Synthesis of androgen receptor antagonists containing a pentafluorosulfanyl (SF5) moiety

Article abstract of DOI:10.1002/ardp.201800175

A novel scaffold of pentafluorosulfanyl (SF₅)-containing enzalutamide analogues was discovered for potent androgen receptor (AR) antagonists through rational drug design. Several compounds showed good biological profiles in AR binding. Of the derivatives studied, compound 8a had potent AR antagonist activity (IC₅₀ = 7.1 ± 1.0 μM) and high efficacy (104.5 ± 12.8%). It exhibited an inhibitory effect comparable to that of enzalutamide (inhibition = 66.0 and 77.9%, respectively) in a prostate cancer cell line. The results point to the potential of using this scaffold to develop new AR antagonists.

Full text of DOI:10.1002/ardp.201800175

Received: 6 June 2018
Revised: 12 September 2018
Accepted: 26 September 2018
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DOI: 10.1002/ardp.201800175
FULL PAPER
Synthesis of androgen receptor antagonists containing a
pentafluorosulfanyl (SF5) moiety
Jiyu Jin1
Wei Lu1
Yan Zhou2
Dehua Yang2
Qiumeng Zhang1
Ming-Wei Wang2
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1 Shanghai Engineering Research Center of
Molecular Therapeutics and New Drug
Development, School of Chemistry and
Molecular Engineering, East China Normal
University, Shanghai, P. R. China
Abstract
A novel scaffold of pentafluorosulfanyl (SF5)-containing enzalutamide analogues was
discovered for potent androgen receptor (AR) antagonists through rational drug
design. Several compounds showed good biological profiles in AR binding. Of
the derivatives studied, compound 8a had potent AR antagonist activity
(IC50 = 7.1 1.0 µM) and high efficacy (104.5 12.8%). It exhibited an inhibitory
effect comparable to that of enzalutamide (inhibition = 66.0 and 77.9%, respectively) in
a prostate cancer cell line. The results point to the potential of using this scaffold to
develop new AR antagonists.
2 The National Center for Drug Screening and
the CAS Key Laboratory of Receptor
Research, Shanghai Institute of Materia
Medica, Chinese Academy of Sciences (CAS),
Shanghai, P. R. China
Correspondence
Prof. Ming-Wei Wang, The National Center
for Drug Screening and the CAS Key
Laboratory of Receptor Research, Shanghai
Institute of Materia Medica, Chinese Academy
of Sciences (CAS), 189 Guo Shou Jing Road,
Shanghai 201203, P. R. China
K E Y W O R D S
androgen receptor, antagonist, enzalutamide, pentafluorosulfanyl
Email: mwwang@simm.ac.cn
Wei Lu, Shanghai Engineering Research
Center of Molecular Therapeutics and New
Drug Development, School of Chemistry and
Molecular Engineering, East China Normal
University, 3663 North Zhongshan Road,
Shanghai 200062, P. R. China
Email: wlu@chem.ecnu.edu.cn
Funding information
Chinese Academy of Sciences, Grant numbers:
XDA12020347, XDA12040308; Thousand
Talents Program in China; Shanghai Science
and Technology Council, Grant number:
16DZ2280100
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1
INTRODUCTION
will progress and develop to castration-resistant prostate cancer
(CRPC) at last.[1] Enzalutamide is a second-generation nonsteroidal
The incidence of prostate cancer has been rising rapidly in recent years
due to environmental and lifestyle changes. Androgen deprivation
therapy (ADT) has been proven to be effective at first, but the tumor
anti-androgen with high affinity binding for the androgen receptor (AR)
ligand-binding domain (Figure 1).[2–4] It received FDA approval in
2012 to treat patients with metastatic castration-resistant prostate
cancer.[5]
The SF5 group is an organic derivative of the hypervalent
molecule sulfur hexafluoride (SF6). In the last 10 years, it has been
Jiyu Jin and Yan Zhou contributed equally to this work.
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Arch Pharm Chem Life Sci. 2018;e1800175.
wileyonlinelibrary.com/journal/ardp
© 2018 Deutsche Pharmazeutische Gesellschaft
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https://doi.org/10.1002/ardp.201800175

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JIN ET AL.
