Discovery and structural optimization of 4-(4-(benzyloxy)phenyl)-3,4-dihydropyrimidin-2(1H)-ones as RORc inverse agonists

Article abstract of DOI:10.1038/aps.2016.32

Aim: Retinoic acid receptor-related orphan nuclear receptors (RORs) are orphan nuclear receptors that show constitutive activity in the absence of ligands. Among 3 subtypes of RORs, RORc is a promising therapeutic target for the treatment of Th17-mediated autoimmune diseases. Here, we report novel RORc inverse agonists discovered through structure-based drug design. Methods: Based on the structure of compound 8, a previously described agonist of RORa, a series of 4-(4-(benzyloxy)phenyl)-3,4-dihydropyrimidin-2(1H)-one derivatives were designed and synthesized. The interaction between the compounds and RORc was detected at molecular level using AlphaScreen assay. The compounds were further examined in 293T cells transfected with RORc and luciferase reporter gene. Thermal stability shift assay was used to evaluate the effects of the compounds on protein stability. Results: A total of 27 derivatives were designed and synthesized. Among them, the compound 22b was identified as the most potent RORc inverse agonist. Its IC₅₀ values were 2.39 μmol/L in AlphaScreen assay, and 0.82 μmol/L in inhibition of the cell-based luciferase reporter activity. Furthermore, the compound 22b displayed a 120-fold selectivity for RORc over other nuclear receptors. Moreover, a molecular docking study showed that the structure-activity relationship was consistent with the binding mode of compound 22b in RORc. Conclusion: 4-(4-(Benzyloxy)phenyl)-3,4-dihydropyrimidin-2(1H)-one derivatives are promising candidates for the treatment of Th17-mediated autoimmune diseases, such as rheumatoid arthritis, psoriasis, and multiple sclerosis.

Full text of DOI:10.1038/aps.2016.32

Acta Pharmacologica Sinica 2016: 1–9
© 2016 CPS and SIMM All rights reserved 1671-4083/16
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Original Article
Discovery and structural optimization of
4-(4-(benzyloxy)phenyl)-3,4-dihydropyrimidin-2(1H)-
ones as RORc inverse agonists
Xi-shan WU1, 2, #, Rui WANG2, 3, #, Yan-li XING2, 4, #, Xiao-qian XUE2, 3, Yan ZHANG2, Yong-zhi LU2, Yu SONG2, 4, Xiao-yu LUO2,
2,
Chun WU2, Yu-lai ZHOU4, Jian-qin JIANG1, , Yong XU
*
*
1Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing 211198, China; 2Institute of Chemical Biology,
Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China; 3University of Chinese
Academy of Sciences, Beijing 100049, China; 4Department of Bioengineering, School of Pharmaceutical Sciences, Jilin University,
Changchun 130021, China
Aim: Retinoic acid receptor-related orphan nuclear receptors (RORs) are orphan nuclear receptors that show constitutive activity
in the absence of ligands. Among 3 subtypes of RORs, RORc is a promising therapeutic target for the treatment of Th17-mediated
autoimmune diseases. Here, we report novel RORc inverse agonists discovered through structure-based drug design.
Methods: Base on the structure of compound 8, a previously described agonist of RORa, a series of 4-(4-(benzyloxy)phenyl)-3,4-
dihydropyrimidin-2(1H)-one derivatives were designed and synthesized. The interaction between the compounds and RORc was
detected at molecular level using AlphaScreen assay. The compounds were further examined in 293T cells transfected with RORc and
luciferase reporter gene. Thermal stability shift assay was used to evaluate the effects of the compounds on protein stability.
Results: A total of 27 derivatives were designed and synthesized. Among them, the compound 22b was identified as the most potent
RORc inverse agonist. Its IC50 values were 2.39 μmol/L in AlphaScreen assay, and 0.82 μmol/L in inhibition of the cell-based luciferase
reporter activity. Furthermore, the compound 22b displayed a 120-fold selectivity for RORc over other nuclear receptors. Moreover,
a molecular docking study showed that the structure-activity relationship was consistent with the binding mode of compound 22b in
RORc.
