Identification of 3,5-Dimethylisoxazole Derivatives as BRD4 Inhibitors for the Treatment of Colorectal Cancer

Article abstract of DOI:10.1021/acsmedchemlett.0c00294

Bromodomain-containing protein 4 (Brd4) plays a critical regulatory role in gene transcription that has been recently recognized as a promising strategy for cancer therapy. Based on the BRD4 protein containing two tandem bromodomain structures, BD1 and BD2, we designed and synthesized a series of 3,5-dimethylisoxazole derivative dimers targeting both bromodomains simultaneously to enhance protein binding potency. Among them, compound 22 significantly inhibited the proliferation of colorectal cancer cells HCT116 (IC₅₀= 162 nM), with a 20-fold increase in antiproliferative activity compared to inhibitor 14. The results of WesternBlot showed that compound 22 could down-regulate c-MYC protein levels and up-regulate HEXIM1 expression and modulate apoptosis through intrinsic pathways. In addition, compound 22 exhibited outstanding antitumor efficacy in the CT-26 tumor mouse model with a tumor suppression rate of 56.1%. Taken together, 3,5-dimethylisoxazole derivative dimer 22 has remarkable protein inhibitory effect and antitumor activity in vitro and in vivo. A protein binding model of compound 22 is being further analyzed, which will facilitate the development of bivalent BRD4 inhibitors and probe the biological function of BRD4.

Full text of DOI:10.1021/acsmedchemlett.0c00294

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Letter
Identification of 3,5-Dimethylisoxazole Derivatives as
BRD4 Inhibitors for the Treatment of Colorectal Cancer
Yifei Yang, Lincheng Fang, Pan Chen, Huibin Zhang, and jinpei zhou
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.0c00294 • Publication Date (Web): 31 Aug 2020
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ACS Medicinal Chemistry Letters
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Identification of 3,5-Dimethylisoxazole Derivatives as BRD4
Inhibitors for the Treatment of Colorectal Cancer
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a, b
c
a
c,*
a,*
Yifei Yang , Lincheng Fang , Pan Chen , Huibin Zhang , Jinpei Zhou
a
Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, PR China.
b
Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock,
Arkansas 72205, United States
c
Center of Drug Discovery, Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, China Pharmaceutical
University, Nanjing 210009, PR China.
ABSTRACT: Bromodomain-containing protein 4 (Brd4) plays a
critical regulatory role in gene transcription that has been recently
recognized as a promising strategy for cancer therapy. Based on the
BRD4 protein containing two tandem bromodomain structures, BD1
and BD2, we designed and synthesized
a series of 3,5-
dimethylisoxazole derivatives dimers targeting both bromodomains
simultaneously to enhance protein binding potency. Among them,
compound 22 significantly inhibited the proliferation of colorectal
cancer cells HCT116 (IC50 = 162 nM), with a 20-fold increase in anti-proliferative activity compared to inhibitor 14. The results of
WesternBlot showed that compound 22 could down-regulate c-MYC protein levels and up-regulate HEXIM1 expression and
modulate apoptosis through intrinsic pathways. In addition, compound 22 exhibited outstanding anti-tumor efficacy in the CT-26
tumor mouse model with a tumor suppression rate of 56.1%. Taken together, 3,5-dimethylisoxazole derivatives dimer 22 has
remarkable protein inhibitory effect and antitumor activity in vitro and in vivo. Protein binding model of compound 22 is being further
analyzed, which will facilitate the development of bivalent BRD4 inhibitors and probe the biological function of BRD4.
KEYWORDS: BRD4 inhibitors, Colorectal cancer, Bivalent ligands, Epigenetics
The Bromodomain (BRD)-containing protein plays a critical
role in recognizing epigenetic acetylated lysine marks on
bivalent BRD4 molecule inhibitor, MT1 (6) and AZD5153 (7)
respectively, exhibiting promising biological activities in vitro
1
17, 20
histones . In humans, 46 proteins contain bromodomains
and in vivo
. MT1 is an intramolecular bivalent BRD4
2
segregated into eight groups . Among them, the bromodomain
binder with more potent proliferation activity than the
17
and extra-terminal domain (BET) family of proteins, consisting
of BRD2, BRD3, BRD4, and testis-specific BRDT members
corresponding monovalent antagonist, JQ1 . However,
AZD5153, a clinical candidate bivalent BET inhibitor, is a single
inhibitor molecule containing two acetyl lysine mimicking
share
a common domain architecture, two N-terminal
3
-5
21
bromodomains and an extra-terminal domain . It has been
proposed that BET proteins are crucial for cancer pathogenesis
via modulating diverse transcriptional programs .
moieties simultaneously engaging both bromodomains . Given
that no bivalent BRD4 inhibitors are currently available in
clinics or in the market, developing bivalent BRD4 inhibitors
with diverse core structures and excellent pharmacological
profile is essential for therapeutic studies.
