Binding pocket-based design, synthesis and biological evaluation of novel selective BRD4-BD1 inhibitors

Article abstract of DOI:10.1016/j.bmc.2019.03.037

Bromodomain-containing protein 4 (BRD4), consisting of two tandem bromodomains (BD1 and BD2), is key epigenetic regulator in fibrosis and cancer, which has been reported that BD1 and BD2 have distinct roles in post-translational modification. But there ar

Full text of DOI:10.1016/j.bmc.2019.03.037

Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Binding pocket-based design, synthesis and biological evaluation of novel
selective BRD4-BD1 inhibitors
Junlong Maa,b, Heng Chena, Jie Yanga, Zutao Yua, Pan Huanga, Haofeng Yanga, Bifeng Zhenga,
⁎
Rangru Liuc, Qianbin Lia, Gaoyun Hua, Zhuo Chena,
a Department of Medicinal Chemistry, Xiangya School of Pharmaceutical Sciences, Central South University, Changsha 410013, Hunan, PR China
b Department of Good Clinical Practice, Taihe Hospital, Hubei University of Medicine, Shiyan 442000, Hubei, China
c Key Laboratory of Tropical Diseases and Translational Medicine of the Ministry of Education & Hainan Provincial Key Laboratory of Tropical Medicine, Hainan Medical
College, Haikou, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Bromodomain-containing protein 4 (BRD4), consisting of two tandem bromodomains (BD1 and BD2), is key
epigenetic regulator in fibrosis and cancer, which has been reported that BD1 and BD2 have distinct roles in post-
translational modification. But there are few selective inhibitors toward those two domains. Herein, this study
designed and synthesized a series of novel selective BRD4-BD1 inhibitors, using computer-aided drug design
(CADD) approach focused on exploring the difference of the binding pockets of BD1 and BD2, and finding the
His437 a crucial way to achieve BRD4-BD1 selectivity. Our results revealed that the compound 3u is a potent
selective BRD4-BD1 inhibitor with IC50 values of 0.56 μM for BD1 but > 100 μM for BD2. The compound ex-
hibited a broad spectrum of anti-proliferative activity against several human cancer and fibroblastic cell lines,
which might be related to its capability of reducing the expression of c-Myc and collagen I. Furthermore, it could
induce apoptosis in A375 cells. To the contrary, the selective BD2 inhibitor, RVX-208, did not indicate any of
these activities. Our findings highlight that the function of BRD4-BD1 might be predominant in fibrosis and
cancer. And it is rational to further develop novel selective BRD4-BD1 inhibitors.
BRD4
BD1 inhibitor
Cancer
Fibrosis
Computer-aided drug design
Structure-activity relationship
1. Introduction
fibrosis.4,12 Therefore, targeting BRD4 would be a potential strategy in
cancer and fibrosis therapy.
Bromodomain-containing protein 4 (BRD4), firstly identified in
1988 as a subset of epigenetic “reader”, is a member of the bromodo-
main and extra-terminal (BET) protein family.1 In the past few years,
compared with other BET family proteins (BRD2, BRD3 and BRDT),
BRD4 has been most extensively studied because of its unique relevance
with a number of human diseases including cancer, inflammation,
cardiovascular diseases and multiple organ fibrosis.2–4 Normally, BRD4
localizes in the nucleus and recruits positive transcriptional elongation
factor complex (P-TEFb) to regulate RNA polymerase II activity, via
binding to ε-N-acetylated lysine residues of histone.5–7 In this way,
BRD4 has the advantages of directly controlling downstream gene ex-
pression responsible for cellular proliferation and inflammatory path-
ways. For instance, BRD4 can directly promote transcription of the c-
Myc oncogene and further increase the expression of c-Myc targeting
genes, which play a crucial role in cancer pathogenesis.8–11 In addition,
BRD4 is also reported to play important roles in fibrosis via directly
binding to the enhancer of collagen I, which is the main inducement of
Structurally, BRD4 contains a pair of bromodomains (BD1 and
BD2), both of which are composed of a conserved fold consisting of four
antiparallel helices (αZ, αA, αB, and αC) and two loop regions (ZA and
BC loops) (Fig. 1).13–15 The loop regions make up the pocket of the
acetyl lysine (KAc) binding site, which is located at one end of the helix
bundle. As for the difference of two tandem bromodomains, the amino
acid sequence of BD1 and BD2 are conserved, achieving > 40%
homology.16 Especially at the site of binding pocket, there are only
three crucial residues different from each other and show 95% sequence
identity.17 Although BD1 and BD2 show a high sequence similarity in
the substrate-binding site, they specifically recognize different acet-
ylation substrates in H3 and H4 histone tails.18 And there have been
some reports about the divergent function of BD1 and BD2. For ex-
ample, the selective inhibition of BD1 can accelerate the differentiation
of oligodendrocyte, which is opposite to the effect of non-selective in-
hibitor.19 And it has been reported that BD2 could have broader effects
toward other acetylated substrates, whereas BD1 tends to merely
⁎ Corresponding author.
E-mail address: cz_job@csu.edu.cn (Z. Chen).
https://doi.org/10.1016/j.bmc.2019.03.037
Received 30 January 2019; Received in revised form 14 March 2019; Accepted 19 March 2019
0968-0896/©2019ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:JunlongMa,etal.,Bioorganic&MedicinalChemistry,https://doi.org/10.1016/j.bmc.2019.03.037

J. Ma, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
BD2. Here, we report the design, synthesis and biological evaluation of
a new class of selective BRD4-BD1 inhibitors, thieno[2,3-d]pyrimidin-
2(1H)-ones, which highlight the selective inhibition of BRD4-BD1
might be more efficient in the therapies of cancer and fibrosis.
2. Results and discussion
2.1. Design of target compounds
To design selective BRD4 inhibitor, the 3D crystal structure of the
BRD4-BD1 and BRD4-BD2 at the binding pocket were superimposed
(Fig. 3). The main structural difference is that the Asp144 in the BC loop
of BRD4-BD1 is replaced by a histidine in BRD4-BD2 (His 437). Because
the unique His437 in BRD4-BD2 flips into the KAc binding pocket, the
BRD4-BD2 cavity is narrower than that of BRD4-BD1. This transfor-
mation of space can be exploited to design selective inhibitors. For
example, RVX-208 exhibits tighter affinity towards BRD4-BD2 for its
structural complementarity with narrow binding pocket.23 To the
contrary, few molecules such as Olionone prefers to bind to BRD4-BD1
because of structural clash with His437 in BRD4-BD2.27
Fig. 1. The protein structure of BRD4-BD1 (PDB id: 2YEL).
recognizing H4 acetylation marks.20,21 Consequently, in view of the
different roles of BDs, it would be significant to design selective BRD4
inhibitors to explore whether BRD4-BD1 and BRD4-BD2 have different
functions in diseases (such as cancer and fibrosis), which is beneficial to
achieve more selective therapeutic effects.
Hence, it was hypothesized that an additional insert of steric hin-
drance moiety towards the His437 in BRD4-BD2 on the non-selective
BRD4 inhibitors might generate BRD4-BD1 selectivity. Firstly, (+)-JQ1
was chosen as the lead compound core for its high potency25. As seen
from cocrystal binding mode of (+)-JQ1 with BRD4 (Fig. 4a, b), a tert-
butyl acetate part exposed to the cavity, which have no binding effect
with receptor. The main interaction is that triazole in (+)-JQ1 extends
into the inside of pocket and acts as H-bond acceptor with the Asn140
of BRD4-BD1 or the Asn433 of BRD4-BD2. Meanwhile the chlor-
obenzene part has hydrophobic interaction with WPF region (Trp81/
374, Pro82/375 and Phe83/376). Hence, in our design, the H-bond
acceptor part and phenyl group were reserved in the novel scaffold
building. To avoid probable adverse effects in central nervous system of
azapine scaffold, the azapine ring was directly replaced by pyr-
imidinone group. Consequently, the alkyl group, a steric hindrance
moiety clashing with His437 of BRD4-BD2, was imported into the
thiophene moiety, which expected to achieve BRD4-BD1 selectivity.
The initial structure conception is shown in Fig. 4c.
Because of its comprehensive effects in disease, there have been
several potent BRD4 inhibitors, some of which have been enrolled into
different phases of human clinical trials (Fig. 2).22–26 All of those in-
hibitors own a KAc mimic as hydrogen bond receptor, such as triazole,
isoxazole and amide. Among them, RVX-208, as the first reported BD2
inhibitor, is now in phase III trial for the treatment of cardiovascular
diseases owing to its upregulation of HDL-cholesterol and apolipopro-
tein A1 levels.23 And Olionone, as one of few reported BD1 inhibitor,
had potential off-target effect because of weak binding potency toward
BRD4-BD1. However, except RVX-208 and Olionone, most of them are
non-selective BRD4 inhibitors which can’t discriminate the function of
each bromodomain. Hence, it makes sense to design novel selective
BRD4 inhibitor to explore the discrepant roles of BRD4-BD1 and BRD4-
Fig. 2. Chemical structures of typical BRD4 inhibitors.
2

J. Ma, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Fig. 3. Superimposition of BRD4-BD1 (PDB id:
2YEL) with BRD4-BD2 (PDB id: 2YEM) by residues
(a, BD1 in green, and BD2 in red), binding pockets
(b, BD1 in soild, and BD2 in line) and amino acid
sequences(c). (For interpretation of the references to
colour in this figure legend, the reader is referred to
the web version of this article.)
2.2. Chemistry
2.3. BRD4 inhibitory assay and SAR study
Target compounds 3a–3z were synthesized as outlined in Scheme 1.
All compounds were purified by flash chromatography and purity was
checked by high performance liquid chromatography (HPLC) before
biological evaluation (purity was > 97%). The structures were con-
firmed by 1H NMR, 13C NMR spectrum and mass spectrometry.