Conclusion: 4-(4-(Benzyloxy)phenyl)-3,4-dihydropyrimidin-2(1H)-one derivatives are promising candidates for the treatment of Th17-
mediated autoimmune diseases, such as rheumatoid arthritis, psoriasis, and multiple sclerosis.
Keywords: nuclear receptor; RORc; inverse agonist; SAR; structure-based optimization; autoimmune disease
Acta Pharmacologica Sinica advance online publication, 4 Jul 2016; doi: 10.1038/aps.2016.32
modular structure composed of an N-terminal domain (AF1),
Introduction
Nuclear receptors (NRs) are a large family of ligand-regulated a DNA-binding domain (DBD), a hinge region, a ligand bind-
transcriptional factors that control the expression of target ing domain (LBD) and a transcriptional activation function
genes involved in a range of physiological processes such as domain 2 (AF2). Binding of ligands in the LBD results in the
development, metabolism, and immunity[1]. The human NR recruitment of transcriptional co-activators (eg, steroid recep-
superfamily is composed of 48 members. Approximately half tor co-activator 1, SRC1), or co-repressors (eg, nuclear receptor
of them have been characterized as ligand-activated transcrip- co-repressor, NCoR)[3, 4]
tion factors regulating the expression of target genes. The
.
Retinoic acid receptor-related orphan nuclear receptors
remaining receptors are considered to be orphan NRs because (RORs) are examples of orphan nuclear receptors that show
they do not have well-characterized ligands[2]. NRs share a constitutive activity in the absence of ligands. There are
three subtypes of RORs: RORα (NR1F1), RORβ (NR1F2) and
RORγ (NR1F3), which are also known as RORa, b and c in
# These authors contributed equally to this work.
To whom correspondence should be addressed.
E-mail xu_yong@gibh.ac.cn (Yong XU);
njjjq@aliyun.com (Jian-qin JIANG)
Received 2016-03-16 Accepted 2016-04-05
humans[5]. The three RORs share a high degree of sequence
*
similarity but display distinct tissue distribution patterns and
distinct functional roles in the regulation of many physiologi-
cal processes[6]. Two forms of RORγ are found in both humans

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Wu XS et al
and mice, termed RORγ1 and RORγt; they only differ in their 8 was assessed for its inhibitory effect on RORc. Compound
amino terminal domain. RORγ1 is expressed in a variety 8 was identified using an AlphaScreen assay (IC50=20.89
of tissues, including the thymus, muscle, kidney and liver, μmol/L). This result provided us with confidence that 8 was
whereas RORγt (with a truncated N-terminus) is exclusively a genuine RORc inverse agonist. Thus, compound 8 was
expressed in the cells of the immune system, eg, T helper cells selected as the starting structure for further SAR study. Exten-
and lymphoid tissue inducer cells[7, 8]. RORγt has been shown sive optimization was conducted, which led to the discovery
to have key functions in the differentiation of Th17 cells and of a potent and selective RORc inverse agonist.
the secretion of inflammatory cytokines such as interleukin
17 (IL-17)[9]. Recent studies indicate that the modulation of
Materials and methods
IL-17 levels in autoimmune diseases may be beneficial in treat- General chemistry
ing diseases, including multiple sclerosis, psoriasis, rheuma- All reagents were purchased from commercial sources and
toid arthritis and inflammatory bowel disease. Thus, RORc used without further purification. For the synthesized com-
is a promising therapeutic target for the treatment of Th17- pounds described below, flash chromatography was per-
mediated autoimmune diseases[10, 11]
.
formed using silica gel (300–400 mesh). All of the reactions
Since the discovery of the first small molecule T0901317 were monitored by TLC using silica gel plates (fluorescence
(1, Figure 1), many RORc ligands with agonistic and inverse F254, UV light). 1H NMR spectra were recorded on a Bruker
agonistic activity have been disclosed in the literature[12, AV-400 spectrometer (Bruker, Fallanden, Switzerland) at
13]
.