6
-8
BRD4 has been identified highly enriched in large clusters of
enhancers that drive the expression of genes involved in the
N
N
N
N
N
9, 10
pathogenesis of cancer . Recent studies have shown that
BRD4 directly regulates the expression of MYC genes as a
N
O
N
N
O
N
N
O
O
O
O
S
N
S
N
O
NH
NH
NH2
O
N
N
N
HN
O
O
N
N
1
1, 12
therapeutic target in cancer
. Further, BRD4 protein plays an
OH
N
essential role in recruiting the positive transcription elongation
factor p-TEFb complex (CDK9 and cyclin T1), which is
required for RNA polymerase II-dependent transcription
elongation . Recently, a number of BRD4 inhibitors which
target BET family proteins for cancer therapy have been
Cl
Cl
Cl
Cl
1
(JQ1)
2 (OTX015)
3 (I-BET762)
4 (I-BET151)
5 (CPI-0610)
N
N
O
N
H
N
H
N
13-15
N
N
S
N
N
N
N
N
N
O
H
O
N
N
O
N
F
N
O
7
O
S
O
S
O
O
N
N
O
N
H
reported, including JQ1(1), OTX015(2), ABBV075(8), etc.
F
Cl
1
6-19
(Figure 1) . To our knowledge, most of the reported BRD4
6 (MT1)
Bivalent inhibitor
7 (AZD5153)
Bivalent inhibitor
8 (ABBV-075)
Cl
inhibitors do not show selectivity for individual family
members and bind BRD4 protein in a monovalent fashion .
Almost at the same time, Tanaka and Waring disclosed the first
2
0
Figure 1. Selected examples of known BET inhibitors.
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Here we detailed the identification and characterization of a
Page 2 of 8
containing triazole as linker (21, 22) exhibited outstanding
activities against BRD4 protein, which was greatly superior to
that of other types of dimer inhibitors. Subsequently,
compounds 28~32 were designed and synthesized by further
optimization, including the removal of methyl group, the
introduction of carbon chains, etc. It was noteworthy that,
compared to compounds 21 and 22, compounds (28~32)
showed considerably more potent BRD4 inhibition activities
(IC50 < 2.1 nM).
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6
new class of bivalent BET inhibitors containing 3,5-
Dimethylisoxazole motif. We found that compound 22 was a
highly potent and efficacious bivalent inhibitor against
colorectal cancer, which was also stronger than its monovalent
counterparts.
Our previously reported quinoxalinone BRD4 inhibitor,
compound 5i, effectively inhibited BRD4 protein (IC50 = 73
2
2
nM) . The docking model showed that the ethyl in compound
i extended into the solvent region outside the pocket.
5
Compound 5i had no room for optimization and was not
suitable for further development as a lead compound (Figure
0
1
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5
6
7
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0
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3
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5
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9
0
1
2
3
4
5
6
7
8
9
0
2
). We used the Skeleton Hopping technique to replace the
quinoxalinone with quinazolinone motif to obtain compound 14
with BRD4 protein activity IC50 = 32 nM, which was
remarkably higher than that of compound 5i. Molecular
docking models implied that the 3-nitrogen atom of compound
14 faced the extra-pocket solvent region, which provided an
advantage for further design of bivalent ligand (Figure 3).
Therefore, the above results suggested that compound 14
deserved further development for the design of dimerized
BRD4 inhibitors.
Figure 3. Docking pose for compound 14 without specific chirality
(yellow, A) in BRD4(1) protein. (B) Compounds 14 without
specific chirality (yellow) and 5i (green) were superimposed in the
docking models (PDB code 2YEL).
Compounds 15~22 were synthesized as shown in Scheme 1.
Bromination of the starting material 2-aminobenzophenone,
followed by amine reduction with tert-butyl subsulfinate, led to
the formation of intermediate 10. Then, compound 10 was
reduced using sodium borohydride to give 11, and subsequent
treatment with triphosgene afforded the quinazolinone 12,
which was then subjected to methylation to give the key
intermediate compound 13. Finally, the target compound 14
was prepared by Suzuki cross-coupling reaction to introduce
H
1
N
O
N
O
Skeleton Hopping
3
NH
N
N
N
O
O
Compound 5i
BRD4(1)IC50 = 73 nM
14
BRD4(1)IC50 = 32 nM
3
1
,5-dimethylisoxazole group. Compound 14 was treated with
,4-Dibromobutane, 1,5-Dibromopentane, 1,6-
Figure 2. Design and modification strategy of our lead compound
4.