As depicted in Scheme 1, an efficient route was developed to syn-
thesize the compounds 3a–3z. Firstly, via Gewald reaction, ketone re-
acted with benzoyl acetonitrile in ethanol to give the compounds
1a–1q. The compounds 2a–2k were obtained by cyclization of inter-
mediate 1a–1q with urea. By using the CeN coupling reaction between
the intermediates 2a–2k and substrates responsible for hydrogen bond
receptors, the target compounds 3a–3z were obtained. However, the
defect of reaction C was the collateral products, O- substituted deriva-
tives, which lead to the low yield. The order of reaction B and reaction
C was reversed to get the single N- substituted compounds. Through the
reaction D to reaction E, the target compounds were obtained with high
yields.
AlphaScreen assay was used to evaluate BRD4 inhibitory activity of
our compounds. (+)-JQ1 and RVX-208 were chosen as positive control.
Initially, compound 3a–3h were synthesized to preliminarily explore
the influence of the spatial factors at thiophene part and the different
hydrogen bond receptors at R2 part. The results were shown in Table 1.
With the increase in the bulk of part A from 3a to 3d, the inhibition rate
and the selectivity between two N-terminal tandem bromodomains
demonstrated that six-membered ring (3c) was the optimal substituent
with 46.4% inhibition of BRD4-BD1 but 26.2% inhibition of BRD4-BD2
at 100 μM. If the ring was too small, it would have weak potency for
BRD4. And if too big as seven members ring, it would lose the se-
lectivity Then, the influence of different hydrogen bond receptors at 3c
and 3e–3h were compared. It’s obvious to find that heterocyclic iso-
xazole might be a preferable group to form hydrogen bond with as-
paraginate. Therefore, through the above initial structure-activity re-
lationship (SAR) analysis, the compound 3c was identified as the lead
compound for further structural modification. Analyzing the docking
conformation of 3c with BRD4-BD1 and BRD4-BD2 (Fig 5a), we found
Fig. 4. (a) Docking conformation of (+)-JQ1 in BRD4-BD1 (PDB id: 2YEL). (b) Docking conformation of (+)-JQ1 in BRD4-BD2 (PDB id: 2YEM). (c) The conception
of designing of the novel BRD4 inhibitors.
3

J. Ma, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Scheme 1. General synthesis of 3a–3z. Reagents and conditions: (A) S, EtOH, morpholine, 60 °C (B) NH2CONH2, 110 °C (C) K2CO3, DMSO, R2Cl, 110 °C (D) K2CO3,
CH3CN, KI, R2Cl 80 °C (E) NH2CONH2, MV 140 °C.
R1 might be a crucial part to achieve selectivity because of its close
distance with His437. Hence, R1 was replaced by methyl formate, di-
methyl, isopropyl, phenyl and tert-butyl. As shown in Table 1, 3o with a
tert-butyl group owned satisfactory potency and selectivity, with the
IC50 value of 2.4 μM for BD1 and > 100 μM for BD2. To verify whether
the selectivity is achieved by the reason of the clash with His437, the
optimal conformation of 3o was docked into the superposed BRD4-BD1
with BRD4-BD2 pocket (Fig 5b). It was obvious to find out that tert-
butyl group was too closed with His437(BRD4-BD2) to be compatible
with the pocket. Consequently, tert-butyl group was indispensable for
the selectivity in structure.
In order to further improve the potency towards BRD4-BD1, we paid
our attention to the WPF region of BRD4. It has been generally ac-
knowledged that WPF region is crucial for the binding potency of in-
hibitors. As shown in Fig. 5a, b, a phenyl group was exactly located in
this region. Hence, some hydrophobic groups (methoxy, methyl for-
mate and halogen) were introduced. By comparing with the activity of
3o–3r (Table 2), the electron donating group (3p) was weaken than the
Table 1
Chemical structure and BRD4 (BD1 and BD2) inhibitory activity of compounds 3a–3o.
Comp.
Structure
n
IC50 (μM)a or (inhibitory rateb)
R1
R2
R3
BRD4-BD1
BRD4-BD2
3a
3b
(9.5%)
(17.8%)
(19.8%)
0
1
2
-H
-H
-H
-H
-H
-H
(23.0%)
3c
3d
(46.4%)
(84.4%)
(26.2%)
(81.2%)
3e
3f
1
1
-H
-H
-H
-H
(23.7%)
(30.0%)
(17.2%)
(13.6%)
3g
1
-H
-H
(38.5%)
(3.1%)
3h
3i
1
1
-H
-Me
-H
-H
(42.6%)
(19.0%)
(66.2%)
(9.0%)
-COOMe
3j
(86.6%)
(44.9%)
(15.1%)
3k
1
1
-Ph
-Ph
-Cl
-Cl
6.0
0.7
3l
(44.9%)
(82.9%)
(14.5%)
3m
3n
(66.6%)
(41.2%)
1
1
-tBu
-tBu
-H
8.2
2.4
0.4
0.2
0.02
3o
-Cl
(23.4%)
(+)-JQ1c
0.17
0.10
0.08
0.01
RVX-208c
3.1
0.12
0.01
a
b
c
Data presented is mean SD value of three independent experiments.
Inhibition rate mean values at a concentration of 100 µM were obtained from three independent experiments.
Used as positive control.
4

J. Ma, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Fig. 5. (a) Docking conformation of 3c in super-
imposition of BRD4-BD1 (PDB id: 2YEL) and BRD4-
BD2 (PDB id: 2YEM). (b) Docking conformation of
3o in in superimposition of BRD4-BD1 (PDB id:
2YEL) and BRD4-BD2 (PDB id: 2YEM). (c) Overlap
of docking conformation of (+)-JQ1(green) and 3u
(red) in BRD4-BD1 (PDB id: 2YEL). (For interpreta-
tion of the references to colour in this figure legend,
the reader is referred to the web version of this ar-
ticle.)
electron withdrawing groups (3o, 3q and 3r) and 4-chlorophenyl
moiety (3o) was identified as the best substituent, which is the same
group as (+)-JQ1′s. For further modification, the substituents of
chlorine were altered to the ortho and meta position of the phenyl
group. Eventually, the compound 3u, containing a 3,4-dichlorophenyl
group, came out as the most suitable compound. Analyzing the docking
conformation of (+)-JQ1 and 3u with BRD4-BD1 (Fig. 5c), the 3,5-di-
methylisoxazole of 3u overlapped with the 3-methyl-1,2,4-triazole of
(+)-JQ1, both of which acted as a KAc mimic. The introduction of 3,4-
dichloro substituent made the plane of the 3,4-dichlorophenyl tend to
be perpendicular to the plane of the pyrimidinone, which could have a
π-π stacking interaction with the Trp81. To verify 3,5-dimethylisox-
azole moiety is an effective KAc mimic, several different heterocycles
were also designed as hydrogen bond receptors (3v–3y) (Table 2).
Meanwhile, the compound 3z, as the O-substituted isomer of the 3u,
was synthesized. By comparing the activities of these compounds, 3u
was reconfirmed as the most suitable compound for further biological
activity evaluation.
Table 2
Chemical structure and BRD4 (BD1 and BD2) inhibitory activity of compounds
3o–3z.
Comp.
Structure
R2
IC50 (μM)a or (inhibitory rateb)
R3
BRD4-BD1
BRD4-BD2
3o
3p
3q
3r
p-Cl
2.4
0.2
0.8
(23.4%)
(42.2%)
(49.7%)
(33.2%)
(52.3%)
(39.5%)
(29.9%)
(66.6%)
(28.3%)
(57.2%)
n.d.d
In general, tert-butyl group was optimal at alkyl part (R1) for BRD4-
BD1 selectivity and 3,5-dimethylisoxazole moiety was the most effec-
tive KAc mimic which interacted with the crucial Asn140. As for WPF
region, 3,4-dichloride might be the most suitable at phenyl substituent
group (R3) for improving potency.
p-OMe
p-COOMe
p-F
11.4
1.5
0.2
0.5
0.2
0.2
3.6
3s
m-Cl
3.1
2.4. Anti-cancer evaluation in vitro
3t
o-Cl
1.6
3u
3v
3w
3x
3y
p-Cl, m-Cl
-H
0.56
0.04
It has been widely recognized that BRD4 is a desired target for the
treatment of cancer. Accordingly, the anti-cancer activity of our com-
pounds was firstly evaluated. Lung cancer cell line (A549), colon cancer
cell line (HT-29), hepatoma cell line (HepG2) and melanoma cell line
(A375) were employed to investigate the cancer cell growth-inhibitory
activity. As shown in Table 3, a large portion of compounds could ef-
fectively inhibit the proliferation of these cancer cell lines at IC50 values
ranging from 2.4 to 19.4 μM, which is similar to (+)-JQ1, the classic
pan-BRD4 inhibitor. While, RVX-208, as the representative of selective
BD2 inhibitor, showed low inhibition potency on these cancer cell lines,
which initially implied different functions of BRD4-BD1 and BRD4-BD2
in cancer. And breast cancer cell line (MCF-7), which has been reported
to be BRD4 inhibitor-resistant cell line,28–30 was also employed to
evaluate synthetic compounds. Compared the cell growth-inhibitory
activity towards other cell lines, most of the synthetic compounds as
(70.4%)
22.7
-H
1.2
1.4
p-OMe
p-Cl
18.5
n.d.d
3z
(32.6%)
0.17
(9.3%)
(+)-JQ1c
RVX-208c
0.02
0.08
0.10
0.01
0.01
3.1
0.12
a
b
Data presented is mean
SD value of three independent experiments.
Inhibition rate mean values at a concentration of 100 µM were obtained
from three independent experiments.
c
d
Used as positive control.
n.d. = not determined.
5

J. Ma, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Table 3
Growth-inhibitory activities towards various cancer cell lines.
Comp.