Some structurally complex natural products, such as 400 or 500 MHz. 13C NMR spectra were recorded at 400 or
digoxin (2) and ursolic acid (UA, 3), have been reported to 500 MHz. CDCl3 is deuterochloroform, and DMSO-d6 is
be RORc inverse agonists[14-16]. Using the T0901317 scaffold hexadeuterodimethylsulfoxide. All coupling constants were
as a lead compound, a series of synthetic RORc inverse ago- measured in hertz, and the chemical shifts (δH and δC) were
nists were developed, including SR1001 (4), SR1555 (5), and quoted in parts per million (ppm) relative to TMS (δ 0), which
SR2211 (6)[17–19]. A team from Genentech identified N-isobutyl- was used as the internal standard. Abbreviations for NMR
N-((5-(4-(methylsulfonyl)phenyl)thiophen-2-yl)methyl)- data are as follows: s=singlet, d=doublet, t=triplet, q=quartet,
1-phenylmethanesulfonamide as RORc inverse agonist (7, Fig- m=multiplet, dd=doublet of doublets, dt=doublet of triplets,
ure 1) via a biochemical screening campaign[20]. Although the app=apparent, and br=broad. The low or high resolution
development of RORc inverse agonists has shown significant mass spectra (LRMS and HRMS) were measured on an Agilent
promise[21, 22], the development of new RORc selective modula- 1200 HPLC-MSD mass spectrometer or an Applied Biosystems
tors with therapeutic potential still remains an urgent need.
Q-STAR Elite ESI-LC-MS/MS mass spectrometer, respectively.
In this paper, we report the discovery of novel 4-(4-(ben- The experimental procedures and characterization of all com-
zyloxy)phenyl)-3,4-dihydropyrimidin-2(1H)-ones as potent pounds are provided in the Supplementary information.
RORc inverse agonists. Compound 8 has been previously
described as an agonist of RORa[23]. Considering the sequence Biological assays
and structural similarity of RORa and RORc and their crucial Protein and peptide preparation
role in the development of Th17-related diseases, compound The human RORc LBD (residues 262–507) was expressed as
Figure 1. Structures of RORc inverse agonists (1–7) and RORa agonist (8) from the literature.
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a His6-fusion protein using the pET24a expression vector method to identify small molecule ligands. In this study, TSA
(Novagen, Madison, WI, USA)[24]. BL21 (DE3) cells that were were carried out using the Bio-Rad CFX96 Real-Time PCR
transformed with this expression plasmid were grown in LB system. All reactions were buffered in 10 mmol/L HEPES,
broth at 25°C until an OD600 of approximately 1.0 was reached, pH 7.5, 150 mmol/L NaCl and 5% (v/v) glycerol at a final con-
and the cells were then induced with 0.1 mmol/L isopropyl- centration of 10 μmol/L protein and 200 μmol/L compounds.
β-D-1-thiogalactopyranoside (IPTG) at 16°C overnight. Cells The 20 μL reaction mix was added to the wells of 96-well PCR
were harvested, resuspended, and high-pressure homog- plates. SYPRO Orange (ABI, Sigma) was added as a fluores-
enized in 200 mL of extract buffer (20 mmol/L Tris (pH 8.0), cence probe at a dilution of 1:1000 and incubated with com-
500 mmol/L NaCl, 10% glycerol, and 25 mmol/L imidazole) pounds on ice at least 30 min. Total DMSO concentration was
per 6 L of cells. The lysate was centrifuged at 20000 rounds restricted to 2% or less. The temperature was raised with a
per minute for 30 min, and the supernatant was loaded onto a step of 0.5°C per minute from 30°C to 80°C, and fluorescence
5-mL NiSO4-loaded HisTrap HP column (GE Healthcare, Pis- readings were taken at each (0.5°C) interval. All experiments
cataway, NJ, USA). The column was washed with extract buf- were performed in triplicates for the RORc inverse agonist
fer, and the protein was eluted with a 25–500 mmol/L imidaz- screen. Melting temperatures (Tm) were calculated by fitting
ole gradient. A gel filtration column (HiLoad S75, 16/60, GE the sigmoidal melt curve to the Boltzmann equation using
Healthcare) was used for a second purification.