First, we selected carbon chains, polyethylene glycol chains,
Dibromohexane, 1,10-Dibromodecane respectively to gain
compounds 15~18. In addition, compound 14 reacted with
Triethylene glycol ditoluenesulfonate or Tetraethylene glycol
ditoluenesulfonate to obtain compounds 19~20. Compounds
21~22 were yielded by the reaction of compound 14 with 3-
Bromopropyne, followed by a click reaction with N3-PEG1-N3
1
and triazole polyethylene glycol chains as the linkers and
obtained dimeric BRD4 inhibitors 15 ~ 22 by connecting two
molecules of the compounds 14. Interestingly, compounds
or N3-PEG3-N3.
H
NH2
NH2
NH2
NH2
N
O
O
S
H
O
S
O
a
O
b
N
c
N
d
NH
Br
Br
Br
Br
8
9
10
11
12
N
O
N
O
O
N
O
e
NH
f
NH
g
N
Br
N
O
N
n
N
N
O
N
O
1
5~18
1
3
14
15, n=1
16, n=2
N
O
N
O
O
N
1
7, n=3
NH
g
N
N
O
n
18, n=7
O
O
O
N
N
N
19, 20
1
4
19, n=2
20, n=3
N
O
N
N
N
O
N
N
O
h, i
NH
n
O
O
N
O
N
N
N
N
N
N
N
2
2
1, 22
1, n=1
22, n=3
O
1
4
Scheme 1. Reagents and conditions: (a) NBS, DCM, 0 ˚C; (b) tert-Butanesulfinamide, Tetraethyl Titanate, THF, reflux; (c) NaBH
O, r.t; (d) Bis(trichloromethyl) Carbonate, THF, r.t; (e) Dimethylsulfate；Cs CO , acetonitrile, r.t; (f) phenylboronic acids, Pd(P(Ph)
Na CO , N , H O/EtOH/Toluene, 80 ˚C; (g) Diverse linkers, NaH, DMF, 0 ˚C to r.t; (h) 3-Bromopropyne, Cs CO , DMF, 0 ˚C to r.t; (i)
Diverse linkers, CuSO , Sodium erythorbate, MeOH, H O, r.t;
4
, THF,
H
2
2
3
3 4
) ,
2
3
2
2
2
3
4
2
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The synthesis of compounds 28~32 was described in Scheme
Bromopropyne to generate intermediate 26. The key
intermediate 27 was prepared by treatment of compound 26
with triphosgene. Lastly, intermediate 27 was made react with
different linkers to obtain the target compounds 28~32 via click
reaction.
1
2
3
4
5
6
7
8
9
2. Compound 9 was used as the starting material to obtain
compound 23 via Suzuki cross-coupling reaction, which was
treated with tert-butylenesulfinate to produce intermediates 24.
Then, compound 24 was reduced by sodium borohydride to
produce compound 25, followed by substitution via 3-
NH2
NH2
NH2
NH2
H
a
O
b
N
c
N
d
O
S
S
Br
O
O
O
N
N
O
N
O
9
23
24
2
5
H
N
O
N
N
H
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
NH2
O
N
N
O
N
N
n
H
S
e
f
O
O
N
O
O
N
N
N
O
N
N
N
N
N
O
2
8~31
2
2
8, n=0
9, n=3
30, n=2
31, n=1
2
6
27
H
N
O
H
N
O
N
N
O
N
N
f
N
N
O
N
N
N
N
O
N
O
N
N
H
2
7
32
Scheme 2. Reagents and conditions: (a) phenylboronic acids, Pd(P(Ph)
Butanesulfinamide, Tetraethyl Titanate, THF, reflux; (c) NaBH , THF, H
trichloromethyl)Carbonate, THF, r.t; (f) Diverse Linkers, CuSO , Sodium erythorbate, MeOH, H
3
)
4
, Na
2
CO
3
, N
2
2
, H O/EtOH/Toluene, 80 ˚C; (b) tert-
4
2
O, r.t; (d) 3-Bromopropyne, NaH, DMF, 0 ˚C to r.t; (e) Bis
O, r.t.
(
4
2
In the initial phase of our study, compounds 15~22 were
(15~18) with carbon chains as the linker exhibited relatively
weak inhibitory activities, except for compound 15 (Table 1).
designed and synthesized using compound 14 as the lead
compound. The results of Alphascreen BRD4 assay
demonstrated that most of the compounds showed more potent
activities than the lead compound 14. Among them, the
compounds (21, 22) containing triazoles displayed prominent
inhibitory activities with BRD4(1) IC50 = 4.2 nM and 7.7 nM,
respectively. The BRD4 inhibitory activities of compounds (19,
20) using polyethylene glycol as the linker were more valid than
that of analogs with other linkers. Furthermore, compounds
Based on the above activity results, we further optimized the
bivalent inhibitors (21, 22) using linkers containing a triazole
group. The methyl on the nitrogen atom was also removed and
the polyethylene glycol chain was replaced by a carbon chain to
produce compounds 28~32. The BRD4(1) protein activities of
compounds (28~32) (BRD4(1) IC50 < 2.1 nM) were much
superior to those of other bivalent compounds (Table 1).
