Discovery and Characterization of XY101, a Potent, Selective, and Orally Bioavailable RORγInverse Agonist for Treatment of Castration-Resistant Prostate Cancer

Article abstract of DOI:10.1021/acs.jmedchem.9b00327

We report the design, optimization, and biological evaluation of nuclear receptor RORγinverse agonists as therapeutic agents for prostate cancer treatment. The most potent compound 27 (designated as XY101) exhibited cellular activity with an IC₅₀

Full text of DOI:10.1021/acs.jmedchem.9b00327

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Article
Discovery and Characterization of XY101, a Potent,
Selective, and Orally Bioavailable ROR# Inverse Agonist for
Treatment of Castration-Resistant Prostate Cancer (CRPC)
Yan Zhang, Xishan Wu, Xiaoqian Xue, Chenchang Li, Junjian Wang, Rui Wang,
cheng Zhang, Chao Wang, Yudan Shi, Lingjiao Zou, Qiu Li, Zenghong Huang,
Xiaojuan Hao, Kerry M. Loomes, Donghai Wu, Hongwu Chen, Jinxin Xu, and Yong Xu
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 09 Apr 2019
Downloaded from http://pubs.acs.org on April 9, 2019
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Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese
Academy of Sciences; Guangzhou Medical University; University of the
Chinese Academy of Sciences
Zou, Lingjiao; Guangzhou Institutes of Biomedicine and Health,
Guangdong Provincial Key Laboratory of Biocomputing, Joint School of
Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese
Academy of Sciences; Guangzhou Medical University; University of the
Chinese Academy of Sciences
Li, Qiu; Guangzhou Institutes of Biomedicine and Health, Guangdong
Provincial Key Laboratory of Biocomputing, Joint School of Life Sciences,
Guangzhou Institutes of Biomedicine and Health, Chinese Academy of
Sciences; Guangzhou Medical University; University of the Chinese
Academy of Sciences
Huang, Zenghong; University of California Davis, Department of
Biochemistry and Molecular Medicine, School of Medicine
Hao, Xiaojuan; Commonwealth Scientific and Industrial Research
Organization (CSIRO)
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Loomes, Kerry; University of Auckland, School of Biological Sciences &
Maurice Wilkins Centre
Wu, Donghai; Guangzhou Institutes of Biomedicine and Health,
Chen, Hongwu; University of California Davis, Department of
Biochemistry and Molecular Medicine, School of Medicine; University of
California Davis, UC Davis Comprehensive Cancer Center
Xu, Jinxin; Guangzhou Institutes of Biomedicine and Health,
Xu, Yong; Guangzhou Institutes of Biomedicine and Health, Guangdong
Provincial Key Laboratory of Biocomputing, Joint School of Life Sciences,
Guangzhou Institutes of Biomedicine and Health, Chinese Academy of
Sciences; Guangzhou Medical University; Guangzhou Regenerative
Medicine and Health Guangdong Laboratory (GRMH-GDL)
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Discovery and Characterization of XY101, a Potent, Selective, and Orally
Bioavailable RORγ Inverse Agonist for Treatment of Castration-Resistant Prostate
Cancer (CRPC)
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Yan Zhang, a,b,c,# Xishan Wu,a,b,c,# Xiaoqian Xue,a,d,# Chenchang Li, Junjian Wang,
a
e,f
Rui Wang, Cheng Zhang, Chao Wang,a,b Yudan Shi,a,b Lingjiao Zou,a,b Qiu Li,a,b
a
a,g
f
h
i
j
f,k
Zenghong Huang, Xiaojuan Hao, Kerry Loomes, Donghai Wu, Hong-Wu Chen,
j
a,c,*
Jinxin, Xu, and Yong Xu
aGuangdong Provincial Key Laboratory of Biocomputing, Joint School of Life
Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of
Sciences; Guangzhou Medical University, Guangzhou, 510530, China.
bUniversity of Chinese Academy of Sciences, No. 19 Yuquan Road, Beijing 100049,
China.
cGuangzhou Regenerative Medicine and Health Guangdong Laboratory
(GRMH-GDL), Guangzhou, 510530, China
dSchool of Life Science, Huizhou University, Huizhou, 516007, China
eSchool of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, 510006,
China.
fDepartment of Biochemistry and Molecular Medicine, School of Medicine,
University of California, Davis, Sacramento, California 95817, USA.
gSchool of Pharmaceutical Sciences, Jilin University, Changchun, China, No.1266
Fujin Road, Chaoyang District, Changchun, Jilin, 130021, China.
hManufacturing, Commonwealth Scientific and Industrial Research Organization
(CSIRO), Clayton, Vic 3168, Australia.
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iSchool of Biological Sciences & Maurice Wilkins Centre, University of Auckland,
Auckland 1010, New Zealand.
jGuangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences;
Guangzhou, 510530, China
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kUC Davis Compreehensive Cancer Center, University of California, Davis,
Sacramento, California 95817, USA.
*Corresponding Author: Yong Xu, PhD, E-mail: xu_yong@gibh.ac.cn
#These authors contributed equally to this work.
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ABSTRACT
We report the design, optimization and biological evaluation of nuclear receptor
RORγ inverse agonists as therapeutic agents for prostate cancer treatment. The most
potent compound 27 (designated as XY101) exhibited cellular activity with an IC50
value of 30 nM in a cell-based reporter gene assay with good selectivity against other
NRs subtypes. The cocrystal structure of 27 in complex with the RORγ ligand binding
domain (LBD) provided solid structural basis for its antagonistic mechanism. 27
potently inhibited cell growth, colony formation, and the expression of AR, AR-V7
and PSA. 27 also exhibited good metabolic stability and pharmacokinetic profile with
oral bioavailability of 59% and half-life of 7.3 h. Notably, 27 demonstrated promising
therapeutic effects with significant tumor growth inhibition in a prostate cancer
xenograft model in mice. The potent, selective, metabolically stable and orally
available RORγ inverse agonists represent a new class of compounds as potential
therapeutics against prostate cancer.
