Discovery of 2-oxo-1,2-dihydrobenzo[cd]indole-6-sulfonamide derivatives as new RORγ inhibitors using virtual screening, synthesis and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2014.03.065

Retinoic acid receptor-related orphan receptor γ (RORγ), a member of the nuclear hormone receptor superfamily, is a promising therapeutic target for treating Th17-mediated autoimmune diseases. We performed structure-based virtual screening targeting the RORγ ligand-binding domain. Among the tested compounds, s4 demonstrated RORγ antagonistic activities with micromolar IC₅₀ values in both an AlphaScreen assay (20.27 μM) and a cell-based reporter gene assay (11.84 μM). Optimization of the s4 compound led to the identification of compounds 7j, 8c, 8k, and 8p, all of which displayed significantly enhanced RORγ inhibition with IC ₅₀ values of 40-140 nM. These results represent a promising starting point for developing potent small molecule RORγ inhibitors.

Full text of DOI:10.1016/j.ejmech.2014.03.065

Accepted Manuscript
Discovery of 2-oxo-1,2-dihydrobenzo[cd]indole-6-sulfonamide derivatives as new
RORγ inhibitors using virtual screening, synthesis and biological evaluation
Zhang Yan, Xue Xiaoqian, Jin Xiangyu, Song Yu, Li Jing, Luo Xiaoyu, Song Ming,
Yan Weiqun, Song Hongrui, Xu Yong, PhD
PII:
S0223-5234(14)00279-7
10.1016/j.ejmech.2014.03.065
EJMECH 6849
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 February 2014
Revised Date: 19 March 2014
Accepted Date: 22 March 2014
Please cite this article as: Z. Yan, X. Xiaoqian, J. Xiangyu, S. Yu , L. Jing, L. Xiaoyu, S. Ming, Y.
Weiqun, S. Hongrui, X. Yong, Discovery of 2-oxo-1,2-dihydrobenzo[cd]indole-6-sulfonamide derivatives
as new RORγ inhibitors using virtual screening, synthesis and biological evaluation, European Journal of
Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.03.065.
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ACCEPTED MANUSCRIPT
Benzo[cd]indol-2(1H)-one derivatives were developed as new RORγ inhibitors for
developing therapeutic drug treating Th17 mediated autoimmune diseases.

ACCEPTED MANUSCRIPT
RORγ is a promising drug target for treating Th17-mediated autoimmune diseases.
A hit compound was obtained by structure-based virtual screening.
7
7 new compounds were designed and prepared starting from the hit compound.
Several compounds acted as RORγ inhibitors and exhibited promising activities.
SARs were analyzed based on predicted binding modes and assessed activity.

ACCEPTED MANUSCRIPT
Discovery of 2-oxo-1,2-dihydrobenzo[cd]indole-6-sulfonamide derivatives as new
RORγ inhibitors using virtual screening, synthesis and biological evaluation
a,1
a,b,1
a,c
a,c
a
a
Zhang Yan , Xue Xiaoqian , Jin Xiangyu , Song Yu , Li Jing , Luo Xiaoyu ,
a
c
b
a,*
Song Ming , Yan Weiqun , Song Hongrui , Xu Yong
a
Institute of Chemical Biology, Guangzhou Institutes of Biomedicine and Health,
Chinese Academy of Sciences, No.190 Kaiyuan Avenue, Guangzhou Science Park,
Guangzhou, Guangdong 510530, China
b
School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, No.103
Wenhua Road, Shenhe District, Shenyang, Liaoning 110016, China
c
Department of Bioengineering School of Pharmaceutical Sciences, Jilin University,
No.1266 Fujin Road, Chaoyang District, Changchun, Jilin 130021, China
*
Corresponding Author: Yong Xu, PhD; E-mail: xu_yong@gibh.ac.cn
1
These authors contributed equally to this work.
Key words:
RORγ inhibitor, Th17 cell differentiation, virtual screening, nuclear hormone receptor.
Abbreviations:
RORγ, Retinoic acid-related orphan receptor γ; LBD, ligand binding domain; LBP,
ligand binding pocket; IC50, 50% inhibitory concentration; SAR, structure activity
relationship; AlphaScreen, Amplified Luminescence Proximity Homogeneous Assay
1

ACCEPTED MANUSCRIPT
Abstract:
Retinoic acid-related orphan receptor γ (RORγ), a member of the nuclear hormone
receptor superfamily, is a promising therapeutic target for treating Th17-mediated
autoimmune diseases. We performed structure-based virtual screening targeting the
RORγ ligand-binding domain. Among the tested compounds, s4 demonstrated RORγ
antagonistic activities with micromolar IC50 values in both an AlphaScreen assay
(20.27 µM) and a cell-based reporter gene assay (11.84 µM). Optimization of the s4
compound led to the identification of compounds 7j, 8c, 8k, and 8p, all of which
displayed significantly enhanced RORγ inhibition with IC50 values of 40-140 nM.
These results represent a promising starting point for developing potent small
molecule RORγ inhibitors.
2

ACCEPTED MANUSCRIPT
1
. Introduction
T-helper 17 (Th17) cells are crucial effector cells that have been implicated in the
pathology of a variety of human inflammatory and autoimmune diseases, including
multiple sclerosis, rheumatoid arthritis, psoriasis, and inflammatory bowel disease [1].
In Th17 cells, interleukin-17 (IL-17) transcription is mediated by Th17-specific
transcriptional regulators retinoic acid receptor-related orphan receptor α and γ
(RORα and RORγ) [2,3]. In the experimental autoimmune encephalomyelitis (EAE)
mouse model, suppression of IL-17 activity reduced severity of the inflammation
symptom. Inhibition of RORγ may lead to enhanced anti-inflammatory activity.
RORγ is ligand-dependent transcriptional factor that belongs to the nuclear hormone
receptor superfamily. A large number of drugs have been developed to target nuclear
receptor superfamily members. Given its crucial role in suppression of IL-17 activity,
RORγ is a promising therapeutic target for treating Th17-mediated autoimmune
diseases [4].
Since the identification of benzenesulfonamide liver X receptor (LXR) agonist
T0901317 as an inverse RORγ agonist [5], several small molecule RORγ ligands have
been disclosed in the literature (Figure 1) [6,7]. Digoxin and its less toxic analogues
selectively antagonize RORγ and suppress Th17 cell differentiation [8,9]. Ursolic acid,
a natural carboxylic acid that is ubiquitously present in plants, also suppressed IL-17
production by selectively inhibiting RORγ [10]. However, digoxin is a cardiac
glycoside used for treating various heart conditions with a narrow therapeutic index,
and ursolic acid also activates the glucocorticoid receptor, another nuclear receptor.
Thus, the utility of those two natural products as candidates for further development is
limiting. Using the T0901317 scaffold as a lead compound, Griffin el al. developed a
series of synthetic RORγ antagonists, including SR1001, SR2211, and SR1555
[
11-13]. Littman et al. identified diphenylpropanamide derivatives (Figure 1, ML209)
as RORγ antagonists via quantitative high-throughput screening using a cell-based
RORγ gene reporter assay [14]. Although significant progress has been made in
3