Table 1. BRD4(1) inhibitory activities of compounds 14 ~ 22, 28~32
R1
R1
N
O
O
N
N
N
Linker
O
O
N
N
Compound No.
Linker
R
1
BRD4(1)
BRD4(1) IC50a,b
μM
0
.5μM(%)
1
1
5
6
Me
100
0.013±0.011
Me
Me
Me
Me
91
78
9
0.056±0.003
0.042±0.012
NT c
17
18
O
1
2
9
0
98
0.031±0.008
O
O
O
O
Me
98
0.019±0.010
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N
N
2
1
N
N
N
N
Me
100
0.0042±0.0012
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
O
N
N
2
2
2
8
N
N
N
N
Me
H
100
100
0.0077±0.0006
<0.0021
O
O
N
N
N
N
N
N
N
N
2
9
N
N
N
N
H
H
100
100
<0.0021
<0.0021
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
O
N
N
N
30
O
O
N
N
N
N
N
3
3
1
2
N
N
H
H
100
100
<0.0021
<0.0021
N
N
O
N
N
N
N
N
N
1
4
100
100
100
0.032±0.006
0.042±0.004
0.0025±0.0011
JQ1
ABBV-075
a
b
c
Data are expressed as the mean ± SD from the dose−response curves of at least three independent experiments.
IC50 values are shown as mean values of at least three determinations unless specified otherwise.
Not test.
The binding model of compound 14 and BRD4BD1 protein
Considering that the above compounds have noticeable
was predicted, and the molecular docking model showed that
the 3,5-dimethylisoxazole group in compound 14 could bind to
the Kac pocket of BRD4 Bromodomain as a Kac mimic. The
nitrogen atom in 3,5-dimethylisoxazole formed a hydrogen
bond to the key amino acid ASN140 located in the Kac pocket,
anchoring compound 14 in the Kac pocket. Benzene ring at
position 4 extended into the WPF hydrophobic pocket formed
by Trp81, Pro82, and Phe83. In addition, nitrogen atom at the
BRD4 protein inhibitory activities, we evaluated the
antiproliferative activities of all compounds 14~22, 28~32 at the
cellular level. Primary screening tests were performed on
pancreatic cancer cells BxPc3, Mia-capa2, and colorectal
cancer cells HCT116, respectively
that both pancreatic and colon cancer cells were sensitive to
most compounds.
2
3, 24
. As a result, it was found
3
-position of compound 14 faced the solvent region outside the
pocket, providing a modification site for post-modification
optimization. Compound 14 overlapped perfectly with our
previously announced compound 5i. The monomer moiety
(
compound 14) of bivalent ligand 22 could manage to bind to
the BRD4Kac pocket, which was similar to the binding pattern
of single-molecule compound 14. 3,5-dimethylisoxazole filled
the Kac pocket forming a hydrogen bond with Asn140 and the
-position benzene ring extended into the WPF hydrophobic
pocket formed by Trp81, Pro82, and Phe83. In addition, the
Figure 4. Docking pose for compound 22 without specific chirality
(purple, A) in BRD4(1) protein. Compounds 14 without specific
chirality (yellow) and 22 without specific chirality (purple) were
superimposed in the docking models (PDB code 2YEL).
4
triazole moiety formed weak π–π stacking interactions with the
-position benzene ring located in WPF shelf and Trp81.The
4
In particular, compounds 17 and 22 exhibited exceptional
activities against all three tumor cell lines, with an ideal
inhibition rate of 59.7% and 83.6% at 2 μM against colon cancer
cell HCT116, respectively. However, compound 14 only
exhibited a 14% inhibition rate against HCT116 at 2 μM.
Therefore, anti-proliferative activities of compounds 17 and 22
against HCT116 cells were further tested at gradient
another monomer moiety connected with nitrogen atoms at the
3-position via Linker, extended into the outer solvent region of
the BRD4Kac pocket (Figure 4). Moreover, we speculated that
ligand 22 could bind to another bromodomain in tandem
according to a previous report, enhancing protein binding
1
7
activity .
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concentrations. The results indicated that compounds 17 and 22
more than that of compound 14. In addition, the anti-
proliferative activity of compound 22 was better than that of the
positive compounds JQ1 and ABBV-075. Thus, our next task
was to further investigate the biological mechanisms of
compound 22.
1
2
3
4
5
6
7
8
9
had antiproliferative potency with an IC50 value of 1.1 μM and
0.16 μM against HCT116 cells, respectively (Table 2). Both
compounds 17 and 22 exhibited significantly more potent anti-
proliferative activities than the lead compound 14, and
particularly the inhibitory activity of compound 22 was 20-fold
Table 2. Anti-proliferation activities of compounds 14 ~ 22, 28~32
Compound No.
