Development of 4,5-dihydro-benzodiazepinone derivatives as a new chemical series of BRD4 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.05.057

Bromodomains (BRDs) are protein interaction modules that selectively recognize ?-N-lysine residues, serving as key epigenetic readers and play a key role in epigenetic regulation of gene transcription. Bromodomain-containing protein 4 (BRD4), a protein containing two BRDs termed BD1 and BD2, has emerged as an attractive candidate for the development of inhibitors targeting gene transcription in several types of cancers. In this study, we made structural modifications of previously reported BRD4 inhibitors, to develop new chemical scaffold 3,4-dihydroquinoxalin-2(1H)-one. Four series of compounds (compounds 7e10) were synthesized, and the BRD4-inhibitory activity and anti-proliferative effect of these compounds were evaluated. We found compound 10d has remarkable anti-proliferative activities toward leukemia cells and could induce apoptosis by mitochondrial pathways. Notably, the analysis of molecular docking suggested that hydrophobic interaction was essential for compound 10d to bind to BD1. In conclusion, these results demonstrate the potential of compound 10d to be utilized as a BRD4 inhibitor with apoptosis inducing effect in future leukemia therapy.

Full text of DOI:10.1016/j.ejmech.2016.05.057

European Journal of Medicinal Chemistry 121 (2016) 294e299
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Development of 4,5-dihydro-benzodiazepinone derivatives as a new
chemical series of BRD4 inhibitors
Jie Li a, Peiqi Wang c, Bihui Zhou a, Jianyou Shi b, Jie Liu b, Xiangrong Li a, Limei Fan a,
,
, *
Yaxin Zheng b **, Liang Ouyang b
a School of Medicine, Zhejiang University City College, Hangzhou 310015, Zhejiang, China
b State Key Laboratory of Biotherapy & Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center of Biotherapy, Chengdu
610041, Sichuan, China
c State Key Laboratory of Stomatology, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Bromodomains (BRDs) are protein interaction modules that selectively recognize ε -N-lysine residues,
serving as key epigenetic readers and play a key role in epigenetic regulation of gene transcription.
Bromodomain-containing protein 4 (BRD4), a protein containing two BRDs termed BD1 and BD2, has
emerged as an attractive candidate for the development of inhibitors targeting gene transcription in
several types of cancers. In this study, we made structural modifications of previously reported BRD4
inhibitors, to develop new chemical scaffold 3,4-dihydroquinoxalin-2(1H)-one. Four series of compounds
(compounds 7e10) were synthesized, and the BRD4-inhibitory activity and anti-proliferative effect of
these compounds were evaluated. We found compound 10d has remarkable anti-proliferative activities
toward leukemia cells and could induce apoptosis by mitochondrial pathways. Notably, the analysis of
molecular docking suggested that hydrophobic interaction was essential for compound 10d to bind to
BD1. In conclusion, these results demonstrate the potential of compound 10d to be utilized as a BRD4
inhibitor with apoptosis inducing effect in future leukemia therapy.
Received 22 March 2016
Received in revised form
23 May 2016
Accepted 25 May 2016
Available online 27 May 2016
Keywords:
Bromodomain inhibitors
BRD4
Dihydrobenzodiazepinone
Apoptosis
Epigenetics
© 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
domain [3]. Notably, it has been reported that BET family mem-
bers played a crucial role in tumorigenesis, and inhibition of them
Bromodomains (BRDs) are protein interaction modules that are
exclusively recruited to acetylated lysine in the N-terminal tails of
histones and function as important regulators of transcriptional
activity and chromatin remodeling [1]. BRDs can modulate enzyme
activities, protein conformations and interactions via lysine acety-
lation, thus controlling many downstream cellular processes such
as cell growth [2]. BRD-containing proteins have been implicated in
diverse human diseases, and the acetyl-lysine (KAc) binding
pockets have made them attractive targets for the development of
selective inhibitors. The bromodomain and extra terminal (BET)
family of bromodomain proteins consists of 4 members in humans
(BRD2, BRD3, BRD4 and BRDT), each of which contains two
conserved N-terminal BRDs (BD1 and BD2) and an extra-terminal
could transcriptionally down-regulate a number of oncogenes. For
instance, BRD4 can directly promote transcription of the c-MYC
oncogene, or increase the expression of MYC target genes [4,5].
