Y08060: A Selective BET Inhibitor for Treatment of Prostate Cancer

Article abstract of DOI:10.1021/acsmedchemlett.8b00003

Prostate cancer is a commonly diagnosed cancer and a leading cause of cancer-related deaths. The bromodomain and extra terminal domain (BET) family proteins have emerged as potential therapeutic targets for the treatment of castration-resistant prostate cancer. A series of 2,2-dimethyl-2H-benzo[b][1,4]oxazin-3(4H)-one derivatives were designed and synthesized as selective bromodomain containing protein 4 (BRD4) inhibitors. The compounds potently inhibit BRD4(1) with nanomolar IC₅₀ values and exhibit high selectivity over most non-BET subfamily members. One of the representative compounds 36 (Y08060) effectively suppresses cell growth, colony formation, and expression of androgen receptor (AR), AR regulated genes, and MYC in prostate cancer cell lines. In in vivo studies, 36 demonstrates a good PK profile with high oral bioavailability (61.54%) and is a promising lead compound for further prostate cancer drug development.

Full text of DOI:10.1021/acsmedchemlett.8b00003

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Letter
Y08060: A Selective BET Inhibitor for Treatment of Prostate Cancer
Qiuping Xiang, Yan Zhang, Jiaguo Li, Xiaoqian Xue, Chao Wang, Ming Song, Cheng Zhang, Rui
Wang, Chenchang Li, Chun Wu, Yulai Zhou, Xiaohong Yang, Guohui Li, Ke Ding, and Yong Xu
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00003 • Publication Date (Web): 13 Feb 2018
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Y08060: A Selective BET Inhibitor for Treatment of Prostate Cancer
Qiuping Xiang,†,‡,# Yan Zhang,†,‡,# Jiaguo Li,§,# Xiaoqian Xue,†,‡ Chao Wang,† Ming Song,† Cheng
Zhang,†,§ Rui Wang,† Chenchang Li,†,§ Chun Wu,† Yulai Zhou,§ Xiaohong Yang,§ Guohui Li,⊥ Ke Ding,
¶ and Yong Xu†*
†Guangdong Provincial Key Laboratory of Biocomputing, Joint School of Life Sciences, Guangzhou Institutes of Biomedi-
cine and Health, Chinese Academy of Sciences; Guangzhou Medical University, Guangzhou, China.
‡University of Chinese Academy of Sciences, No. 19 Yuquan Road, Beijing 100049, China.
§College of Pharmacy, Jilin University, Changchun, China, No. 1266 Fujin Road, Chaoyang District, Changchun, Jilin
130021, China.
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⊥Laboratory of Molecular Modeling and Design, State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of
Chemical Physics, The Chinese Academy of Sciences, Dalian, Liaoning 116023, China.
¶School of Pharmacy, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, China.
KEYWORDS: bromodomain, BRD4, prostate cancer, epigenetics
ABSTRACT: Prostate cancer is a commonly diagnosed cancer and a leading cause of cancer-related deaths. The bromodomain and
extra terminal domain (BET) family proteins have emerged as potential therapeutic targets for the treatment of castration-resistant
prostate cancer. A series of 2,2-dimethyl-2H-benzo[b][1,4]oxazin-3(4H)-one derivatives were designed and synthesized as selective
bromodomain containing protein 4 (BRD4) inhibitors. The compounds potently inhibit BRD4(1) with nanomolar IC50 values, and
exhibit high selectivity over most non-BET subfamily members. One of the representative compounds 36 (Y08060) effectively
suppresses cell growth, colony formation, and expression of androgen receptor (AR), AR regulated genes, and MYC in prostate
cancer cell lines. In in vivo studies, 36 demonstrates a good PK profile with high oral bioavailability (61.54%), and is a promising
lead compound for further prostate cancer drug development.