Antiproliferative activity (IC50, μM)a
A549
HT-29
HepG2
A375
MCF-7
3n
29.6
66.5
18.9
19.3
49.9
18.9
16.6
24.5
31.9
95.1
53.1
13.1
1.8
3.9
0.1
1.4
5.1
2.4
0.3
0.6
2.1
7.6
2.8
1.0
20.1
33.8
13.0
17.8
10.7
19.7
19.2
19.4
54.4
98.0
235.8
19.6
2.6
2.7
1.0
3.2
1.3
2.0
1.6
0.1
6.3
1.6
10.9
26.4
15.1
12.8
63.7
3.6
2.0
3.6
2.8
2.5
7.6
8.0
0.4
1.2
1.8
> 100
78.3
3o
13.6
14.2
8.7
2.0
0.4
1.7
1.3
0.4
3.0
0.4
3p
10.4
3q
1.5
38.6
3r
14.2
15.9
11.1
4.9
1.0
0.8
1.5
33.3
3s
1.7
25.7
3t
30.0
14.5
2.4
1.2
3.3
69.2
3u
1.4
16.6
3w
1.1
14.7
6.5
2.7
0.7
51.2
0.3
> 100
> 100
> 300
38.0
3x
14.5
103.0
11.3
1.3
1.4
0.2
RVX-208b
(+)-JQ1b
36.4
1.9
240.9
7.5
1.1
a
b
Data presented is mean
Used as positive control.
SD value of three independent experiments.
well as (+)-JQ1 are both insensitive to MCF-7 cells, which indicated
that the compounds inhibited the growth of cancer cells via binding to
BRD4.
To further verify discrepant functions in cancer, compound 3u, as
the most potent selective BD1 inhibitor we’ve got, was proceeded to test
the efficacy in A375 cells by measuring reduction of c-Myc, a well-
known proto-oncogene in cancer progression which directly regulated
by BRD4. A375 cells were treated with 3u, (+)-JQ1 and RVX-208 at the
concentration of 10 μM. As shown in Fig. 6a, 3u reduced the expression
of the c-Myc in A375 cells at protein level in a dose-dependent manner.
Significantly, it could be concluded that the selective inhibition of BD1
was sufficient to regulate the expression of c-Myc in A375 cells. These
results were in consistent with the decrease of c-Myc at mRNA level
(Fig. 6b). Both 3u and (+)-JQ1 could inhibit the transcription of target
gene at 10 μM, while RVX-208 was as the same as vehicle control group.
In brief, these results suggested that the effect of BRD4-BD1 and BRD4-
BD2 might be discrepant in cancer cells, and the inhibition of BRD4-
BD1 rather than BRD4-BD2 could play a vital role in the regulation of
oncogene.
Furthermore, the apoptosis-inducing activity related to c-Myc sup-
pression on A375 cells was investigated, by which conducted double-
staining with Annexin V-FITC and PI- and detected by the fluorescence
microscope. As shown in Fig. 7, 3u and (+)-JQ1 displayed similar
ability to induce early apoptosis (the cytomembrane could be stained by
Annexin V-FITC while cell nucleus could not be stained by PI in this
state) of the cells both at the concentration of 10 μM. RVX-208 had no
effect on apoptosis at the same concentration, which was in line with
the weak inhibitory activity of c-Myc and the cell-viability data. In
general, these results indicate the discrepant functions of BRD4-BD1
Fig. 7. Detection of apoptotic cells by Annexin V-FITC and PI double staining.
and BRD4-BD2 in the occurrence of cancer. And specific targeting
single bromodomain of BRD4-BD1 is sufficient in the therapy of cancer.
2.5. Anti-fibrotic evaluation in vitro
The vital role of BRD4 in fibrosis has been revealed in recent years.
However, the detail functions of BRD4-BD1 and BRD4-BD2 on fibrotic
development have not be specified yet. Here, it was explored whether
the selective BRD4-BD1 inhibitors also have the abilities of anti-fibrosis
as well as anti-cancer effects. Pulmonary fibroblastic cell line (HLF-1),
renal fibroblastic cell line (NRK-49F) and liver fibroblastic cell line (LX-
2) were chosen to investigate the fibrotic cell growth-inhibitory activity
using MTT assay (Tables S1 and S2). As shown in Table 4, most of the
compounds displayed potent anti-proliferative activities with average
IC50 value ranging from 4.2 to 19.6 μM, which were more effective than
(+)-JQ1 and RVX-208. And these data imply that BRD4 -BD1 may also
be the primary factor in fibrosis.
In order to investigate the precise mechanism of anti-fibrosis, the
inhibitory effect of 3u to collagen I in HLF-1 cell line was assessed,
which is the main component of the extracellular matrix (ECM) and a
key factor in governing the course of fibrosis. The results displayed that
3u dose-dependently inhibited the expression of collagen I and was
more potent than (+)-JQ1 at the same concentration of 10 μM. To the
Fig. 6. Western blot and quantitative RT-PCR analysis of c-Myc.
6

J. Ma, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Table 4
BD2 by the introduction of tert-butyl group, which would clash with the
unique His437 of BRD4-BD2. SAR study led to the identification of 3u
as the most potent BRD4-BD1 inhibitory activities, which possessed
high selectivity with IC50 values of 0.56 μM for BRD4-BD1
but > 100 μM for BRD4-BD2 Moreover, it showed a significant growth-
inhibitory effect on multiple cancer cell lines as well as fibroblastic cell
lines. The mechanism studies of anti-cancer and anti-fibrotic effects
proved that 3u could inhibit the transcription and translation of c-Myc
and collagen I, while a selective BD2 inhibitor, RVX-208, had no sig-
nificant inhibition. Hence, these results suggest that the BRD4-BD1
might be predominant in fibrosis and cancer, which encourage us to
discovery the more potent selective BRD4-BD1 inhibitors to avoid any
unwanted BRD4-BD2 dependent cellular effects that are unrelated to
the treatments of cancer and fibrosis. Finally, 3u could be used as a
novel lead compound to further develop potent selective BRD4 in-
hibitors against organ fibrosis or cancer.
Growth-inhibitory activities towards various fibroblastic cell lines.
Comp.
Antiproliferative activity (IC50, μM)a
HLF-1
NRF-49F
LX-2
3n
21.3
18.9
14.1
6.6
2.6
0.9
2.0
23.1
26.3
22.2
8.1
3.6
21.6
10.4
12.9
19.6
17.3
15.3
11.3
9.1
3.4
0.8
1.3
1.6
1.3
1.9
4.2
3o
0.8
0.8
3p
3q
0.9
0.9
3r
14.5
12.8
12.0
13.6
7.6
0.4
2.0
0.9
0.9
23.3
8.5
0.8
3s
2.0
3t
17.2
16.0
6.0
2.3
1.1
3u
0.3
0.2
3w
0.6
1.6
4.2
3x
14.6
52.0
20.4
1.5
1.5
1.8
11.2
42.0
21.1
2.4
2.9
0.4
12.4
79.7
28.6
3.8
RVX-208b
(+)-JQ1b
3.4
4.1
a
b
Data presented is mean
Used as positive control.
SD value of three independent experiments.
4. Experimental section
4.1. Chemistry section
4.1.1. A general method for synthesis of compounds 1a–1q
3-oxo-3-phenylpropanenitrile (2.96 g, 20.4 mmol) and pentan-3-one
(2.14 mL, 20.4 mmol) were put into 100 mL round bottom flask, then
50 mL ethyl alcohol chloride as solvent and 1 mL morpholine used as
catalyst for the reaction were added. The reaction mixture was stirred at
room temperature for 15 mins. Then sulphur (0.85 g, 26.6 mmol) was
added to above mixture. The reaction mixture was heated at 60 °C for
12 h. The organic solvent was evaporated and the residue dissolved in
ethyl acetate and purified with silica gel column chromatography
(product eluted at 10% [v/v] ethyl acetate/petroleum ether) to afford
(2-amino-4-ethyl-5-methylthiophen-3-yl)(phenyl) methanone(1a) as a
yellow crystal.
4.1.2. A General method for synthesis of compounds 2a–2k
(2-amino-4-ethyl-5-methylthiophen-3-yl)(phenyl)methanone(1a,
2 g, 8.2 mmol) and urea (0.98 g, 16.3 mmol) were put into 100 mL
round bottom flask, then 40 mL acetic acid as solvent was added. The
reaction mixture was heated at 110 °C for 12 h. The reaction mixture
was then diluted with 150 mL ethyl acetate and washed with brine
(75 mL × 3). The organic solvent was removed under vacuum. The
mixture was dried to get the crude product of 5-ethyl-6-methyl-4-phe-
nylthieno[2,3-d] pyrimidin-2(1H)-one (2a). The crude product was re-
crystallized with EtOH, filtered, and dried in vacuum to give pure
product of 5-ethyl-6-methyl-4-phenylthieno[2,3-d] pyrimidin-2(1H)-
one (2a) as a yellow crystal.
Fig. 8. Western blot and quantitative RT-PCR analysis of collagen I.
contrary, RVX-208 had no inhibition effect on the expression of col-
lagen I (Fig. 8a). And quantitative RT-PCR assay was subsequently
performed to verify the mRNA results. As expected, 3u at 1 μM was as
the potent as (+)-JQ1 at 10 μM in the inhibition transcription of col-
lagen I, while this effect was not observed with RVX-208 treatment
(Fig. 8b). Collectively, the mRNA level were in keeping with protein
level, which may account for its anti-proliferative activity. In summary,
all the experiments highlight that BRD4-BD1 plays the dominate role in
the process of fibrosis and selectively inhibiting BRD4-BD1 can de-
monstrate more potent anti-fibrotic activity than the inhibition of both
BRD4-BD1 and BRD4-BD2.