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FIGURE 1 Design of enzalutamide analogues containing the pentafluorosulfanyl group
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investigated extensively, resulting in a large number of building
blocks and reagents bearing this substituent.[3,6] SF5-substituted
aromatic compounds have a high degree of thermal stability and
inertia to hydrolysis, displaying a better effect than that of the CF3
group.[7] Due to its unique properties, the SF5 group has special
utility as fluorine-containing substituents.[8,9] Compared with CF3,
the high electronegativity of SF5-containing compounds[10] is
capable of increasing polarity while displaying excellent lipophilic-
ity.[11] Exchange of CF3 with SF5 in drugs and crop protection agents
showed good outcomes, leading to improved pharmacological
characteristics,[12] such as mefloquine,[13] fenfluramine,[14] triflura-
lin,[15] and fipronil.[16]
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RESULTS AND DISCUSSION
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2.1 Chemistry
Pentafluorosulfanyl derivatives 8a–d and 10a–d were prepared as
illustrated in Scheme 1. 3-(Pentafluorosulfanyl)aniline (1) was
bromized with 1,3-dibromo-5,5-dimethylhydantoin to form
4-bromo-3-(pentafluorosulfanyl)aniline (2). The second step consisted
of the aromatic nucleophilic displacement of bromide from compound
2 using copper cyanide, leading to the formation of 4-amino-2-
(pentafluorosulfanyl)benzonitrile (3). Then, compound 3 was reacted
with thiophosgene to form 4-isothiocyanato-2-(pentafluorosulfanyl)
benzonitrile (4).
Since there are no reported AR antagonists bearing the SF5, we
designed and biologically evaluated eight SF5-containing enzalutamide
analogues as described in this paper.
The 2-bromo-2-methylpropanoic acid (6) was reacted with four
different arylamines (5a–d) using TEA to produce compounds 7a–d.
SCHEME 1 Synthesis of enzalutamide derivatives 8a–d and 10a–d. Reagents and conditions: (a) DBDMH, DMAc, −20°C; (b) CuCN, NMP,
180°C; (c) thiophosgene, CCl4/water, rt; (d) TEA, DCM, rt; (e) TEA, CHCl3, reflux

JIN ET AL.
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Arylated aminoacids 7a–d were further cyclized upon the treatment
2.2.2 Anti-proliferative activity of compounds 8a–d
and 10a–d in prostate cancer cell lines
with isothiocyanate
4 to make the desired pentafluorosulfanyl
derivatives 8a–d.
We performed experiments accordingly to detect the anti-proliferative
effect of compounds using prostate cancer LNCaP cell line. Measure-
ment of proliferative activity was performed after treatment with a
concentration range of each compound for 72 h. The results
demonstrated the inhibitory effect of compounds on growth of
LNCaP cell line (Table 2).
Pentafluorosulfanyl derivatives 10a–d were prepared in a similar
manner by coupling thioisocyanate 9 with appropriately arylated
amino acids 7a–d. Compound
9 was prepared by reacting 3-
(pentafluorosulfanyl)aniline (1) and thiophosgene (Scheme 1).
All of enzalutamide derivatives containing SF5 group exhibited
high anti-proliferative activity in prostate cancer LNCaP cell line. The
compound 8a exhibited a comparable inhibitory effect with enzalu-
tamide (inhibition = 66.0 and 77.9%, respectively) on growth of LNCaP
cell line.
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2.2 Biological activity
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2.2.1 Antagonist affinity of compounds 8a–d and
10a–d
We examined the AR antagonist activity of the representative
compounds 8a–d and 10a–d with a luciferase gene reporter assay in
mouse myoblast C2C12 cells. As shown in Table 1, these enzalutamide
analogues with para-cyano and meta-SF5 moieties displayed
different degrees of AR antagonist activity. Compound 8a
exhibited a comparable AR antagonist activity with enzalutamide
(IC50 = 7.1 1.0 µM and 3.0 1.4 µM, efficacy = 104.5 and 100%,
respectively), without causing cytotoxicity demonstrated by viability of
C2C12 myotubes (Supporting Information). AR antagonistic potency
for 8c and 8d was similar with IC50 values of 5.0 2.8 and 5.5 5.9 µM,
respectively, accompanied by weak efficacies (60.3 and 76.1%,
respectively). When an ester group was introduced (8b), potency
was further reduced.