Prostate cancer (PC) is the most common cancer in men
worldwide.1-3 The activation of androgen receptor (AR) signal-
ing has a central role in the initiation and progression of pros-
tate cancer, even after castration.4, 5 Endocrine therapy for pros-
tate cancer, which is based on the classic work of Huggins and
Hodges,6 has been used to reduce androgen synthesis or block
AR activation. Initially, prostate cancer is sensitive to androgen
deprivation therapy (ADT), which can induce marked tumor
regression and normalize prostate specific antigen (PSA) in
serum. Most patients, however, acquire resistance to treatment,
thus leading to castrate-resistant prostate cancer (CRPC). Men
who develop CRPC always succumb to the disease.7 Although
some success has been achieved with therapies such as Abi-
raterone and the second generation anti-androgen agent Enzalu-
tamide, which target AR signaling, durable responses to these
therapies are limited, presumably due to acquired resistance.8-12
Clinically, there is an urgent need for alternative therapeutic
strategies for CRPC.
Notwithstanding the discovery of these BET inhibitors, chemi-
cals that have entered clinical trials for CRPC are still limited.
Among these, 2, OTX-015, ZEN003694 and GS-5829, are cur-
rently being evaluated either as a single agent or in combina-
tion with AR-antagonists (Enzalutamide or ARN-509)20-23 in
clinical trials in patients with CRPC, but potent and specific
BET inhibitors with different chemotypes are still in high de-
mand to assist in our understanding of the therapeutic potential
of BET inhibition. Herein, we describe the discovery and opti-
mization of 2,2-dimethyl-2H-benzo[b][1,4]oxazin-3(4H)-one
derivatives and the efforts which yielded 5-bromo-2-methoxy-
N-(6-methoxy-2,2-dimethyl-3-oxo-3,4-dihydro-2H-
benzo[b][1,4]oxazin-7-yl)benzenesulfonamide (Y08060, 36) as
a specific BRD4 inhibitor with promising therapeutic activity
in vivo.
X-ray crystallographic analysis reveals that 3 binds to the
BRD4(1) bromodomain with three key interactions, which are
shown in Figure 1C. The lactam of the quinazolinone forms
two hydrogen bonds with the conserved residue Asn140. Oxy-
gen interacts with the conserved residue Tyr97 through a water-
mediated hydrogen bond. The sulfonamide forms two addition-
al hydrogen bonds with two water molecules.
Asangani and colleagues reported that BRD4 (bromodomain-
containing protein 4) can physically interact with the N-
terminal domain of AR and this interaction can be disrupted by
14
(+)-JQ1 (1).13,
Bromodomain and extra terminal domain
(BET) inhibitors may have a promising therapeutic potential
for the treatment of CRPC.7 Several classes of BET bromo-
domain inhibitors have been discovered, including 1 ((+)-JQ1),
2 (I-BET-762), 3 (PFI-1), and 4 (Y02224) (Figure 1A).14-19
In this paper, we described further optimization of 3 with the
aim of improving its oral absorption property (compound 3,
after oral dosing in the rat, showed a low oral bioavailability of
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32%). Based on this analysis, we rationally designed two heter-
ocyclic structures, 2,2-dimethyl-2H-benzo[b][1,4]oxazin-
nitrophenols (5a−5b) through subsequent procedures including
standard acylation and cyclization.24 Compounds 7a−7d were
obtained after an alkylation or hydrocarboxylation reaction of
6a−6b with amine or sodium alcoholate, respectively. Reduc-
tion of the nitro group was achieved with Pd/C in a hydrogen
atmosphere or with iron and ammonium chloride. Direct sul-
fonylation of 8a−8f with different sulfonyl chlorides yielded
the final sulfonamides (13−26 and 32−37) with good to moder-
ate yields.
1
2
3
4
5
6
7
8
3(4H)-ones (I) and 2,2-dimethyl-2H-pyrido[3,2-b][1,4]oxazin-
3(4H)-ones (II), as BRD4 bromodomain inhibitors by scaffold
hopping (Figure 1B). In both series, the 2,2-
dimethylmorpholin-3-one scaffold was utilized as the acetylat-
ed lysine (KAc) mimics and forms important hydrogen bonds
with the BRD4(1) protein. The dimethyl group mimics the ter-
minal methyl group of the acetyl lysine and occupies the small
pocket surrounded by residues Pro82, Phe83, Val87, and Ile146
(Figure 1C). The sulfonamide moiety was maintained to form
hydrogen bond networks with water. To develop compounds
with improved affinity for BRD4(1), we performed an exten-
sive structure-activity relationship (SAR) study to analyze the
protein-ligand interactions from three different areas, the WPF
shelf (which is composed of Trp81, Pro82, Phe83), the acety-
lated lysine binding region and the BC loop.