4.1.3. A general method for synthesis of compounds 2l to 2s
(2-amino-6-(tert-butyl)-4,5,6,7-tetrahydrobenzo[b]thiophen-3-yl)
(3,4-dichlorophenyl) methanone(1p, 2 g, 5.2 mmol) and 4-(chlor-
omethyl)-3,5-dimethylisoxazole (0.78 mL, 6.2 mmol) were put into
100 mL round bottom flask. Then 50 mL acetonitrile as solvent, 0.1 g
potassium carbonate and 0.1 g potassium iodide used as catalyst for the
reaction were added. The reaction mixture was heated at 80 °C for 4 h.
The organic solvent was removed under vacuum. The mixture was dried
to get the crude product of 5-ethyl-6-methyl-4-phenylthieno[2,3-d]
pyrimidin-2(1H)-one (2a). The crude product was recrystallised with
EtOH, filtered, and dried in vacuum to give pure product of (6-(tert-
butyl)-2-(((3,5-dimethylisoxazol-4-yl)methyl)amino)-4,5,6,7-tetra-
hydrobenzo [b]thiophen-3-yl)(3,4-dichlorophenyl)methanone (2s) as a
yellow crystal.
3. Conclusions
BRD4 plays a critical role in the development of cancer and fibrosis.
And the close relevance of cancer and fibrosis, which is like the re-
lationship between the seed and the soil, highlights the advantages of
BRD4 and makes it a promising drug target. However, the specific
functions of BRD4-BD1 and BRD4-BD2 are unclear in the occurrence of
cancer and fibrosis. Until now, most of BRD4 inhibitors are non-selec-
tive. Especially for BD1, there are no potent selective inhibitors. Thus,
there remains an urgent need to develop potent and selective BRD4-
BD1 inhibitors for biological studies and disease therapeutics.
4.1.4. A General method for synthesis of compounds 3a–3z
Herein, through preliminarily combining molecular docking and
molecular dynamic study, a series of novel BRD4 inhibitors were de-
signed and synthesized with high selectivity for BRD4-BD1 over BRD4-
5-ethyl-6-methyl-4-phenylthieno[2,3-d] pyrimidin-2(1H)-one (2a,
2 g, 7.4 mmol) and 4-(chloromethyl)-3,5-dimethylisoxazole (0.94 mL,
8.9 mmol) were put into 100 mL round bottom flask, then 50 mL
7

J. Ma, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
dimethylsulfoxide as solvent and 0.1 g potassium carbonate used as
catalyst for the reaction were added. The reaction mixture was heated
at 110 °C for 1.5 h. The reaction mixture was then diluted with 100 mL
dichloromethane and washed with brine (50 mL × 3). The organic
solvent was removed under vacuum. The residue dissolved in ethyl
acetate and purified with silica gel column chromatography (product
eluted at 100% [v/v] ethyl acetate/petroleum ether) to afford 1-((3,5-
dimethylisoxazol-4-yl)methyl)-5-ethyl-6-methyl-4-phenylthieno[2,3-d]
pyrimidin-2(1H)-one (3a) as a yellow crystal.
406.1597.
4.1.5.5. 1-(2-oxopropyl)-4-phenyl-5,6,7,8-tetrahydrobenzo[4,5]thieno
[2,3-d]pyrimidin-2(1H)-one (3e). Yellow solid, yield 77%, m.p.
234.4–236.6 °C; 1H NMR (500 MHz, DMSO) δ 7.56–7.47 (m, 5H),
5.08 (d, J = 11.6 Hz, 2H), 2.69 (s, 2H), 2.32 (s, 3H), 1.90 (s, 2H),
1.72 (d, J = 4.3 Hz, 2H), 1.49 (d, J = 4.7 Hz, 2H); 13C NMR (126 MHz,
DMSO) δ 200.96, 169.05, 159.62, 154.25, 138.50, 130.13, 129.83,
129.24, 128.47, 128.34, 118.05, 57.93, 28.03, 26.91, 24.81, 22.59,
22.18; HR-ESI-MS m/z: calcd for C19H19N2O2S{[M+H]+}339.1167,
found 339.1189.
4.1.5. A General method for synthesis of compounds 3p–3q and 3s–3u
(6-(tert-butyl)-2-(((3,5-dimethylisoxazol-4-yl)methyl)amino)-
4,5,6,7-tetrahydrobenzo[b] thiophen-3-yl)(3,4-dichlorophenyl)metha-
none (2s, 0.5 g, 1.0 mmol) and urea (0.12 g, 2.1 mmol) were put into
25 mL pressure flask, then 15 mL acetic acid as solvent was added. The
reaction mixture was heated at 145 °C under microwave for 25 mins.
The reaction mixture was then diluted with 30 mL ethyl acetate and
washed with brine (20 mL × 3). The organic solvent was removed
under vacuum. The mixture was dried to get the crude product 7-(tert-
butyl)-4-(3,4-dichlorophenyl)-1-((3,5-dimethylisoxazol-4-yl)methyl)-
5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-2(1H)-one (3u).
The crude product was recrystallized with EtOH, filtered, and dried in
vacuum to give pure product of 7-(tert-butyl)-4-(3,4-dichlorophenyl)-1-
((3,5-dimethylisoxazol-4-yl)methyl)-5,6,7,8-tetrahydrobenzo[4,5]
thieno[2,3-d]pyrimidin-2(1H)-one (3u) as a yellow crystal.
4.1.5.6. 1-(3-oxobutan-2-yl)-4-phenyl-5,6,7,8-tetrahydrobenzo[4,5]thieno
[2,3-d]pyrimidin-2(1H)-one (3f). Yellow solid, yield 62%, m.p.
227.5–230.2 °C; 1H NMR (500 MHz, DMSO) δ 7.50 (s, 5H), 4.88 (d,
J = 6.4 Hz, 1H), 2.76 (d, J = 35.3 Hz, 2H), 2.07 (d, J = 37.4 Hz, 3H),
1.90 (s, 2H), 1.74 (s, 2H), 1.55 (t, J = 11.0 Hz, 3H), 1.49 (s, 2H); 13C
NMR (126 MHz, DMSO) δ 201.44, 169.24, 159.12, 153.59, 138.47,
130.19, 130.13, 129.36, 128.42, 118.78, 65.68, 26.94, 26.53, 24.84,
22.61, 22.20, 12.90; HR-ESI-MS m/z: calcd for
C20H21N2O2S{[M
+H]+}353.1324, found 353.1371.
4.1.5.7. N-methyl-2-(2-oxo-4-phenyl-5,6,7,8-tetrahydrobenzo[4,5]thieno
[2,3-d]pyrimidin-1(2H)-yl)acetamide (3g). Brown solid, yield 89%, m.p.
173.4–175.2 °C; 1H NMR (500 MHz, DMSO) δ 8.01 (d, J = 3.6 Hz, 1H),
7.61–7.45 (m, 5H), 4.81 (s, 2H), 2.81 (s, 2H), 2.63 (d, J = 4.4 Hz, 3H),
2.05 (s, 2H), 1.79 (d, J = 4.1 Hz, 2H), 1.56 (d, J = 5.1 Hz, 2H); 13C
NMR (126 MHz, DMSO) δ 171.00, 168.21, 162.79, 160.21, 138.32,
134.83, 129.93, 129.36, 128.36, 127.11, 124.53, 66.03, 27.10, 25.81,
4.1.5.1. 1-((3,5-dimethylisoxazol-4-yl)methyl)-5-ethyl-6-methyl-4-
phenylthieno[2,3-d]pyrimidin-2(1H)-one (3a). Yellow solid, yield 82%,
m.p. 163.6–165.8 °C; 1H NMR (500 MHz, DMSO) δ 7.57–7.48 (m, 5H),
5.20 (d, J = 13.2 Hz, 2H), 2.45 (s, 3H), 2.31 (s, 3H), 2.12 (s, 3H), 2.09
(d, J = 7.2 Hz, 2H), 0.75–0.44 (m, 3H); 13C NMR (126 MHz, DMSO) δ
168.99, 168.53, 159.44, 157.28, 154.37, 138.58, 133.99, 129.99,
128.38, 128.13, 126.26, 118.44, 108.17, 41.09, 20.02, 14.43, 12.83,
25.74, 22.63, 22.40; HR-ESI-MS m/z: calcd for
C19H20N3O2S{[M
+H]+}354.1276, found 354.1284.
4.1.5.8. 1-methyl-4-phenyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]
11.64, 10.58; HR-ESI-MS m/z: calcd for
C
21H22N3O2S{[M+H]+
}
pyrimidin-2(1H)-one
(3h). Yellow
solid,
yield
92%,
m.p.
380.1433, found 380.1442.
278.4–280.1 °C; 1H NMR (500 MHz, DMSO) δ 7.61 (t, J = 9.9 Hz,
3H), 7.53 (d, J = 6.6 Hz, 2H), 3.16 (s, 3H), 2.58 (s, 2H), 1.65 (s, 2H),
1.43 (d, J = 11.6 Hz, 4H); 13C NMR (126 MHz, DMSO) δ 177.20,
154.06, 153.58, 132.01, 131.94, 130.80, 129.24, 128.66, 126.49,
117.50, 35.31, 25.53, 24.94, 22.09; HR-ESI-MS m/z: calcd for
4.1.5.2. 1-((3,5-dimethylisoxazol-4-yl)methyl)-4-phenyl-1,5,6,7-
tetrahydro-2H-cyclopenta[4,5]thieno[2,3-d] pyrimidin-2-one (3b). Yellow
solid, yield 75%, m.p. 166.7–168.8 °C; 1H NMR (500 MHz, DMSO) δ
7.56 (ddd, J = 21.7, 15.1, 7.3 Hz, 5H), 5.20 (s, 2H), 2.84 (t, J = 6.4 Hz,
2H), 2.45 (s, 3H), 2.34 (d, J = 6.3 Hz, 2H), 2.22 (dd, J = 13.7, 6.9 Hz,
2H), 2.11 (s, 3H); 13C NMR (126 MHz, DMSO) δ 168.55, 168.00,
162.43, 159.42, 154.99, 138.87, 137.65, 134.17, 130.74, 129.02,
128.50, 114.96, 108.15, 40.99, 30.78, 29.21, 27.77, 11.63, 10.57;
HR-ESI-MS m/z: calcd for C21H20N3O2S{[M+H]+}378.1276, found
378.1283.
