The design, synthesis and anti-tumor mechanism study of new androgen receptor degrader

Article abstract of DOI:10.1016/j.ejmech.2020.112512

Targeted protein degradation using small molecules is a novel strategy for drug development. In order to solve the problem of drug resistance in the treatment of prostate cancer, proteolysis-targeting chimeras (PROTAC) was introduced into the design of anti-prostate cancer derivatives. In this work, we synthesized two series of selective androgen receptor degraders (SARDs) containing the hydrophobic degrons with different linker, and then investigated the structure-activity relationships of these hybrid compounds. Most of the synthesized compounds exhibited moderate to good activity against all the cancer cell lines selected. Among them, compound A9 displayed potent inhibitory activity against LNCaP prostate cancer cell line with IC₅₀ values of 1.75 μM, as well as excellent AR degradation activity. Primary mechanism studies elucidated compound A9 arrested cell cycle at G0/G1 phase and induced a mild apoptotic response in LNCaP cells. Further study indicated that the degradation of AR was mediated through proteasome-mediated process. For all these reasons, compound A9 held promising potential as anti-proliferative agent for the development of highly efficient SARDs for drug-resistance prostate cancer therapies.

Full text of DOI:10.1016/j.ejmech.2020.112512

Journal Pre-proof
The design, synthesis and anti-tumor mechanism study of new androgen receptor
degrader
Hang Xie, Jian-Jia Liang, Ya-Lei Wang, Tian-Xing Hu, Jin-Yi Wang, Rui-Hua Yang,
Jun-Ke Yan, Qiu-Rong Zhang, Xia Xu, Hong-Min Liu, Yu Ke
PII:
S0223-5234(20)30484-0
DOI:
https://doi.org/10.1016/j.ejmech.2020.112512
EJMECH 112512
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 May 2020
Revised Date: 20 May 2020
Accepted Date: 24 May 2020
Please cite this article as: H. Xie, J.-J. Liang, Y.-L. Wang, T.-X. Hu, J.-Y. Wang, R.-H. Yang, J.-K.
Yan, Q.-R. Zhang, X. Xu, H.-M. Liu, Y. Ke, The design, synthesis and anti-tumor mechanism study of
new androgen receptor degrader, European Journal of Medicinal Chemistry, https://doi.org/10.1016/
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Two series of selective androgen receptor degraders (SARDs) containing the
hydrophobic degrons with different linker were identified. Compound A9 displayed
the most potent inhibition against LNCaP and showed excellent AR degradation
efficacy.

The design, synthesis and anti-tumor mechanism study of new
androgen receptor degrader
Hang Xie a,b,c, 1, Jian-Jia Liang d, 1, Ya-Lei Wang a,b,c, Tian-Xing Hu a,b,c, Jin-Yi Wang a,b,c, Rui-Hua
Yang a,b,c, Jun-Ke Yan a,b,c, Qiu-Rong Zhang a,b,c, Xia Xu a,b *, Hong-Min Liu a,b,c *, Yu Ke a,b,c
*
a School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, Henan, 450001, P. R.
China
b Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan Province;
Zhengzhou 450001, P. R. China
c Key Laboratory of Advanced Drug Preparation Technologies, Ministry of Education of China,
d School of pharmacy, Wuhan University, Wuhan, Hubei, 430072, P. R. China.
1 These authors contributed equally to this work
Corresponding Authors:
Prof. Yu Ke
Zhengzhou University, Zhengzhou, 450001, China
E-mail: ky@zzu.edu.cn (Yu Ke)
Prof. Hong-Min Liu
Zhengzhou University, Zhengzhou, 450001, China
E-mail: liuhm@zzu.edu.cn (Hong-Min Liu)
Prof. Xia Xu
Zhengzhou University, Zhengzhou, 450001, China
E-mail: xuxia@zzu.edu.cn (Xia Xu)

Abstract
Targeted protein degradation using small molecules is a novel strategy for drug
development. In order to solve the problem of drug resistance in the treatment of
prostate cancer, proteolysis-targeting chimeras (PROTAC) was introduced into the
design of anti-prostate cancer derivatives. In this work, we synthesized two series of
selective androgen receptor degraders (SARDs) containing the hydrophobic degrons
with different linker, and then investigated the structure-activity relationships of these
hybrid compounds. Most of the synthesized compounds exhibited moderate to good
activity against all the cancer cell lines selected. Among them, compound A9
displayed potent inhibitory activity against LNCaP prostate cancer cell line with IC50
values of 1.75 μM, as well as excellent AR degradation activity. Primary mechanism
studies elucidated compound A9 arrested cell cycle at G0/G1 phase and induced a
mild apoptotic response in LNCaP cells. Further study indicated that the degradation
of AR was mediated through proteasome-mediated process. For all these reasons,
compound A9 held promising potential as anti-proliferative agent for the development
of highly efficient SARDs for drug-resistance prostate cancer therapies.
Key words: Androgen receptor; PROTACs; 1,2,3-triazole; Protein degradation

1. Introduction
Prostate cancer (PCa) is the most common malignancy and the second leading
cause of cancer death, after lung cancer, in men in developed countries[1, 2].
Epidemiologic studies suggest that PCa risk factors include race, age, family history,
lifestyle, and diet [3, 4]. Currently, the main therapeutic option of advanced PCa is
androgen deprivation therapy (ADT) with limited clinical outcomes[5, 6]. While
common anticancer therapies can treat androgen-dependent PCa, efficacy is limited
for androgen-independent and castration-resistant prostate cancer (CRPC). CRPC
shows resistance to hormonal therapy and causes severe problems, such as metastasis
and recurrence[7]. In the past few years, several drugs for the treatment of CRPC have
been approved, including taxanes such as paclitaxel, docetaxel, cabazitaxel, and new
androgen receptor targeting agents such as abiraterone acetate and enzalutamide.
However, the rapid emergence of drug resistance always eliminates the treatment’s
success[8]. Thus, there is an urgent need for the identification of new therapeutic
targets and molecular regulatory agents for the treatment of CRPC.
Androgen receptor (AR) expression has been shown by numerous studies to be
vital in PCa progression, which controls a variety of physiological and developmental
processes. AR is often overexpressed in CRPC cells and promotes their
androgen-dependent proliferation, which makes it an attractive therapeutic target for
CRPC treatment[9]. The traditional approach focus on regulating AR activity. AR
antagonists such as Nilutamide, Bicalutamide and Enzalutamide are widely used in
clinic (Fig. 1). One of the main drawbacks of this classic strategy is drug
resistance[10-12]. Therefore, downregulation of the AR level is regarded as a
promising strategy to treat CRPC.
Fig. 1. Structures of typical nonsteroidal AR antagonists
Recently, inducing protein degradation by small molecules has become a hot spot

for drug discovery[13-15]. Although degraders to other nuclear receptors are available,
AR degraders have been obscure and been difficult to discover and develop[16]. To
date, there have been two approaches targeting AR protein degradation: proteolysis
targeting chimeras (PROTACs) and selective androgen receptor degraders (SARDs).
The PROTAC technology is a hybrid molecule possesses a known ligand to a binding
pocket and a recognition motif for E3 ubiquitin ligase recruitment that ultimately
promotes ubiquitination-dependent proteasomal degradation of the target
protein[17-20], and several PROTAC molecules targeting AR have been reported (Fig.
2). In 2017, Naito et al developed a complex comprised of AR antagonist and bestatin
(an inhibitor of the cIAP1) via a PEG linker[21]. This complex induced
cIAP1-mediated ubiquitination of AR with proteasomal degradation at 3 μM. More
recently, Wang et al discovered series of AR targeting-PROTAC degraders, which
were designed using several different classes of AR antagonists and a von
Hippel−Lindau (VHL) ligand[9]. The most potent one exhibited excellent degradation
potency with the effective dose for degradation is 0.1 μM in LNCaP cells.
Nonetheless, an apparent disadvantage of these molecules is that their molecular
weight is greater than 1000 Da, which according to Lipinski’s rule of five is an
undesirable drug-like property for small molecules[22].
Fig. 2. Structures of representative AR downregulators
Another method for molecules capable of inducing protein degradation is