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3
CONCLUSION
In summary, a series of SF5-containing enzalutamide derivatives were
designed, synthesized, and evaluated for AR antagonism in vitro.
Compound 8a had potent AR antagonist activity (IC50 = 7.1 1.0 µM)
and high efficacy (104.5 12.8%). It exhibited a comparable inhibitory
effect with enzalutamide (inhibition = 66.0 and 77.9%, respectively) in
prostate cancer cell line. Of the derivatives studies, 8a representing
this novel scaffold showed
characterization.
a profile that warrants further
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4
EXPERIMENTAL
To investigate the importance of the cyano moiety, com-
pounds 10a–d, in which the cyano is absent, were synthesized.
Unfortunately, 10d was completely inactive compared to
enzalutamide, whereas compounds 10b and 10c exhibited
weaker antagonist activity than that of 8b and 8c. Although
10a had a similar potency as 8a, its efficacy was only half that of
8a (Table 1). It is therefore clear that cyano moiety is required for
AR antagonism.
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4.1 Chemistry
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4.1.1 General
All reagents are commercially available and were used without
further purification. The solvents used were of analytical grade.
Melting points were taken on a Fisher Johns melting point apparatus,
TABLE 1 Bioactivity of compounds 8a–d and 10a–d
TABLE 2 Anti-proliferative activity of compounds 8a–d and 10a–d
Antagonist activity in C2C12
Compounds
Anti-proliferative activity in LNCaP (%)
Compounds
IC50 (µM)
3.0 1.4
7.1 1.0
20.5 23.9
5.0 2.8
5.5 5.9
7.0 0.7
26.3 22.9
28.7 2.6
NA
Efficacy (%)
100
Enzalutamide
77.9 3.5
66.0 2.6
102.4 0.7
76.2 2.3
93.5 12.1
75.3 10.8
86.7 5.4
88.4 2.8
129.7 5.2
Enzalutamide
8a
8a
104.5 12.8
83.1 17.4
60.3 25.4
76.1 9.7
51.6 7.3
67.1 30.8
69.6 2.5
30.6 10.7
8b
8b
8c
8c
8d
8d
10a
10b
10c
10d
10a
10b
10c
10d
Data were normalized and plotted relative to DMSO-control treated (no
compound) cells and expressed as the means SD.
Data presented are the means SD from three independent experiments.

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JIN ET AL.
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uncorrected and reported in degrees centigrade. 1H NMR and
13C NMR spectra were scanned on a Bruker DRX-400 (400 MHz)
using tetramethylsilane (TMS) as internal standard and one or two of
the following solvents, DMSO-d6 and CDCl3. Chemical shifts are
given in δ, ppm. Splitting patterns were designated as follows: s:
singlet; d: doublet; t: triplet; q: quartet; m: multiplet. The mass
spectra (MS) were recorded on a Finnigan MAT-95 mass spectrom-
eter. The purity of all testing compounds was established by HPLC to
be >95.0%. HPLC analysis was performed at room temperature using
an Agilent Eclipse XDBC18 (250 mm × 4.6 mm) and as a mobile phase
gradient from 5% MeCN/H2O (1‰ TFA) for 1 min, 5% MeCN/
H2O (1‰ TFA) to 95% MeCN/H2O (1‰ TFA) for 9 min and 95%
MeCN/H2O (1‰ TFA) for 5 min more, a flow rate of 1.0 mL/min and
plotted at 254 nm.
J = 8.3 Hz, 1H), 7.10 (s, 1H), 6.77 (d, J = 8.3 Hz, 1H), 4.47 (s, 2H).
13C NMR (101 MHz, CDCl3) δ 156.35, 150.29, 136.39, 117.38, 116.09,
113.80, 97.05. ESI-MS m/z: 245.25 [M+H]+.