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1
. INTRODUCTION
Prostate cancer is one of the most commonly diagnosed cancers in the world and the
second leading cause of cancer mortality in man.1-3 Androgen deprivation therapy
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(ADT), first demonstrated by Charles Huggins in 1941, is the main option for
4
advanced prostate cancer. Despite an initial benefit, unfortunately, the majority of
patients will progress to castration-resistant prostate cancer (CRPC). CRPC continues
to rely on androgen receptor (AR) signaling with multiple mechanisms including AR
overexpression with or without amplification, AR mutations, AR splice variants and
others. The second-generation anti-androgen therapies have recently emerged for the
treatment of CRPC. These agents either suppress the synthesis of extragonadal
androgens (e.g., abiraterone, a CYP17A1 irreversible inhibitor)5,6 or target the AR
directly (e.g., enzalutamide, an AR antagonist).7,8 Although the discovery of
abiraterone and enzalutamide represent breakthroughs in the treatment of metastatic
CRPC, most of patients who initially have a response to these agents, eventually
acquire secondary resistance.9-14 The development of novel therapies that can
overcome aberrant AR signaling and improve patient outcome is an unmet medical
need for the treatment of lethal prostate cancer.
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Figure 1. Structures of RORγ Inverse Agonists.
The retinoic acid receptor-related orphan receptor γ (RORγ), a member of the nuclear
receptor superfamily, is a ligand-dependent transcriptional factor. RORγt regulates the
differentiation and function of Th17 immune cells that produce the pro-inflammatory
cytokines, interleukin-17 (IL-17), interleukin-17F (IL-17F), and interleukin-22
(
IL-22).15,16 As a result of its importance in multiple inflammatory pathways, RORγ
has emerged as a valuable therapeutic target for the treatment of autoimmune and
metabolic diseases. As such, multiple RORγ inverse agonists are in development for
therapeutic purposes.17-20 The most well-known tool molecule T0901317 (1, Figure. 1)
was identified initially by Tularik Inc. as an agonist of multiple nuclear receptors
(NR
S
) including liver X receptor (LXR),21 pregnane X receptor (PXR),22 and
23
farnesoid X receptor (FXR). Subsequently, T0901317 was identified as an inverse
agonist of RORγ by Griffin’s group at Scripps, Florida.24 Using 1 as a starting
compound, they developed a series of synthetic RORγ inverse agonists, including
2
5
SR2211 (2). Compound 2 (Figure 1), containing a hexafluoroisopropanol group,
showed an excellent selectivity for RORγ over other NR in a cellular luciferase
S
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reporter gene assay. Compound 2 has been evaluated for its therapeutic potential in
suppression of Th17 cell-mediated autoimmune diseases in collagen-induced arthritis
model.26 GSK has disclosed several para-sulfonylphenyl acetamide motifs bearing
variations at the heterocyclic ring amide,27 aryl amide (3, Figure 1),28 and biaryl
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amide. Recently, there has been significant progress in the development of small
molecule RORγ inverse agonists.30-33Some RORγ inverse agonists, including
VTP-43742 (Vitae/Allergan, terminated), ARN-6039 (Arrien Pharmaceuticals),
JTE-451 (Japan Tobacco), RTA 1701 (Reata), and AZD-0284 (AstraZeneca), have
recently been advanced into clinical trials.34-38
Previously, we reported a series of novel and potent RORγ inverse agonists with
diverse
scaffold
including
3,4-dihydropyrimidin-2(1H)-ones,
N-phenyl-2-(N-phenylphenylsulfonamido) acetamides, benzo[cd]indol-2(1H)-ones (4,
Figure 1).39-42 We further found that RORγ is overexpressed and amplified in
metastatic CRPC tumors. RORγ directly stimulates AR gene transcription by binding
to an exonic ROR response element (RORE) that acts as a key determinant of AR
gene expression. RORγ inverse agonists effectively reduced the expression and
activity of full length AR and AR spliced variants that are active in CRPC, thereby
inhibiting tumor growth and metastasis.39 Furthermore, RORγ inverse agonists
displayed enhanced efficacy in enzalutamide-resistant tumors. These findings provide
excciting proof-of-concept and an alternative therapeutic option for the treatment of
CRPC by targeting RORγ, an upstream regulator of AR gene transcription.
In this study, we report the design, optimization, and biological evaluation of a series
of novel RORγ inverse agonists based on the biphenyl-4-yl-amine scaffold as potent,
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selective and orally available candidate for the treatment of prostate cancer. Targeting
RORγ could circumvent secondary resistance problems associated with agents that
directly target AR signaling and benefit patients with CRPC.
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. RESULTS AND DISCUSSION
.1. Structure-based Design for RORγ Inverse agonists.
To design novel and potent RORγ inverse agonists, we overlaid the predicted binding
4
3
models of 2 and 3 (Figure 1) in complex with RORγ LBD (4QM0) derived from
molecular docking as illustrated in Figure 2. Both compounds occupy the whole
binding pocket with some key interactions including H-bond interactions with Arg367
and Phe377, edge-to-face π-π interaction with Phe378, and hydrophobic interaction
with Phe388, Trp317, and other residues. Importantly, the middle phenyl groups of 2
and 3 superimposed nearly identically. By combining the structural features of 2 and
the GSK compound (3) and keeping these interactions between the key
pharmacophore and residues of RORγ LBD, we designed hybridized compounds with
either a sulfonamide or amide linker as shown in Figure 2. Our aim was to obtain the
optimal RORγ inverse agonists with improved potency and pharmacodynamics
properties. To develop compounds with improved potency for RORγ, we focused on
the polar region around Arg367 and performed an extensive SAR study.