SARDs. SARDs are recognized as pure AR antagonists that can mimic a partially
denatured protein folding state, leading to degradation by the ubiquitin–proteasome
system (UPS)[12, 23, 24]. For instance, Miller’s group reported that derivatives of
bicalutamide that contained a basic indolyl were highly potent and efficacious
SARDs[25, 26].The best compound (Fig. 2, UT-155) of this series showed robust
activity and revealed that the AR degradation efficacy of this compound is 10 μM.
However, because of low bioactivity and bioavailabilities, most of the reported AR
degraders are limited for further studies. It is therefore crucial to identify new and
more effective agents to treat PCa.
Despite the different mechanisms for degrading AR by PROTACs and SARDs,
these compounds have a molecular skeleton that induces AR degradation and could be
called a degron[27-29]. Very recently, based on the success of specific degrons, our
group inspired by Crews’s major breakthrough in hydrophobic tagging (HyT), and
this strategy is believed to mimic a partially denatured protein folding state, leading to
chaperone-mediated degradation by the proteasome[30, 31]. In the present report, to
facilitate the discovery process for new SARDs, two series of bifunctional compounds
based on the high-affinity AR agonist RU59063 derivatives connected through a
1,2,3-triazole linker to an adamantyl group were designed, synthesized. Furthermore,
we investigated the effects of length and diversity of linker moiety with the insertion
of PEG linker and other functional group for optimization of these compounds. To our
delight, some compounds of this series exhibited potent AR degradation efficacy and
anti-proliferation activity against LNCaP, C4-2B cell lines. Their anti-proliferative
mechanisms were also investigated. These results provide new insights to the
potential use of HyT as novel and efficient SARDs. The detailed illustration of target
derivatives was shown in Fig 3.

Fig.3. Illustration of the design strategy for target compounds
2. Results and discussion
2.1 Chemistry
The synthesis of the designed SARD derivatives was shown in Schemes 1, 2 and
3. Firstly, we synthesized the key AR ligand intermediates 2-4, 7a-c, 9, 11, 14, and the
synthetic route was shown in Scheme 1. Cyanonilutamide (1) was prepared according
to the former literature report[7]. Compound 2-4 was synthesized via an alkylation of
1 with 1,3-Dibromopropane, 1-azido-4-(bromomethyl) benzene and 3-Bromopropyne
in the presence of sodium hydride (Scheme 1A). In a previous study, it was indicated
that the hydantoin group played an important role for the binding of the SARD
compounds[16, 32]. And as such, commercially available methyl carbamate was
reacted with e-j by using potassium carbonate as base in acetonitrile to provide 5a-c, 8,
10 and 12, which were further reacted with 3-Fluoro-4-methylphenylisothiocyanate to
afford 6a-c, 9, 11 and 13 with good yield. Moreover, the compound 6a-c were treated
with p-TsCl and NaN3 to yield 7a-c. Finally, the key intermediate 14 was gained
through the reaction of 13 with BBr3 in dichloromethane (Scheme 1B).

Scheme 1. Synthesis of AR ligands intermediates a.
a Reagents and conditions: (a) K2CO3, DMF, 45 , 5h, 74%; (b) 1. 1,3-Dibromopropane, NaH, dry
DMF, 0 , 2h; 2. NaN3, acetone, 80 , 6h, 81%; (c, d) 1-azido-4-(bromomethyl)benzene,
3-Bromopropyne, NaH, dry DMF, 0 , 1h, 64%, 72%; (e, h, i, j) Bromo-1-butanol,
1-azido-4-(bromomethyl)benzene, 3-Bromopropyne, 4-Methoxybenzyl bromide, K2CO3, CH3CN,
40 , 2-6h; (f) 3-Fluoro-4-methylphenylisothiocyanate, iPOH, triethylamine, rt, 3h, 53%-82%; (g)
1. TsCl, DABCO, CH2Cl2, 0 , 1h; 2. NaN3, acetone, 80 , 5h, 86%. (k) BBr3, CH2Cl2, -20 , 1.5h,
60%.
Hydrophobic tagging intermediates 16, 18a-d, 21, 24a-d were synthesized as
shown in Scheme 2. The commercially available PEG chain mixed with
3-bromopropyne catalyzed by t-BuOK in THF at room temperature to provide 15 and
17a-d, and then reacted with 1-adamantaneacetic acid in the system of DCC/DMAP
to afford 16 and 18a-d. The same procedure can be used to synthesize the
intermediate 19 and 22a-d, which were further reacted with p-TsCl/DMAP to afford
compounds 20, 23a-d with good yields. Then, following the reaction with NaN3,
azide intermediates 21 and 24a-d were obtained.

Scheme 2. Synthesis of HyT ligands intermediates a.
a
Reagents and conditions: (a) 3-Bromopropyne, t-BuOK, THF, 4-7h, 15%; (b)
1-Adamantaneacetic acid, DCC, DMAP, CH2Cl2, 0 , 2-5h, 88%; (c) TsCl, DMAP, CH2Cl2, 0 ,
1-2h, 75%; (d) NaN3, acetone, 80 , 7h, 82%.
The target product SARDs (A1-29, B1-5), which contained three series types,
were synthesized as shown in Scheme 3. In series I and II, the click reaction of
different azides with corresponding alkynes provided the final products A1-29. All of
the compounds gave moderate to good yields. Based on the preliminary biological
activity of the synthesized compounds, it was supposed that 1,2,3-trizole group may
improve the anticancer activities of compounds. Subsequently, in series III, we
deliberately synthesized the compounds B1-5 with no 1,2,3-trizole group for further
structure activity relationship study.
R2
R2
Yields
(%)
Yield
(%)
Cmpd.
R1
W
Cmpd.
R1
W
A1
A2
A3
A4
A5
A6
A7
A8
A9
A10
A11
(CH2)2
(CH2)2
(CH2)2
O
O
S
78%
85%
73%
84%
67%
71%
72%
59%
65%
46%
82%
A12
A13
A14
A15
A16
A17
A18
A19
(CH2)2
(CH2)2
(CH2)2
(CH2)2
O
O
S
47%
64%
52%
70%
51%
56%
53%
59%
n=3
S
S
O
O
S
O
O
S
n=4
n=5
(CH2)2
(CH2)3
(CH2)4
(CH2)5
n=2
S
S
S
A20
A21
(CH2)2
S
S
67%
73%
S
S

R2
Yield
(%)
R2
Yield
(%)
Cmpd.
W
Cmpd.
W
A22
A23
A24
A25
O
S
83%
78%
75%
86%
A26
A27
A28
A29
O
S
62%
71%
57%
52%
n=3
n=4
O
S
O
S
n=2
Yield
(%)
Cmpd.
W
R2
B1
B2
B3
B4
B5
S
O
S
S
S
69%
54%
62%
47%
49%
n=2
n=3
n=4
Scheme 3. Synthesis of SARD derivatives A1-29, B1-5 a.
a Reagents and conditions: (a) CuSO4, Na-ascorbate, THF/H2O, rt, 2-10h; (b) Cs2CO3, DMF, 80 .
2.2. Biological evaluation
2.2.1. AR Binding Affinity
In order to explore the AR binding affinity of the new SARD derivatives, the
fluorescence polarization (FP) based binding assay by competition with the
fluorescent tracer was carried out, and the results were shown in Table 1. In general,
it was worth noting that these compounds with hydrophobic tagging indeed have
binding affinity to AR. More interestingly, the binding affinity values of the
compounds for AR were closely related to the length of the R2 group, too long or too
short chains reduced the binding affinity of the compounds (A1-21). Comparing the
type of the W substituents, we also found that compounds with thiourea substituents,
numbered as A7-9, A14, A18 and A29 had a strongly increasing affinity. However,
changing the R1 aliphatic substituent to aromatic unit (compound A1-21) led to a
decrease of the affinity, which indicated that substituents on the AR ligand moiety
might play an important role in determining its binding affinity.

