and selective RORγ antagonists. Diphenylpropana-
mide 4n inhibited the transcriptional activity of RORγt, but
not RORα, in cells. In addition, it suppressed human TH17 cell
differentiation at submicromolar concentrations.
KEYWORDS: retinoic acid-related orphan receptor, RORγ antagonist, diphenylpropanamide, TH17-related autoimmune diseases
etinoic acid-related orphan receptor γ (RORγ) is a
pathogenesis of psoriasis as well.11 Treatment of mice with
Rmember of the nuclear hormone receptor superfamily. neutralizing anti-IL-17 antibodies ameliorates autoimmune
inflammation of the central nervous system, whereas transfer
of cells producing IL-17 exacerbates the disease phenotype.9
Despite the potential of RORγ as a therapeutic target for these
diseases, there are relatively few selective small-molecule RORγ
antagonists disclosed in the literature (Figure 1). Digoxin (1)
selectively antagonizes RORγ with an IC50 of 2.0 μM. Because
of the cytotoxicity associated with digoxin, several less toxic
analogues were synthesized and shown to retain RORγ
inhibitory activity.12 Ursolic acid (2), an active component in
many herbs, selectively inhibited RORγ,13,14 and recently
SR2211 (3) was reported as a selective RORγ antagonist.15
In this letter, we report the discovery and preliminary SAR
(structure−activity relationship) studies of a novel series of
potent and selective RORγ antagonists.
To identify selective RORγ antagonists, we performed a
quantitative high-throughput screen (qHTS)16 of 310000
compounds (PubChem AID: 2604) using a cell-based RORγ
gene reporter assay to detect RORγ transcriptional inhibitors
and a VP16 gene reporter assay as a counterscreen to exclude
nonspecific inhibitors.12 RORγ-dependent actives from the
qHTS were tested in the above assays as well as gene reporter
assays using RORα and DHR312 to confirm RORγ activity and
Although some high-affinity ligands for RORγ have been
identified, such as 7-hydroxy cholesterol, RORγ is still
considered an orphan receptor.1,2 A single gene encodes for
two isoforms, RORγ1 and RORγt, generated by alternative
initiation and splicing, which differ only in their amino terminal
domains.3 RORγ1 is widely expressed in many tissues,
including liver, adipose tissue, skeletal muscle, and kidney.
RORγt is expressed in CD4+/CD8+ thymocytes, lymphoid
tissue inducer cells, innate lymphoid cells that produce IL-17
and/or IL-22, and inflammatory T cells, including TH17 and a
subset of TCRγδ-expressing T cells. TH17 cells are T
lymphocytes that populate intestinal lamina propria and
produce interleukin 17a (IL-17a), IL-17f, and IL-22.3 RORγt
is a key transcription factor for the differentiation of TH17
cells,4 which comprise one of the distinct effector T cell
lineages involved in the regulation of host defense, particularly
against extracellular microorganisms at mucosal barriers.5
Recent studies indicate that TH17 cells play key pro-
inflammatory roles in a variety of autoimmune diseases and
in cancer.6,7 For instance, mice lacking IL-17 or the p19 subunit
of IL-23, which is required for the expansion and possibly the
function of TH17 cells in vivo, are resistant to mouse
autoimmune disease models, including experimental auto-
immune encephalomyelitis, collagen-induced arthritis, and
inflammatory bowel disease.8−10 TH17 cells and the cytokines
they produce appear to have essential functions in the
Received: September 17, 2012
Accepted: November 28, 2012
© XXXX American Chemical Society
A
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Table 1. SAR of Region A
*
Values of IC50 are the mean of four replicates from two independent
experiments. All tested analogues showed no cytotoxicity in the RORγ
assay up to 96 μM.
Figure 1. RORγ inhibitors.
selectivity. In agreement with results from two recent
publications,12,14 digoxin and ursolic acid were identified as
selective antagonists in this screen. We chose to focus on a
series of diphenylpropanamides, where the most potent
member, 4a (Figure 1), had an IC50 value of 3.3 μM in the
RORγ assay and showed no activity in the VP16, RORα, and
DHR3 assays up to 96 μM. More importantly, 4a inhibited
mouse TH17 cell differentiation by 84% and had no effect on
Th1 cell differentiation at 20 μM (data not shown). Thus, 4a
was selected as the lead for SAR studies.
Three regions within 4a were probed for the SAR studies:
the amide region A, aryl substitution of B, and aryl substitution
of C (Figure 2). As described in Scheme 1, chromanone 7 was
Figure 2. SAR plan for 4a.
a
Scheme 1. Synthesis of Diphenylpropanamides
a
Reagents and conditions: (i) TFA, 3−12 h, 60−80 °C. (ii) 2,2′-Bipyridine, Pd(OAc)2, HOAc, DMA, 110 °C, 12 h. (iii) Method A: NHR3R4, DMA,
2−16 h, 80 °C; method B: NHR3R4, Me3Al, r.t. to 60 °C, 6 h.
