Design, synthesis and biological evaluation of novel thiohydantoin derivatives as potent androgen receptor antagonists for the treatment of prostate cancer

Article abstract of DOI:10.1016/j.bmc.2020.115953

Prostate cancer (PC) is the most common malignancy in men worldwide. Here, two series of novel thiohydantoin derivatives of enzalutamide as potent androgen receptor (AR) antagonists were designed and synthesized. Among them, compound 31c was identified as an AR antagonist which is 2.3–fold more potent than enzalutamide. Molecular docking studies were performed to explain the improved potency of 31c at AR. In cell proliferation assays, 31c exhibited similar anti-proliferative activities with enzalutamide against hormone sensitive LNCaP cells and AR-overexpressing LNCaP/AR cells. These data indicate that 31c can be a good lead compound for further structure optimization for the treatment of prostate cancer.

Full text of DOI:10.1016/j.bmc.2020.115953

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Design, Synthesis and Biological Evaluation of Novel Thiohydantoin Deriva-
tives as Potent Androgen Receptor Antagonists for the Treatment of Prostate
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Ao Wang, Yawan Wang, Xin Meng, Yushe Yang
PII:
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https://doi.org/10.1016/j.bmc.2020.115953
BMC 115953
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Please cite this article as: A. Wang, Y. Wang, X. Meng, Y. Yang, Design, Synthesis and Biological Evaluation of
Novel Thiohydantoin Derivatives as Potent Androgen Receptor Antagonists for the Treatment of Prostate Cancer,
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Bioorganic & Medicinal Chemistry
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Design, Synthesis and Biological Evaluation of Novel Thiohydantoin Derivatives as
Potent Androgen Receptor Antagonists for the Treatment of Prostate Cancer
Ao Wang a, b, Yawan Wang , Xin Meng and Yushe Yang
c
a
a, b, c,
a
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, P. R.
China
School of Pharmacy, University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049, P. R. China
b
c
School of Chinese Materia Medica, Nanjing University of Chinese Medicine, 138 Xianlin Avenue, Nanjing 210023, P. R. China
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Prostate cancer (PC) is the most common malignancy in men worldwide. Here, two series of novel
thiohydantoin derivatives of enzalutamide as potent androgen receptor (AR) antagonists were
designed and synthesized. Among them, compound 31c was identified as an AR antagonist which
is 2.3−fold more potent than enzalutamide. Molecular docking studies were performed to explain
the improved potency of 31c at AR. In cell proliferation assays, 31c exhibited similar anti-
proliferative activities with enzalutamide against hormone sensitive LNCaP cells and AR-
overexpressing LNCaP/AR cells. These data indicate that 31c can be a good lead compound for
further structure optimization for the treatment of prostate cancer.
Available online
Keywords:
androgen receptor
novel thiohydantoin derivatives
androgen receptor antagonists
prostate cancer

Corresponding author. Tel.: 86-21-50806786; fax: 86-21-50806786 (Y.Y.); e-mail: ysyang@simm.ac.cn

1
. Introduction
develop novel and potent AR antagonists for the treatment of
CRPC.
Prostate cancer (PC) is the most common malignancy in men
worldwide and the second most common cause of cancer-related
death worldwide after lung cancer. According to the International
Agency for Research on Cancer's Globocan data for 2018, the
number of deaths due to prostate cancer was 358,989.1
Fig. 1. Chemical structures of compounds 1−6 (representative AR
antagonists)
In the setting of CRPC, the first-generation AR antagonists
undergo an antagonist-to-agonist switch and stimulate AR activity
induced prostate tumor cell growth because of their low affinity
for the AR relative to testosterone and dihydrotestosterone (DHT).
Therefore, series 1 compounds (Fig. 2) were designed which
Most PCs are initially androgen-dependent, and the androgen
receptor (AR) is highly expressed at all stages of the disease,
playing a key role in the development and progression of PC.
2
,3
Androgen ablation, by surgical or chemical castration in
combination with AR antagonists such as 1 (flutamide) , 2
O
4
O2N
F3C
H HO
N
O O
S
F3C
NC
O N
2
N
NH
NH
5
6
(
bicalutamide) and 3 (nilutamide) (Fig. 1), has been the standard
O
F3C
O
O
7
F
treatment for advanced PC for many years. This therapy is
initially effective in 80−90% of patients, however, more than 50%
of the patients will ultimately develop castration resistant prostate
cancer (CRPC) because tumor cells will spontaneously synthesize
in situ or through the adrenal glands to synthesize low levels of
androgens to resume growth. CRPC is attributed to elevated AR
gene expression which can be driven by AR gene amplification,
AR gene mutation,
1
2
3
F
HO
O
F
N
O
S
NC
NC
F3C
S
N
H
NH
N
N
N
N
N
N
H
F3C
H
N
NC
Cl
N
4
O
N
O
O
5
6
8
adopted a ring-closing strategy in order to simulate the structure of
endogenous steroid ligands and enhance the binding affinity to
AR. In addition, AR agonists and AR antagonists share different
modes of action towards the related transcriptional machinery
inducing the “closed” and “open” conformations of AR-H12 helix
9
,10
1
1,12
or ligand-independent AR activation
through other factors such as increased expression of
1
3,14
transcriptional coactivators.
(
H12). It is supposed that the ectopic of H12 may play an important
Since 2012, three AR antagonists (Figure 1) have been
approved by the FDA, including 4 (enzalutamide) for mCRPC and
role in the antagonist conformation, similar to the estrogen
receptor and glucocorticoid receptor antagonist structures.
2
1-23
So
5
(apalutamide), 6 (darolutamide) for nonmetastatic CRPC
1
5-17
we designed series 2 compounds (Fig. 2) that features the
cyclization of the amide group to the right-hand side benzene ring
of enzalutamide to increase the steric hindrance between the
substituent and H12 to increase the AR antagonistic activity. The
design, synthesis and biological evaluation of these series of
compounds are reported herein.
(nmCRPC).
Despite the promising results achieved in CRPC
patients with androgen deprivation therapies (ADT), such as
1
5,18
enzalutamide,
Recently, an F876L mutation in AR ligand binding domain
AR−LBD) has been identified, which confers an antagonist-to-
agonist switch driving the resistance to enzalutamide and
resistance to such therapies has been observed.
(
1
9,20
apalutamide.
Therefore, there is an unmet and urgent need to
S
NC
R
N
N
Series 1
F3C
F
O
S
O
X
NC
N
X= C, N, S
N
N
H
F3C
O
O
O
S
Further SAR
NC
Y'
S
Y''
NC
Enzalutamide (4)
Series 2
Y
Y
n
N
N
N
N
R1
R2
F3C
n
F3C
O
O
Y = O, NR
n = 0, 1
Y = O, NR
n = 0, 1
Y', Y'' = C, N
Fig. 2. Design of two novel series of thiohydantoin derivatives
2
. Results and discussion
2
.1. Chemistry

Key intermediate EN-2 and target compounds (11, 16, 21) were
prepared as depicted in Scheme 1. Compound 7 was treated with
thiophosgene to provide the intermediate EN-2. Compound 8 was
compound 13, which was treated with benzyl chloroformate
(CbzCl) to obtain the mono-protected compound 14, and 14 was
cyclized with EN-2 to give compound 15. Removal of the Cbz
group yielded compound 16. Compound 17 and ethyl 2,3-
dibromopropanoate underwent a nucleophilic substitution reaction
to provide compound 18, followed by reduction by iron powder to
obtain compound 19. Next, catalytic hydrogenation reduced the
imine to obtain compound 20, which was reacted with EN-2 to
yield compound 21.
reduced
to
tetrahydroquinoline-2-carboxylic
acid
by
hydrogenation in the presence of PtO
2
, which was further
esterified to give intermediate 9. Then it was treated with N-
Bromosuccinimide (NBS) to afford compound 10, which was
cyclized with EN-2 to give compound 11. Compound 12 was
treated with ethyl 2,3-dibromopropanoate at 80°C to get
NC
NC
F3C
S
a
Cl
Cl
F3C
Br
N
NH2
C
EN-2
S
7
NC
S
e
b,c
d
Br
N
N
S
N
H
9
COOCH3
N
COOCH3 F3C
N
8
COOH
H
10
O
O
H
11
Cbz
N
N
NC
NC
S
NH2
f
h
O
g
Br
e
Br
Br
N
N
N
N
Br
N
H
3
O
F3C
F3C
Br
NH2
N
H
12
1
14
O
N
O
NH
O
15
16
Cbz
O
S
NO2
NO2
O
O
NC
F3C
H
e
N
Br
SH
S
O
N
i
j
O
k
N
N
O
O
Br
S
20
S
Br
S
19
O
21
1
7
18
Br
Br
a
a
Scheme 1. Synthesis of key intermediate EN-2 and target compounds (11, 16, 21) . Reagents and conditions: (a)H
MeOH, rt; (c) SOCl , MeOH, 0°C -rt; (d) NBS, DMF, rt; (e) EN-2, DMF, 30°C; (f) ethyl 2,3-dibromopropanoate, Et N, DMF, 80°C; (g)
CbzCl, K CO , CH CN, rt; (h) 33% HBr.CH COOH, rt; (i) ethyl 2,3-bromo-2-oxopropanoate, dithiothreitol, DMF, K CO , 0°C; (j) Fe,
NH Cl, EtOH:H O=12:1, 70°C; (k) H , Pd/C, MeOH, 35°C
2 2 2
O, rt; (b)H , PtO ,
2
3
2
3
3
3
2
3
4
2
2
As for target compounds 27a−27i, the syntheses are depicted in
Scheme 2. Suzuki coupling reactions between Compound 22 and
various boric acid or borate esters yielded compounds 23a−23i.
Then the methyl group was oxidized by selenium dioxide to obtain
the corresponding compounds 24a−24i, followed by Pinnick
oxidation to obtain the corresponding carboxylic acids 25a−25i.
Next, the quinoline core was reduced to tetrahydroquinoline and
the carboxylic acids was converted to corresponding esters
26a−26i, under similar conditions as described for Scheme 1.
Finally, cyclization reactions with EN-2 yielded compounds
27a−27i.

N
N
N
CHO
a
c
b
Br
R
R
22
2
2
2
2
2
3a: R=-H
23f: R=
24a: R=-H
24f: R=
N
N
3b: R=-Ph
3c: R=
24b: R=-Ph
24c: R=
N
Cl
N
Cl
23g: R=
24g: R=
N
O
N
O
N
N
3d: R=
CF3 23h: R=
CF3 23i: R=
N
24d: R=
3
CF 24h: R=
N
N
N
3e: R=
24e: R=
CF3 24i: R=
NC
N
N
N
N
S
N
COOH
H
N
COOCH3
d,e
f
F3C
N
N
R
R
R
O
2
2
2
2
2
5a: R=-H
5b: R=-Ph
5c: R=
25f: R=
26a: R=-H
26f: R=
26g: R=
2
7a: R=-H
27f: R=
27g: R=
N
N
N
N
Cl
26b: R=-Ph
26c: R=
N
Cl
27b: R=-Ph
N
Cl
25g: R=
N
O
N
O
N
O
N
N
N
27c: R=
27d: R=
N
N
N
5d: R=
CF325h: R=
26d: R=
CF326h: R=
N
N
CF3
27h: R=
N
5e: R=
CF3
N
26e: R=
CF3
N
27e: R=
CF3
N
N
N
25i: R=
26i: R=
N
27i: R=
a
a
Scheme 2. Synthesis of series 1 compounds (27a−27i) . Reagents and conditions: (a) various boric acid or borate ester reagents, K
Pd (PPh , dioxane: H O = 4:1, 80°C; (b) SeO , dioxane, 100°C; (c) NaClO , NaH PO .2H O, 2-methyl-2-butene, t-BuOH, rt; (d) H
PtO , MeOH, rt; (e) SOCl , MeOH, 65°C; (f) EN-2, DMF, 30°C
2
CO
3
,
,
3
)
4
2
2
2
2
4
2
2
2
2
The preparation of compounds 31a−31e is depicted in Scheme
. Compound 28 was treated with thiophosgene to get the
intermediate EN-3. Intermediates 29a−29d (prepared as described
in Supplementary material) were treated under Strecker reaction
conditions with acetone and trimethylsilyl cyanide to generate the
cyanoamines 30a−30d. Then cyclization reaction with EN-2 or
EN-3 using lithium bistrimethylsilylamine at the temperature of -
78°C yielded the imine compounds, followed by acidic hydrolysis
to provide target compounds 31a−31e.
3
NC
N
NC
N
S
a
Cl
Cl
F3C
N
F3C
NH2
O
C
S
EN-3
28
NC
M
O
S
O
X
X
X
Y
b
F3C
N
Y
EN-2 or EN-3
c,d
N
Y
H2N
n
n
N
O
n
NC
0a: X=C, Y=O, n=0
30b: X=C, Y=NCH , n=0
0c: X=C, Y=O, n=1
0d: X=C, Y=O, n=0
H
3
1a: X=C, Y=O, n=0, M=C
2
2
2
2
9a: X=C, Y=O, n=0
9b: X=C, Y=NCH , n=0
9c: X=C, Y=O, n=1
9d: X=N, Y=O, n=0
3
3
3
3
3
1b: X=C, Y=NCH , n=0, M=C
3
3
3
1c: X=C, Y=O, n=1, M=C
1d: X=C, Y=O, n=0, M=N
1e: X=N, Y=O, n=0, M=C
3
3
a
a
2 2
Scheme 3. Synthesis of series 2 compounds (31a−31e) . Reagents and conditions: (a) H O, rt; (b) TMSCN, ZnCl , Acetone, 40°C; (c)
LiHMDS, THF, -78°C; (d) 2N HCl, MeOH, 80°C
Compounds 33a−33k and 36 were prepared as depicted in
Scheme 4. The synthetic route of 33a−33k is basically the same
as that of 31a−31e. Through diazotization of 5-
aminoisobenzofuran-1(3H)-one (29a), compound 34 was
obtained. Next, compound 35 was obtained from 34 through a
Copper-catalyzed Ullman coupling reaction and a methylation
reaction with by methyl iodide. Finally it was cyclized with EN-2
to yield compound 36.
NC
M
O
S
O
O
X
X
X
O
a
F3C
N
O
EN-2 or EN-3
N
A
O
H2N
n
n
N
O
A
n
NC
3
H
b,c
2
2
2
9a: X=C, n=0
9c: X=C, n=1
9d: X=N, n=0
2a: X=C, n=0, A=
32b: X=C, n=0, A=
32c: X=C, n=0, A=
33f: X=C, n=0, M=C, A=
33g: X=N, n=0, M=N, A=
33h: X=C, n=1, M=C, A=
33i: X=C, n=0, M=N, A=
33j: X=C, n=0, M=N, A=
33k: X=C, n=0, M=N, A=
O
3
3a: X=C, n=0, M=C, A=
3b: X=C, n=0, M=C, A=
d
3
O
3
2d: X=C, n=0, A=
2e: X=C, n=0, A=
O
33c: X=C, n=0, M=C, A=
3d: X=C, n=0, M=N, A=
O
3
O
O
3
O
Br
32f: X=C, n=0, A=
O
33e: X=C, n=0, M=C, A=
O
3
4
3
2g: X=N, n=0, A=
3
2h: X=C, n=1, A=
e,f
O
NC
S
g
O
O
O
O
F3C
N
N
N
H
5
O
36
O
3