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Figure 2. Design and optimization strategy for novel biphenyl-4-yl-amines
derivatives as RORγ inverse agonist. The binding modes of compounds 2 (magenta)
and 3 (blue) in complex with RORγ ligand binding domain (LBD) (PDB code: 4QM0)
were predicted by molecular docking. The ligands and important residues are shown
as sticks. The hydrogen bonds and π-π interactions are shown as red and green dashed
lines, respectively. Figures were prepared using PyMOL.
2.2. Chemistry.
The synthesis of sulfonamide and amide compounds is depicted in Scheme 1. The
commercially available 2-fluoroaniline (5) reacted with hexafluoroacetone trihydrate
(6) using a catalytic amount of p-toluenelsuphonic acid, which afforded the desired
hexafluoropropanol intermediate 7. A subsequent diazo-reaction using intermediate 7
and potassium iodide under acidic conditions yielded the corresponding 4-iodophenyl
analogue 8. Intermediate
8
was coupled with the Boc-protected
aminophenyl-4-boronic acid to afford 9. Deprotection of the N-Boc functionality
using TFA in DCM provided the unsubstituted amine derivative 10. Compound 10
was treated with the appropriate sulfonyl chloride or carboxylic acids to generate final
sulfonamides 11, 12, 15, 16, 18-21 and amides 22, 23, 26-36. Appropriate sulfonyl
chlorides were synthesized from benzyl halides, extrusion of SO , and chlorination.
2
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Hydrolysis of the carboxylic ester analogue 16 and nitrile analogue 12 afforded the
targeted compound 17 and 13, respectively. Reduction of the nitro analogues 22 and
23 afforded the amino compounds 24 and 25, respectively. The amidation of
compound 17 afforded the targeted compound 14.
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Scheme
1.
Synthesis
of
N-([1,1'-biphenyl]-4-yl)-sulfonamide
and
N-([1,1'-biphenyl]-4-yl)amide derivatives.
Reagents and conditions: (a) p-toluenesulfonic acid, 90 °C, 12 h, 30%; (b) HCl, NaNO
°C, 0.5 h; KI, 0 °C - rt, 12 h, 96%; (c) (4-((tert-butoxycarbonyl)amino)phenyl)boronic acid,
Pd(PPh , K CO , 1,4-dioxane, 80 °C, 5 h, 74 %; (d) TFA, DCM, rt, 3 h, 91%; (e) R -SO Cl,
pyridine, 80 °C, 2 h, 33% − 75%. (f) R -COOH, HATU, DIPEA, DCM, rt, 3 h, 47% − 88%.
2
, DMF, 0 -
5
3
)
4
2
3
1
2
2
2.3. Structure−Activity Relationships of Sulfonamide Derivatives
Dual-luciferase reporter assay and thermal shift assay (TSA) were firstly used for
SAR evaluation.The luciferase reporter assay can test the activity of compounds on
RORγ transcription while the TSA assay can determine the thermodynamic stability
of RORγ LBD in the presence of tested compounds. To find more potent RORγ
ligand based on the modeled complex structures, we incorporated various polar
groups into the para-position on the benzyl group to allow formation of an H-bond
with the carbamidine of Arg367 (Table 1). The substitution at the para-position of the
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benzyl group with methylsulfonyl (11), cyano (12), aminocarbonyl (13), and
methyl-aminocarbonyl (14) showed weak inhibitory activities with IC50 values of less
than 10 μM. Compound 15 bearing a nitro group at para-position of benzyl group
exhibited an IC50 value of 2.22 μM. Significantly, the methyl ester derivative 16
offered significant improvement in both RORγ transcription activities (IC50 = 0.09 μM)
and stabilization effects for the RORγ protein with a temperature shift of 8.0 °C. In
contrast, the corresponding carboxyl acid derivative 17 abolished transcriptional
activity.
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3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 1. Structure–activity relationships of sulfonamide derivatives
Gal4-RORγ-LBDa
IC50 (μM)
TSAb
Cmpd
R
1
cLogP
3.68
ΔT
m
(°C)
1
1
1
2
7.76 ± 0.56
7.55 ± 1.09
8.72 ± 2.11
1.0
0
0
4.75
13
14
3.83
8.05 ± 1.40
0
4.04
1
1
1
5
6
7
2.22 ± 0.51
0.09 ± 0.01
(23%)
1.5
8.0
0
5.06
5.29
5.06
1
8
1.49 ± 0.21
3.5
5.06
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2
2
2
2
2
2
2
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19
7.66 ± 0.06
4.24 ± 1.12
2.96 ± 0.50
1.0
4.0
2.0
4.98
5.29
5.29
2
2
0
1
0
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0
a
All assay results are reported as mean ± SD from at least three independent experiments. IC50
b
values were calculated from the luciferase assay. ΔT
m
values were calculated from the thermal
shift assay.
There are two other important residues Gln286 and Arg364 near Arg367. We therefore
also investigated the effects of substitutions at the meta- or ortho-positions of the
benzyl group (18, 19, 20, 21) to explore whether the H-bond formed or not. The
luciferase reporter assay showed that the meta-substituent analogue 18 afforded an
approximately equipotent transcriptional response to the para-substituent analogue 15,
while the ortho-substituent analogue 19 exhibited weaker activities than those of 18
and 15. For the methyl ester group substitutions, both the meta- (20) and
ortho-positions (21) were less potent than 16. A molecular docking analysis also
supported a favorable ligand binding interaction with 16 through a key H-bond with
Arg367 (Figure 3). From this SAR analysis the most potent compound in this series is
1
6.