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4.1.4 General procedure for the synthesis of 4 and 9
In a 100 mL round-bottomed flask was added thiophosgene (1.1
eq) in 5 mL water to give an orange suspension. Compound 1 or 3
(1.0 eq) was added. The reaction mixture was held at room
temperature with stirring on for 16 h. The aq layer was back
extracted with EA. The mixture was concentrated by rotovap to
give 4 and 9. The crude products were used to next step without
further purification.
The original NMR spectra are provided as Supporting Informa-
tion. The InChI codes of the investigated compounds together
with some biological activity data are also provided as Supporting
Information.
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4.1.5 General procedure for the synthesis of 7a–d
In
a 100 mL round-bottomed flask were added four different
arylamines (5a–d) (1.0 eq), 2-bromo-2-methylpropanoic acid (1.5
eq), and TEA (3.0 eq) in 150 mL DCM to give a colorless suspension.
The reaction mixture was held at room temperature with stirring on for
3 days. The mixture was concentrated by rotovap. One hundred
milliliter water was added. Sixty milliliter 2 M HCl(aq) was used to
adjust pH to 5. The aq layer was back extracted with EA. The organic
was dried with Na2SO4, filtered and concentrated to give crude
product. Twenty milliliter DCM and 50 mL Et2O was added. The
reaction mixture was filtered through sintered glass funnel with 50 mL
Et2O to give 7a–d.
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4.1.2 Synthesis of 4-bromo-3-
(pentafluorosulfanyl)-aniline (2)
In a 100 mL round-bottomed flask was added 3-(pentafluorosulfanyl)-
aniline (1) (2 g, 9.13 mmol, 1.0 eq) in 15 mL DMAc to give a yellow
solution. The reaction mixture was cooled to −5°C with stirring
on.
1,3-Dibromo-5,5-dimethylimidazolidine-2,4-dione
(1.435 g,
5.02 mmol, 0.55 eq) in 10 mL DMAc was added. The reaction
mixture was held at −5°C with stirring on for 16 h. One hundred
milliliter water was added. The aq layer was back extracted with EA.
Combined the organic layers and washed with water and brine. The
organic was dried with Na2SO4, filtered and concentrated to give
crude product 3.3 g. The crude product was purified by column
chromatography to give 4-bromo-3-(pentafluorosulfanyl)aniline (2)
(1.7 g, 62.5% yield) as orange oil. 1H NMR (400 MHz, CDCl3) δ 7.47
(d, J = 8.8 Hz, 1H), 7.12 (s, 1H), 6.97 (d, J = 8.8 Hz, 1H), 4.30 (s, 2H).
13C NMR (101 MHz, CDCl3) δ 154.32, 154.15, 153.98, 145.52,
136.59, 118.89, 116.06, 116.01, 115.96, 115.90, 104.15. ESI-MS
m/z: 297.97, 300.14 [M+H, M+2H]+.
2-((3-Fluoro-4-(methylcarbamoyl)phenyl)amino)-2-
methylpropanoic acid (7a)
White solid (6.8 g, 76% yield). M.p. 210–212°C; 1H NMR (400 MHz,
DMSO) δ 12.68 (s, 1H), 7.62 (s, 1H), 7.45 (t, J = 8.8 Hz, 1H), 6.70 (s,
1H), 6.34 (d, J = 7.2 Hz, 1H), 6.16 (d, J = 14.7 Hz, 1H), 2.73 (d,
J = 4.3 Hz, 3H), 1.44 (s, 6H). 13C NMR (101 MHz, DMSO) δ 176.57,
163.86, 162.05, 159.61, 150.68, 150.56, 131.15, 109.45, 109.32,
109.22, 98.85, 98.58, 56.10, 26.22, 25.46. ESI-MS m/z: 255.25
[M+H]+.
