Y06014 is a selective BET inhibitor for the treatment of prostate cancer

Article abstract of DOI:10.1038/s41401-021-00614-7

Bromodomain and extra-terminal proteins (BETs) are potential targets for the therapeutic treatment of prostate cancer (PC). Herein, we report the design, the synthesis, and a structure?activity relationship study of 6-(3,5-dimethylisoxazol-4-yl)benzo[cd]indol-2(1H)-one derivative as novel selective BET inhibitors. One representative compound, 19 (Y06014), bound to BRD4(1) in the low micromolar range and demonstrated high selectivity for BRD4(1) over other non-BET bromodomain-containing proteins. This molecule also potently inhibited cell growth, colony formation, and mRNA expression of AR-regulated genes in PC cell lines. Y06014 also shows stronger activity than the second-generation antiandrogen enzalutamide. Y06014 may serve as a new small molecule probe for further validation of BET as a molecular target for PC drug development.

Full text of DOI:10.1038/s41401-021-00614-7

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ARTICLE
Y06014 is a selective BET inhibitor for the treatment
of prostate cancer
1,2
2
2,3
2
2
4
2
2
Tian-bang Wu , Qiu-ping Xiang , Chao Wang , Chun Wu , Cheng Zhang , Mao-feng Zhang , Zhao-xuan Liu , Yan Zhang ,
1
2,5,6
Lin-jiu Xiao and Yong Xu
Bromodomain and extra-terminal proteins (BETs) are potential targets for the therapeutic treatment of prostate cancer (PC). Herein,
we report the design, the synthesis, and a structure−activity relationship study of 6-(3,5-dimethylisoxazol-4-yl)benzo[cd]indol-2
(
1H)-one derivative as novel selective BET inhibitors. One representative compound, 19 (Y06014), bound to BRD4(1) in the low
micromolar range and demonstrated high selectivity for BRD4(1) over other non-BET bromodomain-containing proteins. This
molecule also potently inhibited cell growth, colony formation, and mRNA expression of AR-regulated genes in PC cell lines. Y06014
also shows stronger activity than the second-generation antiandrogen enzalutamide. Y06014 may serve as a new small molecule
probe for further validation of BET as a molecular target for PC drug development.
Keywords: prostate cancer; bromodomain inhibitor; BRD4; Y06014; androgen receptor
Acta Pharmacologica Sinica (2021) 0:1–12; https://doi.org/10.1038/s41401-021-00614-7
INTRODUCTION
abrogation of AR-mediated transcription and inhibition of tumor
growth in CRPC xenograft mouse models [8, 15–18]. These studies
revealed that inhibition of BET proteins represents a promising
approach to block AR signaling and overcome antiandrogen
resistance. Thus, targeting BET proteins is an alternative strategy
for the treatment of CRPC.
Prostate cancer (PC) is one of the most common malignancies
diagnosed among men [1, 2]. Since Huggins and Hodges’s
discovery in the 1940s that androgens promote PC growth [3],
the central therapy for patients with locally advanced or
metastatic disease is targeting the androgen receptor (AR). The
standard first-line treatment for metastatic PC is androgen
deprivation therapy (ADT), which can induce marked tumor
regression and normalize serum prostate-specific antigen (PSA).
Initially, patients are sensitive to ADT. However, most patients
become insensitive to these therapies after long-term treatment
and eventually progress to castration-resistant prostate cancer
A number of small molecules BET inhibitors have been reported
to date [19–27] (Fig. 1). JQ1 (1) was the first reported BET
bromodomain inhibitor. This discovery motivated drug develop-
ment efforts in this area. Since then, several BET inhibitors have
been discovered, such as I-BET-762 (2) and I-BET-151 (3) [23, 24].
Recently, our group reported several selective BET inhibitors, such
as Y02224, Y02234, Y06137, and Y08060 [16–18, 28].
However, compounds that have entered clinical trials for CRPC
are still limited. Several BET inhibitors, including I-BET-762, OTX-
015, ZEN003694, and GS-5829, have recently advanced into
human clinical trials for CRPC as a single agent or in combination
with AR antagonists (enzalutamide or ARN-509) [29–32]. Despite
the discovery of these BET inhibitors, finding new, potent, and
specific BET inhibitors with different chemotypes is still highly
desirable to exploit the therapeutic potential of BET inhibition for
CRPC.
(
CRPC), which is predominantly driven by deregulated AR
signaling [4–7]. Unfortunately, most patients who develop CRPC
succumb to the disease [8]. Several drugs targeting AR signaling
against CRPC such as abiraterone acetate, enzalutamide, and
darolutamide have been approved by the FDA. However,
secondary resistance has been observed in patients [9–12]. Taken
together, these data strongly argue for the development of new
strategies to block AR function in CRPC.
The bromodomain and extra-terminal (BET) family is an
important class of epigenetic readers [13]. It consists of
bromodomain-containing protein 2 (BRD2), BRD3, BRD4, and
BRDT and recognizes acetylated histones by tandem domains
In this study, we report that Y06014, a benzo[cd]indol-2(1H)-one
derivative, binds to the BRD4 bromodomain. We found that
Y06014 could inhibit cell growth, proliferation, and colony
formation of PC cell lines. Y06014 also reduces the expression of
the AR-regulated genes PSA, KLK2, and TMPRSS2. Overall, Y06014
(
BD1 and BD2) to regulate gene transcription [14]. Moreover,
BRD2/3/4 can bind to the N-terminal domain of AR directly and
this interaction can be disrupted by BET inhibitors, resulting in
1
College of Science, Key Laboratory of Rare-earth Chemistry and Applying of Liaoning Province, Shenyang University of Chemical Technology, Shenyang 110142, China;
Guangdong Provincial Key Laboratory of Biocomputing, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences,
2
3
4
Guangzhou Medical University, Guangzhou 510530, China; University of Chinese Academy of Sciences, Beijing 100049, China; College of Pharmacy, Taizhou Polytechnic
5
6
College, Taizhou 225300, China; Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory), Guangzhou 510005, China and State Key
Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
Correspondence: Yan Zhang (zhang_yan2012@gibh.ac.cn) or Lin-jiu Xiao (x109@163.com) or Yong Xu (xu_yong@gibh.ac.cn)
These authors contributed equally: Tian-bang Wu, Qiu-ping Xiang, Chao Wang
Received: 23 November 2020 Accepted: 13 January 2021
©
The Author(s), under exclusive licence to CPS and SIMM 2021

Novel BET inhibitors against prostate cancer
TB Wu et al.
2
Fig. 1 Structures of representative BET bromodomain inhibitors.
represents a new class of compounds to develop BET inhibitors
against PC.
chromatography (HPLC) analysis to be >95%. HPLC instrument,
Dionex Summit HPLC (column: Diamonsil C18, 5.0 μm, 4.6 × 250
mm (Dikma Technologies); detector, PDA-100 photodiode array;
injector, ASI-100 autoinjector; pump, p-680A). A flow rate of 1.0
MATERIALS AND METHODS
Computational methods
mL/min was used with a mobile phase of MeOH in H
85:15).
2
O (v/v,
In the molecular docking study, the crystal structure of BRD4(1) in
complex with the inhibitor Y02234 (PDB ID: 5YQX) [17] was used
as the reference structure. Protein structure preparation for
docking studies includes water deletion, hydrogen atom addition,
and protonation state adjustment. All of the ligand and
protein preparation were performed in Maestro (version 9.4,
Schrödinger, LLC, New York, NY, 2013) implemented in the
1,3-Dioxo-1H-benzo[de]isoquinolin-2(3H)-yl 4-methylbenzene-
sulfonate (8)
Benzo[de]isochromene-1,3-dione (7) (6 g, 30 mmol) and hydro-
xylamine hydrochloride (2 g, 30 mmol) were combined as a
solution in pyridine (40 mL). The reaction was conducted under
reflux for 1 h followed by cooling to 80 °C. To the reaction, the
system mixture was added powdered p-toluenesulfonyl chlor-
ide (11.5 g, 60 mmol). After the addition, the reaction was
performed under reflux for 1 h. After cooling to room temperature,
the reaction mixture was poured into ice water (300 mL)
and stirred to precipitate crystals. The precipitate was filtered
and rinsed with additional cool water (100 mL) and saturated
Schrödinger program (http://www.schro
a molecular docking study was performed with the Glide program
version 6.1, Schrödinger, LLC, New York, NY, 2013) using the SP
̈
dinger.com). In this study,
(
score mode.