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0
1
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9
0
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9
0
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0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. 3D presentation of the predicted binding mode of 16 (yellow) with RORγ
LBD (PDB code: 4QM0). For clarity, only important residues (gray) at the binding
site are shown.
2
.4. Structure−Activity Relationships of Amide Derivatives.
To further explore the chemical space, we synthesized compounds with an amide
linker (Table 2). For comparison with the sulfonamide derivatives, the nitro (22, 23),
methyl sulfonyl (26), ethyl sulfonyl (27) derivatives with the amide linker were
designed and synthesized to assess whether the critical interactions with residues
around Arg367 were maintained. When a nitro group was introduced, compound 23
with an ortho-position substituent and compound 22 with a para-position substituent
both showed encouraging activities with IC50 values of 0.19 and 1.49 μM,
respectively (Table 2). The TSA assay also demonstrated that both 23 and 22
stabilized RORγ LBD with temperature shifts of 4-6 °C. When compared to
compounds (15, 19) with sulfonamide linkers, 22 and 23 with amide linkers exhibited
better activities. Their respective reduced products, 24 and 25 exhibited slightly
decreased potencies. Coupling either methyl sulfonyl and ethyl sulfonyl groups to the
benzyl group generated 26 and 27, respectively. Both these compounds showed
significant improved potency (IC50 = 0.12 μM, 0.03 μM) as compared with the
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1
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1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
corresponding compounds with sulfonamide linker (11). Compounds 26 and 27 also
showed good protein stabilizing abilities with temperature shifts of 7.8 and 10.1 °C,
respectively.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. Structure–activity relationships of amide derivatives
Gal4-RORγ-LBDa
IC50 (μM)
TSAb
Cmpd
R
2
cLogP
ΔT
m
(°C)
2
2
2
3
1.49 ± 0.50
0.19 ± 0.02
4.16 ± 2.26
0.96 ± 0.46
6.0
5.13
5.05
4.16
4.11
4.2
1.5
1.5
(
XY018)
2
2
4
5
2
6
0.12 ± 0.03
7.8
3.75
4.28
27
(XY101)
0
.03 ± 0.01
10.1
2
8
4.17 ± 2.10
1.83 ± 0.89
0.82 ± 0.06
3.08 ± 0.17
6.3
5.9
5.8
7.8
5.44
5.97
6.50
4.62
29
30
3
1
32
0.46 ± 0.04
0.93 ± 0.38
1.52 ± 0.36
1.98 ± 0.71
5.2
5.4
5.0
5.2
4.11
4.46
4.15
5.95
3
3
3
3
4
5
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36
1.08 ± 0.28
6.5
7.56
a
All assay results are reported as mean ± SD from at least three independent experiments. IC50
b
values were calculated from the luciferase assay. ΔT
m
values were calculated from the thermal
1
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shift assay.
To further confirm the importance of the physicochemical properties, compounds
with either hydrophobic or polar head groups were synthesized (Table 2). Changing
the substituent from a butyl (28) to a hexyl group (30) led to a 5-fold improvement in
IC50 value from the luciferase reporter assay (4.17 μM to 0.82 μM, respectively) and a
corresponding thermal shift stabilization effect for RORγ LBD as shown by the TSA
assay (6.3 °C and 5.8 °C, respectively). We also synthesized compounds with polar
group in the alkyl tail (32, 33, and 34). Compound 32 included polar atoms at the tail
of flexible alkyl chain and exhibited a good inhibitory effect with an IC50 of 0.46 μM
and temperature shift of 5.2 °C. By comparison, the bulky and nonpolar substituted
analogues, 35 and 36, showed moderate activities. Among them, compound 27 was
one of the most potent compounds with strong biochemical activity.
To explain the structural basis for the SAR, we successfully determined the cocrystal
structure of 27 bound to RORγ LBD. In this cocrystal structure, there are 3 RORγ
LBD-ligand complexes. The overall structures of the three subunits superimposed
very well in the ligand binding pocket (Supporting Information Figure 1). As
expected, the NH of the amide linker makes an H-bond interaction with the backbone
carbonyl of Phe377. The carbonyl oxygen atom of the amide forms indirect H-bond
interactions with residues Gln286, His323, and Glu379 via a water molecule (Figure
4). Also as expected, the middle phenyl ring forms face-to-edge π-π interactions with
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Phe378. The two oxygen atoms of ethyl sulfone form H-bond interactions with the
backbone NH of Leu287 and carbamidine of Arg367. The ethyl sulfone occupies the
hydrophobic cavity surrounded by residues Ala327, Val361, Arg364, and Met365,
which is consistent with the binding modes for GSK compound obtained by Kallen
and Narjes (Supporting information Figure S2).19,27,44 The hexafluoroisopropanol
group, pointing to residues Trp317 and His479, may disrupt the His479-Tyr502
interaction thereby pushing H-12 outward. When comparing the structures of 27 and 1
in complex with RORγ LBD, we can see that the hexafluoroisopropanol doesn’t
superimpose well. However, both molecules push H-12 and contribute to the
antagonistic conformation (Supporting information Figure S2).44,45
0
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9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Cocrystal structure of compound 27 in complex with the RORγ LDB (2.30
Å, PDB code: 6J1L). (A) Compound 27 forms extensive interactions with the RORγ
LDB. Water molecules are shown as spheres in red. Hydrogen bonds and π-π stacking
interactions are indicated by dashed lines in red and green, respectively. For clarity,
only important residues (gray) at the binding site are shown. (B) Compound 27 is
well-defined by the electron density and fits snugly in the ligand binding pocket. The
electrostatic potential surface is shown.