B
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Table 2. SAR of Regions B and C
Figure 3. Inhibition of mouse Th17 cell differentiation with 4n. Flow
cytometry of intracellular staining for IL-17a and IFN-γ (top) or IL-
17a and FoxP3 (bottom) in sorted naïve T cell populations activated
and expanded in Th17 or Treg polarizing culture conditions. DMSO
or 2 μM compound 4n was added on day 1, and analysis was done on
day 5. The staining control of FoxP3 staining in cells that do not
express FoxP3 is provided in Figure 2b in the Supporting Information.
substituent was removed (4p), a significant loss in potency was
observed. A phenyl group at the 5-position (4q) resulted in
about a 2-fold loss of activity in comparison to the lead (4a).
Electron-withdrawing groups, such as 4-CF3 (4s), 4-Cl (4t),
and 3-MeO (4v), as well as electron-donating groups, such as
4-MeO (4x) and 4-Me (4u), were tolerated. All tested
analogues showed no antagonistic activity in the VP16,
RORα, and DHR3 assays up to 96 μM.
Diphenylpropanamides have been reported as T-type
calcium channel blockers21 and glucocorticoid receptor (GR)
modulators.22 The benzylic stereocenter is known to affect the
biological activities of GR modulators. The enantiomers of 4n
were separated by chiral HPLC to afford 4n(−) and 4n(+),
assigned according to their optical rotation. Compound 4n(−)
*
Values of IC50 are the mean of four replicates from two independent
experiments. All tested analogues showed no cytotoxicity in the RORγ
assay up to 96 μM.
was >20-fold more potent (IC50 = 0.5 μM) than 4n(+) (IC50
=
10.5 μM), demonstrating that the chiral center affects potency.
Compound 4n(−) was inactive against RORα, VP16, and
DHR3 at concentrations up to 96 μM, indicating that it is a
highly selective RORγ transcriptional inhibitor. In addition, a
profiling of 4n(−) for antagonistic activity in a panel of 20
nuclear receptors was carried out in human embryonic kidney
293t (HEK293t) cells, which showed that 4n(−) had only weak
activities against ERRα (IC50 = 14 μM), LXRα (IC50 = 10 μM),
TRα (IC50 = 4.5 μM), and TRβ (IC50 = 13 μM) (Table 2 in
the Supporting Information). On the contrary, 4n(−) showed
no activity against other nuclear receptors including GR.
Compound 4n inhibited RORγt transcriptional activity with an
IC50 value of 300 nM when assayed in HEK293t cells.12
We examined next whether these diphenylpropanamides
target RORγ directly. Fluorescein-labeled 25-hydroxycholester-
ol binds to the RORγ ligand binding domain (LBD) with a Kd
of 109 nM.12 Compounds 4a or 4n inhibited fluoroprobe
binding to RORγ with IC50 values of 680 and 110 nM,
respectively. Compound 4k, an inactive derivative, failed to
displace hydroxycholesterol (Figure 1 in the Supporting
Information). In accordance with the cell-based RORγ gene
reporter assay, compound 4n(−) was 12-fold more potent
(IC50 = 51 nM) than compound 4n(+) (IC50 = 605 nM) in the
competition assays (Figure 1 in the Supporting Information).
prepared either by reaction of phenol 5 with cinnamic acid
617−19 or by reaction of coumarin 8 with boronic acid 9.20
Reaction of chromanone 7 with amine NHR3R4 gave amide 4.
All analogues were evaluated in the RORγ, VP16, RORα, and
DHR3 assays.
Primary, secondary, and aromatic amines were explored in
region A. Compounds 4a−4n were selected from over 40
compounds synthesized for SAR analysis of this area (Table 1).
Secondary (4f) or aromatic amides (4g) showed weak or no
activity. The pyrrolidine (4b), morpholine (4k), and acyclic
tertiary amides (4e) showed no activity, while the unsubstituted
piperidine (4c) and the azepane (4d) had weak potency. The 3-
methyl piperidine (4a) was superior to 2-methyl (4h) and 4-
methyl (4i) piperidines. The 3-methyl (4a) and 3,3-dimethyl
(4j) substitutions had similar potency. Two analogues derived
from meso amines, cis-3,5-dimethylpiperidine (4n) and cis-3,5-
dimethylmorpholine (4l), were about 3−4-fold more potent
than the lead (4a), while cis-3,5-dimethylpiperazine (4m) was
inactive.
