Design, synthesis and biological evaluation of indole-2-one derivatives as potent BRD4 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2020.112780

Bromodomain protein 4 (BRD4) plays a crucial role in transcriptional regulation and is considered to be a viable drug target for cancer treatment. Herein, we designed and synthesized a series of indole-2-one derivatives through scaffold hopping drug desig

Full text of DOI:10.1016/j.ejmech.2020.112780

European Journal of Medicinal Chemistry 208 (2020) 112780
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Design, synthesis and biological evaluation of indole-2-one derivatives
as potent BRD4 inhibitors
Yu Xu 1, Xiu-Juan Zhang 1, Wen-Bo Li, Xing-Rong Wang, Shuai Wang, Xue-Peng Qiao,
Shi-Wu Chen*
School of Pharmacy, Lanzhou University, Lanzhou, 730000, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Bromodomain protein 4 (BRD4) plays a crucial role in transcriptional regulation and is considered to be a
viable drug target for cancer treatment. Herein, we designed and synthesized a series of indole-2-one
derivatives through scaffold hopping drug design. Most of the compounds showed potent BRD4 inhib-
itory activities and anti-proliferation activities in cancer cell lines. Especially, compound 12j exhibited
excellent BRD4 inhibitory activities (BD1 IC50 ¼ 19 nM, BD2 IC50 ¼ 28 nM) and anti-proliferation potency
Received 19 July 2020
Received in revised form
16 August 2020
Accepted 19 August 2020
Available online 30 August 2020
with IC50 values of 4.75 mM and 1.35 mM in HT-29 and HL-60 cells, respectively. Additionally, docking
studies showed that the hydrophobic pocket next to KAc region and WPF shelf were critical to the ac-
tivity of the compound. Compound 12j could arrest the cell-cycle progression of HT-29 cells into the G1
phase and reduce the expression of c-Myc. Moreover, compound 12j exhibited favorable oral pharma-
cokinetic properties. All the results demonstrated that compound 12j was a potent BRD4 inhibitor and
had merely potential for colon cancer treatment.
Keywords:
BRD4
Indole-2-one
Scaffold hopping
Cell-cycle
c-Myc
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
Similar to other members of the protein family, BRD4 is composed
of the ET domain, C-terminal domain (CTD) and two highly homol-
Bromodomains are “Readers” of histone acetylated, which can
specifically recognize acetylated lysine (KAc) residues in the N-ter-
minal tails of histone proteins[1] and associatewithvarious diseases
such as cancer, inflammation and obesity [2]. Currently, 61 struc-
turally homologous bromodomains present in 46 different human
proteins have been identified, and these domains consist of roughly
110 amino acid residues [3]. Among them, the bromodomain and
extra terminal (BET) proteins are the most prominent and studied.
BET family consists of bromodomain-containing protein 2 (BRD2),
BRD3, BRD4, and bromodomain testis-specific protein (BRDT), each
of which contains two subtypes of bromodomains (BD1 and BD2)
[4,5]. Notability, all of the BET family proteins localize in the nucleus
[5]. BRD2, BRD3, and BRD4 are ubiquitously expressed in various
cells, whereas BRDT is only specifically expressed in tissues such as
pachytene spermatocytes, diplotene spermatocytes, and round
spermatids [4,6].
ogous N-terminal bromodomains in spatial configuration [7]. As
histone acetylated reader, BRD4 could recognize the acetylated lysine
residues in histone H4 with N-terminal bromodomains and recruit
positive transcriptional elongation factor p-TEFb and cyclin T1/CDK9
complex to phosphorylate RNA polymerase II (RNA pol II) to activate
gene transcription [8]. Additionally, BRD4 could facilitate the tran-
scription of tumor-growth-promoting c-Myc and antiapoptotic genes
Bcl-2, which are key regulators for the proliferation and survival of
human tumor cells [9,10]. Since directly targeting Myc protein is still
elusive, targeting BRD4 to interrupt p-TEFb binding to histones and
decrease the expression of c-Myc were considered as a feasible way
forMycrelatedcancertreatment[11].Hence, BRD4hasbecomeoneof
the hot targets for antitumor drug discovery.
At present, multiple BRD4 inhibitors with different scaffolds have
been reported in the literature and many of them have demonstrated
good therapeutic effects in preclinical researches and clinical trials,
such as 1 ((þ)-JQ1) [12], 2 (I-BET762) [13], 3 (CPI-0610) [14], 4 (ABBV-
075) [15], 5 (PFI-1) [16] and 6 [17] (Fig. 1). (þ)-JQ1 was the first re-
ported BRD4 inhibitor with IC50 values of 77 nM and 33 nM against
BD1 and BD2 in the AlphaScreen assay, respectively [12]. However,
(þ)-JQ1 possessed serious preclinical toxicity in vivo and was widely
* Corresponding author.
E-mail address: chenshw@lzu.edu.cn (S.-W. Chen).
These authors made equal contributions to this work.
1
https://doi.org/10.1016/j.ejmech.2020.112780
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

2
Y. Xu et al. / European Journal of Medicinal Chemistry 208 (2020) 112780
used asa chemicalprobe [18]. Fortunately, its analogues I-BET762and
CPI-0610 have entered clinical trials. I-BET762 is now in a phase II
clinicaltrialfornuclearproteinintestis(NUT)midlinecarcinoma.CPI-
0610 has completed the phase I clinical trial in patients with pro-
gressive lymphoma. Besides, some BRD4 inhibitors have demon-
strated goodapplicationprospectsincombinationtherapiessuchasI-
BET762, CPI-0610 and ABBV-075. Particularly, CPI-0610 in combina-
tion with Ruxolitinib for myelofibrosis is in phase II clinical trial and
ABBV-075 in combination with Venetoclax for breast cancer and lung
cancer has completed the phase I clinical trial. In addition, PFI-1 and 6
are potent BRD4 BD1 inhibitors with IC50 values of 220 nM and
410 nM in the Alpha Screen assay, respectively [16,17]. Besides,
compound 6 with the structure of indolinone demonstrated a good
pharmacokinetic profile [17] and represented a class of potential
antitumor inhibitors. Inspired by these facts, we believe that potent
BRD4 inhibitors with different chemical types may have important
applications in anti-tumor therapies. In the present study, we report
the scaffold hopping design, synthesis, and biological evaluation of
indole-2-one as a new class of potent small molecule BRD4 inhibitors.
KAc, we conducted molecular docking on the compound 11 and
PFI-1 and found that they had a similar combination mode with
BRD4 BD1 (PDB code: 4E96) (Fig. 2A and B). The result showed that
the design was reasonable. To further improving the affinity of
compounds for BRD4 BD1, we evaluated the structure-activity
relationship (SAR) from the hydrophobic pocket and WPF shelf
(Fig. 3).
2.2. Synthesis
The general synthetic routes of novel 2-methoxy-N-(2-
oxoindolin-5-yl)benzene sulfonamide derivatives were illustrated
in Schemes 1. The commercially material 5-nitroindolin-2-one (8)
was treated with iron powder and ammonium chloride in EtOH/
H2O to give 5-aminoindolin-2-one (10), which was reacted with 2-
methoxybenzenesulfonyl chloride to obtain compound 11. Subse-
quently, compounds 12ae12j were prepared by Knoevenagel
condensation of compound 11 with the appropriate aldehydes or
ketones in the presence of piperidine [20,21].
The 5-amino-3-(propan-2-ylidene)indolin-2-one derivatives
were synthesized starting from intermediate 7 as shown in
Schemes 2. The intermediate 10 was obtained by Knoevenagel
condensation of 7 with acetone, followed by reduction of 9 with
iron powder and ammonium chloride. Then the compound 10 was
further converted to target compounds 13ae13b by acylation with
appropriate acids in the presence of tetramethyluronium hexa-
fluorophosphate (HATU). Simultaneously, compounds 14ae14m
were obtained by sulfonylation with appropriate sulfonyl chlorides
in the presence of N-ethyldiisopropylamine (DIPEA) at room
temperature.
2. Results and discussions
2.1. Design strategy
The chemical probe PFI-1 is a potent BRD4 inhibitor and effi-
ciently blocks the interaction of BET BRDs with acetylated histone
tails [19]. Besides, PFI-1 has moderate anti-proliferative effects on
leukemic cell lines (5e10
mM) and could downregulate the
expression of Myc [19]. To understand the binding mode between
BRD4 BD1 (PDB code: 4E96) with PFI-1, we performed docking
studies with Glide docking. Docking studies indicated that the
lactam of the dihydroquinoxalin-2(1H)-one formed two stable
hydrogen bonds with the conserved residue Asn140 and the oxygen
atom in carbonyl group interacted with the conserved residue
Tyr97 through a water-mediated hydrogen bond. The sulfonamide
formed two additional hydrogen bonds with two water molecules
and the methoxyphenyl occupied the WPF shelf (Fig. 2A). To
identify a novel scaffold and improve the BRD4 inhibitor activity,
we selected PFI-1 as the starting point and retained the lactam
through scaffold hopping to obtain compound 11. In order to verify
whether the designed compound could recognize the binding site
2.3. Structure-activity relationship study
The binding affinities against BRD4 BD1 of the synthesized
target compounds were evaluated by TR-FRET assay [22], and
(þ)-JQ1 and PFI-1 were selected as positive controls. As shown in
Table 1, some of the synthesized compounds showed moderate to
high potency against BRD4 BD1. Comparing 11 with 12ae12l, the
results showed that the introduction of appropriate substituents at
the 3-position of indole-2-one could increase the inhibitory activity
of BRD4 BD1. This result revealed that occupying the hydrophobic
Fig. 1. The structures of BRD4 inhibitors.