2-((4-(Methoxycarbonyl)phenyl)amino)-2-methylpropanoic acid
(7b)
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4.1.3 Synthesis of 4-amino-2-(pentafluorosulfanyl)-
benzonitrile (3)
White solid (2.4 g, 76% yield). M.p. 154–156°C; 1H NMR (400 MHz,
DMSO) δ 12.67 (s, 1H), 7.68 (d, J = 8.8 Hz, 2H), 6.77 (s, 1H), 6.51 (d,
J = 8.8 Hz, 2H), 3.74 (s, 3H), 1.47 (s, 6H). 13C NMR (101 MHz, DMSO) δ
176.60, 166.26, 150.85, 130.59, 116.05, 112.28, 55.99, 51.18, 25.57.
ESI-MS m/z: 194.25 [M+H]+.
In a 100 mL round-bottomed flask was added 4-bromo-3-(pentafluor-
osulfanyl)aniline (2) (1.7 g, 5.70 mmol, 1.0 eq) and cyanocopper
(0.613 g, 6.84 mmol, 1.2 eq) in 30 mL NMP to give a brown solution.
The reaction vessel was purged with nitrogen. The reaction was heated
to 180°C with stirring on for 4 h. One hundred milliliter water was
added. The aq layer was back extracted with EA. Combined the organic
layers and washed with water and brine. The organic was dried with
Na2SO4, filtered, and concentrated to give crude product 1.5 g. The
crude product was purified by column chromatography to give
4-amino-2-(pentafluorosulfanyl)benzonitrile (3) (1.05 g, 75% yield) as
white solid. M.p. 149–151°C; 1H NMR (400 MHz, CDCl3) δ 7.54 (d,
2-Methyl-2-(p-tolylamino)propanoic acid (7c)
White solid (1.4 g, 37% yield). M.p. 166–168°C; 1H NMR (400 MHz,
DMSO) δ 8.93 (s, 2H), 6.86 (d, J = 8.2 Hz, 2H), 6.42 (d, J = 8.2 Hz, 2H),
2.13 (s, 3H), 1.40 (s, 6H). 13C NMR (101 MHz, DMSO) δ 177.52,
144.17, 129.00, 124.50, 113.98, 56.07, 25.76, 19.97. ESI-MS m/z:
238.08 [M+H]+.

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2-Methyl-2-((4-(4-(methylamino)-4-oxobutyl)phenyl)amino)-
propanoic acid (7d)
8.09 (d, J = 8.2 Hz, 1H), 7.80 (t, J = 7.9 Hz, 1H), 7.43 (d, J = 10.5 Hz, 1H),
7.34 (d, J = 8.0 Hz, 1H), 2.81 (d, J = 3.5 Hz, 3H), 1.56 (s, 6H). 13C NMR
(101 MHz, DMSO) δ 179.92, 174.57, 163.38, 160.12, 157.64, 138.26,
138.15, 137.88, 136.38, 133.64, 130.87, 130.83, 129.70, 126.07,
125.20, 125.05, 118.07, 117.83, 115.60, 108.13, 66.58, 26.23, 22.90.
HRMS (ESI) m/z calcd. for C19H16F5N3OS2 [M+H]+: 461.0655. Found:
461.0655.
