Discovery of indoline derivatives that inhibit esophageal squamous cell carcinoma growth by Noxa mediated apoptosis

Article abstract of DOI:10.1016/j.bioorg.2019.103190

A series of novel indoline derivatives were synthesized and evaluated for antiproliferative activity against four selected cancer cell lines (Hela, A549, HepG2 and KYSE30). Among them, compound 20 displayed the potent inhibition activity against esophagea

Full text of DOI:10.1016/j.bioorg.2019.103190

BioorganicChemistry92(2019)103190
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Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Discovery of indoline derivatives that inhibit esophageal squamous cell
carcinoma growth by Noxa mediated apoptosis
Dong-Jun Fua,b,c,1, Miaomiao Lia,1, Sai-Yang Zhanga,d,1, Jiang-Feng Lia, Beibei Shaa,
⁎
⁎
Longhao Wanga, Yan-Bing Zhangb,c, Ping Chena,d, , Tao Hua,
a School of Basic Medical Sciences, Zhengzhou University, Zhengzhou 450001, China
b New Drug Research & Development Center, School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, China
c Collaborative Innovation Center of New Drug Research and Safety Evaluation, Zhengzhou 450001, China
d Henan Institutes of Advanced Technology, Zhengzhou University, Zhengzhou 450001, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of novel indoline derivatives were synthesized and evaluated for antiproliferative activity against four
selected cancer cell lines (Hela, A549, HepG2 and KYSE30). Among them, compound 20 displayed the potent
inhibition activity against esophageal cancer cells (Kyse30, Kyse450, Kyse510 and EC109). Cellular mechanism
studies in esophageal squamous cell carcinoma (ESCC) cells elucidated compound 20 inhibited cell growths in
vitro and in vivo, reduced colony formation, arrested cell cycle at M phase, and induced Noxa-dependent
apoptosis in ESCC. Importantly, compound 20 was identified as a novel Noxa mediated apoptosis inducer. These
results suggested that compound 20 might be a promising anticancer agent with potential for development of
further clinical applications.
Indoline
Esophageal squamous cell carcinoma
Cell growths in vitro and in vivo
Noxa-dependent apoptosis
1. Introduction
of the tumor cell specific M2 isoform of pyruvate kinase [17]. Spir-
ooxindole derivative 8 caused restoration of the tumor protein 53
Heterocycle-fused scaffolds as one of the privileged structures in
drug discovery exhibit various biological activities allowing them to act
as anticancer, antiinflammatory, antipsychotic, and antidiabetic agents
[1–7]. Indoline as a simple class of heterocycle-fused scaffold prolifi-
cally designed with strong pharmacological significances in particular
of anticancer ability [8–10]. 1-Aroylindoline-hydroxamic acid 1 (Fig. 1)
as a dual HDAC6/HSP90 inhibitor displayed striking cytotoxic effects
with GI50 values ranging 1.04–1.61 μM against lung A549, colon
HCT116 and leukemia HL60 cell lines [11]. Indoline 2 demonstrated
the significantly anticancer activity in a UACC-62 xenograft model in
mice [12]. 7-Aroyl-aminoindoline-1-sulfonamide 3 inhibited the human
cancer cell growth of gastric MKN45, lung H460, and colon HT29 cells
with an IC50 value of 8.8, 9.4, and 8.6 nM, respectively [13]. Indoline-
pyrimidine hybrid 4 could inhibit protein kinase R-like endoplasmic
reticulum kinase (PERK) activation and suppress the growth of a human
tumor xenograft in mice [14]. 1-Arylsulfonyl indoline 5 exhibited the
potent antiproliferative activity against lung A549 cells with an IC50
value of 79 nM [15]. Compound 6 could inhibit multidrug resistant
cancer cell lines and arrest cell cycle at G2/M phase [16]. 1-(Sulfonyl)-
5-(arylsulfonyl)indoline 7 was designed and synthesized as a activator
function by triggering its nuclear accumulation and preventing its
proteasomal degradation as an anti-breast cancer agent [18]. Indoline
analogue 9 as a novel receptor-interacting protein kinase 1 inhibitor
exhibited the potent antimetastasis activity in the experimental B16
melanoma lung metastasis model [19].
As one of the most aggressive digestive system tumors, esophageal
squamous cell carcinoma is associated with numerous factors, including
advanced age, achalasia, tobacco use, alcohol abuse, high-starch diets
lacking in fruits, and so on [20–22]. In 2012, 400,000 deaths were
reported in the world due to esophageal carcinoma [23]. Chemical
prevention and treatment using synthetic drugs has emerged as an
advanced strategy for esophageal squamous cell carcinoma [24–26].
Noxa as a B-cell lymphoma 2 (Bcl-2) family member plays an important
role in apoptosis induction [27]. Activated Noxa could inhibit the ac-
tivities of Bfl-1/A1 and Mcl-1, then release and directly bind Bak and
Bax to promote cell apoptosis [28]. So, Noxa might be a promising
target for developing new chemotherapeutic agents to treat esophageal
carcinoma.
Here, a series of new anticancer indolines were synthesized and
explored for their antiproliferative activity. In order to investigate the
⁎ Corresponding authors at: School of Basic Medical Sciences, Zhengzhou University, China.
E-mail addresses: zzdx_chenping@zzu.edu.cn (P. Chen), hnhutao@zzu.edu.cn (T. Hu).
1 These authors contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2019.103190
Received 15 May 2019; Received in revised form 27 July 2019; Accepted 8 August 2019
Availableonline10August2019
0045-2068/©2019ElsevierInc.Allrightsreserved.