General chemistry
All reagents and solvents were obtained from commercial
sources and used without further purification. Flash chromato-
graphy was performed using silica gel (200–300 mesh). All
reactions were monitored by TLC, using silica gel plates with
3
NaHCO (100 mL) to give the title compound (8.6 g, 78%) as a
yellow solid.
Benzo[cd]indol-2(1H)-one (9)
1
13
fluorescence F254 and UV light visualization. H NMR and
NMR spectra were recorded on a Bruker AV-400 spectrometer at
00 MHz and the Bruker AV-500 spectrometer at 125 MHz.
C
To a solution of compound 8 (8.6 g, 23.5 mmol) in ethanol (50 mL)
and water (40 mL) was added an aqueous solution of sodium
hydroxide (2.7 mol/L, 30 mL) at room temperature. The mixture
was heated to reflux temperature for 3 h while distilling the
ethanol. After the reaction was completed, the reaction mixture
was cooled to 75 °C, concentrated hydrochloric acid was added
dropwise, and a yellow precipitate was formed. Then, the mixture
was further cooled. The precipitate was collected by filtration and
washed with water (100 mL × 2). The resulting crude product was
purified by silica gel chromatography with dichloromethane
4
Coupling constants (J) are expressed in hertz (Hz). Chemical
shifts (δ) of NMR are reported in parts per million (ppm) units
relative to internal control (TMS). Low-resolution ESI-MS were
recorded on an Agilent 1200 HPLC-MSD mass spectrometer and
high-resolution ESI-MS on an Applied Biosystems Q-STAR Elite
ESI-LC-MS/MS mass spectrometer. The purity of compounds was
determined by reverse-phase high-performance liquid
Acta Pharmacologica Sinica (2021) 0:1 – 12

Novel BET inhibitors against prostate cancer
TB Wu et al.
3
1
(
DCM) to give the title compound (3.3 g, 83%) as a yellow solid. H
NMR (400 MHz, DMSO-d ) δ 10.75 (s, 1H), 8.16 (d, J = 8.0 Hz, 1H),
.01 (d, J = 6.8 Hz, 1H), 7.78 (t, J = 7.6 Hz, 1H), 7.65 (d, J = 8.4 Hz,
1-((2,4-Difluorophenyl)sulfonyl)-6-(3,5-dimethylisoxazol-4-yl)benzo
[cd]indol-2(1H)-one (16) 1
6
8
1
Yellow solid, 45% yield. H NMR (400 MHz, CDCl ) δ 8.42 (m, 1H),
3
H), 7.48 (t, J = 7.2 Hz, 1H), 6.98 (d, J = 7.2 Hz, 1H).
8.12 (d, J = 7.0 Hz, 1H), 7.99 (d, J = 7.6 Hz, 1H), 7.88 (d, J = 8.1 Hz,
1
H), 7.78 (dd, J = 8.2, 7.1 Hz, 1H), 7.46 (d, J = 7.6 Hz, 1H), 7.13 (ddd,
6
-Bromobenzo[cd]indol-2(1H)-one (10)
J = 9.0, 7.9, 1.4 Hz, 1H), 6.89 (ddd, J = 10.5, 8.4, 2.4 Hz, 1H), 2.33 (s,
13
Benz[c,d]indol-2(1H)-one (3.38 g, 20 mmol) was dissolved in DCM
50 mL). Bromine (3.2 g, 20 mmol) was added to the solution and
3H), 2.16 (s, 3H). C NMR (125 MHz, CDCl ) δ 167.96, 166.73,
3
(
165.89, 164.61, 161.57, 159.49, 134.87, 134.20, 134.12, 131.03,
129.32, 129.09, 126.50, 126.02, 123.88, 123.82, 122.87, 113.11,
112.32, 112.18, 111.54, 105.99, 105.78, 105.59, 11.61, 10.65. MS
stirred at room temperature for 24 h. A saturated sodium
thiosulfate aqueous solution (30 mL) was poured into the reaction
mixture. The resulting precipitate was filtered off and washed with
+
(ESI) m/z for C H F N O S [M + H] , calcd: 440.06; found 441.6.
2
2 14 2 2 4
1
DCM to give the title compound a yellow solid (4.0 g, 80%). H
R
HPLC, t = 26.73 min, 95.40% purity.
6
NMR (400 MHz, DMSO-d ) δ 10.88 (s, 1H), 8.11 (d, J = 6.8 Hz, 1H),
8
.09 (d, J = 5.2 Hz, 1H), 7.93 (t, J = 6.0 Hz, 1H), 7.50 (d, J = 6.4 Hz,
6-(3,5-Dimethylisoxazol-4-yl)-1-(phenylsulfonyl)benzo[cd]indol-2
(1H)-one (14)
1H), 7.48 (t, J = 7.2 Hz, 1H), 6.90 (d, J = 6.0 Hz, 1H).
To a stirred solution of 6-(3,5-dimethylisoxazol-4-yl)benzo[cd]indol-
2(1H)-one (11) (100 mg, 0.38 mmol) in DMF (5 mL) was added NaH
(45 mg, 1.14 mmol) at 0 °C. One hour later, Benzenesulfonyl
chloride (100 mg, 0.57 mmol) was added to the solution and
the reaction mixture was stirred at 75 °C for 2 h. Water was added,
the aqueous layer was extracted with ethyl acetate (30 mL × 3), and
the combined organic layer was washed with water and brine,
6
-(3,5-Dimethylisoxazol-4-yl)benzo[cd]indol-2(1H)-one (11)
A mixture of 6-bromobenzo[cd]indol-2(1H)-one (10) (500 mg, 2.01
mmol), 3,5-dimethylisoxazole-4-boronic acid (426 mg, 3.02 mmol),
Pd (pph
DMF/H O = 30:1 was stirred under argon at 100 °C overnight. After
the reaction was complete, the reaction mixture was cooled to
room temperature. The reaction mixture was extracted with EtOAc
3 4 2 3
) (350 mg, 0.3 mmol), and K CO (834 mg, 6.03 mmol) in
2
2 4
dried with Na SO , and evaporated. The residue was purified by
(
50 mL × 3). The organic layer was washed with brine and dried
silica gel chromatography with petroleum ether/EtOAc (3/1, v/v) to
afford the title compound (60 mg, 40%) as a yellow solid. H NMR
1
over Na SO . The solid was filtered off, and the filtrate was
2
4
concentrated under reduced pressure. The resulting crude
product was purified by silica gel chromatography with petroleum
(500 MHz, CDCl ) δ 8.22 (d, J = 7.9 Hz, 2H), 8.12 (d, J = 7.0 Hz, 1H),
3
8.01 (d, J = 7.5 Hz, 1H), 7.83 (d, J = 8.2 Hz, 1H), 7.79 (m, 1H), 7.65 (t,
ether/EtOAc (3/1, v/v) to yield the title compound (170 mg, 32%)
J = 7.4 Hz, 1H), 7.56 (t, J = 7.8 Hz, 2H), 7.44 (d, J = 7.5 Hz, 1H), 2.31
1
13
as a yellow solid. H NMR (500 MHz, CDCl ) δ 8.73 (s, 1H), 8.15 (dd,
(s, 3H), 2.14 (s, 3H). C NMR (125 MHz, CDCl ) δ 166.69, 164.79,
3
3
J = 5.5, 2.0 Hz, 1H), 7.81 (m, 2H), 7.30 (d, J = 7.2 Hz, 1H), 7.08 (d, J =
159.48, 138.47, 135.12, 134.37, 130.96, 130.74, 129.37, 129.25,
7
.2 Hz, 1H), 2.32 (s, 3H), 2.16 (s, 3H). MS (ESI) m/z for C16
H
12
N
2
O
2
129.06, 128.06, 126.50, 125.90, 124.29, 123.78, 113.12, 111.01, 11.59,
+
+
[
M + H] calcd. 264.28; found 265.8. HPLC, t
R
= 22.38 min, 99.00%
10.64. MS (ESI) m/z for C22
H
16
N
2
O
4
S [M + H] , calcd: 404.08; found
purity.
