Design, synthesis, and biological activity evaluation of a series of novel sulfonamide derivatives as BRD4 inhibitors against acute myeloid leukemia

Article abstract of DOI:10.1016/j.bioorg.2021.104849

Accumulating researches have contributed much effect to discover novel chemotherapeutic drug for leukemia with expeditious curative effect, of which bromodomain-containing protein 4 (BRD4) inhibitor is considered as a eutherapeutic drug which has presented efficient cell proliferation suppression effect. In this study, we disclosed a series of phenylisoxazole sulfonamide derivatives as potent BRD4 inhibitors. Especially, compound 58 exhibited robust inhibitory potency toward BRD4-BD1 and BRD4-BD2 with IC₅₀ values of 70 and 140 nM, respectively. In addition, compound 58 significantly suppressed cell proliferation of leukemia cell lines HL-60 and MV4-11 with IC₅₀ values of 1.21 and 0.15 μM. In-depth study of the biological mechanism of compound 58 exerted its tumor suppression effect via down-regulating the level of oncogene c-myc. Moreover, in vivo pharmacokinetics (PK) study was conducted and the results demonstrated better pharmacokinetics features versus (+)-JQ1. In summary, our study discovers that compound 58 represents as a novel BRD4 inhibitor for further investigation in development of leukemia inhibitor with potentiality.

Full text of DOI:10.1016/j.bioorg.2021.104849

Bioorganic Chemistry 111 (2021) 104849
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis, and biological activity evaluation of a series of novel
sulfonamide derivatives as BRD4 inhibitors against acute myeloid leukemia
,
,
c
,
,
Ziying Fenga 1, Aiping Chenb 1, Jing Shic 1, Daoguang Zhoua, Wei Shia, Qianqian Qiua,
Xinhong Liua, Wenlong Huanga, Jieming Lid *, Hai Qiana *, Wenjie Zhanga
,
,
e
,
,e,*
a Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China
b College of Pharmacy, Nanjing University of Chinese Medicine, 138 Xianlin Avenue, Nanjing 210023, PR China
c Center for Drug Evaluation, NMPA, 128 Jianguo Road, Beijing 100022, PR China
d Henan University of Traditional Chinese Medicine, Zhengzhou, 450046, Henan, PR China
e Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
BRD4
Accumulating researches have contributed much effect to discover novel chemotherapeutic drug for leukemia
with expeditious curative effect, of which bromodomain-containing protein 4 (BRD4) inhibitor is considered as a
eutherapeutic drug which has presented efficient cell proliferation suppression effect. In this study, we disclosed
a series of phenylisoxazole sulfonamide derivatives as potent BRD4 inhibitors. Especially, compound 58
exhibited robust inhibitory potency toward BRD4-BD1 and BRD4-BD2 with IC50 values of 70 and 140 nM,
respectively. In addition, compound 58 significantly suppressed cell proliferation of leukemia cell lines HL-60
Bromodomain
Leukemia
C-myc
Inhibitor
and MV4-11 with IC50 values of 1.21 and 0.15 μM. In-depth study of the biological mechanism of compound
58 exerted its tumor suppression effect via down-regulating the level of oncogene c-myc. Moreover, in vivo
pharmacokinetics (PK) study was conducted and the results demonstrated better pharmacokinetics features
versus (+)-JQ1. In summary, our study discovers that compound 58 represents as a novel BRD4 inhibitor for
further investigation in development of leukemia inhibitor with potentiality.
1. Introduction
domain (CTD)[7]. Each bromodomain comprises the active acetyl-
lysine-binding pocket which is formed by ZA loop and BC loop[8].
Histone covalent modification is a class of sophisticated epigenetics
modification that, in the context of chromatin biology, partakes in
regulating the structure of chromatin and the transcriptional stimulation
of oncogene[1,2]. Bromodomain-containing protein 4 (BRD4) is a pro-
tein that considered as a member of bromodomain and extra-terminal
domain (BET) family, which serves as a “reader” to recognize the spe-
Interfering the interaction between BRD4 and histone via BRD4 inhi-
bition is recently recognized as a promising therapeutic strategy for
various types of carcinomas[5,9–11], inflammation[12,13], and HIV
[14,15]. For its notably “druggable” site of acetyl-lysine-binding
pockets, BRD4 has been seen as a promising target for the further
development of small molecular inhibitor[8].