Y. Xu et al. / European Journal of Medicinal Chemistry 208 (2020) 112780
3
Fig. 2. Binding mode of PFI-1 (yellow) and 11 (green) to BRD4 BD1 (PDB code: 4E96). The binding site residues are shown as sticks. Hydrogen bond interactions are indicated by
yellow dashed lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 3. Design of novel BRD4 inhibitors.
Scheme 1. Reagents and conditions: (i) Fe, NH4Cl, EtOH/H2O, reflux, 1.2 h; (ii) 2-methoxybenzenesulfonyl chloride, DIPEA, CH2Cl2, rt, 12 h; (iii) appropriate aldehydes, CH2Cl2,
piperidine, N2, rt, 8e24 h; or appropriate ketones, piperidine, rt, 8e24 h.
pocket next to KAc region was critical to the activity of the com-
pound. Comparing 12a with 12b and 12k with 12l, the results
indicated that the smaller the substituent of 3-position was, the
higher the enzyme activity of the compound would be. At the same
time, comparing 12c with 12d and 12g with 12h, we found that the
trans configuration had higher inhibition rate than the cis config-
demonstrated that the sulfonamide group had a greater contribu-
tion to the activity of the compound than its isosteric amide group.
In order to study this difference, we analyzed the interaction force
between the compounds and the protein crystal. As depicted in
Fig. 4, N-substitution of the sulfonamide group occupied the hy-
drophobic WPF shelf through the sulfonamide bend angle. How-
ever, the lactam of compounds 13ae13b made the entire molecule
appear linear, so that the terminal substituent could not occupy the
WPF shelf well. Thence, compounds with sulfonamide as the con-
necting chain might have better activity against BRD4 BD1.
Subsequently, comparing 12j with 14a, we found that methox-
yphenyl substituent on the terminal has better activity in the ortho
position than the para position. Comparing 12j with 14be14c, the
results showed that cyclohexyl and phenyl substituents at the tail
had similar activities. Through the comparison of 14de14f, it was
found that when the substituent was introduced to the para posi-
tion of 2-methoxy, the activity was the best when bromine atom
was substituted, followed by the methyl group, and the fluorine
substitution activity was the worst. This result was similar to the
result of introducing a substituent at the para position of 2-fluorine
uration at 1
mM. Remarkably, the compound 12j exhibited the most
potent BRD4 inhibitory activity with the IC50 value of 19 nM. By the
way, almost all of the synthesized compounds had a suitable hy-
drophobic constant (5 > cLogP > 2)and ligand efficiency (LE > 0.30)
[23]. These results indicated that we could further modify the
compound by changing the molecular weight of the compound and
the affinity for the protein to obtain the desired compounds.
Therefore, we chose the propane-2-alkylene substituent at 3-
position to discuss the hydrophobic WPF shelf to the effect of
inhibitory activity.
As shown in Table 2, most compounds exhibited excellent
inhibitory activities against BRD4 BD1, and some compounds dis-
played similar BRD4 BD1 inhibitory activity to compound 12j.
Comparing 13a with 12j and 13b with 14c, the results

4
Y. Xu et al. / European Journal of Medicinal Chemistry 208 (2020) 112780
Scheme 2. Reagents and conditions: (i) acetone, piperidine, rt, 14 h; (ii) Fe, NH4Cl, EtOH/H2O, reflux, 1.2 h; (iii) appropriate acids, HATU, DIPEA, DMF, rt, 12e15 h; (iv) appropriate
sulfonyl chlorides, DIPEA, CH2Cl2, rt, 12e24 h.
at the terminal benzene ring (14g vs 14h). In order to study the
effect of different substituents on the activity of BRD4 BD1, we
introduced some macrocycles such as naphthalene ring and dihy-
drobenzofuran. Nevertheless, we found that when the macrocycles
were introduced, the inhibitory activity and ligand efficiency of the
compounds were reduced (14j vs 14k and 14l vs 14m). Above all,
these results suggested that the designed compounds exhibited
good BRD4 BD1 inhibitory activity.
compound 14d demonstrated the strongest anti-proliferative ac-
tivity against HL-60 cells with IC50 value of 0.62 M, and had similar
inhibitory activity to (þ)-JQ1 in WI-38 cells, indicating that com-
pound 14d might have similar toxicity to (þ)-JQ1. Overall consid-
eration of the data from the above assays, it is necessary for
compounds 12j, 14b and 14d to be further studied.
m
2.5. The inhibitory activities of BRD4 and BRD2
Additionally, we investigated the inhibitory activities of BRD4
and BRD2 for compounds 12j, 14b and 14d with (þ)-JQ1 as control.
As shown in Table 4, the data showed that these compounds did not
potent selectivity for the two tandem bromodomains in BRD2 and
BRD4 or the selectivity of BD1 and BD2, and had slightly weaker
inhibitory activity than (þ)-JQ1. Particularly, compound 12j
demonstrated better enzyme activity than compounds 14b and
14d, and displayed similar inhibitory activity with (þ)-JQ1 in BRD2
(BD1). Based on the facts of the anti-proliferation activity of tumor
cells and the inhibitory activities of BRD2/4, we selected compound
12j to further study its mechanism of anti-tumor action.
2.4. Anti-proliferation activity
Furthermore, the compounds with excellent BRD4 BD1 inhibi-
tory potency were further evaluated in the promyelocytic cell line
(HL-60), human colon cancer cell line (HT-29) and human normal
cell line (WI-38) for their anti-proliferation activities, and the re-
sults were summarized in Table 3. Most of the target compounds
displayed moderate to high effective inhibition on HL-60 and HT-
29 cells proliferation. The anti-proliferative activity of 12j on HL-60
and HT-29 cells was better than that of PFI-1, but weaker than
(þ)-JQ1. In addition, its toxic on WI-38 cells was weaker than that of
PFI-1 and (þ)-JQ1. These results showed that compound 12j
possessed strong anti-proliferative activity and weak toxicity. Be-
sides, compound 14b showed good activity on HL-60 cells and HT-
29 and low toxic on WI-38 cells, indicating that compound 14b may
have cell selectivity inhibition and low toxic activity. Moreover,
2.6. Docking studies
In order to explain the interaction between the compound 12j
with BRD4,
a docking analysis was carried out for further

Y. Xu et al. / European Journal of Medicinal Chemistry 208 (2020) 112780
5
Table 1
BRD4 BD1 inhibitory activities of compounds 11 and 12.
2.7. Cell cycle analysis
Literatures showed that BRD4 could control the M phase to G1
phase transition by retaining P-TEFb key regulatory genes
throughout mitosis [25e27]. As shown in our above study, the HT-
29 cell line was responsive to compound 12j, and accordingly we
treated the HT-29 cells with different concentrations of 12j for 24 h
and performed cell cycle analysis using Annexin-V/PI staining and
flow cytometry [28]. As shown in Fig. 6, the results indicated that
compound 12j could arrest the cell-cycle progression of the cell line
into the G1 phases compared with vehicle treatment. The per-
centage of cells in G1 phase after treatment by compound 12j under
Compds
Inhibition rate at 1 m
M (%)a IC50 (nM)a cLogPb LEc
(þ)-JQ1
PFI-1
11
e
e
e
101
100
15
12
88
ND
36
4.82
1.27
0.94
2.28
0.36
0.41
ND
concentrations of 2.5, 5.0 and 10.0 mM were 85.98%, 86.49% and
86.05%, respectively, compared with 64.63% of vehicle control.
12a
101
0.43
12b
12c
100
27
114
ND
2.81
2.72
0.39
ND
2.8. Effects on c-Myc protein expression
12d
12e
41
4
ND
ND
2.72
3.84
ND
ND
Previous studies showed that BRD4 inhibitors could effectively
down-regulate the transcription of c-Myc, which was an oncogene
overexpressed in various tumors [9,29,30]. Meanwhile, Western
blot analysis was performed to examine the expression of c-Myc
protein. As shown in Fig. 7, compound 12j displayed profound
inhibitory effects on c-Myc protein expression. Therefore, we
concluded that the inhibitory effects of compound 12j on HT-
29 cells proliferation were at least in part concerned with the
downregulation of c-Myc expression.