9
The synthesis of 2,2-dimethyl-2H-pyrido[3,2-b][1,4]oxazin-
3(4H)-one derivatives (27−31) is shown in Scheme 2. Com-
pound 10 was synthesized using procedures similar to those
used with amides 6a−6b, then reacted with nitric acid and con-
centrated sulfuric acid to produce the nitro-substituted product
(11). Compounds 27−31 could be synthesized similarly using
the procedures outlined in Scheme 1.
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60
To optimize the interactions with the WPF shelf, various sub-
stituents at the R1 position of 2,2-dimethyl-2H-
benzo[b][1,4]oxazin-3(4H)-one were designed to explore the
chemical space with a view to improving the affinity (Table 1).
The 2,2-dimethyl-2H-benzo[b][1,4]oxazin-3(4H)-one deriva-
tives were designed and synthesized as shown in Scheme 1.
The amides (6a−6b) were prepared from 2-amino-5-
A
B
D
C
E
Figure 1. (A) Structures of representative BRD4(1) bromodomain inhibitors. IC50 values for the inhibitors are shown. (B) Design of new BRD4 bromo-
domain inhibitors. (C) Superimposition of the binding modes of compounds I, II and 3. The crystal structures of BRD4(1) protein and compound 3 were
taken from PDB ID 4E96. (D) Co-crystal structure of compound 25 in a complex with BRD4(1) protein (PDB ID: 5Z1R). (E) Co-crystal structure of com-
pound 36 in a complex with the BRD4(1) protein (PDB ID: 5Z1S). The binding site residues are shown as sticks. Hydrogen bond interactions are indicated
by yellow dashed lines.
To ensure the compounds reach the WPF shelf and become
involved in hydrophobic interactions with residues Trp81,
Pro82, and Phe83 of the WPF shelf, alkyl, cycloalkyl or aro-
matic groups were introduced and compounds 13−17 were
synthesized (Table 1). Compounds 13 and 14 show moderate
activity with temperature shifts of 3.9 and 4.5 °C in the thermal
shift assay (TSA) and IC50 values of 12.01 and 11.87 μM in the
AlphaScreen assay, respectively. Compound 15 in which R1 is
a cyclohexyl group, exhibits 3-fold increase of activity with an
IC50 value of 3.87 μM. Replacement of the cyclohexyl group in
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15 with a thienyl group led to 16, which in the TSA, exhibits
1
2
3
potency similar to that of 15 (5.1 °C vs 5.5 °C). Replacement of
the cyclohexyl group in 15 with a phenyl group resulted in 17,
which shows activity (IC50 = 3.34 μM) similar to that of 15.
*Reagents and conditions：(a) (i) 2-bromo-2-methylpropanoyl
bromide, DCM, Et3N, 0 °C, 4 h; (ii) K2CO3, DMF, 80 °C, 4 h,
50%; (b) HNO3, H2SO4, rt, 2 h, 17%; (c) Pd/C, CH3OH, H2, rt,
5 h, 90%; (e) sulfonyl chloride, pyridine, 80 °C, 2 h, 50−70%.