White solid (1.4 g, 97% yield). M.p. 108–110°C; 1H NMR (400 MHz,
DMSO) δ 11.32 (s, 1H), 7.91 (s, 1H), 7.46 (d, J = 8.0 Hz, 2H), 7.36 (d,
J = 8.0 Hz, 2H), 2.63–2.57 (m, 2H), 2.55 (d, J = 9.2 Hz, 3H), 2.10 (t,
J = 7.3 Hz, 2H), 1.88–1.74 (m, 2H), 1.50 (s, 6H), 1.23 (dd, J = 14.4,
7.3 Hz, 1H). 13C NMR (101 MHz, DMSO) δ 172.13, 172.05, 143.30,
129.90, 129.11, 125.69, 64.31, 45.34, 34.59, 34.03, 26.64, 25.38,
21.77. ESI-MS m/z: 279.24 [M+H]+.
4-(4-(3-(4-Cyano-3-(pentafluoro-l6-sulfanyl)phenyl)-5,5-
dimethyl-4-oxo-2-thioxoimidazolidin-1-yl)phenyl)-N-
methylbutanamide (8d)
White solid (120 mg, 79% yield). M.p. 77–79°C; 1H NMR (400 MHz,
CDCl3) δ 8.15 (s, 1H), 7.96 (d, J = 8.2 Hz, 1H), 7.83 (d, J = 8.2 Hz, 1H),
7.36 (d, J = 7.9 Hz, 2H), 7.21 (d, J = 7.7 Hz, 2H), 5.52 (s, 1H), 2.82 (d,
J = 4.6 Hz, 3H), 2.74 (t, J = 7.6 Hz, 2H), 2.24 (t, J = 7.3 Hz, 2H), 2.09–
1.96 (m, 2H), 1.59 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 179.62,
174.96, 173.02, 143.64, 137.00, 135.24, 132.75, 131.57, 129.94,
129.34, 128.96, 115.59, 109.63, 66.42, 35.76, 34.94, 26.75, 26.34,
23.70. HRMS (ESI) m/z calcd. for C23H23F5N4O2S2 [M+H]+: 546.1183.
Found: 546.118.
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4.1.6 General procedure for the synthesis of 8a–d
and 10a–d
In a 100 mL round-bottomed flask was added 4 or 9 (1.0 eq) and
TEA (1.5 eq) in 30 mL CHCl3 to give a yellow solution. The reaction
vessel was purged with nitrogen. The reaction was heated to
65°C with stirring on for 1 h. The reaction mixture was cooled to
25°C with stirring on. 7a–d (1.0 eq) were added. The reaction was
heated to 65°C with stirring on for 16 h. The mixture was
concentrated by rotovap. The crude product was purified by
column chromatography to give 8a–d and 10a–d.
4-(5,5-Dimethyl-4-oxo-3-(3-(pentafluoro-l6-sulfanyl)phenyl)-2-
thioxoimidazolidin-1-yl)-2-fluoro-N-methylbenzamide (10a)
White solid (73 mg, 77% yield). M.p. 88–90°C; 1H NMR (400 MHz,
CDCl3) δ 8.24 (t, J = 8.4 Hz, 1H), 7.83 (d, J = 5.9 Hz, 2H), 7.59 (q,
J = 8.0 Hz, 2H), 7.25 (d, J = 4.3 Hz, 1H), 7.15 (d, J = 11.8 Hz, 1H), 6.71 (d,
J = 5.6 Hz, 1H), 3.04 (d, J = 4.6 Hz, 3H), 1.58 (s, 6H). 13C NMR
(101 MHz, CDCl3) δ 179.79, 173.85, 161.77, 160.58, 158.09, 152.84,
138.40, 138.29, 132.23, 132.08, 130.58, 128.19, 125.63, 125.26,
125.23, 121.49, 121.37, 117.09, 116.83, 65.42, 25.96, 22.82. HRMS
(ESI) m/z calcd. for C19H17F6N3O2S2 [M+H]+: 497.0666. Found:
497.0666.
4-(3-(4-Cyano-3-(pentafluoro-l6-sulfanyl)phenyl)-5,5-dimethyl-
4-oxo-2-thioxoimidazolidin-1-yl)-2-fluoro-N-methylbenzamide
(8a)
White solid (320 mg, 57% yield). M.p. 145–147°C; 1H NMR
(400 MHz, DMSO) δ 8.52 (s, 1H), 8.45 (d, J = 3.7 Hz, 1H), 8.42 (d,
J = 8.5 Hz, 1H), 8.09 (d, J = 8.2 Hz, 1H), 7.80 (t, J = 7.9 Hz, 1H),
7.43 (d, J = 10.5 Hz, 1H), 7.34 (d, J = 8.0 Hz, 1H), 2.81 (d,
J = 3.5 Hz, 3H), 1.56 (s, 6H). 13C NMR (101 MHz, DMSO) δ
179.92, 174.57, 163.38, 160.12, 157.64, 138.26, 138.15, 137.88,
136.38, 133.64, 130.87, 130.83, 129.70, 126.07, 125.20, 125.05,
118.07, 117.83, 115.60, 108.13, 66.58, 26.23, 22.90. HRMS
(ESI) m/z calcd. for C20H16F6N4O2S2 [M+H]+: 522.0619. Found:
522.0619.