12f
1
ND
4.40
ND
12g
12h
12i
20
36
9
ND
ND
ND
2.64
2.64
1.29
ND
ND
ND
2.9. In vivo pharmacokinetic studies
Based on the facts that the combination of cellular potency and
binding affinities, compound 12j was further evaluated for its
in vivo metabolic profile in rats. Upon administration of compound
12j at 5 mg/kg of intravenous dosing (iv) or 15 mg/kg of oral dosing
(po), the plasma concentrations were determined by liquid
chromatography-tandem mass spectrometric (LC-MS/MS). As
shown in Table 5, data analysis showed that compound 12j had a
12j
100
100
19
26
2.68
2.66
0.43
0.41
12k
12l
21
ND
3.78
ND
Cmax value of 2175 mg/L, an AUC value of 14 600 mg/L h, an appro-
a
All results were measured twice; ND means not determined.
cLogP values were calculated using ChemDraw 18.0.
LE ¼ 1.40 ꢀ (pIC50/Heavy atoms), ND means not calculated.
priate T1/2 value of 3.59 h, and an oral bioavailability (F) value of
66%. Collectively, our results indicated that compound 12j had
more favorable pharmacokinetic properties in rats than those of
(þ)-JQ1 in mouse [31].
b
c
illustrating the possible binding modes of the compound 12j on
BRD4 BD1 (PDB code: 4E96) and BD2 (PDB code: 5UVX) using
€
3. Conclusion
Schrodinger Suite [24]. As illustrated in Fig. 5A and B, the lactam of
the indole-2-one formed two stable molecular key hydrogen bonds
with Asn140 and indirectly established another molecular key
hydrogen bond with the water molecule and Tyr97. Besides, the
methoxyphenyl group of compound 12j extended to the WPF shelf
via a sulfonamide linker and the sulfonamide formed two hydrogen
bonds with waters. Moreover, the propane-2-alkylene group at the
3-position could penetrate into the hydrophobic pocket beside the
KAc. As described in Fig. 5C and D, we could found that the binding
mode for compound 12j to BRD4 BD2 was similar to that of BRD4
BD1. The only difference was that the sulfonamide group did not
form hydrogen bonds with surrounding water molecules. There-
fore, the binding force of compound 12j to BRD4 BD2 was slightly
weaker than that of BRD4 BD1, and this predicted binding force was
consistent with its activity. Above all, compound 12j exhibited
excellent binding ability with BRD4 and the binding modes pointed
us the key interactions between synthesized compounds and BRD4
which will guide us in the future design.
In summary, we reported a series of indole-2-one derivatives as
potent BRD4 inhibitors. The specific structure-activity relationship
of these compounds showed that the hydrophobic pocket next to
KAc region was critical to their activity and the smaller the sub-
stituent of 3-position or 5-position was, the higher the enzyme
activity of the compound would be. The enzymatic assay revealed
that the synthesized compounds exhibited an excellent potency of
inhibiting BRD4. The representative compound 12j demonstrated
the supreme BRD4 inhibitory activity with the IC50 value of 19 nM
and preferable anti-proliferation activity against HT-29 cell lines
with the IC50 value of 4.75 mM. The compound 12j could arrest the
cycle of HT-29 cells in G1 phase and the inhibitory effects of com-
pound 12j on HT-29 cells proliferation might be concerned with the
downregulation of c-Myc expression. Additionally, compound 12j
showed favorable oral pharmacokinetic properties. All of these
results demonstrated that compound 12j could be utilized as a
BRD4 inhibitor for further investigation.

6
Y. Xu et al. / European Journal of Medicinal Chemistry 208 (2020) 112780
Table 2
BRD4 BD1 inhibitory activities of compounds 13 and 14.
Compds
R3/R4
Inhibition ratio at 1
18
m
M (%)a
IC50 (nM)a
ND
cLogPb
3.08
LEc
ND
13a
13b
14a
14b
15
ND
94
25
3.49
3.16
3.54
ND
97
0.39
0.44
100
14c
14d
100
101
22
23
3.28
3.74
0.47
0.41
14e
101
34
3.02
0.40
14f
100
100
30
56
3.17
3.57
0.41
0.41
14g
14h
101
20
3.82
0.43
14i
14j
31
98
ND
92
4.77
3.29
ND
0.38
14k
14l
77
98
203
62
4.05
4.05
0.35
0.37
14m
90
208
4.38
0.31
a
All results were measured twice; ND means not determined.
cLogP values were calculated using ChemDraw 18.0.
LE ¼ 1.40 ꢀ (pIC50/Heavy atoms), ND means calculated.
b
c
4. Experimental section
1525 Binary HPLC Pump, and in each case, the major peak
accounted for ꢁ95% of the combined total peak area when moni-
tored by a UV detector. 1H NMR and 13C NMR spectra were recorded
with a Agilent NMR inova 400 or 600 spectrometer with TMS as the
4.1. Chemistry: general procedures
All starting materials and regents were commercially available
and used without further purified, unless otherwise stated. All re-
actions were monitored by thin layer chromatograph (TLC) on sil-
icagel GF254 (0.25 mm, Qingdao Ocean Chemical Ltd., China).
Column chromatography (CC) was performed on Silica Gel 60
(230e400 mesh, Qingdao Ocean Chemical Ltd., China). Melting
points were determined in Kofler apparatus and were uncorrected.
The purities of all target compounds were estimated by Waters
internal standard, all chemical shift d were given in partsper million
(ppm). Mass spectra were recorded on a Bruker Dalton APEXII49e
with ESI source as ionization, respectively.
4.1.1. Procedure for the synthesis of 5-aminoindolin-2-one (8)
A mixture of 5-nitroindolin-2-one (7, 2.81 mmol), iron powder
(14.00 mmol) and ammonium chloride (1.67 mmol) in EtOH/
H2O ¼ 4:1 (10 mL) was refluxed for 1.2 h. After the reaction was

Y. Xu et al. / European Journal of Medicinal Chemistry 208 (2020) 112780
7
4.1.2. Procedure for the synthesis of 5-nitro-3-(propan-2-ylidene)
indolin-2- one (9)
mixture of
A
7
(11.22 mmol) and piperidine (0.67 mL,
6.73 mmol) in acetone (6 mL) was stirred at room temperature for
14 h. Next, the insoluble matter in the solution was filtered, and the
filter cake was dried at room temperature to obtain product 9.
Yellow solid; yield: 86%; 1H NMR (600 MHz, DMSO‑d6)
d 11.16 (s,
1H), 8.27 (d, J ¼ 6.0 Hz, 1H), 8.15e8.14 (m, 1H), 6.99e6.97 (m, 1H),
2.49 (s, 3H), 2.42 (s, 3H).
4.1.3. Procedure for the synthesis of 5-amino-3-(propan-2-ylidene)
indolin-2- one (10)
Then using compound 9 as a raw material, the compounds 10
was synthesized using the similar method as 8. Yellow solid; yield:
90%; 1H NMR (600 MHz, DMSO‑d6)
d 9.98 (s, 1H), 6.91 (s, 1H),
8.50e8.37 (m, 2H), 5.19 (brs, 2H), 2.44 (s, 3H), 2.21 (s, 3H).
4.1.4. Procedure for the synthesis of 2-methoxy-N-(2-oxoindolin-5-
yl) benzenesulfonamide (11)
Fig. 4. The surface binding mode of 12j, 14c (yellow) and 13ae13b (green) to BRD4
BD1 (PDB code: 4E96). (For interpretation of the references to colour in this figure
legend, the reader is referred to the Web version of this article.)
O-methoxybenzenesulfonyl chloride (8.90 mmol, 1.1 equiv) was
added to a solution of compound 8 (8.90 mmol) in DIPEA (1.64 mL,
9.46 mmol) at room temperature for 12 h. After the reaction was
completed, the insoluble matter in the solution was filtered and the
filter cake was dried at room temperature to obtain product 11.
Table 3
Black
solid;
yield:
60%;
m.p.
205e207
ꢂC;
HPLC
Anti-proliferation effects of the selected compounds against three cell lines.
(MeOH:H2O ¼ 85:15, 1.0 mL/min) tR ¼ 3.92 min, 99.5%. 1H NMR
Compds
IC50 (m
M)a
(600 MHz, DMSO‑d6)
d
10.24 (s, 1H), 7.65 (d, J ¼ 7.2 Hz, 1H), 7.54 (t,
HL-60b
HT-29c
WI-38c
J ¼ 7.2 Hz, 1H), 7.17 (d, J ¼ 8.4 Hz, 1H), 6.99 (t, J ¼ 7.2 Hz, 1H), 6.95 (s,
1H), 6.86 (d, J ¼ 8.4 Hz, 1H), 6.61 (d, J ¼ 7.8 Hz, 1H), 3.91 (s, 3H), 3.37
(þ)-JQ1
PFI-1
12a
12b
12j
12k
14a
14b
14c
14d
14e
14f
14g
14h
14j
0.16 0.01
3.99 0.09
4.64 0.17
1.15 0.47
1.35 0.33
2.93 0.64
16.66 1.88
1.52 0.27
9.60 0.22
0.62 0.04
1.73 0.24
1.56 0.36
19.19 1.88
5.30 0.80
4.65 0.71
3.36 0.99
20.31 2.02
6.37 0.50
0.48 0.16
14.72 3.26
10.5 0.79
3.20 0.95
4.75 0.63
>50
6.68 0.63
>50
>50
4.97 0.27
19.80 1.82
8.55 2.96
5.85 0.49
44.07 1.89
12.94 1.61
9.98 2.14
84.42 7.25
24.78 2.46
5.11 0.65
32.77 1.69
8.95 1.92
47.14 2.27
16.14 1.13
28.75 5.24
14.62 1.04
10.89 0.39
9.97 1.15
(s, 2H); 13C NMR (150 MHz, DMSO‑d6)
d 176.1, 156.3, 140.4, 134.8,
131.3, 130.1, 126.3, 120.6, 119.9, 118.4, 112.7, 110.3, 109.0, 56.0, 35.8.