4
5
6
7
8
9
The results suggest that 17 may serve as a lead compound for
discovery of new BRD4(1) inhibitors. An extensive SAR in-
vestigation was then conducted, revealing that various substitu-
tions at different positions of the phenyl ring in 17 could have
diverse impacts on the BRD4(1) bromodomain inhibitory activ-
ity. For instance, the ortho-methyl ester substituted analogue
Next, we further investigated another head group, 2,2-
dimethyl-2H-pyrido[3,2-b][1,4]oxazin-3(4H)-one, which is
similar to 2,2-dimethyl-2H-benzo[b][1,4]oxazin-3(4H)-one
except that a nitrogen atom has been introduced at 10-C posi-
tion (Figure 1B). We combined the 2,2-dimethyl-2H-
pyrido[3,2-b][1,4]oxazin-3(4H)-one with the optimal alkyl and
aromatic motifs obtained above, leading to compounds 27−31
(Table 1). These compounds show significant loss of inhibition.
Compound 28 binds to BRD4(1) with an IC50 value of 25.43
μM, and is approximately 4-fold weaker than 16. Compound 31
is 14-fold less potent than the corresponding compound (22)
(40.1 μM vs 2.73 μM ). Compounds 27, 29 and 30 have maxi-
mum inhibitions of less than 40% at 20 μM. These results indi-
cate that adding the nitrogen atom at the 10-C position on the
head group is detrimental to potency. Therefore, we focused on
the scaffold of 2,2-dimethyl-2H-benzo[b][1,4]oxazin-3(4H)-
one as described below.
10
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12
13
14
15
16
17
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(18) shows moderate activity (IC50 = 9.32 μM and ΔTm
=
4.0 °C), but when a Br atom is introduced at the ortho-position,
the resulting compound 19 has no activity. Further study
showed that compounds bearing methyl or F atom substitutents
at the para-position of the phenyl ring exhibit inhibitory activi-
ty similar to 17 with IC50 values of 4.63 and 4.79 μM, respec-
tively.
Scheme
1.
Synthesis
of
2,2-dimethyl-2H-
benzo[b][1,4]oxazin-3(4H)-one Derivatives*
To understand the structural basis for the activity, we deter-
mined the co-crystal structure of 25 bound to BRD4(1). The
crystal structure of 25 shows that the binding mode of the head
group is similar to that of 3. As shown in Figure 1D, the 2,2-
dimethyl-2H-benzo[b][1,4]oxazin-3(4H)-one scaffold binds to
and forms extensive hydrophobic interactions with the deep
and narrow acetyl lysine binding pocket formed by residues
Val87, Leu92, Leu94, Tyr97 and Pro82, Phe83, Ile146. The
key hydrogen bond interactions with Asn140 and Tyr97 are
maintained. The sulfonamide linker provides a suitable angle
turn for the 1-bromo-4-methylbenzene group to reach the hy-
drophobic WPF shelf, as is also seen in the crystal structure of
4 in a complex with BRD4(1). The NH of the sulfonamide
linker participates in a hydrogen bond interaction with external
water. The water molecule presents a suitable geometry ena-
bling good contact with the methoxyl group on the phenyl ring.
All the structure information demonstrates that 2,2-dimethyl-
2H-benzo[b][1,4]oxazin-3(4H)-one is a good starting point for
bromodomain inhibitor development.
*Reagents and conditions: (a) (i) 2-bromo-2-methylpropanoyl bromide,
Et3N, DCM, 0 °C, 4 h. (ii) K2CO3, DMF, 80 °C, 4 h, 50%; (b) amine,
K2CO3, DMSO 100 °C, 4 h, 40−70% or CH3ONa, CuI, DMF, 100 °C, 18 h,
60%; (c) Pd/C, H2, CH3OH, rt, 5 h, 70−80%. or Fe, NH4Cl, AcOH, EtOH,
H2O, 80 °C, 30 min, 90%; (d) sulfonyl chloride, pyridine, 80 °C, 2 h,
50−90%; (e) BBr3, DCM, 0 °C to rt, 5 h, 90%.
When two halogen atoms were added to the phenyl ring at
different positions, improved potency was obtained. The 2,4-
difluorophenyl compound (22) exhibits improved activity, with
an IC50 of 2.73 μM. The TSA assay also demonstrated stabiliza-
tion of the BRD4(1) protein with a temperature shift of 6.3 °C.