a
a
Scheme 4. Synthesis of series 2 compounds (33a−33k, 36) . Reagents and conditions: (a) TMSCN, ZnCl
C; (b) LiHMDS, THF, -78°C; (c) 2N HCl, MeOH, 80°C; (d) NaNO , 47% HBr, CuBr, 0°C -rt; (e) N,N-Dimethylglycine hydrochloride,
CuI, DMSO, K CO , 120°C; (f) K CO , CH I, DMF, rt; (g) EN-2, LiHMDS, THF, -78°C
2
, various cyclic ketones, 40°
2
2
3
2
3
3
2
.2. Androgen receptor (AR) antagonist assay
synthesized and evaluated. It was surprised to find that the activity
of 31a is better than 31b and enzalutamide (Table 2). So the right
side was chosen as a lactone instead of a lactam. In order to further
explore the ring size of the benzolide compounds, compounds 31c
and 33h were then synthesized. It was found that 31c exhibit
weaker activity than that of 31a, and 33h is also weaker than 33b.
These results indicate that the five-membered ring lactone is
preferred.
All the synthesized compounds were evaluated for their AR
antagonistic activity using the GeneBLAzer® Betalactamase
reporter technology for nuclear receptors (NRs) (see
Experimental Section). Enzalutamide (4) was used as the
reference, and the data are shown in Table 1 and Table 2.
As shown in Table 1, the activity of the tested compounds
varies in a wide range. Compounds 11, 16 and 21 were first tested
for their AR antagonistic activity, and it was found that their
activities were all weaker than enzalutamide (Table 1). However,
among them, compound 11 showed relatively better activity than
that of 16 and 21 for which the X is a carbon. Therefore, we set the
X of the tricyclic series compounds as a carbon, and explored the
substituent R at the 3-position of the right-side benzene ring and
synthesized compounds 27a−27i, but the activity is still far weaker
than enzalutamide. So series 1 compounds were discontinued for
further structural modifications.
At the same time, in order to prevent the right benzene ring from
24
metabolism via oxidation of the electron-rich aromatic ring, we
also replaced the right benzene ring with a pyridine ring and
synthesized 31e. It was found that the activity was greatly
diminished, which means that the right benzene ring cannot be
replaced.
In addition, compounds 33a−33c, 33e, 33f and 36 were
synthesized. Because part A plays an important role in AR
antagonist activity based on the structure-activity relationship
study of enzalutamide. It was found that their activities are better
than enzalutamide except for 36, and the substituent with
cyclopentane shows the best activity.
Table 1. AR Antagonist Effect of series 1 compounds
S
NC
R
N
N
Since the benzene ring on the left side of the enzalutamide was
replaced with a pyridine ring in apalutamide (5), which showed
improved activity, we next synthesized compounds 31d, 33d, and
F3C
O
X
Compd.
X
C
N
S
R
IC50/nM
4377
3
3i−33k. Among them, the activity of 33j and 33k are much better
than enzalutamide, but 31d, 33d and 33i are weaker than
enzalutamide.
1
1
2
2
2
2
2
2
2
2
2
2
1
Br
Br
Br
H
6
9862
Finally, we also synthesized 33g, where the left and right
benzene rings were both replaced with pyridine rings, and found
that the activity was reduced by nearly 5-fold compared to
enzalutamide, this is further confirmed that the right benzene ring
cannot be replaced.
1
18140
2837
7a
7b
7c
7d
7e
7f
7g
7h
7i
C
C
C
C
C
C
C
C
C
2
.3. Antiproliferative assay
Ph
3438
Compounds with potent AR antagonist activity were selected
for further studies in antiproliferative assays toward normal
LNCaP (hormone sensitive) cells and LNCaP/AR cells, which
were engineered (using viral infection with a cDNA encoding for
the AR) to express 3- to 5-fold higher levels of the AR to mimic
the clinical setting of CRPC. The data were listed in Table 3.
Enzalutamide (4) was used as the reference drug. For LNCaP cells,
the antiproliferative activity of 33h is better than that of the control
drug enzalutamide, while 31a and 31c are equivalent as
enzalutamide, and the other compounds (33a−33c, 33e, 33f, 33j,
and 33k) are weaker. For LNCaP/AR cells, the anti-proliferative
activity of 33e is the most potent, which is slightly better than
compound enzalutamide. Compound 31c showed comparable
activity to that of enzalutamide, while the activities of other
compounds (31a, 33b, 33c, 33f−33k) are weaker. Taken together,
the anti-proliferative activity of compound 31c is comparable to
that of compound enzalutamide in both types of cells, and it has
great development potential.
3906
N
CF3
2183
N
CF3
>50000
8953
N
N
Cl
8075
N
O
N
5978
N
4527
N
enzalutamide
646.9
Table 2. AR Antagonist Effect of series 2 compounds
In the discovery process of enzalutamide, it was reported that
compounds with an ester substituent on the right benzene ring are
NC
M
S
2
4
O
more active than compounds with an amide substituent. Since the
ester group is easier to metabolize than an amide, ring-closing
strategy was adopted by closing the ester group to the benzene
ring, forming lactones, in order to improve metabolic properties
and increase the steric hindrance to push H12 away at the same
time. Based on this, compounds 31a and 31b were first
X
F3C
N
N
Y
O
A
n

Compd.
M
C
C
C
N
C
C
C
C
N
C
C
N
C
N
N
N
C
A
X
C
C
C
C
N
C
C
C
C
C
C
N
C
C
C
C
C
Y
O
n
0
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
IC50/nM
148.6
590.2
195.5
941.1
2292
216.7
80.7
31c with AR were analyzed by Schrödinger. The key interactions
of 31c with the key residues are shown in Fig. 3A. Overall, the
binding pose of compound 31c is very similar to that of
enzalutamide. The cyano group of 31c formed two important
hydrogen bonds with Arg752 and Gln711, and there is a π-π
interaction between the benzene ring and Phe764, which is similar
to enzalutamide (Fig. 3B). In addition, the carbonyl group and the
ester oxygen of 31c formed two hydrogen bonds with Arg779,
while only the amide NH group of enzalutamide formed a
hydrogen bond with Ser778. One more hydrogen bond in binding
patterns may be the reason for the high potency 31c toward AR.
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
1a
1b
1c
1d
1e
3a
3b
3c
3d
3e
3f
NCH
O
3
O
O
O
O
O
95.2
O
26735
238.8
140.9
2219
134.6
516.2
63.8
O
O
O
O
O
3g
3h
3i
O
O
O
O
3j
O
3k
6
O
93.3
O
669.6
454.6
Enzaluta
mide
Table 3. Antiproliferative activity for novel thiohydantoin
Derivatives
Compd.
1a
1c
3b
IC50/µM(LNCaP) IC50/µM(LNCaP/AR)
3
1.982
1.936
3.687
2.192
2.663
1.287
4.77
1.472
0.730
1.086
17.00
0.569
5.588
2.036
0.730
3
3
Fig. 3. The predicted binding modes and 2D diagram of (A)
compound 31c and (B) enzalutamide in complex with AR (PDB
ID: 2OZ7)
3
3
3c
3e
3
3
3h
3k
3
. Conclusion
In conclusion, two series of novel thiohydantoin derivatives
enzalutamide
1.900
were designed, synthesized and evaluated for AR antagonist effect
and antiproliferative activities. series 2 compounds showed
excellent AR antagonistic activity, ten compounds (31a, 31c,
2
.4. Molecular modelling studies
Since the AR antagonistic activity of 31c is 2.3−fold more
3
3a−33c, 33e, 33f, 33h,33j, 33k) are 2−7 fold more potent than
the control drug enzalutamide. Among them, the compound 31c
exhibited comparable anti-proliferative activities to that of
enzalutamide in both LNCaP and LNCaP/AR cell proliferation
assays. In addition, Molecular docking studies were performed to
show that one more hydrogen bond in binding patterns may be the
reason for the high potency 31c toward AR. These data indicate
potent than enzalutamide (4) and its anti-cell proliferation activity
for LNCaP cells and LNCaP/AR cells is equivalent to that of
enzalutamide, we investigated the possible binding modes of 31c
to AR (PDB ID: 2OZ7) which was obtained from Protein Database
online(www.rcsb.org). The possible binding modes of compound