BxPc3
.5μM (%)
46.4
27.9
44.0
45.2
7.1
Mia-capa2
2.5μM (%)
68.2
HCT116
2.0μM (%)
59.3
HCT116
IC50 (μM) a,b
NTc
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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9
0
2
1
1
5
6
71.3
52.0
NT
17
70.9
59.7
1.101±0.023
NT
1
1
2
8
9
0
65.2
48.8
67.3
43.4
NT
42.1
23.2
61.3
53.0
42.2
21.8
52.1
37.1
11.2
9.1
73.4
35.3
NT
21
64.0
49.6
NT
2
2
2
2
8
9
68.4
83.6
0.162±0.012
NT
62.2
60.3
66.4
60.4
NT
30
63.2
60.5
NT
3
3
1
1
2
4
54.4
55.6
NT
57.2
60.1
NT
59.1
14.0
3.341±0.011
4.212±0.015
0.550±0.009
JQ1
56.2
23.2
ABBV-075
36.8
64.3
69.5
a
b
c
Data are expressed as the mean ± SD from the dose−response curves of at least three independent experiments.
IC50 values are shown as mean values of at least three determinations unless specified otherwise.
Not test.
The cell cycle arrest potency of compound 22 was evaluated
in HCT116 cells. Cell cycle arrest experiments revealed that
compound 22 dramatically increased the percentage of HCT116
cells in the G0/G1 phase at 0.16 μM for 48 h. The G0/G1 phase
arrest effect of compound 22 was significantly more than that
of the lead compound 14 (Figure 5).
Figure 5. Effects of compounds 22 and 14 on the cell cycle arrest
of HCT116 cells.
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We further explored the potency of compound 22 on clone-
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forming against HCT116 cells. The results showed that 22
significantly inhibited the proliferation of HCT116 cells in a
concentration-dependent manner for 12 days. In addition, the
effect of compound 22 on suppressing the proliferation of
HCT116 cells was notably exceeded the lead compound 14
(Figure 6).
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Figure 7. Effects of compounds 22 (1 μM), 14 (1 μM) and ABBV-
075 (1 μM) on protein expression in HCT116 cancer cells.
Figure 6. Plate clone-forming effects of compounds 22 and 14 in
HCT116 cell lines.
Based on the inhibitory activity of 22 against human
colorectal cancer cell line HCT116, the effect of compound 22
on tumor growth inhibition in vivo was further investigated in
the CT-26 (mouse colorectal cancer cell line) model mouse. The
results showed that the intraperitoneal administration of
compound 22 at 20mg/kg for 14 days prominently inhibited
tumor growth in CT-26 model mice, and tumor suppression rate
was 56.1% significantly superior to 11.5% of the lead
compound 14 (Figure 8 and S2). There were also no serious
toxic side effects in the tumor mice and no obvious weight loss
in the rats, confirming the feasibility of bivalent BET inhibitors.
In addition, we conducted immunohistochemical analysis of
tumor tissue in sections. The results demonstrated that
compound 22 notably promoted the expression of HEXIM1,
Because compound 22 exhibited excellent biological activity
at both protein and cellular levels, we moved on to understand
its intrinsic mechanism of inducing apoptosis in HCT116 cells.
After treatment of HCT116 cells with compound 22 for 48 h,
the content of relevant proteins such as Bcl-2, HEXIM1, BRD4
and c-Myc were determined by WesternBlot. The results
showed that compound 22 was able to significantly down-
regulate c-Myc protein expression and decreased Bcl-2 protein
expression in HCT116 cells compared to lead compound 14. In
addition, compound 22 remarkably up-regulated the expression
level of HEXIM1 protein and enriched BRD4 protein,
consistent with the findings reported in previous literature
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5, 26
(Figure 7)
. Moreover, compound 22 could activate
downstream apoptotic effectors such as caspase 3 (Figure S1).
Mechanism studies indicated that compound 22 promoted
apoptosis in HCT116 cells via an intrinsic pathway, which was
conducive to further study of the mechanism of BET protein in
cells.
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and decreased the expression of c-Myc and KI67
.
Meanwhile, HE staining implied the promotion of
morphological changes in cell necrosis (Figure 9). In addition,
the pharmacokinetic parameters of compound 22 were
predicted using SwissADME and Discovery Studio 3.0, which
indicated that the compound 22 showed poor absorption and
3
0, 31
bioavailability (Figure S3 and Table S1)
.
Figure 8. Anticancer effects of Compound 22 in CT-26 xenograft model of colorectal cancer.
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ACS Medicinal Chemistry Letters
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Figure 9. Immunohistochemistry of CT-26 tumor tissues treated with compound 22.