The activity of 22 was better than that of other dihalogen sub-
stituents. Compound 23, with a 2-chloro-4-fluorophenyl group,
has an IC50 value of 1.44 μM and is 2 times more potent than
compound 17. The 2-fluoro-3-chlorophenyl compound (24) is
essentially inactive, its maximum inhibition being less than 20%
at 20 μM. This result indicates that substitution at this position
is unfavorable. Compounds 25 and 26 were also synthesized,
and bind to BRD4(1) with IC50 values of 1.99 and 2.96 μM,
respectively, and are equipotent with 22 and 23, respectively.
Table 1. Structure-activity relationship of compounds with
modifications of the WPF shelf, acetylated lysine binding re-
gion and the BC loop.
TSA
AlphaScreen
Compd
R1
--
R2
X
△Tm
Inhibition rate
at 20 μM (%)
IC50
(°C)a
(μM)c
1d
13
14
12.0
98.2
0.12
12.01
11.87
--
H
H
--
C
C
Scheme 2. Synthesis of 2,2-dimethyl-2H-pyrido[3,2-
b][1,4]oxazin-3(4H)-one Derivatives*
3.9
4.5
61.1
73.4
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a hydroxyl group (37) without any obvious effect on the
BRD4(1) inhibitory potency. The crystal structures of 36 and
37 (Figure 1E and Figure S1) bound to BRD4(1) indicates that
a size restriction exists in this area. These results suggest that 5-
bromo-2-methoxybenzene substitution was most favorable in
the WPF region. In fact, this substituent had been validated for
compound 4 in our previous study.19 Our modifications at this
site have therefore yielded compounds 36 and 37 which have
much improved binding affinities over that of our initial com-
pound (25).
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
H
C
C
C
C
C
C
C
C
C
C
C
C
N
N
N
N
N
C
C
C
C
C
C
6.5
5.1
5.5
4.0
2.7
2.9
4.5
6.3
5.7
2.4
4.5
4.5
1.4
2.3
1.1
2.6
2.3
4.2
3.6
1.8
0
97.1
81.7
80.0
76.0
26.2
70.6
60.3
98.2
97.7
<20
3.87
6.54
3.34
9.32
>50
1
2
3
4
5
6
7
8
H
H
H
H
9
H
4.63
4.79
2.73
1.44
>50
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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34
35
36
37
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Bromodomain proteins have a conserved module, and share a
common 3D structure pattern of three long helices (helix A−C)
and one short helix (helix Z). The acetylated lysine can recog-
nize the bromodomains by forming several hydrogen bonds
with the conserved residue Asn140. This selectivity is critical
for the success of drug discovery. To investigate the selectivity
profile, representative compounds were tested against 8 bro-
modomain-containing proteins by thermal stability shift assay.
As shown in Figure 2A, the results illustrate that all the com-
pounds displayed excellent selectivity for BET subfamily over
other non-BET family with the exception of their moderate
inhibition of CBP/EP300.
H
H
H
H
H
46.0
42.0
19.7
37.9
15.2
33.7
42.8
99.0
97.0
79.0
0
1.99
2.96
>50
H
H
The data from the above assays show that 36 and 37 have
good profiles for further evaluation. The binding abilities of 36,
37 and reference compound 1 to BRD4(1) were then deter-
mined by an isothermal titration calorimetry (ITC) experiment
(Figure 2B, Figure S2). Compound 1 showed a Kd value of 92
nM. The Kd of 36 and 37 were determined as 302 and 498 nM,
respectively, which is consistent with the data from the Al-
phaScreen and TSA assays (Table 1).