Table 1. Binding affinity of compound A1-29 and B1-5 to AR a.
AR Affinity (%)
at 10 μM
88.60
AR Affinity (%)
at 10 μM
33.89
AR Affinity (%)
at 10 μM
52.79
Cmpd.
Cmpd.
Cmpd.
Enz.
A1
A12
A13
A14
A15
A16
A17
A18
A19
A20
A21
A22
A23
A24
A25
A26
A27
A28
A29
B1
24.30
20.13
22.22
A2
19.09
79.51
46.67
A3
31.59
25.34
35.09
A4
25.97
47.04
29.78
A5
41.93
51.75
69.09
A6
36.67
69.30
37.85
A7
74.56
39.93
B2
26.32
A8
87.85
33.72
B3
34.21
A9
85.29
39.14
B4
32.02
A10
A11
32.30
43.39
B5
35.76
35.76
52.79
a AR binding affinity was shown as a ratio to the R1881 control and the data represent the mean of
at least three independent experiments.
2.2.2. Cell Viability Assay
To evaluate their inhibition of prostate cancer cell proliferation, we estimated the
anti-proliferative activity of these compounds in LNCaP (AR-positive, dependent),
C4-2B (AR-positive, independent), PC-3 (AR-negative) and WPMY-1 (human normal
prostate stromal cell) cell lines, and the cytotoxic activity of target compounds were
detected by SRB (Sulforhodanime B) assay. The IC50 values (concentration required
to inhibit tumor cell proliferation by 50%) were listed in Table 2, with Enzalutamide
used as positive control.
Initially, we evaluated the effect of series I and II on the cell viability of LNCaP
cells. It was found that the linker type of the compounds had significant effect on the
cell viability. And the preliminary structure and activity relationships (SARs) could be
concluded as follows: (1) Various R1 groups had certain influence on potency. Hybrids
with alkyl chains possessed higher anti-proliferative activity than those with phenyl
group (A1 vs A2, A5 vs A6, A12 vs A13). Compounds with 5 carbons exhibited
stronger anti-cancer capability than those with 3, 4 or 6 carbons, and once the length

of alkyl chain reached 6 carbons, the inhibitory effects were reduced sharply (A9 >
A8 > A7 > A10). (2) Under the condition that the compounds had the same R1 group,
we found that the R2 group between 1,2,3-triazole and adamantyl also slightly
regulated the activity. For example, compound A7 with a 2-PEG linker (IC50 = 4.06
μM) and A14 with a 3-PEG linker (IC50 = 3.97 μM) were higher than that of A20 with
a 5-PEG linker (IC50 = 6.92 μM). (3) Comparing the atom of the W, it was interesting
to find out compounds with S atoms were more favorable for activity than those
containing O atoms (A1 vs A3, A5 vs A7, A12 vs A14, A16 vs A18). This
phenomenon occurred with most of target compounds in this study. (4) The presence
of 1,2,3-triazole group had remarkable effect on the activity of the compounds. For
example, compound A22-29 with a 1,2,3-triazole group, most of these compounds
displayed moderate to good anti-proliferative activity against the selected AR-positive
cell lines (IC50 = 6.31-17.40 μM). While, for the compounds B1-5 with a phenyl
group exhibited no inhibitory activity on LNCaP cells at low concentrations. The
phenyl group at R1 position was designed to investigate whether it had the function to
increase the affinity of the pocket like Enzalutamide. However, we only treated it as a
common substituent group due to the experimental results.
Encouraged by the above interesting results, we also investigated the possible
toxicity of these SARDs toward PC-3 (AR negative) and WPMY-1(AR negative).
One can see that all target compounds had no cellular toxicity (IC50 > 80 μM). These
results indicated that the compounds had good selectivity between AR positive and
negative cells.
Table 2. Inhibitory activities of compounds against the tested cancer cell lines.a
IC50(μmol/L)
Compd.
LNCaP (AR+)
36.68±0.02
C4-2B(AR+)
52.67±0.06
PC-3(AR-)
WPMY-1(AR-)
Enzalutamide
＞80
＞80
A1
A2
＞80
＞80
＞80
＞80
4.62±0.30
11.20±0.94
＞80
＞80

A3
A4
A5
A6
A7
A8
A9
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
6.64±1.70
10.62±2.47
＞80
＞80
7.28±2.73
12.35±2.71
＞80
＞80
4.06±1.47
1.86±0.31
1.75±0.42
6.03±2.02
12.34±1.25
16.57±1.8
＞80
A10
A11
A12
A13
A14
A15
A16
A17
A18
A19
A20
A21
A22
A23
A24
33.89±3.4
＞80
＞80
6.66±2.33
9.95±3.21
＞80
＞80
3.97±1.35
5.80±1.14
＞80
＞80
9.96±2.98
11.67±2.64
＞80
＞80
4.25±1.19
7.64±1.43
＞80
＞80
6.92±3.01
6.61±0.99
＞80
12.20±2.84
＞80
＞80
＞80
＞80
＞80
11.92±3.37

＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
A25
A26
A27
17.40±4.48
8.34±1.122
7.70±3.99
＞80
9.13±0.02
6.87±0.96
A28
A29
7.76±2.24
6.31±0.58
11.22±0.81
B1
B2
B3
B4
B5
＞80
＞80
＞80
＞80
＞80
＞80
＞80
＞80
14.60±1.71
＞80
a MTT methods, cell lines were treated with target compounds for 72 h. The average value of the
three independent experiments was expressed by mean ± SDs.
As can be seen in Table 3, some compounds with better growth inhibitory effects
were chosen to evaluate for their cytotoxic activity against another three cancer cell
lines in SGC-7901 (human stomach cancer line, AR positive), MCF-7(human breast
cancer cell line, AR positive) and GES-1 (human normal gastric cells, AR negative).
To our delight, four selected products exerted good inhibitory activity against
SGC-7901 cells with the IC50 values of 4.11, 7.79, 6.93 and 4.98 μM, respectively. In
contrast, compounds A7, A8, A9 and A14 were less potent against MCF-7 cells with
the IC50 values of 9.12, 13.33, 50.50 and 7.23 μM, respectively. We also found that
these four compounds had no cytotoxicity against normal gastric GES-1 cells.
Predictably, target cytotoxcity by the SARDs is selective for the AR. Furthermore, A9
showed equal or better anti-proliferative activity compared to other compounds. Thus,
A9 was selected for further investigation. Moreover, LNCaP cell lines were chosen
for subsequent experiments as they were the most sensitive to A9.
Table 3. Inhibitory activities of compounds against the tested cancer cell lines.a

IC50(μmol/L)
Compd.
SGC-7901(AR+)
MCF-7(AR+)
GES-1(AR-)
＞80
A7
A8
4.112±0.312
7.793±1.789
6.932±0.984
4.982±0.774
9.115±2.174
13.326±1.32
50.5±0.375
7.729±1.774
＞80
＞80
＞80
A9
A14
a MTT methods, cell lines were treated with target compounds for 72 h. The average value of the
three independent experiments was expressed by mean ± SDs.
2.2.2. Cell cycle arrest effects of A9 on LNCaP cells
To determine whether this anticancer effect is caused by a cell cycle that
accumulates at a certain phase, the effects of different concentrations of compound A9
on cell cycle progression were examined with LNCaP cell line. LNCaP cells slightly
interfered with cell cycle distribution after treatment with compound A9 for 24 h. As
shown in Fig. 4A and B, the percentage of cells in the G0/G1 phase after the
treatment with compound A9 at various concentrations (1, 2 and 10 µmol/L) were
59.30%, 67.54% and 74.56% when compared with the control (47.99%). The results
suggested that A9 caused the G0/G1 arrest pattern in a concentration dependent
manner, with a concomitant decrease of cells in other phases of the cell cycle, but this
result is not significant.
Fig. 4. Effect of compound A9 on the cell cycle distribution of LNCaP cells. Cells were treated

with different concentrations (0, 1, 2, 10 µmol/L) for 24 h. Then the cells were fixed and stained
with PI to analyze DNA content by flow cytometry. (A) Diagram of the cell cycle for each phase
based on flow cytometry. (B) The proportions of cells in G1, S, and G2/M phases were calculated.
The data were analyzed by Student's t-test.
2.2.4. Apoptosis effects of A9 on LNCaP cells
To further investigate the mechanism of the inhibitory effect caused by A9 on
prostate cancer. Annexin V/PI staining was used to quantitatively measure the effect
of A9 on LNCaP cells apoptosis. After treatment with compound A9 for 24h at
indicated concentrations (0, 1, 2, 10 µmol/L), LNCaP cells were labeled with the two
dyes, and the resulting red (PI) and green (FITC) fluorescence was monitored by flow
cytometry. As shown in cytometry apoptosis diagram (Fig. 5A and B), the percentage
of Total rate of apoptosis were increased from 2.76% (DMSO control) to 19.22% after
treatment with 10μM of A9.
Fig. 5. Compound A9 induced apoptosis of LNCaP cells. (A) LNCaP cells apoptosis was detected
by Annexin V/PI on flow cytometry. Cells treated with 0, 1, 2 and 10 µM A9 for 24 h were
collected and processed for analysis. (B) The proportion of apoptotic cells increased dependently
with A9 treatment concentration.
2.2.5. AR degradation assay.
AR-dependent transcription is a major driver of prostate tumor cell proliferation.
It has become clear that an increased level of AR protein is present in the majority of