that compound 31c can be a good lead compound for further
structural optimization for the treatment of prostate cancer.
protection of argon, then NBS (0.19 g, 3.34 mmol, 3.19 equiv)
which was dissolved in anhydrous DMF was slowly added. The
reaction was stirred at room temperature for 1.5 h, then the solution
was added some ice water, extracted with EtOAc. The organic
4
. Experimental Section
2 4
phase was washed with saturated brine, dried over Na SO , filtered
4
.1. General Chemistry Methods
and concentrated under reduced pressure to give a light yellow
1
All solvents and chemicals were used as purchased without
solid 252 mg in 89.2% yield. H NMR (400 MHz, Chloroform-d)
further purification. Room temperature refers to 20−25°C.
Intermediates not described below were purchased from
commercial vendors and were used as supplied unless stated
otherwise. All reactions were monitored using thin-layer
chromatography (TLC) on silica gel F-254 TLC plates. Column
chromatography was carried out using silica gel (200−300 mesh).
δ 7.12 – 7.08 (m, 2H), 6.50 – 6.47 (m, 1H), 4.41 (s, 1H), 4.05 (dd,
J = 8.6, 3.8 Hz, 1H), 3.80 (s, 2H), 2.77 (dddt, J = 22.2, 16.5, 11.4,
5.5 Hz, 2H), 2.33 – 2.25 (m, 1H), 2.00 (dtd, J = 13.0, 8.8, 5.2 Hz,
1H).
4
3
.1.4. Synthesis
,3a,4,5-tetrahydroimidazo[1,5-a]quinolin-2(1H)-
of
4-(7-bromo-3-oxo-1-thioxo-
1
13
H and C NMR spectra were recorded on a Bruker 400, a Bruker
00, or a Bruker 600 NMR spectrometer using solvent residual as
yl)-2-(trifluoromethyl)benzonitrile (11)
5
Methyl
6-bromo-1,2,3,4-tetrahydroquinoline-2-carboxylate
an internal standard. Chemical shifts (δ) are reported in parts per
million (ppm), and coupling constants (J) are reported in hertz
(10) (0.25 g, 925.49 umol, 1 equiv) was reacted with EN-2 (0.211
g, 925.49 umol, 1 eqviu) in 20 mL anhydrous DMF at room
temperature for 1 h. After work up, the crude product was poured
into ice water. Then the solution was extracted with EtOAc, the
(Hz). Electron ionization-mass spectra were obtained on a
Finnigan MAT95 spectrometer, and electrospray ionization (ESI)
mass spectra were obtained on a Krats MS 80 mass spectrometer.
All final compounds were purified to >95% purity as determined
by analytical high-performance liquid chromatography
organic phase was washed with saturated brine, dried over Na
filtered, concentrated and purified by silica gel chromatography
20%-50% EtOAc/hexanes) to give the desired product. It was
2 4
SO ,
(
(PLATISIL ODS 250 mm × 4.6 mm, particle size 5 μm) with
1
obtained as a white solid 0.288 g in 66.74% yield. H NMR (400
MHz, Chloroform-d) δ 8.76 – 8.71 (m, 1H), 8.02 (d, J = 8.2 Hz,
1H), 7.91 (d, J = 2.0 Hz, 1H), 7.81 – 7.77 (m, 1H), 7.47 – 7.42 (m,
2H), 4.47 (dd, J = 12.4, 3.2 Hz, 1H), 3.17 (dd, J = 9.2, 4.2 Hz, 2H),
2.75 – 2.68 (m, 1H), 2.25 – 2.16 (m, 1H). C NMR (126 MHz,
DMSO) δ 177.30, 170.87, 138.09, 136.31, 134.43, 134.26, 131.94,
131.43, 128.60, 128.30, 123.26, 117.86, 115.03, 108.81, 60.50,
acetonitrile/water as the mobile phase.
4
.1.1. Synthesis of
4-isothiocyanato-2-
(
trifluoromethyl) benzonitrile (EN-2)
1
3
Thiophosgene (0.45 mL, 5.91 mmol, 1.1 equiv) was dissolved
in 20 mL water stirred at room temperature, 7 (1 g, 5.37 mmol, 1
equiv) was added to the solution in batches. The reaction mixture
was stirred at room temperature for 4 h and TLC analysis indicated
the reaction was completed. Then the solution was extracted with
25.39, 23.57. HRMS (ESI) m/z calcd for C19
463.969, found: 463.9686.
3 3
H10BrF N OS
CH
over Na
2
Cl
2
, the organic phase was washed with saturated brine, dried
SO , filtered, concentrated and purified by silica gel
4
3
.1.5. Synthesis
,3a,4,5-tetrahydroimidazo[1,5-a]quinoxalin-2(1H)-
of
4-(7-bromo-3-oxo-1-thioxo-
2
4
chromatography (0−10% EtOAc/hexanes) to give the desired
yl)-2-(trifluoromethyl)benzonitrile (16)
product. It was obtained as a yellow oil 898 mg in 73.25% yield.
Ethyl 6-bromo-1,2,3,4-tetrahydroquinoxaline-2-carboxylate
1
H NMR (400 MHz, Chloroform-d) δ 7.87 (d, J = 8.3 Hz, 1H),
(13)
7
.61 (d, J = 2.1 Hz, 1H), 7.51 (dd, J = 8.3, 2.1 Hz, 1H).
1
3
2 (3 g, 16.04 mmol, 1 equiv) was dissolved in DMF, Et N
4
.1.2. Synthesis of Methyl
1,2,3,4-
(
(
4.46 ml, 32 mmol, 2 equiv) and ethyl 2,3-dibromopropanoate
2.33 mL, 16.04 mmol, 1 equiv) were added to the solution, then
the reaction mixture was slowly warmed to 80℃. The reaction
mixture was stirred at this temperature for 24h and TLC analysis
indicated the reaction was completed. Then the solution was
extracted with EtOAc, the organic phase was washed with
saturated brine, dried over Na SO , filtered, concentrated and
2 4
purified by silica gel chromatography (10%-15% EtOAc/hexanes)
to give the mixture brown oil 1.63 g.
tetrahydroquinoline-2-carboxylate (9)
(10 g, 57.75 mmol, 1 equiv) and PtO (0.656 g, 2.88 mmol,
.05 equiv) were dissolved in methanol stirred at room
8
2
0
temperature under the protection of Hydrogen. The reaction
mixture was stirred at room temperature for 4 h and TLC analysis
indicated the reaction was completed. Then the solution was
filtered through celite, the filtrate was collected and concentrated,
2
the reaction mixture was cooled to 0°C, and 6.28 mL SOCl was
added dropwise over 10 min. Then the reaction mixture was
allowed to warm slowly to rt. The reaction mixture was stirred at
room temperature for 6.5 h and TLC analysis indicated the reaction
was completed. Then the solution was extracted with EtOAc, the
organic phase was washed with saturated brine, dried over
Na SO , filtered, concentrated and purified by silica gel
2 4
chromatography (5% EtOAc/hexanes) to give the desired product.
It was obtained as a yellow oil 3.58 g in 32.42% yield. H NMR
1-benzyl 3-ethyl 7-bromo-3,4-dihydroquinoxaline-1,3(2H)-
dicarboxylate (14)
The mixture was dissolved in CH
temperature, then K CO (1.18 g, 8.61 mmol, 1.5 equiv) and 0.8
mL CbzCl were added to the solution, the reaction mixture was
stirred at room temperature for 4 h and TLC analysis indicated the
reaction was completed. Then the solution was extracted with
EtOAc, the organic phase was washed with saturated brine, dried
over Na SO , filtered, concentrated and purified by silica gel
2 4
chromatography (10%-30% EtOAc/hexanes) to give the desired
product. It was obtained as a yellow oil 908 mg.
3
CN stirred at room
2
3
1
(
1
400 MHz, Chloroform-d) δ 7.07 – 7.01 (m, 1H), 7.00 (dt, J = 7.6,
.2 Hz, 1H), 6.69 (td, J = 7.4, 1.2 Hz, 1H), 6.62 (dd, J = 7.9, 1.1
Hz, 1H), 4.41 (s, 1H), 4.08 (dd, J = 8.7, 3.8 Hz, 1H), 3.81 (s, 3H),
2
5
.83 (dddt, J = 21.9, 16.4, 11.2, 5.5 Hz, 3H), 2.32 (dtd, J = 12.9,
.7, 3.8 Hz, 1H), 2.09 – 1.99 (m, 1H).
Benzyl 7-bromo-2-(4-cyano-3-(trifluoromethyl)phenyl)-3-
oxo-1-thioxo-2,3,3a,4-tetrahydroimidazo[1,5-a]quinoxaline-
4
.1.3. Synthesis
of
Methyl
6-bromo-1,2,3,4-
tetrahydroquinoline-2-carboxylate (10)
5
(1H)-carboxylate (15)
Methyl 1,2,3,4-tetrahydroquinoline-2-carboxylate (9) (0.2 g,
1
.05 mmol, 1 eqviu) was put into a three-necked flask, dissolved
The mixture (2.478 g, 5.91 mmol, 1 equiv) was reacted with
in anhydrous DMF stirred at room temperature under the
EN-2 (1.6 g, 7.01 mmol, 1.19 eqviu) in 12 mL anhydrous DMF

for 5 h. After work up, the crude product was poured into ice water.
Then the solution was extracted with EtOAc, the organic phase
desired product. It was obtained as a light yellow solid 60 mg in
12.79% yield. H NMR (400 MHz, Chloroform-d) δ 7.16 – 7.13
1
was washed with saturated brine, dried over Na
2
SO
4
, filtered,
(m, 0H), 7.04 (ddd, J = 8.6, 2.4, 1.0 Hz, 1H), 6.49 (dd, J = 8.6, 1.0
Hz, 1H), 4.63 (s, 0H), 4.34 (d, J = 2.4 Hz, 0H), 4.32 – 4.26 (m,
1H), 3.29 (ddd, J = 12.6, 3.1, 0.9 Hz, 1H), 3.07 (ddd, J = 12.6, 7.9,
1.0 Hz, 1H), 1.33 (td, J = 7.1, 1.0 Hz, 2H).
concentrated and purified by silica gel chromatography (10%-20%
EtOAc/hexanes) to give the desired product. It was obtained as a
yellow oil 2.226 g in 62.74% yield for the next step.
Then the previous step compound was added to 33%
4.1.9. Synthesis of 4-(7-bromo-3-oxo-1-thioxo-3a,4-
dihydro-1H-benzo[b]imidazo[1,5-d][1,4]thiazin-
2(3H)-yl)-2-(trifluoromethyl)benzonitrile (21)
HBr.CH
analysis indicated the reaction was completed. Then slowly add
0% NaOH solution to adjust the pH of the solution to alkaline,
then the solution was extracted with EtOAc. The organic phase
was washed with saturated brine, dried over Na SO , filtered,
concentrated and purified by silica gel chromatography
3
COOH, stirred at room temperature for 0.5 h and TLC
1
Ethyl
7-bromo-3,4-dihydro-2H-benzo[b][1,4]thiazine-3-
carboxylate (20) (56 mg, 0.185 mmol, 1 equiv) was reacted with
EN-2 (50 mg, 0.219 mmol, 1.19 eqviu) in 10 mL anhydrous DMF
for 5.5h. After work up, the crude product was poured into ice
water. Then the solution was extracted with EtOAc, the organic
phase was washed with saturated brine, dried over Na SO , filtered,
2
4
1
(
50%−100% EtOAc/hexanes) to give the desired products. H
NMR (400 MHz, DMSO-d6) δ 8.71 (d, J = 2.3 Hz, 1H), 8.41 (d, J
8.2 Hz, 1H), 8.24 (d, J = 1.8 Hz, 1H), 8.03 (dd, J = 8.3, 1.9 Hz,
H), 7.18 (dd, J = 8.8, 2.3 Hz, 1H), 6.91 (d, J = 4.4 Hz, 1H), 6.75
d, J = 8.7 Hz, 1H), 4.72 (dd, J = 10.9, 4.4 Hz, 1H), 3.81 (dt, J =
1.6, 4.5 Hz, 1H), 3.64 (t, J = 11.3 Hz, 1H). 13C NMR (126 MHz,
DMSO) δ 177.07, 169.52, 138.29, 137.36, 136.83, 134.83, 129.53,
28.68, 124.27, 121.56, 116.59, 115.47, 109.33, 105.27, 57.00.
2
4
=
1
(
1
concentrated and purified by silica gel chromatography (10%-20%
EtOAc/hexanes) to give the desired product. It was obtained as a
1
white solid 55 mg in 61.28% yield. H NMR (400 MHz, DMSO-
d6) δ 8.60 (d, J = 8.9 Hz, 1H), 8.42 (d, J = 8.2 Hz, 1H), 8.26 (d, J
= 1.8 Hz, 1H), 8.04 (dd, J = 8.3, 1.9 Hz, 1H), 7.64 (d, J = 2.2 Hz,
1H), 7.40 (dd, J = 8.9, 2.2 Hz, 1H), 4.89 (dd, J = 11.6, 3.5 Hz, 1H),
1
1
3
HRMS (ESI) m/z calcd for C18
H
11BrF
3
N
4
OS 466.9779, found:
3.87 (t, J = 12.0 Hz, 1H), 3.61 (dd, J = 12.4, 3.5 Hz, 1H). C NMR
4
66.9784.
(126 MHz, DMSO) δ 178.00, 169.14, 138.56, 136.94, 134.81,
1
2
31.74, 128.86, 127.18, 125.63, 119.02, 115.67, 109.33, 57.98,
6.77. HRMS (EI) m/z calcd for C18 ON BrF 482.9317,
4
.1.6. Synthesis
of
Ethyl
3-((5-bromo-2-
H
9
3
3 2
S
nitrophenyl)thio)-2-oxopropanoate (18)
found: 482.9306.
1
7 (2.82 g, 12.05 mmol, 1 equiv) and dithiothreitol (1.85 g, 12
mmol, 1 equiv) were put into a three-necked flask, dissolved in
anhydrous DMF stirred at 0℃ under the protection of argon, then
4.1.10. Synthesis of 4-2-methyl-6-phenylquinoline
(23b)
K
2
CO
3
(1.99 g, 18.25 mmol, 1.5 equiv) and ethyl 2,3-
22 (0.3 g, 1.35 mmol, 1 equiv) was coupled to Phenylboronic
dibromopropanoate (1.8 mL, 14.46 mmol, 1.2 equiv) were added
to the solution. The reaction mixture was stirred at 0℃ for 2 h and
TLC analysis indicated the reaction was completed. Then the
solution was extracted with EtOAc, the organic phase was washed
acid (0.198 g, 1.62 mmol, 1.2 equiv) using K CO (0.559 g, 4.86
2
3
mmol, 3 equiv) and Pd(PPh ) (0.156 g, 0.162 mmol, 0.1 equiv) in
3
4
12 mL dioxane and 3 mL water for 16 h at the temperature of 80℃.
After work up, some of water was poured into the solution,
extracted with EtOAc. The organic phase was washed with
saturated brine, dried over Na SO , filtered, concentrated and
2 4
with saturated brine, dried over Na SO , filtered, concentrated and
purified by silica gel chromatography (10%-20% EtOAc/hexanes)
to give the desired product. It was obtained as a yellow solid 1.65g
2
4
purified by silica gel chromatography (10% EtOAc/hexanes) to
give the desired product. It was obtained as a white solid 0.221g
1
in 39.34% yield. H NMR (400 MHz, Chloroform-d) δ 8.11 (d, J
1
=
8.8 Hz, 1H), 7.65 (d, J = 2.0 Hz, 1H), 7.48 (dd, J = 8.8, 2.0 Hz,
in 74.61% yield. H NMR (400 MHz, Chloroform-d) δ 8.13 – 8.09
1
2
H), 6.46 (s, 1H), 6.43 (d, J = 1.3 Hz, 1H), 4.43 (q, J = 7.1 Hz,
H), 1.43 (t, J = 7.1 Hz, 3H).
(m, 2H), 8.00 – 7.96 (m, 2H), 7.76 – 7.71 (m, 2H), 7.55 – 7.49 (m,
2H), 7.44 – 7.39 (m, 1H), 7.33 (d, J = 8.4 Hz, 1H), 2.79 (s, 3H).
4
.1.7. Synthesis
of
Ethyl
7-bromo-2H-
4.1.11. Synthesis
carbaldehyde (24b)
of
6-phenylquinoline-2-
benzo[b][1,4]thiazine-3-carboxylate (19)
Ethyl 3-((5-bromo-2-nitrophenyl)thio)-2-oxopropanoate (18)
2-methyl-6-phenylquinoline (23b) (221 mg, 1.01 mmol,1 equiv)
(
1
550 mg, 1.58 mmol, 1 equiv) was dissolved in 12 mL EtOH and
mL water stirred for a few minutes, Fe (352 mg, 6.32 mmol, 4
equiv) and NH Cl (422 mg, 7.88 mmol, 5 equiv) were added to the
and SeO (450 mg, 4.06 mmol, 4 equiv) were dissolved in dioxane,
the reaction was slowly warmed to 100℃. The reaction mixture
2
4
was stirred at 100℃ for 4 h and TLC analysis indicated the reaction
was completed. Then the solution was cooled to room temperature
and extracted with EtOAc, the organic phase was washed with
saturated brine, dried over Na SO , filtered and concentrated under
solution, then the reaction mixture was slowly warm to 70℃. The
reaction mixture was stirred at 70℃ for 2.5 h and TLC analysis
indicated the reaction was completed. The solution was filtered
with celite to get filtrate, extracted with EtOAc, the organic phase
2
4
reduced pressure to give a yellow solid 230 mg in 97.83% yield.
1
was washed with saturated brine, dried over Na
2
SO
4
, filtered,
H NMR (400 MHz, DMSO-d6) δ 10.14 (d, J = 0.8 Hz, 1H), 8.64
concentrated and purified by silica gel chromatography (5%
EtOAc/hexanes) to give the desired product. It was obtained as a
white solid 200 mg in 42.18% yield for the next step.
(d, J = 8.4 Hz, 1H), 8.43 (d, J = 2.0 Hz, 1H), 8.32 – 8.22 (m, 2H),
8.02 (d, J = 8.4 Hz, 1H), 7.91 – 7.86 (m, 2H), 7.56 (dd, J = 8.3, 6.8
Hz, 3H), 7.50 – 7.45 (m, 1H).
4
.1.8. Synthesis of Ethyl 7-bromo-3,4-dihydro-2H-
4.1.12. Synthesis of 6-phenylquinoline-2-carboxylic
acid (25b)
benzo[b][1,4]thiazine-3-carboxylate (20)
Ethyl 7-bromo-2H-benzo[b][1,4]thiazine-3-carboxylate (19)
The compounds NaClO (0.87 g, 9.62 mmol, 9 equiv) and
2
(466 mg, 1.55 mmol, 1 equiv) was dissolved in methanol stirred at
NaH PO .2H O (1 g, 6.90 mmol, 7 equiv) were dissolved in 8 mL
2
4
2
room temperature, and then Pd/C (170 mg, 1.55 mmol, 1 equiv)
was added to the solution. The reaction mixture was stirred at room
temperature for 8 h under the protection of hydrogen and TLC
analysis indicated the reaction was completed. After completed,
filter off Pd/C with celite to get filtrate, concentrated and purified
by silica gel chromatography (5% EtOAc/hexanes) to give the
of water, and then slowly added to the tert-butanol solution of 6-
phenylquinoline-2-carbaldehyde (24b) (230 mg, 0.986 mmol, 1
equiv), after the dropwise addition, 5 mL of 2-methyl-2-butene
was added and reacted at room temperature. The reaction mixture
was stirred at room temperature for 12 h and TLC analysis
indicated the reaction was completed. The solution was