GraphPad Prism. ΔTm is the difference in Tm values calculated
for reactions with and without compounds[26, 27]
.
Transient transfection assays
Transient transfection assays were performed as described Molecular docking simulation
previously[11, 24]. 293T cells were maintained in DMEM con- The crystal structure of RORc in complex with compound
taining 10% fetal bovine serum (FBS). Cells were transiently 7 (PDB code: 4QM0) was used as the reference structure in
transfected in Opti-MEM using Lipofectamine 2000 (Invitro- the docking study. Protein structure preparation for dock-
gen), according to the manufacturer’s protocol. The 96-well ing studies included water deletion, hydrogen atom addi-
plates were plated 24 h prior to transfection (15 000 cells tion and protonation state adjustment. All of the ligand and
per well). For all of the experiments, each well of cells was protein preparations were performed in Maestro (version 9.4,
transfected in Opti-MEM with 25 ng of reporter plasmids Schrödinger, LLC, New York, NY, USA. 2013), implemented
and 5 ng of Renilla luciferase expression plasmids. For Gal4- in the Schrödinger program (http://www.schrodinger.com).
driven reporter assays, the cells were transfected with 25 ng The Glide docking program with SP score was used for bind-
of Gal4-RORg LBD (residues 262–507) and 25 ng of pG5Luc ing mode prediction[28]
reporter. For native promoter reporter assays, the cells were
.
co-transfected with the Pcp2/RORE-Luc reporter along with
the plasmids encoding full-length RORc. The cells were then Design rationality
Results
transfected with the indicated expression and reporter plas- In this study, we try to develop new RORc inverse agonists
mids. The media was changed 24 h after transfection, and the starting from compound 8 which was identified as RORa
compounds were added. Cells were incubated for another agonist[23]. In order to predict RORc-8 binding modes, we
24 h followed by harvesting for a luciferase assay using the analyzed known RORc-ligand complex structures. In a previ-
dual-luciferase reporter assay system (Promega). Luciferase ous study from Genentech, compound 7 (Figure 1) was dem-
data were normalized to Renilla luciferase data, which was onstrated to be a potent RORc inverse agonist. In the RORc-
used as an internal transfection control.
LBD-7 complex structure (PDB code: 4QM0), the benzylic ring
of 7 formed a π–π interaction with His479 and disturbed the
secondary structure for helices 11’–12 in the RORc-LBD co-
AlphaScreen assay
Interactions between RORc and ligands were assessed by structure, which may account for the RORc inverse agonist
luminescence-based AlphaScreen technology (Perkin Elmer) profile of 7. The N-isobutyl group of 7 resided in a shallow
using a histidine detection kit from PerkinElmer (Norwalk, hydrophobic pocket lined with Val376, Phe388, and Phe401. It
CT, USA) as previously described[24, 25]. All of the reactions is worth noting that access to the shallow hydrophobic pocket
contained 200 nmol/L receptor LBD bound to nickel acceptor may help to achieve potency improvement[20]
.
beads (5 μg/mL) and 50 nmol/L biotinylated SRC1-4 peptide
Because there is an asymmetric center in this scaffold, we
bound to streptavidin donor beads (5 μg/mL) in the presence first investigated the differences in the RORc binding modes of
or absence of the indicated concentration of control compound the S and R configurations of 8. The molecular docking result
SR2211 or candidate compounds. The N-terminal biotinylated showed that the (S) enantiomer had a repeatable and stable
coactivator peptide SRC1-4 sequence was QKPTSGPQTPQA- binding mode. Thus, we selected 8 (S) for a docking study,
QQKSLLQQLLTE. Compound concentrations varied from and all further docking studies were carried out with 8 (S). In
150 nmol/L to 200 μmol/L in the dose-response assay.