PCa, and many therapeutic strategies focus on regulating AR activity. Meanwhile,
PSA is a key protein of PCa drug resistance. To determine whether SARDs can
circumvent resistance associated with these two mechanisms, LNCaP cells were
treated with compound A9 at three different concentrations (1, 2, and 10 µM) for 24 h.
Untreated cells were applied as the control. Fig 6A showed that compound A9 could
dose-dependently down-regulate the AR, PSA and TMPRSS2 (Two proteins
associated with AR)[33, 34]. Then we evaluated the kinetics of A9 in induction of AR
degradation at 10 µM in the LNCaP cell lines, our data showed that A9 effectively
reduced the AR protein level and achieved near-complete AR depletion with a 72 h
treatment (Fig 6B). Gratifyingly, the progesterone receptor (PR), another steroid
receptor not recognized by the parent ligand RU59063[35], was not degraded in T47D
cells under conditions that result in near-complete degradation of the androgen
receptor. These results indicated that target degradation by the A9 was selective for
the AR, and we no longer see any effects on PR-A and PR-B levels (Fig.6C).
Moreover, the prostate cancer cells used in this study do not express detectable levels
of the PR and as such, any A9 induced signaling or proliferation effects can be solely
attributed to AR degradation and not PR-A or PR-B suppression.
Furthermore, to investigated the specific mechanism of action of AR degradation
mediated by compound A9, we pre-treated LNCaP cells with the proteasome inhibitor
MG132, the lysosomal inhibitor E-64 (Fig. 6D). Our data show that AR degradation
was blocked by MG132, but was unaffected by E-64, it suggested that compound A9
induces the degradation of AR in cells via the proteasome.

Fig. 6. Compound A9 induced AR protein degradation. (A) Compound A9 down-regulated AR,
PSA, TMPRSS2 protein in a dose-dependent. (B) Compound A9 down-regulated AR protein in a
time-dependent. (C) Treatment of T47D breast cancer cells for 24 h with 10
near-complete degradation of the AR and no loss of progesterone receptors. (D) A9-mediated AR
degradation (10 M) at 24 h was blocked by 3h pretreatment with proteasome inhibitor MG132
(10 M) and was unaffected by 3h pretreatment with lysosomal inhibitor E-64 (10 M).
µM A9 showed
µ
µ
µ
2.2.7. Molecule docking.
To better elucidate the mechanism of compound A9 degradation at the molecular
level, we performed molecular docking using sybyl-x2.1.1 software. In the case of the
compound A9, similar to other AR ligands[36], the cyano group on the A ring forms
two hydrogen bonds with the side chain of Arg752 and Gln711. At the same time, the
hydrophobic tagging side chain, as a protein degrading degron, appeared to display
important interactions with AR. It formed a hydrogen bond between the ester group of

the side chain with Tyr-739 on helix 4 (Fig 7A and B). These noncovalent interactions
may distort the position of helix 12 (red) so that it could not fold properly, and the
ubiquitination of AR would proceed. Moreover, docking studies indicated that the
aliphatic chain group on the hydantoin moiety had obvious effect on the binding
affinity. Importantly, hydrophobic tagging was exposed resulting in destabilization of
the AR, subsequently caused the receptor ubiquitination and degradation.
Fig. 7. Docking analysis (A) HyT compound with long side chain A9 (yellow) bound to AR (PDB:
2AXA). (B) The hydrophobic interactions and hydrogen bond interaction between A9 and the
backbone residues of AR.
3. conclusion
In this study, based on the hydrophobic tagging strategy of developing novel
anti-androgen drugs, two series of SARDs for selective degradation of AR were
designed and synthesized. Optimization of the Linkers of these SARDs resulted in
compounds with promising inhibitory activities of cell viability and AR degradation
activity. The SAR research demonstrated that Hybrids with alkyl chains possessed
higher anti-proliferative activity than those with phenyl group, and the presence of
1,2,3-triazole group had remarkable effect on the activity of the compounds. Most of
these derivatives showed AR binding affinity and could inhibit the growth of the
prostate cancer cell with AR expression. Among them, compound A9 displayed
similar AR binding affinity with Enzalutamide and excellent inhibitory activity
against LNCaP cells. A9 also could inhibit the growth of other tumor cells with AR
expression (SGC-7901 and MCF-7), but had no effect on the cells without AR
expression, showing good AR selectivity. Compound A9 could dose-dependently
down-regulate the AR, PSA and TMPRSS2, but it had no effect on the PR protein,

meanwhile the degradation of AR is mediated through proteasome-mediated process.
Molecular docking analysis illustrated the interaction of long hydrophobic tagging
chain of these compounds with AR may cause effective impact on the degradation of
these compounds. These findings provided new possibilities for discovering novel
scaffolds in the development of novel SARDs for drug resistance prostate cancer
therapies.
4. Experimental section
4.1. General
If not stated otherwise, the commercially available starting materials were used
directly. All solvents were dried according to appropriate protocols. Thin-layer
chromatography (TLC) was carried out on glass plates coated with silica gel (Qingdao
Haiyang Chemical Co., G60F-254) and visualized by UV light (254 nm). The
products were purified by column chromatography over silica gel (200-300 mesh).
Melting point were determined on a WRS-1A melting-point apparatus and are
uncorrected. 1H NMR and 13C NMR spectra were recorded on a Bruker 400 MHz and
101 MHz spectrometer, respectively. High resolution mass spectra (HRMS) of all
derivatives were recorded on a Waters Micromass Q-T of Micromass spectrometer by
electrospray ionization (ESI).
4.1.1. The general method for the synthesis of compound 1.
Compound 1 was synthesized by following the previously reported method[7].
4-Fluoro-2-(trifluoromethyl)benzonitrile (2.01 g, 10.3 mmol) was added to a mixture
of hydantoin (6.8 g, 53.1 mmol) and potassium carbonate (2.20 g, 10.3 mmol) in
DMF (30 mL) and stirred at 45 °C for 24 h. Reaction mixture was diluted in ethyl
acetate and washed three times with water. The organic layer was dried over sodium
sulfate, filtered and concentrated in vacuo. Column chromatography (eluent 30:1
1
DCM/Methanol) gave 1 as a white solid (2.30 g, 74%). H NMR (400 MHz,
DMSO-d6) δ 8.83 (s, 1H), 8.30 (d, J = 8.4 Hz, 1H), 8.19 (d, J = 1.6 Hz, 1H), 8.04 (dd,
J = 8.4, 1.8 Hz, 1H), 1.43 (s, 6H). 13C NMR (100 MHz, DMSO-d6) δ 175.7, 152.9,
136.9, 135.9, 131.1, 130.8, 129.9, 124.1, 123.9, 123.6, 120.8, 115.2, 106.5, 57.9, 24.5.
4.1.2. The general method for the synthesis of 2, 3 and 4.