concentrated, followed by adding 15 mL water，then filtered to
collect the filter cake. Finally put the filter cake into a vacuum
(trifluoromethyl)benzonitrile (27c): It was obtained as a white
solid. H NMR (400 MHz, DMSO-d6) δ 9.01 (d, J = 8.7 Hz, 1H),
1
drying oven to dry. It was obtained as a white solid 218 mg in
8.95 (d, J = 2.4 Hz, 1H), 8.59 (dd, J = 4.8, 1.6 Hz, 1H), 8.41 (d, J
= 8.2 Hz, 1H), 8.29 (d, J = 1.9 Hz, 1H), 8.12 (dt, J = 8.1, 2.0 Hz,
1H), 8.08 – 8.04 (m, 1H), 7.75 (d, J = 2.2 Hz, 1H), 7.69 (dd, J =
8.6, 2.3 Hz, 1H), 7.50 (dd, J = 8.0, 4.7 Hz, 1H), 4.88 (dd, J = 12.0,
3.6 Hz, 1H), 3.21 (pd, J = 15.1, 13.2, 5.3 Hz, 2H), 2.40 (dtt, J =
1
8
8.70% yield. H NMR (400 MHz, DMSO-d6) δ 8.58 (d, J = 8.5
Hz, 1H), 8.39 (d, J = 2.0 Hz, 1H), 8.27 (d, J = 8.8 Hz, 1H), 8.23 –
.19 (m, 1H), 8.16 (d, J = 8.5 Hz, 1H), 7.91 – 7.86 (m, 2H), 7.56
t, J = 7.7 Hz, 2H), 7.49 – 7.43 (m, 1H).
8
(
1
3
3
1
1
1
1.2, 12.4, 6.6 Hz, 1H). C NMR (126 MHz, DMSO) δ 176.81,
70.98, 148.58, 147.53, 138.15, 136.30, 134.90, 134.55, 134.45,
34.04, 133.89, 129.38, 128.37, 127.81, 124.11, 123.88, 121.99,
15.05, 108.77, 60.69, 25.77, 23.85. HRMS (ESI) m/z calcd for
4
.1.13. Synthesis of Methyl
6-phenyl-1,2,3,4-
tetrahydroquinoline-2-carboxylate (26b)
-phenylquinoline-2-carboxylic acid (25b) (218 mg, 0.87
mmol, 1 equiv) and PtO (10 mg, 0.04 mmol, 0.05 equiv) were
6
2
C
24
H
16
F
3
N
4
OS 465.0983, found: 465.0991.
added to the methanol solution, and then hydrogen gas was
bubbled to react at room temperature for 8 h. Then the reaction
4-(3-oxo-1-thioxo-7-(6-(trifluoromethyl)pyridin-3-yl)-
mixture was cooled to 0°C, and 0.1 mL SOCl
2
was added dropwise,
3,3a,4,5-tetrahydroimidazo[1,5-a]quinolin-2(1H)-yl)-2-
next the reaction mixture was slowly warmed to 65℃. The reaction
mixture was stirred at this temperature for 4h and TLC analysis
indicated the reaction was completed. Then the solution was
extracted with EtOAc, the organic phase was washed with
saturated brine, dried over Na SO , filtered, concentrated and
2 4
purified by silica gel chromatography (8% EtOAc/hexanes) to give
(trifluoromethyl)benzonitrile (27d): It was obtained as a white
solid. H NMR (400 MHz, DMSO-d6) δ 9.16 (d, J = 2.2 Hz, 1H),
1
9.06 (d, J = 8.7 Hz, 1H), 8.42 (dd, J = 8.2, 3.7 Hz, 2H), 8.29 (d, J
= 1.8 Hz, 1H), 8.07 (dd, J = 8.2, 1.9 Hz, 1H), 8.01 (d, J = 8.3 Hz,
1H), 7.86 (d, J = 2.2 Hz, 1H), 7.80 (dd, J = 8.7, 2.3 Hz, 1H), 4.90
(dd, J = 12.0, 3.6 Hz, 1H), 3.31 – 3.17 (m, 2H), 2.50 – 2.32 (m,
1
3
the desired product. It was obtained as a yellow solid 55 mg in
1H). C NMR (126 MHz, DMSO) δ 177.45, 171.38, 148.55,
138.60, 138.31, 136.78, 136.24, 136.15, 134.91, 132.86, 130.03,
128.84, 125.09, 122.50, 121.35, 115.50, 109.26, 61.11, 26.29,
1
2
3.53% yield. H NMR (400 MHz, Chloroform-d) δ 7.58 – 7.52
(
m, 3H), 7.45 – 7.38 (m, 3H), 7.33 – 7.23 (m, 4H), 6.70 (d, J = 8.3
Hz, 1H), 4.49 (s, 1H), 4.12 (dd, J = 8.9, 3.7 Hz, 1H), 3.82 (s, 4H),
.98 – 2.79 (m, 3H), 2.35 (dtd, J = 12.7, 5.5, 3.7 Hz, 1H), 2.08 (dtd,
J = 12.6, 8.9, 5.2 Hz, 1H).
24.21. HRMS (ESI) m/z calcd for C25
531.072.
13 6 4
H F N OS 531.0716, found:
2
4
-(3-oxo-1-thioxo-7-(4-(trifluoromethyl)phenyl)-3,3a,4,5-
4
3
.1.14. Synthesis of 4-(3-oxo-7-phenyl-1-thioxo-
,3a,4,5-tetrahydroimidazo[1,5-a]quinolin-2(1H)-
tetrahydroimidazo[1,5-a]quinolin-2(1H)-yl)-2-
(trifluoromethyl)benzonitrile (27e): It was obtained as a white
1
yl)-2-(trifluoromethyl)benzonitrile (27b)
solid. H NMR (400 MHz, DMSO-d6) δ 9.02 (d, J = 8.6 Hz, 1H),
Methyl
6-phenyl-1,2,3,4-tetrahydroquinoline-2-carboxylate
8.41 (d, J = 8.2 Hz, 1H), 8.28 (s, 1H), 8.07 (d, J = 8.3 Hz, 1H), 7.95
(d, J = 8.1 Hz, 2H), 7.83 (d, J = 8.1 Hz, 2H), 7.76 (s, 1H), 7.71 (d,
J = 8.9 Hz, 1H), 4.91 – 4.83 (m, 1H), 3.22 (dq, J = 17.6, 11.2, 8.6
Hz, 2H), 2.49 – 2.30 (m, 2H). C NMR (126 MHz, DMSO) δ
177.31, 171.42, 143.55, 138.61, 136.76, 135.90, 135.63, 134.91,
129.82, 128.83, 128.49, 127.73, 126.26, 124.78, 123.77, 122.41,
115.50, 109.24, 61.15, 26.23, 24.29. HRMS (ESI) m/z calcd for
(
0
26b) (49 mg, 0.18 mmol, 1 equiv) was reacted with EN-2 (50 mg,
.22 mmol, 1.2 eqviu) in 5 mL anhydrous DMF for 4 h. After work
up, the crude product was poured into ice water. Then the solution
was extracted with EtOAc, the organic phase was washed with
2 4
saturated brine, dried over Na SO , filtered, concentrated and
purified by silica gel chromatography (20%-50% EtOAc/hexanes)
to give the desired product. It was obtained as a white solid 62 mg
in 72.98% yield. H NMR (400 MHz, DMSO-d6) δ 8.97 (d, J = 8.6
Hz, 1H), 8.41 (d, J = 8.2 Hz, 1H), 8.28 (d, J = 1.9 Hz, 1H), 8.06
dd, J = 8.2, 1.9 Hz, 1H), 7.75 – 7.68 (m, 2H), 7.68 – 7.60 (m, 2H),
1
3
C
26
H
14
F
6
N
3
OS 530.0764, found: 530.0767.
1
4
-(3-oxo-7-(quinolin-3-yl)-1-thioxo-3,3a,4,5-
tetrahydroimidazo[1,5-a]quinolin-2(1H)-yl)-2-
trifluoromethyl)benzonitrile (27f): It was obtained as a light
(
(
7
3
3
1
1
2
4
.48 (t, J = 7.7 Hz, 2H), 7.41 – 7.35 (m, 1H), 4.87 (dd, J = 12.0,
.6 Hz, 1H), 3.20 (pd, J = 15.5, 13.4, 5.5 Hz, 2H), 2.47 – 2.27 (m,
1
yellow solid. H NMR (400 MHz, DMSO-d6) δ 9.32 (d, J = 2.3
Hz, 1H), 9.06 (d, J = 8.7 Hz, 1H), 8.71 (d, J = 2.4 Hz, 1H), 8.42
1
3
H). C NMR (126 MHz, DMSO) δ 176.65, 171.02, 139.12,
38.18, 137.19, 136.28, 134.45, 134.32, 129.12, 128.97, 128.38,
27.58, 126.52, 123.90, 121.86, 115.06, 108.74, 60.73, 25.78,
(d, J = 8.2 Hz, 1H), 8.30 (d, J = 1.8 Hz, 1H), 8.07 (dd, J = 8.2, 1.2
Hz, 3H), 7.92 (d, J = 2.2 Hz, 1H), 7.86 (dd, J = 8.7, 2.3 Hz, 1H),
7
1
.79 (ddd, J = 8.3, 6.8, 1.5 Hz, 1H), 7.67 (ddd, J = 8.2, 6.9, 1.2 Hz,
H), 4.91 (dd, J = 12.0, 3.6 Hz, 1H), 3.25 (ddd, J = 17.0, 11.8, 7.9
3.94. HRMS (ESI) m/z calcd for C25
62.0893.
15 3 3
H F N OS 462.0896, found:
1
3
Hz, 1H), 2.43 (ddd, J = 24.3, 12.4, 6.3 Hz, 1H). C NMR (126
MHz, DMSO) δ 177.29, 171.44, 149.71, 147.27, 138.63, 136.78,
135.41, 134.92, 134.47, 133.14, 132.24, 130.10, 129.95, 129.12,
4
.1.15. General procedure for the preparation of
compounds (27a, 27c-27i)
1
6
5
28.86, 128.53, 128.12, 127.59, 124.84, 122.52, 115.51, 109.23,
The experimental procedure for synthesizing compounds (27a,
7c-27i) is basically the same as 27b.
1.18, 26.27, 24.33. HRMS (ESI) m/z calcd for C28
14.1070, found: 514.1069.
4 3
H17ON F S
2
4
-(3-oxo-1-thioxo-3,3a,4,5-tetrahydroimidazo[1,5-a]quinolin-
4
-(7-(2-chloropyrimidin-5-yl)-3-oxo-1-thioxo-3,3a,4,5-
tetrahydroimidazo[1,5-a]quinolin-2(1H)-yl)-2-
trifluoromethyl)benzonitrile (27g): It was obtained as a light
2
(1H)-yl)-2-(trifluoromethyl)benzonitrile (27a): It was obtained
1
as a white solid. H NMR (400 MHz, Chloroform-d) δ 8.80 (dd, J
8.3, 1.1 Hz, 1H), 8.01 (d, J = 8.2 Hz, 1H), 7.92 (d, J = 2.0 Hz,
H), 7.81 (dd, J = 8.2, 2.0 Hz, 1H), 7.37 – 7.23 (m, 4H), 4.49 (dd,
(
=
1
1
yellow solid. H NMR (400 MHz, DMSO-d6) δ 9.18 (s, 2H), 9.04
d, J = 8.7 Hz, 1H), 8.42 (d, J = 8.2 Hz, 1H), 8.28 (d, J = 1.8 Hz,
1H), 8.06 (dd, J = 8.2, 1.9 Hz, 1H), 7.87 (d, J = 2.2 Hz, 1H), 7.79
dd, J = 8.7, 2.2 Hz, 1H), 4.89 (dd, J = 12.0, 3.5 Hz, 1H), 3.21 (dtd,
J = 23.1, 17.5, 17.0, 6.1 Hz, 2H), 2.40 (ddp, J = 25.0, 12.6, 6.7, 6.2
(
J = 12.3, 3.3 Hz, 1H), 3.23 – 3.14 (m, 2H), 2.75 – 2.67 (m, 1H),
.28 – 2.17 (m, 1H). 13C NMR (126 MHz, DMSO) δ 176.79,
2
1
1
(
71.10, 138.20, 136.27, 134.91, 134.45, 129.54, 128.62, 128.36,
25.67, 121.54, 115.06, 108.72, 60.61, 25.54, 24.01. HRMS (ESI)
1
3
Hz, 1H). C NMR (126 MHz, DMSO) δ 177.43, 171.32, 159.32,
1
1
m/z calcd for C19
11 3 3
H F N OS 386.0573, found: 386.058.
58.24, 138.54, 136.75, 136.17, 134.87, 131.77, 130.01, 129.75,
28.79, 128.41, 124.67, 122.43, 115.46, 109.21, 61.05, 26.16,
4
-(3-oxo-7-(pyridin-3-yl)-1-thioxo-3,3a,4,5-
tetrahydroimidazo[1,5-a]quinolin-2(1H)-yl)-2-