405.1. HPLC, t = 28.16 min, 98.03% purity.
R
1
-(Cyclopentylsulfonyl)-6-(3,5-dimethylisoxazol-4-yl)benzo[cd]
6-(3,5-Dimethylisoxazol-4-yl)-1-((4-fluorophenyl)sulfonyl)benzo[cd]
indol-2(1H)-one (15)
indol-2(1H)-one (12)
General procedure for syntheses of 12, 13, 16. To a stirred solution
of 6-(3,5-dimethylisoxazol-4-yl)benzo[cd]indol-2(1H)-one (11) (150
mg, 0.57 mmol) in DMF (5 mL) was added NaH (68 mg, 1.71 mmol)
at 0 °C. One hour later, cyclopentanesulfonyl chloride (144 mg,
General procedure for syntheses of 15, 17, and 18. To a stirred
solution of 6-(3,5-dimethylisoxazol-4-yl)benzo[cd]indol-2(1H)-
one (11) (90 mg, 0.34 mmol) in THF (10 mL) was added NaH
(45 mg, 1.42 mmol) at 0 °C. One hour later, 4-
fluorobenzenesulfonyl chloride (162 mg, 0.57 mmol) was added
to the solution and the reaction mixture was stirred at rt for 3 h.
Water was added, the aqueous layer was extracted with ethyl
acetate (30 mL × 3), and the combined organic layer was washed
0
.85 mmol) was added to the solution and the reaction mixture
was stirred at 0 °C for 3 h. Water was added, the aqueous layer was
extracted with ethyl acetate (30 mL × 3), and the combined
organic layer was washed with water and brine, dried with
Na
2
SO
4
, and evaporated. The residue was purified by silica gel
2 4
with water and brine, dried with Na SO , and evaporated. The
residue was purified by silica gel chromatography with
chromatography with petroleum ether/EtOAc (3/1, v/v) to afford
the title compound (101 mg, 45%) as a yellow solid. H NMR (500
MHz, CDCl ) δ 8.22 (d, J = 6.8 Hz, 1H), 7.84 (ddd, J = 15.2, 12.3, 7.5
Hz, 3H), 7.40 (d, J = 7.5 Hz, 1H), 4.42 (m, 1H), 2.32 (s, 3H), 2.26 (td,
J = 14.5, 7.2 Hz, 2H), 2.16 (s, 3H), 2.09 (dt, J = 13.1, 7.5 Hz, 2H), 1.96
1
petroleum ether/EtOAc (2/1, v/v) to afford the title compound
1
(60 mg, 42%) as a yellow solid. H NMR (500 MHz, CDCl ) δ 8.28
3
3
(m, 2H), 8.12 (d, J = 7.0 Hz, 1H), 7.99 (d, J = 7.5 Hz, 1H), 7.85 (d,
J = 8.2 Hz, 1H), 7.81 (m, 1H), 7.44 (d, J = 7.5 Hz, 1H), 7.22 (t, J =
13
13
(
1
1
m, 2H), 1.76 (m, 2H). C NMR (125 MHz, CDCl
3
) δ 166.69, 165.81,
59.50, 135.97, 131.00, 130.87, 129.39, 129.02, 126.31, 126.02,
24.33, 123.63, 113.13, 111.02, 63.41, 27.47, 25.93, 11.60, 10.64. MS
3
8.5 Hz, 2H), 2.31 (s, 3H), 2.14 (s, 3H). C NMR (125 MHz, CDCl ) δ
167.23, 166.72, 165.18, 164.82, 159.46, 134.98, 134.45, 131.18,
131.10, 130.96, 130.86, 129.43, 129.10, 126.52, 126.00, 124.19,
123.95, 116.68, 116.50, 113.08, 111.02, 11.58, 10.63. MS (ESI) m/z
+
(
ESI) m/z for C21
20 2 4
H N O S [M + H] , calcd: 396.11; found 396.9.
+
HPLC, t = 37.62 min, 96.11% purity.
for C H FN O S [M + H] , calcd: 422.07; found 423.4. HPLC,
R
22 15
2 4
tR = 33.17 min, 96.16% purity.
1
-(Cyclohexylsulfonyl)-6-(3,5-dimethylisoxazol-4-yl)benzo[cd]
indol-2(1H)-one (13)
Yellow solid, 86% yield. H NMR (500 MHz, CDCl ) δ 8.22 (d, J =
1-((5-Chloro-2-methoxyphenyl)sulfonyl)-6-(3,5-dimethylisoxazol-4-
1
yl)benzo[cd]indol-2(1H)-one (17)
Yellow solid, 56% yield. H NMR (500 MHz, CDCl
3
1
6
7
2
.9 Hz, 1H), 7.88 (d, J = 8.2 Hz, 1H), 7.82 (dd, J = 9.0, 7.4 Hz, 2H),
.39 (d, J = 7.5 Hz, 1H), 3.77 (tt, J = 12.1, 3.4 Hz, 1H), 2.32 (s, 3H),
.22 (d, J = 11.1 Hz, 2H), 2.15 (s, 3H), 1.91 (d, J = 13.6 Hz, 2H), 1.76
3
) δ 8.26 (d, J = 2.6
Hz, 1H), 8.12 (d, J = 6.9 Hz, 1H), 7.92 (d, J = 7.6 Hz, 1H), 7.86 (d, J =
8.2 Hz, 1H), 7.78 (dd, J = 8.2, 7.1 Hz, 1H), 7.53 (dd, J = 8.8, 2.6 Hz, 1H),
7.46 (d, J = 7.6 Hz, 1H), 6.86 (d, J = 8.9 Hz, 1H), 3.54 (s, 3H), 2.33 (s,
1
3
(
ddd, J = 24.6, 12.3, 3.1 Hz, 2H), 1.39 (m, 4H). C NMR (125 MHz,
13
CDCl ) δ 166.68, 165.77, 159.48, 136.10, 130.98, 130.88, 129.38,
3
3
3H), 2.16 (s, 3H). C NMR (125 MHz, CDCl ) δ 166.62, 164.67, 159.45,
1
2
29.03, 126.26, 126.04, 124.30, 123.62, 113.13, 111.14, 62.96,
156.01, 135.95, 135.82, 131.56, 130.86, 130.73, 129.24, 128.95, 127.53,
126.4, 125.93, 125.83, 124.16, 123.32, 113.60, 113.21, 111.48, 56.27,
5.72, 24.95, 24.88, 11.60, 10.64. MS (ESI) m/z for C H N O S
2
2 22 2 4
+
+
[
M + H] , calcd: 410.13; found 411.2. HPLC, t = 41.17 min,
11.63, 10.64. MS (ESI) m/z for C H ClN O S [M + H] , calcd: 468.05;
R
23 17
2 5
9
9.62% purity.
R
found 469.2. HPLC, t = 22.69 min, 98.69% purity.
Acta Pharmacologica Sinica (2021) 0:1 – 12

Novel BET inhibitors against prostate cancer
TB Wu et al.
4
1
-((5-Bromo-2-methoxyphenyl)sulfonyl)-6-(3,5-dimethylisoxazol-4-
3
CDCl ) δ 168.60, 167.61, 166.49, 159.67, 138.99, 130.41, 129.46,
yl)benzo[cd]indol-2(1H)-one (18)
Yellow solid, 41% yield. H NMR (500 MHz, CDCl ) δ 8.39 (d, J = 2.5
Hz, 1H), 8.12 (d, J = 7.0 Hz, 1H), 7.92 (d, J = 7.6 Hz, 1H), 7.86 (d, J =
129.26, 128.68, 126.41, 125.79, 125.08, 121.87, 113.56, 104.99,
1
+
52.62, 41.43, 11.56, 10.64.MS (ESI) m/z for C H N O [M + H] ,
3
19 16 2 4
calcd: 336.11; found 337.1. HPLC, t = 24.06 min, 97.27% purity.