ACCEPTED MANUSCRIPT
developing RORγ antagonists [15-17], the identification of novel, non-steroidal RORγ
antagonists for therapeutic use still remains an urgent need.
In this study, we report the successful application of structure-based virtual screening
in novel RORγ antagonist discovery. We screened a commercial small molecule
database with approximately 220,000 compounds that was provided by Specs Ltd.
Several non-steroidal compounds were identified as RORγ antagonists in a biological
assay.
Of
these,
the
s4
hit
compound
bearing
a
2
-oxo-1,2-dihydrobenzo[cd]indole-6-sulfonamide scaffold was selected as a starting
point for further structural optimization. In total, 77 derivatives were designed,
synthesized or purchased, and these were assessed in a bioassay. Several derivatives
had better suppression of the RORγ basal transcriptional activity than the starting
compound s4. The most potent compounds 7j, 8c, 8k, and 8p inhibited RORγ with
IC50 values of 40-140 nM. Their RORγ antagonism was 300-fold higher than the hit
compound s4. These compounds thus represent a promising starting point for
developing potent small molecule RORγ antagonist with the potential for treatment of
autoimmune diseases.
2
2
. Results and discussion
.1. Virtual screening
Structure-based virtual screening (SBVS) has been widely used in drug discovery.
There are several successful examples of SBVS, particularly in nuclear receptor
ligand discovery [18-20]. A schematic representation of the hit discovery strategy
used in this work is presented in Figure 2. In this study, SBVS was first performed
with the molecular docking program Glide, implemented in Schrödinger. After
hierarchical molecular docking, post-processing and expert inspection, 24 compounds
were selected based on their docking score, docking pose, hydrogen bond and
hydrophobic interaction characterizations. Candidates were purchased from Specs Ltd.
for further biological assay.
4

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All of the selected compounds were assessed for their inhibitory activity using the
AlphaScreen biochemical assay, which is a widely used assay for assessing
ligand-dependent interactions between nuclear receptors and their co-regulators
[
21-23]. In the present study, we monitored RORγ ligand binding domain (LBD)
interactions with the fourth LXXLL motif of coactivator SRC1 (SRC1-4) in the
presence of the assessed compounds. Among the 24 molecules (Supplementary
Figure S1, Table S1), 13 compounds demonstrated over 50% inhibition at 50 µM
concentration in the AlphaScreen assay (Supplementary Figure S2). Compound s4
was characterized by a completely new scaffold (Figure 1), and it demonstrated
moderate antagonistic activity in the AlphaScreen assay (IC50 = 20.27 µM) and a
cell-based luciferase reporter gene assay (IC50 = 11.84 µM) (Supplementary Table
S2). To investigate the receptor-ligand interaction details, the binding mode was
predicted by molecular docking, as demonstrated in Figure 3. In compound s4, the
carbonyl oxygen on the amide group forms a strong hydrogen bond with Arg367 of
helix H5, and the NH of the sulfonamide group forms a direct hydrogen bond with
Phe377. A π-π interaction exists between the ligand and the phenyl group in region B
(Figure 1). These structure and interaction characterizations are common for nuclear
receptor ligands. The compound anchors to the hydrophobic LBD through some
conserved hydrogen bonds. s4 was therefore selected as the starting point for further
structural optimization.
2
.2. Chemistry
Compound s4 (Figure 1) features a 2-oxo-1,2-dihydrobenzo[cd]indole-6-sulfonamide
scaffold and was selected as a template for new RORγ antagonist optimization.
Considering the predicted interactions between s4 and the residues in the RORγ LBD,
we kept the common benzo[cd]indol-2(1H)-one moiety and modified two regions of
this molecule to improve antagonistic activity (Figure 1). First, the hydrogen atom in
region A was replaced with an ethyl to explore whether a larger group was tolerable.
The amine group in region B was then replaced with different primary and secondary
amines to explore how shape, hydrophobicity, and flexibility affect potency.
5

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A general procedure for the synthesis of the designed compounds (6, 7, 8) is outlined
in Scheme 1. The commercially available benzo[de]isochromene-1,3-dione (1)
reacted with PTSA in the presence of pyridine to produce compound 2, which was
then reacted with NaOH followed by HCl treatment to produce compound 3.
Subsequently, compound 3 was reacted with ethyl iodide in DMF to produce
compound 4, and compound 4 was then reacted with chlorosulfonic acid in
chloroform to obtain compound 5. Finally, the target compounds (6, 7, 8) were
obtained from the reactions of compound 5 with different amines.
Scheme 1. Synthesis of 2-oxo-1,2-dihydrobenzo[cd]indole-6-sulfonamide derivatives.
Reagents and conditions: (a) NH
mol/L NaOH, EtOH, H O, reflux, 3 h; (2) HCl, rt 30 min; (c) NaH, C
to rt 3 h; (d) HSO Cl, CHCl , 0 ˚C to 50 ˚C, 6.0 h; (e) R -NH-R , DMAP, TEA, DMF,
4
OH·HCl, pyridine, PTSA, 95 ˚C, 1 h; (b) (1) 2.7
2
2 5
H
I, DMF, 0 ˚C
3
3
2
3
rt overnight or DIPEA, DCM, rt overnight or 2,6-lutidine, THF/acetone=1:1, 65 ˚C,
overnight or pyridine, rt overnight.
2
.3. SAR study
Starting from the hit compound s4, we purchased and synthesized 77 compounds.
First, we investigated the influence of different fused heterocyclic ring derivatives in a
protein-based AlphaScreen assay and a cell-based reporter gene assay. For the
cell-based reporter gene assay, we determined the RORγ transcriptional activity using
a widely used Gal4-driven promoter. We also assessed compounds for their
suppression of the transcriptional activity of the RORγ reporter gene that contained
the natural ROR response element (RORE), which was derived from the Purkinje cell
protein 2 gene (see experimental section for details) [23]. Dose response curves for
T0901317 and compounds in this study are demonstrated in Supplementary Figure
6