the following discussion, we explored the SAR of this series,
with a concentration on the right-hand side of the molecule,
which does not involve the asymmetric center. Therefore,
Thermal stability shift assay
Thermal stability shift assay (TSA) is an increasingly popular compounds were synthesized as racemates to rapidly produce
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Wu XS et al
structure-activity data.
The molecular docking study of 8 (S) (Figure 2A) and 8 (S)
overlaid with 7 (Figure 2B) was performed. In compound 8,
the carbonyl oxygen of the urea and ester moiety interacted
with the amino acid residues Gly380 and Gln286 through
H-bonds. Two π-π interactions exist between compound 8
and His323 and Phe388. Upon careful examination of the
binding mode of 8 (S) overlaid with 7, we considered the
effects of increasing the size of the terminal aromatic ring of
8 by introducing substituents that would interfere with the
packing of helix12. In addition, we also considered the effects
of introducing substituents on the terminal aryl ring of 8 to
access the hydrophobic pocket lined with Val376, Phe388, and
Phe401. We investigated whether this modification (Figure
3) on the terminal aryl ring of 8 could improve the activity of
RORc.
Figure 3. Strategy for structural optimization.
Synthesis
Compounds 15a-l and 18a-l were synthesized as shown in
Scheme 1. The key intermediates 12a-c were synthesized
from 4-hydroxybenzaldehyde (9a-c), urea (10) and ethyl ace-
toacetate (11) with anhydrous ZnCl2 by means of a Biginelli
multicomponent reaction[23]. Compounds 13a-l and 16a-l were
reduced with NaBH4 in the presence of ethanol, followed by
treatment with phosphorus tribromide in DMF, which gener-
ated intermediates 14a-l and 17a-l, respectively. Condensation
of intermediates 14a-l and 17a-l with intermediates 12a-b was
accomplished using the Williamson ether synthesis to afford
SAR analysis
We first explored the introduction of additional steric bulk
on the terminal aryl ring of 8 in an effort to interfere with the
packing of helix12. As demonstrated in Table 1, the introduc-
tion of a fluorine atom (15a) at the 4-position of the terminal
phenyl ring of 8 resulted in a significant loss of potency.
Naphthalene (15b) and quinoxaline (15c) derivatives also
resulted in a complete loss of activity, whereas benzodioxole
(15d) derivatives showed better activity than 8. To further
investigate the impact of introducing larger substituents onto
the terminal aromatic ring, compound 15e was synthesized.
However, compound 15e had no activity. Replacement of
the phenyl ring in compound 15e with a thiophene (15f)
resulted in a slight potency decrease compared to 8. Interest-
ingly, inserting an oxygen atom between biphenyl groups
at the R1 position dramatically improved RORc inhibition
(15g, 15h, 15i vs 15e). Compound 15g (IC50=3.47 μmol/L)
the desired target compounds 15a-l and 18a-l[29]
.
Compounds 22a and 22b were synthesized as shown in
Scheme 2. Compounds 19a-b were reacted with phenol by
using the Williamson ether synthesis in the presence of Cs2CO3
to give intermediates 20a-b. Compounds 20a-b were reduced
with NaBH4, followed by treatment with phosphorus tribro-
mide, which resulted in intermediates 21a-b. Condensation
of the obtained intermediates 21a-b with the intermediate 12b
was accomplished using the Williamson ether synthesis to
afford the desired target compounds 22a and 22b.
Figure 2. Predicted binding modes of ligands with RORc. (A) The docking models of compound 8 (green) in the RORc ligand binding pocket (orange).