2.5. Selectivity over Other Nuclear Receptors.
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To investigate the selectivity profile, compounds 16, 23, 26, 27 and 30 were assessed
using the cellular reporter gene assay against RORα, RORβ, LXRα, and FXR, which
are widely used in nuclear receptor ligand discovery studies.40-42 RORγ, RORα, RORβ，
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LXRα, and FXR are categorized into the same subgroup of NRs based on their
sequence homology. As shown in Table 3, the tested compounds displayed different
nuclear receptor activity profiles. Compound 23 displayed very weak RORα
antagonistic activity (7.57 μM with a maximum of 37% inhibition), and compound 30
exhibited similar potencies for RORα, FXR with RORγ. On the other hand,
compounds 16, 26, 27 exhibited potent RORγ activity in the reporter gene assay and
excellent selectivity for RORγ versus the other NRs in our selectivity panel.
2.6. Evaluation of Binding Affinities by AlphaScreen and Isothermal Titration
Calorimetric (ITC) Assays.
To further confirm the ability of ligands to bind to RORγ LBD and disrupt its
interaction with co-regulators, potent compounds were selected for AlphaScreen assay
based on the luciferase and TSA assay datas (Table 3). By definition, any compound
disrupting the recruitment of the SRC1 co-activator peptide acts as an RORγ inverse
agonist. Most of the tested compounds exhibited IC50 values in the micromolar range.
The most active compound 27 exhibited an IC50 value of 0.75 μM in the AlphaScreen
assay. These results suggest that these compounds potently disrupt the interaction of
RORγ LBD and SRC1 and function as inverse agonists.
Table 3. Potency and selectivity profiles
IC50 (μM) IC50 (μM) IC50 (μM)
RORγ RORα RORβ
IC50 (μM)
LXRα
IC50 (μM) IC50 (μM)
d
K (μM)
Cmpd
FXR
AlphaScreen ITC
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2
2
2
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3
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5
6
1
0.33
2.6 (55)
NA
NA
NA
NA
NA
NA
NA
0.05 (agonist) 5.3 (agonist)
1
6
3
0.09 ± 0.01
NA
NA
NA
NA
NA
NA
NA
NA
NA
4.83 ± 0.08
3.46 ± 1.32
2.28 ± 0.05
0.75 ± 0.09
2
0.19 ± 0.02 7.57 (37)
0.78
0.56
0.38
0
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0
26
27
30
0.12 ± 0.03
0.03 ± 0.01
NA
NA
0.82 ± 0.06 1.74 (80)
1.25 (93) 5.31 ± 0.22
NA = not active. Transcriptional activities were measured using reporter gene assays in 293T cells
with Gal4-RORγ LBD, Gal4-RORα-LBD, Gal4-RORβ-LBD, Gal4-LXRα-LBD, Gal4-FXR-LBD
expression vectors. IC50 values are reported as means ± SD. In vitro binding to the RORγ LBD
was measured using AlphaScreen and ITC assays.
Furthermore, compounds 23, 26, and 27 were selected for binding affinity
determination using isothermal titration calorimetry (ITC). All these tested
compounds exhibited K
d
values in the nanomolar range, which further validated their
binding affinities to RORγ LBD. Compound 27 was identified as the most active
compound with a K value of 380 nM (Table 3 and Supporting Information Figure
d
S3).
2.7. Inhibitory Effects on Cell Growth, Colony Formation and Gene Expression
in Prostate Cancer Cells by inverse agonists.
Given their encouraging potencies and selectivities, representative compounds were
assessed for their inhibitory effects on cell growth on a panel of human prostate
cancer cell lines such as LNCaP, 22Rv1, C4-2B with distinct expression levels of AR
and AR variants, and the AR-negative cell lines, DU145 and PC-3 (Table 4). The
second-generation androgen receptor antagonist enzalutamide was used as a reference.
In AR-positive LNCaP cells, these RORγ inverse agonists showed stronger cell
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growth inhibition as compared with enzalutamide. All the tested RORγ inverse
agonists were more effective than the second generation antiandrogen enzalutamide in
cell growth inhibition, further supporting the promising therapeutic potential of RORγ
inhibition on CRPC. In particular, the RORγ selective inverse agonist 27 displayed a
1
1
1
1
1
1
1
1
1
1
2
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2
2
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3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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8
9
0
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0
7
-fold higher cell growth inhibition activity than enzalutamide.
In the other two CRPC cells, C4-2B and 22Rv1, which express high levels of AR-V7,
these compounds were also more potent than enzalutamide. However, when compared
to their effects on LNCaP cells, the growth inhibition activities of several of these
compounds were slightly weaker in C4-2B and 22Rv1 cells. Notably, in AR-negative
DU145 cell line, these compounds showed much weaker growth inhibition activity,
3
9
which is consistent with our previous study. Otherwise, these compounds exhibited
similar potencies for 22Rv1 and PC-3 cells. Overall, these compounds exhibited
reasonable potency in the three AR-positive prostate cancer cell lines LNCaP, C4-2B,
and 22Rv1.