ACCEPTED MANUSCRIPT
S4. For most compounds, the suppression effects of the basal transcriptional activity
for both RORγ LBD and full length RORγ are similar. As demonstrated in Table 1,
compound 6a demonstrated 11-fold improved inhibition compared with s4 in a
reporter gene assay, while adding one flexible carbon atom between the scaffold and
the fused heterocyclic ring completely abolished inhibition (6b, 6c, 6d, 6e vs. 6a).
Compound 6f contained an isoquinoline group on the right side and demonstrated
significant improvement with an IC50 value of 0.10 µM. Naphthalene (6g), benzofuran
(6h), or indoline (6i) derivatives resulted in complete loss of RORγ inhibition.
To further explore SAR, substituents with different rings that were linked by linkers
of various lengths were attached to sulfonamide (Table 2). Introduction of a
4
-morpholinophenyl moiety increased RORγ potency by more than 35-fold (7a vs. s4).
An ethyl substituent at the R position did not greatly alter the potency (7b vs. 7a),
1
while increasing linker flexibility by adding 3 carbon atoms to the morpholine group
resulted in a loss of inhibition (7d vs. 7a). Modifying the substituent R from
3
4
-morpholinophenyl (7a) to biphenyl-4-yl (7e) also resulted in a loss of inhibition.
Introducing of a 3-morpholinophenyl (7f) to the R position completely abolished
3
inhibition, suggesting that the substituent position and hydrophobic properties are
critical for binding (7f vs. 7a). Similar to 7f, compounds 7g, 7h and 7i did not
demonstrate any inhibition. Interestingly, inserting an oxygen atom between biphenyl
groups dramatically improved RORγ inhibition and had an IC50 of 0.14 µM (7j vs. 7e).
The compounds with relatively rigid diazene (7k) or amide (7m) linkers demonstrated
moderate gains in cellular activity.
To investigate the spatial effect, smaller compounds were also obtained and assessed
for SAR analysis (Table 3). There was a slight increase in inhibition with the
introduction of a tert-butyl carbamate group at the meta-position of the phenyl group
(8a vs. s4), which was completely lost with a similar group attached to piperidine ring
at the R position (8b). An acetyl group attached to both the phenyl and piperidine
3
groups resulted in significant gains in cellular activity; however, an aromatic phenyl
7

ACCEPTED MANUSCRIPT
group was preferred. Compound 8f, which has an acetyl group at the ortho-position of
the phenyl group, was inactive, while 8g and 8c have an acetyl group at the meta- or
para-position and were ranked as the most potent RORγ antagonists. Modifying the
substituent from 3-tert-butyl phenyl (8i) to 2-tert-butyl phenyl (8j) caused a loss of
inhibition. Compound 8k, which bears a 3,4-dimethoxybenzyl group, demonstrated
2
comparable activity to 8c and 8g. Adding another substituent group at the R position
resulted in similar inhibition (8p vs. 8k). Adding one atom before the aromatic ring
resulted in a loss of inhibition (8m, 8r, 8t, 8u), but adding small substituents, such as
a halogen atom, caused minor gains in activity (8s, 8q and others not listed in this
table), which is presumably because of the disruption of the π-π stacking interaction
between the ligand and LBD residues.
To rationalize the SAR between these novel inhibitors and RORγ, we investigated
ligand binding modes within the LBD using a molecular docking method. The crystal
structure demonstrated that digoxin bound in the ligand-binding pocket of RORγ [9].
Three O atoms of digoxin made direct hydrogen bonds with Phe377 in β-strand 1,
Arg367 in helix H5 and His479 in helix H11. Two O atoms of digoxin formed
hydrogen bonds via water molecules with Glu379 in loop s1-s2 and Val367 in the H5.
A recently reported crystal structure demonstrated that T0901317 (PDB code
4
NB6.pdb) is bound in the RORγ ligand-binding pocket. In the ligand binding pocket,
the phenyl-sulfonamide group on T0901317 forms a unique π-π stacking interaction
with Phe378, Phe388 and Phe401 [15]. In the present study, compound s4 directly
forms two hydrogen bonds with residues in the LBD. The carbonyl oxygen on the
amide forms a strong hydrogen bond with Arg367 of H5, while the NH of the
sulfonamide group makes a direct hydrogen bond with the carboxyl oxygen atom of
the Phe377 backbone. Strong π-π interactions also exist between the inhibitors and
Phe378, Phe388 and Phe401. All of the compounds presented here bear similar
structural characteristics but differ in shape and flexibility on the right side. The
predicted binding modes of the most potent compounds, 7j, 8c, 8k, 8p, are
demonstrated in Figure 4 (see Supplementary Figure S3 for other compounds). The
8

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2
-oxo-1,2-dihydrobenzo[cd]indole-6-sulfonamide moiety on the left side fits snugly
into the hydrophobic pocket, and the polar head forms a conserved hydrogen bond
with Arg367. This bulky group linked with a sulfonamide restrains the direction of the
3
R group. The phenyl group close to the sulfonamide NH forms a strong π-π
interaction; however, the rigid substituent group connected to this phenyl group
reduces potency because of steric conflicts with Ile397 (7e, 7f, 7g). This phenyl group
is important for potency because of the strong π-π interaction, and two aromatic rings
linked with a flexible atom are able to maintain the potency of the compounds (7k,
7
m, 7j). The compounds bearing a flexible linker with different carbon chain lengths
or piperidine rings lose inhibition in both the reporter gene assay and the
protein-based AlphaScreen assay (7c, 7d, 8e, 8m, 8q, 8r, 8t, 8u and 7h, 8b). The
most potent and simple substitute is a small aromatic phenyl ring (8g, 8k, 8p). Further
medicinal chemistry optimization should be performed with small or flexible
substituents on the right side or a small substituent on the left side.
3
. Conclusion
In summary, we have discovered a new series of RORγ antagonists using a
structure-based virtual screening approach in conjunction with medicinal chemistry
optimization and biological evaluation. Based on the initial hit compound s4, 77
derivatives were synthesized or purchased and assessed with the AlphaScreen assay
and the luciferase reporter gene assays. Of these, 20 derivatives demonstrated
remarkably improved activity; the most potent compounds, 7j, 8c, 8k, 8p,
demonstrated 300-fold improvement compared with compound s4. SAR analysis
3
demonstrated that changes in R group substituents critically influenced antagonistic
activity. Further left side structural optimization and in vivo studies are currently in
progress and will be reported in due course.
4
4
. Experimental section
.1. Molecular docking
The crystal structure of RORγ in complex with the antagonist digoxin (PDB code:
9