BRD4 has also been suggested to stimulate G1 gene transcription
and promote cell cycle progression to S phase [6,16]. Hence, BRD4 is
becoming a new therapeutic candidate for cancer drug discovery
that targets gene transcription, and development of BRD4 in-
hibitors is of great clinical significance [20e22].
To date, several small molecule inhibitors of BET bromodomains
have been identified, such as JQ-1, PFI-1, RVX-208, etc (Fig. 1)
[7e11]. PFI-1 (Fig. 1, compound 1) occupies the acetyl-lysine
binding site in BD1 and BD2 and serves a cytotoxic role in leuke-
mia cell lines, indicating that BRD inhibitors have a potential to be
used in novel cancer therapy [7]. Another example RVX-208 (Fig. 1,
compound 2), is now under clinical trial for treatment of nuclear
protein in testis (NUT) midline carcinoma (NMC) [12]. The struc-
turally related triazolodiazepine compounds (þ)-JQ1 (Fig. 1, com-
pound 3) are the most potent (nanomolar) [10].
* Corresponding author.
** Corresponding author.
E-mail addresses: zhengyaxinxyz@163.com (Y. Zheng), ouyangliang@scu.edu.cn
(L. Ouyang).
http://dx.doi.org/10.1016/j.ejmech.2016.05.057
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

J. Li et al. / European Journal of Medicinal Chemistry 121 (2016) 294e299
295
O
N
N
N
O
O
N
N
O
NH
OH
S
O
O
O
O
N
O
S
N
H
NH
2,RVX-208
Cl
1, PFI-1
3, (+)-JQ1
O
O
O
N
N
N
N
H
NH
NH
N
4
O
5
Fig. 1. Known BET bromodomain inhibitors.
Since the exact role of BET bromodomains in gene transcription
regulation remains to be elucidated, further development of
domain-specific inhibitors is needed for more detailed-descriptions
of the BET family. Recently, new chemical series of BRD4 inhibitors
(Fig. 1, compound 4 and 5) with aromatic heterocyclic amide
structure were discovered by using protein-ligand docking and
structure-guided design [23]. These lead compounds exhibited the
potential to be new chemical scaffold BRD4 inhibitors in future
cancer therapy. In this study, we synthesized a series of compounds
(compounds 7aee, 8aee, 9aee, 10aee) via structural modifications
of this known leading compound. After preliminarily determining
their inhibitory activity against BD1 and/or BD2, five compounds
(7e, 8d, 8e, 10d, and 10e) with relatively higher potency were
selected as candidate compounds. Then, we further tested their
inhibitory activity towards BD1 and BD2, and their anti-
proliferative activity against three leukemia cell lines (U937,
HL60, and MV-411), using the typical dual-inhibitor PFI-1, RVX-208
and (þ)-JQ1 as control. The results proposed compound 10d as the
most effective inhibitor of BRD4. In vitro biological evaluations were
conducted to further demonstrate the apoptosis-inducing efficacy
of compound 10d. Moreover, docking analysis compared the
conformation of compound 10d and PFI-1 in BRD4(BD1). Alto-
gether, this study identifies compound 10d as a potent and selective
BD1 inhibitor of BRD4 with apoptosis-inducing effect in leukemia
cells.
2. Results
2.1. Chemical synthesis and screening of candidate BRD4 inhibitors
The chemical synthesis and structures of compounds 7e10 are
shown in Tables 1e4. Benzoic acid 6a could react with oxalyl
chloride to produce acyl chloride which was subsequently
condensed with 3,5-dimethyl-4-isoxazolamine to form 7a. Also,
aryl amides 7bee, 8aee, 9aee, and 10aee were prepared in the
same manner. Then, all of the products were purified and fully
characterized by NMR, mass spectrometry. For further structur-
eeactivity relationship studies, we investigated the effect of sub-
stitution site. Compounds 7e, 8d, 8e, 10d, and 10e showed relatively
higher inhibition rate against BRD4 than other compounds at a
Table 1
Synthesis of compounds 7aee and their BRD4-inhibitory activity.