Compounds 2-4 were prepared following the method as described for 2.
Compound 1 (1.00 g, 3.3 mmol) was dissolved in DMF (40 mL) under N2, followed
by addition of NaH (60% in mineral oil, 0.20 g, 5.1 mmol) and stirring for 1 h at room
temperature. Then dibromo alkane (0.85 g, 5.1 mmol) was added and the reaction was
stirred for 2 h at 55 °C. To the reaction was added EtOAc (200 mL) and the mixture
was successively washed with brine (5 ×50 mL) and H2O (3 × 25 mL). The organic
layer was dried over sodium sulfate, filtered and concentrated in vacuo. Column
chromatography (eluent 5:1 PE/EtOAc) gave intermediates as a clear oil (5.21 g,
81%). After that, intermediate was put in the system of acetone with NaN3 reflux 6h at
1
80 to obtain compound 2. Compound 2, yellow oil, H NMR (400 MHz, CDCl3) δ
8.15 (s, 1H), 8.00 (dd, J = 8.4, 1.8 Hz, 1H), 7.92 (d, J = 8.4 Hz, 1H), 3.65 (t, J = 6.0
Hz, 2H), 3.57 – 3.45 (m, 2H), 2.36 – 2.16 (m, 2H), 1.56 (d, J = 2.5 Hz, 6H). 13C NMR
(100 MHz, DMSO-d6) δ 172.6, 155.8, 139.0, 138.7, 132.1, 130.8, 129.9, 125.1, 123.5,
123.6, 121.8, 114.3, 106.5, 57.6, 44.1, 30.0, 30.1, 23.5.
Compound 3, yellow solid, 1H NMR (400 MHz, DMSO-d6) δ 8.33 (d, J = 8.4 Hz, 1H),
8.26 (s, 1H), 8.10 (d, J = 8.3 Hz, 1H), 7.51 (d, J = 8.3 Hz, 2H), 7.10 (d, J = 8.3 Hz,
2H), 4.62 (s, 2H), 1.41 (s, 6H). 13C NMR (100 MHz, DMSO-d6) δ 174.5, 153.1, 138.3,
136.8, 135.9, 134.8, 131.5, 131.1, 130.8, 130.5, 129.9, 129.3, 126.3, 124.1, 124.0,
123.5, 120.8, 118.9, 115.1, 106.7, 106.7, 61.9, 41.9, 22.6.
4.1.3. The general method for the synthesis of 5a-c, 8, 10 and 12.
Compounds 5a-c, 8, 10 and 12 were prepared following the method as described
for 5a. To the solution of 2-Aminoisobutyric acid methyl ester (2.50 g, 21.34mmol)
and K2CO3 (5.90 g, 42.68 mmol) in CH3CN, 3-Bromo-1-propanol was added slowly
under ice bath conditions. the mixture was heated to 60
and kept for 4 h. The
filtrate was concentrated under vacuum and then dissolved in EtOAc. The resulting
aqueous mixture was extracted with EtOAc (4 × 30 mL) and the organic layers were
washed by saturated aqueous NH4Cl (3×10 mL). The organic layers were dried over
MgSO4 and concentrated under reduced pressure to give the white solid 5a, which
was used directly without further purification.
4.1.4. The general method for the synthesis of 6a-c, 9, 11 and 13.

Compounds 6a-c, 9, 11 and 13 were prepared following the method as described
for 6a. 5a (1 g, 5.26 mmol) reacted with 3-Fluoro-4-methylphenylisothiocyanate (1.23
g, 4.38 mmol) and TEA (0.65 g, 6.57 mmol) in iPrOH (10 mL) under reflux for 3-6 h.
Upon completion, the reaction mixture was cooled to room temperature and the
colorful precipitate formed was filtered and washed with water to afford the desired
compound 6a. 6a then reacted with TsCl at 0 , the mixture was kept overnight. Upon
completion of the reaction, intermediate was put in the system of acetone with NaN3
reflux 6h at 80 to obtain 7a-c.
Compound 7a, yellow solid, 1H NMR (400 MHz, CDCl3) δ 8.00 – 7.93 (m, 1H), 7.92
(s, 1H), 7.79 (d, J = 8.2 Hz, 1H), 3.78 (dd, J = 17.0, 8.7 Hz, 2H), 3.48 (t, J = 6.2 Hz,
2H), 2.14 (d, J = 5.8 Hz, 2H), 1.68 (s, J = 64.4 Hz, 6H). 13C NMR (100 MHz, CDCl3)
δ 178.6, 175.1, 137.1, 135.1, 133.8, 133.4, 133.1, 132.8, 132.1, 130.1, 127.1, 127.0,
126.9, 126.9, 126.0, 123.2, 120.5, 117.8, 114.8, 109.8, 65.2, 49.1, 41.7, 27.4, 23.1.
Compound 9, yellow solid, 1H NMR (400 MHz, DMSO-d6) δ 8.35 (d, J = 8.4 Hz, 1H),
8.23 (s, 1H), 8.14 (d, J = 8.3 Hz, 1H), 7.41 (d, J = 8.3 Hz, 2H), 7.08 (d, J = 8.3 Hz,
2H), 4.61 (s, 2H), 1.41 (s, 6H). 13C NMR (100 MHz, DMSO-d6) δ 172.5, 151.0, 137.3,
136.8, 135.9, 133.8, 131.5, 131.1, 130.8, 130.6, 119.9, 129.3, 126.3, 124.1, 124.0,
123.5, 120.8, 116.9, 115.1, 106.7, 106.7, 61.9, 41.9, 22.6.
4.1.5. The general method for the synthesis of 14.
The compound 13 (1g, 0.8 equiv.) was added in dichloromethane under protection
of N2. Slowly add BBr3 (0.7g, 3.0 equiv.) at -20 °C and then stirred for 12 h. The
reaction was quenched with 10 mL of water, and extracted with ethyl acetate (3 × 25
mL). The resulting solution was concentrated under reduced pressure, and the residue
was purified by silica gel column chromatography (Petroleum ether: EtOAc, 3: 1) to
give 14. White solid, 60% yield. 1H NMR (400 MHz, DMSO-d6) δ 9.37 (s, 1H), 8.33
(d, J = 8.6 Hz, 1H), 8.24 (d, J = 1.3 Hz, 1H), 8.08 (d, J = 8.5 Hz, 1H), 7.25 (d, J = 8.5
Hz, 2H), 6.73 (dd, J = 8.5, 3.5 Hz, 2H), 4.50 (s, 2H), 1.36 (s, 6H).
4.1.6. The general method for the synthesis of 15 and 17a-d.
Compounds 15 and 17a-d were prepared following the method as described for
17a. To a suspension of KOt-Bu (116 g, 1 mol) in dry THF (1 L) was added

diethylene glycol (2.00 g, 1.9 mmol) at 0 °C under N2 atmosphere. The reaction
mixture was allowed to stir at 20 °C for 30 min then propargyl bromide (1.40 g, 0.94
mmol) was added dropwise. The resulting mixture was allowed to stir at 80 °C for 18
h. The mixture was filtered and concentrated under reduced pressure and the resulting
residue was purified by column chromatography (Petroleum ether: EtOAc = 50/1 to
0:1) furnish 17a (0.40 g, 0.27 mmol, 15% yield) as a yellow oil.
4.1.7. The general method for the synthesis of 16, 18a-d, 21 and 24a-d.
Compounds 16, 18a-d, 21 and 24a-d were prepared following the method as
described for 18a. To a solution of 1-adamantaneacetic acid (1.00 g, 5.15 mmol) and
17a (1.27 g, 6.18 mmol) in dichloromethane (25 mL) at room temperature was added
DMAP (0.1 g, 0.82 mmol). The reaction mixture was cooled to 0 °C and DCC (1.27 g,
6.18 mmol) was added to the mixture. The resulting mixture was stirred at 0 °C for 30
min and at room temperature overnight. The reaction mixture was quenched with H2O
and the aqueous layer was extracted twice with EtOAc. The combined extracts were
washed with brine, dried over Na2SO4, filtered, and concentrated. The crude product
was chromatographed on silica gel and was isolated as a colorless oil (1.90 g, 88%).
Compound 18a, colorless oil, 1H NMR (400 MHz, CDCl3) δ 4.26 – 4.22 (m, 2H), 4.22
– 4.20 (m, 2H), 3.78 – 3.61 (m, 6H), 2.44 (t, J = 2.3 Hz, 1H), 2.10 (s, 2H), 1.97 (s,
3H), 1.82 – 1.62 (m, 12H). 13C NMR (100 MHz, CDCl3) δ 171.7, 79.5, 74.5, 70.3,
69.3, 69.2, 62.9, 58.4, 48.8, 42.6, 36.7, 32.7, 28.7, 28.6, 28.6, 26.2, 25.4, 25.4, 24.7.
1
Compound 18b, colorless oil, H NMR (400 MHz, CDCl3) δ 4.24 – 4.21 (m, 2H),
4.21 (d, J = 2.3 Hz, 2H), 3.72 – 3.64 (m, 10H), 2.45 (t, J = 2.4 Hz, 1H), 2.10 (s, 2H),
1.97 (s, 3H), 1.74 – 1.58 (m, 12H). 13C NMR (100 MHz, CDCl3) δ 171.7, 79.6, 74.5,
70.6, 70.5, 70.4, 69.2, 69.0, 62.9, 58.3, 48.8, 42.3, 36.7, 32.7, 28.6.
Compound 18c, colorless oil, 1H NMR (400 MHz, CDCl3) δ 4.41 – 4.11 (m, 5H), 3.88
– 3.46 (m, 12H), 2.11 (d, J = 9.0 Hz, 2H), 2.08 (s, 1H), 1.97 (s, 3H), 1.75 – 1.58 (m,
12H). 13C NMR (100 MHz, CDCl3) δ 171.7, 79.7, 74.5, 70.5, 70.5, 70.3, 69.1, 69.0,
62.8, 58.3, 48.7, 42.2, 36.5, 32.6, 27.1.
Compound 18d,1H NMR (400 MHz, CDCl3) δ 4.26 – 4.10 (m, 2H), 3.76 (dd, J = 5.9,
3.2 Hz, 2H), 3.73 – 3.51 (m, 14H), 2.08 (s, 2H), 1.96 (s, 3H), 1.77 – 1.55 (m, 12H).