Table 4. Anti-proliferation effects of inverse agonists against the prostate cancer cell
lines LNCaP, 22Rv1, C4-2B, DU145, and PC-3
AR-positive cell lines (IC50
)
AR-negative cell lines (IC50
DU145 PC-3
44.70 ± 0.93 53.38 ± 0.47
)
Cmpd
LNCaP
22Rv1
C4-2B
Enzalutamide
42.37 ± 2.37
6.79±1.18
14.83 ± 2.01
7.13 ± 0.8
36.66 ± 4.21
23.56 ± 0.61
10.06
2
6.42±1.03
23.59 ± 0.47
5.64 ± 0.14
9.00 ± 0.33
18.21 ± 0.99
45.69±2.1
～60
24.82±2.41
1
3
2
2
6
0
3
6
23.79
30.88 ± 0.95
5.53
25.64 ± 1.04 12.36 ± 2.40
28.43 ± 0.89 11.14 ± 1.78
41.23 ± 0.47 21.84 ± 0.01
5.14 ± 0.36
11.10 ± 0.42
9.20
13.82
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5.83 ± 1.39
14.17 ± 1.10
10.57
36.92 ± 2.43 19.25 ± 0.31
Cell viability, as measured by Cell-Titer GLO (Promega) of prostate cancer cells treated with the
indicated concentrations of compounds for 72-96 h. Experiments were performed independently
two times.
0
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To evaluate the long-term cell growth inhibitory effects, colony formation assays were
performed for the representative compound 27 over a period of 14 days. Consistent
with the cell viability assay, treatment of C4-2B cells with 27 reduced colony
formations in a dose-dependent manner (Figure 5A). Colony formation was reduced
to less than 10% in C4-2B cells at the concentration of 8 μM.
Figure 5. RORγ inverse agonists suppress colony formation, and expression of AR
and AR regulated genes. (A) Compound 27 inhibited colony formation of C4-2B
prostate cancer cells. Cells were cultured and treated with vehicle (DMSO), 2 μM, 4
μM, or 8 μM 27 for 14 days followed by staining. (B) qRT-PCR analysis for mRNA
expression of full length AR (AR-FL), AR variant (AR-V7), prostate-specific antigen
(
PSA), KLK2, ERG, TMPRSS2, and C-MYC in LNCaP cells treated with vehicle, 5
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μM compounds 2, 23, or 27 for 48 h. (C) PSA transcriptional activity was evaluated
using luciferase reporter assays in LNCaP cells transfected with a PSA-luc reporter
plasmid. Cells were treated with vehicle or with 0.4 μM, 2.0 μM, or 10.0 μM 23, 26 or
1
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2
7. Data represent mean ± standard error of the mean (s.e.m.) (n = 3) from one of
three independent experiments. *P≤0.05, **P≤0.01 by two-tailed Student’s t-test.
D) Western blot analysis of AR (AR-FL) and AR variants (AR-vs) levels in 22Rv1
(
cells treated with vehicle or different doses of 26, and 27 for 48h. Lysates from 22Rv1
cells were analyzed by western blot analysis using an antibody specific for the AR
NTD. Representative densitometry data for both full length AR (AR-FL) and AR
variants (AR-vs) are shown with GAPDH as a loading control.
To explore whether the RORγ inverse agonists regulate the expression of AR,
AR-regulated genes and other oncogenes in prostate cancer cells, qRT-PCR analysis
was performed in LNCaP cells. As shown in Figure 5B, the AR-regulated genes PSA
(also known as KLK3), KLK2 and TMPRSS2 were strongly suppressed at the mRNA
level upon 27 treatment. MYC, a known oncogene in many types of cancer including
prostate cancer, was also significantly down-regulated by 27. Compound 27 also
strongly suppressed the mRNA expression of full length AR (AR-FL). As
prostate-specific antigen (PSA) is used extensively as a biomarker in the clinical
management of prostate cancer, a PSA promoter driven luciferase reporter assay was
also used as a surrogate to further evaluate the potential impact of compounds on the
4
6
transcription of PSA. Compounds 26 and 27 significantly reduced the transcriptional
activity of PSA in a dose-dependent manner (Figure 5C). Furthermore, western blot
analysis indicated that treatment of 22Rv1 cells with compound 26 and 27 resulted in
significant down regulation of both AR-FL and AR variants levels (Figure 5D). These
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results indicated that the RORγ inverse agonists 26 and 27 effectively inhibited cell
growth and related gene expression in prostate cancer cells.
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.8. Assessment of Metabolic Stability and Pharmacokinetic (PK) Properties for
RORγ Inverse Agonists 23, 26, and 27.
To assess the potential of this series of RORγ inverse agonists in vivo, we further
investigated the metabolic stability and pharmacokinetic properties of compounds 23,
2
6 and 27 (Table 5 and Supporting Information Figure S4). An in vitro metabolic
stability study with rat liver microsomes (RLM) indicated that all 3 tested compounds
exhibited excellent metabolic stability. After incubation with rat liver microsomes for
6
0 min, 79%, 78%, 93% compound remaining were detected for 23, 26, and 27,
respectively. Compound 27 is very stable in this study with very large half-life
(Supporting Information Figure S4).
In a PK study, plasma levels of these inverse agonists were monitored after a single
oral dose (po) of 10 mg/kg or an intravenous (iv) dose of 2 mg/kg (Table 5).
Compound 23 exhibited reasonable PK profiles with high plasma exposure AUC(0-∞)
value of 6444 (μg/L·h) and maximum plasma concentration (Cmax) value of 839
(μg/L), after a 2 mg/kg iv administration. However, 23 demonstrated relatively low
oral bioavailability of 19% after an oral administration. In contrast to 23, both of the
compounds 26 and 27 exhibited significantly improved PK profiles with high plasma
exposure AUC(0-∞) values of 24481 and 19577 μg/L·h, reasonable oral bioavailability
values of 52% and 59%, and good oral half-life values of 8.67 h and 7.32 h,
respectively, after a 10 mg/kg oral administration. In addition, compounds 26 and 27
possessed low clearance and a modest volume of distribution (V ). The above results
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suggested that 26 and 27 may have good in vivo efficacies when orally administered.