D.-J. Fu, et al.
BioorganicChemistry92(2019)103190
Fig. 1. The chemical structures of anticancer indolines.
broad-spectrum anti-cancer activity, four different cancer cell lines
(Hela, A549, HepG2 and KYSE30) were selected to obtain IC50 values.
Importantly, the anticancer mechanisms in vitro and in vivo against
esophageal squamous cell carcinoma of indolines were evaluated.
assay [32–36]. Representative chemical features of indoline derivatives
are the 3,4,5-trimethoxyphenyl moiety linked to a substituted indoline
ring through a carbonyl and methylene group spacer (Fig. 2). On the
basis of that scaffold, we designed different structures depicted in Fig. 2
to determine the relationship between chemical structures and anti-
proliferative activity. Several substituents such as bromine atom, nitro,
or methyl were introduced in the heterocycle core or the indoline ring.
Moreover, the influence of the nature of the heterocyclic moiety in
antiproliferative activity was studied. Thus, the indoline scaffold was
changed by indole, phenothiazine, 1,2,3,4-tetrahydroquinoline, and
2,3,4,5-tetrahydro-1H-benzo[b]azepine. Finally, the importance of
3,4,5-trimethoxyphenyl moiety was also analyzed.
2. Results and discussion
2.1. Chemistry
The synthetic route of indoline derivatives were shown in Scheme 1.
5-(Bromomethyl)-1,2,3-trimethoxybenzene 11 was synthesized by re-
acting (3,4,5-trimethoxyphenyl)methanol 10 with phosphorus tri-
bromide in the chloroform system according to previous references
[29–31]. With the intermediate 11 in hand, we next synthesized in-
doline derivatives bearing a 3,4,5-trimethoxyphenyl scaffold 12–13 by
the nucleophilic substitution reaction. Treatment of substituted benzoyl
chloride and thiophene-2-carbonyl chloride with aromatic amine in the
presence of sodium hydroxide furnished indoline derivatives 15–17 and
29–32. The structure confirmation of all indoline derivatives were de-
scribed in the Supporting Information.
The carbonyl and methylene group spacer between 3,4,5-tri-
methoxyphenyl moiety and indoline ring affected the antiproliferative
activity against four cancer cell lines. When the methylene group was
replaced by a carbonyl group, the antiproliferative activity against
Hela, A549 and KYSE30 cells were improved (12 Vs 15, 13 Vs 16). Data
collected in Table 1 show that the inhibitory potency against Hela and
A549 cells is decreased when the indoline ring was replaced by a
1,2,3,4-tetrahydroquinoline ring or 2,3,4,5-tetrahydro-1H-benzo[b]
azepine ring, indicating that the size of indoline ring played an im-
portant role for antiproliferative activity. Especially, indoline 16 dis-
played the potent activity against Hela, A549, HepG2 and KYSE30 cells
with IC50 values of 4.32 μM, 18.41 μM, 23.72 μM and 12.76 μM, re-
spectively.
2.2. Antiproliferative activity of indoline derivatives
In order to discovery novel Noxa mediated apoptosis inducers, we
evaluated the antiproliferative activity of all indoline derivatives
against Hela (Human cervical cancer cell line), A549 (Human lung
carcinoma cell line), HepG2 (Human liver cancer cell line), and KYSE30
(Human esophagus cancer cell line) using the CCK8 assay. In this paper,
the anticancer drug 5-FU was used as the reference drugs in the CCK8
Modification of the nature of the heterocyclic moiety present in the
target analogues by introduction of indole (compound 18) or phe-
nothiazine (compound 19) led to potent activity results against four
cancer cell lines with IC50 values ranging from 1.35 μM to 19.45 μM. All
2