Compound
Substitution
R1
Inhibition rate (30
BD1a
mM)
Ar
BD2a
7a
7b
7c
7d
7e
H
H
H
Ph
Ph
Ph
<20%
n.d.b
n.d.
n.d.
n.d.
2-naphthyl
3-pyridyl
Ph
23.1 2.1%
32.6 4.3%
46.2 7.2%
64.5 3.7%
3-OMe-Ph
36.3 5.1%
a
Each compound was tested in triplicate; the data are presented as the mean SD.
n.d. means not determined.
b

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J. Li et al. / European Journal of Medicinal Chemistry 121 (2016) 294e299
Table 2
Synthesis of compounds 8aee and their BRD4-inhibitory activity.
Compound
Substitution
R1
Inhibition rate (30
BD1a
mM)
Ar
BD2a
8a
8b
8c
8d
8e
H
H
H
Ph
Ph
Ph
<20%
<20%
<20%
72.4 2.4%
88.7 1.3%
n.d.b
n.d.
n.d.
23.1 2.4%
36.2 3.6%
2-naphthyl
3-pyridyl
Ph
3-OMe-Ph
a
Each compound was tested in triplicate; the data are presented as the mean SD.
n.d. means not determined.
b
Table 3
Synthesis of compounds 9aee and their BRD4-inhibitory activity.
Compound
Substitution
R1
Inhibition rate (30
BD1a
mM)
Ar
BD2a
9a
9b
9c
9d
9e
H
H
H
Ph
Ph
Ph
<20%
n.d.b
n.d.
n.d.
n.d.
n.d.
2-naphthyl
3-pyridyl
Ph
23.1 5.3%
34.2 2.2%
32.1 1.7%
22.8 3.1%
3-OMe-Ph
a
Each compound was tested in triplicate; the data are presented as the mean SD.
n.d. means not determined.
b
concentration of 30
were selected as candidate compounds for further studies.
m
M (Tables 1e4). Therefore, these compounds
selected compound 10d to validate its anti-proliferative activity,
and to decipher the underlying mechanisms.
To investigate the effect of compound 10d on the proliferation of
leukemia cells, the MTT assay was conducted with compound 10d
in U937 and HL-60 cells, respectively (Fig. 2A). As shown in Fig. 3A,
compound 10d caused an anti-proliferative effect on U937 and HL-
60 cell growth in a dose-dependent manner, and the IC50 was
2.2. BRD4 inhibitors induce apoptosis via the mitochondrial
pathways in leukemia cells
BRD4-inhibitory activity of the candidate compounds was
investigated by BRD4 bromodomain 1 TR-FRET assay kit, and was
expressed as IC50 values (Table 5). Among the tested compounds,
compound 10d showed the most intense activity against BD1 with
approximately 4 mM concentration in HL-60 cells. To characterize
the compound 10d-induced HL-60 cell growth inhibition, we
observed the morphologic changes in the cells. When the cells were
cultured with different concentrations of compound 10d, the
apoptotic alterations were observed by Hoechst 33258 staining
under the inverted microscopy (Fig. 2B). Moreover, compound 10d-
induced apoptosis was also measured by flow cytometry after
Annexin V-PI staining (Fig. 2C and D). To further clarify the mech-
anism of compound 10d, the expression pattern of BRD4 and
an IC50 value of 1.25
mM, similar to the typical inhibitor PFI-1
(IC50 ¼ 1.24
m
M). Moreover, compound 10d performed as a more
selective inhibitor than PFI-1, since its inhibitory activity against
BD1 was nearly 10-fold higher than that against BD2
(IC50 ¼ 11.23
mM). In addition, compound 10dalso exhibited anti-
viability activity against HL60and MV-411 cells. Herein, we

J. Li et al. / European Journal of Medicinal Chemistry 121 (2016) 294e299
297
Table 4
Synthesis of compounds 10aee and their BRD4-inhibitory activity.