cific
ε
-N acetylated lysine residues on histone tails[3]. The recognition
Many BRD4 inhibitors with different scaffolds have been reported in
the literature, such as the compounds 1–12 (Fig. 1). (+)-JQ-1 (1) with
triazolothienodiazepine was the first potent BET inhibitor with an IC50
value of 77 nM against BRD4 in the AlphaScreen assay[16]. Its analog I-
BET762 (2) has entered clinical trials for cancer treatment[17]. I-
BET151 (3) bearing with isoxazoloquinaoline was discovered by GSK
through fragment-based approach and it exhibited in vivo efficacy
against MLL-fusion leukemia [18,19]. By the application of the same
and binding of the acetylated lysine sequences on histones of BRD4
could modulate multifarious downstream cell processes, including cell
cycle, proliferation and apoptosis[4,5]. After combining to acetylate
histones, BRD4 interacts with the positive transcription elongation fac-
tor complex (P-TEFb) and thereby affects the activity of RNA polymerase
II [6]. The constitution of BRD4 includes two bromodomains (BD1 and
BD2), N-terminal extra-terminal (NET) domain, and a C-terminal
* Corresponding authors at: Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing
210009, PR China (H. Qian and W. Zhang). Henan University of Traditional Chinese Medicine, Zhengzhou, 450046, Henan, PR China (J. LI).
E-mail addresses: jiemingli@hactcm.edu.cn (J. Li), qianhai24@163.com (H. Qian), zhangwjcpu@163.com (W. Zhang).
1
These authors made equal contribution to this work.
https://doi.org/10.1016/j.bioorg.2021.104849
Received 27 December 2020; Received in revised form 18 March 2021; Accepted 19 March 2021
Available online 22 March 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

Z. Feng et al.
Bioorganic Chemistry 111 (2021) 104849
approach, ABBV-075 (7) and PFI-1 (8) were identified as the selective
sulfonamide inhibitors with IC50 values of 20 nM and 220 nM against
BRD4, respectively [20,21]. As an alternative way, structrue-based
screening methods have also been widely used to find BRD4 inhibitors
for different chemotypes. Compound 10 with 3,5-dimethylisoxazole
scaffold[22] was described as a specific BRD4 inhibitor with signifi-
cant activity, high selectivity and good pharmacokinetics profile derived
from the optimization of a structrue-based screening hit. Studies
recently reported two series of potent inhibitors 11 and 12, which were
identified as BRD4 inhibitors through the application of high-
throughput screening and crystallography[23,24].
2. Results and discussion
2.1. Design strategy for the development of novel BRD4 inhibitors
Based on our previous literature investigational study, we noted that
the representatively typical drug for BRD4 inhibition and the com-
pounds published recently, all collectively presented that the nitrogen
heterocycles (including methylisoxazole, methylpyridine, thiazolidi-
none and triazole) are a kind of crucial and functional groups that are
necessary for activity, the core structure of the compound is highly
conserved, and monocyclic, bicyclic and tricyclic rings could retain the
activity of inhibiting BET proteins. The cores of these compounds also
contain hydrophobic side chains. The main purpose of our research is to
design and synthesize a series of BRD4 inhibitor with high selectivity
and novel chemical structures with various kinds of new functional
groups appended to a known scaffold with effective combination ability
to BET proteins. Compound 11 bearing 3,5-dimethylisoxazole was
discovered using computational research and it showed combination
ability toward BET family proteins including BRD2, BRD3 and BRD4
[29]. Here in this study, compound 11 was selected as our lead com-
pound for the development of potential BRD4 inhibitor via structural
modification mainly for two reasons. The first reason is that compound
11 has been validated to own BET affinity toward BRD2/3/4 and the
binding mode of 11 has been well investigated via crystallographic data.