MS (ESI) m/z 317.2 for [M‒H]‒.
4.1.5. General procedure for the synthesis of compounds 12ae12i
A
mixture of 11 (0.31 mmol) and piperidine (0.67 mL,
6.73 mmol) in DCM (2 mL) was stirred at room temperature. And
under the protection of nitrogen, appropriate aldehydes
(3.10 mmol) was added to the mixture. After the reaction was
completed, the solvent was evaporated in vacuo and the residue
was purified by silica gel column chromatography to obtain the
products 12ae12i.
9.47 1.29
>50
13.04 4.21
16.59 1.11
24.42 7.71
19.65 1.90
>50
14k
14l
14m
14.48 0.59
23.20 3.66
4 .1. 5 .1. ( Z ) - N - ( 3 - E t h y l i d e n e - 2 - o x o i n d o l i n - 5 - y l ) - 2 -
methoxybenzenesulfonamide (12a). Yellow solid; yield: 40%; m.p.
195e197 ꢂC; HPLC (MeOH:H2O ¼ 85:15, 1.0 mL/min) tR ¼ 4.04 min,
a
All results were measured three times.
Determined by CCK8 method.
Determined by MTT method.
b
c
99.3%. 1H NMR (400 MHz, DMSO‑d6)
d 10.28 (s,1H), 9.60 (s,1H), 7.63
(d, J ¼ 8.0 Hz, 1H), 7.51 (t, J ¼ 8.0 Hz, 1H), 7.26 (s, 1H), 7.14 (d,
J ¼ 8.4 Hz,1H), 6.95 (t, J ¼ 7.6 Hz,1H), 6.89 (d, J ¼ 8.4 Hz,1H), 6.79 (q,
J ¼ 8.4 Hz, 1H), 6.63 (d, J ¼ 8.4 Hz, 1H), 3.88 (s, 3H), 2.07 (d,
Table 4
Inhibitory activities of the selected compounds against BRD4/2 of BET family.
J ¼ 8.4 Hz, 3H); 13C NMR (100 MHz, DMSO‑d6)
d
168.2, 156.7, 139.3,
Compds
IC50 (nM)a
136.7, 135.5, 131.7, 130.7, 129.2, 126.6, 122.9, 122.8, 120.5, 117.9, 113.1,
110.2, 56.5, 15.2. MS (ESI) m/z 343.2 for [M‒H]‒.
BRD4 (BD1)
BRD4 (BD2)
BRD2 (BD1)
BRD2 (BD2)
12j
14b
14d
(þ)-JQ1
19 1.2
25 0.9
23 2.1
12 2.6
28 1.1
43 3.1
21 2.8
3.9 0.4
24 0.5
40 1.8
39 1.3
29 2.1
18 1.6
25 1.7
22 0.5
6.7 1.3
4.1.5.2. (Z)-2-Methoxy-N-(2-oxo-3-propylideneindolin-5-yl)benze-
nesulfonamide (12b). Yellow solid; yield: 31%; m.p. 164e166 ꢂC;
HPLC (MeOH:H2O ¼ 85:15, 1.0 mL/min) tR ¼ 3.76 min, 99.1%. 1H
a
NMR (600 MHz, DMSO‑d6)
d 10.32 (s, 1H), 9.62 (s, 1H), 7.66 (d,
All results were measured twice.
J ¼ 8.4 Hz, 1H), 7.56 (t, J ¼ 7.8 Hz, 1H), 7.24 (s, 1H), 7.19 (d, J ¼ 8.4 Hz,
1H), 6.99 (t, J ¼ 7.8 Hz, 1H), 6.94 (d, J ¼ 8.4 Hz, 1H), 6.73 (t, J ¼ 7.8 Hz,
1H), 6.67 (d, J ¼ 8.4 Hz, 1H), 3.92 (s, 3H), 2.48e2.45 (m, 2H), 1.13 (t,
completed, filtered while hot and concentrated under reduced
pressure to give the product 8. Yellow solid; yield: 99%; 1H NMR
J ¼ 7.2 Hz, 3H); 13C NMR (150 MHz, DMSO‑d6)
d 156.3, 142.5, 134.9,
(600 MHz, DMSO‑d6)
d
9.95 (s, 1H), 6.54 (s, 1H), 6.51 (d, J ¼ 7.8 Hz,
131.3, 130.2, 127.5, 122.4, 122.1, 112.6, 109.8, 55.9, 21.8, 18.5. MS (ESI)
m/z 381.3 for [MþNa]þ.
1H), 6.41 (d, J ¼ 7.8 Hz, 1H), 5.03 (s, 2H), 3.26 (s, 2H).

8
Y. Xu et al. / European Journal of Medicinal Chemistry 208 (2020) 112780
Fig. 5. Possible binding mode of compound 12j to BRD4. (A) Predicted binding mode for 12j to BRD4 BD1 (PDB code: 4E96). (B) Surface of 12j and BRD4 BD1. (C) Predicted binding
mode for 12j to BRD4 BD2 (PDB code: 5UVX). (D) Surface of 12j and BRD4 BD2. The binding site residues are shown as sticks. Hydrogen bond interactions are indicated by yellow
dashed lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
4.1.5.3. (Z)-N-(3-(Cyclopropylmethylene)-2-oxoindolin-5-yl)-2-
methoxybenzene sulfonamide (12c). Yellow solid; yield: 43%; m.p.
184e186 ꢂC; HPLC (MeOH:H2O ¼ 85:15, 1.0 mL/min) tR ¼ 3.90 min,
1.50e1.47 (m, 2H); 13C NMR (150 MHz, CDCl3)
d 169.6, 156.1, 148.9,
138.8, 134.9, 131.0, 130.9, 126.3, 123.9, 123.4, 120.9, 119.5, 112.1,
110.0, 56.5, 39.7, 33.1, 25.7. MS (ESI) m/z 397.2 for [M‒H]‒.
99.2%. 1H NMR (600 MHz, DMSO‑d6)
d 10.27 (s,1H), 9.58 (s,1H), 7.66
(d, J ¼ 7.2 Hz, 1H), 7.56 (d, J ¼ 7.2 Hz, 1H), 7.36 (s, 1H), 7.19 (d,
J ¼ 9.0 Hz,1H), 6.99 (t, J ¼ 7.2 Hz,1H), 6.89 (d, J ¼ 8.4 Hz,1H), 6.67 (d,
J ¼ 9.0 Hz, 1H), 6.19 (d, J ¼ 11.4 Hz, 1H), 3.91 (s, 3H), 1.95e1.93 (m,
1H), 1.22 (brs, 2H), 0.89 (brs, 2H); 13C NMR (150 MHz, DMSO‑d6)
4.1.5.6. (Z)-N-(3-(Cyclohexylmethylene)-2-oxoindolin-5-yl)-2-
methoxybenzene sulfonamide (12f). Yellow solid; yield: 31%; m.p.
122e124 ꢂC; HPLC (MeOH:H2O ¼ 85:15, 1.0 mL/min) tR ¼ 4.69 min,
98.1%. 1H NMR (600 MHz, CDCl3)
d
8.15 (s, 1H), 7.77 (d, J ¼ 7.2 Hz,
d
167.8, 156.3, 146.8, 138.4, 134.9, 131.0, 130.2, 126.4, 125.6, 122.6,
1H), 7.50 (t, J ¼ 7.2 Hz, 1H), 7.33 (s, 1H), 7.19 (s, 1H), 7.03 (d,
J ¼ 7.8 Hz, 1H), 6.98 (t, J ¼ 7.8 Hz, 1H), 6.88e6.85 (m, 2H), 6.67 (d,
J ¼ 8.4 Hz, 1H), 4.06 (s, 3H), 2.79e2.77 (m, 1H), 1.86e1.79 (m, 3H),
1.65 (brs, 2H), 1.48e1.42 (m, 1H), 1.37e1.29 (m, 4H); 13C NMR
122.3, 120.1, 116.7, 112.6, 109.6, 56.0, 12.8, 10.0. MS (ESI) m/z 369.2
for [M‒H]‒.