C
17H17N2OS{[M+H]+}297.1062, found 297.1071.
4.1.5.9. Methyl
1-((3,5-dimethylisoxazol-4-yl)methyl)-2-oxo-4-phenyl-
1,2,5,6,7,8-hexahydrobenzo[4,5]thieno[2,3-d] pyrimidine-7-carboxylate
(3i). White solid, yield 62%, m.p. 94.1–96.1 °C; 1H NMR (500 MHz,
DMSO) δ 7.58–7.45 (m, 5H), 5.19 (dd, J = 68.6, 15.7 Hz, 2H), 3.61 (s,
3H), 2.98 (d, J = 12.6 Hz, 1H), 2.89–2.80 (m, 2H), 2.46 (s, 3H), 2.12 (s,
3H), 2.08 (s, 1H), 1.83 (d, J = 16.2 Hz, 2H), 1.53 (d, J = 8.4 Hz, 1H);
13C NMR (126 MHz, DMSO) δ 174.51, 168.78, 168.63, 159.44, 157.95,
154.57, 138.41, 130.19, 128.99, 128.46, 128.43, 127.78, 118.15,
108.12, 52.20, 41.15, 38.75, 26.95, 25.67, 24.94, 11.65, 10.57; HR-
4.1.5.3. 1-((3,5-dimethylisoxazol-4-yl)methyl)-4-phenyl-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-2(1H)-one
(3c). Yellow
solid, yield 82%, m.p. 184.1–186.6 °C; 1H NMR (500 MHz, DMSO) δ
7.57–7.46 (m, 5H), 5.19 (s, 2H), 2.66 (d, J = 19.5 Hz, 2H), 2.44 (s, 3H),
2.11 (s, 3H), 1.87 (s, 2H), 1.70 (d, J = 4.7 Hz, 2H), 1.47 (d, J = 4.7 Hz,
2H); 13C NMR (126 MHz, DMSO) δ 168.78, 168.53, 159.44, 157.72,
154.60, 138.57, 130.09, 129.53, 129.47, 128.41, 118.57, 108.18,
41.23, 26.83, 24.78, 22.60, 22.20, 11.64, 10.58; HR-ESI-MS m/z:
calcd for C22H22N3O2S{[M+H]+}392.1433, found 392.1477.
ESI-MS m/z: calcd for
C
24H24N3O4S{[M+H]+}450.1488, found
450.1500.
4.1.5.10. 1-((3,5-dimethylisoxazol-4-yl)methyl)-7,7-dimethyl-4-phenyl-
5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d] pyrimidin-2(1H)-one (3j). White
solid, yield 79%, m.p. 180.0–182.6 °C; 1H NMR (500 MHz, DMSO) δ
7.56–7.48 (m, 5H), 5.19 (s, 2H), 2.47 (s, 2H), 2.45 (s, 3H), 2.12 (s, 3H),
1.88 (s, 2H), 1.25 (s, 2H), 0.92 (s, 6H); 13C NMR (126 MHz, DMSO) δ
168.83, 168.56, 159.45, 157.77, 154.60, 138.58, 130.06, 128.88, 128.44,
128.38, 128.02, 118.36, 108.20, 41.12, 38.17, 34.88, 30.23, 27.84, 24.28,
4.1.5.4. 1-((3,5-dimethylisoxazol-4-yl)methyl)-4-phenyl-1,5,6,7,8,9-
hexahydro-2H-cyclohepta[4,5]thieno[2,3-d] pyrimidin-2-one (3d). Rufous
solid, yield 72%, m.p. 159.8–161.5 °C; 1H NMR (500 MHz, CDCl3) δ 7.38
(d, J = 6.9 Hz, 2H), 7.27 (dd, J = 15.9, 8.6 Hz, 3H), 5.03 (s, 2H), 2.59 (d,
J = 4.1 Hz, 2H), 2.31 (s, 3H), 2.06 (s, 3H), 2.05–1.99 (m, 2H), 1.60 (s, 2H),
1.50 (s, 2H), 1.24 (s, 2H); 13C NMR (126 MHz, CDCl3) δ 168.82, 168.07,
159.13, 155.51, 154.85, 137.72, 135.28, 132.77, 130.12, 128.59, 127.92,
119.75, 107.40, 40.70, 31.72, 29.14, 28.82, 27.01, 26.38, 11.58, 10.53;
11.65, 10.59; HR-ESI-MS m/z: calcd for
C
24H26N3O2S{[M+H]+
}
420.1746, found 420.1752.
4.1.5.11. 4-(4-chlorophenyl)-1-((3,5-dimethylisoxazol-4-yl)methyl)-7-
phenyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]
pyrimidin-2(1H)-one
HR-ESI-MS m/z: calcd for
C
23H24N3O2S{[M+H]+}406.1589, found
(3k). Yellow solid, yield 90%, m.p. 123.1–125.6 °C; 1H NMR
8

J. Ma, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
(500 MHz, DMSO) δ 7.60–7.52 (m, 4H), 7.28 (dt, J = 14.7, 7.4 Hz, 4H),
7.20 (t, J = 7.0 Hz, 1H), 5.19 (dd, J = 78.7, 15.8 Hz, 2H), 3.03–2.90
(m, 2H), 2.83–2.73 (m, 1H), 2.44 (d, J = 10.6 Hz, 3H), 2.34–2.25 (m,
1H), 2.14 (d, J = 12.9 Hz, 3H), 1.92–1.76 (m, 2H), 1.63 (qd, J = 12.2,
4.9 Hz, 1H); 13C NMR (126 MHz, DMSO) 168.59, 167.57, 159.45,
158.28, 154.51, 145.60, 137.25, 135.01, 130.53, 129.40, 129.05,
128.88, 128.59, 127.23, 126.84, 118.25, 108.12, 41.32, 40.04, 32.46,
29.62, 27.36, 11.66, 10.60; HR-ESI-MS m/z: calcd for C28H25ClN3O2S
{[M+H]+}502.1356, found 502.1357.
J = 43.7, 14.0 Hz, 2H), 1.47 (dd, J = 11.1, 6.7 Hz, 1H), 1.05–0.96
(m, 1H), 0.86 (s, 9H); 13C NMR (126 MHz, DMSO) δ 168.51, 168.28,
161.02, 159.39, 157.78, 154.64, 130.68, 130.56, 130.06, 129.64,
118.24, 113.73, 108.23, 55.72, 44.64, 40.96, 32.61, 28.27, 27.37,
26.63, 24.06, 11.61, 10.55. HR-ESI-MS m/z: calcd for C27H32N3O3S{[M
+H]+}478.2146, found 478.2170.
4.1.5.17. methyl 4-(7-(tert-butyl)-1-((3,5-dimethylisoxazol-4-yl)methyl)-
2-oxo-1,2,5,6,7,8-hexahydrobenzo [4,5]thieno [2,3-d]pyrimidin-4-yl)
benzoate (3q). Yellow solid, yield 64%, m.p. 208.1–210.6 °C; 1H NMR
(500 MHz, DMSO) δ 8.08 (d, J = 8.1 Hz, 2H), 7.64 (d, J = 8.0 Hz, 2H),
5.29–5.10 (m, 2H), 3.90 (s, 3H), 2.76 (dd, J = 16.6, 4.3 Hz, 1H), 2.45
(s, 3H), 2.40 (d, J = 13.9 Hz, 1H), 2.10 (d, J = 14.6 Hz, 3H), 2.05 (t,
J = 13.1 Hz, 1H), 1.72 (d, J = 11.2 Hz, 2H), 1.42 (td, J = 11.7, 4.5 Hz,
1H), 0.97 (qd, J = 12.6, 4.3 Hz, 1H), 0.84 (s, 9H); 13C NMR (126 MHz,
DMSO) δ 168.64, 167.58, 166.28, 159.44, 158.29, 154.50, 142.74,
130.93, 130.57, 129.31, 129.15, 128.99, 118.14, 108.09, 52.82, 44.59,
41.26, 32.60, 27.71, 27.39, 26.56, 23.91, 11.65, 10.58; HR-ESI-MS m/z:
calcd for C28H32N3O4S{[M+H]+}506.2114, found 506.2111.
4.1.5.12. 4-(4-chlorophenyl)-1-((2-chlorothiazol-5-yl)methyl)-7-phenyl-
5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d] pyrimidin-2(1H)-one (3l). Yellow
solid, yield 79%, m.p. 203.9–205.0 °C; 1H NMR (500 MHz, DMSO) δ 7.93
(s, 1H), 7.56 (dd, J = 21.3, 8.4 Hz, 4H), 7.34–7.25 (m, 4H), 7.22 (t,
J = 6.9 Hz, 1H), 5.43 (s, 2H), 3.07–2.95 (m, 2H), 2.89–2.82 (m, 1H), 2.28
(d, J = 11.6 Hz, 1H), 1.89–1.79 (m, 2H), 1.65 (dt, J = 11.6, 7.3 Hz, 1H);
13C NMR (126 MHz, DMSO) δ 168.06, 158.25, 154.13, 152.51, 145.61,
143.05, 137.20, 135.05, 133.85, 130.47, 129.61, 128.92, 128.63, 127.30,
126.88, 118.45, 45.70, 40.60, 32.49, 29.63, 27.34; HR-ESI-MS m/z: calcd
for C26H20Cl2N3OS2{[M+H]+}524.0425, found 524.0420.