[29] The second reason is that compound 11 with simple structure
provides a platform that is easy to investigate the effect of alteration of
functional group distinctly. According to the reported research by
Christopher et al[30] we noted that 1,2,3-triazole could be used as po-
tential acetyl-lysine mimetic heterocycle to improve the molecular
interaction with BRD4 protein. Herein, based on compound 11, the
1,2,3-triazole five-ring system with multifarious hydrogen-bond
So far, majority of researchers have done much effort to discover that
the inhibition of BRD4 is a curative strategy for human acute myeloid
leukemia (AML)[25]. However, drug-resistance against BRD4 inhibitor
gradually occurred with the long-term usage in clinical therapy. Lucu-
bration of the concrete mechanism of BRD4 inhibitor demonstrated that
NF-κB signaling activation[26], overexpression of DUB3[27], and the
aberrant suppression of PRC2 complex[28] all contribute to the primary
and acquired BRD4 inhibitor resistance. Therefore, seeking for effective
BRD4 inhibitor with novel chemical structure is still of great importance
for the AML therapeutic field. Although there are many reported in-
hibitors, the discovery of novel potent inhibitors still attracts much
attention due to their therapeutic potentiality for various human dis-
eases. The diverse inhibitors will also provide pharmacological tools to
investigate the fine structure and the different functions of BRD4 pro-
tein. Here in this study, we disclosed a novel serious of compounds
bearing 1,2,3-triazole and sulfonamide groups as potential BRD4 in-
hibitors for acute myelogenous leukemia (AML).
Fig. 1. Representative BRD4 inhibitors. KAc (Acetylated lysine) mimicking motif highlighted in red. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
2

Z. Feng et al.
Bioorganic Chemistry 111 (2021) 104849
receptors was introduced on the side chains to aim at improving its
binding potency with BRD4 protein via promoting the formation of a
hydrogen bond (Fig. 2). Simultaneously, the 1-position of 1,2,3-triazole
moiety was found probable to be extended into the ZA channel, which
might further increase the affinity to BRD4 protein, and the introduction
of a 1,2,3-triazole five-ring system might improve the stability in liver
microsomes[31] and thereby improve the pharmacokinetics feature.
Different substituents were also introduced into the 1-position of the
1,2,3-triazole to investigate structure–activity relationship. Subse-
quently, using the principle of bioelectricity isotactic, the structure of
acetylated lysine active center was modified, of which 3,5-dimethyl
isoxazole structure was replaced by N-methylpyrimidine parent nu-
cleus. Based on these two structural modification strategies, we designed
and synthesized a series of compounds (compounds 19–40, 51–65) for
further evaluation of the biological function.
(Fig. 3). Additionally, the experimental consequences exhibited that
compounds 51–65 bearing methylpyridine manifested roughly 10 folds
of potentiated binding potency with BRD4-BD1 versus compounds
19–40 bearing methylisoxazole. These results collectively illustrated
that methylpyridine group acts as a KAc mimic group with higher
binding affinity with BRD4-BD1 versus methylisoxazole. Next, the
introduction of 1,2,3-triazole group enabled the compound to extend to
the ZA channel and thereby enhancing its combination ability to BRD4.
To understand the binding mode of the series of methylpyridine
¨
compounds, Schrodinger was adopted to analyze the possible interac-
tion mechanism with BRD4-BD1. As shown in Fig. 3A, compound 58 was
docked into BRD4-BD1 crystal complex (PDB ID: 4BJX), and the analysis
of the docking conformation of 58 with BRD4-BD1 revealed that 58
binds in well-defined pocket and forms interactions with ASN140 and a
second interaction with TYR97, via a structured water molecule.
Meanwhile, it has a second interaction with ASP81, which is similar to
the binding mode of the lead compound 11 (Fig. 3B). Altogether,
compound 58 exhibits efficient binding affinity to BD1 domain of BRD4,
which may support its efficiently inhibitory effect against BRD4.
2.2. Chemistry
The synthetic routes of target compounds 19–40 and 51–65 were
outlined in Schemes 1-2. Flash chromatography was conducted for the
purification of compounds and the purity was verified using HPLC
(purity was > 95%). 1H NMR, 13C NMR spectrum, infrared spectrum,
and mass spectrometry were applied to further confirm the chemical
structure.
2.4. Compound 58 presented promising BRD4 inhibitory effect in AML
cells
BRD4-BD2 binding affinity of the preferred compounds was also
detected. Compound 58 showed relatively lower inhibitory rate against
BRD4-BD1 than other compounds, which suggested that the phenyl
group was important for compound activity. R1 was replaced with N-
methyl pyridone-2(1H)-one, of which the inhibition data identified it as
the optimal substituent. Furthermore, compounds with high BRD4-BD1
affinity were selected to further investigate their IC50 values against
BRD4-BD2, of which compound 58 exhibited better affinity toward
BRD4-BD2 compared with I-BET151, and similar affinities toward
BRD4-BD2 versus (+)-JQ1 (Table 2).