(150 MHz, CDCl3)
d 169.5, 156.1, 148.5, 138.7, 134.9, 131.1, 131.0,
4.1.5.4. (E)-N-(3-(Cyclopropylmethylene)-2-oxoindolin-5-yl)-2-
methoxybenzene sulfonamide (12d). Yellow solid; yield: 18%; m.p.
217e219 ꢂC; HPLC (MeOH:H2O ¼ 85:15, 1.0 mL/min) tR ¼ 3.96 min,
125.9, 123.5, 123.2, 120.9, 118.9, 112.2, 109.9, 56.6, 38.1, 31.5, 29.7,
25.7, 25.5. MS (ESI) m/z 411.2 for [M‒H]‒.
99.4%. 1H NMR (600 MHz, DMSO‑d6)
d 10.30 (s,1H), 9.54 (s,1H), 7.62
(d, J ¼ 5.4 Hz, 1H), 7.54 (brs, 1H), 7.17 (d, J ¼ 7.2 Hz, 1H), 7.14 (s, 1H),
6.97 (brs, 1H), 6.76 (brs, 1H), 6.59 (brs, 1H), 6.30 (d, J ¼ 10.8 Hz, 1H),
3.91 (s, 3H), 2.01 (brs, 1H), 1.09 (brs, 2H), 0.83 (brs, 2H); 13C NMR
4.1.5.7. (Z)-N-(3-(furan-2-ylmethylene)-2-oxoindolin-5-yl)-2-
methoxybenzene sulfonamide (12g). Yellow solid; yield: 48%; m.p.
232e234 ꢂC; HPLC (MeOH:H2O ¼ 85:15, 1.0 mL/min) tR ¼ 3.97 min,
96.8%. 1H NMR (600 MHz, DMSO‑d6)
d 10.42 (s,1H), 9.76 (s,1H), 8.23
(150 MHz, DMSO‑d6)
d 168.7, 156.3, 148.6, 136.6, 134.8, 130.9, 130.1,
124.8, 123.7, 121.9, 119.9, 113.4, 112.6, 109.3, 56.0, 11.9, 9.97. MS (ESI)
(s, 1H), 8.06 (s, 1H), 7.69 (d, J ¼ 7.8 Hz, 1H), 7.50 (t, J ¼ 7.8 Hz, 1H),
7.26e7.24 (m, 2H), 7.14 (d, J ¼ 7.8 Hz, 1H), 7.00 (d, J ¼ 7.8 Hz, 1H),
6.95 (d, J ¼ 7.8 Hz, 1H), 6.83 (s, 1H), 6.66 (d, J ¼ 8.4 Hz, 1H), 3.90 (s,
m/z 369.2 for [M‒H]‒.
3H); 13C NMR (150 MHz, DMSO‑d6)
d 169.1,156.3,150.5,146.9,139.2,
4.1.5.5. (Z)-N-(3-(Cyclopentylmethylene)-2-oxoindolin-5-yl)-2-
methoxybenzene sulfonamide (12e). Yellow solid; yield: 32%; m.p.
156e158 ꢂC; HPLC (MeOH:H2O ¼ 85:15, 1.0 mL/min) tR ¼ 4.36 min,
134.9, 131.6, 130.2, 122.2, 121.5, 120.9, 119.9, 119.6, 118.1, 113.7, 112.7,
109.6, 56.1. MS (ESI) m/z 395.2 for [M‒H]‒.
98.1%. 1H NMR (600 MHz, CDCl3)
d
8.31 (s, 1H), 7.76 (d, J ¼ 7.8 Hz,
1H), 7.51 (t, J ¼ 7.8 Hz, 1H), 7.35 (s, 1H), 7.21 (s, 1H), 7.04 (d,
J ¼ 9.0 Hz, 1H), 6.99 (t, J ¼ 7.8 Hz, 1H), 6.6.93 (d, J ¼ 9.6 Hz, 1H), 6.84
(d, J ¼ 7.6 Hz, 1H), 6.67 (d, J ¼ 8.4 Hz, 1H), 4.05 (s, 3H), 3.20e3.16 (m,
1H), 2.01e1.98 (m, 2H), 1.83e1.80 (m, 2H), 1.75e1.72 (m, 2H),
4.1.5.8. (E)-N-(3-(furan-2-ylmethylene)-2-oxoindolin-5-yl)-2-
methoxybenzene sulfonamide (12h). Yellow solid; yield: 12%; m.p.
>260 ꢂC; HPLC (MeOH:H2O ¼ 85:15, 1.0 mL/min) tR ¼ 4.14 min,
99.7%. 1H NMR (600 MHz, DMSO‑d6)
d 10.52 (s, 1H), 9.61 (s, 1H),

Y. Xu et al. / European Journal of Medicinal Chemistry 208 (2020) 112780
9
Fig. 6. Cellular effects of compound 12j in HT-29 cell lines. (A) Cell cycle analysis was performed on HT-29 cells cultured with DMSO; (B, C, D) Cell cycle analysis was performed on
HT-29 cells cultured with varied concentration of compound 12j (2.5, 5.0, 10.0 M) for 24 h.
m
1H), 3.92 (s, 3H); 13C NMR (100 MHz, DMSO‑d6)
d 167.4,156.8,150.9,
147.1, 138.2, 135.4, 131.6, 130.7, 126.7, 124.7, 123.4, 122.7, 121.7, 120.5,
119.8, 114.3, 114.2, 113.1, 110.1, 56.5. MS (ESI) m/z 395.2 for [M‒H]‒.
4.1.5.9. (Z)-N-(3-((1H-imidazole-2-yl)methylene)-2-oxoindolin-5-
yl)-2-methoxy benzene sulfonamide (12i). Yellow solid; yield: 25%;
m.p. >260 ꢂC; HPLC (MeOH:H2O ¼ 85:15,1.0 mL/min) tR ¼ 4.05 min,
95.9%. 1H NMR (600 MHz, DMSO‑d6)
d 11.08 (s, 1H), 9.74 (s, 1H), 9.64
(s, 1H), 7.69 (d, J ¼ 7.2 Hz, 1H), 7.55e7.40 (m, 5H), 7.18 (d, J ¼ 8.4 Hz,
Fig. 7. Western blotting analysis of the c-Myc protein in HT-29 cells treated with
compound 12j and (þ)-JQ1 for 24 h. GAPDH was used as the loading control.
1H), 6.98 (t, J ¼ 7.2 Hz,1H), 6.93 (d, J ¼ 7.8 Hz,1H), 6.74 (d, J ¼ 7.8 Hz,
1H), 3.93 (s, 3H); 13C NMR (100 MHz, DMSO‑d6)
d 181.7, 169.9, 156.8,
146.1,143.2,140.2,135.1,131.4,130.6,127.0,125.3,125.1,122.9,122.5,
121.3, 120.3, 112.9, 109.6, 56.6. MS (ESI) m/z 395.2 for [M‒H]‒.
Table 5
Pharmacokinetic properties of compound 12j in rats.
4.1.6. General procedure for the synthesis of compounds 12je12l
PK Parameters
Cmax g/L)
iv (5 mg/kg)
po (15 mg/kg)
A
mixture of 11 (0.31 mmol) and piperidine (0.67 mL,
(
m
e
2175
1.00
3.59
6.73 mmol) in appropriate ketones (1.5 mL) was stirred at room
temperature. After the reaction was completed, the insoluble
matter in the solution was filtered and the filter cake was dried at
room temperature to obtain products 12je12l.
Tmax (h)
e
T1/2 (h)
CLz/F (L/h/kg)
1.56
0.68
7296
7340
e
1.03
AUC
AUC
F (%)
(
m
g/L h)
14 384
14 600
66%
0-t
(
mg/L h)
0-∞
4.1.6.1. 2-Methoxy-N-(2-oxo-3-(propan-2-ylidene)indolin-5-yl)ben-
zenesulfonamide (12j). Yellow solid; yield: 70%; m.p. 180e182 ꢂC;
HPLC (MeOH:H2O ¼ 85:15, 1.0 mL/min) tR ¼ 3.72 min, 95.8%. 1H
8.22 (s, 1H), 7.97 (s, 1H), 7.65 (d, J ¼ 7.2 Hz, 1H), 7.52 (t, J ¼ 7.2 Hz,
1H), 7.42 (s, 1H), 7.34 (s, 1H), 7.16 (d, J ¼ 8.4 Hz, 1H), 6.96 (t,
J ¼ 7.8 Hz,1H), 6.83 (d, J ¼ 9.0 Hz,1H), 6.74 (s,1H), 6.63 (d, J ¼ 9.0 Hz,
NMR (600 MHz, DMSO‑d6)
d 10.31 (s, 1H), 9.56 (s, 1H), 7.65 (d,
J ¼ 8.4 Hz, 1H), 7.54 (d, J ¼ 7.2 Hz, 1H), 7.25 (s, 1H), 7.17 (d, J ¼ 7.8 Hz,
1H), 7.00e6.97 (m, 1H), 6.91e6.89 (m, 1H), 6.63 (d, J ¼ 8.4 Hz, 1H),

10
Y. Xu et al. / European Journal of Medicinal Chemistry 208 (2020) 112780
3.92 (s, 3H), 2.46 (s, 3H), 2.17 (s, 3H); 13C NMR (150 MHz, DMSO‑d6)
168.4, 156.3, 154.6, 137.3, 134.9, 130.8, 130.2, 126.3, 123.7, 122.5,
J ¼ 8.4 Hz, 1H), 6.64 (d, J ¼ 7.8 Hz, 1H), 3.77 (s, 3H), 2.46 (s, 3H), 2.15
(s, 3H); 13C NMR (150 MHz, DMSO‑d6)
168.4, 162.3, 154.7, 137.5,
d
d
121.5, 120.0, 117.9, 112.6, 108.9,56.0, 24.4, 22.2. MS (ESI) m/z 357.2
for [M‒H]‒.