4.1.5.18. 7-(tert-butyl)-1-((3,5-dimethylisoxazol-4-yl)methyl)-4-(4-
4.1.5.13. 1-((3,5-dimethylisoxazol-4-yl)methyl)-7-isopropyl-4-(4-
fluorophenyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno
[2,3-d]pyrimidin-
methoxyphenyl)-5,6,7,8-
Tetrahydropyrido
[4′,3′:4,5]thieno[2,3-d]
2(1H)-one (3r). Yellow solid, yield 92%, m.p. 241.4–243.4 °C; 1H
NMR (500 MHz, DMSO) δ 7.56 (dd, J = 8.0, 5.7 Hz, 2H), 7.33 (t,
J = 8.7 Hz, 2H), 5.18 (dd, J = 98.8, 15.7 Hz, 2H), 2.76 (dd, J = 16.6,
4.1 Hz, 1H), 2.45 (s, 3H), 2.41 (d, J = 14.3 Hz, 1H), 2.13 (d,
J = 14.7 Hz, 1H), 2.11 (s, 3H), 1.85–1.70 (m, 2H), 1.43 (td, J = 11.7,
4.3 Hz, 1H), 1.00 (tt, J = 12.1, 5.9 Hz, 1H), 0.85 (s, 9H); 13C NMR
(126 MHz, DMSO) δ 168.59, 167.63, 164.33, 162.37, 159.42, 158.04,
154.56, 134.86, 131.10, 131.04, 130.33, 129.37, 118.29, 115.54,
115.36, 108.15, 44.62, 41.12, 32.61, 27.94, 27.39, 26.59, 24.00,
pyrimidin-2(1H)-one (3m). Brown solid, yield 86%, m.p. 69.1–71.6 °C;
1H NMR (500 MHz, DMSO) δ 7.49 (t, J = 8.4 Hz, 2H), 7.05 (d,
J = 8.4 Hz, 2H), 5.18 (s, 2H), 3.84 (s, 3H), 3.63 (s, 2H), 2.80 (dt,
J = 13.0, 6.5 Hz, 1H), 2.44 (s, 3H), 2.15 (dd, J = 15.7, 6.8 Hz, 2H),
2.10 (s, 3H), 1.59 (d, J = 6.2 Hz, 2H), 0.98 (t, J = 10.3 Hz, 6H); 13C
NMR (126 MHz, DMSO) δ 168.47, 161.11, 159.42, 157.91, 154.71,
130.62, 130.52, 128.52, 127.94, 118.07, 114.91, 113.82, 108.18,
55.75, 53.78, 47.10, 45.79, 41.12, 18.73, 14.41, 11.61, 10.54; HR-
ESI-MS m/z: calcd for
C
25H29N4O3S{[M+H]+}465.1960, found
11.64, 10.57; HR-ESI-MS m/z: calcd for C26H29FN3O2S{[M+H]+
}
465.1963.
466.1965, found 466.1960.
4.1.5.14. 7-(tert-butyl)-1-((3,5-dimethylisoxazol-4-yl)methyl)-4-phenyl-
5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d] pyrimidin-2(1H)-one (3n). White
solid, yield 74%, m.p. 245.8–247.6 °C; 1H NMR (500 MHz, DMSO) δ 7.51
(dd, J = 16.4, 8.4 Hz, 5H), 5.19 (dd, J = 86.0, 15.7 Hz, 2H), 2.76 (d,
J = 14.6 Hz, 1H), 2.45 (s, 3H), 2.41 (d, J = 15.4 Hz, 1H), 2.12 (s, 3H), 2.07
(d, J = 13.2 Hz, 1H), 1.74 (dd, J = 25.9, 13.8 Hz, 2H), 1.43 (t, J = 9.5 Hz,
1H), 0.99 (dd, J = 16.9, 7.7 Hz, 1H), 0.85 (s, 9H);13C NMR (126 MHz,
DMSO) δ 168.69, 168.57, 159.43, 157.91, 154.64, 138.46, 130.25, 130.12,
129.45, 128.47, 128.43, 118.26, 108.17, 44.61, 41.14, 32.62, 27.80, 27.41,
26.57, 23.96, 11.64, 10.57; HR-ESI-MS m/z: calcd for C26H30N3O2S{[M
+H]+}448.2059, found 448.2067.
4.1.5.19. 7-(tert-butyl)-4-(3-chlorophenyl)-1-((3,5-dimethylisoxazol-4-yl)
methyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno [2,3-d]pyrimidin-2(1H)-one
(3s). Yellow solid, yield 79%, m.p. 198.8–201.0 °C; 1H NMR
(500 MHz, DMSO) δ 7.60 (t, J = 5.9 Hz, 1H), 7.57 (d, J = 13.5 Hz,
1H), 7.53 (t, J = 7.7 Hz, 1H), 7.46 (d, J = 7.5 Hz, 1H), 5.30–5.07 (m,
2H), 2.77 (dd, J = 16.5, 3.9 Hz, 1H), 2.45 (s, 3H), 2.41 (d, J = 14.3 Hz,
1H), 2.10 (d, J = 12.7 Hz, 3H), 2.09–2.00 (m, 1H), 1.86–1.71 (m, 2H),
1.47–1.40 (m, 1H), 1.01 (dt, J = 13.2, 8.5 Hz, 1H), 0.85 (s, 9H); 13C
NMR (126 MHz, DMSO) δ 168.64, 166.96, 159.44, 158.23, 154.50,
140.36, 133.21, 130.52, 130.43, 129.98, 129.16, 128.25, 127.28,
118.18, 108.10, 44.57, 41.21, 32.62, 27.74, 27.40, 26.56, 23.96,
11.65, 10.58; HR-ESI-MS m/z: calcd for C26H29ClN3O2S{[M+H]+
}
4.1.5.15. 7-(tert-butyl)-4-(4-chlorophenyl)-1-((3,5-dimethylisoxazol-4-yl)
methyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno [2,3-d]pyrimidin-2(1H)-one
(3o). Yellow solid, yield 89%, m.p. 154.2–156.1 °C; 1H NMR
(500 MHz, DMSO) δ 7.56 (dd, J = 21.8, 8.5 Hz, 4H), 5.29–5.08 (m,
2H), 2.77 (dd, J = 16.7, 4.7 Hz, 1H), 2.45 (s, 3H), 2.44–2.37 (m, 1H),
2.11 (s, 3H), 2.11–2.05 (m, 1H), 1.79 (t, J = 11.1 Hz, 2H), 1.44 (td,
482.1669, found 482.1671.
4.1.5.20. 7-(tert-butyl)-4-(2-chlorophenyl)-1-((3,5-dimethylisoxazol-4-yl)
methyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno [2,3-d]pyrimidin-2(1H)-one
(3t). Yellow solid, yield 75%, m.p. 108.1–110.6 °C; 1H NMR
(500 MHz, DMSO) δ 7.70–7.40 (m, 4H), 5.22 (dt, J = 34.5, 15.7 Hz,
2H), 2.74 (t, J = 14.0 Hz, 1H), 2.44 (d, J = 2.1 Hz, 3H), 2.38 (d,
J = 17.1 Hz, 1H), 2.06 (d, J = 35.4 Hz, 3H), 2.03–1.89 (m, 1H),
1.77–1.66 (m, 2H), 1.39–1.30 (m, 1H), 1.03 (ddd, J = 20.0, 14.5,
6.9 Hz, 1H), 0.80 (d, J = 32.4 Hz, 9H); 13C NMR (126 MHz, DMSO) δ
168.39, 165.92, 159.17, 157.53, 154.71, 137.31, 131.57, 131.04,
130.69, 129.76, 129.66, 128.95, 127.83, 118.79, 108.03, 44.56,
41.33, 32.67, 27.42, 26.36, 25.18, 23.58, 11.61, 10.40; HR-ESI-MS
m/z: calcd for C26H29ClN3O2S{[M+H]+}482.1669, found 482.1669.
J = 11.7, 4.4 Hz, 1H), 1.00 (dt, J = 12.3, 7.5 Hz, 1H), 0.86 (s, 9H); 13
C
NMR (126 MHz, DMSO) δ 168.64, 167.42, 159.45, 158.17, 154.54,
137.22, 134.97, 130.56, 130.45, 129.25, 128.56, 118.20, 108.12,
44.60, 41.17, 32.63, 27.91, 27.41, 26.59, 23.97, 11.65, 10.59; HR-
ESI-MS m/z: calcd for
C
26H29ClN3O2S{[M+H]+}482.1669, found
482.1673.
4.1.5.16. 7-(tert-butyl)-1-((3,5-dimethylisoxazol-4-yl)methyl)-4-(4-
methoxyphenyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno
[2,3-d]pyrimidin-
2(1H)-one (3p). Yellow solid, yield 67%, m.p. 120.1–122.6 °C; 1H
4.1.5.21. 7-(tert-butyl)-4-(3,4-dichlorophenyl)-1-((3,5-dimethylisoxazol-
4-yl)methyl)-5,6,7,8-tetrahydrobenzo[4,5] thieno[2,3-d]pyrimidin-2(1H)-
one (3u). Yellow solid, yield 93%, m.p. 202.1–204.4 °C; 1H NMR
(500 MHz, DMSO) δ 7.81 (t, J = 4.9 Hz, 1H), 7.77 (d, J = 8.2 Hz,
1H), 7.51 (dd, J = 8.2, 1.4 Hz, 1H), 5.19 (dd, J = 89.6, 15.7 Hz, 2H),
NMR (400 MHz, DMSO)
δ 7.47 (d, J = 8.6 Hz, 2H), 7.05 (d,
J = 8.7 Hz, 2H), 5.18 (dd, J = 93.1, 15.8 Hz, 2H), 3.84 (s, 3H), 2.77
(dd, J = 16.6, 4.3 Hz, 1H), 2.46 (d, J = 7.8 Hz, 1H), 2.43 (d,
J = 16.5 Hz, 3H), 2.26–2.17 (m, 1H), 2.10 (s, 3H), 1.83 (dd,
9

J. Ma, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
2.77 (dd, J = 16.6, 4.2 Hz, 1H), 2.45 (s, 3H), 2.41 (d, J = 14.2 Hz, 1H),
2.11 (s, 3H), 2.06 (s, 1H), 1.88–1.76 (m, 2H), 1.43 (td, J = 11.6, 4.3 Hz,
1H), 1.05–0.96 (m, 1H), 0.86 (d, J = 9.1 Hz, 9H); 13C NMR (126 MHz,
DMSO) δ 168.69, 165.91, 159.45, 158.41, 154.41, 138.82, 132.93,
131.38, 130.80, 130.64, 130.59, 129.06, 128.99, 118.16, 108.06,
44.56, 41.21, 32.62, 27.85, 27.39, 26.58, 23.97, 11.66, 10.60; HR-
ESI-MS m/z: calcd for C26H28Cl2N3O2S{[M+H]+}516.1279, found
516.1274.