2.3. Structure-activity relationship study
To develop compounds with improved affinity for BRD4-BD1, we
focused on the ZA channel region and KAc (Acetylated lysine) pocket
region, and performed an extensive structure activity relationship (SAR)
study in an effort to enhance the protein–ligand interactions. The BRD4
protein binding affinity of compounds (19–40, and 51–65) were eval-
uated utilizing TR-FRET assay in vitro. (þ)-JQ-1 and I-BET151 were
applied as positive control. The binding affinity against BRD4-BD1 at a
Further investigation of the effect of compound 58 on AML was
conducted via estimating the anti-proliferative capacity on AML cell
lines HL-60 and MV4-11 (Table 3). The reported sensitive cell lines
human promyelocytic leukemia HL-60 and acute myeloid leukemia
MV4-11 were utilized to test the cellular proliferation inhibition effects.
Our compounds manifested reasonable potency against HL-60 and MV4-
11 cells. Overall, considering the data from the above assays, compound
58 has good profiles for further evaluation.
concentration of 1 μM was estimated and the results were presented in
Table 1. Obviously, the synthetic compounds 19–40, and 51–65 all
indicated conspicuously higher affinity to BRD4-BD1 compared to the
previously reported compound 11. Comparing our synthetic novel de-
rivatives to 11 (lead compound), the introduction of 1,2,3-triazole
significantly potentiated the inhibitory effect toward BD1, which indi-
rectly validated our previous assumption that the introduction of tri-
azole side chain could improve the selectivity toward BRD4. Especially
To study the specific mechanism of 58, we subsequently applied flow
cytometry (FCM) assay to explore its capacity to induce cell cycle arrest
and apoptosis in AML cells (MV4-11). As shown in Fig. 4A and 4B,
compound 58 had the effect in inducing cell cycle arrest in G1 phase in a
dose dependent, and generating cell apoptosis with the increasing con-
centration. Furthermore, multiple studies demonstrated that BET bro-
modomain proteins act as upstream modulatory factors for c-myc[32],
compound 58 with an IC50 value of 0.07
μM toward BRD4-BD1 is
considered as the best among all preferred compound and it exerted
similar activity to (þ)-JQ-1 and better activity than I-BET151. Docking
study further demonstrated that compound 58 exposed to the ZA
channel via triazole side chain, which further explains that compounds
with 1,2,3-triazole group own augmented binding affinity with BRD4
Fig. 2. Schematic representation of key KAc (Acetylated lysine) mimetic binding interaction of 11 with BRD4-BD1 and SAR investigations on the KAc mimicking
group and ZA Channel region. Fg represents functional groups.
3

Z. Feng et al.
Bioorganic Chemistry 111 (2021) 104849
Scheme 1. General synthesis of 3,5-dimethylisoxazole derivatives 19–40. Reagents and conditions: (i) 4-iodo-3,5-dimethylisoxazole, Pd(PPh3)4, K2CO3, 1,4-
dioxane/H2O (4/1), 110℃; (ii) ClSO3H, PCl5, CH2Cl2, 0℃; (iii) cyclopentylamine, pyridine, CH2Cl2, rt; (iv) BBr3, CH2Cl2, 0℃; (v) 3-bromo-1-propyne, K2CO3,
acetonitrile, reflux; (vi) azides, CuSO4⋅5H2O, sodium L-ascorbate, H2O/CH3OH (3:1), r.t., 6–15 h.
Scheme 2. General synthesis of N-methyl pyridone-2(1H)-one derivatives 51–65. Reagents and conditions: (i) ClSO3H, PCl5, CH2Cl2, 0℃; (ii) cyclopentylamine,
pyridine, CH2Cl2, rt; (iii) bis(pinacolato)diboron, KOAc, Pd(dppf)2Cl2, 1,4-dioxane, 95 ◦C; (iv) 6 M HCl, reflux for 8 h; (v)CH3I, NaH, DMF, rt; (vi) the intermediate
44, Pd(PPh3)4, K2CO3, 1,4-dioxane/H2O (4/1), 110℃; (vii) BBr3, CH2Cl2, 0℃; (viii) 3-bromo-1-propyne, K2CO3, DMF, reflux; (ix) azides, CuSO4⋅5H2O, sodium L-
ascorbate, H2O/CH3OH (3:1), r.t., 6–15 h.
and BRD4 inhibitor (+)-JQ1 induces reduction on c-myc expression to
make an anti-proliferative effect[33]. Hence, to further assess whether
compound 58 exerted its effect via impeding the expression of c-myc, we
conducted the western blot experiment to study the cellular effect
related to c-myc, and (+)-JQ-1 was used as positive control. As shown in
Fig. 4C, the abundance of c-myc was presented in dose-dependent
reduction both in (+)-JQ1 group and compound 58 group when
compared with negative control, which indicated that compound 58 is
4

Z. Feng et al.
Bioorganic Chemistry 111 (2021) 104849
Table 1
Structures and BRD4-BD1 binding affinity of compounds 19–40 and 51–65.
Compd
R1
R2
BRD4-BD1
Compd
R1
R2
BRD4-BD1
IC50
(μ
M) a
IC50
(μ
M) a
19
H
1.61 ± 0.06
39
4-NO2
1.22 ± 0.06
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
2-Cl
1.22 ± 0.07
3.15 ± 0.01
2.01 ± 0.03
1.18 ± 0.06
2.12 ± 0.03
0.89 ± 0.06
1.87 ± 0.04
1.85 ± 0.17
0.56 ± 0.01
4.72 ± 0.05
1.12 ± 0.02
1.93 ± 0.11
1.54 ± 0.01
1.93 ± 0.02
6.42 ± 0.12
5.58 ± 0.17
2.10 ± 0.04
2.87 ± 0.08
1.04 ± 0.05
40
4-OCF3
H
2.61 ± 0.13
0.11 ± 0.01
0.21 ± 0.01
0.39 ± 0.02
0.14 ± 0.01
0.42 ± 0.02
0.18 ± 0.01
0.16 ± 0.01
0.070 ± 0.01
0.24 ± 0.01
0.27 ± 0.03
0.29 ± 0.01
0.20 ± 0.01
0.26 ± 0.01
0.45 ± 0.04
0.48 ± 0.03
>10
3-Cl
51
4-Cl
52
2-Cl
4-F
53
4-Cl
2,4-di-F
54
4-F
2-OCH3
55
3-Cl
4-OCH3
56
3-OCH3
4-OCH3
3,4-di-OCH3
4-NO2
4-Br
3-OCH3
57
3,4-di- OCH3
3,4-dioxolane
3,4,5-tri-OCH3
3-CH3
58
59
60
61
2,4-di-Cl
2-NO2
4-OH
4-tertiary butyl
4-OCF3
–
2-CH2CH3
3-isopropyl
3-tertiary butyl
4-tertiary butyl
4-butyl
62
63
64
65
11
–
–
–
2-CH3-5-Cl
2-NO2
(þ)-JQ-1
I-BET151
–
0.062 ± 0.01
0.086 ± 0.02
–
a
The IC50 in the table was calculated from the TR-FRET assay. The data were expressed as the means ± SD, representing the data from at least three independent
experiments.
an effective BRD4 selective inhibitor via down-regulating c-myc.
as well as tumor inhibitory function of compound 58, further investi-
gation of the in vivo pharmacokinetics study was conducted to estimate
its metabolic feature in rats. 10 mg/kg dose and 20 mg/kg dose of
compound 58 for intravenous injection (i.v.) and oral administration (p.
o.), respectively. Subsequently, LC-MS/MS was applied for the analysis
2.5. Assessment of pharmacokinetics (PK) properties
According to the conspicuous binding affinity toward BD1 and BD2
5

Z. Feng et al.
Bioorganic Chemistry 111 (2021) 104849
Fig. 3. (A) Docking conformation of 58 in BRD4-BD1 (PDB ID: 4BJX). (B) Overlay of compound lead compound 11 (Blue) and 58 (Green) bound to BRD4-BD1 (PDB
ID: 4BJX). Key residues are labeled in red, and hydrogen bonding interactions are represented by green dashed lines. (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.)
of compound 58 plasma concentrations. Depicted in Table 4 were the
Table 2
analytical results of compound 58. Data showed that the Cmax value was
The affinity to BRD4-BD2 of the selected preferred compounds.
88.73 ng/ml, the AUC value was 553.38 ng*h /ml, the T1/2 value was
Compounds
BRD4-BD2
Compounds
BRD4-BD2
10.85 h, and the oral bioavailability was 36.88%. Conclusively, our
experimental data exhibited that compound 58 is a potential BRD4 in-
hibitor with better pharmacokinetics features compared to those of
(+)-JQ1[34].
IC50
(μ
M) b
IC50
(μ
M) b
26
29
51
52
53
54
55
56
57
58
1.65 ± 0.07
1.12 ± 0.03
0.26 ± 0.01
0.55 ± 0.06
0.94 ± 0.02
0.22 ± 0.03
0.76 ± 0.04
0.42 ± 0.03
0.30 ± 0.02
0.14 ± 0.05
59
0.51 ± 0.03
0.46 ± 0.01
0.57 ± 0.04
0.44 ± 0.03
0.57 ± 0.07
0.86 ± 0.06
0.84 ± 0.03
0.12 ± 0.04
NT
60
61
62
3. Conclusion
63
64
65
In summary, we designed and synthesized a series of novel 3,5-dime-
thylisoxazole derivatives as BRD4 inhibitors via structural modification
of a previously reported molecule. In vitro experiments demonstrated
conspicuous anti-proliferative capacity of compound 58, and 58 also
exhibited robust binding affinity toward both BRD4-BD1 and BRD4-BD2
with IC50 values of 70 and 140 nM, respectively. Docking studies were
performed to determine structure–activity relationships. Moreover,
compound 58 presented proliferative effect toward HL-60 and MV4-11
(þ)-JQ-1
I-BET151
NT = not tested.
b
The IC50 in the table was calculated from the TR-FRET assay. The data were
expressed as the means ± SD, representing the data from at least three inde-
pendent experiments.
leukemia cells with IC50 values of 1.21 and 0.15 μM, respectively. In
addition, it can further down-regulated c-myc levels in MV4-11 cells.
These findings demonstrate that compound 58 might be a potent BRD4
inhibitor in the cellular environment and is worthy of further
investigation.
Table 3
The IC50 values of 17 optimization compounds against HL-60 and MV4-11 cell
lines.
Compounds
HL-60
MV4-11
Compounds
HL-60
MV4-11
IC50
(
μ
M)a
IC50
(
μ
M)
IC50
(
μM)
IC50
(
μ
M)
a
a
a
4. Experimental section
26
29
51
52
53
54
55
56
57
58
>10
1.47 ±
0.11
59
4.45 ±
0.22
0.44 ±
0.05
4.1. Chemistry
2.85 ±
0.21
0.86 ±
0.07
60
>10
0.89 ±
0.11
4.1.1. General
1.56 ±
0.15
0.42 ±
0.08
61
6.42 ±
0.16
0.48 ±
0.06
The hydrogen and carbon nuclear magnetic resonance (NMR)
spectra were measured by Bruker ACF300/400 MHz nuclear magnetic
resonance instrument, the internal standard was tetramethylsilane
(TMS); the mass spectrometry (MS) was measured by Waters UPLC/MS
liquid-mass spectrometry system; experiment The middle column
chromatography uses 200–300 mesh silica gel produced by Qingdao
Ocean Chemical Co., Ltd. as the stationary phase, and thin layer chro-
matography (GF254) plates are purchased from Yantai Jiangyou Silica
Gel Development Co., Ltd. The chemical reagents are all commercially
available analytical or chemically pure products. Unless otherwise
specified, reagents are used directly without treatment.
1.55 ±
0.09
0.37 ±
0.14
62
4.57 ±
0.08
0.42 ±
0.07
5.54 ±
0.14
0.65 ±
0.22
63
6.57 ±
0.10
0.54 ±
0.12
2.10 ±
0.22
0.28 ±
0.02
64
>10
0.93 ±
0.15
3.12 ±
0.13
0.58 ±
0.13
65
5.86 ±
0.23
1.18 ±
0.22
1.76 ±
0.05
0.37 ±
0.06
(þ)-JQ-1
I-BET151
1.12
0.13
1.87 ±
0.11
0.32 ±
0.10
NT
NT
1.21 ±
0.02
0.15 ±
0.02
4.1.2. Synthetic procedures
NT = not tested.
Compounds 13, 41 and 45 were purchased. Other compounds were
prepared by one of two schemes.
a
The IC50 value in the table were expressed as the means ± SD, representing
the data from at least three independent experiments.