2
4
4.11. HRMS (EI) m/z calcd for C23
99.0479.
H
13ON
5
ClF
3
S 499.0476, found:
4.1.18. Synthesis of 5-amino-2-methylisoindolin-1-
one (29b)
Methyl 2-methyl-4-nitrobenzoate (5g, 25.62mmol, 1equiv) and
4
-(7-(3,5-dimethylisoxazol-4-yl)-3-oxo-1-thioxo-3,3a,4,5-
tetrahydroimidazo[1,5-a]quinolin-2(1H)-yl)-2-
trifluoromethyl)benzonitrile (27h): It was obtained as a light
NBS (6.8 g, 38.2 mmol, 1.5 equiv) were dissolved in CCl
temperature under the protection of Argon, then AIBN (420 mg,
.56 mmol, 0.1 equiv) was added to the solution. The reaction
4
at room
(
2
1
yellow solid. H NMR (400 MHz, DMSO-d6) δ 8.96 (d, J = 8.5
Hz, 1H), 8.41 (d, J = 8.2 Hz, 1H), 8.28 (d, J = 1.9 Hz, 1H), 8.06
dd, J = 8.2, 1.9 Hz, 1H), 7.38 – 7.30 (m, 2H), 4.88 (dd, J = 11.9,
.6 Hz, 1H), 3.17 (ddt, J = 24.2, 17.7, 8.5 Hz, 2H), 2.41 (s, 0H),
.35 (dq, J = 12.4, 6.0 Hz, 1H), 2.25 (s, 3H). C NMR (126 MHz,
DMSO) δ 177.28, 171.47, 165.72, 158.58, 138.59, 136.75, 134.91,
34.66, 130.23, 129.72, 128.73, 127.62, 126.65, 122.22, 115.69,
15.50, 109.21, 61.08, 26.09, 24.35, 11.86, 10.98. HRMS (EI) m/z
was slowly warmed to 80 ℃ and stirred for 11.5 h. After
completed, the mixture was concentrated and purified by silica gel
chromatography (5%−20% EtOAc/hexanes) to give the desired
product (EN-4). It was obtained as a white solid 6.3 g in 89.73%
(
3
2
1
3
1
yield. H NMR (400 MHz, Chloroform-d) δ 8.35 (d, J = 2.3 Hz,
1
(
H), 8.21 (dd, J = 8.6, 2.2 Hz, 1H), 8.13 (d, J = 8.5 Hz, 1H), 4.99
s, 2H), 4.02 (d, J = 0.9 Hz, 3H).
1
1
calcd for C24
H
17
O
2
N
4
F
3
S 482.1019, found: 482.1023.
EN-4 (6 g, 21.89 mmol, 1 equiv) was dissolved in MeOH
2
stirred at room temperature, and then 10 mL 2M MeNH /MeOH
4
-(7-(1-cyclopropyl-1H-pyrazol-4-yl)-3-oxo-1-thioxo-
was added to the solution. The reaction mixture was stirred at room
temperature for 12 h and TLC analysis indicated the reaction was
completed. Then the solution was concentrated and extracted with
EtOAc, the organic phase was washed with saturated brine, dried
over Na SO , filtered, concentrated and purified by silica gel
2 4
chromatography (30%−50% EtOAc/hexanes) to give the desired
3
,3a,4,5-tetrahydroimidazo[1,5-a]quinolin-2(1H)-yl)-2-
trifluoromethyl)benzonitrile (27i): It was obtained as a white solid.
H NMR (400 MHz, DMSO-d6) δ 8.84 (d, J = 8.6 Hz, 1H), 8.40
d, J = 8.2 Hz, 1H), 8.27 (d, J = 1.1 Hz, 2H), 8.04 (dd, J = 8.2, 1.9
(
1
(
Hz, 1H), 7.89 (d, J = 0.8 Hz, 1H), 7.56 (d, J = 2.1 Hz, 1H), 7.50
(
dd, J = 8.6, 2.1 Hz, 1H), 4.83 (dd, J = 11.9, 3.7 Hz, 1H), 3.75 (tt,
J = 7.4, 3.9 Hz, 1H), 3.23 – 2.99 (m, 2H), 2.38 (ddq, J = 31.0, 12.4,
.8 Hz, 2H), 1.13 – 1.05 (m, 2H), 1.02 – 0.96 (m, 2H). 13C NMR
126 MHz, DMSO) δ 176.28, 171.02, 138.19, 136.27, 136.12,
product (EN-5). It was obtained as a light yellow solid 1.6 g in
1
3
8.03% yield. H NMR (400 MHz, Chloroform-d) δ 8.39 – 8.33
6
(
(m, 2H), 8.00 (d, J = 8.2 Hz, 1H), 4.53 (s, 2H), 3.27 (s, 2H).
1
1
34.43, 132.84, 129.93, 128.99, 128.39, 127.01, 125.61, 122.22,
21.78, 120.83, 115.06, 108.68, 60.72, 32.82, 25.66, 23.98, 6.29.
EN-5 (1.1 g, 5.72 mmol, 1 equiv) was dissolved in methanol
stirred at room temperature, and then Pd/C (150 mg, 0.014 mmol,
0.05 equiv) was added to the solution. The reaction mixture was
stirred at room temperature for 8 h under the protection of
hydrogen and TLC analysis indicated the reaction was completed.
After completed, filter off Pd/C with celite to get filtrate,
concentrated to give the desired product (29b). It was obtained as
HRMS (EI) m/z calcd for C25
4
H
18ON
5
F
3
S 493.1179, found:
93.1185.
4
.1.16. Synthesis
of
5-isothiocyanato-3-
(
trifluoromethyl)picolinonitrile (EN-3)
Thiophosgene (0.489 mL, 6.4 mmol, 1.2 equiv) was dissolved
1
a gray solid 0.9 g in 96.94% yield. H NMR (400 MHz, DMSO-
in 20 mL water stirred at room temperature, 28 (1 g, 5.37 mmol, 1
equiv) was added to the solution in batches. The reaction mixture
was stirred at room temperature for 4 h and TLC analysis indicated
the reaction was completed. Then the solution was extracted with
d6) δ 7.28 (d, J = 8.1 Hz, 1H), 6.62 – 6.56 (m, 2H), 5.72 (s, 2H),
.24 (s, 2H), 2.97 (s, 3H).
4
4.1.19. Synthesis
of
3-aminofuro[3,4-b]pyridin-
CH
over Na
2
Cl
2
, the organic phase was washed with saturated brine, dried
SO , filtered, concentrated and purified by silica gel
7(5H)-one (29d)
2
4
Methyl 3-methylpicolinate (5g, 33.08mmol, 1equiv) was
chromatography (0-10% EtOAc/hexanes) to give the desired
products. It was obtained as a light yellow oil 540 mg in 44.09%
yield. H NMR (400 MHz, Chloroform-d) δ 8.76 (d, J = 2.3 Hz,
dissolved in CH Cl stirred at 0℃ under the protection of nitrogen,
2
2
then Bu NNO (11 g, 36.13 mmol, 1.1 equiv) which was dissolved
4
3
1
2 2
in CH Cl and trifluoroacetic anhydride (4.6 mL, 33 mmol, 1
1
H), 7.88 (d, J = 2.3 Hz, 1H).
equiv) were added to the solution stirred for 2 h. Then the reaction
mixture was stirred at room temperature for 10 h, the solution was
concentrated, extracted with EtOAc, the organic phase was
washed with saturated brine, dried over Na SO , filtered,
2 4
concentrated and purified by silica gel chromatography (10%-15%
4
.1.17. Synthesis of 5-aminoisobenzofuran-1(3H)-
one (29a)
CuSO (35 mg, 0.22 mmol, 0.007 equiv) was dissolved in 1.5
4
mL water, then Zn (5.5 g, 84.11 mmol, 2.8 equiv) was added to the
solution followed by 20% NaOH solution 12.75 g, the reaction was
stirred at ice bath, next 5-aminoisoindoline-1,3-dione (5.00 g,
EtOAc/hexanes) to give the desired product (EN-6). It was
1
obtained as a white solid 3.79 g in 58.41% yield. H NMR (400
MHz, Chloroform-d) δ 9.33 – 9.30 (m, 1H), 8.44 (dq, J = 2.2, 0.7
Hz, 1H), 4.05 (s, 3H), 2.73 (t, J = 0.7 Hz, 3H).
3
0.84 mmol, 1 equiv) was added after a few minutes. The reaction
mixture was stirred at 60℃ for 4 h and TLC analysis indicated the
reaction was completed. The reaction was cooled to room
temperature for suction filtration, and then the filter cake was
washed with a small amount of water to obtain a filtrate, and then
the pH was adjusted to 3-4 with concentrated hydrochloric acid, at
which time a large amount of solid precipitated out, and suction
filtration was performed to obtain a yellow solid. It was dissolved
with 10 mL water and 10 mL concentrated hydrochloric acid,
slowly raise the temperature to 100°C for 1 h, and then adjust the
pH to 8-9 with 20% NaOH solution. At this time, a large amount
of solid is precipitated, and suction filtration is performed to obtain
EN-6 (2.96 g, 15.09 mmol, 1 equiv) and NBS (4.03 g, 22.63
mmol, 1.5 equiv) were dissolved in CCl at room temperature
4
under the protection of argon, then AIBN (246mg, 1.5mmol,
0.1equiv) was added to the solution. The reaction was slowly
warmed to 80℃ and stirred for 11.5 h. After completed, the
mixture was concentrated and purified by silica gel
chromatography (10%−20% EtOAc/hexanes) to give the desired
product (EN-7). It was obtained as a white solid 1.84 g in 44.33%
1
yield. H NMR (400 MHz, Chloroform-d) δ 9.41 (d, J = 2.4 Hz,
1H), 8.71 (d, J = 2.4 Hz, 1H), 4.97 (s, 2H), 4.09 (s, 3H).
a pink solid, which is placed in a vacuum drying cabinet at 50 °C,
EN-7 (3.27 g, 11.89 mmol, 1 equiv) was dissolved in 20 mL
dioxane and 7 mL water stirred at 100℃, the reaction mixture was
stirred at this temperature for 12 h, then the solution was cooled to
room temperature, extracted with EtOAc, the organic phase was
1
3
.2 g of solid was finally obtained with a yield of 69.58%. H NMR
(400 MHz, DMSO-d6) δ 7.45 (d, J = 8.4 Hz, 1H), 6.67 (dd, J = 8.4,
2
.0 Hz, 1H), 6.59 (d, J = 1.8 Hz, 1H), 6.26 (s, 2H), 5.16 (s, 2H).