(B) The superimposition of the binding modes of 7 (cyan) and 8 (green). The ligands and important residues are shown as sticks. Hydrogen bonds are
depicted as red dashed lines, and π–π interactions indicted by green dashed lines.
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Scheme 1. Reagents and conditions: (1a) ZnCl2, 80ºC, 1 h; (1b) 1. NaBH4, EtOH, rt, 1 h; 2. PBr3, DCM, rt, 1 h; (1c) 12, K2CO3, DMF, 80ºC, 4 h.
Scheme 2. Reagents and conditions: (2a) phenol, Cs2CO3, DMF, 75ºC, 4 h; (2b) 1. NaBH4, EtOH, rt, 1 h; 2. PBr3, DCM, rt, 1 h; (2c) 12b, K2CO3, DMF,
80ºC, 4 h.
demonstrated a 6-fold improved inhibition compared with 8 ring of the compounds (15g, 15h, 15i) and His479 residues,
(IC50=20.89 μmol/L), whereas adding one flexible carbon atom which interfere with the packing of helix12 in the AF2 region.
between the oxygen atom and the terminal aryl ring led to a We further synthesized compounds 15k and 15l. The results
slight reduction in RORc potency (15j vs 15g). These results demonstrated that both compounds resulted in a complete
indicated that the presence of two aromatic rings linked with loss of activity.
a flexible atom, such as an oxygen atom, at the R1 position
We next investigated the modification of substituent R2
increases the potency of the compounds, which is presumably (Table 2) to probe the hydrophobic pocket lined with Val376,
due to the π-π stacking interaction between the terminal aryl Phe388, and Phe401. Taking into account the synthetic chal-
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Table 1. SAR at the R1 position.
Table 2. SAR at the R2, R3, and R4 positions.
AlphaScreen
TSA
ΔTm
(ºC)b
AlphaScreen
IC50 (µmol/L)a
(% max inhibition)
TSA
ΔTm
(ºC)b
Compound
8
R1
IC50 (µmol/L)a
Compound R2
R3
R4
(% max inhibition)
20.89±0.68 (99)
NA
NA
1.9
NA
4.0
NA
18a
18b
18c
18d
18e
18f
18g
18h
18i
H
Cl
CH3
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
NA
NA
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
21.24±1.22 (67)
NA
5.2
4.9
6.9
4.5
5.8
5.2
4.7
6.4
7.3
7.5
7.0
15a
NA
OCH3
OC2H5
O-nPr
O-nBu
OBn
Cl
O-nPr
O-nBu
OBn
NA
NA
6.38±0.38 (69)
8.86±2.85 (65)
8.87±0.24 (86)
4.72±0.06 (89)
5.51±0.75 (67)
4.74±0.25 (91)
5.10±0.95 (87)
15b
NA
15c
NA
18j
18k
18l
15d
8.71±1.33 (87)
NA
All assay results are reported as the arithmetic mean±SD from at least
two independent experiments, NA=not active.
15e
a Inhibition of RORc LBD recruitment of the biotin-SRC1–4 co-activator
peptide.
b Inhibition of TSA activity of RORc by tested compounds.
15f
29.38±5.81 (65)
3.47±0.07 (75)
4.0
3.4
lenges, we also selected 8 as the starting point for further
structural optimization. We first examined the importance of
the methoxy substitution at the R3 position. When comparing
compounds 18a and 8, it appeared that methoxy substitution
at the R3 position favored efficacy. We then explored a range
of substituents at the R2 position (Table 2). With the methoxy
group maintained at the R3 position, R2 was modified from H
to halogen, small alkyls, and small alkoxyl groups. The results
of these modifications showed that the small substituents at
the R2 position did not provide any potency improvement
(18b, 18c, 18d, 18e vs 8). In the R2 position, substituents with
different groups that were linked by an oxygen atom were
attached to the aryl ring as a means of probing the intended
cavity. Compounds 18f (O-nPr), 18g (O-nBu) and 18h (OBn)
were synthesized and exhibited improved activity. Com-
pound 18f exhibited the best activity, with an IC50 of 6.38
μmol/L and a temperature shift of 5.8ºC in the thermal shift
assay. This result indicated that the propoxy group at the R2
position is the optimal size and fits very well in the hydropho-
bic pocket. Additionally, when comparing compounds 18b,
18f, 18g and 18h with compounds 18i, 18j, 18k and 18l, respec-
tively, it appeared that the introduction of a chlorine atom at
the R4 position increased RORc potency.