13C NMR (100 MHz, CDCl3) δ 171.6, 79.6, 74.5, 71.4, 70.5, 70.3, 69.2, 69.1, 62.8,
59.0, 58.3, 48.7, 46.6, 42.3, 36.3, 32.5, 27.2, 25.7.
4.1.8. The general method for the synthesis of A1-29.
Into a round bottom flask, alkyne derivatives (1.0 equiv) with different side chain
length (4, 11, 16, 18a-d) and appropriate azides (2, 3, 7a-c and 9) or adamantaneacetic
azides (21, 24a-c, 1.2equiv) were dissolved in THF (4mL), then put 1mL H2O in it,
finally, CuSO4 (1.0 equiv) and Na-ascorbate (3.0 equiv) as catalytic agent to react
8.5h. Then insoluble matter was filtered and condensed, subsequently, extracted with
ethyl acetate and washed with brine, dried over anhydrous Na2SO4, filtered,
evaporated and the crude product was purified by column chromatography to give
compounds.
3-((1-(3-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-2,4-dioxoimidazolidin-1
-yl)propyl)-1H-1,2,3-triazol-4-yl)methoxy)propyl2-((3r,5r,7r)-adamantan-1-yl)acetate
(A1).
Colorless oil,74% yield. 1H NMR (400 MHz, CDCl3) δ 8.14 (d, J = 1.5 Hz, 1H), 8.00
(dd, J = 8.5, 1.8 Hz, 1H), 7.93 (d, J = 8.4 Hz, 1H), 7.67 (s, 1H), 4.63 (s, 2H), 4.48 (t, J
= 6.7 Hz, 2H), 4.14 (t, J = 6.5 Hz, 2H), 3.67 – 3.58 (m, 2H), 3.51 – 3.43 (m, 2H), 2.44
– 2.33 (m, 2H), 2.05 (s, 2H), 2.00 – 1.88 (m, 5H), 1.76 – 1.59 (m, 12H), 1.52 (s, 6H).
13C NMR (100 MHz, CDCl3) δ 174.3, 171.1, 168.5, 167.0, 165.8, 156.5, 153.4, 136.6,
136.2, 135.4, 133.7, 133.4, 128.1, 123.1, 123.0, 119.1, 117.1, 115.9, 114.9, 108.4,
77.3, 66.5, 66.1, 64.4, 62.1, 49.1, 47.8, 37.6, 31.4, 29.9, 29.2, 23.4, 23.3, 22.6.
ESI-HRMS: m/z cacld. For C34H41F3N6O5 [M+H]+: 671.7342, found 671.7339.
3-((1-(4-((3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-2,4-dioxoimidazolidin-
1-yl)methyl)phenyl)-1H-1,2,3-triazol-4-yl)methoxy)propyl
2-((3r,5r,7r)-adamantan-1-yl)acetate (A2).
Colorless oil, 58% yield. 1H NMR (400 MHz, CDCl3) δ 8.00 (s, 1H), 7.97 (d, J = 10.5
Hz, 1H), 7.84 (d, J = 8.2 Hz, 1H), 7.77 (d, J = 8.4 Hz, 1H), 7.61 (d, J = 8.3 Hz, 1H),
5.18 (s, 1H), 4.71 (d, J = 9.2 Hz, 1H), 4.18 (t, J = 6.4 Hz, 1H), 3.67 (t, J = 6.2 Hz, 2H),
2.05 (s, 2H), 1.97 (dd, J = 12.6, 6.1 Hz, 5H), 1.69 (d, J = 11.8 Hz, 5H), 1.60 (d, J =
12.7 Hz, 7H), 1.51 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 174.3, 171.1, 168.5, 167.0,
165.8, 156.5, 153.4, 136.6, 136.2, 135.4, 133.7, 133.4, 128.1, 123.1, 123.0, 119.1,

117.1, 115.9, 115.0, 108.4, 77.3, 66.5, 66.1, 64.4, 62.1, 49.1, 47.8, 37.6, 31.4, 29.9,
29.2, 23.4, 23.3, 22.6. ESI-HRMS: m/z cacld. For C38H41F3N6O5 [M+H]+: 719.7782,
found 719.7762.
3-((1-(3-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-
thioxoimidazolidin-1-yl)propyl)-1H-1,2,3-triazol-4-yl)methoxy)propyl
2-((3r,5r,7r)-adamantan-1-yl)acetate (A3).
Colorless oil, 73% yield. 1H NMR (400 MHz, CDCl3) δ 7.96 (d, J = 8.2 Hz, 1H), 7.89
(s, 1H), 7.80 – 7.74 (m, 1H), 7.63 (s, 1H), 4.63 (d, J = 5.1 Hz, 2H), 4.50 (t, J = 6.5 Hz,
2H), 4.14 (t, J = 6.4 Hz, 2H), 3.86 – 3.75 (m, 2H), 3.63 (t, J = 6.2 Hz, 2H), 2.57 –
2.47 (m, 2H), 2.05 (d, J = 5.0 Hz, 2H), 2.00 – 1.89 (m, 5H), 1.85 – 1.60 (m, 12H),
1.55 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 174.3, 171.1, 168.5, 167.0, 165.8, 156.5,
153.4, 136.6, 136.2, 135.4, 133.7, 133.4, 128.1, 123.1, 123.0, 119.1, 117.1, 115.9,
115.0, 108.4, 77.25, 66.5, 66.1, 64.4, 62.1, 49.1, 47.8, 37.6, 31.4, 29.9, 29.2, 23.4,
23.3, 22.6. ESI-HRMS: m/z cacld. For C34H41F3N6O4S [M+H]+: 687.7952, found
687.7942.
3-((1-(4-((3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thioxoimidazol
idin-1-yl)methyl)phenyl)-1H-1,2,3-triazol-4-yl)methoxy)propyl
2-((3r,5r,7r)-adamantan-1-yl)acetate (A4).
Colorless oil, 79% yield. 1H NMR (400 MHz, CDCl3) δ 8.20 (s, 1H), 8.06 (dd, J = 8.4,
1.8 Hz, 1H), 8.00 (s, 1H), 7.94 (d, J = 8.4 Hz, 1H), 7.76 (d, J = 8.4 Hz, 2H), 7.56 (d, J
= 8.4 Hz, 2H), 4.71 (d, J = 7.5 Hz, 4H), 4.17 (t, J = 6.4 Hz, 2H), 3.67 (t, J = 6.2 Hz,
2H), 2.05 (s, 2H), 1.97 (dd, J = 12.6, 6.2 Hz, 5H), 1.73 – 1.55 (m, 12H), 1.47 (s, 6H).
13C NMR (100 MHz, CDCl3) δ 174.4, 171.9, 153.4, 146.3, 137.7, 136.8, 136.4, 135.3,
129.4, 127.0, 123.1, 123.0, 120.9, 120.5, 115.0, 108.4, 67.3, 64.5, 62.3, 60.9, 48.0,
43.1, 42.4, 36.7, 32.7, 29.7, 29.1, 28.6, 23.7. ESI-HRMS: m/z cacld. For
C38H41F3N6O4S [M+H]+: 735.8392, found 735.8389.
2-(2-((1-(3-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-2,4-dioxoimidazolidi
n-1-yl)propyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)ethyl
2-((3r,5r,7r)-adamantan-1-yl)acetate (A5).
Colorless oil, 56% yield. 1H NMR (400 MHz, CDCl3) δ 8.14 (d, J = 1.8 Hz, 1H), 8.00
(dd, J = 8.4, 1.9 Hz, 1H), 7.93 (d, J = 8.5 Hz, 1H), 7.69 (s, 1H), 4.70 (s, 2H), 4.48 (t, J
= 6.7 Hz, 2H), 4.25 – 4.20 (m, 2H), 3.69 (tt, J = 4.3, 2.4 Hz, 6H), 3.50 – 3.43 (m, 2H),
2.38 (dt, J = 13.8, 6.9 Hz, 2H), 2.09 (d, J = 3.1 Hz, 2H), 1.95 (s, 3H), 1.73 – 1.60 (m,