Scheme 1 Firstly, we used 4-methoxyphenylboronic acid (13) as raw
6

Z. Feng et al.
Bioorganic Chemistry 111 (2021) 104849
Fig. 4. Compound 58 was validated the effects of cell apoptosis promotion, G1 phase arrest, and c-myc silencing. (A) Compound 58 caused apoptosis in MV4-11 cells
after 48 h of incubation. (B) Compound 58 caused cell cycle arrest in MV4-11 cells after 24 h of incubation. (C) Western blot assay indicated that the down-regulation
of c-myc is presented concentration dependent with compound 58. *p < 0.05, ***p < 0.001 compared with control group. #p < 0.05, ###p < 0.001 compared with
&
&&&
JQ-1 0.5
μ
M group. p < 005,
p < 0.001 compared with 58 0.5
μ
M group. ns means no significant difference.
7

Z. Feng et al.
Bioorganic Chemistry 111 (2021) 104849
Table 4
In vivo pharmacokinetic (PK) data for compound 58.
Route of administration
Dose
Tmax
(h)
Cmax
AUC0-t
T1/2
(h)
CL
F (%)
(mg/kg)
(ng/mL)
(ng*h/mL)
(ng*h/mL)
p.o.
i.v.
20
10
0.083
2
88.73
553.38
768.98
10.85
10.65
0.030
0.011
36.88
302.35
–
material, which reacted with 4-iodo-3,5-dimethylisoxazole via Suzuki
coupling reaction to obtain compound 14. Then, in the presence of
chlorosulfonic acid and PCl5, compound 14 underwent ortho chlor-
osulfonation of benzene ring to give intermediate 15 of benzenesulfonyl
chloride, of which compound 15 was reacted with cyclopentamine to
form compound 16 of benzenesulfonamide. Then we used boron tri-
bromide to demethylate to give compound 17. The intermediate 18 was
obtained by nucleophilic substitution of 17 with 3-bromopropyne.
Finally, the target compound 19–40 was obtained by click reaction. In
the process of the synthesis of compound 19–40, we first selected 25%
methanol as the reaction solvent for the click chemistry reaction. In this
reaction solvent, the click chemistry reaction was catalyzed by anhy-
drous copper sulfate and sodium ascorbate.
Add 10
384-well plate. After incubating for 15 min at room temperature in the
dark, 5 L of BRD4 ligand/APC receptor complex was added to each
μL of BRD4-BD1 europium chloride solution to each well of a
μ
well. The plate was read by a microplate reader after incubating at
ambient temperature for 1 h avoids light. The difference between the
absorbance of the negative control and the compound group is
compared with the absorbance of the negative control, which is the
BRD4 inhibition rate. The concentration of small molecule compounds
corresponding to 50% inhibition rate is the half inhibitory concentration
(IC50).
4.3.2. Cell culture and proliferation inhibition assays.
HL-60 or MV4-11 cells were seeded in 96-well plates at a concen-
Scheme 2 Compound 51–65 was synthesized from 4-methoxybromo-
benzene (41) by chlorosulfonation to give compound 42, which was
then reacted with cyclopentamine to give compound 43. Finally, the key
intermediate 44 of Suzuki coupling reaction was obtained by 43 and
pinacol diboride. On the other hand, compound 46 was synthesized by
refluxing 5-bromo-2-methoxypyridine (45) in 6 M hydrochloric acid.
Then, compound 46 was methylated to give compound 47. Intermediate
44 and 47 was mixed to perfume Suzuki reaction to give 48. Finally,
compound 49 was obtained by compound 48 demethylation. After
nucleophilic substitution of compound 49, compound 50 was obtained.
The target compounds 51–65 were obtained by click reaction.
For extended syntheses methods please see Supplementary
materials.
tration of 1 × 104 cells per well. Cells were grown in 100
μ
L of IMDM
containing 20% fetal bovine serum. After 12 h, 50
μL of which con-
taining various concentrations of compounds (triple diluted) was added.
The measurement was conducted 72 h after seeding, and 10 L of Cell-
μ
counting kit-8 (CCK-8) reagent was added to each well and incubated in
37 ◦C for 4 h. The spectrophotometric absorbance of each well was
measured by a multi-detection microplate reader at a wavelength of 450
nm. The inhibition rate was calculated as ((A450 treated - A450 blank)/
(A450 control - A450 blank)) × 100. The IC50 was calculated by GraphPad
Prism 5 statistical software.