15g
15h
5.99±1.12 (55)
2.7
15i
15j
6.32±0.70 (63)
7.37±0.16 (73)
NA
3.1
15k
15l
NA
NA
NA
NA
All assay results are reported as the arithmetic mean±SD from at least
two independent experiments, NA=not active.
a Inhibition of RORc LBD recruitment of the biotin-SRC1–4 co-activator
peptide.
Finally, all the beneficial changes were combined, and com-
pounds 22a and 22b were designed and synthesized (Table 3).
b Inhibition of TSA activity of RORc by tested compounds.
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Table 3. SAR at the R2 position.
AlphaScreen
Luciferase
TSA
ΔTm
(ºC)c
Compound R2
IC50 (µmol/L)a
IC50 (µmol/L)b
(% max inhibition)
(% max inhibition)
22a
22b
Cl
O-nPr
4.60±0.35 (92)
2.39±0.11 (98)
2.84±0.42
0.82±0.00 (93)
4.9
5.2
All assay results are reported as the arithmetic mean±SD from at least
two independent experiments, NA=not active.
a Inhibition of RORc LBD recruitment of the biotin-SRC1–4 co-activator
peptide.
b Inhibition of Luciferase reporter activity of RORc by tested compounds.
c Inhibition of TSA activity of RORc by tested compounds.
Furthermore, we also evaluated their cell activity in a reporter
gene assay. As expected, both compounds showed good
RORc inhibition activity. Compound 22b was the most potent
RORc inverse agonist among all the compounds. The IC50 of
22b was 2.39 μmol/L and 0.82 μmol/L in the protein-based
AlphaScreen assay and the reporter gene assay, respectively
(Figure 4). The TSA assay also showed a stabilizing effect on
the RORc protein with a temperature shift of 5.2ºC.
Figure 4. Dose-response curves for suppression of the derivative activity
of RORc by 22b in the Dual Luciferase assay (A) and AlphaScreen assay (B).
The IC50 values are 2.39 and 0.82 μmol/L, respectively.
tivity for RORc versus the other NRs in our selectivity panel.
One highlight from this work was that 22b was the most
potent RORc inverse agonist, with more than a 120-fold selec-
Selectivity profile
To assess the selectivity profile for the three isoforms of ROR, tivity over the other NRs. 22b showed weak activity at FXR
we profiled the potent inverse agonist compounds with 293T and LXRα. The results indicated that 22b gave a significantly
cell-based assays using Gal4-NR constructs (Table 4). In improved selectivity window over the other NRs.
addition, the selectivity profiles of these compounds for the
farnesoid X receptor (FXR) and liver X receptor (LXR)-α were
investigated using the same cell-based luciferase reporter gene To rationalize the SAR between these novel inverse agonists
assay[30]
and RORc, a molecular docking study was performed with
Discussion
.
Compounds 15g, 18f, 18k, 18l and 22b exhibited potent the most potent compound 22b (S) (Figure 5). The binding
RORc activity in the reporter gene assay and excellent selec- model of 22b (S) (Figure 5A) was similar to that of 8 (S) (Fig-
Table 4. RORc potency and selectivity profiles in Gal4 human transcription assaysa.