Taken together, based on the BRD4 inhibitor compound 5i
2013ZX09301303-002) and China Scholarship Council
previously reported by our group, we designed and synthesized
the lead compound 14 utilizing the skeleton-hopping principle
and the BRD4(1) protein activity of compound 14 significantly
increased with an IC50 value of 32 nM. Since the structural
characteristics of BRD4 contained two tandem Bromodomains,
we designed and synthesized a series of bivalent BRD4
inhibitors by using different types and lengths of linkers for
connecting two molecules 14. Among them, compound 22
exhibited excellent BRD4 protein inhibitory activity with an
IC50 value of 7.7 nM and showed significant anti-proliferative
activity with an IC50 value of 162 nM against HCT116 cells.
Meanwhile, compound 22 resulted in cell cycle arrest at the
G0/G1 phase. WesternBlot results indicated that compound 22
interfered with apoptosis via intrinsic pathways. CT26 mouse
model in vivo experiments implied that compound 22
effectively inhibited tumor growth in vivo with a tumor
suppression rate of 56.1%. In addition, immunohistochemistry
results showed that compound 22 achieved antitumor effects in
vivo by inhibiting the BRD4 signaling pathway and thus might
be a powerful chemical probe for further investigation of BRD4
Bromodomains.
(201907060014).
ABBREVIATIONS
Brd4, Bromodomain-containing protein 4; HEXIM1,
Hexamethylene bis-acetamide inducible protein; MYC,
Myelocytomatosis oncogene; pTEFb, Positive transcription
elongation factor b; BET, Bromodomain and extra-terminal
domain;
REFERENCES
(1)Liu, Z.; Wang, P.; Chen, H.; Wold, E.A.; Tian, B.; Brasier,
A.R.; Zhou, J. Drug Discovery Targeting Bromodomain-
Containing Protein 4. J Med Chem 2017, 60, 4533-4558.
(2)French, C.A. Pathogenesis of NUT midline carcinoma. Annu
Rev Pathol 2012, 7, 247-65.
(3)Wyce, A.; Ganji, G.; Smitheman, K.N.; Chung, C.W.;
Korenchuk, S.; Bai, Y.; Barbash, O.; Le, B.; Craggs, P.D.;
McCabe, M.T.; Kennedy-Wilson, K.M.; Sanchez, L.V.;
Gosmini, R.L.; Parr, N.; McHugh, C.F.; Dhanak, D.; Prinjha,
R.K.; Auger, K.R.; Tummino, P.J. BET inhibition silences
expression of MYCN and BCL2 and induces cytotoxicity in
neuroblastoma tumor models. PLoS One 2013, 8, e72967.
ASSOCIATED CONTENT
Supporting Information
(4)Sanchez, R.; Meslamani, J.; Zhou, M.M. The bromodomain:
from epigenome reader to druggable target. Biochim Biophys
Acta 2014, 1839, 676-85.
(5)Toure, M.; Crews, C.M. Small-Molecule PROTACS: New
Approaches to Protein Degradation. Angew Chem Int Ed Engl
Compound synthesis, spectroscopic characterization and biology
experimental procedures (PDF)
2
016, 55, 1966-73.
AUTHOR INFORMATION
(6)Chapuy, B.; McKeown, M.R.; Lin, C.Y.; Monti, S.; Roemer,
M.G.; Qi, J.; Rahl, P.B.; Sun, H.H.; Yeda, K.T.; Doench, J.G.;
Reichert, E.; Kung, A.L.; Rodig, S.J.; Young, R.A.; Shipp,
M.A.; Bradner, J.E. Discovery and characterization of super-
enhancer-associated dependencies in diffuse large B cell
lymphoma. Cancer Cell 2013, 24, 777-90.
Corresponding Author
* Huibin Zhang Email: zhanghb80@163.com
*
Jinpei Zhou Email: jpzhou668@163.com
ORCID:
(7)Shi, J.; Wang, Y.; Zeng, L.; Wu, Y.; Deng, J.; Zhang, Q.;
Lin, Y.; Li, J.; Kang, T.; Tao, M.; Rusinova, E.; Zhang, G.;
Wang, C.; Zhu, H.; Yao, J.; Zeng, Y.X.; Evers, B.M.; Zhou,
M.M.; Zhou, B.P. Disrupting the interaction of BRD4 with
diacetylated Twist suppresses tumorigenesis in basal-like
breast cancer. Cancer Cell 2014, 25, 210-25.
Jinpei Zhou: 0000-0002-5904-6164
Author Contributions
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the
manuscript.
(8)Delmore, J.E.; Issa, G.C.; Lemieux, M.E.; Rahl, P.B.; Shi, J.;
Jacobs, H.M.; Kastritis, E.; Gilpatrick, T.; Paranal, R.M.; Qi, J.;
Chesi, M.; Schinzel, A.C.; McKeown, M.R.; Heffernan, T.P.;
Vakoc, C.R.; Bergsagel, P.L.; Ghobrial, I.M.; Richardson, P.G.;
Young, R.A.; Hahn, W.C.; Anderson, K.C.; Kung, A.L.;
Bradner, J.E.; Mitsiades, C.S. BET bromodomain inhibition as
a therapeutic strategy to target c-Myc. Cell 2011, 146, 904-17.