The SAR study was also investigated for regions B and C
(Table 2). In region C, there were negligible changes in
potency by removal of the 4-methoxy substituent (4o) or
methylation of the phenol (4r). However, when the 6-methoxy
C
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Figure 5. Inhibition of IL-17 expression from human memory Th17
cells. Human memory (CD45RO+CD45RA−CD3+CD4+-
CCR6+CD161+) cells were purified from healthy donor peripheral
blood samples and were cultured in the presence of IL-1β, IL-23, and
IL-2 for 6 days with or without 3 μM compound 4n. Intracellular
staining for IFN-γ or IL-17A in memory CD4+ T cells from multiple
donors (n = 11 or n = 9) performed on day 6. (Top) Representative
FACS plots from one donor are shown. (Bottom) Each symbol
indicates a separate donor. Statistical analysis was done by a two-tailed
unpaired Student's t test; IL-17A−IFN-γ+, not significant; and IL-
17A+IFN-γ , p < 0.01.
These results indicate that the diphenylpropanamide antago-
nists directly interact with the RORγ LBD.
Because RORγt is a key regulator for Th17 cell polarization,
compound 4n was evaluated further for antagonistic activity in
Th17 differentiation assays. Compound 4n selectively inhibited
murine Th17 cell differentiation (Figure 3 and Figure 2a in the
Supporting Information) without affecting the differentiation of
+
̈
naive CD4 T cells into other lineages, including Th1 and
regulatory T cells (data not shown and Figure 3). Moreover,
compound 4n inhibited RORγt-directed expression of IL-17A
in human T cells but had no effect on expression induced by
the closely related RORα (Figure 4a), confirming its selectivity
for RORγ. We showed previously that RORγt activity is
important both for induction and for maintenance of human
Th17 cells.12 Indeed, compound 4n significantly reduced
Figure 4. Compound 4n inhibits human Th17 cell differentiation. (a)
FACS-sorted naive human CD4 T cells were transduced with
+
̈
lentiviral vectors encoding human RORα-IRES-GFP or RORγt-IRES-
GFP on day 1 (16 h after TCR stimulation), and GFP expressing cells
were gated for anyalsis on day 6. DMSO or 0.5 μM compound 4n was
added 6 h after transduction. (b) Flow cytometry of the production of
̈
IL-17A and IFN-γ by human naive cord blood T cells cultured for 6
induction of IL-17A expression upon in vitro differentiation
days in the presence of IL-2, IL-23, and IL-1β with various
concentrations of TGF-β (ng/mL).
+
̈
of naive cord blood CD4 T cells (Figure 4b) and maintenance
D
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of IL-17A in ex vivo-isolated memory human Th17 cells, with
little effect on IFN-γ expressing cells (Figure 5).
In summary, we have identified a series of diphenylpropa-
namides as novel and selective RORγ antagonists. Preliminary
SAR studies of the active compound identified in the initial
screen, 4a, led to the identification of 4n(−),23 which can serve
as a valuable pharmacological tool for studying various cellular
activities controlled by RORγ.24 Further lead optimization and
in vivo studies are currently ongoing and will be reported in
due course.
ASSOCIATED CONTENT
* Supporting Information
■
S
RORγ, VP16, RORα, and DHR3 assays description; NR
profiling data; in vitro RORγ binding assay; in vitro mouse T
cell and human T cell assays; synthetic procedures; and
analytical data. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*Tel: 301-217-5740. E-mail: huangwe@mail.nih.gov (W.H.).
Tel: 212-263-5711. E-mail: Dan.Littman@med.nyu.edu
(D.R.L.).
Present Address
⊥Division of Cancer Biology, National Cancer Institute.
Author Contributions
J.R.H., J.I., C.P.A., F.R., R.L.J., W.H., and D.R.L. designed the
experiments. J.R.H., E.E.E., W.H., and D.R.L. wrote the
manuscript with input from the coauthors. J.R.H, E.E.E.,
H.W., P.H., R.L.J., and W.H. performed experiments. J.R.H., J.I.,
R.H., R.L.J., W.H., and D.R.L. analyzed the data.
Funding
This research was supported by the NIH Roadmap for Medical
Research and the Intramural Research Program of the National
Human Genome Research Institute, National Institutes of
Health, NIH Grants K99DK091508 (J.R.H.) and R03DA26211
(D.R.L.).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the Kansas University Chemical Methodology and
Library Development program for providing compounds to the
NIH Molecular Libraries Small Molecule Repository. We thank
Paul Shinn and Danielle Van Leer for compound management,
the NCATS analytical group for analytical chemistry support,
and the New York Cord Blood Center for providing samples.
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