1
washed with saturated brine, dried over Na
concentrated and purified by silica gel chromatography
20%−50% EtOAc/hexanes) to give the desired product (EN-8). It
2
SO
4
, filtered,
oil 267 mg in 43.98% yield. H NMR (400 MHz, DMSO-d6) δ
9.68 (s, 1H), 7.75 (d, J = 8.7 Hz, 1H), 7.44 (d, J = 2.2 Hz, 1H), 7.40
(dd, J = 8.6, 2.2 Hz, 1H), 3.78 (s, 3H), 3.54 (td, J = 7.2, 5.4 Hz,
3H), 3.08 – 3.00 (m, 2H), 1.48 (s, 9H).
(
1
was obtained as a light yellow solid 880 mg in 41.10% yield. H
NMR (400 MHz, DMSO-d6) δ 9.58 (dd, J = 2.3, 0.9 Hz, 1H), 9.03
EN-12 (276 mg, 0.934 mmol, 1 equiv) was dissolved in 9 mL
dioxane and 3 mL water, 0.5 mL 2N HCl was added to the solution,
the mixture was slowly warmed to 100℃ stirred for 2 h. After the
reaction was completed, the mixture was cooled to room
(dd, J = 2.3, 0.8 Hz, 1H), 5.56 (t, J = 0.8 Hz, 2H).
EN-8 (0.88 g, 4.89 mmol, 1 equiv) was dissolved in methanol
stirred at room temperature, and then Pd/C (26 mg, 0.244 mmol,
.05 equiv) was added to the solution. The reaction mixture was
0
temperature and adjusted to neutral by NaHCO
with EtOAc. The organic phase was washed with saturated brine,
dried over Na SO , filtered, concentrated and purified by silica gel
3
, then extracted
stirred at room temperature for 8 h under the protection of
hydrogen and TLC analysis indicated the reaction was completed.
After completed, filtered off Pd/C with celite to get filtrate,
concentrated and purified by silica gel chromatography (5%
2
4
chromatography (30%−60% EtOAc/hexanes) to give the desired
product (29c). It was obtained as a white solid 70 mg in 45.90%
1
MeOH/CH
2
Cl
2
) to give the desired product (29d). It was obtained
yield. H NMR (400 MHz, DMSO-d6) δ 7.58 (d, J = 8.5 Hz, 1H),
1
as a light yellow solid 343 mg in 46.76% yield. H NMR (400
6.51 (dd, J = 8.5, 2.3 Hz, 1H), 6.37 (d, J = 2.2 Hz, 1H), 6.14 (s,
2H), 4.34 (t, J = 6.0 Hz, 2H), 2.84 (t, J = 6.0 Hz, 2H).
MHz, DMSO-d6) δ 8.10 (d, J = 2.4 Hz, 1H), 6.95 (d, J = 2.4 Hz,
1
H), 6.52 (s, 2H), 5.23 (s, 2H).
4
.1.21. Synthesis
dihydroisobenzofuran-5-yl)amino)propanenitrile
30a)
29a (0.5 g, 3.35 mmol, 1 equiv) was reacted with TMSCN (1.26
mL, 10.06 mmol, 3 eqviu) using ZnCl (46 mg, 0.33 mmol, 0.1
of
2-methyl-2-((1-oxo-1,3-
4
.1.20. Synthesis of 6-aminoisochroman-1-one (29c)
(
Methyl 2-bromo-4-nitrobenzoate (10 g, 38.46 mmol, 1 equiv)
was dissolved in 80 mL ethanol and 20 mL water stirred for a few
minutes, Fe (6.44 g, 115.3 mmol, 3 equiv) and NH Cl (8.23 g,
53.8 mmol, 4 equiv) were added to the solution, then the reaction
4
2
1
equiv) in 2mL Acetone at the temperature of 40℃ for 2 h. After
work up, the solution was concentrated. Then the solution was
extracted with EtOAc, the organic phase was washed with
saturated brine, dried over Na SO , filtered, concentrated and
2 4
purified by silica gel chromatography (30%-60% EtOAc/hexanes)
to give the desired product. It was obtained as a white solid 576
mg in 78.22% yield. H NMR (400 MHz, DMSO-d6) δ 7.63 (d, J
= 9.0 Hz, 0H), 7.18 (s, 0H), 6.97 (d, J = 2.1 Hz, 0H), 6.96 – 6.94
(m, 0H), 5.29 (s, 0H), 1.72 (s, 1H).
mixture was slowly warm to 70 ℃ . The reaction mixture was
stirred at 70℃ for 5.5 h and TLC analysis indicated the reaction
was completed. The solution was filtered with celite to get filtrate,
extracted with EtOAc, the organic phase was washed with
saturated brine, dried over Na
give the desired product (EN-9). It was obtained as a white solid
2 4
SO , filtered and concentrated to
1
1
8
.05 g in 90.99% yield. H NMR (400 MHz, DMSO-d6) δ 7.64 (d,
J = 8.6 Hz, 1H), 6.87 (d, J = 2.2 Hz, 1H), 6.55 (dd, J = 8.6, 2.2 Hz,
1
H), 6.17 (s, 2H), 3.73 (s, 3H).
4
.1.22. Synthesis of 4-(4,4-dimethyl-5-oxo-3-(1-
oxo-1,3-dihydroisobenzofuran-5-yl)-2-
thioxoimidazolidin-1-yl)-2-
EN-9 (844 mg, 3.67 mmol, 1 equiv), DMAP (45 mg, 0.367
mmol, 0.1 equiv) and Et
dissolved in anhydrous THF stirred at room temperature, then
Boc O (1.68 mL, 7.34 mmol, 2 equiv) was added to the solution
3
N (1.53 mL, 11.01 mmol, 3 equiv) were
(
trifluoromethyl)benzonitrile (31a)
2-methyl-2-((1-oxo-1,3-dihydroisobenzofuran-5-
2
drop by drop until completed. The reaction mixture was purified
by silica gel chromatography (10%−15% EtOAc/hexanes) to give
the desired products 523 mg for the next step.
yl)amino)propanenitrile (30a) (300 mg, 1.39 mmol, 1 equiv) was
dissolved in anhydrous THF stirred at -78℃，then 1M LiHMDS
in THF (1.39 mL, 1.39 mmol, 1 equiv) was added to the solution
drop by drop until completed. The mixture was stirred at -78℃ for
The above compounds were dissolved in 5 mL DMF,
tributyl(vinyl)stannane (0.5 mL, 1.72 mmol, 1.5 equiv) and
Pd(PPh ) (0.13 g, 0.11 mmol, 0.1 equiv) were added to the
3 4
solution stirred at 110℃ in microwave at a rate of 100 W for 2.5
h. then the solution was cooled to room temperature, extracted
with EtOAc, the organic phase was washed with saturated brine,
0
.5 h, then EN-2 (380 mg, 1.66 mmol, 1.2 equiv) which was
dissolved in THF was added to the solution. Then the reaction
mixture was stirred at room temperature for 1h and TLC analysis
indicated the reaction was completed. Then the solution was
purified by silica gel chromatography (30%-60% EtOAc/hexanes)
to give the compound. Then 8 mLMeOH and 5 mL 2N HCl were
added stirred at 80℃ for 2 h. The solution was extracted with
EtOAc, the organic phase was washed with saturated brine, dried
2 4
dried over Na SO , filtered, concentrated and purified by silica gel
chromatography (0-10% EtOAc/hexanes) to give the desired
1
product (EN-11). It was obtained as a white solid 570 mg. H NMR
2 4
over Na SO , filtered, concentrated and purified by silica gel
(
7
400 MHz, DMSO-d6) δ 9.75 (s, 0H), 7.79 (d, J = 6.4 Hz, 0H),
.78 (s, 0H), 7.51 (dd, J = 8.7, 2.2 Hz, 0H), 7.44 (dd, J = 17.5, 10.9
Hz, 0H), 5.59 (dd, J = 17.4, 1.4 Hz, 0H), 5.34 (dd, J = 10.9, 1.4 Hz,
chromatography (30%-100% EtOAc/hexanes) to give the desired
1
product. It was obtained as a white solid 36 mg in 4.85% yield. H
NMR (400 MHz, DMSO-d6) δ 8.42 (d, J = 8.3 Hz, 1H), 8.33 (d, J
0
H), 3.79 (s, 0H), 1.49 (s, 1H).
=
1.9 Hz, 1H), 8.12 (dd, J = 8.2, 1.9 Hz, 1H), 8.07 (d, J = 8.1 Hz,
1
5
1
1
2
4
H), 7.75 (dd, J = 1.8, 0.8 Hz, 1H), 7.63 (dd, J = 8.1, 1.7 Hz, 1H),
EN-11 (570 mg, 2.06 mmol, 1 equiv) was dissolved in
1
3
.52 (s, 2H), 1.56 (s, 6H). C NMR (126 MHz, DMSO) δ 181.90,
76.61, 171.61, 150.56, 142.51, 139.82, 138.12, 135.87, 132.96,
29.89, 127.97, 127.46, 126.45, 116.89, 110.59, 71.71, 68.62,
anhydrous THF stirred at ice bath, then 0.5M 9-BBN in THF (20
mL, 10.3 mmol, 5 equiv) was added to the solution drop by drop
until completed. Then the mixture was slowly warmed to room
4.90. HRMS (EI) m/z calcd for C21
45.0693.
14 3 3 3
H O N F S 445.0702, found:
temperature stirred for 12 h, next 1N NaOH (1 mL) and 30% H
2 2
O
(
1.8 mL) were added to the solution. The reaction mixture was
stirred at room temperature for 2 h and TLC analysis indicated the
reaction was completed. Then the solution was concentrated,
extracted with EtOAc. The organic phase was washed with
saturated brine, dried over Na SO , filtered, concentrated and
2 4
purified by silica gel chromatography (20%−50% EtOAc/hexanes)
to give the desired product (EN-12). It was obtained as a colorless
4
.1.23. General procedure for the preparation of
compounds (31b-31e, 33a-33k)
The experimental procedure for synthesizing compounds (31b-
1e, 33a-33k) is basically the same as 25a.
3