(126 MHz, DMSO) δ 171.48, 168.62, 160.23, 160.11, 160.05, 138.62,
136.01, 132.83, 131.33, 130.66, 129.85, 126.76, 124.10, 110.59,
100.04, 58.59, 44.51, 32.62, 28.02, 27.53, 27.39, 24.11, 11.31,
10.15; HR-ESI-MS m/z: calcd for C26H28Cl2N3O2S{[M+H]+
}
516.1279, found 516.1277.
4.2. Docking study
All ligands were drawn in ChemDraw 2014, and saved as sdf style.
Ligand molecules were prepared using the MOE2018 to get low-energy
3D conformations. Protein (PDB code: 2YEM, 2YEL) was retrieved from
RSCB database and prepared using the protein preparation wizard from
MOE2018. Molecular docking was performed using a flexible ligand
and fixed receptor models. Ligand conformations are generated with
the bond rotation method. These are then placed in the site with the
Triangle Matcher method, and ranked with the London dG scoring
function. The Retain option specifies the number of poses (2 0 0) to pass
to the Refinement, for energy minimization in the pocket, before re-
scoring with the GBVI/WSA dG scoring function. At last, only the best-
scoring ligand protein complexes were kept for analyses
4.1.5.22. 7-(tert-butyl)-4-phenyl-1-(pyrimidin-2-ylmethyl)-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-2(1H)-one
(3v). Yellow
solid, yield 78%, m.p. 185.5–186.3 °C; 1H NMR (500 MHz, DMSO) δ
8.77 (d, J = 4.8 Hz, 2H), 7.55–7.36 (m, 6H), 5.69–5.56 (m, 2H), 2.81
(dd, J = 16.9, 3.7 Hz, 1H), 2.52 (d, J = 11.8 Hz, 1H), 2.24 (t,
J = 13.0 Hz, 1H), 1.87 (d, J = 15.0 Hz, 1H), 1.76 (d, J = 11.4 Hz,
1H), 1.55–1.44 (m, 1H), 1.06–0.97 (m, 1H), 0.85 (s, 9H); 13C NMR
(126 MHz, DMSO) δ 171.28, 165.80, 162.55, 160.56, 157.80, 138.19,
135.32, 129.89, 129.34, 128.32, 127.00, 124.03, 120.61, 68.80, 44.54,
32.58, 28.10, 27.50, 27.38, 24.10; HR-ESI-MS m/z: calcd for
C
25H27N4OS{[M+H]+}431.1906, found 431.1898.
4.1.5.23. 7-(tert-butyl)-1-((2-chlorothiazol-5-yl)methyl)-4-phenyl-5,6,7,8-
4.3. Biological evaluation
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-2(1H)-one
(3w). White
solid, yield 90%, m.p. 225.2–226.7 °C; 1H NMR (500 MHz, DMSO) δ
7.92 (s, 1H), 7.56–7.45 (m, 5H), 5.45–5.38 (m, 2H), 2.78 (dd, J = 16.5,
4.4 Hz, 1H), 2.45 (d, J = 13.5 Hz, 1H), 2.07 (t, J = 14.2 Hz, 1H), 1.73
(t, J = 14.5 Hz, 2H), 1.45 (td, J = 11.5, 4.2 Hz, 1H), 0.97 (tt, J = 11.4,
5.8 Hz, 1H), 0.85 (s, 9H); 13C NMR (126 MHz, DMSO) δ 169.16, 157.94,
154.22, 152.50, 143.04, 138.34, 133.91, 130.46, 130.20, 130.05,
128.46, 128.43, 118.44, 45.61, 44.60, 32.63, 27.83, 27.40, 26.62,
23.91; HR-ESI-MS m/z: calcd for C24H25ClN3OS2{[M+H]+}470.1128,
found 470.1133.
4.3.1. Binding affinity of target compounds to BRD4-BD1 and BRD4-BD2
by AlphaScreen assay.
The bead-based Amplifed Luminescent Proximity Homogeneous
Assay (ALPHA) was used to measure the binding of a tetra-acetylated
histone H4 tail peptide to BRD4 BD1 and BD2. BRD4(1) (No. AL609C,
Perkin Elmer), BRD4(2) (No. AL610C, Perkin Elmer), (+)-JQ1(No.
BD229144, Bidepharm), RVX-208(No. BD229143, Bidepharm). Prepare
1X Alpha Assay Buffer (50 mM HEPES, 01% BSA); serial dilution of
sample (Preparation of 10X of compound dilutions in 1X Assay Buffer,
final concentration of DMSO in these compound dilutions will be 1%,
start from 100 μM and 1:5 dilution); 5X BRD4(BD1 or BD2); 5X H4
K5,8,12,16Ac; 4X Histidine Conjugated Acceptor Beads with 1X Assay
Buffer and 4X Streptavidin (SA) Donor Beads with 1X Assay Buffer.
Transfer 2 µl compounds to 384 assay plates. Then mix 4 µl BRD4(BD1
or BD2), 4 µl H4 K5,8,12,16Ac and 5 µl Histidine Conjugated Acceptor
Beads. Incubate at room temperature for 60 min. At last, add 5ul SA-
Donor Beads and incubate 30 min at RT in the dark. Read endpoint with
Bio Tek Cytation™ 5 Cell Imaging Multi-Mode Reader with Alpha mode.
The IC50 was calculated by GraphPad Prism 5 statistical software.
4.1.5.24. 7-(tert-butyl)-1-((2-chlorothiazol-5-yl)methyl)-4-(4-
methoxyphenyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-
2(1H)-one (3x). White solid, yield 91%, m.p. 239.9–241.7 °C; 1H NMR
(500 MHz, DMSO) δ 7.91 (s, 1H), 7.45 (d, J = 8.6 Hz, 2H), 7.04 (d,
J = 8.7 Hz, 2H), 5.44–5.34 (m, 2H), 3.83 (s, 3H), 2.77 (dd, J = 16.5,
4.4 Hz, 1H), 2.49–2.40 (m, 1H), 2.20 (t, J = 13.9 Hz, 1H), 1.80 (dd,
J = 55.4, 13.8 Hz, 2H), 1.46 (td, J = 11.6, 4.6 Hz, 1H), 0.97 (tt,
J = 12.2, 6.2 Hz, 1H), 0.85 (s, 9H); 13C NMR (126 MHz, DMSO) δ
168.76, 161.08, 157.82, 154.25, 152.49, 143.01, 133.98, 130.57,
130.53, 130.29, 130.26, 118.42, 113.80, 55.74, 45.52, 44.64, 32.63,
28.30, 27.38, 26.68, 24.02; HR-ESI-MS m/z: calcd for
4.3.2. Cell culture
C
25H27ClN3O2S2{[M+H]+}500.1233, found 500.1238.
MCF-7 cells, A549 cells and HT-29 cells were cultured in RPMI 1640
supplemented with 10% FBS and penicillin/streptomycin mixture.
HepG2 cells, A375 cells, NRK-49F cells and LX-2 cells were cultured in
D-MEM (High Glucose) supplemented with 10% FBS and penicillin/
streptomycin mixture. HLF-1 cells were cultured in D-MEM/F-12(1:1)
supplemented with 10% FBS and penicillin/streptomycin mixture. All
cells were incubated at 37 °C in a humidified incubator (5% CO2 in air).
4.1.5.25. 7-(tert-butyl)-4-(4-chlorophenyl)-1-((2-chlorothiazol-5-yl)
methyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-2(1H)-one
(3y). Yellow solid, yield 65%, m.p. 270.3–272.1 °C; 1H NMR (500 MHz,
DMSO) δ 7.91 (s, 1H), 7.61–7.49 (m, 4H), 5.41 (s, 2H), 2.79 (dd,
J = 16.4, 4.1 Hz, 1H), 2.46 (d, J = 14.0 Hz, 1H), 2.11 (t, J = 13.7 Hz,
1H), 1.78 (d, J = 15.2 Hz, 2H), 1.48–1.41 (m, 1H), 1.03–0.95 (m, 1H),
0.87 (d, J = 17.5 Hz, 9H); 13C NMR (126 MHz, DMSO) δ 167.88,
158.20, 154.13, 152.51, 143.06, 137.09, 135.06, 133.83, 130.63,
130.51, 129.86, 128.60, 118.38, 45.63, 44.60, 32.62, 27.95, 27.39,
4.3.3. Cell proliferative inhibition assay
Briefly, cells were seeded into 96-well plates at a density appro-
priate for exponential growth at the start of the assay, and treated with
a range of concentrations of compounds for 48 h. Fresh MTT (10 µl) was
added to each well and incubated at 37 °C for 4 h. The formazan crystals
in each well were dissolved in 150 µl DMSO. The spectrophotometric
absorbance of each well was measured by a multi-detection microplate
reader at a wavelength of 490 nm. The IC50 was calculated by GraphPad
Prism 5 statistical software.
26.64, 23.92; HR-ESI-MS m/z: calcd for C24H24Cl2N3OS2{[M+H]+
}
504.0738, found 504.0740.