R
8
.2 Hz, 1H), 7.78 (dd, J = 8.2, 7.1 Hz, 1H), 7.67 (dd, J = 8.8, 2.5 Hz,
H), 7.46 (d, J = 7.6 Hz, 1H), 6.80 (d, J = 8.8 Hz, 1H), 3.54 (s, 3H), 2.33
1
2-(6-(3,5-Dimethylisoxazol-4-yl)-2-oxobenzo[cd]indol-1(2H)-yl)
acetic acid (23)
13
(
s, 3H), 2.16 (s, 3H). C NMR (125 MHz, CDCl ) δ 166.61, 164.66,
3
1
1
1
59.45, 156.50, 138.74, 135.96, 134.31, 130.86, 130.72, 129.24,
28.95, 127.89, 126.42, 125.83, 124.17, 123.32, 114.00, 113.21,
Methyl 2-(6-(3,5-dimethylisoxazol-4-yl)-2-oxobenzo[cd]indol-1(2H)-
yl)acetate (150 mg, 0.45 mmol) was dissolved in MeOH (5 mL) and
2 mol/L NaOH aqueous solution (5 mL). The mixture was stirred at
room temperature for 2 h. The solvent was removed and diluted
hydrochloric acid was added dropwise, and a yellow precipitate
was formed. The precipitate was collected by filtration and
washed with water (10 mL × 2). The resulting crude product was
purified by recrystallization with petroleum and ether/ethyl
2 5
12.66, 111.48, 56.22, 11.63, 10.64. MS (ESI) m/z for C23H17BrN O S
+
[
M + H] , calcd: 512.00; found 513.1. HPLC, t = 30.81 min, 98.75%
R
purity.
6-(3,5-Dimethylisoxazol-4-yl)-1-ethylbenzo[cd]indol-2(1H)-one (19)
General procedure for the synthesis of 19, 20, and 21. To a stirred
solution of 6-(3,5-dimethylisoxazol-4-yl)benzo[cd]indol-2(1H)-one
acetate to afford the final product (100 mg, 70%) as a yellow
1
(
11) (80 mg, 0.3 mmol) in DMF (5 mL) was added NaH (35 mg, 0.9
solid. H NMR (400 MHz, DMSO-d
Hz, 1H), 7.86 (q, J = 8.0 Hz, 2H), 7.49 (d, J = 7.3 Hz, 1H), 7.29 (d, J =
7.3 Hz, 1H), 4.72 (s, 2H), 2.29 (s, 3H), 2.10 (s, 3H). C NMR (125 MHz,
CDCl ) δ 171.60, 167.95, 166.60, 159.70, 138.81, 130.45, 129.60,
6
) δ 13.12 (s, 1H), 8.15 (d, J = 6.2
mmol) at rt. 30 min later, chloroethane (71 mg, 0.45 mmol) was
added to the solution and the reaction mixture was stirred at rt for
13
3
h. Water was added, the aqueous layer was extracted with ethyl
3
acetate (20 mL × 3), and the combined organic layer was washed
with water and brine, dried with Na SO , and evaporated. The
residue was purified by silica gel chromatography with petroleum
129.34, 128.63, 126.23, 125.69, 125.32, 121.94, 113.61, 105.29,
+
41.36, 11.56, 10.61. MS (ESI) m/z for C H N O [M + H] , calcd:
2
4
18 14 2 4
R
322.10; found 323.1. HPLC, t = 14.59 min, 98.98% purity.
ether/EtOAc (2/1, v/v) to yield the desired product (80 mg, 90%) as
1
a yellow solid. H NMR (400 MHz, CDCl
3
) δ 8.13 (m, 1H), 7.72 (d,
6-(3,5-Dimethylisoxazol-4-yl)-1-(2-(pyrrolidin-1-yl)ethyl)benzo[cd]
indol-2(1H)-one (24)
J = 3.3 Hz, 2H), 7.30 (d, J = 7.2 Hz, 1H), 6.98 (d, J = 7.2 Hz, 1H), 4.01
(
q, J = 7.2 Hz, 2H), 2.31 (s, 3H), 2.15 (s, 3H), 1.41 (t, J = 7.2 Hz, 3H).
General procedure for the synthesis of 24–28. To a stirred solution
of 6-(3,5-dimethylisoxazol-4-yl)benzo[cd]indol-2(1H)-one (11) (100
mg, 0.38 mmol) in DMF (5 mL) was added NaH (46 mg, 1.14 mmol)
at 0 °C. 30 min later, 1-(2-chloroethyl)pyrrolidine (97 mg, 0.57
mmol) was added to the solution and the reaction mixture was
stirred at 100 °C overnight. Water was added, the aqueous layer
was extracted with ethyl acetate (20 mL × 3), and the combined
organic layer was washed with water and brine, dried with
1
3
3
C NMR (125 MHz, CDCl ) δ 167.58, 166.41, 159.71, 139.51, 130.45,
1
29.14, 128.98, 128.58, 127.31, 125.59, 124.56, 121.24, 113.68,
1
04.76, 35.04, 14.04, 11.56, 10.64. MS (ESI) m/z for C H N O
M + H] , calcd: 292.12; found 293.0. HPLC, t
18 16 2 2
+
[
R
= 21.95 min, 98.52%
purity.
6
-(3,5-Dimethylisoxazol-4-yl)-1-propylbenzo[cd]indol-2(1H)-one
20)
Yellow solid, 75% yield. H NMR (400 MHz, CDCl
.75 (m, 2H), 7.29 (d, J = 7.2 Hz, 1H), 6.97 (d, J = 7.2 Hz, 1H), 3.92 (t,
J = 7.2 Hz, 2H), 2.31 (s, 3H), 2.16 (s, 3H), 1.91 (m, 2H), 1.04 (t, J = 7.4
(
2 4
Na SO , and evaporated. The residue was purified by silica gel
1
3
) δ 8.13 (m, 1H),
chromatography with petroleum ether/EtOAc (1/1, v/v) to yield
1
7
the desired product (59 mg, 43%) as a yellow solid. H NMR (400
MHz, CDCl
3
) δ 8.13 (m, 1H), 7.72 (d, J = 3.6 Hz, 2H), 7.29 (d, J = 7.2
1
3
Hz, 3H). C NMR (125 MHz, CDCl
3
) δ 167.93, 166.41, 159.72,
39.97, 130.44, 129.13, 128.98, 128.53, 127.21, 125.51, 124.58,
21.19, 113.68, 104.94, 41.98, 22.13, 11.58, 11.51, 10.66. MS (ESI)
Hz, 1H), 7.03 (d, J = 7.2 Hz, 1H), 4.11 (t, J = 7.3 Hz, 2H), 2.89 (t, J =
7.4 Hz, 2H), 2.67 (s, 4H), 2.31 (s, 3H), 2.15 (s, 3H), 1.82 (s, 4H).
1
3
1
1
C
NMR (125 MHz, CDCl ) δ 167.80, 166.42, 159.71, 139.77, 130.48,
3
+
m/z for C19
= 25.79 min, 99.35% purity.
H
18
N
2
O
2
[M + H] , calcd: 306.14; found 307.1. HPLC,
129.13, 129.05, 128.57, 127.11, 125.60, 124.64, 121.30, 113.66,
t
R
105.02, 54.40, 54.26, 39.75, 23.59, 11.56, 10.64.MS (ESI) m/z for
+
C H N O [M + H] , calcd: 361.18; found 362.1. HPLC, t = 30.69
2
2
23
3
2
R
1
-Butyl-6-(3,5-dimethylisoxazol-4-yl)benzo[cd]indol-2(1H)-one (21)
min, 99.89% purity.
1
Yellow solid, 52% yield. H NMR (400 MHz, CDCl
.76 (m, 2H), 7.29 (d, J = 7.2 Hz, 1H), 6.97 (d, J = 7.2 Hz, 1H), 3.95 (t,
J = 7.2 Hz, 2H), 2.31 (s, 3H), 2.16 (s, 3H), 1.85 (m, 2H), 1.52 (m, 2H),
3
) δ 8.13 (m, 1H),
7
6-(3,5-Dimethylisoxazol-4-yl)-1-(2-morpholinoethyl)benzo[cd]
indol-2(1H)-one (25)
Yellow solid, 52% yield. H NMR (400 MHz, CDCl
1
3
1
0
1
1
1
3
.99 (t, J = 7.4 Hz, 3H). C NMR (125 MHz, CDCl
59.72, 139.94, 130.44, 129.13, 128.97, 128.53, 127.23, 125.53,
24.56, 121.19, 113.69, 104.91, 40.13, 30.91, 20.24, 13.78, 11.57,
0.65. MS (ESI) m/z for C H N O [M + H] , calcd: 320.15; found
21.1. HPLC, t
3
) δ 167.88, 166.41,
3
) δ 8.10 (d, J = 3.4
Hz, 1H), 7.73 (d, J = 3.3 Hz, 2H), 7.29 (d, J = 7.1 Hz, 1H), 7.00 (d, J =
7.2 Hz, 1H), 4.09 (t, J = 6.8 Hz, 2H), 3.70 (s, 4H), 2.76 (t, J = 6.8 Hz,
+
13
2H), 2.59 (s, 4H), 2.31 (s, 3H), 2.15 (s, 3H). C NMR (125 MHz, CDCl3)
2
0 20 2 2
R
= 29.68 min, 97.74% purity.