4
-(4,4-dimethyl-3-(2-methyl-1-oxoisoindolin-5-yl)-5-oxo-2-
4-(4-oxo-1-(1-oxo-1,3-dihydroisobenzofuran-5-yl)-2-thioxo-
1,3-diazaspiro[4.5]decan-3-yl)-2-(trifluoromethyl)benzonitrile
(33c): It was obtained as a white solid. H NMR (400 MHz,
thioxoimidazolidin-1-yl)-2-(trifluoromethyl)benzonitrile (31b): It
was obtained as a white solid. H NMR (400 MHz, DMSO-d6) δ
1
1
8
1
.42 (d, J = 8.3 Hz, 1H), 8.32 (d, J = 1.9 Hz, 1H), 8.11 (dd, J = 8.2,
.9 Hz, 1H), 7.86 (d, J = 8.0 Hz, 1H), 7.64 (d, J = 1.7 Hz, 1H), 7.50
DMSO-d6) δ 8.42 (d, J = 8.3 Hz, 1H), 8.31 (d, J = 1.8 Hz, 1H),
8.13 – 8.09 (m, 1H), 8.07 (d, J = 8.1 Hz, 1H), 7.69 (s, 1H), 7.55 (d,
J = 8.1 Hz, 1H), 5.52 (s, 2H), 2.43 (d, J = 12.8 Hz, 2H), 2.00 – 1.84
(
dd, J = 8.1, 1.7 Hz, 1H), 4.57 (s, 2H), 3.11 (s, 2H), 1.55 (s, 6H).
1
3
13
C NMR (126 MHz, DMSO) δ 180.00, 174.86, 166.36, 142.92,
38.03, 137.90, 136.21, 134.00, 133.11, 129.77, 128.07, 124.90,
23.64, 115.03, 108.66, 66.56, 51.29, 29.09, 23.01. HRMS (EI)
(m, 2H), 1.67 – 1.45 (m, 5H), 0.98 (d, J = 13.5 Hz, 1H). C NMR
1
1
(126 MHz, DMSO) δ 180.02, 173.52, 169.74, 148.66, 140.25,
137.92, 136.18, 134.15, 131.89, 128.12, 126.04, 125.72, 125.51,
115.05, 108.61, 69.83, 67.52, 31.56, 23.62, 20.38. HRMS (EI) m/z
m/z calcd for C22
17 2 4 3
H O N F S 458.1019, found: 458.1016.
18 3 3 3
calcd for C24H O N F S 485.1015, found: 485.1015.
4
-(4,4-dimethyl-5-oxo-3-(1-oxoisochroman-6-yl)-2-
thioxoimidazolidin-1-yl)-2-(trifluoromethyl)benzonitrile (31c): It
was obtained as a white solid. H NMR (600 MHz, DMSO-d6) δ
5-(8-oxo-5-(1-oxo-1,3-dihydroisobenzofuran-5-yl)-6-thioxo-
2-oxa-5,7-diazaspiro[3.4]octan-7-yl)-3-
1
8
0
6
1
1
2
.41 (d, J = 8.2 Hz, 0H), 8.32 (d, J = 2.0 Hz, 0H), 8.14 – 8.09 (m,
H), 7.48 (d, J = 6.9 Hz, 0H), 4.58 (t, J = 6.0 Hz, 0H), 3.17 (t, J =
.0 Hz, 0H), 1.55 (s, 1H). C NMR (126 MHz, DMSO) δ 180.30,
75.23, 164.20, 142.22, 140.43, 138.43, 136.70, 134.48, 131.34,
29.49, 129.32, 128.58, 126.13, 115.48, 109.19, 67.71, 67.12,
(trifluoromethyl)picolinonitrile (33d): It was obtained as a white
solid. H NMR (600 MHz, DMSO-d6) δ 9.18 (d, J = 2.1 Hz, 1H),
1
1
3
8.58 (d, J = 2.1 Hz, 1H), 8.05 (d, J = 8.2 Hz, 1H), 8.02 (d, J = 1.7
Hz, 1H), 7.90 (dd, J = 8.1, 1.8 Hz, 1H), 5.50 (s, 2H), 3.74 (d, J =
1
3
11.4 Hz, 2H), 3.66 (d, J = 11.4 Hz, 2H). C NMR (151 MHz,
DMSO) δ 171.17, 170.28, 152.05, 149.33, 139.56, 132.31, 132.10,
128.76, 126.68, 125.04, 122.47, 121.11, 114.82, 70.30, 67.51,
16 3 3 3
7.39, 23.51. HRMS (EI) m/z calcd for C22H O N F S 459.0859,
found: 459.0860.
3
4
3.13. HRMS (EI) m/z calcd for C20
60.0444.
11 4 4 3
H O N F S 460.0448, found:
5
-(4,4-dimethyl-5-oxo-3-(1-oxo-1,3-dihydroisobenzofuran-5-
yl)-2-thioxoimidazolidin-1-yl)-3-(trifluoromethyl)picolinonitrile
31d): It was obtained as a white solid. H NMR (400 MHz,
1
(
4-(4-oxo-1-(1-oxo-1,3-dihydroisobenzofuran-5-yl)-2-thioxo-
DMSO-d6) δ 9.28 (d, J = 2.0 Hz, 1H), 8.85 (d, J = 2.0 Hz, 1H),
7-oxa-1,3-diazaspiro[4.4]nonan-3-yl)-2-
8
1
.08 (d, J = 8.1 Hz, 1H), 7.76 (d, J = 1.7 Hz, 1H), 7.62 (dd, J = 8.1,
.7 Hz, 1H), 5.52 (s, 2H), 1.58 (s, 7H). 13C NMR (126 MHz,
(trifluoromethyl)benzonitrile (33e): It was obtained as a white
solid. H NMR (400 MHz, DMSO-d6) δ 8.42 (d, J = 8.3 Hz, 1H),
1
DMSO) δ 180.03, 174.93, 170.16, 154.17, 149.21, 140.84, 136.35,
8.29 (d, J = 1.8 Hz, 1H), 8.10 (dd, J = 8.3, 1.9 Hz, 1H), 8.05 (d, J
= 8.1 Hz, 1H), 7.86 (d, J = 1.6 Hz, 1H), 7.72 (dd, J = 8.2, 1.6 Hz,
1H), 5.52 (s, 2H), 4.41 (d, J = 10.6 Hz, 1H), 3.98 (d, J = 10.6 Hz,
1H), 3.75 (q, J = 7.8 Hz, 1H), 3.49 (q, J = 7.5 Hz, 1H), 2.59 (p, J =
1
2
4
33.64, 131.43, 126.63, 126.17, 124.98, 114.70, 70.31, 67.45,
3.47. HRMS (EI) m/z calcd for C20
46.0649.
13 3 4 3
H O N F S 446.0655, found:
1
3
6
1
1
7
4
.8 Hz, 2H). C NMR (126 MHz, DMSO) δ 181.04, 173.38,
70.18, 149.07, 141.81, 138.47, 136.70, 134.33, 131.57, 128.29,
28.26, 126.52, 126.07, 125.11, 115.48, 109.10, 75.44, 74.79,
4
-(4,4-dimethyl-5-oxo-3-(7-oxo-5,7-dihydrofuro[3,4-
b]pyridin-3-yl)-2-thioxoimidazolidin-1-yl)-2-
trifluoromethyl)benzonitrile (31e): It was obtained as a white
(
0.26, 68.17, 36.97. HRMS (EI) m/z calcd for C22
73.0652, found: 473.0653.
14 4 3 3
H O N F S
1
solid. H NMR (400 MHz, DMSO-d6) δ 8.90 (d, J = 2.0 Hz, 1H),
.44 (d, J = 8.2 Hz, 1H), 8.32 (dd, J = 15.0, 2.0 Hz, 2H), 8.13 (d, J
8.0 Hz, 1H), 5.56 (s, 2H), 1.60 (s, 6H). 13C NMR (126 MHz,
DMSO) δ 180.77, 174.61, 167.66, 153.36, 143.54, 142.88, 137.79,
8
=
4-(4-oxo-1-(1-oxo-1,3-dihydroisobenzofuran-5-yl)-2-thioxo-
8-oxa-1,3-diazaspiro[4.5]decan-3-yl)-2-
1
6
36.34, 135.52, 133.95, 133.40, 127.96, 114.99, 108.86, 68.04,
6.93, 22.92. HRMS (EI) m/z calcd for C20 S 446.0655,
(trifluoromethyl)benzonitrile (33f): It was obtained as a white
solid. H NMR (400 MHz, DMSO-d6) δ 8.43 (d, J = 8.3 Hz, 1H),
1
H
13
O
3
N
4
F
3
found: 446.0655.
8.32 (d, J = 1.9 Hz, 1H), 8.13 (dd, J = 8.2, 1.9 Hz, 1H), 8.09 (d, J
=
8.1 Hz, 1H), 7.70 (d, J = 1.6 Hz, 1H), 7.56 (dd, J = 8.0, 1.7 Hz,
4
-(8-oxo-5-(1-oxo-1,3-dihydroisobenzofuran-5-yl)-6-thioxo-
1
2
H), 5.53 (s, 2H), 4.00 – 3.87 (m, 2H), 3.80 (dd, J = 11.6, 4.9 Hz,
H), 2.43 (d, J = 13.2 Hz, 2H), 1.80 (td, J = 12.9, 5.2 Hz, 2H). C
5
(
,7-diazaspiro[3.4]octan-7-yl)-2-(trifluoromethyl)benzonitrile
33a): It was obtained as a white solid. H NMR (400 MHz,
DMSO-d6) δ 8.41 (d, J = 8.3 Hz, 1H), 8.28 (s, 1H), 8.10 (dd, J =
.5, 5.8 Hz, 2H), 7.79 (s, 1H), 7.65 (d, J = 8.1 Hz, 1H), 5.55 (s,
H), 2.66 (t, J = 10.1 Hz, 3H), 2.50 – 2.41 (m, 2H), 2.04 – 1.91 (m,
H), 1.55 (dd, J = 11.0, 5.8 Hz, 1H). C NMR (126 MHz, DMSO)
1
3
1
NMR (126 MHz, DMSO) δ 180.75, 174.02, 170.18, 149.30,
1
1
40.35, 138.21, 136.67, 134.58, 132.37, 128.49, 126.63, 126.33,
26.00, 115.48, 109.18, 70.31, 65.26, 62.33, 31.72. HRMS (EI)
8
2
1
16 4 3 3
m/z calcd for C23H O N F S 487.0808, found: 487.0805.
1
3
δ 180.59, 175.06, 170.22, 149.32, 141.28, 138.45, 136.64, 134.28,
5-(4-oxo-1-(7-oxo-5,7-dihydrofuro[3,4-b]pyridin-3-yl)-2-
thioxo-1,3-diazaspiro[4.4]nonan-3-yl)-3-
1
3
31.84, 126.72, 126.16, 125.44, 115.51, 108.93, 70.34, 68.03,
1.62, 13.90. HRMS (EI) m/z calcd for C22 S 457.0702,
H
14
O
3
N
3
F
3
(trifluoromethyl)picolinonitrile (33g): It was obtained as a white
1
found: 457.0701.
solid. H NMR (400 MHz, DMSO-d6) δ 9.31 – 9.22 (m, 1H), 8.97
–
2
1
8.87 (m, 1H), 8.86 – 8.80 (m, 1H), 8.41 – 8.33 (m, 1H), 5.57 (s,
H), 2.39 (dt, J = 13.8, 6.6 Hz, 2H), 2.21 (dt, J = 14.0, 6.5 Hz, 3H),
.73 (dt, J = 16.5, 8.4 Hz, 2H), 1.49 – 1.35 (m, 2H). C NMR (126
4
-(4-oxo-1-(1-oxo-1,3-dihydroisobenzofuran-5-yl)-2-thioxo-
1
(
,3-diazaspiro[4.4]nonan-3-yl)-2-(trifluoromethyl)benzonitrile
33b): It was obtained as a white solid. H NMR (400 MHz,
DMSO-d6) δ 8.41 (d, J = 8.3 Hz, 1H), 8.32 (d, J = 1.9 Hz, 1H),
.11 (dd, J = 8.3, 1.9 Hz, 1H), 8.06 (d, J = 8.1 Hz, 1H), 7.82 (s,
H), 7.68 (dd, J = 8.1, 1.7 Hz, 1H), 5.52 (s, 2H), 2.33 (dt, J = 14.0,
.8 Hz, 2H), 2.19 (dt, J = 13.8, 6.0 Hz, 2H), 1.78 – 1.65 (m, 2H),
.43 – 1.30 (m, 2H). 13C NMR (126 MHz, DMSO) δ 182.09,
77.54, 171.60, 150.60, 143.17, 139.92, 138.04, 135.78, 133.27,
29.83, 127.99, 127.49, 126.81, 116.91, 110.42, 77.30, 71.69,
1
3
1
MHz, DMSO) δ 181.02, 175.75, 168.06, 153.89, 144.26, 143.40,
1
1
36.37, 136.15, 134.13, 133.63, 129.52, 129.44, 129.17, 123.12,
14.71, 76.13, 68.50, 36.28, 25.04. HRMS (EI) m/z calcd for
8
1
6
1
1
1
3
C
21
H
14
O
3
N
5
F
3
S 473.0764, found: 473.0764.
4-(4-oxo-1-(1-oxoisochroman-6-yl)-2-thioxo-1,3-
diazaspiro[4.4]nonan-3-yl)-2-(trifluoromethyl)benzonitrile (33h):
It was obtained as a white solid. H NMR (400 MHz, DMSO-d6)
δ 8.40 (d, J = 8.3 Hz, 0H), 8.30 (s, 0H), 8.10 (dd, J = 8.0, 4.5 Hz,
1
7.48, 26.42. HRMS (EI) m/z calcd for C23
H
16
O
3
N
3
F
3
S 471.0859,
found: 471.0858.
1H), 7.53 (d, J = 10.4 Hz, 1H), 4.57 (t, J = 6.0 Hz, 1H), 3.16 (t, J