12H), 1.52 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 174.3, 171.1, 168.5, 167.0, 165.8,
156.5, 153.4, 136.6, 136.2, 135.4, 133.7, 133.4, 128.1, 123.1, 123.0, 119.1, 117.1,
115.9, 115.0, 108.4, 77.3, 66.5, 66.1, 64.4, 62.1, 49.1, 47.8, 37.6, 31.4, 29.9, 29.2,
23.4, 23.3, 22.6. ESI-HRMS: m/z cacld. For C35H43F3N6O6 [M+H]+: 701.7602, found
701.7612.
2-(2-((1-(4-((3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-2,4-dioxoimidazolidi
n-1-yl)methyl)phenyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)ethyl
2-((3r,5r,7r)-adamantan-1-yl)acetate (A6).
1
Colorless oil, 77% yield. H NMR (400 MHz, CDCl3) δ 8.05 (s, 1H), 7.99 (d, J = 8.3
Hz, 1H), 7.96 (d, J = 1.3 Hz, 1H), 7.85 (dd, J = 8.2, 1.8 Hz, 1H), 7.77 (d, J = 8.5 Hz,
2H), 7.64 – 7.57 (m, 2H), 5.19 (s, 2H), 4.78 (s, 2H), 4.27 – 4.18 (m, 2H), 3.75 (dd, J =
5.9, 3.1 Hz, 2H), 3.70 (dd, J = 9.6, 4.7 Hz, 4H), 2.08 (s, 2H), 1.94 (s, 3H), 1.71 – 1.59
(m, 12H), 1.51 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 174.3, 171.1, 168.5, 167.0,
165.8, 156.5, 153.4, 136.6, 136.2, 135.4, 133.7, 133.4, 128.0, 123.0, 123.0, 119.1,
117.1, 115.9, 115.0, 108.4, 77.3, 66.5, 66.1, 64.4, 62.1, 49.1, 47.8, 37.6, 31.4, 29.8,
29.2, 23.4, 23.3, 22.6. ESI-HRMS: m/z cacld. For C39H43F3N6O6 [M+H]+: 749.8042,
found 749.8046.
2-(2-((1-(3-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thioxoimidaz
olidin-1-yl)propyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)ethyl
2-((3r,5r,7r)-adamantan-1-yl)acetate (A7).
Brown oil, 77% yield. 1H NMR (400 MHz, CDCl3) δ 7.96 (d, J = 8.3 Hz, 1H), 7.89 (d,
J = 1.5 Hz, 1H), 7.77 (dd, J = 8.3, 1.9 Hz, 1H), 7.68 (s, 1H), 4.70 (d, J = 6.4 Hz, 2H),
4.50 (t, J = 6.5 Hz, 2H), 4.22 (dd, J = 10.1, 5.1 Hz, 2H), 3.80 (dd, J = 9.1, 6.6 Hz, 2H),
3.69 (tdd, J = 8.7, 5.9, 2.7 Hz, 7H), 2.59 – 2.48 (m, 2H), 2.09 (s, 2H), 1.95 (s, 3H),
1.73 – 1.60 (m, 12H), 1.55 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 174.3, 171.1, 168.5,
167.0, 165.8, 156.5, 153.4, 136.6, 136.2, 135.4, 133.7, 133.4, 128.1, 123.1, 123.0,
119.1, 117.1, 115.9, 115.0, 108.4, 77.3, 66.5, 66.1, 64.4, 62.1, 49.1, 47.8, 37.6, 31.4,
29.9, 29.2, 23.4, 23.3, 22.6. ESI-HRMS: m/z cacld. For C35H43F3N6O5S [M+H]+:
717.8212, found 717.8232.
2-(2-((1-(4-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thioxoimidaz
olidin-1-yl)butyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)ethyl
2-((3r,5r,7r)-adamantan-1-yl)acetate (A8).

Colorless oil, 47% yield. 1H NMR (400 MHz, CDCl3) δ 7.96 (d, J = 8.3 Hz, 1H), 7.89
(d, J = 1.6 Hz, 1H), 7.76 (dd, J = 8.3, 1.8 Hz, 1H), 7.62 (s, 1H), 4.69 (s, 2H), 4.45 (t, J
= 6.8 Hz, 2H), 4.51 – 4.39 (m, 2H), 4.27 – 4.18 (m, 2H), 4.25 – 4.16 (m, 2H), 3.79 –
3.61 (m, 8H), 2.09 (s, 2H), 2.04 (dd, J = 14.3, 7.1 Hz, 2H), 1.95 (s, 2H), 1.91 – 1.85
13
(m, 12H), 1.73 – 1.60 (m, 6H), 1.56 (s, 3H). C NMR (100 MHz, CDCl3) δ 178.6,
175.1, 171.7, 145.5, 137.0, 135.2, 133.7, 132.8, 127.0, 126.0, 122.6, 114.8, 110.1,
70.5, 69.9, 69.3, 65.2, 64.8, 62.9, 49.4, 48.9, 43.3, 42.4, 36.7, 32.8, 28.6, 27.6, 25.0,
23.1. ESI-HRMS: m/z cacld. For C36H45F3N6O5S [M+H]+: 731.8212, found 731.8232.
2-(2-((1-(5-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thioxoimidaz
olidin-1-yl)pentyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)ethyl
2-((3r,5r,7r)-adamantan-1-yl)acetate (A9).
Colorless oil, 52% yield. 1H NMR (400 MHz, CDCl3) δ 7.95 (d, J = 8.3 Hz, 1H), 7.89
(s, 1H), 7.81 – 7.74 (m, 1H), 7.58 (s, 1H), 4.69 (s, 2H), 4.40 (t, J = 6.9 Hz, 2H), 4.29 –
4.16 (m, 3H), 3.76 – 3.64 (m, 8H), 2.09 (s, 2H), 2.02 (dd, J = 14.9, 7.3 Hz, 2H), 1.96
(s, 3H), 1.92 – 1.86 (m, 2H), 1.66 (dd, J = 29.9, 10.3 Hz, 12H), 1.57 (s, 6H), 1.42 (dt,
J = 15.0, 7.6 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 178.4, 175.2, 171.7, 145.3,
137.1, 135.2, 133.4, 132.1, 127.0, 127.0, 122.5, 114.8, 70.5, 69.9, 69.3, 65.1, 64.8,
62.9, 49.9, 48.9, 43.9, 42.4, 36.7, 32.8, 29.6, 28.6, 27.3, 23.9, 23.2. ESI-HRMS: m/z
cacld. For C37H47F3N6O5S [M+H]+: 745.8122, found 745.8132.
2-(2-((1-(6-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thioxoimidaz
olidin-1-yl)hexyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)ethyl
2-((3r,5r,7r)-adamantan-1-yl)acetate (A10).
Brown oil, 43% yield. 1H NMR (400 MHz, CDCl3) δ 8.13 (s, 1H), 7.96 (d, J = 8.3 Hz,
1H), 7.89 (s, 1H), 7.78 (d, J = 8.2 Hz, 1H), 4.55 – 4.45 (m, 4H), 4.26 – 4.19 (m, 3H),
3.88 – 3.80 (m, 2H), 3.77 – 3.71 (m, 3H), 3.69 – 3.61 (m, 3H), 2.09 (s, 2H), 2.04 (dd,
J = 14.4, 6.9 Hz, 2H), 1.95 (s, 4H), 1.88 (dd, J = 13.9, 6.3 Hz, 3H), 1.72 – 1.60 (m,
13
12H), 1.58 (s, 6H), 1.42 (dt, J = 15.2, 7.7 Hz, 3H). C NMR (100 MHz, CDCl3) δ
178.4, 175.1, 171.7, 160.6, 139.9, 137.0, 135.2, 133.7, 133.3, 132.1, 127.6, 126.0,
69.2, 68.9, 65.1, 64.1, 62.9, 50.2, 48.8, 43.8, 42.3, 36.7, 32.8, 29.5, 28.6, 27.2, 23.7,
23.2. ESI-HRMS: m/z cacld. For C38H49F3N6O5S [M+H]+: 759.8021, found 745.8041.
2-(2-((1-(4-((3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thioxoimida