Compound
Substitution
R1
Inhibition rate (30
BD1a
mM)
Ar
BD2a
10a
10b
10c
10d
10e
H
H
H
Ph
Ph
Ph
32.4 1.3%
25.3 3.3%
45.2 2.1%
96.1 2.1%
88.8 2.2%
n.d.b
n.d.
n.d.
54.2 1.2%
67.1 4.3%
2-naphthyl
3-pyridyl
Ph
3-OMe-Ph
a
Each compound was tested in triplicate; the data are presented as the mean SD.
n.d. means not determined.
b
Table 5
several key proteins in apoptotic pathway were examined by
western blot (Fig. 2E). Bax expression was increased whereas Bcl-2
expression was decreased in HL-60 cells, indicating compound 10d-
induced apoptosis in HL-60 cells is mediated by a mitochondrial
pathway. Next, we measured the active form of caspase-3 to
investigate the involvement of caspase-3in compound 10d-induced
apoptosis. Additionally, we showed that BRD4 expression was
down-regulated in compound 10d-treated HL-60 cells. Altogether,
these results indicate that compound 10d can effectively inhibit
BRD4 and induce apoptosis via the mitochondrial pathways in HL-
60 cells.
BRD4 inhibition profile and anti-viability activity of compound 10d.
Compound
Enzyme inhibition (IC50
,
m
M)a
Anti-viability (IC50, mM)
BD1b
BD2b
U937
HL-60
MV-411
7e
8d
8e
10d
8.24 1.22
7.42 1.42
6.25 1.01
1.25 0.27
2.74 0.74
0.98 0.24
>10
>20
>20
>20
11.23 1.41
9.14 1.01
3.54 0.61
0.61 0.02
0.021 0.002
>20
>20
>20
>20
18.2
>20
>20
>20
17.1
11.4
9.25
4.02
8.24
2.19
>20
5.3
19.2
13.2
17.61
4.21
9.61
7.75
>20
10e
PFI-1
RVX-208
(þ)-JQ1
0.091 0.003
6.4
a
Each compound was tested by FRET binding method.
b
2.3. Molecular docking of compound 10d with BD1
Data represent the mean and standard deviation of three independent
experiments.
Molecular docking is a widely used computational approach to
Fig. 2. BRD4 inhibitor compound 10d induces apoptosis in leukemia cells: A. Cell viability was measured by the MTT assay in compound 10d-treated U937 and HL-60 cells; B.
Apoptosis was determined by the analysis of Hoechst staining; C and D. Apoptosis was detected via Annexin V-FITC/PI staining in HL-60 cells treated with indicated concentrations
of compound 10d by flow cytometry analysis; *, P < 0.01; E. HL-60 cells were treated with compound 10d for 24 h, and then the expression of Bax, Bcl-2, pro-caspase 3, caspase 3
and Brd4 were examined by western blot.

298
J. Li et al. / European Journal of Medicinal Chemistry 121 (2016) 294e299
Fig. 3. Docking pose of PFI-1 (left) and compound 10d (right) in the active site of BD1.
screen potential active structures based on their interactions with
the binding site [13]. Compound 10d was selected as a potential
BRD-4 inhibitor for leukemia treatment, based upon the consider-
ation of kinase inhibitory activity and anti-tumor efficacy. In order
to understand the conformation of compound 10d in the active site
of BD1, the analysis of molecular docking was conducted to
compare the similarities and differences between compound 10d
and the reported BRD4 inhibitor PFI-1.