(9)Mertz, J.A.; Conery, A.R.; Bryant, B.M.; Sandy, P.;
Funding Sources
This study was supported by the Natural Science Foundation of
Jiangsu Province (No. BK 20141349), the China National Key
HiTech Innovation Project for the R&D of Novel Drugs (No.
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Balasubramanian, S.; Mele, D.A.; Bergeron, L.; Sims, R.J., 3rd.
Page 8 of 8
Optimization of a Series of Bivalent Triazolopyridazine Based
Bromodomain and Extraterminal Inhibitors: The Discovery of
(3R)-4-[2-[4-[1-(3-Methoxy-[1,2,4]triazolo[4,3-b]pyridazin-
6-yl)-4-piperidyl]phenoxy]ethyl]-1,3-dimethyl-piperazin-2-one
(AZD5153). J Med Chem 2016, 59, 7801-17.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Targeting MYC dependence in cancer by inhibiting BET
bromodomains. Proc Natl Acad Sci U S A 2011, 108, 16669-
74.
(10)Chatterjee, A.; Rodger, E.J.; Eccles, M.R. Epigenetic
drivers of tumourigenesis and cancer metastasis. Semin Cancer
Biol 2018, 51, 149-159.
(22)Yang, Y.; Zhao, L.; Xu, B.; Yang, L.; Zhang, J.; Zhang,
H.; Zhou, J. Design, synthesis and biological evaluation of
dihydroquinoxalinone derivatives as BRD4 inhibitors. Bioorg
Chem 2016, 68, 236-44.
(23)Zhang, P.; Li, R.; Xiao, H.; Liu, W.; Zeng, X.; Xie, G.;
Yang, W.; Shi, L.; Yin, Y.; Tao, K. BRD4 Inhibitor AZD5153
Suppresses the Proliferation of Colorectal Cancer Cells and
Sensitizes the Anticancer Effect of PARP Inhibitor. Int J Biol
Sci 2019, 15, 1942-1954.
(11)Hogg, S.J.; Vervoort, S.J.; Deswal, S.; Ott, C.J.; Li, J.;
Cluse, L.A.; Beavis, P.A.; Darcy, P.K.; Martin, B.P.; Spencer,
A.; Traunbauer, A.K.; Sadovnik, I.; Bauer, K.; Valent, P.;
Bradner, J.E.; Zuber, J.; Shortt, J.; Johnstone, R.W. BET-
Bromodomain Inhibitors Engage the Host Immune System and
Regulate Expression of the Immune Checkpoint Ligand PD-L1.
Cell Rep 2017, 18, 2162-2174.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(12)Lu, J.; Qian, Y.; Altieri, M.; Dong, H.; Wang, J.; Raina, K.;
Hines, J.; Winkler, J.D.; Crew, A.P.; Coleman, K.; Crews, C.M.
Hijacking the E3 Ubiquitin Ligase Cereblon to Efficiently
Target BRD4. Chem Biol 2015, 22, 755-63.
(24)Cheng, X.; Huang, Z.; Long, D.; Jin, W. BET inhibitor
bromosporine enhances 5-FU effect in colorectal cancer cells.
Biochem Biophys Res Commun 2020, 521, 840-845.
(25)Yang, Y.; Chen, P.; Zhao, L.; Zhang, F.; Zhang, B.; Xu, C.;
Zhang, H.; Zhou, J. Exploiting the 7-methylimidazo[1,5-
a]pyrazin-8(7H)-one scaffold for the development of novel
chemical inhibitors for Bromodomain and Extraterminal
Domain (BET) family. Bioorg Chem 2019, 90, 103044.
(26)Chen, P.; Yang, Y.; Yang, L.; Tian, J.; Zhang, F.; Zhou, J.;
Zhang, H. 3-Hydroxyisoindolin-1-one derivates: Synthesis by
palladium-catalyzed CH activation as BRD4 inhibitors against
human acute myeloid leukemia (AML) cells. Bioorg Chem
2019, 86, 119-125.
(27)Ba, M.; Long, H.; Yan, Z.; Wang, S.; Wu, Y.; Tu, Y.; Gong,
Y.; Cui, S. BRD4 promotes gastric cancer progression through
the transcriptional and epigenetic regulation of c-MYC. J Cell
Biochem 2018, 119, 973-982.
(28)Bowry, A.; Piberger, A.L.; Rojas, P.; Saponaro, M.;
Petermann, E. BET Inhibition Induces HEXIM1- and RAD51-
Dependent Conflicts between Transcription and Replication.
Cell Rep 2018, 25, 2061-2069.e4.