130.8, 128.9, 123.8, 122.5, 122.0, 118.4, 114.2, 109.0, 55.5, 24.4, 22.2.
MS (ESI) m/z 357.2 for [M‒H]‒.
4.1.6.2. (Z)-2-Methoxy-N-(2-oxo-3-propylideneindolin-5-yl)benze-
nesulfonamide (12k). Yellow solid; yield: 60%; m.p. 148e150 ꢂC;
HPLC (MeOH:H2O ¼ 85:15, 1.0 mL/min) tR ¼ 3.93 min, 98.8%. 1H
4.1.8.2. 2-Bromo-N-(2-oxo-3-(propan-2-ylidene)indolin-5-yl)benze-
nesulfonamide (14b). Yellow solid; yield: 65%; m.p. 145e147 ꢂC;
HPLC (MeOH:H2O ¼ 85:15, 1.0 mL/min) tR ¼ 4.19 min, 99.2%. 1H
NMR (600 MHz, DMSO‑d6)
d
10.20 (s, 1H), 9.53 (brs, 1H), 7.62 (brs,
NMR (600 MHz, DMSO‑d6) d 10.37 (s, 1H), 10.19 (s, 1H), 7.94 (d,
1H), 7.54 (brs, 1H), 7.16 (brs, 1H), 6.97 (brs, 1H), 6.87 (brs, 2H), 6.61
(brs, 1H), 3.90 (s, 3H), 3.21 (brs, 2H), 3.07 (brs, 2H), 2.25 (brs, 2H);
J ¼ 7.2 Hz, 1H), 7.81 (d, J ¼ 7.8 Hz, 1H), 7.50 (brs, 2H), 7.24 (s, 1H),
6.90 (d, J ¼ 7.8 Hz, 1H), 6.65 (d, J ¼ 9.0 Hz, 1H), 2.45 (s, 3H), 2.14 (s,
13C NMR (150 MHz, DMSO‑d6)
d
167.5, 161.8, 156.3, 137.6, 134.9,
3H); 13C NMR (150 MHz, DMSO‑d6)
d 168.3, 154.8, 138.3, 137.6,
131.0, 130.2126.3, 123.0, 121.5, 120.0, 115.7, 112.6, 109.3, 55.9, 33.4,
135.3, 134.4, 131.8, 129.8, 128.1, 123.8, 122.4, 121.6, 119.2, 117.8, 109.1,
32.5, 18.9. MS (ESI) m/z 369.2 for [M‒H]‒.
24.4, 22.2. MS (ESI) m/z 405.1 for [M ꢃ H]- and 407.1 for [MþH]þ.
4 .1. 6 . 3 . N - ( 3 - C yc lo h e x yl i d e n e - 2 - o x o i n d o l i n - 5 - yl ) - 2 -
methoxybenzenesulfonamide (12l). Yellow solid; yield: 57%; m.p.
119e121 ꢂC; HPLC (MeOH:H2O ¼ 85:15, 1.0 mL/min) tR ¼ 4.24 min,
4.1.8.3. N-(2-Oxo-3-(propan-2-ylidene)indolin-5-yl)cyclo-
hexanesulfonamide (14c). Yellow solid; yield: 28%; m.p.
156e158 ꢂC; HPLC (MeOH:H2O ¼ 85:15, 1.0 mL/min) tR ¼ 4.50 min,
96.7%. 1H NMR (600 MHz, DMSO‑d6)
d
10.31 (s,1H), 9.50 (s, 1H), 7.61
95.5%. 1H NMR (600 MHz, CDCl3)
d 10.40 (s, 1H), 10.22 (s, 1H), 7.78
(d, J ¼ 7.2 Hz, 1H), 7.54 (t, J ¼ 7.8 Hz, 1H), 7.26 (s, 1H), 7.17 (d,
J ¼ 7.8 Hz,1H), 6.97 (t, J ¼ 7.2 Hz,1H), 6.89 (d, J ¼ 8.4 Hz,1H), 6.60 (d,
J ¼ 8.4 Hz, 1H), 3.90 (s, 3H), 3.19 (brs, 2H), 2.61 (brs, 2H), 1.69 (brs,
(s,1H), 7.02 (d, J ¼ 7.8 Hz,1H), 6. 95 (d, J ¼ 8.4 Hz,1H), 2.35e2.31 (m,
5H), 1.85e1.79 (m, 5H), 1.65e1.65 (m, 1H), 1.45e1.39 (m, 3H),
1.30e1.22 (m, 3H); 13C NMR (150 MHz, CDCl3)
d 169.8, 158.8, 136.8,
2H), 1.61 (brs, 4H); 13C NMR (150 MHz, DMSO‑d6)
d168.7, 156.2,
131.3, 125.2,122.8,121.9,119.2,110.1, 60.2, 29.7, 26.9, 26.5, 25.6, 25.1,
137.5, 134.9, 130.6, 130.2, 123.4, 121.9, 120.0, 119.8, 117.9, 112.5, 55.9,
23.6. MS (ESI) m/z 333.2 for [M‒H]‒.
32.0, 28.9, 27.8, 27.4, 25.1. MS (ESI) m/z 397.2 for [M‒H]‒.
4.1.8.4. 5-Bromo-2-methoxy-N-(2-oxo-3-(propan-2-ylidene)indolin-
5-yl)benzene sulfonamide (14d). Yellow solid; yield: 43%; m.p.
143e145 ꢂC; HPLC (MeOH:H2O ¼ 85:15, 1.0 mL/min) tR ¼ 4.39 min,
4.1.7. General procedure for the synthesis of compounds 13ae13b
A mixture of appropriate acids (0.64 mmol), HATU (1.06 mmol)
and DIPEA (0.19 mL, 1.06 mmol) in DMF (2 mL) was stirred at room
temperature for 5 min. Then compound 10 (0.53 mmol) was added
to the mixture. After the reaction was completed, an appropriate
amount of distilled water was added to the solution, and a pre-
cipitate was precipitated. Allow to stand for half an hour, the
insoluble matter in the solution was filtered, and the filter cake was
dried at 60 ꢂC to obtain products 13a and 13b.
96.6%. 1H NMR (600 MHz, DMSO‑d6)
d 10.36 (s,1H), 9.78 (s,1H), 7.72
(d, J ¼ 8.4 Hz, 1H), 7.68 (s, 1H), 7.21 (s, 1H), 7.16 (d, J ¼ 8.4 Hz, 1H),
6.89 (d, J ¼ 7.8 Hz, 1H), 6.65 (d, J ¼ 8.4 Hz, 1H), 3.90 (s, 1H), 2.46 (s,
3H), 2.17 (s, 3H); 13C NMR (100 MHz, DMSO‑d6)
d 168.8, 156.0, 155.3,
138.0, 137.8, 132.5,130.7, 128.6, 124.3, 122.9, 122.2, 118.4, 115.7, 111.4,
109.6, 56.9, 24.9, 22.7. MS (ESI) m/z 435.1 for [M ꢃ H]- and 437.1 for
[MþH]þ.
4.1.7.1. 2-Methoxy-N-(2-oxo-3-(propan-2-ylidene)indolin-5-yl)ben-
zamide (13a). Yellow solid; yield: 82%; m.p. 238e240 ꢂC; HPLC
(MeOH:H2O ¼ 85:15, 1.0 mL/min) tR ¼ 4.61 min, 97.7%. 1H NMR
4.1.8.5. 5-Fluoro-2-methoxy-N-(2-oxo-3-(propan-2-ylidene)indolin-
5-yl)benzene sulfonamide (14e). Yellow solid; yield: 17%; m.p.