Methyl 4-(5,5-dimethyl-4-oxo-3-(3-(pentafluoro-l6-sulfanyl)-
phenyl)-2-thioxoimidazolidin-1-yl)benzoate (10b)
White solid (75 mg, 82% yield). M.p. 93–95°C; 1H NMR (400 MHz,
CDCl3) δ 8.19 (d, J = 8.4 Hz, 2H), 7.85 (s, 1H), 7.82 (d, J = 5.0 Hz,
1H), 7.59 (d, J = 4.0 Hz, 2H), 7.40 (d, J = 8.4 Hz, 2H), 3.93 (s, 3H),
1.57 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 180.81, 175.15, 165.95,
153.84, 139.58, 133.27, 131.68, 131.25, 131.11, 129.86, 129.16,
126.70, 126.65, 126.60, 126.55, 126.51, 66.38, 52.50, 23.82.
HRMS (ESI) m/z calcd. for C19H17F5N2O3S2 [M+H]+: 480.0601.
Found: 480.060.
Methyl 4-(3-(4-cyano-3-(pentafluoro-l6-sulfanyl)phenyl)-5,5-
dimethyl-4-oxo-2-thioxoimidazolidin-1-yl)benzoate (8b)
White solid (30 mg, 34% yield). M.p. 122–124°C; 1H NMR (400 MHz,
DMSO) δ 8.52 (s, 1H), 8.45 (d, J = 3.7 Hz, 1H), 8.42 (d, J = 8.5 Hz, 1H),
8.09 (d, J = 8.2 Hz, 1H), 7.80 (t, J = 7.9 Hz, 1H), 7.43 (d, J = 10.5 Hz, 1H),
7.34 (d, J = 8.0 Hz, 1H), 2.81 (d, J = 3.5 Hz, 3H), 1.56 (s, 6H). 13C NMR
(101 MHz, DMSO) δ 179.92, 174.57, 163.38, 160.12, 157.64, 138.26,
138.15, 137.88, 136.38, 133.64, 130.87, 130.83, 129.70, 126.07,
125.20, 125.05, 118.07, 117.83, 115.60, 108.13, 66.58, 26.23, 22.90.
HRMS (ESI) m/z calcd. for C20H16F5N3O3S2 [M+H]+: 505.0553. Found:
505.0553.
5,5-Dimethyl-3-(3-(pentafluoro-l6-sulfanyl)phenyl)-2-thioxo-1-
(p-tolyl)imidazolidin-4-one (10c)
White solid (74 mg, 89% yield). M.p. 103–105°C; 1H NMR (400 MHz,
CDCl3) δ 7.85 (d, J = 12.5 Hz, 2H), 7.61 (s, 2H), 7.34 (d, J = 6.6 Hz, 2H),
7.20 (d, J = 6.5 Hz, 2H), 2.43 (s, 3H), 1.57 (s, 6H). 13C NMR (101 MHz,
CDCl3) δ 181.02, 175.58, 153.81, 139.83, 133.45, 132.64, 131.76,
130.52, 129.34, 129.07, 126.72, 126.44, 66.17, 23.66, 21.33. HRMS
(ESI) m/z calcd. for C18H17F5N2OS2 [M+H]+: 436.0702. Found:
436.0702.
4-(4,4-Dimethyl-5-oxo-2-thioxo-3-(p-tolyl)imidazolidin-1-yl)-2-
(pentafluoro-l6-sulfanyl)benzonitrile (8c)
White solid (35 mg, 43% yield). M.p. 93–95°C; 1H NMR (400 MHz,
DMSO) δ 8.52 (s, 1H), 8.45 (d, J = 3.7 Hz, 1H), 8.42 (d, J = 8.5 Hz, 1H),

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4-(4-(5,5-Dimethyl-4-oxo-3-(3-(pentafluoro-l6-sulfanyl)phenyl)-
2-thioxoimidazolidin-1-yl)phenyl)-N-methylbutanamide (10d)
White solid (60 mg, 60% yield). M.p. 86–87°C; 1H NMR (400 MHz,
DMSO) δ 8.15 (s, 1H), 8.03 (d, J = 6.4 Hz, 1H), 7.85–7.70 (m, 3H), 7.37
(d, J = 8.3 Hz, 2H), 7.30 (d, J = 8.2 Hz, 2H), 2.66–2.60 (m, 2H), 2.57 (d,
J = 4.5 Hz, 3H), 2.12 (t, J = 7.4 Hz, 2H), 1.90–1.80 (m, 2H), 1.49 (s, 6H).