H
25.43
>50
H
H
H
>50
40.1
3.59
4.77
4.73
NA
Cl
A
B
(CH3)2N-
CH3O-
HO-
6.6
7.2
100.0
100.0
0.69
0.63
The BC loop is also an active site. To find more potent ana-
logues based on the experimentally determined co-crystal struc-
ture of 25 and BRD4(1), we designed various substituents on
the R2 group to explore the chemical space of the BC loop for
affinity improvement (Table 1). When R2 in compound 25 is
replaced by Cl, analogue 32 shows slightly weaker activity than
25 in the TSA and AlphaScreen assays (ΔTm = 4.2 °C and IC50
= 3.59 μM). Compounds 33 and 34, bearing a larger group,
dimethylamine or pyrrolidine respectively, show moderate
activity. However compound 35, which bears a morpholine
group, is inactive. The biochemical activity results showed that
a larger group may have steric conflict with residues around
this pocket. Then we synthesized compound 36 (Table 1), in
which the H atom at the R2 position was replaced by a methox-
yl group. Compound 36 has an IC50 value of 0.69 μM and tem-
perature shift of 6.6 °C in the TSA assay. It was also shown
that the methoxyl group at R2 position in 36 can be replaced by
Figure 2. Compounds 36 and 37 are potent and selective BRD4(1) inhibi-
tors. (A) The bromodomain selectivity profiles were determined by a TSA
assay. Compound concentration, 200 μM; protein concentration, 10 μM.
The heat map shows the relative ΔTm values. Red indicates large ΔTm , and
green indicates small ΔTm . (B) ITC assay.
Recent studies demonstrated that small molecule inhibitors of
BET proteins exhibit in vitro efficacy in models of CRPC and
are currently being evaluated in several clinical trials. Potent
and specific BET inhibitors such as 1 and 2 effectively inhibit
cell growth in prostate cancer cell lines such as C4-2B, LNCaP,
and 22Rv1. Our representative compounds were evaluated for
their cellular antiproliferative activity and specificity in the C4-
2B, LNCaP, 22Rv1, PC3 and DU145 cell lines (Table 2). They
exhibit reasonable potency against C4-2B, LNCaP and 22Rv1
cell lines but could not be well correlated with the other cell
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lines, indicating that these analogues are more active against
To assess the potential of this series of BRD4(1) inhibitors in
vivo, compound 36 was selected for preliminary pharmacoki-
netic (PK) analysis (Table 3). The plasma level of 36 was de-
termined after an i.v. dose of 2 mg/kg or a single oral dose of
10 mg/kg. Encouragingly, compound 36 exhibits reasonable
PK properties with an oral AUC value of 3765.71 μg/L·h, a T1/2
value of 1.96 h, and an oral bioavailability of 61.5%. The re-
sults indicate that 36 could be advanced for in vivo pharmaco-
logical studies.
1
2
3
4
5
6
7
8
AR-dependent prostate cancers. For example, 36 has IC50 val-
ues of 3.23 and 4.41 μM in inhibition of cell viability in the C4-
2B and LNCaP cell lines, respectively. Compound 37 has simi-
lar potencies in the same cell lines when compared to 36. The
results illustrated that our BET inhibitors, similar to compound
1, display excellent cellular specificity.
To further investigate the growth inhibitory effects, colony
formation assays were performed for the representative com-
pound 36. As in the cell viability assay, 36 reduced colony
formation in a dose-dependent manner (Figure 3A and Figure
S3). Colony formations were reduced to less than 10% in
22Rv1 and C4-2B cells at 5 μM. Thus, 36 significantly inhibits
growth of prostate cancer cells.
9
Table 3. Intravenous (iv) and oral (po) pharmacokinetic pro-
files of compound 36 in ratsa
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11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
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43
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Route
ivb
poc
Cmax (µg/L)
Tmax (h)
AUC(0-t) (µg/L·h)
AUC(0-∞) (µg/L·h)
T1/2 (h)
1000.37
0.083
1199.17
1223.82
1.65
926.40
1.67
3520.79
3765.71
1.96
Table 2. Antiproliferative effects against prostate cell lines C4-
2B, LNCaP, 22Rv1, PC3 and Du145
MRT(0-t) (h)
Cl (L/h/kg)
Vz (L/kg)
1.17
1.64
4.01
2.60
--
--
Cell growth inhibition (IC50, μM)a
Compd
F（%）
--
61.54
C4-2B
0.19
LNCaP
0.16
22Rv1
0.071
PC-3
3.01
Du145
2.52
aCompounds were formulated in 5% DMSO, 40% PEG400 and 55% (sa-
1
b
c
line). A dose of 2 mg/kg. A dose of 10 mg/kg. Values are given as mean
of 3 independent experiments.