=
1
6.1 Hz, 1H), 2.38 – 2.28 (m, 1H), 2.18 (dt, J = 13.9, 6.8 Hz, 1H),
.72 (s, 1H), 1.37 (d, J = 27.7 Hz, 1H). 13C NMR (126 MHz,
DMSO) δ 180.49, 176.16, 164.19, 142.26, 141.05, 138.53, 136.62,
hydrochloride (210 mg, 1.5 mmol, 0.8 equiv) were dissolved in
DMSO under the protection of Argon, then the reaction was slowly
warmed to 120℃. The reaction mixture was stirred for 15 h and
TLC analysis indicated the reaction was completed. Then some
water was added to the solution, extracted with EtOAc. The
aqueous phase was collected, then 2N HCl was added to the
solution drop by drop to adjust PH to about 6. Then the solution
was extracted with EtOAc, the organic phase was washed with
1
1
34.38, 131.36, 129.80, 129.69, 128.44, 128.41, 126.08, 115.50,
09.01, 75.77, 67.73, 36.04, 27.35, 25.04. HRMS (EI) m/z calcd
for C24
H
18
O
3
N
3
F
3
S 485.1015, found: 485.1016.
5
-(4-oxo-1-(1-oxo-1,3-dihydroisobenzofuran-5-yl)-2-thioxo-
7
-oxa-1,3-diazaspiro[4.4]nonan-3-yl)-3-
2 4
saturated brine, dried over Na SO , filtered, concentrated for the
(trifluoromethyl)picolinonitrile (33i): It was obtained as a white
next step.
1
solid. H NMR (400 MHz, DMSO-d6) δ 9.24 (d, J = 2.0 Hz, 0H),
.78 (d, J = 2.1 Hz, 0H), 8.06 (d, J = 8.1 Hz, 0H), 7.87 (d, J = 1.6
Hz, 0H), 7.72 (dd, J = 8.1, 1.7 Hz, 0H), 4.42 (d, J = 10.6 Hz, 0H),
8
The collected compound was dissolved in anhydrous DMF,
then K CO (777 mg, 5.64 mmol, 3 equiv) and CH I (0.24 mL,
2
3
3
4
0
1
1
.01 (d, J = 10.5 Hz, 1H), 3.81 – 3.71 (m, 0H), 3.51 (q, J = 7.5 Hz,
3.76 mmol, 2 equiv) were added to the solution stirred at 30℃ for
4 h. Then the solution was extracted with EtOAc, the organic phase
was washed with saturated brine, dried over Na SO , filtered,
2 4
1
3
H), 2.70 – 2.53 (m, 1H). C NMR (126 MHz, DMSO) δ 180.55,
73.20, 170.15, 153.96, 149.14, 141.53, 136.04, 133.70, 131.47,
26.61, 126.21, 125.06, 114.70, 75.61, 74.81, 70.28, 68.20, 36.99.
concentrated and purified by silica gel chromatography (30%-50%
EtOAc/hexanes) to give the desired product. It was obtained as a
13 4 4 3
HRMS (EI) m/z calcd for C21H O N F S 474.0604, found:
4
1
74.0604.
light yellow solid 135 mg in 29.08% yield. H NMR (400 MHz,
DMSO-d6) δ 7.60 (s, 1H), 7.54 (d, J = 8.4 Hz, 1H), 6.74 (dd, J =
5
-(4-oxo-1-(1-oxo-1,3-dihydroisobenzofuran-5-yl)-2-thioxo-
8
=
.5, 2.0 Hz, 1H), 6.68 (d, J = 1.9 Hz, 1H), 5.22 (s, 2H), 3.59 (d, J
1.1 Hz, 2H), 1.55 (d, J = 4.5 Hz, 2H), 1.14 (d, J = 4.3 Hz, 2H).
1
(
,3-diazaspiro[4.4]nonan-3-yl)-3-(trifluoromethyl)picolinonitrile
1
33j): It was obtained as a light yellow solid. H NMR (400 MHz,
DMSO-d6) δ 9.27 (d, J = 2.1 Hz, 0H), 8.83 (d, J = 2.1 Hz, 0H),
.07 (d, J = 8.1 Hz, 0H), 7.82 (dd, J = 1.7, 0.8 Hz, 1H), 7.67 (dd, J
8.1, 1.7 Hz, 1H), 5.53 (s, 1H), 2.36 (dt, J = 13.6, 6.6 Hz, 1H),
.25 – 2.14 (m, 1H), 1.73 (q, J = 6.4 Hz, 1H), 1.38 (dt, J = 18.3,
.7 Hz, 1H). C NMR (126 MHz, DMSO) δ 180.22, 175.89,
70.15, 154.11, 149.26, 141.48, 136.21, 133.75, 131.75, 126.67,
26.21, 125.34, 114.72, 76.07, 70.30, 36.10, 25.01. HRMS (EI)
4.1.26. Synthesis of 4-(7-oxo-4-(1-oxo-1,3-
dihydroisobenzofuran-5-yl)-5-thioxo-4,6-
diazaspiro[2.4]heptan-6-yl)-2-
8
=
2
9
1
1
(
trifluoromethyl)benzonitrile (36)
1
3
Methyl 1-((1-oxo-1,3-dihydroisobenzofuran-5-
yl)amino)cyclopropanecarboxylate (35) (100 mg, 0.4 mmol, 1
equiv) was dissolved in anhydrous THF stirred at -78℃，then 1M
LiHMDS in THF (0.4 mL, 0.4 mmol, 1 equiv) was added to the
solution drop by drop until completed. The mixture was stirred at
m/z calcd for C22
15 3 4 3
H O N F S 472.0811, found: 472.0807.
5
-(4-oxo-1-(1-oxo-1,3-dihydroisobenzofuran-5-yl)-2-thioxo-
-78℃ for 0.5h, then EN-2 (111 mg, 0.486 mmol, 1.2 equiv) which
1
(
,3-diazaspiro[4.5]decan-3-yl)-3-(trifluoromethyl)picolinonitrile
33k): It was obtained as a white solid. H NMR (400 MHz,
DMSO-d6) δ 9.27 (d, J = 2.0 Hz, 1H), 8.84 (d, J = 2.1 Hz, 1H),
.07 (d, J = 8.1 Hz, 1H), 7.69 (d, J = 1.6 Hz, 1H), 7.54 (dd, J = 8.1,
.7 Hz, 1H), 5.52 (s, 2H), 2.43 (d, J = 12.5 Hz, 2H), 1.99 – 1.84
m, 3H), 1.57 (ddd, J = 29.8, 13.9, 4.5 Hz, 6H). C NMR (126
MHz, DMSO) δ 180.03, 173.67, 170.16, 154.34, 149.19, 140.46,
was dissolved in THF was added to the solution. Then the reaction
mixture was stirred at room temperature for 1 h and TLC analysis
indicated the reaction was completed. Then the solution was
purified by silica gel chromatography (30−60% EtOAc/hexanes)
to give the desired product. It was obtained as a white solid 57 mg
1
8
1
(
1
3
1
in 31.78% yield. H NMR (400 MHz, DMSO-d6) δ 8.42 (d, J = 8.3
Hz, 1H), 8.31 (d, J = 1.9 Hz, 1H), 8.11 (dd, J = 8.2, 1.9 Hz, 1H),
1
7
C
36.43, 133.61, 132.23, 129.39, 126.59, 126.32, 125.91, 114.73,
0.31, 68.29, 32.03, 24.03, 20.86. HRMS (EI) m/z calcd for
8
1
2
.04 (d, J = 8.1 Hz, 1H), 7.73 (d, J = 1.6 Hz, 1H), 7.64 (dd, J = 8.1,
.7 Hz, 1H), 5.50 (s, 2H), 1.65 – 1.58 (m, 2H), 1.47 – 1.41 (m,
H). C NMR (126 MHz, DMSO) δ 179.83, 171.86, 169.69,
23
H
17
O
3
N
F
4 3
S 486.0968, found: 486.0966.
1
3
4
.1.24. Synthesis of 5-bromoisobenzofuran-1(3H)-
148.81, 139.27, 138.16, 136.34, 133.79, 130.73, 127.62, 126.28,
125.75, 124.41, 114.74, 108.36, 70.01, 49.67, 14.12. HRMS (EI)
one (34)
m/z calcd for C21
.2. Molecular Docking
All docking procedures were completed with programs as
12 3 3 3
H O N F S 443.0546, found: 443.0552.
2
9a (2 g, 13.41 mmol, 1 equiv) was dissolved in 3 mL 47%HBr
2
solution, stirred for a few minutes at ice bath, then NaNO (1.02 g,
4.74 mmol, 1.1 equiv) dissolved in some water was added drop
by drop. 1h later, CuBr (2.11 g, 14.74 mmol, 1.1 equiv) which was
dissolved in 3 mL 47%HBr solution was added to the solution drop
by drop, then reaction mixture was stirred at room temperature for
4
1
implemented in Schrodinger Suite.
The crystal structure of androgen receptor (PDB: 2OZ7) was
prepared in Protein Preparation Wizard with all water molecules
deleted and bond orders assigned. The 2D structures of 31c and
Enzalutamide subjected to LigPrep and possible tautomeric states
at pH 7.0 ± 2.0 were generated using Epik. Induced-fit docking
was performed using the program Induced Fit and briefly consisted
of several steps: first, the initial docking using Glide, and then
sampling and minimization of sidechain residues within 5 Å of the
docked ligand using Prime, followed by re-docking using Glide.
In the initial docking, the receptor van der Waals radii scaling was
set at 0.50 and the ligand van der Waals radii was scaled at 0.5 to
soften the potentials. Finally, the binding energy for each output
pose were estimated as IFDScore.
3
h and TLC analysis indicated the reaction was completed. Then
the solution was extracted with EtOAc, the organic phase was
washed with saturated brine, dried over Na SO , filtered,
2
4
concentrated and purified by silica gel chromatography (0-20%
EtOAc/hexanes) to give the desired product. It was obtained as a
pink solid 1.38 g in 48.31% yield. H NMR (400 MHz,
Chloroform-d) δ 7.81 (d, J = 8.5 Hz, 1H), 7.70 (dtd, J = 4.8, 1.7,
0
1
.8 Hz, 2H), 5.33 (s, 2H).
4
.1.25. Synthesis
of
Methyl
1-((1-oxo-1,3-
dihydroisobenzofuran-5-
yl)amino)cyclopropanecarboxylate (35)
-bromoisobenzofuran-1(3H)-one (34) (400 mg, 1.88 mmol, 1
equiv), 1-aminocyclopropane -1-carboxylic acid (380 mg, 3.76
mmol, 2 equiv), K CO (777 mg, 5.64 mmol, 3 equiv), CuI (215
mg, 1.12 mmol, 0.6 equiv) and N,N-Dimethylglycine
5
4
.3. Cell culture and antiproliferative assays
2
3

LNCaP/AR cells were cultured in RPMI-1640 supplemented
with 10% Fetal Bovine Serum (FBS) and 1% Pen-Step-Solution
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3
.
(
PSS) at 37℃ in a 5% CO
culture were plated into 96-well plates at 3000 cells per well in
00ul and incubated for 24 h. After 24 h, an equal volume of
2
humidified Incubator. Cultivated
1
0.2174/1389450111314040002
1
4.
Yamaguchi N, Morizane S, Yumioka T, Iwamoto H, Hikita K,
Sejima T, Honda M, Takenaka A. Flutamide as an Alternative
Anti-androgen Agent and Predictor of the Efficacy of Novel
Androgen Receptor-targeted Agents. Anticancer Res. 2019,
medium containing various concentrations of compounds was
added to each well (9-point and 3 replicate wells per
concentration). Cells were treated for 6 days and then 100 ul
CellTiter-Glo was added to each well. The reagents were mixed on
a decolorizing shaker for 2 minutes to induce cell lysis. The culture
plate was incubated at room temperature for 10 minutes to stabilize
the fluorescence signal value. Finally, the chemiluminescence
signal value was measured in an automatic microplate reader. The
proliferation inhibition rate was calculated. Inhibition rate (%) =
3
9(7), 3879-3885.DOI:10.21873/anticanres.13538
5
.
Koukourakis MI, Kakouratos C, Kalamida D, Mitrakas A,
Pouliliou S, Xanthopoulou E, Papadopoulou E, Fasoulaki V,
Giatromanolaki A. Comparison of the effect of the antiandrogen
apalutamide (ARN-509) versus bicalutamide on the androgen
receptor pathway in prostate cancer cell lines. Anticancer Drugs.
2
018, 29(4), 323-333.DOI:10.1097/CAD.0000000000000592
6
.
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Nilutamide in Patients with Metastatic Prostate Cancer who
Underwent Orchiectomy: A Systematic Review and
Metaanalysis. Curr Clin Pharmacol. 2019, 14(2), 108-
(Lum negative control-Lum test product)/ (Lum negative control-
Lum blank control) *100 GraphPad Prism 6 software was used for
drawing, and SPSS software calculated IC50 (half the inhibitory
concentration).
1
15.DOI:10.2174/1574884714666190112151202
7
8
9
.
.
.
Crawford E D , Deantoni E P . Current status of combined
androgen blockade: optimal therapy for advanced prostate
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LNCaP cells were cultured in RPMI-1640 supplemented with
1
2
0% Fetal Bovine Serum (FBS) at 37℃ in a 5% CO humidified
1
995, 80(4), 1062-1066. DOI: 10.1210/jcem.80.4.7714067
incubator. Cells were trypsinized and diluted to 37,500 cells/mL
with medium. This cell suspension was seeded in 384-well plates
at a volume of 40 uL (1,500 cells/well), then the plate was
incubated at 37℃ under 5% CO for 24 h. A 10uL aliquot of drug
2
solution, which supplemented with 4-fold serial dilutions of
Dalesio O . Maximum androgen blockade in advanced prostate
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Linja M J , Savinainen K J , Saramki O R , et al. Amplification
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compounds or DMSO, was added to each well, and incubated at
3
2
7℃ in a CO humidified incubator for 6 days. A 25 uL aliquot of
CellTiter-Glo was added into each well of the plate, shaked the
plate at room temperature for 10 minutes, and read the plateon
Envision. IC50 is defined as the drug concentration causing 50%
cell growth inhibition, determined by dose-response curves.
10. Edwards J , Krishna N S , Grigor K M , Bartlett J M. Androgen
receptor gene amplification and protein expression in hormone
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1
0.1038/sj.bjc.6601127
1
1
1. Grasso, C., Wu, YM., Robinson, D. et al. The mutational
landscape of lethal castration-resistant prostate cancer. Nature,
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.4. Cell culture and AR antagonistic activity assay
2
012, 487, 239–243. https://doi.org/10.1038/nature11125
2. Takahito, Hara, Jun-ichi, Miyazaki, Hideo, Araki, et al. Novel
Mutations of Androgen Receptor A Possible Mechanism of
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HEK293 cells were cultured in DMEM supplemented with 10%
Fetal Bovine Serum (FBS) at 37℃ in a 5% CO humidified
2
incubator. Cells were trypsinized and counted. Then 10uL/well
transfection solution was prepared with opti-MEM containing 5 ng
androgen receptor clone, 100 ng pGL4.36 vector and 315 nL
fugene. Cell suspension was diluted with DMEM without phenol
red supplemented with 10% Dialyzed FBS and 1% GlutaMax, and
mixed with transfection solution to reach 444,444 cells/mL. This
diluted cell suspension was seeded in 96-well plates at a volume
of 90 uL (40,000 cells/well), and then the plate was incubated for
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5. DOI: 10.1002/jcb.10683
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K , Mulders P ,et al. Increased survival with enzalutamide in
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Medicine, 2012, 367, 1187–1197. DOI:
2
4 h. A 10 uL of drug solution, which supplemented with serial
dilutions of compounds or DMSO, was added. Then the plate was
incubated for 30 mins. 10 nL of DHT (1.5 nM final) was added to
each well of the plate, then the plate was incubated at 37℃ under
1
0.1056/NEJMoa1207506
16. Smith M R , Saad F , Chowdhury S , et al. Apalutamide Treatment
and Metastasis-free Survival in Prostate Cancer.[J]. New England
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5
2
% CO . After 24 h of incubation, 100 uL of Steady-Glo was
1
0.1056/NEJMoa1715546
added into each well of the plate, shaked the plate at room
temperature for 20 minutes, and read the plate on Envision. IC50 is
defined as the drug concentration causing 50% luciferase
expression inhibition, determined by dose-response curves.
1
7. Brave M , Weinstock C , Brewer J R , et al. An FDA Review of
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clincanres.3835.2019. DOI: 10.1158/1078-0432.CCR-19-3835
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metastatic prostate cancer before chemotherapy[J]. New England
1
Acknowledgments
Journal of Medicine, 2014, 371(5), 424-433. DOI：
1
0.1056/NEJMoa1405095
We gratefully acknowledge the financial support from National
Natural Science Foundation of China (82073684).
1
2
9. Korpal M , Korn J M , Gao X , et al. An F876L Mutation in
Androgen Receptor Confers Genetic and Phenotypic Resistance
to MDV3100 (Enzalutamide)[J]. Cancer Discovery, 2013,
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M. Molecular basis of agonism and antagonism in the oestrogen
receptor. Nature. 1997, 389, 753–758. DOI: 10.1038/39645
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Agard D A , Greene G L. The Structural Basis of Estrogen
Receptor/Coactivator Recognition and the Antagonism of This
Interaction by Tamoxifen. Cell. 1998, 95, 927–937. DOI:
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Structures of Antagonistic and Agonistic Forms of the
Glucocorticoid Receptor Ligand-binding Domain RU-486
INDUCES A TRANSCONFORMATION THAT LEADS TO
ACTIVE ANTAGONISM[J]. Journal of Biological Chemistry,
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4. Michael, E, Jung, et al. Structure−Activity Relationship for
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https://doi.org/10.1021/jm901488g
Supplementary Material
1
13
Synthesis of intermediate 29b-29d. H NMR, C NMR data of
all target compounds

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Design, Synthesis and Biological Evaluation of Novel Thiohydantoin Derivatives as Potent Androgen
Receptor Antagonists for the Treatment of Prostate Cancer
a, b
c
a
a, b,c, 
Ao Wang , Yawan Wang , Xin Meng and Yushe Yang
a
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,
55 Zuchongzhi Road, Shanghai 201203, P. R. China
School of Pharmacy, University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049, P. R.
5
b
China
c
School of Chinese Materia Medica, Nanjing University of Chinese Medicine, 138 Xianlin Avenue, Nanjing
10023, P. R. China
2
NC
NC
F
S
S
O
O
Fused ring
F3C
N
F3C
N
N
N
N
O
H
O
O
3
1c
Enzalutamide(4)
AR antagonist effect: IC50:454.6nM
IC50:195.5nM
2.3-fold improvement

Declaration of interests
The authors declare that they have no known competing
☒
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
☐
The authors declare the following financial
interests/personal relationships which may be
considered as potential competing interests: