Design, synthesis and biological evaluation of benzo[cd]indol-2(1H)-ones derivatives as BRD4 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2018.04.048

Compound 1 bearing with benzo [cd]indol-2(1H)-one scaffold was identified as an effective BRD4 inhibitor through the AlphaScreen-based high-throughput screening and its high-resolution crystal structure with BRD4_BD1 protein. A series of 48 compounds were designed and synthesized by structural optimization on compound 1. All the compounds have been evaluated for their BRD4 inhibitory activities. The results showed that compounds 23, 24, 28 and 44 are the most potential ones with the IC₅₀ values of 1.02 μM, 1.43 μM, 1.55 μM and 3.02 μM, respectively. According to their co-crystal structures in complex with BRD4_BD1 and the protein thermal shift assays, the binding modes were revealed that the additional indirect hydrogen bonds and hydrophobic interactions make such four compounds more active than 1 against BRD4. Furthermore, compounds 1, 23 and 44 were chosen to evaluate for their antiproliferative activities on the MLL-AF4-expression acute leukemia cell line (MV4-11), other cancer cell lines (MDA-MB-231, A549, 22Rv1) and the non-cancer cell lines (HUV-EC-C, MRC5, RPTEC). The results showed that these compounds exhibited good and selective inhibitory activities against MV4-11 cells with the IC₅₀ values of 11.67 μM, 5.55 μM, and 11.54 μM, respectively, and could act on the cell proliferation by blocking cell cycle at G1 phase. They could markedly down-regulate the expressions of the c-Myc, Bcl-2 and CDK6 oncogenes in MV4-11 in the qRT-PCR and western blot studies, which further demonstrated that compound 1 and its derivatives could serve as a promising therapeutic strategy for MLL leukemia by targeting BRD4_BD1 protein.

Full text of DOI:10.1016/j.ejmech.2018.04.048

Accepted Manuscript
Design, synthesis and biological evaluation of benzo[cd]indol-2(1H)-ones derivatives
as BRD4 inhibitors
Yuxin Feng, Senhao Xiao, Yantao Chen, Hao Jiang, Na Liu, Cheng Luo, Shijie Chen,
Hua Chen
PII:
S0223-5234(18)30388-X
10.1016/j.ejmech.2018.04.048
EJMECH 10397
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 February 2018
Revised Date: 10 April 2018
Accepted Date: 23 April 2018
Please cite this article as: Y. Feng, S. Xiao, Y. Chen, H. Jiang, N. Liu, C. Luo, S. Chen, H. Chen,
Design, synthesis and biological evaluation of benzo[cd]indol-2(1H)-ones derivatives as BRD4 inhibitors,
European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.04.048.
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ACCEPTED MANUSCRIPT
Design, Synthesis and Biological Evaluation of Benzo[cd]indol-
2(1H)-ones Derivatives as BRD4 inhibitors
Yuxin Feng, Senhao Xiao , Yantao Chen, Hao Jiang, Na Liu, Cheng Luo, Shijie
Chen* and Hua Chen*
A series of novel derivatives 4-9 and 11-54 bearing with benzo[cd]indol-2(1H)-
one scaffold have been designed and synthesized through structure-based
optimization on a lead BRD4 inhibitor 1. Compounds 23, 24, 28 and 44 are the most
potential BRD4 inhibitors and compounds 23 and 44 exhibit significantly inhibitory
activities against MLL-rearranged MV4-11 cells proliferation.

ACCEPTED MANUSCRIPT
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research Paper
Design, Synthesis and Biological Evaluation of Benzo[cd]indol-2(1H)-ones
Derivatives as BRD4 inhibitors
Yuxin Fenga, Senhao Xiaob,c,d,# , Yantao Chenb,c, Hao Jiangb,c, Na Liua, Cheng Luob,c, Shijie Chenb,c* and
Hua Chena*
aKey Laboratory of Chemical Biology of Hebei Province, College of Chemistry and Environmental Science, Hebei University, Baoding 071002, China
bDrug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of MateriaMedica, Chinese Academy of Sciences, Shanghai
201203, China
cUniversity of Chinese Academy of Sciences, Beijing 100049, China
dSchool of Life Science and Technology, Shanghai Tech University, Shanghai 200031, China
ARTICLE INFO
ABSTRACT
Article history:
Received
Compound 1 bearing with benzo[cd]indol-2(1H)-one scaffold was identified as an effective BRD4
inhibitor through the AlphaScreen-based high-throughput screening and its high-resolution crystal
structure with BRD4_BD1 protein. A series of 48 compounds were designed and synthesized by
structural optimization on compound 1. All the compounds have been evaluated for their BRD4
inhibitory activities. The results showed that compounds 23, 24, 28 and 44 are the most potential ones
with the IC50 values of 1.02 µM, 1.43 µM, 1.55 µM and 3.02 µM, respectively. According to their co-
crystal structures in complex with BRD4_BD1 and the protein thermal shift assays, the binding modes
were revealed that the additional indirect hydrogen bonds and hydrophobic interactions make such four
compounds more active than 1 against BRD4. Furthermore, compounds 1, 23 and 44 were chosen to
evaluate for their antiproliferative activities on the MLL-AF4-expression acute leukemia cell line
(MV4-11), other cancer cell lines (MDA-MB-231, A549, 22Rv1) and the non-cancer cell lines (HUV-
EC-C, MRC5, RPTEC). The results showed that these compounds exhibited good and selective
inhibitory activities against MV4-11 cells with the IC50 values of 11.67 µM, 5.55 µM, and 11.54 µM,
respectively, and could act on the cell proliferation by blocking cell cycle at G1 phase. They could
markedly down-regulate the expressions of the c-Myc, Bcl-2 and CDK6 oncogenes in MV4-11 in the
qRT-PCR and western blot studies, which further demonstrated that compound 1 and its derivatives
could serve as a promising therapeutic strategy for MLL leukemia by targeting BRD4_BD1 protein.
Revised
Accepted
Available online
Keywords:
Benzo[cd]indol-2(1H)-one
Sulfonamide
BRD4 inhibitor
Bromodomain and extra-terminal domain
Acetyl-lysine binding pocket
2018 Elsevier Ltd. All rights reserved
.
#Co-first author; *Correspondence: E-mail:hua-todd@163.com; Tel & Fax.: 0086-312-5971116;
E-mail:shijiechen@simm.ac.cn; Tel & Fax.: 0086-021-50806600.

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Compound F, having benzo[cd]indol-2(1H)-one acaffold[19]
,
1. Introduction
was described as a specific BRD4 inhibitor with significant
activity, high selectivity and good pharmacokinetic profile
derived from the optimization of a structrue-based screening
hit[20]. Luo et al recently reported two series of potent inhibitors
G and H, which were identified as BRD4 inhibitors through the
Bromodomain-containing protein 4 (BRD4), belonging to the
bromodomain and extra-terminal domain (BET) family of
proteins, is responsible for recognizing the specific ε-N
acetylated lysine residues on histone tails[1-3]. As the ‘readers’
of lysine acetylation state, BRD-containing proteins play a key
role in gene transcription and epigenetics by binding acetylated
lysines (KAc) on chromatin structures. These proteins have been
implicated in diverse human diseases, such as cancer, diabetes,
inflammation and cardiovascular diseases[4-6]. For instance,
BRD4 can promote transcription of the c-MYC and Bcl-2
oncogenes, which are key regulators for the proliferation and
survival of tumor cells[7-9]. Hence, BRD4 is becoming a hot
target for cancer drug discovery, and its acetyl-lysine (KAc)
binding pockets provide a ‘druggable’ site which is suitable for
small molecule inhibitor development[10-12].
application
of
high-throughput
screening
and
crystallography[21,22]. Although there are many reported
inhibitors, the discovery of novel potent inhibitors still attracts
many attentions due to their therapeutic potential for various
human diseases[23,24]. The diverse inhibitors will also provide
pharmacological tools to investigate the fine structure and the
different functions of BRD4 protein.
Generally, the small-molecule inhibitors can anchor into the
KAc binding pocket of BRD4 as an acetylated lysine mimic
through forming a direct hydrogen bond with the conserved
asparagine 140 (N140) at the bottom of the pocket and indirect
hydrogen bonds via water molecule. They are also able to
interact with the WPF shelf composed of W81, P82 and F83 or
the ZA channel region which constitute the active KAc binding
pocket[20,25,26]. Recently, we found a new BRD4 ligand 1 (Fig.
1) bearing with benzo[cd]indol-2(1H)-one scaffold through high-
throughput screening. On the basis of a good understanding the
X-ray crystallograpthic support available, herein, we would like
to report the optimization of the derivatives of 1 (Scheme 1) to
explore its chemistry space for the potent drug discovering and
the more detailed structure activity relationships (SAR)
investigation. All of the new compounds were evaluated for their
BRD4 inhibitory activities.
Fig. 1 The structures of BRD4 inhibitors.
Many BRD4 inhibitors with different scaffolds have been
reported in the literature, such as the compounds A-H (Fig.
1)[13-24]. Compound A ((+)-JQ1) with triazolothienodiazepine
was the first potent BET inhibitor with an IC50 value of 77 nM
against BRD4 in the AlphaScreen assay[13]. Its analog I-
BET762 (compound B) has entered clinical trials for cancer
treatment[14]
.
Compound
C
(I-BET151) bearing with
isoxazoloquinaoline was discovered in GSK through fragment-
based approach and expressed in vivo efficacy against MLL-
fusion leukemia[15,16]. By the application of the same
approach, compounds D (PFI-1) and E were identified as the
selective sulfonamide inhibitors with IC50 values of 220 nM and
140 nM against BRD4, respectively[17,18]. As an alternative
way, structrue-based screening methods have also been widely
used to find BRD4 inhibitors for different chemotypes.
Scheme 1. The design and synthesis of compounds 1, 4-9 and 11-54.
Conditions and reagents: (a) 1) HSO3Cl, CHCl3, 0-50 oC; 2) Secondary
amines or amino acid esters, CH2Cl2, DIPEA, rt., two steps: 40-80% yields;
(b) Chloroalkane or bromoalkane, NaH, DMF, rt., 46-82% yields; (c) 90%
CF3COOH-H2O, rt., 68-82% yields; (d) NaOH, MeOH, rt., 85% yields; (e)

ACCEPTED MANUSCRIPT
NH2NH2 (or NH2OH, aromatic amine), HATU, DIPEA, 50 oC, 38-75%
yields.
structure of the first bromodomain of BRD4 (BRD4_BD1) in
complex with compound 1 (PDB code: 5Z90). As shown in Fig
2B, the acetylated lysine binding pocket of BRD4_BD1 was
occupied by compound 1 and the ligand fitted in the electron-
density map well. Previous studies indicated that N140 played a
crucial role in the substrates binding to BRD4_BD1[6]. The
carbonyl of lactam of compound 1 did form a hydrogen-bond
with N140 (Fig 2C). Besides, another indirect hydrogen-bond
was formed between the carbonyl and tyrosine 97 (Y97) via a
water molecule. Both the direct and indirect hydrogen-bonds
contributed to the affinity of compound 1 to the protein.
Moreover, compound 1 was also able to interact with the WPF
shelf by means of hydrophobic interactions (Fig 2C and 2D).
Except for the WPF shelf, residues (V87, L92, Y139, I146 and
M149) surrounding the ligand also formed hydrophobic
interactions with 1 and contributed to its activity (Fig 2D). In
conclusion, the obtained crystal structure of compound 1 in
complex with BRD4 was a direct and powerful proof of that
compound 1 could compete with the endogenous substrate for
active site binding. The structure of the complex also set up the
basis for us to conduct further structure-based optimization.
2. Results and Discussions
2.1 Discovery of a potent BRD4_BD1 inhibitor.
In this study, we performed AlphaScreen based high-
throughput screening[27] from our in-house library which
contained 20,000 small molecules (Fig S1). The primary
screening was performed with all 20,000 compounds at
concentration of 100 µM. Then, we chose 37 compounds with
inhibition rate higher than 90% for further validation. After two
rounds of screening, 17 candidate compounds were selected for
futher IC50 determination. The structures and inhibitory activities
were shown in Table S1, which exhibited compound 1 had the
best avtivity. Therefore, we identified 1 as a hit compound and
subsequently determine its IC50 value of 6.83 ± 1.46 µM by
AlphaScreen assay in three independent experiments. (Fig 2A).
To confirm the scaffold authenticity of the compound 1, and,
also to delineate the bind mode for performing structure-based
optimization, we determined a high resolution X-ray crystal
Fig 2. (A) Inhibition activity determination for compound 1 disrupting the interaction of BRD4_BD1 and peptide substrates. (B) Crystal structure of the first
bromodomain of BRD4 in complex with compound 1 (PDB ID: 5Z90). (C) Molecular interactions between compound 1 and key residues of BRD4_BD1.
Protein residues are shown in grey sticks with labeled names, structural waters in red spheres, respectively. Hydrogen bonds are shown as dotted yellow lines. (D)
2D diagram of interaction between compound 1 and the surrounding residues within the binding pocket of BRD4_BD1.

ACCEPTED MANUSCRIPT
that of 1, while 12 and 13 with morpholine and diethylamine,
respectively, showed good activities at 20 µM. Compound 14
with the ortho-methoxylphenylamine had poor activity.
2.2 Chemistry
Synthesis of compound 1 and its derivatives 4-9 and 11-54
were outlined in Scheme 1. Briefly, the commercially available
benzo[cd]indol-2(1H)-one 2 reacted with chlorosulfonic acid
followed by pyrrolidine treatment to generate the intermediate 3.
The nucleophilic substitutions of 3 with a variety of chloroalkane
or bromoalkane provided the N-alkyl substitution products 1 and
4-9.
To further explore SAR, 3-hydroxyl and 3-/4-amino groups
were incorporated into the pyrrolidine and piperidine (15-32),
respectively. As shown in Table 2, compounds 23, 24 and 28
with 3-hydroxyl or 3-amino on piperidine expressed very
significant inhibitory activities. Overall, the activities of
compounds bearing with the naked hydroxyl or amino, such as
15, 16, 20, 22, 23, 24, 26, 28 and 30, were more better than those
of the corresponding ones with Boc protection group, which
suggested that the hydroxyl or amino were more favorable for
their inhibitory activities possibly through forming an additional
hydrogen-bond as the donor. This postulation was supported by
the compounds 17 and 18 with 3-methoxyl on pyrrolidine that
showed poor activities. Furthermore, SAR analysis suggested
that the compounds containing hydroxyl were more active than
those having amino except 28, for examples 15, 16 versus 21,
22, and 23, 24 versus 26. Compounds 26, 30 and 32 exhibited
similar inhibition ratio, suggesting that the position of amino on
piperidine seemed to have little influence on their inhibitions.
As an alternative way, compound 10 was firstly obtained by
treatment with 2 and ethyl chloride. Then, compound 10 reacted
with chlorosulfonic acid to give the sulfonyl chloride
intermediate which was subsquently ammonolyzed with various
secondary amines or amino acid esters to afford the desired
sulfonamide products 11-18, 19, 21, 23-25, 27, 29, 31, 33, 43-46,
47, 49, 51 and 53.
t-Butyloxycarbonyl (Boc) group in 19, 21, 25, 27, 29 and 31
were removed in 90% CF3COOH to achieve the products 20, 22,
26, 28, 30 and 32, respectively.
The ester in 33, 47, 49, 51 and 53 were hydrolyzed in the
presence of base to afford the acid derivatives 34, 48, 50, 52 and
54, respectively. The condensation of 34 with hydrazine, or
hydroxylamine or aromatic amine using tetramethyluronium
hexafluorophosphate (HATU) and N-ethyldiisopropylamine
(DIPEA) as catalysts at room temperature gave the products 35-
42.
the pyrrolidine of the sulfonamide were displaced by proline
and its derivatives to give compounds (33-44). As shown in
Table 2, compound 33 with proline methyl ester exhibited
similar inhibitory activity against BRD4 with that of 1 at both 20
µM and 5 µM. Compounds 34-36 with more polar groups on
proline showed moderate activities, while the aromatic amide
derivatives 37-42 had no activities. In this series, the reductive
products 43 and 44, in which the carboxyl was reduced to
hydroxyl, showed good BRD4 inhibitory activities, especially,
the later was more active than 1 at 5 µM. This observation also
indicated that the hydroxyl group on the pyrrolidine and
piperidine had an important role in the binging between
BRD4_BD1 and the ligands.
The structures of all the newly compounds were determined
by NMR, (HR) MS and element analysis. Both analytical and
spectral data of compounds are in agreement with the proposed
structures.
2.3. BRD4_BD1 inhibitory activity
The pyrrolidine was further changed with amino alcohol chain
or amino acid (ester) to afford compounds 45-54 based on above
SAR analysis. However, most of the compounds 45-54 had no or
poor inhibitions possibly due to their flexibility, except 46 and
All the newly synthesized compounds 1, 4-9 and 11-54 were
evaluated for their BRD4_BD1 inhibitory activities at the
concentrations of 20 µM and 5 µM via AlphaScreen assay (Table
1 and 2). Compound 1 was used as the positive control.
52. Both compounds containing 3-amino-propanol and
D-valine,
respectively, had moderate activities against BRD4_BD1.
Since the lactam in 1 could interact with Asn140 and the
adjacent water molecule by hydrogen-bonds, we initiated the
structural modification on this fragment for affinity
improvement. Compounds 4-9 with various substituents attached
to the lactam were designed to increase the binding interaction.
However, no matter the change of the length of alky chain or the
introduction of some polar groups, such as carbonyl, hydroxyl,
carboxyl, or amide groups, the anti-BRD4 activities of 4-9
reduced markedly (Table 1). The results were consistent with the
reported results[20], suggesting that the ethyl group attached to
the lactam in compound 1 is the most favorable group to
accommodate well into the acetyl lysine site of the KAc binding
pocket.
The compounds with the inhibition rate above that of 1 at 5
µM were chosen to further evaluate their IC50 values (Table 2,
Fig S2). It could be seen that compounds 23, 24, 28 and 44
exhibited the most potential activities against BRD4_BD1 with
the IC50 values of 1.02 ± 0.08, 1.43 ± 0.23, 1.55 ± 0.43 and 3.02
± 1.26, respectively.
Above insights should be helpful to guide further design and
synthesis of the more potent BRD4_BD1 inhibitors derived from
compound 1.
Subsequently, we focused our optimization on the pyrrolidine
of the sulfonamide which occupied the hydrophobic WPF shelf.
The results are shown in Table 2. We preferentially evaluated
some different secondary amines (11-13) having similar size
with pyrrolidine and an aromatic amine (14). Compound 11 with
piperidine (six-membered ring) exhibited significant BRD4
inhibitory activity at both 20 µM and 5 µM, nearly same with
Table 1. BRD4_BD1 inhibitory activities of 1 and 4-9.
Compds.
R1
Inhibition ratio (%)
IC50 (µM)

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Fig 3. The melting curves of BRD4_BD1 protein with the compounds. The
thermal shift assay displayed the stabilization of the BRD4_BD1 by adding
compound 1 and its derivatives at a concentration ratio of 1:20 (100 µM).
20 µM
5 µM
(mean
± SD)
1
4
5
99
25
15
59
5
6.83 ± 1.46
N. D. b
2.5. Co-crystal structures of BRD4_BD1 inhibitors
a
In order to further explore the structure-activity relationships
of compound 1 and its analogs, we determined the co-crystal
structures of compound 23, 24, 28 and 44 in complex with
BRD4-BD1, respectively. According to the structures (Fig 4), the
same direct and indirect hydrogen bonds formed between N140,
Y97 and compound 1, as well as the hydrophobic interactions
with the WPF shelf existed in the all four co-crystal structures.
As expected, an additional indirect hydrogen bond was formed
between isoleucine 146 (I146) and the hydroxyl group in 23, 44
and the amino group in 28 via another water molecule (Fig 4A,
4C and 4D). This extra interaction further strengthened the
interactions between compounds 23, 28 and 44 and the binding
site of the protein. However, because of the opposite orientation
of the hydroxyl group of compound 24, the excess hydrogen
bond did not exist between the compound and I146 due to
overlarge distance (Fig 4B). This may partly explain that the
activity of compound 24 was slightly lower than that of
compound 23. Additionally, 24 also formed hydrophobic
interactions with residues L94 and D145 except those existed in
the interactions between compound 1 and protein (Fig 2B and
Fig S4B), which may make it have better activity than that of 1.
In terms of compound 44, although it formed the extra hydrogen
bond, residues participated in the hydrophobic interactions were
less than those of compound 23 and 28 (Fig S4A, C, D), which
possibly result in its the weakest inhibitory activity among these
three compounds.
-
N. D.
6
9
7
N. D.
7
8
8
4
-
-
N. D.
N. D.
9
-
-
N. D.
[a]: No activity;
[b]: Not determined.
2.4. Protein Thermal shift
To confirm the binding between BRD4_BD1 protein and our
compounds, we next carried out protein thermal shift assay[20].
As shown in Fig 3, compared to DMSO control, the melting
temperature (Tm values) of BRD4_BD1 protein was evidently
increased due to the addition of compound 1 and its derivatives
at a concentration of 100 µM. The results indicated that the
ligands had interaction with BRD4_BD1 protein and could
strengthen its stability in vitro.
Table 2. BRD4_BD1 inhibitory activities of 11-54.
Inhibition ratio (%)
20 µM 5 µM
Inhibition ratio (%)
20 µM 5 µM
IC50 (µM)
IC50 (µM)
Compds.
R2
Compds.
R2
(mean ± SD)
(mean ± SD)

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MeOOC
11
12
13
14
99
82
67
8
58
29
21
17
N. D.b
N. D.
N. D.
N. D.
33
34
35
36
97
82
88
80
55
33
35
28
N. D.
N. D.
N. D.
N. D.
N
15
95
57
N. D.
37
-
3
N. D.
16
17
18
19
20
88
27
20
20
45
37
9
N. D.
N. D.
N. D.
N. D.
N. D.
38
39
40
41
42
-
14
-
-
-
-
-
-
N. D.
N. D.
N. D.
N. D.
N. D.
9
7
-
18
16
a
21
22
23
18
61
-
N. D.
N. D.
43
44
45
74
98
50
23
66
7
N. D.
3.02 ± 1.26
N. D.
27
93
87
100
1.02 ± 0.08
24
25
26
27
100
39
1.43 ± 0.23
N. D.
46
47
48
49
89
51
-
42
8
N. D.
N. D.
N. D.
N. D.
-
NH2
63
18
11
N. D.
-
N
22.4
N. D.
4
9
28
29
30
31
100
12
66
89
71
98
-
1.55 ± 0.43
N. D.
50
51
52
53
54
-
53
60
-
-
-
N. D.
N. D.
N. D.
N. D.
N. D.
29
21
37
N. D.
41
-
N. D.
32
N. D.
-
4
[a]: No activity;

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[b]: Not determined.
cell lines (HUV-EC-C, MRC5, RPTEC) and other cancer cell
lines (MDA-MB-231, A549, 22Rv1). As shown in Fig S5, the
three compounds showed lower inhibitory activities on the non-
leukemia cancer cell lines than that of MV4-11 and minimal
effects on normal cell lines. The results suggested that
compounds 1, 23 and 44 have potent selectivity over other cell
lines, especially the non-cancer cells lines.
2.6. MLL-rearranged cell activity and cell cycle analysis
As BET family was determined as a therapeutic target in acute
myeloid leukemia (AML)[28], tens of inhibitors have been
proceed in phase or clinical trial these years[29]. In this study,
we used MLL-AF4-expression acute leukemia cell line (MV4-
11) to perform cell-based assays. Firstly, we performed a 3-days
cell viability assay against MV4-11 cell lines. Besides the parent
compound 1, 23 and 44 were chosen for cellular activity
evaluation, which show the best inhibitory activity among its
classification. As shown in Fig 5A, after treated with compounds
1, 23 and 44, reduction of viable cells was observed with an IC50
value of 11.67 µM, 5.55 µM, and 11.54 µM, respectively. The
results showed that the compounds had effective inhibition on
MV4-11 cell proliferation. In addition, we also carried the cell
proliferation assays of these active compounds on the non-cancer
To further investigate the mechanism of the anti proliferation
effects, we performed cell cycle analysis and treated the MV4-11
cells with different concentrations of compounds for 24 h. The
results showed these compounds could induce evidently cell
cycle arrest at G1 phase in a dose-dependent manner (Fig 5B and
Fig S6).
Fig 4. Co-crystal structures of compounds 23 (A), 24 (B), 28 (C), and 44 (D) with BRD4_BD1 (PDB IDs: 5Z8G, 5Z8R, 5Z8Z, and 5Z9K, respectively). The
ligands are shown as sticks, and the protein is shown as a cartoon. The binding site residues are shown as sticks, structural waters are in red spheres, respectively.
Hydrogen bonds interactions are indicated by dashed lines in yellow.
The BET family plays a crucial role on the transcription of c-
Myc, Bcl-2, CDK6 oncogenes, thus BRD4 inhibitors could
2.7. Down regulation of the expression of BET family target
genes

target ACCEPTED MANUSCRIPT
authenticated as a BRD4 inhibitor through the AlphaScreen-
induce down regulation of these
genes in both
transcription and translation level[30]. To further confirm the
anti proliferation effect was caused by the inhibition of BRD4,
we further performed real-time quantative flouresence PCR and
western Blot in MV4-11 cells to detect these target genes. As
shown in Fig 5C and 5D, remarkable dose-dependent decreases
of target genes expression were observed at mRNA and protein
level, respectively. The results demonstrated that the anti
proliferation effect of our compounds was, at least in part, due to
the inhibition of BRD4 in leukemia cell lines. Based on these
data, compound 1 and its derivatives might serve as a promising
therapeutic strategy for MLL leukemia by targeting BRD4_BD1
protein.
based high-throughput screening and its high-resolution crystal
structure with BRD4_BD1 protein. Among them, compounds 23,
24, 28 and 44 are the most potential inhibitors against BRD4
with the IC50 values of 1.02 µM, 1.43 µM, 1.55 µM and 3.02 µM,
respectively. The co-crystal structures of the four compounds
revealed that the additional indirect hydrogen bond with I146
and the hydrophobic interactions with residues L94 and D145
make such compounds more active than 1 against BRD4.
Compounds 1, 23 and 44 exhibited good and selective inhibitory
activities against MV4-11 cells, and could act on the cell
proliferation by blocking cell cycle at G1 phase. The direct
cellular inhibition activities were found that compounds 1, 23
and 44 also could effectively decrease the expressions of the c-
Myc, Bcl-2 and CDK6 genes in MV4-11 in the qRT-PCR and
western blot studies. All the results demonstrated that compound
1 and its derivatives could serve as the promising BRD4
inhibitors for MLL leukemia treatment.
3. Conclusions
A series of 48 compounds have been designed and synthesized
through structure-based optimization on the parent compound 1
containing benzo[cd]indol-2(1H)-one scaffold, which was
Fig 5. Cellular activities of compound 1, 23, and 44. (A) Cell proliferation inhibition curves of compounds for the MV4-11 cell lines at 72 hours. (B) MV4-11
cells were arrested at G1 phase by compounds at 24 hours. (C) Quantitative RT-PCR ananlysis revealed compound 1, 23, and 44 could down regulate the
transcription of c-Myc, Bcl-2 and CDK6 genes in MV4-11 cells after treating with them for 6 hours. (D) Treatment with compounds for 6 hours could dose-
dependently decrease the protein of c-Myc in MV4-11 cells.
4.1. General
4. Experimental section

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Column chromatography was carried out on flash silica gel
130.4 (CH), 130.3 (CH), 127.0, 126.5, 125.8 (2C), 125.2 (CH),
103.0 (CH), 47.5 (2CH2), 35.1(CH2), 25.3 (2CH2), 13.9 (CH3);
HRMS (ESI): Calcd for C17H18N2O3SNa ([M+Na]+): 353.0936,
Found: 353.0932. Anal. Calcd for C17H18N2O3S: C, 61.80; H,
5.49; N, 8.48. Found: C, 61.78; H, 5.52; N, 8.49.
(300-800 mesh). TLC analysis was conducted on silica gel plates
(Silica G UV254). Melting points were measured in an open
capillary on a SGW X-4 melting point apparatus and are
uncorrected. NMR spectra were recorded at 400 MHz and 600
1
MHz for H on a Bruker instrument. Chemical shifts (δ values)
and coupling constants (J values) are given in ppm and hertz,
respectively, using TMS (1H NMR) solvents as internal
standards. The High Resolution Mass Spectra (HRMS) were
carried out on a FTICR-MS (Ionspec 7.0T) mass spectrometer
with electrospray ionization (ESI). Element analysis was
performed using a Heraeus (CHNO, rapid) elemental analyzer.
The silica gel (300-400 mesh) for flash column chromatography
was from Qingdao Marine Chemical (China).
4.3. General procedure for the synthesis of compounds 11-18,
19, 21, 23, 24, 25, 27, 29, 31, 33, 43-46, 47, 49, 51 and 53
Following the above synthetic procedure of 1 from 3, the N-
ethyl intermediate 10 (9 g, yield 80%) were obtained using 2 (10
g, 60 mmol) as the starting material. The sulfonamide compound
11 was prepared by the ammonolysis reactions of the
corresponding sulfonyl chloride subsititution with piperidine
according to the above procedure from 2 to 3.
4.2. General procedure for the synthesis of compounds 1 and
4-9
Under the same conditions, compounds 12-18, 19, 21, 23, 24,
25, 27, 29, 31, 33, 43-46, 47, 49, 51 and 53 were obtained (for
detail, see Supporting information 1 and 2).
To a solution of benzo[cd]indol-2(1H)-one 2 (10 g, 60 mmol)
in chloroform (150 mL) was added batches of chlorosulfonic
acid (22 mL, 6.0 equiv.) at 0 °C for 30 min. The reaction mixture
was heated at 50 °C for 5 h. The mixture was then poured into
ice water and extracted with CHCl3 (100 mL × 3). The organic
layer was washed with brine and dried over Na2SO4. The solid
was filtered off, and the filtrate was concentrated under reduced
pressure to afford 2-oxo-1,2-dihydrobenzo[cd]indole-6-sulfonyl
chloride (10 g, yield 50%). The resulting crude product was
resolved in dichloromethane (100 mL), then the solution was
added into pyrrolidine (3.4 mL, 1.2 equiv.) and N-ethyldi
isopropylamine (DIPEA, 18 mL, 3.0 equiv.). The reaction
mixture was stirred at room temperature for 5 h. After
completion of the reaction as monitored by TLC, dilute HCl was
added, the aqueous layer was extracted with DCM (80 mL × 3),
and the organic layer was washed with water and brine, dried
with Na2SO4, and evaporated to give 6-(pyrrolidin-1-ylsulfonyl)
benzo[cd]indol-2(1H)-one 3 (9 g, yield 80%).
1-ethyl-6-(piperidin-1-ylsulfonyl)benzo[cd]indol-2(1H)-one
(11):
o
1
Yellow-green solid, yield 78%, m.p. 126.2 - 126.7 C; H
NMR (600 MHz, CDCl3) δ (ppm): 8.70 (d, J = 8.4 Hz, 1H), 8.08
(d, J = 7.2 Hz, 1H), 8.07 (d, J = 7.2 Hz, 1H), 7.80 (t, J = 7.8 Hz,
1H), 6.95 (d, J = 7.8 Hz, 1H), 3.97 (q, J = 7.2 Hz, 2H), 3.07 (t, J
= 5.4 Hz, 4H), 1.61 - 1.58 (m, 4H), 1.40 - 1.36 (m, 5H); 13C
NMR (150 MHz, CDCl3) δ (ppm): 167.5 (C=O), 143.7, 133.4
(CH), 130.4 (CH), 130.3 (CH), 126.5, 126.1, 125.7, 125.6, 125.2
(CH), 103.1 (CH), 46.5 (2CH2), 35.1 (CH2), 25.2 (2CH2), 23.4
(CH2), 13.9 (CH3); HRMS (ESI): Calcd for C18H20N2O3SNa
([M+Na]+): 367.1092, Found: 367.1090. Anal. Calcd for
C18H20N2O3S: C, 62.77; H, 5.85; N, 8.13. Found: C, 62.75; H,
5.88; N, 8.16.
4.4. General procedure for the synthesis of compounds 20, 22,
26, 28, 30 and 32
The product 3 (150 mg, 0.5 mmol) and sodium hydride (NaH,
36 mg, 1.5 mmol) were dissolved in DMF (15 mL).
Bromoethane (65.4 mg, 0.6 mmol) was added dropwise into the
solution and the reaction mixture was stirred at room
temperature. After completion of the reaction as monitored by
TLC, the reaction mixture was extracted with ethyl acetate (10
mL × 2). The organic layer was washed with brine and dried over
Na2SO4. The solid was filtered off, and the filtrate was
concentrated under reduced pressure. The crude product was
purified by silica gel chromatography with petroleum ether/ethyl
acetate (3/1, v/v) to yield compound 1.
Compound 19 (100 mg, 0.2 mmol) was dissolved in 2 mL
DCM and 2 mL 90% CF3COOH. The mixture was stirred at
room temperature. After completion of the reaction as monitored
by TLC, solid NaHCO3 was added to the solution to neutralize
the acid, and the solution extracted with water (3 mL) and ethyl
acetate (12 mL). The organic solvent was in sequence washed
with the distilled water (10 mL × 4), the saturated brine (10 mL ×
2), dried with MgSO4 for 5 h, then evaporated under vacuum to
afford the crude product. The residue was purified by silica gel
chromatography with dichloromethane/methanol (15/1, v/v) to
afford the compound 20.
Under the same conditions, compounds 4-9 were obtained (for
detail, see Supporting information 1 and 2).
Under the same conditions, compounds 22, 26, 28, 30 and 32
were obtained (for detail, see Supporting information 1 and 2).
1-ethyl-6-(pyrrolidin-1-ylsulfonyl)benzo[cd]indol-2(1H)-one
(1):
(R)-6-((3-aminopyrrolidin-1-yl)sulfonyl)-1-ethylbenzo[cd]indol-
Yellow-green solid, yield 77%, m.p. 168.1-168.9 oC; 1H NMR
(600 MHz, CDCl3) δ (ppm): 8.79 (d, J = 8.4 Hz, 1H), 8.16 (d, J
= 7.2 Hz, 1H), 8.11 (d, J = 7.2 Hz, 1H), 7.83 (t, J = 7.8 Hz, 1H),
6.96 (d, J = 7.8 Hz, 1H), 3.99 (q, J = 7.2 Hz, 2H), 3.31 (t, J = 6.6
Hz, 4H), 1.79 - 1.77 (m, 4H), 1.39 (t, J = 7.2 Hz, 3H); 13C NMR
(150 MHz, CDCl3) δ (ppm): 167.6 (C=O), 143.6, 132.9 (CH),
2(1H)-one (20):
Yellow-green solid, yield 80%, m.p.161.0 - 162.1 oC; 1H NMR
(600 MHz, CD3OD) δ (ppm): 8.75 (d, J = 8.4 Hz, 1H), 8.17 (d, J
= 7.8 Hz, 1H), 8.08 (d, J = 7.0 Hz, 1H), 7.90 (t, J = 7.8 Hz, 1H),

ACCEPTED MANUSCRIPT
7.24 (d, J = 7.2 Hz, 1H), 3.99 (q, J = 7.2 Hz, 2H), 3.71 - 3.68 (m,
1H), 3.54 - 3.52 (m, 1H), 3.45 (dd, J = 10.8, 6.6 Hz, 1H), 3.30
(d, J = 5.4 Hz, 1H), 3.27 - 3.22 (m, 1H), 2.20 - 2.14 (m, 1H),
1.87 - 1.82 (m, 1H), 1.35 (t, J = 7.2 Hz, 3H); 13C NMR (150
MHz, CD3OD) δ (ppm): 169.2 (C=O), 145.2, 135.1 (CH), 131.8
(CH), 131.4 (CH), 127.6, 126.9, 126.8, 126.7, 126.4 (CH), 105.3
(CH), 53.1 (CH), 51.2 (CH2), 46.9 (CH2), 36.1 (CH2), 31.4
(CH2), 14.1 (CH3); HRMS (ESI): Calcd for C17H19N3O3SNa
([M+Na]+): 368.1045, Found: 368.1041. Anal. Calcd for
C17H19N3O3S; C, 59.11; H, 5.54; N, 12.17. Found: C, 59.12; H,
5.56; N, 12.14.
1-((1-ethyl-2-oxo-1,2-dihydrobenzo[cd]indol-6-
yl)sulfonyl)pyrrolidine-2-carbohydrazide (35):
1
Yellow-green oil, yield 68%; H NMR (600 MHz, CDCl3) δ
(ppm): 8.76 (d, J = 8.4 Hz, 1H), 8.15 (d, J = 7.8 Hz, 1H), 8.09 (d,
J = 6.6 Hz, 1H), 7.83 (t, J = 7.8 Hz, 1H), 6.96 (d, J = 7.2 Hz,
1H), 4.25 (dd, J = 8.4, 2.4 Hz, 1H), 3.96 (q, J = 7.2 Hz, 2H),
3.60 - 3.57 (m, 1H), 3.25 - 3.21 (m, 1H), 2.11- 2.08 (m, 1H),
1.81 - 1.74 (m, 1H), 1.62 - 1.55 (m, 2H), 1.36 (t, J = 7.2 Hz, 3H);
13C NMR (150 MHz, CDCl3 ) δ (ppm): 171.6 (C=O), 167.4
(C=O), 144.4, 134.0 (CH), 131.0 (CH), 130.0 (CH), 126.5, 125.7
(2C), 125.6 (CH), 125.0, 103.2 (CH), 61.3 (CH), 49.3 (CH2),
35.1 (CH2), 30.4 (CH2), 24.4 (CH2), 13.8 (CH3); HRMS (ESI):
Calcd for C18H20N4O4SNa ([M+Na]+): 411.1103, Found:
411.1108. Anal. Calcd for C18H20N4O4S: C, 55.66; H, 5.19; N,
14.42. Found: C, 55.69; H, 5.15; N, 14.46.
4.5. General procedure for the synthesis of compounds 34, 48,
50, 52 and 54
To a solution of 33 (300 mg, 0.8 mmol) in 25 mL methanol
was added NaOH (93mg, 2.4mmol). The reaction mixture was
stirred at 50 oC. After completion of the reaction as monitored by
TLC, 12N HCl was added to the solution to neutralize the base.
The solvent was removed under reduced pressure. The residue
was purified by silica gel chromatography with dichloromethane/
methanol (3/1, v/v) to afford the compound 34.
4.7 Protein purification
His-tagged BRD4_BD1 construct was cloned into a modified
pET28a vector and expressed in the E. Coli strain BL21(DE3).
Cells were grown at 37ꢀ°C until OD600 reach a value of 0.6,
induced with 0.4ꢀmM IPTG and incubated overnight at 16ꢀ°C.
The protein was initially purified by Ni-NTA affinity
chromatography, and followed by an anion exchange
chromatography without cleave the 6x His-Tag. The protein was
further purified using a Superdex 75 (10/300) column (GE
Healthcare) equilibrated with a buffer containing 20 mM HEPES
(pH 7.4), 100 mM NaCl and 1 mM DTT. The protein was
concentrated and flash-frozen in liquid nitrogen and stored at -
80ꢀ°C.
Under the same conditions, compounds 48, 50, 52 and 54
were obtained (for detail, see Supporting information 1 and 2).
1-((1-ethyl-2-oxo-1,2-dihydrobenzo[cd]indol-6-
yl)sulfonyl)pyrrolidine-2-carboxylic acid (34)：
Yellow solid, yield 85%, m.p. 199.5 - 200.7 oC; 1H NMR (600
MHz, CD3OD) δ (ppm): 8.81 (d, J = 8.4 Hz, 1H), 8.21 (d, J = 7.8
Hz, 1H), 8.09 (d, J = 7.2 Hz, 1H), 7.89 (t, J = 7.8 Hz, 1H), 7.22
(d, J = 7.8 Hz, 1H), 4.28 (brs, 1H), 4.00 (q, J = 7.2 Hz, 2H), 3.53
(d, J = 6.6 Hz, 1H), 3.36 (brs, 1H), 1.97 - 1.92 (m, 3H), 1.67 -
1.65 (m, 1H), 1.36 (t, J = 7.2 Hz, 3H); 13C NMR (150 MHz,
CD3OD) δ (ppm): 180.0 (C=O), 169.4 (C=O), 144.5, 134.4
(CH), 132.2 (CH), 131.5 (CH), 129.4, 127.4, 126.9, 126.7, 126.2
(CH), 105.4 (CH), 64.4 (CH), 49.9 (CH2), 36.1 (CH2), 32.7
(CH2), 25.8 (CH2), 14.1 (CH3); HRMS (ESI): Calcd for
C18H18N2O5SNa ([M+Na]+): 397.0834, Found: 397.0832. Anal.
Calcd for C18H18N2O5S: C, 57.74; H, 4.85; N, 7.48. Found: C,
57.79; H, 4.82; N, 7.51.
4.8 Crystallization
For crystallization, the 6x His-Tag need to be removed by
TEV protease at 4 °C overnight. The protein was further purified
by size exclusion chromatography (GE Healthcare) in a buffer
containing 10 mM HEPES (pH 7.4), 100 mM NaCl and 5 mM
DTT. The peak fraction was collected and concentrated to
7ꢀmg/mL for crystallization. Crystals were obtained in a
crystallization reagent (4 M sodium formate and 15% glycerol)
at 16 °C by sitting-drop vapour-diffusion method. The crystals
were soaked in the crystallization reagents plus compounds and
1% DMSO for 4 or 5 days. The X-ray diffraction data of the
small molecule and BRD4_BD1 complex was collected at
Shanghai Synchrotron Radiaton Facilities (SSFR), BL19U. The
data was analyzed as previously described[31].
4.6. General procedure for the synthesis of compounds 35-42
To a solution of 34 (15 mg, 0.4 mmol), HATU (213 mg, 0.7
mmol) and hydrazine (16 mg, 0.5 mmol) in dry DMF (15 mL)
was added DIPEA (0.21 mL, 1.2 mmol). The mixture was added
for stirring at room temperature until the completion of reaction
(followed by TLC).The mixture was neutralized with 0.5N HCl
solution, then the residue was extracted with ethyl acetate (10
mL × 3). The organic layer was then sequentially washed with
water, brine, and dried over anhydrous Na2SO4, evaporated to
dryness under reduced pressure. The residue was purified by
silica gel chromatography with dichloromethane/methanol (10/1,
v/v) to afford the compound 35.
4.9 AlphaScreen assay
The amplified luminescent proximity homogeneous assay
(AlphaScreen) was performed as previously described[31]. In
general, the acceptor and donor beads were purchased at Perkin
Elmer. The sequence of biotin-labelled substrate peptide was
SGRG-K(Ac)-GG-K(Ac)-GLG-K(Ac)-GGA-K(Ac)-
Under the same conditions, compounds 36-42 were obtained
(for detail, see Supporting information 1 and 2).
RHRKVGG-K-Biotin (synthesized by Shanghai China Peptide
Corporation, purity > 95%). The assay was performed in an

assay buffer containing 20 mM HEPES, pH A7.4C, C0.1E%PbToEvinDe MANUSCRIPT
4.13 Quantitative real time PCR
serum albumin (w/v), 0.01% Triton X-100 and 1 mM DTT at a
volume of 20 µL. The BRD4_BD1 protein need to be incubated
with compounds at room temperature first. After 15 min, biotin-
labelled peptide was added into the plate (OptiPlate-384,
PerkinElmer) for 10 min. Then the streptavidin donor beads and
nickel chelate acceptor beads were added and incubated for 60
min. The signal counts were then measured by EnVision
(PerkinElmer) and analyzed by Graphpad Prism 5.0.
MV4-11 cells were cultured in 6 well plates at the density of
5×105/mL. Different concentrations of compounds or DMSO
control were added into the plates and incubateted for 6 hours.
The cells were then harvested and washed by PBS twice. The
total RNA was extracted by TRIzol Reagent kit (Vazyme
Viotech) and immediately performed reverse transcription using
HiScript II Q RT SuperMix Kit (Vazyme Biotech). Then, the
qRT-PCR assay was performed on the Quant Studio 6 Flex Real-
Time PCR system (Applied Biosystems) using SYBR Green
Real-Time PCR Master Mix (VazymeViotech). The data was
4.10 Protein thermal shift
The thermal shift assay was performed in an assay buffer
containing 20 mM HEPES, pH 7.4, 100 mM NaCl, 1 mM DTT.
The reaction system contained 5×SYPRO® Orange dye
(Invitrogen, 5000×stock in DMSO), 5 µM Brd4_BD1 protein
plus 0.5% DMSO or 100 µM compounds to make the final
DMSO concentration equal. The melting curve was generated by
analysized by the ∆∆Ct = ∆Ct (GENE-B2M)
(GENE-B2M) cancer method.
– ∆Ct
normal
4.14 Western Blot
MV4-11 cells were plated in six wells plate at the density of
5×105/mL at a volume of 2 mL. The cells were treated with
different concentrations of compounds or DMSO control for 6
hours. The primer antibodies (c-Myc, CST) were diluted in a
ratio of 1:1000 and incubated at 4 °C overnight. Then, the
secondary antibodies were diluted in a ratio of 1:10000 (Anti-
Rabbit, CST) and incubated for 1 h at room temperature.
Imaging was performed on ChemiScope3400 imaging system
(Amersham).
o
heating the plate with a slope of 2.5 C/min from 25 oC to 95oC.
The assay was performed on QuantStudioTM 6 Flex real-time
PCR machine (Applied Biosystems) and analyzed by the
software Protein Thermal Shift™v1.3 (Applied Biosystems).
4.11 Cell Proliferation assay
MV4-11 cells were obtained from the American Type Culture
Collection and cultured in RPMI 1640 medium supplemented
with 10% FBS (Life Technologies) and 1% penicillin/
streptomycin (Life Technologies). For the cell proliferation
assay, MV4-11 cells were plated in 96-well plates at a density of
1×105 /mL in a volume of 100 µL. About one hour later, different
concentrations of diluted compounds or DMSO control were
added into the plates in a volume of 25 µL and incubateted for 72
h. Normal cell lines HUV-EC-C (normal vascular endothelium
cells), MRC5 (normal lung fibroblast cells), RPTEC (normal
kidney epithelial cells) and cancer cell lines A549 (non-small
cell lung cancer cells), MDA-MB-231 (triple negative breast
cancer cells), 22Rv1 (prostate cancer cells) were also obtained
from the American Type Culture Collection and plated in 96-well
plates at a density of 3×104 /mL in a volume of 100 µL. The cell
viability was determined by CellTiter-Glo Luminescent Cell
Viability Assay (Promega) as the manufacture insturcted. The
luminescence was measured by EnVision (Perkin Elmer). Every
dilution point was run in triplicate, the data of which was
averaged and analyzed in Graphpad Prism 5.0.
Acknowledgments
We thank the staffs from BL19U1 beam line of National
Facility for Protein Science Shanghai (NFPS) at Shanghai
Synchrotron Radiation Facility, for assistance during data
collection. This work is supported by the National Natural
Science Foundation of China (81703415 to S.C., 21472208 and
81625022 to C.L.). This work is also sponsored by Shanghai
Sailing Program (17YF1423100 to S.C.).
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Highlights
> Compound 1 bearing with benzo[cd]indol-2(1H)-one scaffold was
identified as an effective BRD4 inhibitor.
> Novel derivatives 4-9 and 11-54 were designed and synthesized by
structural optimization on compound 1.
> Compounds 23, 24, 28 and 44 are the most BRD4 inhibitors.
> The co-crystal structures of the inhibitors in complex with BRD4_BD1
revealed the binding mode.
> Compounds 1, 23 and 44 inhibit MV4-11 cells proliferation by
blocking cell cycle at G1 phase.
> Compounds 1, 23 and 44 could decrease the expressions of the c-Myc,
Bcl-2 and CDK6 genes in MV4-11.