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3
B0W.pdb) was used as the reference structure in the docking study. Protein structure
preparation for docking studies included water deletion, hydrogen atom addition and
protonation state adjustment. All of the ligand and protein preparation were performed
in Maestro (version 9.4, Schrödinger, LLC, New York, NY, 2013) implemented in the
Schrödinger program (http://www.schrödinger.com). The Specs database subset with
nearly 220,000 compounds was selected and downloaded from the ZINC website
(http://zinc.docking.org) for virtual screening. In this study, structure-based virtual
screening was performed with the Glide molecular docking program (version 6.1,
Schrödinger, LLC, New York, NY, 2013) using the high-throughput virtual screening
(HTVS), Glide SP, and Glide XP modes. For all of the methods, glide docks flexible
ligands into a rigid receptor structure. Final ranking from the docking was based on
the docking score, which combines the Epik state penalty with the Glide Score. Glide
HTVS docking without using any constraints output the top 20,000 structures for next
step evaluation. Using hydrogen bond constraints with Arg367, Glide SP output 5000
structures based on the docking score. These 5000 structures were further evaluated
with Glide XP, and 115 compounds were kept according to their binding mode and
docking scores (lower than -9.0 kcal/mol). Finally, 24 compounds were selected and
purchased from Specs Ltd. for subsequent biological evaluation (Supplementary
Figure S1 and Table S1). For these compounds, which were obtained from the
substructure search or were optimized from s4, molecular docking was performed to
predict the plausible binding mode and binding affinity.
4
.2. Substructure and similarity search
To obtain structurally similar compounds to s4 from the Specs database, a
substructure search or similarity search was performed using the Canvas module as
implemented in Schrödinger. Part of s4 was used as a query structure, and 292
compounds were obtained; the resulting structures were clustered according to their
functional-class fingerprints using the hierarchical cluster protocol in Canvas. Of
these, 37 compounds were selected based on their Fpscreen score and structural
diversity, and they were then purchased from Specs Ltd.
4
.3. Chemistry
The reagents were purchased from commercial sources and used without further
10

ACCEPTED MANUSCRIPT
purification. For the synthesized compounds described below, flash chromatography
was performed using silica gel (300-400 mesh). All of the reactions were monitored
1
by TLC using silica gel plates (fluorescence F254, UV light). H-NMR spectra were
recorded on a Bruker AV-400 spectrometer at 400 MHz. Coupling constants (J) were
1
3
expressed in hertz (Hz). C-NMR spectra were recorded at 400 or 500 MHz.
Chemical shifts (δ) were reported in parts per million (ppm) (using TMS as an
internal control). Signals were described as singlet (s), doublet (d), triplet (t), quartet
(q), multiplet (m), and broad (br). The low- or high-resolution mass spectra (LRMS
and HRMS) were measured on an Agilent 1200 HPLC-MSD mass spectrometer or an
Applied Biosystems Q-STAR Elite ESI-LC-MS/MS mass spectrometer, respectively.
1
13
(
See Supplementary Figure S5 for H-NMR and C-NMR spectra of compounds 6f,
7
a, 7k, 8a, 8c, 8i, 8k and 8s).
4
.3.1. 1,3-dioxo-2,3-dihydro-1H-phenalen-2-yl 4-methylbenzenesulfonate (2).
To a solution of compound 1 (11.9 g, 60 mmol) in pyridine (70 mL) was added
hydroxylamine hydrochloride (4.18 g, 60 mmol). The reaction mixture was heated to
reflex temperature for 1 h followed by cooling to 80 ˚C. Powdered p-toluenesulfonyl
chloride (22.9 g, 120 mmol) was added to the system mixture refluxed for 1 h. After
cooling to room temperature, the reaction mixture was poured into ice water (1000
mL) and stirred to precipitate crystals. The precipitate was filtered and rinsed with
additional cool water (100 mL) and saturated NaHCO (100 mL) to give the title
3
compound (17.2 g, 78% yield). The compound was used for the next step without
+
further purification. MS (ESI), m/z for C20
H
14
O
5
S ([M + H] ): Calcd 366.06, found
3
67.0.
4
.3.2. Benzo[cd]indol-2(1H)-one (3).
To a solution of compound 2 (17.2 g, 47 mmol) in ethanol (50 mL) and water (40 mL)
was added an aqueous solution of sodium hydroxide (2.7 mol/L, 60 mL) at room
temperature. The mixture was heated to reflux temperature for 3 h while distilling the
ethanol. After the reaction was completed, the reaction mixture was cooled to 75 ˚C,
concentrated hydrochloric acid was added dropwise, and a yellow precipitate was
11

ACCEPTED MANUSCRIPT
formed. Then, the mixture was further cooled. The precipitate was collected by
filtration and washed with water (100 mL×2). The resulting crude product was
purified by silica gel chromatography with dichloromethane to give the title product
+
as a yellow solid (6.65 g, 82%). MS (ESI), m/z for C11
H
7
NO ([M + H] ): Calcd
1
69.05, found 170.0.
4
.3.3. 1-ethylbenzo[cd]indol-2(1H)-one (4).
Compound 3 (6.65 g, 39 mmol) and sodium hydride (2.81 g, 117 mmol) were
dissolved in DMF (100 mL) and cooled to 0 ˚C. Ethyl iodide (7.33 g, 47 mmol) was
added dropwise. The reaction mixture was stirred at 0 ˚C for approximately 0.5 h. The
cooling bath was removed, and the reaction mixture was stirred at room temperature.
TLC demonstrated that the reaction was complete, and ice water (200 mL) was added.
The reaction mixture was extracted with ethyl acetate (150 mL×2). The organic layer
2 4
was washed with brine and dried over Na SO . The solid was filtered off, and the
filtrate was concentrated under reduced pressure. The resulting crude product was
purified by silica gel chromatography with petroleum ether/ethyl acetate (10/1, v/v) to
yield the title product as a yellow oil (6.46 g, 84%). MS (ESI), m/z for C11H11NO ([M
+
+
H] ): Calcd 197.08, found 198.0.
4
.3.4. 1-ethyl-2-oxo-1,2-dihydrobenzo[cd]indole-6-sulfonyl chloride (5).
To a solution of compound 4 (6.46 g, 33 mmol) in chloroform (100 mL) was added
chlorosulfonic (11.5 g, 99 mmol) dropwise at 0 ˚C for 10 min. The reaction mixture
was heated at 50 ˚C for 6 h. The mixture was then poured into ice water. The reaction
mixture was extracted with DCM (150 mL×2). The organic layer was washed with
brine and dried over Na SO . The solid was filtered off, and the filtrate was
2
4
concentrated under reduced pressure. The resulting crude product was purified by
silica gel chromatography with petroleum ether/ethyl acetate (5/1, v/v) to yield the
3
title product as a yellow solid (5.75 g, 59%). MS (ESI), m/z for C13H10ClNO S ([M +
+
H] ): Calcd 295.01, found 296.0.
12