13C NMR (101 MHz, DMSO) δ 180.59, 175.38, 172.06, 142.83,
134.24, 133.37, 133.24, 129.70, 129.53, 129.22, 127.01, 126.23,
65.98, 40.10, 34.77, 34.28, 26.63, 25.40, 22.94. HRMS(ESI) m/z calcd.
for C22H24F5N3O2S2 [M+H]+: 521.1230. Found: 521.1230.
4.2.4 Anti-proliferative activity assay
LNCaP cells were cultured in RPMI-1640 medium (Life Technolo-
gies) supplemented with 10% (v/v) fetal bovine serum, 1% sodium
pyruvate, and 1% L-glutamine. The cells were seeded into 96-well
plates at a density of 8 × 104 cells per well. After 24 h incubation,
the culture medium was removed and the cells were cultivated in
the medium supplemented with 2% (v/v) fetal bovine serum for
2 days and then treated with different concentrations of test
compounds for 3 days. Cell viability was measured with the CCK-8
kit (Dojindo).
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4.2 Biological assays
ACKNOWLEDGMENTS
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4.2.1 Cell preparation
We thank Yang Feng for technical assistance. This work was partially
supported by the “Personalized Medicines – Molecular Signature-
based Drug Discovery and Development Strategic Priority Research
Program” of Chinese Academy of Sciences grants (XDA12020347
to M.-W.W.; XDA12040308 to D.Y.) and the Thousand Talents
Program in China (to M.-W.W.). We are grateful for the
financial support for Shanghai Science and Technology Council
(16DZ2280100).
C2C12 cells were provided and certified by the Cell Bank at Shanghai
Institutes for Biological Sciences, Chinese Academy of Sciences and
confirmed as negative for mycoplasma contamination. C2C12 was
cultured in phenol red-free HG-DMEM medium (Life Technologies,
Carlsbad, CA, USA) supplemented with 10% (v/v) fetal bovine serum,
50 IU/mL penicillin, 50 µg/mL streptomycin, and 1% sodium pyruvate.
Cells were maintained at 37°C in 5% CO2 incubator and seeded onto
10 cm cell culture dishes before transfection. After overnight culture,
the cells were transiently co-transfected with AR expressing plasmid
(pSVAR0) and luciferase reporter gene vector (MMTV-Luc) using
FuGENE® HD Transfection Reagent (Promega, Madison, WI, USA)
while cells reaching 80–90% confluence. After 18 h, the transfected
cells were distributed to 384-well plates at a density of 15000 cells
per well and incubated for further 6 h at 37°C before compound
treatment.
CONFLICT OF INTEREST
The authors have declared no conflict of interest.
ORCID
Wei Lu
http://orcid.org/0000-0003-3041-9200
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4.2.2 Antagonist assay
Enzalutamide was used as positive control. Antagonist activity of
testing compounds was measured by Steadylite plus Reporter Gene
Assay System (PerkinElmer, Boston, MA, USA) according to the
manufacturer's instructions. In brief, after incubation for 6 h as above
mentioned, 5 µL testing compounds diluted in culture medium with
eight different working concentrations (384 pM to 30 µM; enzaluta-
mide (128 pM to 10 µM)) were added to cell well followed by 5 µL DHT
(final concentration as EC80). After 24 h incubation, Steadylite reagent
(50 µL, equal volume) was introduced, gently shaked for 2 min, and
kept at room temperature for 15 min before luminescence 384
measurement on an EnSpire multilabel plate reader (PerkinElmer).
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