4.3.3. Cell cycle and apoptosis analysis.
Flow cytometry (FCM) was used to analyze the effects on cell cycle
and apoptosis treated by compounds. Annexin V-FITC Apoptosis
Detection Kit (Cat.NO: KGA107) and Cell Cycle Detection Kit (Cat.NO:
KGA512) purchased from KeyGEN BioTECH were used to complete the
cell cycle experiment and apoptosis evaluation based on the provided
protocol. Cells treated with the preferred compound for 24 h were
subsequently applied for the FCM examination.
4.2. Docking studies
All ligand molecules were drawn in ChemDraw 2014, and saved as
sdf style. Then ligands were processed at a simulated pH of 7.4 ± 1.0 to
generate all possible tautomers, stereoisomers, and protonation states
and were finally minimized at the OPLS 2005 force field with Ligand
preparation protocol of Maestro 10.2. The crystal complex (PDB id:
4BJX) was selected as the BRD4-BD1 docking protein. Docking study
was applied to investigate the molecular interaction mechanism via
4.3.4. Western blotting
MV4-11 cells in the logarithmic phase were harvested and BCA
Protein Assay Kit (Beyotime) was adopted for the calculation of
extracted proteins’ concentration. After that, SDS-polyacrylamide gel
was prepared for protein separation. After transferring the protein strap
onto the PVDF membrane, TBST solution of 5% fat-free milk was used to
block the non-specific binding sites. Subsequently, primary antibodies
against c-myc and β-actin was diluted and used to deal with the PVDF
membrane, following the co-incubation of secondary-antibody. Finally,
proteins were developed and the gray density was subsequently
analyzed using ImagJ and Graphpad software.
¨
Schrodinger software. Using Protein Data Bank, the published crystal
structure of BRD4 (PDB ID: 4O74) was obtained and prepared via Pro-
tein Preparation Wizard based on the force field OPLS3 (optimized po-
tentials for liquid simulations). The related 3 dimension structures of
compound 58 were computed through the LigPrep module. Ultimately,
¨
the schrodinger XP precision was adopted for the calculation and the
chosen structure with the lowest-energy was used for the result.
4.3. Biological evaluation
4.3.5. In vivo PK study
Compound 58 dissolved in 1% Tween80/water to a concentration of
1 mg/mL, and was given to ICR mice (Male, 180–220 g, n = 3) by gavage
administration. Blood samples were collected at 0.25, 0.5, 1, 2, 4, 8, and
24 h after administration (anticoagulant: EDTA-Na2)0.100 mL of sol-
vent of methanol: acetonitrile (1:1, v/v) with internal standard was
added to 10 mL of plasma and vortexed thoroughly. It was centrifuged
for 5 min, and then 20 mL of the supernatant was mixed with 20 mL of
water for analysis. Samples were analyzed by Xevo TQ-S triple quad-
rupole mass spectrometer (Waters, USA). The ACQUITY UPLC BEH C18
(1.7 mm, 2.0 mm × 50 mm, Waters, USA) was used for the analysis.
Gradient elution was applied consisting of 5 mM ammonium acetate
aqueous solution containing 0.1% formic acid and acetonitrile
4.3.1. Binding affinities toward BRD4-BD1 and BRD4-BD2 from TR-FRET
assay
TR-FRET Assay Kit for BRD4 bromodomain 1 (Item No. 600520) and
BRD4 bromodomain 2 (Item No. 600520) (Cayman, Ann Arbor, MI,
USA) were applied for the BRD4 binding affinity evaluation.
The BRD4 inhibition assay was performed by using BRD4 bromo-
domain 1 TR-FRET Assay Kit (Item No. 600520) and BRD4 bromodo-
main 2 TR-FRET Assay Kit (Item No. 600520) obtained from Cayman
(Ann Arbor, MI, USA). Dilute the sample solution with the buffer solu-
tion, and prepare the sample concentrations at 100
10 M, 5 M, 1 M, 0.5 M, 0.2 M, 0.1 M, respectively. Add 5
each well, and set up 3 replicate wells for each concentration gradient.
μM, 50
μ
M, 20
μ
M,
μ
μ
μ
μ
μ
μ
μ
L to
8

Z. Feng et al.
Bioorganic Chemistry 111 (2021) 104849
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Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
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9