In this study, ligands with the most favorable binding free en-
ergies and reasonable orientations were chosen as the optimal
docked conformation. We showed that PFI-1 and compound 10d
could bind to BD1 domain of BRD4 stably (Fig. 3A and B). PFI-1
could form hydrogen bond with residue Pro82 of BRD4. For com-
pound 10d, the hydrogen bond was found between compound 10d
and residue Gln85of the BRD4. Moreover, PFI-1 can form hydro-
phobic interactions in the hydrophobic pocket with lipophilic res-
idues Phe83, Val87, Leu92, Cys136 and Ile146. Interestingly,
compound 10d, same as PFI-1, could form equal number of hy-
drophobic interactions with identical residues in the hydrophobic
pocket. The hydrophobic interactions between compound 10d and
BRD4 were also formed with lipophilic residues Pro82, Val87,
Leu92, Cys136 and Ile146. Altogether, compound 10d exhibits
efficient binding to BD1 domain of BRD4, which may support its
inhibitory effect against BRD4.
tandem BRDs (BD1 and BD2) [18]. With a histone H3 tail poly-
peptide encompassing BD1 residues 12e19, BD1 primarily binds
Nzeta-acetylated lysine 14 to the conserved asparagine 140 of Brd4,
whereas BD2 interact with the binding site for acetylated lysines of
the adjacent molecule via the N-terminal linker sequence [19].
Previous studies clarified that dual-inhibition of BD1 and BD2 had
effect on expression of 754 genes while only 46 genes were affected
when BD2 was inhibited solely [8]. In this study, we reported a
novel selective inhibitor on BD1, compound 10d, which has shown
a potent anti-viability effect in leukemia cells. Therefore, we may
suppose that inhibition of BD1 plays a pivotal role in cancer sup-
pression and thus can be implicated in future leukemia therapy.
The results of bioinformatics analysis and in vitro studies
demonstrated that compound 10d could effectively inhibit BD1 and
induce apoptosis in leukemia cells. Through further analysis, we
found that hydrophobic group and carbonyl group were necessary
for BRD4 inhibitors to form pi-sigma conjugation and hydrogen
bond respectively.
4. Conclusion
In this study, we report our structural modification chemical
synthesis and evaluation of a new class of BET bromodomain BRD4
inhibitors. Upon the basis of the previously reported BRD4 in-
hibitors, a new chemical scaffold 3,4-dihydroquinoxalin-2(1H)-one
was synthesized. The BRD4-inhibitory activity and anti-
proliferative effect of these compounds were evaluated. We found
compound 10d has remarkable anti-proliferative activities toward
leukemia cells and could induce apoptosis by mitochondrial path-
ways. Therefore, further investigation aiming at the potential
mechanisms behind the effects of BRD inhibitors is indispensable
when it comes to developing novel BRD4 inhibitors for leukemia
therapy.
3. Discussion
Lysine acetylation is a frequently observed posttranslational
modification which regulates chromatin structure and transcrip-
tion. Acetyl-lysine modifications create docking sites for BRDs, and
can be modulated by small interaction modules in diverse proteins
[14]. BET proteins are involved in the expression of key oncogenes
and anti-apoptotic proteins via their two N-terminal BRDs [15].
Recent studies have shown that potent and specific inhibitors of
BET bromodomains have a variety of effects, particularly in the field
of oncology [15]. PFI-1, a highly selective inhibitor, has been found
to efficiently occupy the KAc binding site in BRD4 and BRD2 and has
exhibit effective anti-proliferative activity against leukemia cells
[7,17]. RVX-208, a compound in phase II clinical trials, is a BD2
specific inhibitor, and has been demonstrated to regulate tran-
scription [8]. Since BRD inhibitors show a great potential to be
translated into clinical trial, further studies of the underlying
inhibitory mechanisms and discoveries of effective BRD inhibitors
still need to be accomplished.
Acknowledgements
We wish to thank Huawei Bioscience (Shanghai) for the help
with kinase assay, and we also thank Dr. Bo Liu and Dr. Shouyue
Zhang (Sichuan University) for their critical reviews on this
manuscript. Financial supports from National Natural Science
Foundation of China (No. 81473091 & 81302668); Distinguished
Young Scholars of Sichuan University (No. 2015SCU04A41) and
Hangzhou Science and Technology Information Institute of China
(No. 20150633B45) are gratefully acknowledged.
BRD4, a member of BET family, has been found to contain two

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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.05.057.
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