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Table 5. Intravenous (iv) and oral (po) pharmacokinetic parameters for 23, 26, and 27 in rats
AUC(0-t)
AUC(0-∞)
(µg/L·h)
Cl
Vz
(mg/kg)
Route
C
max (µg/L)
Tmax (h)
T
1/2 (h)
F (%)
(µg/L·h)
(mL/h/kg)
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23 iv (2 mg/kg)
839 ± 38
0.19 ± 0.10
8.00 ± 0.00
3335 ± 495
6444 ± 1719
-
7.67 ± 2.36
-
326.67 ± 92.57
193.73 ± 12.28
272.26 ±28.23
3425 ± 594
3544 ± 271
3935 ± 693
23 po (10 mg/kg)
721 ± 120
980 ± 53
1425 ± 161
818 ± 19
3217 ± 781
7660 ± 329
19
52
59
2
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2
6 iv (2 mg/kg)
6 po (10 mg/kg)
7 iv (2 mg/kg)
7 po (10 mg/kg)
0.08 ± 0.00
5.33 ±2.31
0.14 ± 0.10
10352 ± 664
24481 ± 5807
7398 ± 757
19577 ± 1879
12.71 ± 1.21
8.67 ± 0.05
9.98 ± 0.88
7.32 ± 1.08
21645 ± 3950
6105 ± 719
1200 ± 182
6.00 ± 2.00
18829 ± 3083
The pharmacokinetic parameters were tested in 6 rats. Compounds were formulated in 5% DMSO, 40% PEG400, 55% 20% HP-β-CD. Values are reported as means
SD.
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.9. Compound 27 Inhibits Prostate Cancer Tumor Growth.
To further evaluate the effect of RORγ inverse agonists on prostate cancer tumor
growth, we generated xenograft tumors using a 22Rv1 prostate cancer cell line that
expresses high levels of multiple AR variants. Mice were treated with either vehicle
or the RORγ inverse agonist 27 (five times per week) via intraperitoneal injection (IP,
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mg/kg) or oral gavage (PO, 50 mg/kg). The efficacy data show that compound 27
exhibited significant antitumor activities during the treatment period with tumor
growth inhibition (TGI) of 53% or 69% (Figure 6). Furthermore, the treatment with
2
7 was well tolerated without obvious body weight loss (data not shown).
Figure 6. Antitumor activity of compound 27 in a 22Rv1 xenograft mouse model (n =
6 mice per group). Mice were treated with vehicle or 27 (5 mg/kg i.p. or 50 mg/kg
orally) 5 times per week. Treatment started when 22Rv1 tumors reached
3
approximately 100 mm . Mean tumor volume ± s.e.m.
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. CONCLUSIONS
In summary, we report the design, synthesis, and optimization of a large series of
novel RORγ inverse agonists for the treatment against prostate cancer. A
structure-based drug design approach was successfully utilized to generate the
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promising compound, 27, which disrupted the recruitment of the co-activator SRC1 to
RORγ and displayed remarkable inhibition of the RORγ transcription activity.
Determination of the high-resolution crystal structure of 27-RORγ complex provided
direct structural evidence on how the compound 27 disrupts RORγ LBD and functions
as inverse agonist. In vitro, compound 27 exhibited potent inhibitory activities on cell
growth, colony formation, and expression of AR and AR regulated genes in
AR-positive prostate cancer cell lines. Compound 27 exhibited good metabolic
stability, favorable pharmacokinetic profiles in rat with excellent oral bioavailability
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(F = 59%) and half-life time (7.32 h). In vivo, oral administration of compound 27
significantly suppressed tumor growth in 22Rv1 tumor xenograft model. The potent,
selective, metabolic stable and orally bioavailable RORγ inverse agonist 27 (XY101)
was selected as a candidate compound for future preclinical studies for the treatment
of advanced prostate cancer and other diseases.
4
4
. EXPERIMENTAL SECTION
.1. Molecular Docking Studies.
The crystal structure of RORγ in complex with the inverse agonist (PDB code:
QM0.pdb) was downloaded from Protein Data Bank (http://www.pdb.org). All the
4
ligand and protein preparations were performed in Maestro (version 9.4, Schrꢀdinger,
LLC, New York, NY, 2013) implemented in the Schrꢀdinger program. The proteins
were prepared using the Protein Preparation Wizard within Maestro 9.4 (Schrꢀdinger,
LLC). Hydrogens were added, bond orders were assigned, and missing side chains for
some residues were added using Prime. The added hydrogens were subjected to
energy minimization until the root-mean-square deviation (RMSD) relative to the
starting geometry reached 0.3 Å. The Glide docking program in Maestro 9.4 was used
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for docking studies. For Glide docking, the grid was defined using a 20 Å box
centered on the ligand, and the important water molecules around ligand were kept.
All parameters were kept as default. The designed molecules were docked using Glide
SP mode, and the predicted binding modes of all the compounds were ranked
according to their glidescores.
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.2. General Chemistry.
All commercial reagents were used without further purification unless otherwise
specified. Enzalutamide were purchased from Selleck. Final compounds were purified
1
either by silica gel chromatography (300-400 mesh) or by recrystallization. H-NMR
and 13C-NMR spectra were recorded on Bruker AV-400 or AV-500 spectrometer.