4.1.5.26. 4-(((7-(tert-butyl)-4-(3,4-dichlorophenyl)-5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-2-yl)oxy)methyl)-3,5-
dimethylisoxazole (3z). White solid, yield 88%, m.p. 80.7–82.6 °C; 1H
NMR (500 MHz, DMSO) δ 7.84 (s, 1H), 7.78 (d, J = 8.0 Hz, 1H), 7.52
(d, J = 8.1 Hz, 1H), 5.33–5.22 (m, 2H), 2.85 (d, J = 15.4 Hz, 1H),
2.60–2.51 (m, 1H), 2.39 (d, J = 15.3 Hz, 3H), 2.24 (s, 3H), 2.19 (s, 1H),
1.95 (d, J = 15.3 Hz, 1H), 1.84 (d, J = 11.3 Hz, 1H), 1.51 (d,
J = 9.9 Hz, 1H), 1.07 (d, J = 9.8 Hz, 1H), 0.87 (s, 9H); 13C NMR
4.3.4. Western blot analysis
A375 or HLF-1 cells (2.5 × 105 cells/well) incubated in 6-well
plates were treated with compounds for 24 h. The treated cells were
collected, lysed on ice, and then centrifuged at 4 °C, 12,000g, 15 min,
10

J. Ma, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
leaving the supernatant. The protein concentration was measured with
BCA Protein Assay kit (No. BL521A, Biosharp). 20–40 μg protein was
electrophoresed on SDS-PAGE and transferred to PVDF membranes.
The membrane was blocked with 5% skim milk, and incubated with
primary antibodies c-myc (No. 13987, Cell Signaling), collagen I (No.
34710, Abcam) and β-Actin (No. 4970, Cell Signaling) overnight. Then
it was incubated with the appropriate secondary antibodies (No.
A21020, Abbkine) for 1 h. At last, the bands were detected by western
fluorescent detection reagent (No. WBKLS0100, Millipore) and imaged
within the ChemiDoc XRS+c imaging system.
transcriptional regulation and its possible role as an endpoint of signal transduction
pathways. Proc Natl Acad Sci USA. 1998;95:8538–8543.
2. Belkina AC, Denis GV. BET domain co-regulators in obesity, inflammation and
cancer. Nat Rev Cancer. 2012;12:465–477.
3. Padmanabhan B, Mathur S, Manjula R, et al. Bromodomain and extra-terminal (BET)
family proteins: new therapeutic targets in major diseases. J Biosci.
2016;41:295–311.
4. Ding N, Hah N, Yu RT, et al. BRD4 is a novel therapeutic target for liver fibrosis. Proc
Natl Acad Sci USA. 2015;112:15713–15718.
5. Fukazawa H, Masumi A. The conserved 12-amino acid stretch in the inter-bromo-
domain region of BET family proteins functions as a nuclear localization signal. Biol
Pharm Bull. 2012;35:2064–2068.
6. Jonkers I, Lis JT. Getting up to speed with transcription elongation by RNA poly-
merase II. Nat Rev Mol Cell Biol. 2015;16:167–177.
7. You JS, Han JH. Targeting components of epigenome by small molecules. Arch
Pharmacal Res. 2014;37:1367–1374.
4.3.5. RNA isolation and quantitative RT-PCR
A375 or HLF-1 cells (2.5 × 105 cells/well) incubated in 6-well
plates were treated with compounds for 24 h. Total RNA were extracted
using Total RNA Extraction Reagent (No. RN01005S, Monad) according
to the manufacturer’s instructions. cDNA was synthesized from total
RNA using MonScript™ RTIII all-in-one Mix (No. RN05004S, Monad).
Expression levels of specific genes were quantified by real-time PCR
(No. 788BR07164, CFX Connect™ Optics Module) using MonAmp™
ChemoHS qPCR Mix (No. RN04001M, Monad). The primer sequences
are shown below.
8. Wu X, Liu D, Tao D, et al. BRD4 regulates EZH2 transcription through upregulation of
C-MYC and represents a novel therapeutic target in bladder cancer. Mol Cancer Ther.
2016;15:1029–1042.
9. Mertz JA, Conery AR, Bryant BM, et al. Targeting MYC dependence in cancer by
inhibiting BET bromodomains. Proc Natl Acad Sci USA. 2011;108:16669–16674.
10. Delmore J, Issa G, Lemieux M, et al. BET Bromodomain Inhibition as a Therapeutic
Strategy to Target c-Myc. Cell. 2011;146.
11. Dang Chi V. MYC, microRNAs and glutamine addiction in cancers. Cell Cycle.
2009;8:3243–3245.
12. Liu L, You Z, Yu H, et al. Mechanotransduction-modulated fibrotic microniches re-
veal the contribution of angiogenesis in liver fibrosis. Nat Mater.
2017;16:1252–1261.
13. Dhalluin C, Carlson JE, Zeng L, et al. Structure and ligand of a histone acetyl-
transferase bromodomain. Nature. 1999;399:491–496.
Gene
Primer sequence (5′–3′)
14. Owen DJ, Ornaghi P, Yang Ji-Chun, et al. The structural basis for the recognition of
acetylated histone H4 by the bromodomain of histone acetyltransferase Gcn5p.
EMBO J. 2000;19:6141–6149.
c-Myc Forward
GGCTCCTGGCAAAAGGTCA
CTGCGTAGTTGTGCTGATGT
CAGCCGCTTCACCTACAGC
TTTTGTATTCAATCACTGTCTTGCC
C-Myc Reverse
15. Morinière Jeanne, Rousseaux S, Steuerwald U, et al. Cooperative binding of two
acetylation marks on a histone tail by a single bromodomain. Nature.
2009;461:664–668.
Collagen I Forward
Collagen I Reverse
16. Filippakopoulos P, Picaud S, Mangos M, et al. Histone recognition and large-scale
structural analysis of the human bromodomain family. Cell. 2012;149.
17. Gacias M, Gerona-Navarro G, Plotnikov AN, et al. Selective chemical modulation of
gene transcription favors oligodendrocyte lineage progression. Chem Biol.
2014;21:841–854.
4.3.6. Cell apoptosis
A375 cells (1 × 105 cells/well) incubated in 24-well plates were
treated with compounds for 24 h. After 24 h, cell apoptosis was de-
termined by using the Annexin V-FITC Apoptosis Detection Kit (No.
A211-01, Vazyme). Briefly, the cells were washed twice with ice-cold
PBS, and then 6 μL of Annexin V-FITC and 3 μL of PI were applied to
stain cells. After incubated for 15 min at room temperature in dark, the
stained cells were analyzed using fluorescence microscope (Bio Tek
Cytation™ 5 Cell Imaging Multi-Mode Reader, USA).
18. Lei-Lei F, Mao T, Xiang L, et al. Inhibition of BET bromodomains as a therapeutic
strategy for cancer drug discovery. Oncotarget. 2015;6:5501–5516.
19. Vollmuth F, Blankenfeldt W, Geyer M. Structures of the dual bromodomains of the P-
TEFb-activating protein Brd4 at atomic resolution. J Biol Chem.
2009;284:36547–36556.
20. Filippakopoulos P, Picaud S, Mangos M, et al. Histone recognition and large-scale
structural analysis of the human bromodomain family. Cell. 2012;149:214–231.
21. Shi J, Wang Y, Zeng L, et al. Disrupting the interaction of BRD4 with diacetylated
Twist suppresses tumorigenesis in basal-like breast cancer. Cancer Cell.
2014;25:210–225.
22. Albrecht BK, Gehling VS, Hewitt MC, et al. Identification of a benzoisoxazoloazepine
inhibitor (CPI-0610) of the bromodomain and extra-terminal (BET) family as a
candidate for human clinical trials. J Med Chem. 2016;59:1330–1339.
23. Picaud S, Wells C, Felletar I, et al. RVX-208, an inhibitor of BET transcriptional
regulators with selectivity for the second bromodomain. Proc Natl Acad Sci USA.
2013;110:19754–19759.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (No. 81573287 & 81773640), Hunan Province
Strategic Emerging Industry Science and Technology Key Project (No.
2016GK4028) and Postgraduates innovation subject of Central South
University (No. 2018zzts875). Key research and development project of
Hainan Province (project number: ZDYF2017092), Haikou, China.
Hainan Province Key Research and Development Project
(ZDYF2016128) Haikou, China. We also would like to thank the
Nuclear Magnetic Resonance Laboratory of Advanced Research Center
in Central South University for the technological assistance in chemical
characterization.
24. Mar G, Guillermo GN, et al. Selective chemical modulation of gene transcription
favors oligodendrocyte lineage progression. Chem Biol. 2014;21:841–854.
25. Filippakopoulos P, Qi J, Picaud S, et al. Selective inhibition of BET bromodomains.
Nature. 2010;468:1067–1073.
26. Baud MG, Lin-Shiao E, Zengerle M, et al. New synthetic routes to triazolo-benzo-
diazepine analogues: expanding the scope of the bump-and-hole approach for se-
lective bromo and extra-terminal (BET) bromodomain inhibition. J Med Chem.
2016;59:1492–1500.
27. Galdeano C, Ciulli A. Selectivity on-target of bromodomain chemical probes by
structure-guided medicinal chemistry and chemical biology. Future Med Chem. 2016
fmc-2016-0059.
28. Sahni JM, Keri RA. Targeting bromodomain and extraterminal proteins in breast
cancer. Pharmacol Res. 2017 S1043661817314032.
Appendix A. Supplementary data
29. Bihani T, Ezell SA, Ladd B, et al. Resistance to everolimus driven by epigenetic
regulation of MYC in ER+ breast cancers. Oncotarget. 2015;6:2407–2420.
30. Feng Q, Zhang Z, Shea MJ, et al. An epigenomic approach to therapy for tamoxifen-
resistant breast cancer. Cell Res. 2014;24:809–819.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmc.2019.03.037.
References
1. Jiang YW, Veschambre P, Erdjument-Bromage H, et al. Mammalian mediator of
11