(29)Wilson, A.J.; Stubbs, M.; Liu, P.; Ruggeri, B.; Khabele, D.
The BET inhibitor INCB054329 reduces homologous
recombination efficiency and augments PARP inhibitor activity
in ovarian cancer. Gynecol Oncol 2018, 149, 575-584.
(30)Daina, A.; Michielin, O.; Zoete, V. SwissADME: a free web
tool to evaluate pharmacokinetics, drug-likeness and medicinal
chemistry friendliness of small molecules. Sci Rep 2017, 7,
42717.
(31)Yang, Y.; Shi, C.Y.; Xie, J.; Dai, J.H.; He, S.L.
Identification of Potential Dipeptidyl Peptidase (DPP)-IV
Inhibitors among Moringa oleifera Phytochemicals by Virtual
Screening, Molecular Docking Analysis, ADME/T-Based
Prediction, and In Vitro Analyses. Molecules 2020, 25, 189.
(
13)Zhang, G.; Smith, S.G.; Zhou, M.M. Discovery of Chemical
Inhibitors of Human Bromodomains. Chem Rev 2015, 115,
1625-68.
14)Boi, M.; Gaudio, E.; Bonetti, P.; Kwee, I.; Bernasconi, E.;
1
(
Tarantelli, C.; Rinaldi, A.; Testoni, M.; Cascione, L.; Ponzoni,
M.; Mensah, A.A.; Stathis, A.; Stussi, G.; Riveiro, M.E.; Herait,
P.; Inghirami, G.; Cvitkovic, E.; Zucca, E.; Bertoni, F. The BET
Bromodomain Inhibitor OTX015 Affects Pathogenetic
Pathways in Preclinical B-cell Tumor Models and Synergizes
with Targeted Drugs. Clin Cancer Res 2015, 21, 1628-38.
(15)Li, Y.; Sabari, B.R.; Panchenko, T.; Wen, H.; Zhao, D.;
Guan, H.; Wan, L.; Huang, H.; Tang, Z.; Zhao, Y.; Roeder,
R.G.; Shi, X.; Allis, C.D.; Li, H. Molecular Coupling of Histone
Crotonylation and Active Transcription by AF9 YEATS
Domain. Mol Cell 2016, 62, 181-193.
(16)Rhyasen, G.W.; Hattersley, M.M.; Yao, Y.; Dulak, A.;
Wang, W.; Petteruti, P.; Dale, I.L.; Boiko, S.; Cheung, T.;
Zhang, J.; Wen, S.; Castriotta, L.; Lawson, D.; Collins, M.; Bao,
L.; Ahdesmaki, M.J.; Walker, G.; O'Connor, G.; Yeh, T.C.;
Rabow, A.A.; Dry, J.R.; Reimer, C.; Lyne, P.; Mills, G.B.;
Fawell, S.E.; Waring, M.J.; Zinda, M.; Clark, E.; Chen, H.
AZD5153: A Novel Bivalent BET Bromodomain Inhibitor
Highly Active against Hematologic Malignancies. Mol Cancer
Ther 2016, 15, 2563-2574.
(17)Tanaka, M.; Roberts, J.M.; Seo, H.S.; Souza, A.; Paulk, J.;
Scott, T.G.; DeAngelo, S.L.; Dhe-Paganon, S.; Bradner, J.E.
Design and characterization of bivalent BET inhibitors. Nat
Chem Biol 2016, 12, 1089-1096.
(18)McLure, K.G.; Gesner, E.M.; Tsujikawa, L.; Kharenko,
O.A.; Attwell, S.; Campeau, E.; Wasiak, S.; Stein, A.; White,
A.; Fontano, E.; Suto, R.K.; Wong, N.C.; Wagner, G.S.;
Hansen, H.C.; Young, P.R. RVX-208, an inducer of ApoA-I in
humans, is a BET bromodomain antagonist. PLoS One 2013, 8,
e83190.
(
19)Filippakopoulos, P.; Knapp, S. Targeting bromodomains:
epigenetic readers of lysine acetylation. Nat Rev Drug Discov
014, 13, 337-56.
20)Waring, M.J.; Chen, H.; Rabow, A.A.; Walker, G.; Bobby,
2
(
R. Potent and selective bivalent inhibitors of BET
bromodomains. Nat Chem Biol 2016, 12, 1097-1104.
(21)Bradbury, R.H.; Callis, R.; Carr, G.R.; Chen, H.; Clark, E.;
Feron, L.; Glossop, S.; Graham, M.A.; Hattersley, M.; Jones,
C.; Lamont, S.G.; Ouvry, G.; Patel, A.; Patel, J.; Rabow, A.A.;
Roberts, C.A.; Stokes, S.; Stratton, N.; Walker, G.E.; Ward, L.;
Whalley, D.; Whittaker, D.; Wrigley, G.; Waring, M.J.
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