205e207 ꢂC; HPLC (MeOH:H2O ¼ 85:15, 1.0 mL/min) tR ¼ 4.11 min,
(600 MHz, DMSO‑d6)
d
10.38 (s,1H), 9.99 (s,1H), 8.03 (s,1H), 7.61 (d,
99.9%. 1H NMR (600 MHz, DMSO‑d6)
d 10.35 (s, 1H), 9.75 (s, 1H),
J ¼ 7.2 Hz, 1H), 7.54 (d, J ¼ 7.8 Hz, 1H), 7.48 (s, 1H), 7.16 (d, J ¼ 8.4 Hz,
7.43e7.39 (m, 2H), 7.21 (d, J ¼ 6.0 Hz, 2H), 6.89 (d, J ¼ 7.2 Hz, 1H),
1H), 7.05 (d, J ¼ 7.2 Hz,1H), 6.77 (d, J ¼ 7.8 Hz,1H), 3.88 (s, 3H), 2.49 (s,
6.64 (d, J ¼ 8.4 Hz, 1H), 3.90 (s, 3H), 2.46 (s, 3H), 2.17 (s, 3H); 13C
3H), 2.31 (s, 3H); 13C NMR (100 MHz, DMSO‑d6)
d
169.1, 164.6, 156.8,
NMR (150 MHz, DMSO‑d6) d 168.3, 153.9, 152.8, 137.5, 130.3, 123.7,
154.7,136.8,133.4,132.3,130.0,125.6,123.9,123.3,120.9,119.9,116.6,
122.5, 121.7, 117.9, 116.7, 114.4, 109.0, 56.6, 24.4, 22.2. MS (ESI) m/z
112.4, 109.1, 56.3, 25.1, 22.7. MS (ESI) m/z 323.2 for [MþH]þ.
375.2 for [M‒H]‒.
4.1.7.2. N-(2-Oxo-3-(propan-2-ylidene)indolin-5-yl)cyclo-
hexanecarboxamide (13b). Yellow solid; yield: 75%; m.p.
200e202 ꢂC; HPLC (MeOH:H2O ¼ 85:15,1.0 mL/min) tR ¼ 4.94 min,
4.1.8.6. 2-Methoxy-5-methyl-N-(2-oxo-3-(propan-2-ylidene)indolin-
5-yl)benzene sulfonamide (14f). Yellow solid; yield: 61%; m.p.
231e233 ꢂC; HPLC (MeOH:H2O ¼ 85:15, 1.0 mL/min) tR ¼ 3.95 min,
95.7%. 1H NMR (600 MHz, DMSO‑d6)
d
10.31 (s, 1H), 9.64 (s, 1H),
98.8%. 1H NMR (600 MHz, DMSO‑d6)
d 10.32 (s, 1H), 9.51 (brs, 1H),
7.91 (s, 1H), 7.42 (d, J ¼ 7.8 Hz,1H), 6.71 (d, J ¼ 7.8 Hz,1H), 2.30e2.27
7.45 (s, 1H), 7.32 (d, J ¼ 6.6 Hz, 1H), 7.22 (s, 1H), 7.05 (d, J ¼ 8.4 Hz,
1H), 6.89 (d, J ¼ 7.2 Hz, 1H), 6.62 (d, J ¼ 7.8 Hz, 1H), 3.86 (s, 1H), 2.45
(s, 3H), 2.19 (s, 3H), 2.16 (s, 3H); 13C NMR (100 MHz, DMSO‑d6)
(m, 5H), 1.79e1.74 (m, 5H), 1.66e1.64 (m, 1H), 1.43e1.37 (m, 2H),
1.29e1.17 (m, 4H); 13C NMR (100 MHz, DMSO‑d6)
d 174.3, 169.1,
154.4, 136.3, 133.8, 123.9, 123.4, 119.1, 115.9, 109.1, 45.4, 29.7, 25.9,
d 168.8, 155.0, 154.6, 137.7, 135.7, 131.3, 130.7, 129.5, 126.4, 124.2,
25.7, 25.0, 22.7. MS (ESI) m/z 299.2 for [MþH]þ.
123.0, 121.9, 118.2, 112.9, 109.5, 56.5, 24.9, 22.7, 20.2. MS (ESI) m/z
371.2 for [M‒H]‒.
4.1.8. General procedure for the synthesis of compounds 14ae14m
Then using compound 10 as a raw material, the compounds
14ae14m were synthesized using the similar method as 12ae12i.
4.1.8.7. 2,5-Difluoro-N-(2-oxo-3-(propan-2-ylidene)indolin-5-yl)
benzenesulfonamide (14g). Yellow solid; yield: 17%; m.p.
238e240 ꢂC; HPLC (MeOH:H2O ¼ 85:15, 1.0 mL/min) tR ¼ 4.22 min,
4.1.8.1. 4-Methoxy-N-(2-oxo-3-(propan-2-ylidene)indolin-5-yl)ben-
zenesulfonamide (14a). White solid; yield: 74%; m.p. 218e220 ꢂC;
HPLC (MeOH:H2O ¼ 85:15, 1.0 mL/min) tR ¼ 4.05 min, 99.6%. 1H
95.8%. 1H NMR (600 MHz, DMSO‑d6)
d 10.41 (s, 1H), 10.39 (brs, 1H),
7.56 (brs, 1H), 7.51e7.48 (m, 2H), 7.24 (s, 1H), 6.89 (d, J ¼ 8.4 Hz, 1H),
6.69 (d, J ¼ 8.4 Hz, 1H), 2.46 (s, 3H), 2.17 (s, 3H); 13C NMR (150 MHz,
NMR (600 MHz, DMSO‑d6)
d
10.35 (s, 1H), 9.71 (s, 1H), 7.59 (d,
DMSO‑d6) d 168.4, 157.9, 155.2, 138.0, 129.5, 123.9, 122.4, 122.2,
J ¼ 9.0 Hz, 2H), 7.20 (brs, 1H), 7.02 (d, J ¼ 9.0 Hz, 2H), 6.82 (d,
119.3, 118.4, 116.7, 109.2, 24.4, 22.3. MS (ESI) m/z 363.2 for [M‒H]‒.

Y. Xu et al. / European Journal of Medicinal Chemistry 208 (2020) 112780
11
4.1.8.8. 2-Fluoro-5-methyl-N-(2-oxo-3-(propan-2-ylidene)indolin-5-
4.2. Biology evaluation
4.2.1. TR-FRET assay
yl)benzenee sulfonamide (14h). Yellow solid; yield: 39%; m.p.
240e242 ꢂC; HPLC (MeOH:H2O ¼ 85:15, 1.0 mL/min) tR ¼ 4.18 min,
96.6%. 1H NMR (600 MHz, DMSO‑d6)
d
10.39 (s, 1H), 10.17 (s, 1H),
The assays were carried out by Sundia (Shanghai, China) Med-
iTech Company, Ltd. At first, 1 ꢀ assay buffer was prepared and
compounds were transferred to assay plate by Echo550. Then
4 ꢀ protein solution with 1 ꢀ reaction solution were prepared and
7.54 (d, J ¼ 5.4 Hz, 1H), 7.46 (d, J ¼ 3.0 Hz, 1H), 7.29 (d, J ¼ 8.4 Hz,
1H), 7.25 (s, 1H), 6.90 (d, J ¼ 7.2 Hz, 1H), 7.68 (d, J ¼ 8.4 Hz, 1H), 2.48
(s, 3H), 2.28 (s, 3H), 2.17 (s, 3H); 13C NMR (150 MHz, DMSO‑d6)
d
168.3, 157.1, 155.5, 154.8, 137.7, 135.9, 134.3, 130.1, 130.0, 123.9,
add 5
m
L of 4 ꢀ protein solution to each well, centrifuge at 1000 rpm
122.5, 121.7, 117.9, 116.9, 116.8109.1, 24.3, 22.2, 19.9. MS (ESI) m/z
359.2 for [M‒H]‒.
for 1 min, and incubate at room temperature for 15 min. At the
same time, 4 ꢀ peptide solution with 1 ꢀ reaction solution were
prepared and 5
m
L of 4 ꢀ peptide solution to each well of the re-
4.1.8.9. N-(2-Oxo-3-(propan-2-ylidene)indolin-5-yl)-[1,10-biphenyl]-
4-sulfonamide (14i). Yellow solid; yield: 30%; m.p. 224e226 ꢂC;
HPLC (MeOH:H2O ¼ 85:15, 1.0 mL/min) tR ¼ 4.84 min, 95.8%. 1H
action plate was add and the mixture was centrifuged at 1000 rpm
for 1 min. Next, 10 L of detection solution was added and the
m
mixture was centrifuged at 1000 rpm for 60 s and incubated at
room temperature for 60 min. Finally, read the conversion rate with
NMR (600 MHz, DMSO‑d6)
d 10.38 (s, 1H), 9.93 (s, 1H), 7.83 (d,
J ¼ 7.8 Hz, 2H), 7.75 (d, J ¼ 7.8 Hz, 2H), 7.68 (d, J ¼ 7.2 Hz, 2H), 7.48 (t,
J ¼ 7.8 Hz, 2H), 7.41 (d, J ¼ 7.2 Hz,1H), 7.23 (s,1H), 6.89 (d, J ¼ 8.4 Hz,
1H), 6.67 (d, J ¼ 8.4 Hz, 1H), 2.44 (s, 3H), 2.13 (s, 3H); 13C NMR
EnVision and calculated the IC50.