δ 167.83, 166.41, 159.67, 139.74, 130.39, 129.18, 129.10, 128.57,
1
27.05, 125.60, 124.67, 121.37, 113.63, 104.92, 66.96, 56.77, 53.84,
+
Methyl 2-(6-(3,5-dimethylisoxazol-4-yl)-2-oxobenzo[cd]indol-1(2H)-
yl)acetate (22)
37.81, 11.58, 10.65.MS (ESI) m/z for C H N O [M + H] , calcd:
377.17; found 378.1. HPLC, t = 29.17 min, 99.48% purity.
22 23 3 3
R
To a stirred solution of 6-(3,5-dimethylisoxazol-4-yl)benzo[cd]
indol-2(1H)-one (11) (200 mg, 0.76 mmol) in DMF (10 mL) was
added K CO (314 mg, 2.27 mmol) and iodoethane (174 mg, 1.14
mmol) at rt. The reaction mixture was stirred at 80 °C for 4 h. Water
was added, the aqueous layer was extracted with ethyl acetate
6-(3,5-Dimethylisoxazol-4-yl)-1-(3-morpholinopropyl)benzo[cd]
indol-2(1H)-one (26)
Yellow solid, 43% yield. H NMR (400 MHz, CDCl
2
3
1
3
) δ 8.13 (m, 1H),
7.73 (d, J = 3.6 Hz, 2H), 7.28 (d, J = 7.2 Hz, 1H), 7.02 (d, J = 7.3 Hz,
(
20 mL × 3), and the combined organic layer was washed with
1H), 4.03 (t, J = 6.8 Hz, 2H), 3.69 (m, 4H), 2.47 (t, J = 7.0 Hz, 2H), 2.42
13
water and brine, dried with Na SO , and evaporated. The residue
(s, 4H), 2.31 (s, 3H), 2.15 (s, 3H), 2.04 (m, 2H). C NMR (125 MHz,
CDCl ) δ 168.01, 166.41, 159.68, 139.95, 130.40, 129.18, 129.07,
2
4
was purified by silica gel chromatography with petroleum ether/
3
EtOAc (3/1, v/v) to yield the desired product (200 mg, 78%) as a
128.54, 127.18, 125.54, 124.60, 121.29, 113.64, 104.97, 66.91, 55.98,
1
yellow solid. H NMR (400 MHz, CDCl ) δ 8.15 (dd, J = 4.6, 2.9 Hz,
53.66, 38.48, 25.53, 11.57, 10.65.MS (ESI) m/z for C H N O [M +
3
23 25 3 3
+
1
4
H), 7.78 (m, 2H), 7.30 (d, J = 7.3 Hz, 1H), 6.91 (d, J = 7.3 Hz, 1H),
H] , calcd: 391.19; found 392.1. HPLC, t = 21.93 min, 95.72%
R
1
3
.73 (s, 2H), 3.81 (s, 3H), 2.31 (s, 3H), 2.15 (s, 3H). C NMR (125 MHz,
purity.
Acta Pharmacologica Sinica (2021) 0:1 – 12

Novel BET inhibitors against prostate cancer
TB Wu et al.
5
6
(
-(3,5-Dimethylisoxazol-4-yl)-1-(3-phenylpropyl)benzo[cd]indol-2
1H)-one (27)
Yellow solid, 69% yield. H NMR (400 MHz, DMSO-d ) δ 8.09 (dd, J
(5 μg/mL) in the presence or absence of the indicated amounts of
control compound (+)-JQ1 (1), or candidate compounds. The C-
terminal biotinylated tetra-acetylated histone peptide H4
(bH4KAc4) sequence was H-SGRGK(Ac)GGK(Ac)GLGK(Ac)GGAK
(Ac)RHRK-Biotin-OH (synthesized by Genscript). The experiment
was conducted with protein/peptide ratio as follows for sensitive
signal: BRD4(1): bH4KAc4 = 50 nM: 50 nM. The reagents were
diluted in the buffer (50 mM MOPS, pH 7.4, 50 mM NaF, 50 μM
CHAPS, and 0.1 mg/mL bovine serum albumin) and allowed to
equilibrate at room temperature prior to addition to low-volume
384-well plates (ProxiPlate-384 Plus, PerkinElmer, USA). Plates
were foil-sealed to protect from light, incubated at room
temperature for 2.5 h, and read on an EnSpire plate reader
(PerkinElmer, USA). When excited by a laser beam of 680 nm, the
donor beam emits singlet oxygen that activates thioxene
derivatives in the acceptor beads, which releases photons of
520–620 nm as the binding signal. All experiments were carried
out in triplicate on the same plate. The results were based on an
average of three experiments with standard errors typically less
than 10% of the measurements.
1
6
=
5.1, 2.4 Hz, 1H), 7.86 (m, 2H), 7.47 (d, J = 7.3 Hz, 1H), 7.24 (q, J =
6
.9 Hz, 5H), 7.15 (td, J = 6.2, 2.6 Hz, 1H), 3.96 (t, J = 7.1 Hz, 2H), 2.73
13
(
m, 2H), 2.29 (s, 3H), 2.09 (s, 3H), 2.04 (dd, J = 14.9, 7.4 Hz, 2H).
C
NMR (125 MHz, CDCl ) δ 167.92, 166.42, 159.70, 141.10, 139.72,
3
1
1
1
3
30.42, 129.16, 129.04, 128.54, 128.42, 128.32, 127.14, 126.04,
25.54, 124.63, 121.30, 113.66, 104.92, 39.96, 33.19, 30.15, 11.57,
0.65. MS (ESI) m/z for C25
83.2. HPLC, t = 36.10 min, 99.88% purity.
+
H
22
N
2
O
2
[M + H] , calcd: 382.17; found
R
6
-(3,5-Dimethylisoxazol-4-yl)-1-(1-phenylethyl)benzo[cd]indol-2
(
1H)-one (28)
1
Yellow solid, 64% yield. H NMR (400 MHz, CDCl ) δ 8.15 (dd, J =
3
6
.0, 1.5 Hz, 1H), 7.78 (m, 2H), 7.48 (d, J = 7.7 Hz, 2H), 7.36 (t, J = 7.5
Hz, 2H), 7.29 (t, J = 7.2 Hz, 1H), 7.08 (d, J = 7.3 Hz, 1H), 6.61 (d, J =
.3 Hz, 1H), 6.04 (q, J = 7.1 Hz, 1H), 2.27 (d, J = 4.4 Hz, 3H), 2.12 (d,
7
1
3
J = 3.4 Hz, 3H), 1.95 (d, J = 7.2 Hz, 3H). C NMR (125 MHz, CDCl ) δ
3
1
1
1
67.77, 166.41, 159.69, 140.11, 138.02, 130.33, 129.20, 129.00,
28.64, 128.61, 127.52, 126.96, 126.69, 125.84, 124.70, 121.12,
13.57, 107.40, 49.26, 17.50, 11.57, 10.66.MS (ESI) m/z for
Thermal shift assay (TSA)
+
C
24
H
20
N
2
O
2
[M + H] , calcd: 368.15; found 369.3. HPLC, t
R
=
TSAs were carried out using the Bio-Rad CFX96 Real-Time PCR
system. All reactions were buffered in 10 mM HEPES, pH 7.5, 150
mM NaCl at a final concentration of 10 μM proteins and 200 μM
compounds. The 20 μL reaction mix was added to the wells of the
96-well PCR plate. SYPRO Orange (ABI, Sigma) was added as a
fluorescence probe at a dilution of 1:1000 and incubated with
compounds on ice for 30 min. Total DMSO concentration was
restricted to 1% or less. Excitation and emission filters for the
SYPRO orange dye were set to 465 and 590 nm, respectively. The
temperature was raised with a step of 0.3 °C per minute from 30 to
75 °C, and fluorescence readings were taken at each (0.3 °C)
interval. All experiments were performed in triplicates. Melting
42.40 min, 99.35% purity.