zolidin-1-yl)methyl)phenyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)ethyl
2-((3r,5r,7r)-adamantan-1-yl)acetate (A11).
Brown oil, 54% yield. 1H NMR (400 MHz, CDCl3) δ 8.05 (s, 1H), 7.99 (d, J = 8.3 Hz,
1H), 7.96 (d, J = 1.3 Hz, 1H), 7.85 (dd, J = 8.2, 1.8 Hz, 1H), 7.77 (d, J = 8.5 Hz, 2H),
7.64 – 7.57 (m, 2H), 5.19 (s, 2H), 4.78 (s, 2H), 4.27 – 4.18 (m, 2H), 3.75 (dd, J = 5.9,
3.1 Hz, 2H), 3.70 (dd, J = 9.6, 4.7 Hz, 4H), 2.08 (s, 2H), 1.94 (s, 3H), 1.71 – 1.59 (m,
12H), 1.51 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 174.3, 171.1, 168.5, 167.0, 165.8,
156.5, 153.4, 136.6, 136.2, 135.4, 133.7, 133.4, 128.1, 123.1, 123.0, 119.1, 117.1,
115.9, 115.0, 108.4, 77.3, 66.5, 66.1, 64.4, 62.1, 49.1, 47.8, 37.6, 31.4, 29.9, 29.2,
23.4, 23.3, 22.6. ESI-HRMS: m/z cacld. For C39H43F3N6O5S [M+H]+: 765.8652, found
765.8649.
2-(2-(2-((1-(3-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-2,4-dioxoimidazol
idin-1-yl)propyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)ethoxy)ethyl
2-((3r,5r,7r)-adamantan-1-yl)acetate (A12).
Brown oil, 47% yield. 1H NMR (400 MHz, CDCl3) δ 8.14 (d, J = 1.5 Hz, 1H), 8.00
(dd, J = 8.4, 1.9 Hz, 1H), 7.93 (d, J = 8.5 Hz, 1H), 7.69 (s, 1H), 4.69 (d, J = 6.2 Hz,
2H), 4.48 (t, J = 6.6 Hz, 2H), 4.21 (dd, J = 9.7, 5.0 Hz, 2H), 3.77 – 3.61 (m, 10H),
3.50 – 3.42 (m, 2H), 2.43 – 2.34 (m, 2H), 2.08 (s, 2H), 1.96 (s, 3H), 1.66 (dd, J = 30.5,
11.4 Hz, 12H), 1.52 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 174.3, 171.7, 159.2,
145.4, 138.6, 136.4, 136.3, 135.7, 135.3, 128.0, 127.9, 123.2, 123.0, 77.3, 70.6, 69.9,
69.2, 64.7, 62.9, 62.1, 48.8, 47.8, 42.3, 37.7, 36.7, 32.8, 30.0, 28.6, 23.4. ESI-HRMS:
m/z cacld. For C37H47F3N6O7 [M+H]+: 745.8132, found 745.8125.
2-(2-(2-((1-(4-((3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-2,4-dioxoimidazo
lidin-1-yl)methyl)phenyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)ethoxy)ethyl
2-((3r,5r,7r)-adamantan-1-yl)acetate (A13).
Brown oil, 43% yield. 1H NMR (400 MHz, CDCl3) δ 8.11 (s, 1H), 7.99 (d, J = 8.3 Hz,
1H), 7.96 (d, J = 1.4 Hz, 1H), 5.18 (s, 2H), 4.78 (s, 2H), 4.25 – 4.16 (m, 2H), 3.84 –
3.63 (m, 14H), 2.07 (s, 2H), 1.93 (d, J = 15.2 Hz, 3H), 1.73 – 1.59 (m, 12H), 1.51 (s,
6H). 13C NMR (100 MHz, CDCl3) δ 174.3, 171.1, 168.5, 167.0, 165.8, 156.5, 153.4,
136.6, 136.2, 135.4, 133.7, 133.4, 128.1, 123.1, 123.0, 119.1, 117.1, 115.9, 115.0,

108.4, 77.3, 66.5, 66.1, 64.4, 62.1, 49.1, 47.8, 37.6, 31.4, 29.9, 29.2, 23.4, 23.3, 22.6.
ESI-HRMS: m/z cacld. For C41H47F3N6O7 [M+H]+: 793.8572, found 793.8576.
2-(2-(2-((1-(3-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thioxoimi
dazolidin-1-yl)propyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)ethoxy)ethyl
2-((3r,5r,7r)-adamantan-1-yl)acetate (A14).
Brown oil, 36% yield. 1H NMR (400 MHz, CDCl3) δ 7.95 (d, J = 8.2 Hz, 1H), 7.88 (s,
1H), 7.76 (d, J = 8.1 Hz, 1H), 7.66 (s, 1H), 4.71 (d, J = 5.6 Hz, 2H), 4.49 (t, J = 6.6
Hz, 2H), 4.24 – 4.18 (m, 2H), 3.85 – 3.77 (m, 2H), 3.69 (ddd, J = 14.6, 7.9, 4.6 Hz,
8H), 2.57 – 2.46 (m, 2H), 2.08 (s, 2H), 1.96 (s, 3H), 1.74 – 1.60 (m, 12H), 1.55 (s,
6H). 13C NMR (100 MHz, CDCl3) δ 178.8, 175.0, 171.7, 145.6, 137.0, 135.2, 132.1,
127.0, 127.0, 123.0, 114.8, 77.3, 70.6, 70.6, 70.5, 69.9, 69.3, 65.2, 64.7, 62.9, 48.8,
48.0, 42.4, 41.5, 36.7, 32.8, 28.6, 28.3, 23.1. ESI-HRMS: m/z cacld. For
C37H47F3N6O6S [M+H]+: 761.8742, found 761.8739.
2-(2-(2-((1-(4-((3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thioxoim
idazolidin-1-yl)methyl)phenyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)ethoxy)ethyl
2-((3r,5r,7r)-adamantan-1-yl)acetate (A15).
Brown oil, 39% yield. 1H NMR (400 MHz, CDCl3) δ 8.20 (d, J = 1.8 Hz, 1H), 8.09 –
8.03 (m, 2H), 7.94 (d, J = 8.4 Hz, 1H), 7.76 (d, J = 8.5 Hz, 2H), 7.55 (d, J = 8.5 Hz,
2H), 4.78 (s, 2H), 4.69 (s, 2H), 4.20 (dd, J = 9.6, 4.7 Hz, 2H), 3.75 (dd, J = 5.9, 3.0
Hz, 3H), 3.73 – 3.65 (m, 9H), 2.07 (s, 3H), 1.95 (s, 4H), 1.65 (dd, J = 30.0, 11.6 Hz,
13H), 1.47 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 174.4, 171.7, 153.4, 146.2, 137.7,
136.8, 136.4, 135.3, 133.8, 129.4, 128.6, 128.0, 123.1, 123.0, 121.0, 120.9, 120.8,
115.0, 77.3, 70.6, 70.5, 70.0, 69.2, 64.7, 62.9, 62.3, 48.8, 43.1, 42.3, 36.7, 32.8, 28.6,
23.7. ESI-HRMS: m/z cacld. For C41H47F3N6O6S [M+H]+: 809.9182, found 809.9190.
1-(1-(3-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-2,4-dioxoimidazolidin-1-
yl)propyl)-1H-1,2,3-triazol-4-yl)-2,5,8,11-tetraoxatridecan-13-yl
2-((3r,5r,7r)-adamantan-1-yl)acetate (A16).
Brown oil, 41% yield. 1H NMR (400 MHz, CDCl3) δ 8.14 (s, 1H), 8.00 (dd, J = 8.4,
1.8 Hz, 1H), 7.93 (d, J = 8.4 Hz, 1H), 7.71 (d, J = 6.8 Hz, 1H), 4.69 (d, J = 6.3 Hz,
2H), 4.47 (t, J = 6.7 Hz, 2H), 4.24 – 4.18 (m, 2H), 3.77 – 3.64 (m, 14H), 3.45 (dd, J =