4.2.2. Anti-proliferative activity
(100 MHz, DMSO‑d6)
d
168.9, 155.4, 144.6, 138.8, 138.7, 138.2, 131.0,
Anti-proliferative activities of the target compounds were car-
ried out based on MTT or CCK8 assays. Cells were incubated at 37 ꢂC
in a 5.0% CO2 atmosphere. MTT assay: The HT-29 and WI-38 cells
were seeded in 96-well plates at a density of 5 ꢀ 103 cells/well in
129.6, 128.9, 127.9, 127.7, 124.4, 122.9, 122.7, 119.0, 109.7, 55.4, 24.9,
22.7. MS (ESI) m/z 403.2 for [M‒H]‒.
4.1.8.10. N-(2-Oxo-3-(propan-2-ylidene)indolin-5-yl)-2,3-
150 mL of the medium with 10% FBS for 24 h. The target compounds
dihydrobenzofuran-5- sulfonamide (14j). Yellow solid; yield: 61%;
and positive controls were dissolved to different concentrations in
the medium and then added them to the well and incubated at
m.p. 159e161 ꢂC; HPLC (MeOH:H2O
¼
85:15, 1.0 mL/min)
tR ¼ 4.05 min, 98.1%. 1H NMR (600 MHz, DMSO‑d6)
d
10.35 (s, 1H),
37 ꢂC for 72 h, then the media was aspirated, and 10
mL of 5 mg/mL
9.67 (s, 1H), 7.54 (s, 1H), 7.42 (d, J ¼ 8.4 Hz, 1H), 7.21 (s, 1H), 6.83 (d,
J ¼ 7.2 Hz, 2H), 6.65 (d, J ¼ 8.4 Hz, 1H), 4.58 (t, J ¼ 8.4 Hz, 2H), 3.17 (t,
J ¼ 8.4 Hz, 2H), 2.46 (s, 3H), 2.16 (s, 3H); 13C NMR (100 MHz,
MTT solution (dilute in sterile PBS) diluted in serum-free media was
added to each well. After 4 h, the supernatant was discarded and
100 mL of DMSO was added to each well. The mixture was shaken
DMSO‑d6)
d
168.9, 163.5, 155.2, 137.9, 131.5, 131.4, 129.1, 128.7, 124.6,
on an oscillator and measured at 490 nm using universal microplate
reader (Infinite M200 Pro, Tecan Inc.). CCK8 assay: The cell culture
and dosing methods were similar to the MTT assay. The difference
was that when using the CCK8 assay, the CCK8 reagent was added,
and the absorbance can be directly measured after cultivation [32].
HL-60 cells were determined by this method. IC50 values were
determined from a log plot of percent of control versus concen-
tration. All samples and controls were tested in triplicate [33].
124.3, 122.9, 122.4, 118.8, 109.6, 109.4, 72.6, 55.4, 28.8, 24.9, 22.7. MS
(ESI) m/z 369.2 for [M‒H]‒.
4.1.8.11. N-(2-Oxo-3-(propan-2-ylidene)indolin-5-yl)naphthalene-2-
sulfonamide (14k). Yellow solid; yield: 60%; m.p. 244e246 ꢂC; HPLC
(MeOH:H2O ¼ 85:15, 1.0 mL/min) tR ¼ 4.37 min, 97.0%. 1H NMR
(600 MHz, DMSO‑d6)
d 10.35 (s, 1H), 9.99 (s, 1H), 8.32 (s, 1H), 8.08
(d, J ¼ 8.4 Hz, 2H), 7.99 (d, J ¼ 7.2 Hz,1H), 7.74 (d, J ¼ 8.4 Hz,1H), 7.66
(d, J ¼ 7.8 Hz, 1H), 7.62 (d, J ¼ 7.2 Hz, 1H), 7.20 (s, 1H), 6.85 (d,
4.2.3. Docking studies
J ¼ 8.4 Hz, 1H), 6.61 (d, J ¼ 7.8 Hz, 1H), 2.41 (s, 3H), 2.05 (s, 3H); 13
C
The docking simulationwas performed using the Glide module of
€
NMR (150 MHz, DMSO‑d6)
d
168.3, 154.7, 137.7, 136.5, 134.1, 131.5,
Schrodinger. Before docking, the ligand 12j was prepared firstly
€
130.4, 129.2, 129.1, 128.8, 127.9, 127.7, 127.6, 123.8, 122.4, 122.2,
using the LigPrep module in Schrodinger. The X-ray crystal structure
118.6, 109.5, 109.0, 24.3, 22.1. MS (ESI) m/z 377.2 for [M‒H]‒.
of BRD4 (http://www.rscb.org./pdb, BRD4 BD1 PDB code: 4E96 and
BRD4 BD2 PDB code: 5UVX) were derived from the PDB Database
and minimized using the Protein Preparation Wizard module in the
Schrodinger Suite [24]. Then, a grid was generated at the active site
of the prepared protein structure using the Glide docking module of
Schrodinger. The Glide docking module of Schrodinger was used to
investigate the interactions between the synthesized compounds
and BRD4. And all the figures displaying the docking results were
obtained using the scientific software Pymol [34].
4.1.8.12. N-(2-Oxo-3-(propan-2-ylidene)indolin-5-yl)naphthalene-1-
sulfonamide (14l). Yellow solid; yield: 50%; m.p. 135e137 ꢂC; HPLC
(MeOH:H2O ¼ 85:15, 1.0 mL/min) tR ¼ 4.27 min, 98.8%. 1H NMR
€
€
€
(600 MHz, DMSO‑d6)
d
10.29 (s, 1H), 10.23 (s, 1H), 8.73 (d, J ¼ 8.4 Hz,
1H), 8.18 (d, J ¼ 9.0 Hz,1H), 8.05 (d, J ¼ 6.0 Hz, 2H), 7.72 (d, J ¼ 7.8 Hz,
1H), 7.66 (d, J ¼ 7.2 Hz,1H), 7.55 (d, J ¼ 7.8 Hz,1H), 2.39 (s, 3H),1.92 (s,
3H); 13C NMR (100 MHz, DMSO‑d6)
d 168.8,155.2,137.9,134.8,134.7,
134.1,130.7,130.5,129.5,128.5,128.0,127.4,124.9,124.2,122.8,122.3,
118.4, 109.5, 55.4, 24.7, 22.6. MS (ESI) m/z 377.2 for [M‒H]‒.
4.2.4. Cell cycle analysis
HT-29 cells were seeded in 6-well plates (3 ꢀ 105 cells/well),
incubated in the presence or absence of compound 12j at the
indicated concentrations for 24 h. After incubation, cells were then
washed three times with PBS, incubated for 1 h with 1 mg/mL
4.1.8.13. 5-(Dimethylamino)-N-(2-oxo-3-(propan-2-ylidene)indolin-
5-yl)naphthalene- 1- sulfonamide (14m). Yellow solid; yield: 28%;
m.p. 220e222 ꢂC; HPLC (MeOH:H2O
¼
85:15, 1.0 mL/min)
tR ¼ 4.70 min, 97.4%. 1H NMR (600 MHz, DMSO‑d6)
d
10.30 (s, 1H),
RNase A and 20 mg/mL propidium iodide at room temperature, and
10.17 (s, 1H), 8.41e8.37 (m, 2H), 8.04 (d, J ¼ 6.6 Hz, 1H), 7.60 (d,
J ¼ 7.8 Hz, 1H), 7.17 (t, J ¼ 7.8 Hz, 1H), 7.24 (d, J ¼ 7.2 Hz, 1H), 6.96 (s,
1H), 6.79 (d, J ¼ 7.8 Hz, 1H), 6.58 (d, J ¼ 7.8 Hz, 1H), 2.79 (s, 6H), 2.39
analyzed with a flow cytometer. Almost 5000 events were collected
for each sample and analyzed by flow cytometry (Beckman Coulter,
Epics XL).
(s, 3H), 1.90 (s, 3H); 13C NMR (100 MHz, DMSO‑d6)
d 168.8, 155.1,
151.9, 137.9, 135.2, 130.8, 130.4, 130.3, 129.6, 129.3, 128.5, 124.2,
123.9,122.8,122.3,119.3,118.4,115.6, 109.5, 45.5, 24.7, 22.7. MS (ESI)
m/z 420.3 for [M‒H]‒.
4.2.5. Western blot analysis
HT-29 cells (5.0 ꢀ 105 cells/dish) were incubated with 12j at
various concentrations for 24 h. After incubation, the cells were

12
Y. Xu et al. / European Journal of Medicinal Chemistry 208 (2020) 112780
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30 min, intermittently vortexed every 5 min, and centrifuged at
12 000 r for 10 min to harvest the supernatants. Next, the protein
concentrations were determined by a BCA Protein Assay Kit. The
protein extracts were reconstituted in loading buffer containing
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Declaration of competing interest
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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.
Acknowledgment
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We are grateful to the National Natural Sciences Foundations of
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thank Professor Yan-bin Shi (Lanzhou University) for his valuable
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(2016) 47e54.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112780.
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