Coupling constants (J) are expressed in hertz (Hz). NMR chemical shifts (δ) are
reported in parts per million (ppm) units relative to the internal control (TMS). Low or
high resolution ESI-MS were recorded on an Agilent 1200 HPLC-MSD mass
spectrometer or an Applied Biosystems Q-STAR Elite ESI-LC-MS/MS mass
spectrometer, respectively. Compound purities were determined by reverse-phase
high-performance liquid chromatography (HPLC) with 20% solvent A (H
2
O) and 80%
solvent B (MeOH or 0.5‰ NH in MeOH) as eluents. HPLC analysis uses a Dionex
3
Summit HPLC column (Inertsil ODS-SP, 5.0 μm, 4.6 mm × 250 mm (GL Sciences
Inc.)) with a UVD170U detector, and a manual injector, a P680 pump with a detection
wavelength of 254 nm and a flow rate of 1.0 mL/min. The purity of all the final
compounds was determined by HPLC to be >95%.
General Procedure for Synthesis of Sulfonyl Chlorides. Step 1. A mixture of halide
(
1.0 equiv) and sodium sulfite (1.2 equiv) in H O (0.1 M) and EtOH (0.2 M) was
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heated to reflux overnight. The mixture was cooled to room temperature and
concentrated until a precipitate began to form. The product was collected by filtration
and azeotroped with toluene. The resulting solid was used in the next step. Step 2. To
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a suspension of the sodium sulfonate (1.0 equiv) in CH
2
Cl (0.1 M) were added DMF
2
(
0.7 equiv) and SOCl (3.9 equiv). After 1.5 h, the white suspension was concentrated
2
and azeotroped with toluene. The sulfonyl chlorides thus formed were used without
further purification.
2
-(4-Amino-3-fluorophenyl)-1,1,1,3,3,3-hexafluoropropan-2-ol
(7).
To
2-fluoroaniline (5, 6.0 g, 54 mmol) in a pressure vessel was added hexafluoroacetone
trihydrate (6, 12.5 g, 56.7 mmol) and p-toluenelsuphonic acid (0.85 g, 5.4 mmol). The
reaction mixture was stirred at 90 °C for 12 h. Water was added, and the mixture was
extracted with EtOAc (3 × 150 mL). The organic layer was washed with saturated
NaHCO
3
solution and brine and dried over Na
2
SO . The solid was filtered off, and the
4
filtrate was concentrated under reduced pressure. The resulting crude product was
purified by silica gel chromatography with petroleum ether/EtOAc (10/1, v/v) and
dried to give 7 as white solid (4.45 g, 30 % yield). MS (ESI), m/z for C
9
H
6
F NO ([M
7
+
+
1] ): Calcd 277.14, found 278.0.
1,1,1,3,3,3-hexafluoro-2-(3-fluoro-4-iodophenyl)propan-2-ol (8). To a solution of 7
4.45 g, 16.1 mmol) in DMF (50 mL) was added concentrated HCl (18 mL, 73 mmol)
at 0 °C, stirred 5 min, and sodium nitrite (1.7 g, 24 mmol) was added, dissolved in
O (20 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 30 min, then added
(
H
2
potassium iodide (4.0 g, 24 mmol) in portions, and then the reaction mixture was
stirred at room temperature for overnight. Water was added, and the mixture was
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extracted with EtOAc (3 × 150 mL). The organic layer was washed with saturated
NaHCO
3
solution and brine and dried over Na
2
SO . The solid was filtered off, and the
4
filtrate was concentrated under reduced pressure. The resulting crude product was
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purified by silica gel chromatography with petroleum ether/EtOAc (50/1, v/v) to
1
obtain 8 (6.2 g, 96 % yield). H NMR: (400 MHz, DMSO-d
6
) δ 7.31 (d, J = 8.4 Hz,
1
H), 7.48 (d, J = 1.6 Hz, 9.2 Hz, 1H), 8.05 (dd, J = 6.8 Hz, 8.4 Hz, 1H), 9.07 (s, 1H).
Tert-butyl(2'-fluoro-4'-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-[1,1'-biphe
nyl]-4-yl)carbamate (9). To a solution of 8 (6.2 g, 16 mmol) in 1,4-dioxane (100 mL)
and water (20 mL) was added (4-((tert-butoxycarbonyl)amino)phenyl)boronic acid
(
4.2 g, 17.6 mmol), followed by addition of potassium carbonate (6.6 g, 48 mmol) and
Pd(PPh (0.9 g, 0.78 mmol), the vessel was purged with argon, sealed and heated to
80 °C for 5 h. Water was added, and the mixture was extracted with EtOAc (3 × 150
mL). The organic layer was washed with saturated NaHCO solution and brine and
dried over Na SO . The solid was filtered off, and the filtrate was concentrated under
)
3 4
3
2
4
reduced pressure. The resulting crude product was purified by silica gel
chromatography with petroleum ether/ EtOAc (20/1, v/v) to yield 9 (5.4 g, 74 %
1
yield). H NMR (400 MHz, CDCl
3
) δ 7.54 – 7.44 (m, 6H), 7.40 (d, J = 8.4 Hz, 1H),
6
.57 (s, 1H), 3.82 (s, 1H), 1.53 (d, J = 3.2 Hz, 9H).
2
-(4'-amino-2-fluoro-[1,1'-biphenyl]-4-yl)-1,1,1,3,3,3-hexafluoro propan-2-ol (10).
To a solution of 9 (5.35 g, 11.8 mmol) in DCM (50 mL) was added trifluoroacetic
acid (7 mL, 96 mmol) dropwise at 0 °C. The reaction mixture was stirred at room
temperature for 3 h. The mixture was concentrated under reduced pressure and
recrystallization was then carried out in petroleum ether/EtOAc to afford 10 (3.8 g,
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