15
17
23
25
36
37
7.04
13.26
7.16
6.56
3.23
7.75
8.40
7.52
7.80
6.28
4.41
7.70
8.02
13.08
7.05
4.85
3.38
7.59
30.40
40.13
26.95
21.01
17.22
22.85
35.93
92.56
54.50
30.93
23.19
33.24
In
summary,
a
series
of
2,2-dimethyl-2H-
benzo[b][1,4]oxazin-3(4H)-one derivatives have been designed
and synthesized as new selective BRD4(1) inhibitors. These
compounds potently inhibit BRD4(1) with nanomolar IC50 val-
ues and selectively inhibit non-BET bromodomain-containing
proteins. One of the most promising compounds (36) has na-
nomolar IC50 value against BRD4(1) and potently inhibits the
proliferation of a panel of prostate cancer cell lines, C4-2B,
LNCaP and 22Rv1. This compound also suppresses the gene
expression in LNCaP cells and demonstrates favorable pharma-
cokinetic properties. Our study provides a new research probe
and lays a basic foundation for further drug discovery efforts
targeting prostate cancer and other cancers. Further optimiza-
tion of the lead compound (36) based on these SAR results and
in vivo tumor xenograft data will be reported in due course.
aThe IC50 was calculated from cell viability assay by Cell-Titer GLO
(Promega).
To investigate whether BRD4(1) inhibitors suppress the AR-
mediated gene and other oncogene expression in prostate can-
cer-related cell lines, qRT-PCR was performed in LNCaP cells.
As shown in Figure 3B, compound 36 strongly inhibits mRNA
expression of full length AR, AR-regulated genes PSA, KLK2
and TMPRSS2. MYC is a known oncogenic driver in prostate
cancers. c-MYC mRNA level is also significantly down-
regulated by 36. The results demonstrated that the BRD4(1)
inhibitor (36), by functioning downstream of AR, may have a
profound effect on CRPC as it suppress AR-, PSA-, and c-
Myc- mediated pathways.
ASSOCIATED CONTENT
Supporting Information
Experimental procedures for the synthesis, 1H NMR and 13C NMR
for final compounds, and details of in vitro assays.
A
B
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: +86-20-32093612; Fax: 86-20-32093612; Email:
xu_yong@gibh.ac.cn (Y. X.)
Author Contributions
#These authors contributed equally to this work
Figure 3. Growth inhibitory effects of CBP inhibitors in different prostate
cancer cell lines. (A) Compound 36 inhibits 22Rv1 cancer cell colony
formation. (B) qRT–PCR analysis of full length AR, PSA, KLK2,
TRPRSS2 and c-MYC expression in LNCaP cell treated with vehicle, 1 (1
μM or 5 μM), or 36 (5 μM) for 48 h. Data represent mean ± s.d. (n = 3)
from one of three independent experiments. NS, not significant.* P ≥ 0.05,
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We gratefully acknowledge financial support from the National
Natural Science Foundation of China (grant 21602222 and
81673357), the “Personalized Medicines − Molecular Signature-
** P ≤ 0.005 by two-tailed Student’s t test.
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ACS Medicinal Chemistry Letters
Page 6 of 7
Thian Kheoh, Ph.D.,; Christopher M. Haqq M. D., Ph.D., and Howard
based Drug Discovery and Development”, Strategic Priority Re-
search Program of the Chinese Academy of Sciences (grant No.
XDA12020363), the Chinese National Programs for Key Research
and Development (grant 2016YFB0201701), the Natural Science
Foundation of Guangdong Province (2015A030312014). The au-
thors thank the staffs from BL17U1, BL18U1, BL19U1 beamlines
of National Facility for Protein Science Shanghai (NFPS) at
Shanghai Synchrotron Radiation Facility, for assistance during
data collection. The authors gratefully acknowledge support from
the Guangzhou Branch of the Supercomputing Center of Chinese
Academy of Sciences.
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