Cmpd
Gal4-RORγ-LBD
Gal4-RORα-LBD
Gal4-RORβ-LBD
Gal4-LXRα-LBD
Gal4-FXR-LBD
15g
18f
18k
18l
1.22±0.06 (91)
0.39±0.00 (95)
1.06±0.06 (81)
0.99±0.12 (89)
0.82±0.00 (93)
>100 (44)
>100 (49)
>100 (22)
>100 (27)
>100 (19)
>100 (26)
>100 (8)
>100 (13)
>100 (9)
>100 (5)
>100 (33)
>100 (15)
>100 (5)
>100 (6)
>100 (7)
>100 (13)
>100 (6)
>100 (10)
>100 (8)
>100 (8)
22b
All assay results are reported as the arithmetic mean±SD from at least two independent experiments.
a All assays were conducted in 293T cells transiently transfected with Gal4-NR-LBD luciferase plasmids. All NR assays monitored the suppression of
their respective basal transcriptional activities, an outcome consistent with the inverse agonist activity of the ligands. In this table, positive % max
indicates suppression of the basal reporter signal relative to DMSO-treated cells.
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Wu XS et al
Figure 5. Predicted binding modes of ligands with the RORc protein. (A) 3D presentation of the predicted binding mode of 22b (magenta)
superimposed with 8 (green) in the ligand binding pocket. (B) 2D schematic presentation of the predicted binding mode of 22b in the ligand binding
pocket. The ligands and important residues are shown as sticks. Hydrogen bonds are depicted as red dashed lines, and π-π interactions indicted by
green dashed lines.
ure 2A). For compound 22b, the carbonyl oxygen of the urea Research (XXH12503-05-06), the National Key Basic Research
and ester moiety form two hydrogen bonds with the backbone Program of China (973 Program, 2013CB910601) and Bureau
of Gly380 and Gln286. Three π-π interactions exist between of Science and Information Technology of Guangzhou Munici-
compound 22b and His323, Phe388 and His479. The propoxy pality (2013J4500008). The authors gratefully acknowledge
group of compound 22b resided in a shallow lipophilic pocket support from the Guangzhou Branch of the Supercomputing
lined with Val376, Phe388 and Phe401. Docking results dem- Center of Chinese Academy of Sciences.
onstrated that the SAR was consistent with the binding mode
of compound 22b. Therefore, the docking simulations further
Author contribution
confirmed that 22b is a favorable RORc inverse agonist. As Yong XU, Jian-qin JIANG, and Yu-lai Zhou designed the
discussed above, we showed that two aromatic rings linked experiments; Xi-shan WU, Rui WANG, and Yan-li XING,
with a flexible atom at the R1 position were crucial for RORc performed the experiments. All authors participated in data
inhibition and that the size of the substituent at the R2 posi- acquisition, analysis, and interpretation. Xi-shan WU, Rui
tion was correlated with potency. These findings provide an WANG, Yan-li XING, Xiao-qian XUE, Yan ZHANG, and Yong
important structural basis for further ligand optimization and XU wrote the paper. All authors reviewed the final manu-
suggest that inverse agonists for RORc with even higher phar- script.
macological utility can be obtained in the near future.
In summary, we designed and optimized a new series of
Supplementary information
RORc inverse agonists comprising a 4-(4-(benzyloxy)phenyl)- Supplementary information is available at the Acta Pharmaco-
3,4-dihydropyrimidin-2(1H)-one scaffold. A total of 27 target logica Sinica’s website.
compounds were synthesized and evaluated for their inhibi-
tory activity against RORc. Compound 22b was the most
potent RORc inverse agonist in this series and displayed more
than a 120-fold selectivity for RORc over other NRs as assessed
by cellular assays. It is notable that 22b lacked inhibitory
activity for RORa. The most potent and selective compound
may serve as a useful tool for developing RORc inverse ago-
nists. Further structural optimization and in vivo studies are
currently in progress and will be reported in due course.
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Acknowledgements
We gratefully acknowledge financial support partly from the
National Natural Science Foundation of China (81373325),
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Acta Pharmacologica Sinica