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4
.3.5. 1-ethyl-N-(isoquinolin-7´-yl)-2-oxo-1,2-dihydrobenzo[cd]indole-6-sulfonamide
(6f).
A reaction mixture of compound 5 (70 mg, 0.237 mmol) and isoquinolin-7-amine (68
mg, 0.473 mmol) in pyridine (4 mL) was stirred at room temperature overnight. Water
was added, the aqueous layer was extracted with ethyl acetate (50 mL×3), and the
2 4
organic layer was washed with water and brine, dried with Na SO and evaporated.
The residue was purified by silica gel chromatography with petroleum ether/ethyl
1
acetate (2/1, v/v) to afford 6f (76 mg, 96%) as a yellow solid. H-NMR (400 MHz,
DMSO-d
6
) δ 11.04 (s, 1H, SO
.32 (d, J = 5.6 Hz, 1H, 7-H), 8.29 (d, J = 8.0 Hz, 1H, Ar-H), 8.09 (d, J = 7.2 Hz, 1H,
-H), 7.93 (t, J = 7.6 Hz, 1H, 4-H), 7.78 (d, J = 8.8 Hz, 1H, Ar -H), 7.72 (s, 1H, Ar
2
NH), 9.15 (s, 1H, 8´-H), 8.74 (d, J = 8.4 Hz, 1H, 5-H),
8
3
-H), 7.62 (d, J = 6.0 Hz, 1H, Ar -H), 7.47 (d, J = 9.2 Hz, 1H, Ar -H), 7.23 (d, J = 8.0
1
3
2 3
Hz, 1H, 8-H), 3.84 (q, J = 7.2 Hz, 2H, CH ), 1.18 (t, J = 7.2 Hz, 3H, CH ). C-NMR
(500 MHz, DMSO-d
6
) δ 167.57, 152.46, 144.42, 142.79, 137.32, 134.77, 132.63,
1
1
31.69, 129.91, 129.39, 128.90, 128.22, 126.98, 126.17, 125.76, 124.84, 124.80,
-
17 3 3
20.78, 114.67, 104.85, 35.50, 14.47. MS (ESI), m/z for C22H N O S ([M - H] ):
Calcd 403.45, found 402.0.
4
1
.3.6.
-ethyl-N-(4´-morpholinophenyl)-2-oxo-1,2-dihydrobenzo[cd]indole-6-sulfonamide
(
7a).
To a solution of compound 5 (100 mg, 0.338 mmol) and 4-morpholinoaniline (84 mg,
.473 mmol) in THF (10 mL) and acetone (10 mL) was added 2,6-lutidine (1 mL),
0
and the reaction mixture was stirred at room temperature overnight. Water was added,
and the aqueous layer was extracted with ethyl acetate (50 mL×3). The organic layer
2 4
was washed with brine, dried with Na SO and evaporated. The residue was purified
by silica gel chromatography with petroleum ether/ethyl acetate (2/1, v/v) to afford 7a
1
(
79 mg, 53%) as a yellow solid. H-NMR (400 MHz, DMSO-d
6
) δ 10.08(s, 1H,
2
SO NH), 8.65 (d, J = 8.4 Hz, 1H, 5-H), 8.11 (d, J = 6.8 Hz, 1H, 7-H), 8.00 (d, J = 7.6
Hz, 1H, 3-H), 7.86 (t, J = 8.4 Hz, 1H, 4-H), 7.20 (d, J = 7.6 Hz, 1H, 8-H), 6.87 (d, J =
13

ACCEPTED MANUSCRIPT
8
2
.8 Hz, 2H, Ar-H), 6.70 (d, J = 9.2 Hz, 2H, Ar-H), 3.87 (q, J = 6.8 Hz, 2H, CH ), 3.62
13
(
m, 4H, 2´-H), 2.91 (m, 4H, 3´-H), 1.22 (t, J = 7.2 Hz, 3H, CH
3
). C-NMR (500 MHz,
DMSO-d
6
) δ 167.63, 149.16, 143.91, 133.95, 131.34, 130.31, 129.54, 129.20, 126.88,
1
26.02, 125.65, 125.06, 123.45, 116.39, 104.90, 66.88, 49.32, 35.51, 14.54. MS (ESI),
-
m/z for C H N O S ([M - H] ): Calcd 437.51, found 436.0.
23
23
3
4
4
.3.7.
(E)-1-ethyl-2-oxo-N-(4´-(phenyldiazenyl)phenyl)-1,2-dihydrobenzo[cd]indole-6-sulfo
namide (7k).
A reaction mixture of compound 5 (70 mg, 0.237 mmol) and (E)-4- (phenyldiazenyl)
aniline (111 mg, 0.473 mmol) in pyridine (4 mL) was stirred at room temperature
overnight. Water was added, and the aqueous layer was extracted with ethyl acetate
2 4
(50 mL×3). The organic layer was washed with water and brine, dried with Na SO
and evaporated. The residue was purified by silica gel chromatography with
petroleum ether/ethyl acetate (2/1, v/v) to afford 7k (51 mg, 47%) as a yellow solid.
1
6 2
H-NMR (400 MHz, DMSO-d ) δ 11.10 (s, 1H, SO NH), 8.74 (d, J = 8.4 Hz, 1H,
5
-H), 8.26 (d, J = 7.6 Hz, 1H, 7-H), 8.11 (d, J = 7.2 Hz, 1H, 3-H), 7.95 (t, J = 7.8 Hz,
H, 4-H), 7.73 (m, J = 9.0 Hz, 4H, 8-H, Ar-H), 7.50 (m, 3H, Ar-H), 7.27 (m, 3H,
1
13
2 3
Ar-H), 3.86 (q, J = 7.2 Hz, 2H, CH ), 1.20 (t, J = 7.2 Hz, 3H, CH ). C NMR (500
MHz, DMSO-d
6
) δ 167.30, 152.77, 148.68, 144.24, 141.25, 134.04, 131.51, 131.26,
1
29.82, 129.65, 128.57, 126.78, 125.67, 124.32, 122.78, 119.86, 104.41, 35.28, 14.00.
-
20 4 3
MS (ESI), m/z for C25H N O S ([M - H] ): Calcd 456.52, found 455.1.
4
.3.8.
Tert-butyl(3´-(1-ethyl-2-oxo-1,2-dihydrobenzo[cd]indole-6-sulfonamido)phenyl)carba
mate (8a).
To a solution of compound 5 (60 mg, 0.203 mmol), DAMP (5 mg, 0.041 mmol) and
tert-butyl(3-aminophenyl)carbamate (85 mg, 0.406 mmol) in DMF (10 mL) was
added TEA (0.08 mL, 0.61 mmol), and the reaction mixture was stirred at room
14