Plasmid construction, protein expression, and purification
The bromodomains were expressed as a His6-fusion protein with a
TEV cleavage site between His6 tag and bromodomain using the
pET24a expression vector (Novagen). cDNA encoding bromodo-
main of human BRD4(1) (residues N44-E168), EP300 (residues
A1040-G1161), CREBBP (residues R1081-G1197), BAZ2B (residues
S1858-S1972), TIF(1) (residues G896-E1014), PCAF (residues G715-
D831), BRD9 (residues L14-Q134), and TAF1(1) (residues R1377-
D1503) were synthesized by Genscript. BL21 (DE3) cells trans-
formed with these expression plasmids were grown in LB broth at
temperatures (T
curve to the Boltzmann equation using GraphPad Prism. ΔT
m
) were calculated by fitting the sigmoidal melt
is
25 °C to an OD600 nm of approximately 1.0 and then induced with
0.1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) at 16 °C for
16 h. Cells were harvested by centrifugation (6000 × g for 15 min
m
the difference in Tm values calculated for reactions with and
without compounds.
at 4 °C, JLA 81000 rotor, on a Beckman Coulter Avanti J-20 XP
centrifuge) and were frozen at −80 °C as pellets for storage. Cells
were resuspended in extract buffer (50 mM HEPES, pH 7.5 at 25 °C,
Cell culture, cell viability, and cell colony formation assays
Prostate cell lines C4–2B, LNCaP, 22Rv1, Du145, and leukemia
cell line MV4;11 were cultured in RPMI-1640, plus 10% FBS. Cells
5
00 mM NaCl, 5 mM imidazole, 5% glycerol, and 0.5 mM TCEP (Tris
2-carboxyethyl)phosphine hydrochloride)) and high-pressure
homogenized using a JN3000 PLUS high-pressure homogenizer
JNBIO, Guangzhou, China) at 4 °C. The lysate was collected on ice
and centrifuged at 12000 × g for 40 min. The supernatant was
loaded onto a 5 mL NiSO -loaded HisTrap HP column (Ni-NTA, GE
(
2
were grown at 37 °C in 5% CO incubators. For cell viability, cells
were seeded in 384-well plates at 500–1000 cells per well
(optimum density for growth) in a total volume of 20 μL of
media. After 12 h, 10 μL chemical compounds with two fold
serial dilution was added in a final volume of 30 μL of media
each well with a final concentration from 5 nM to 100 μM. The
measurement was conducted 96 h after seeded for LNCaP,
C4–2B, and 22Rv1, 72 h after seeded for Du145, and 120 h after
seeded for MV4;11. Then, 25 μL of Cell-Titer GLO reagents
(Promega) was added, and luminescence was measured on
GLOMAX microplate luminometer (Promega), according to the
manufacturer’s instructions. The estimated in vitro half-maximal
inhibitory concentration (IC ) values were calculated using
(
4
Healthcare, NJ). The column was washed with 20 mL of extract
buffer (50 mM HEPES, pH 7.5 at 25 °C, 500 mM NaCl, 50 mM
imidazole). The protein was eluted with a 50–500 mM imidazole
gradient in elute buffer with 50 mM HEPES, pH 7.5 at 25 °C, 500
mM NaCl, 500 mM imidazole. The protein was concentrated and
further purified by a gel filtration column (HiLoad, Superdex 75,
16/60, GE Healthcare). The sample purity of each fraction was
examined by SDS-PAGE, and the sample concentration was
determined by Bradford assay. Purified proteins were concen-
trated and stored in the gel filtration buffer (10 mM Hepes pH 7.5
at 25 °C, 150 mM NaCl, 0.5 mM TCEP) and were used for
crystallization or stored at −80 °C for AlphaScreen or TSA assays.
5
0
GraphPad Prism 6 software.
For colony formation assay, 1500 cells per well were seeded in a
6-well plate for C4–2B, respectively. Cells were cultured with
vehicle or indicated concentrations of compounds for 14 days
with a 3 mL medium. When the cell colony grew visible, the
medium was removed and the plates were washed with 2 mL PBS
for one time. The cell colonies were stained with 2.5% crystal
violet (in MeOH) for 2 h. The plates were scanned with an HP
scanner.
AlphaScreen assay
Interactions between bromodomain-containing proteins (BCP)
and ligands were assessed by luminescence-based AlphaScreen
technology (Perkin Elmer) as previously described in refs. [17, 18]
using a histidine detection kit from PerkinElmer (Norwalk, CT). All
of the reactions contained bromodomain-containing protein
bound to nickel acceptor beads (5 μg/mL) and biotinylated
acetylated histone H4 peptide bound to streptavidin donor beads
RNA isolation and quantitative real-time PCR
22Rv1 cells were plated at 200,000 cells per well in 12-well plates.
Twenty-four hours later, cells were treated with vehicle or 19
Acta Pharmacologica Sinica (2021) 0:1 – 12

Novel BET inhibitors against prostate cancer
TB Wu et al.
6
Fig. 2 Binding mode analysis of 6, 14, and 19 in complex with the BRD4(1) protein. The crystal structure 5YQX from the PDB was used as a
template for molecular docking for compounds (a) 6, (b) 14, and (c) 19. All compounds are shown as sticks, and all residues are shown as lines.
Water molecules are shown as spheres. In addition, an electrostatic potential surface is shown. Hydrogen bonds are shown as yellow
dashed lines.
(
5 μM) for 48 h. Total RNA was then isolated with the Eastep®
Briefly, benzo[de]isochromene-1,3-dione (7) reacted with hydro-
xylamine hydrochloride and p-toluene sulfonic acid (PTSA) in the
presence of pyridine to produce compound 8, which was further
reacted with NaOH followed by HCl treatment to produce
intermediate 9. Direct bromination of 9 with bromine provided
fragment 10, which was followed by coupling with 3,5-dimethy-
lisoxazole-4-boronic acid under palladium catalysis to deliver 11.
The final sulfonamides (12–18) were synthesized from com-
pound 11 using the corresponding sulfonyl chlorides. For
compounds 19–22 and 24–28, the R group was introduced by
reaction with chloro-, bromo- or iodo-substituents. Compound 23
was synthesized by a simple hydrolysis reaction.
Super Total RNA Extraction Kit and cDNA was synthesized from
1
TM
000 ng total RNA using the All-in-one
First-Strand cDNA
Synthesis Kit. qPCRs were performed in triplicate using standard
SYBR green reagents on a Bio-Rad CFX96 real-time PCR system.
The analysis was performed on triplicate PCR data for each
biological duplicate (normalized to β-actin). The primer sequences
for qPCR used are as follows: AR-FL_qPCR_fwd, 5′-ACA TCA AGG
AAC TCG ATC GTA TCA TTG C-3′; AR-FL_qPCR_rev, 5′-TTG GGC ACT
TGC ACA GAG AT-3′; AR-V7_qPCR_fwd, 5′-CCA TCT TGT CGT CTT
CGG AAA TGT TAT GAA GC-3′; AR-V7_qPCR_rev, 5′-TTT GAA TGA
GGC AAG TCA GCC TTT CT-3′. PSA_fwd, 5′-CAC AGG CCA GGT ATT
TCA GGT-3′; PSA_rev, 5′-GAG GCT CAT ATC GTA GAG CGG-3′;
KLK2_fwd, 5′-CAA CAT CTG GAG GGG AAA GGG-3′; KLK2_rev, 5′-
AGG CCA AGT GAT GCC AGA AC-3′; TMPRSS2_fwd, 5′-CAA GTG
CTC CAA CTC TGG GAT-3′; TMPRSS2_rev, 5′-AAC ACA CCG ATT CTC
GTC CTC-3′; C-MYC_fwd, 5′-GGC TCC TGG CAA AAG GTC A-3′;
C-MYC_rev, 5′-CTG CGT AGT TGT GCT GAT GT-3′; β-Actin_fwd,
Structure-activity relationship (SAR) studies
To optimize the interactions with the WPF shelf, various
substituents at the R position of 6-(3,5-dimethylisoxazol-4-yl)
benzo[cd]indol-2(1H)-one were designed to explore the chemical
space for affinity improvement (Table 1). All synthesized
compounds were evaluated for their BRD4(1) inhibitory activities
via a TSA and an AlphaScreen assay.