ACCEPTED MANUSCRIPT
temperature overnight. Water was added, and the aqueous layer was extracted with
ethyl acetate (50 mL×3). The organic layer was washed with brine, dried with Na SO
2
4
and evaporated. The residue was purified by silica gel chromatography with
petroleum ether/ethyl acetate (5/1, v/v) to afford 8a (55.3 mg, 58%) as a yellow solid.
1
H-NMR (400 MHz, CDCl ) δ 8.58 (d, J = 8.4 Hz, 1H, 5-H), 8.17 (d, J = 8.0 Hz, 1H,
3
7
7
-H), 8.00 (d, J = 7.2 Hz, 1H, 3-H), 7.68 (t, J = 7.6 Hz, 1H, 4-H), 7.17 (s, 1H, 2´-H),
.01 (m, 2H, 8-H, 5´-H), 6.83 (d, J = 7.6 Hz, 1H, Ar-H), 6.73 (d, J = 7.2 Hz, 1H,
Ar-H), 6.63 (s, 1H, CONH), 3.91 (q, J = 7.0 Hz, 2H, CH
2
), 1.43 (s, 9H, C(CH
3 3
) ),
1
3
1
1
1
.25 (t, J = 7.0 Hz, 3H, CH
3
). C-NMR (400 MHz, DMSO-d ) δ 167.66, 153.44,
6
44.16, 141.12, 138.83, 134.48, 131.51, 130.20, 130.03, 128.83, 126.96, 126.07,
25.76, 124.96, 114.38, 113.77, 109.99, 104.84, 79.98, 35.52, 28.97, 14.54. MS (ESI),
-
m/z for C H N O S ([M - H] ): Calcd 467.15, found 466.1.
24
25
3
5
4
.3.9. N-(4´-acetylphenyl)-1-ethyl-2-oxo-1,2-dihydrobenzo[cd]indole-6-sulfonamide
8c).
To a solution of compound 5 (100 mg, 0.338 mmol) and 1-(4-aminophenyl) ethanone
64 mg, 0.473 mmol) in THF (10 mL) and 10 mL acetone (10 mL), and the reaction
(
(
mixture was stirred at room temperature overnight. Water was added, and the aqueous
layer was extracted with ethyl acetate (50 mL×3). The organic layer was washed with
2 4
brine, dried with Na SO and evaporated. The residue was purified by silica gel
chromatography with petroleum ether/ethyl acetate (5/1, v/v) to afford 8c (90 mg,
1
6
7%) as a yellow solid. H-NMR (400 MHz, DMSO-d ) δ 10.82 (s, 1H, SO NH), 8.69
6
2
(d, J = 8.4 Hz, 1H, 5-H), 8.19 (d, J = 7.6 Hz, 1H, 7-H), 8.09 (d, J = 7.2 Hz, 1H, 3-H),
7
.94 (t, J = 8.0 Hz, 1H, 4-H), 7.57 (m, 2H, 3´-H, 5´-H), 7.31 (d, J = 5.6 Hz, 2H, 2´-H,
6
´-H), 7.25 (d, J = 7.6 Hz, 1H, 8-H), 3.86 (q, J = 6.8 Hz, 2H, CH ), 2.43 (s, 3H,
2
13
3 3 6
COCH ), 1.22 (t, J = 6.8 Hz, 3H, CH ). C-NMR (400 MHz, DMSO-d ) δ 197.57,
1
1
67.30, 144.12, 138.69, 138.44, 133.81, 131.15, 130.07, 129.73, 128.68, 126.73,
25.63, 124.76, 124.55, 124.37, 119.28, 104.38, 35.26, 26.95, 13.98. MS (ESI), m/z
-
for C21
H
18
N
2
O
4
S ([M - H] ): Calcd 394.1, found 393.0.
15

ACCEPTED MANUSCRIPT
4
.3.10.
N-(2´-(tert-butyl)phenyl)-1-ethyl-2-oxo-1,2-dihydrobenzo[cd]indole-6-sulfonamide
8i).
A reaction mixture of compound 5 (100 mg, 0.338 mmol) and 2-(tert-butyl) aniline
92 mg, 0.617 mmol) in pyridine (4 mL) was stirred at room temperature overnight.
Water was added, and the aqueous layer was extracted with ethyl acetate (50 mL×3).
The organic layer was washed with water and brine, dried with Na SO and
(
(
2
4
evaporated. The residue was purified by silica gel chromatography with petroleum
1
ether/ethyl acetate (5/1, v/v) to afford 6i (50 mg, 36%) as a yellow solid. H-NMR
(
400 MHz, DMSO-d
7.2 Hz, 1H, 7-H), 8.05 (d, J = 7.6 Hz, 1H, 3-H), 7.94 (t, J = 8.0 Hz, 1H, 4-H), 7.44
d, J = 8.0 Hz, 1H, 8-H), 7.32 (d, J = 7.6 Hz, 1H, Ar-H), 7.15 (t, J = 7.6 Hz, 1H Ar-H),
6 2
) δ 9.37(s, 1H, SO NH), 8.76 (d, J=8.4 Hz, 1H, 5-H), 8.21 (d, J
=
(
6
.90 (t, J = 7.6 Hz, 1H, Ar-H), 6.36 (d, J = 7.6 Hz, 1H, Ar-H), 3.97 (q, J = 7.2 Hz, 2H,
13
2 3 3
CH ), 1.45 (s, 9H, C(CH )), 1.29 (t, J = 7.2 Hz, 3H, CH ). C-NMR (400 MHz,
DMSO-d ) δ 167.48, 148.58, 143.62, 134.99, 132.94, 131.90, 130.79, 130.72, 129.92,
6
1
1
28.31, 127.92, 126.72, 126.65, 125.71, 125.52, 124.96, 104.59, 35.90, 35.31, 32.28,
-
4.05. MS (ESI), m/z for C23
H
24
N
2
O
3
S ([M - H] ): Calcd 408.15, found 407.1.
4
.3.11.
N-(3´,4´-dimethoxyphenyl)-1-ethyl-2-oxo-1,2-dihydrobenzo[cd]indole-6-sulfonamide
8k).
To a solution of compound 5 (70 mg, 0.237 mmol), DAMP (6 mg, 0.047 mmol) and
(
3
0
,4-dimethoxyaniline (73 mg, 0.473 mmol) in DMF (10 mL) was added TEA (0.1 mL,
.71 mmol), and the reaction mixture was stirred at room temperature overnight.
Water was added, and the aqueous layer was extracted with ethyl acetate (50 mL×3).
The organic layer was washed with brine, dried with Na SO and evaporated. The
2
4
residue was purified by silica gel chromatography with petroleum ether/ethyl acetate
1
(
5/1, v/v) to afford 8k (47.6 mg, 49%) as a yellow solid. H-NMR (400 MHz, CDCl
3
)
δ 8.55 (d, J = 8.4 Hz, 1H, 5-H), 8.04 (t, J = 3.6 Hz, 2H, 7-H, 3-H), 7.72 (t, J = 7.4 Hz,
H, 4-H), 6.82 (d, J = 7.6 Hz, 1H, 8-H), 6.64 (s, 1H, 2´-H), 6.55 (d, J = 8.8 Hz, 1H,
1
16

ACCEPTED MANUSCRIPT
5
2
´-H), 6.41 (d, J = 8.4 Hz, 1H, 6´-H), 3.93 (q, J = 7.0 Hz, 2H, CH ), 3.73 (s, 3H,
13
OCH ), 3.65 (s, 3H, OCH ), 1.24 (t, J = 7.0 Hz, 3H, CH ). C-NMR (400 MHz,
3
3
3
6
DMSO-d ) δ 167.39, 149.74, 147.09, 143.83, 133.68, 131.20, 130.98, 130.08, 129.12,
1
26.70, 125.58, 125.00, 114.47, 113.56, 104.49, 56.61, 56.29, 35.29, 14.08. MS (ESI),
-
m/z for C H N O S ([M - H] ): Calcd 412.46, found 411.1.
21
20
2
5
4
.3.12.
N-(2´,5´-dichlorobenzyl)-1-ethyl-2-oxo-1,2-dihydrobenzo[cd]indole-6-sulfonamide
8s).
(
To a solution of compound 5 (100 mg, 0.338 mmol) and (2,5-dichlorophenyl)
methanamine (72 mg, 0.409 mmol) in DCM (10 mL) was added DIPEA (1 mL), and
the reaction mixture was stirred at room temperature overnight. Water was added, and
the aqueous layer was extracted with ethyl acetate (50 mL×3). The organic layer was
2 4
washed with brine, dried with Na SO and evaporated. The residue was purified by
silica gel chromatography with petroleum ether/ethyl acetate (3/1, v/v) to afford 6s
1
(
32 mg, 21%) as a yellow solid. H-NMR (400 MHz, DMSO-d
6
) δ 8.68 (d, J = 8.4 Hz,
1
4
H, 5-H), 8.28 (t, J = 5.2 Hz, 1H, 7-H), 8.11 (m, 1H, 3-H), 7.88 (t, J = 8.0 Hz, 1H,
-H), 7.26 (d, J = 7.6 Hz, 1H, 8-H), 7.19 (d, J = 7.2 Hz, 2H, Ar-H), 7.13 (m, 1H,
) , 3.93 (q, J = 7.2 Hz, 2H, CH ), 1.27 (t, J =
). C-NMR (400 MHz, DMSO-d ) δ 167.48, 143.54, 135.99, 133.09,
Ar-H), 4.25 (d, J = 5.2 Hz, 2H, ArCH
2
2
13
7
1
1
.2 Hz, 3H, CH
3
6
32.42, 130.72, 130.57, 130.50, 128.83, 126.53, 125.40, 124.97, 104.32, 42.20, 35.25,
-
4.13. MS (ESI), m/z for C H Cl N O S ([M - H] ): Calcd 434.03, found 433.0.
20
16
2
2
3
4
4
.4. Biological assays
.4.1. Protein and peptide preparation
The human RORγ LBD (residues 262–507) was expressed as a His6-fusion protein
using the pET24a expression vector (Novagen, Madison, WI) as described in
reference [23]. BL21 (DE3) cells that were transformed with this expression plasmid
were grown in LB broth at 25 ˚C until an OD600 of approximately 1.0 was reached,
and the cells were then induced with 0.1 mM isopropyl-β-D-1-thiogalactopyranoside
17

ACCEPTED MANUSCRIPT
(IPTG) at 16 ˚C overnight. Cells were harvested, resuspended, and high-pressure
homogenized in 200 mL of extract buffer (20 mM Tris (pH 8.0), 500 mM NaCl, 10%
glycerol, and 25 mM imidazole) per 6 liters of cells. The lysate was centrifuged at
2
4
0,000 rpm for 30 min, and the supernatant was loaded onto a 5-mL NiSO -loaded
HisTrap HP column (GE Healthcare, Piscataway, NJ). The column was washed with
extract buffer, and the protein was eluted with a 25–500 mM imidazole gradient. A
gel filtration column (HiLoad S75, 16/60, GE Healthcare) was used for a second
purification.
4
.4.2. AlphaScreen assays
Interactions between RORγ and ligands were assessed by luminescence-based
AlphaScreen technology (Perkin Elmer) as previously described in [22-25] using a
histidine detection kit from PerkinElmer (Norwalk, CT). All of the reactions
contained 100 nM receptor LBD bound to nickel acceptor beads (5 µg/mL) and 20
nM biotinylated SRC1-4 peptide bound to streptavidin donor beads (5 µg/mL) in the
presence or absence of the indicated amounts of control compounds T0901317, UA,
SR2211, or candidate compounds. The N-terminal biotinylated coactivator peptide
SRC1-4 sequence was QKPTSGPQTPQAQQKSLLQQLLTE. Compound
concentrations varied from 150 nM to 200 µM in the dose-response assay.
4
.4.3. Transient transfection assays
2
93T cells were maintained in DMEM containing 10% fetal bovine serum (FBS).
Cells were transiently transfected in Opti-MEM using Lipofectamine 2000
Invitrogen), according to the manufacturer’s protocol. The 96-well plates were plated
4 h prior to transfection (15000 cells per well). For all of the experiments, each well
(
2
of cells was transfected in Opti-MEM with 25 ng of reporter plasmids and 5 ng of
Renilla luciferase expression plasmids. For Gal4-driven reporter assays, the cells were
transfected with 25 ng of Gal4-RORγ LBD (residues 262-507) and 25 ng of pG5Luc
reporter. For native promoter reporter assays, the cells were co-transfected with the
Pcp2/RORE-Luc reporter along with the plasmids encoding full-length RORγ. The
18

ACCEPTED MANUSCRIPT
cells were then transfected with the indicated expression and reporter plasmids. The
media was changed 24 h after transfection, and the compounds were added. Cells
were incubated for another 24 h followed by harvesting for a luciferase assay using
the dual-luciferase reporter assay system (Promega). Luciferase data were normalized
to Renilla luciferase data, which was used as an internal transfection control. All of
the assays were performed in triplicate, and the standard deviations were calculated
using the triplicates.
Acknowledgements
We gratefully acknowledge financial support from the National Natural Science
Foundation of China (Grant 81373325), National Key Basic Research Program of
China (973 Program, Grant 2013CB910601), the “100 Talents Project” of CAS, and
Guangzhou Bureau of Science and Information Technology, China, (Grants
2
012Y2-00051, 2013Y2-00048),“Interdisciplinary Cooperation Team” Program for
Science and Technology Innovation of the CAS. We gratefully thank Professor Yong
Li for advice and related plasmids.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in the online version, at
doi:xxx.
19

ACCEPTED MANUSCRIPT
Figure Legends
Figure 1. Selected examples of known RORγ antagonists as disclosed in the literature
and RORγ virtual screening hit (s4).
Figure 2. Schematic representation of the virtual screening workflow used for RORγ
ligand discovery in this study.
Figure 3. 3D (A) and 2D (B) schematic presentation of the predicted binding mode of
s4 in the RORγ ligand binding pocket.
Figure 4. 3D presentation of the predicted binding modes of 7j (A), 8c (B), 8k (C),
8
p (D) in the RORγ ligand binding pocket.
Table 1. SAR of the sulfonamide with a fused heterocyclic ring.
Table 2. SAR of the sulfonamide with multiple ring substituents.
Table 3. SAR of the sulfonamide with a single ring substituent.
20

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Figure 1. Selected examples of known RORγ antagonists disclosed in the literature
and RORγ virtual screening hit (s4).
Figure 2. Schematic representation of the virtual screening workflow used for RORγ
ligand discovery in this study.
1

ACCEPTED MANUSCRIPT
Figure 3. 3D (A) and 2D (B) schematic presentation of the predicted binding mode of
s4 in RORγ ligand binding pocket. Hydrogen bonds interactions are indicated by dash
lines in red, and π-π interactions indicted by dash lines in green. Fused heterocyclic
ring on the right side is surrounded by hydrophobic residues Phe378, Phe388, and
Phe401.
Figure 4. 3D presentation of predicted binding modes of 7j (A), 8c (B), 8k (C), 8p (D)
in the RORγ ligand binding pocket. Hydrogen bonds interactions are indicated by
dash lines in red, and π-π interactions indicated by dash lines in green. Aromatic ring
on the right side is surrounded by hydrophobic residues Phe378, Phe388, and Phe401.
2

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Table 1. SAR of the sulfonamide with a fused heterocyclic ring.
Luciferase
Alphascreen
Gal4-RORγ-LBD full-length-RORγ
RORγ-LBD
Cmpd
R
1
R
2
R
3
a
a
IC50 (µM)
IC50 (µM)
b
IC (µM)
5
0
(
%max inhibition) (%max inhibition)
T1317
UA
0.54 (82)
0.13 (76)
0.43 (79)
3.5 (76)
1.06 (63)
0.42 (44)
2.50 ± 0.87
9.41 ± 1.07
3.54 ± 0.01
SR2211
s4
H
H
11.84 (79)
7.56 (62)
20.27 ± 6.26
6
a*
Et
Et
Et
H
H
H
0.93 (60)
(10)
1.35 (45)
(10)
6.72 ± 0.89
6
b
-
-
H
N
6c
1.21 (32)
12.76 (34)
F
6d
Et
H
(30)
(20)
-
6
e
Et
Et
Et
H
H
H
(20)
0.10 (70)
(40)
(40)
0.15 (72)
(10)
-
6
f
17.52 ± 4.07
14.09 ± 2.07
6
6
g*
h*
H
H
H
(10)
(40)
(20)
(40)
23.77 ± 4.35
52.26 ± 4.21
6
i
Et
N
O
a
b
*
Inhibition of constitutive activity of RORγ by tested compounds.
Inhibition of RORγ LBD recruitment of the SRC1-4 coactivator peptide.
Denotes compounds from similarity/substructure search.
3

ACCEPTED MANUSCRIPT
Table 2. SAR of the sulfonamide with multiple ring substituents.
Luciferase
Alphascreen
Gal4-RORγ-LBD
full-length-RORγ
RORγ-LBD
Cmpd
R
1
R
2
R
3
a
a
IC50 (µM)
IC50 (µM)
b
IC50 (µM)
(
%max inhibition)
(%max inhibition)
7
a
Et
H
H
H
0.34 (60)
0.55 (71)
12.15 ± 0.05
48.42 ± 2.93
7b*
0.30 (64)
0.41 (54)
7
c
Et
Et
H
H
0.35 (63)
(10)
0.29 (63)
(30)
-
-
7
d
7
7
e*
H
Et
Et
Et
Et
H
H
H
H
H
H
H
H
H
(20)
(53)
(40)
(58)
5.93 ± 0.07
7
f
-
g*
(10)
(20)
-
7
h
(50)
(50)
-
O
7
i
(20)
(30)
156.30 ± 8.7
3.09 ± 0.71
6.05 ± 1.09
8.35 ± 0.45
7
j*
0.14 (70)
0.20 (59)
0.38 (65)
0.15 (60)
0.2 (54)
0.18 (60)
7
k
Et
7
m*
H
a
b
*
Inhibition of constitutive activity of RORγ by tested compounds.
Inhibition of RORγ LBD recruitment of the SRC1-4 coactivator peptide.
Denotes compounds from similarity/substructure search.
4

ACCEPTED MANUSCRIPT
Table 3. SAR of the sulfonamide with a single ring substituent.
Luciferase
Alphascreen
Gal4-RORγ-LBD
full-length-RORγ
RORγ-LBD
Cmpd
R
1
R
2
R
3
a
a
IC50 (µM)
IC50 (µM)
b
IC50 (µM)
(
%max inhibition) (%max inhibition)
8a
8b
8c
8d
8e
Et
Et
Et
Et
Et
H
H
H
H
H
2.51 (79)
(50)
3.39 (80)
-
7.04 ± 1.20
-
0.04 (75)
0.27 (44)
6.03 (44)
0.05 (64)
0.09 (73)
0.59 (27)
70.14 ± 4.39
-
-
8
f
Et
H
(10)
(30)
-
8
g
Et
Et
Et
Et
Et
Et
Et
Et
H
H
H
H
H
H
H
0.01 (67)
0.32 (65)
4.90 (70)
(10)
0.04 (62)
0.09 (67)
9.98 (65)
(20)
-
8
h*
89.76 ± 1.27
18.98 ± 3.71
13.22 ± 3.21
20.10 ± 1.53
174.40
8
i
8
j
8
k
0.05 (72)
(30)
0.02 (71)
(30)
8
m
8
n*
p*
(10)
(10)
-
8
0.09 (70)
0.08 (55)
6.47 ± 0.84
8q
Et
H
2.57 (62)
0.41 (67)
-
8
r
Et
Et
Et
H
H
H
(40)
0.13 (66)
(40)
(40)
1.08 (53)
(50)
-
8
s
31.61 ± 2.14
-
8
t
5