5
′-GAG AAA ATC TGG CAC CAC ACC-3′; β-Actin_rev, 5′-ATA CCC
CTC GTA GAT GGG CAC-3′.
To enhance the hydrophobic interactions with residues Trp81,
Pro82, and Phe83 of the WPF shelf, cycloalkyl substituents were
introduced at the R positions, and compounds 12 and 13 were
synthesized (Table 1). Compounds 12 and 13 displayed BRD4(1)
inhibitory activity similar to that of 6 with temperature shifts of 1.8
and 1.2 °C in the TSA, respectively. Aromatic substituents were also
introduced to extend the SAR study. Replacement of the
cyclohexyl group in 13 with a phenyl group led to 14, which
exhibited a similar inhibitory effect to that of 13 (TSA: 1.2 °C vs
1.2 °C; inhibition rate: 28.6% vs 28.4% for 13 and 14, respectively).
The results suggested that cyclopentyl, cyclohexyl, and phenyl
groups have similar affinities for BRD4(1).
The predicted binding mode of compound 14 in complex
with BRD4(1) obviously showed that the phenyl group is
directly above the WPF hydrophobic area formed by the
hydrophobic residues Trp81, Pro82, Met149, and Ile146 (Fig. 2b).
Thus, various phenyl-substituted derivatives were designed and
synthesized for biological evaluation. The results revealed that
substitutions to different positions of the phenyl ring in 14 could
achieve diverse impacts on BRD4(1) bromodomain inhibitory
activity. As shown in Table 1, para-F-substituted derivative 15 had
no activity. However, when disubstituted derivatives were
designed and synthesized, improved potency was obtained. 2,4-
Difluorophenyl compound 16 exhibited improved activity with a
maximum inhibition of 18.9% at 20 μM. Compound 17 binds to
BRD4(1) with a temperature shift of 1.2 °C in the TSA and was
equipotent to 14. Further study showed that the meta-Cl group in
17 could be replaced by a bromo moiety (18) to display almost
RESULTS AND DISCUSSION
In-house library screening
To screen potent BET inhibitors, a TSA was performed on an in-
house chemical collection. Compound 6 with a 6-(3,5-dimethyli-
soxazol-4-yl)benzo[cd]indol-2(1H)-one scaffold was shown to have
a temperature shift of 2.1 °C. The predicted binding mode of 6
bound to BRD4(1) showed that the 3,5-dimethylisoxazole group
interacted with the conserved residues Asn140 and Tyr97 through
direct and water-mediated hydrogen bonds (Fig. 2a). The methyl
group of the 3,5-dimethylisoxazole group mimicked the terminal
methyl group of acetyl-lysine (KAc) and occupied the small pocket
surrounded by the residues Pro82, Phe83, Val87, and Ile146.
Moreover, the tricyclic aromatic core of compound 6 could
also fill the KAc binding pocket. The butylsulfonyl group of 6 was
able to interact with the WPF shelf (composed of Trp81, Pro82,
and Phe83) by means of hydrophobic interactions (Fig. 2a).
The carbonyl O could form hydrogen bonds with water,
contributing to its activity. These important interactions indicated
that 6 may serve as a good starting point for new BET inhibitors.
To develop compounds with improved affinity for BRD4, an
extensive structure−activity relationship investigation was
conducted.
Chemistry
The 6-(3,5-dimethylisoxazol-4-yl)benzo[cd]indol-2(1H)-one deriva-
tives were designed and synthesized as shown in Scheme 1.
Acta Pharmacologica Sinica (2021) 0:1 – 12

Novel BET inhibitors against prostate cancer
TB Wu et al.
7
4
Scheme 1 Synthesis of 6-(3,5-dimethylisoxazol-4-yl)benzo[cd]indol-2(1H)-one derivatives. Reagents and conditions: (a) NH OH·HCl,
pyridine, PTSA, 95 °C, 1 h, 78%; (b) (i) 2.7 mol/L NaOH, EtOH, H
dimethylisoxazole-4-boronic acid, Pd(PPh , K CO , DMF/H
0%–86%; (f) RI, NaH, DMF, rt, 3 h, 52%–90% or RX (X = Br, Cl), NaH, DMF, 100 °C, overnight, 43%–78%; (g) 2 mol/L NaOH aqueous solution,
MeOH, rt, 2 h, 70%.
2
O, reflux, 3 h, (ii) HCl, rt, 30 min, 83%; (c) Br
2
, DCM, rt, 24 h, 80%; (d) 3,5-
3
)
4
2
3
2
O = 30:1, 100 °C, overnight, 32%; (e) sulfonyl chloride, NaH, DMF 0 °C, 3 h,
4
identical potency against BRD4(1), with an inhibition ratio
value of 11.7% at 20 μM. The TSA also demonstrated a stabilizing
effect from 17 on the BRD4(1) protein with a temperature shift
of 1.2 °C. All of the above results suggested that these
sulfonamide derivatives demonstrated weak binding affinity to
the BRD4(1) protein. It seems that the sulfonamide linker might
provide an unfavorable angle and distance range for
substitution and thus cannot form favorable interactions with
the WPF shelf.
19 is similar but not identical to that of 6 (Fig. 2a). Analysis of the
binding model for compound 19 in complex with the BRD4(1)
domain suggested that replacement of the ethyl group in 19 with
a larger group such as a propyl or phenyl group might further
enhance the hydrophobic interactions with BRD4(1). This dis-
covery encouraged us to design larger substituted compounds.
However, when the ethyl moiety in 19 was replaced with the
relatively large hydrophobic groups propyl (20), butyl (21), or
methyl acetate (22), the resulting molecules displayed a 1- to 2-
fold potency loss. For instance, propyl group-substituted analog
20 exhibited an IC50 value of 9.17 μM against BRD4(1), which was
approximately twofold less potent than 19. The compound with a
hydrophilic substituent (23) showed a dramatic loss of inhibition,
and its maximum inhibition was less than 20% at 20 μM. This
result indicated that the hydrophobic interaction between the
ligand and the protein is potentially a critical interaction. To
illustrate this point, flexible linkers with 2–3 methylenes were used
to attach polar substituents (24, 25, and 26). Not surprisingly,
these compounds exhibited moderate or weak activity. Replace-
ment of the polar substituent in 26 with a phenyl group resulted
in 27 showing a sharp decrease in BRD4(1) inhibitory potency
Next, to find potent 6-(3,5-dimethylisoxazol-4-yl)benzo[cd]indol-
2
(1H)-one derivative, the sulfonamide linker was replaced by an
amide moiety and its derivatives to give compounds 11 and
9–28. These compounds displayed significant improvements in
1
the inhibition of BRD4(1) (Table 1). Interestingly, the sulfonamide
moiety in compound 6 was removed to yield compound 11,
which displayed an obvious potency improvement against BRD4
(
1) with a thermal shift of 6.3 °C and an IC50 value of 4.87 μM.
Further investigation revealed that the hydrogen atom in 11 could
be replaced by an ethyl moiety (19) to display almost identical
potency against BRD4(1), with a thermal shift of 5.9 °C and an IC50
value of 3.79 μM. All of the results showed that compound 19 is a
good starting point for bromodomain inhibitor development.
To better design potent compounds, we predicted the complex
structure of 19 with the BRD4(1) protein to gain a structural
understanding of their interactions (Fig. 2c). The modeled
structure of 19 and BRD4(1) shows that the binding mode of
m
(ΔT = 1.1 °C). Compound 28 was also synthesized. It was found
that 28 bound to BRD4(1) with a temperature shift of 2.7 °C and a
maximum inhibition ratio of less than 30% at 20 μM. Luckily,
the crystal structure of the 28-BRD4(1) complex was obtained
(Fig. 3a, b). We can see that 28 showed an orientation of the
Acta Pharmacologica Sinica (2021) 0:1 – 12

Novel BET inhibitors against prostate cancer
TB Wu et al.
8
Table 1. Structure-activity relationship study.
Compd
R
TSA
AlphaScreen
ΔT
m
(°C)
Inhibition rate at 20 μM (%)
IC50 (μM)
1
6
–
12.0
98.2 ± 0.26
0.12 ± 0.002
2.1
1.8
23.8 ± 1.84
1
1
1
1
1
2
3
4
5
6
36.4 ± 0.90
28.6 ± 0.99
28.4 ± 2.34
NA
1.2
1.2
0.6
0.0
18.5 ± 1.07
1
7
1.2
7.6 ± 1.20
18
11
19
20
21
1.2
6.3
5.9
4.7
3.8
11.7 ± 0.70
94.0 ± 0.14
87.3 ± 0.19
46.6 ± 0.52
62.4 ± 0.52
H
4.87 ± 0.07
3.79 ± 0.02
9.17 ± 0.02
8.30 ± 0.28
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Novel BET inhibitors against prostate cancer
TB Wu et al.
9
Table 1. continued
Compd
R
TSA
ΔT
AlphaScreen
m
(°C)
Inhibition rate at 20 μM (%)
IC50 (μM)
22
23
24
25
26
27
4.4
30.4 ± 2.04
18.1 ± 2.37
3.1 ± 2.94
27.2 ± 0.80
16.1 ± 1.53
NA
7.15 ± 0.13
1.7
2.0
4.1
3.5
1.1
17.19 ± 0.06
38.86 ± 0.49
2
8
2.7
24.4 ± 1.37
Fig. 3 Cocrystal structure of compound 28 with BRD4(1) (PDB ID: 7DHS). a Fo-fc omits map overlapped with the ligand. The density contour
is shown, and the contour level is set to 1.0 sigma. b Compound 28 is shown as sticks, and all residues are shown as lines. Water molecules are
shown as spheres. In addition, an electrostatic potential surface is shown. Hydrogen bonds are shown as yellow dashed lines.
Acta Pharmacologica Sinica (2021) 0:1 – 12

Novel BET inhibitors against prostate cancer
TB Wu et al.
1
0
6
-(3,5-dimethylisoxazol-4-yl)benzo[cd]indol-2(1H)-one
scaffold
these various groups explored, the ethyl moiety remained the best
for binding to the BRD4(1) WPF shelf. Hence, our modifications at
the WPF shelf position yielded compounds 11 and 19 with much
improved binding affinities over initial compound 6.
that was almost identical to that of the other compounds (6, 14,
or 19, Fig. 2a–c). From the above SAR, we can see that among
Evaluation of the binding specificity over other non-BET
bromodomain-containing proteins
Bromodomains have been reported to be a part of numerous
large protein architectures, many of which share a conserved
module and have the common 3D structure pattern of three long
helices (helices A–C) and one short helix (helix Z). The selectivity of
diverse bromodomain inhibitors is critical for the success of drug
discovery. To study the selectivity profile, representative com-
pounds were tested against BRD4(1) domain proteins and seven
representative non-BET bromodomain-containing proteins from
six other subfamilies by thermal stability shift assay. Compound 1
was included in these experiments as a control compound, and
the results are shown in Fig. 4. The results showed that compound
1
9 displayed excellent selectivity for the BET subfamily over other
non-BET families.
Fig. 4 Thermal shift analysis for compounds 1 and 19 against 8
bromodomain-containing proteins. The heat map shows the
Evaluation of the inhibitory effects on cell growth and gene
expression in PC cell lines
relative ΔT
m
, where red indicates a large ΔT
m
and yellow and green
indicate small ΔT
m
values. Compound concentration, 200 µM;
protein concentration, 10 µM.
The BET family has been identified as a novel therapeutic target
for PC. To evaluate the antiproliferative activities of these 6-(3,5-
dimethylisoxazol-4-yl)benzo[cd]indol-2(1H)-one derivative, we
tested our representative compounds for their cellular activity in
AR-positive and AR-negative PC cell lines (C4–2B, LNCaP, 22Rv1,
and Du-145) (Table 2, Fig. 5a, b).
All compounds exhibited reasonable potency against these PC
cell lines. For example, 19 showed cell viability inhibition IC50
values of 1.93, 2.07, and 2.28 μM in the C4–2B, LNCaP, and 22Rv1
cell lines, respectively (Fig. 6a). These activities were stronger than
those of the second-generation antiandrogen enzalutamide
Table 2. Antiproliferation effects against prostate cell lines C4–2B,
LNCaP, 22Rv1, Du145, and leukemia cell line MV4;11.
a
Compd
Cell growth inhibition (IC50, μM)
C4–2B
LNCaP
22Rv1
Du145
MV4;11
1
1
2
2
2
2
2
a
1
9
0
1
2
5
6
10.08
1.93
6.94
2.07
25.38
2.28
30.00
3.56
4.09
0.54
0.45
0.66
10.05
0.32
1.98
(
36.66 μM in 22Rv1 cells) [18, 33]. It is interesting to note that
compounds 20 and 25 displayed less potent inhibitory activity
than 19 at the protein level but showed similar potencies to 19 in
the same cell lines. All of the tested compounds were also active
against MV4;11 leukemia cells (Table 2). It seems that the present
compounds are more sensitive to MV4;11 cells than prostate cells.
The results showed that our BET inhibitors effectively inhibited PC
cell proliferation.
To further confirm the long-term cell growth inhibitory effects, a
colony formation assay was performed with representative
compound 19. As shown in Fig. 6b, compound 19 inhibited
6.88
1.92
1.58
2.53
16.9
6.07
6.24
8.78
31.47
2.48
34.00
2.87
22.51
1.85
28.59
1.88
13.57
13.05
13.68
14.02
The IC50 was calculated from cell viability assay by Cell-Titer GLO
Promega).
(
Fig. 5 BET inhibitors suppress the growth of (a) LNCaP and (b) 22Rv1 prostate cancer cells
Acta Pharmacologica Sinica (2021) 0:1 – 12

Novel BET inhibitors against prostate cancer
TB Wu et al.
1
1
Fig. 6 Compound 19 inhibits prostate cancer cell growth and AR-regulated gene expression. a Inhibition curves for prostate cell growth.
b Compound 19 inhibits C4–2B cell colony formation. c qRT−PCR analysis of full-length AR, AR-V7, PSA, KLK2, TMPRSS2, and c-MYC expression
in 22Rv1 cells treated with vehicle or 19 (5 μM) for 48 h. Data represent the mean ± SD (n = 3) from one of three independent experiments. NS
not significant. *P ≤ 0.05, **P ≤ 0.005 by two-tailed Student’s t test.
C4–2B cell colony formation in a dose-dependent manner. Colony
formation was markedly decreased by compound 19 at 2 μM. The
results showed that 19 significantly inhibited the growth of the
C4–2B cell line.
2018GZR110105016), the Natural Science Foundation of Jiangsu Province (grant
BK20190246) and the Natural Science Foundation of the Jiangsu Higher Education
Institutions of China (grant 19KJB350011). The authors also gratefully acknowledge
support from the Guangzhou Branch of the Supercomputing Center of the Chinese
Academy of Sciences.
To investigate whether the antiproliferative effects were caused
by the inhibition of BRD4(1), we further performed real-time
quantitative fluorescent PCR (qRT-PCR) in 22Rv1 cells to detect the
expression of the AR, AR-regulated genes, and other oncogenes.
As shown in Fig. 6c, the AR-regulated genes PSA (also known as
KLK3), KLK2, and TMPRSS2 were suppressed at the mRNA level
upon treatment with 19 in 22Rv1 cells. The oncogenic driver MYC
was also downregulated by the BET bromodomain inhibitor. The
results showed that c-Myc mRNA levels were suppressed upon
treatment with 19. The results demonstrated that BET inhibitor 19
could effectively suppress cell growth and the related gene
expression in PC cells. Based on these data, compound 19 and its
derivatives may serve as a promising therapeutic strategy for PC
by targeting BRD4(1).
AUTHOR CONTRIBUTIONS
YX and LJX designed the research. TBW, QPX, CW, CW, CZ, MFZ, and ZXL conduct the
research; TBW, QPX, YZ, LJX, and YX analyzed the date and wrote the paper. All
authors read and approved the final manuscript.
ADDITIONAL INFORMATION
Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s41401-021-00614-7.
Competing interests: The authors declare no competing interests.
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