D.-J. Fu, et al.
BioorganicChemistry92(2019)103190
Scheme 1. Reagents and conditions: (a) PBr3, CHCl3, reflux; (b) K2CO3, aromatic amine, acetone, reflux; (c) Aromatic amine, K2CO3, acetone, reflux.
Fig 2. Structural modifications proposed from the hit indolines.
these results investigated that heterocyclic moiety palyed an important
role for the antiproliferative activity. Several indoline-3,4,5-tri-
methoxyphenyl derivatives were then prepared which incorporate di-
verse substituents in different positions of the indoline ring. One
striking observation is the influence that the introduction of a bromine
atom attaching to indoline ring of compound 20 in the inhibitory ac-
tivity. When the hydrogen atom attaching phenyl ring of compound 16
was replaced by a bromine atom, compound 20 displayed more potent
antiproliferative activity against A549, HepG2 and KYSE30 cells. These
results indicated that the bromine atom as an electron-withdrawing
group might affect the inhibitory activity of indolines. In addition, the
importance of 3,4,5-trimethoxyphenyl unit was explored. Target deri-
vatives 24–27 without the 3,4,5-trimethoxyphenyl unit showed the
weak activity results against A549 cancer cells with IC50
values > 80 μM. However, target derivatives 18–23 with the 3,4,5-tri-
methoxyphenyl unit showed the potent activity results against same
cancer cells A549 with IC50 values < 20 μM (see Table 2).
To explore the effect of heterocycle for the inhibitory activity,
thiophene was introduced into the targeted compounds 29–32. A
comparison with the antiproliferative activity results of compound 31,
3

D.-J. Fu, et al.
BioorganicChemistry92(2019)103190
Table 1
2.3.3. Compound 20 induced cell apoptosis
Antiproliferative activity of the synthetic derivatives 12–13 and 15–17.
Flow cytometry analysis was performed after the cells were treated
with compound 20 and stained with Annexin V-FITC/PI to check the
apoptosis induced by compound 20. Results in Fig. 5 shows that com-
pound 20 induced cell apoptosis in both Kyse30 and Kyse450 cell lines
in dose depended manner. In both 2 cell lines, the apoptosis percentage
rise to approximately 50% from control which is under 5% (Fig. 5A).
The activation of Caspase-3 were enhanced by compound 20 treatment
in both cell lines, Caspase-3 in about 45% cells has been activated
(Fig. 5B). Similarly, compound 20 significantly induced the cleavage of
Caspase-9, Caspase-3, and PARP (Fig. 5C). These results suggest that
compound 20 triggered Caspases related apoptosis in ESCC cells.
Compound
n
IC50 (μM)a
Hela
2.3.4. Compound 20 induced apoptosis via up-regulating Noxa
To further check if the apoptosis induced by compound 20 is though
the intrinsic pathway, mitochondrial membrane potential (MMP/ΔΨ)
were analyzed using flow cytometry. The loss of MMP in Kyse30 and
Kyse450 were clearly observed as the concentration of compound 20
increases (Fig. 6A). These suggest that the compound 20 may induce
cell apoptosis through intrinsic apoptosis pathway. Next, we de-
termined the mitochondria apoptosis related proteins the Bcl-2 family
and XIAP. As shown in Fig. 6B, in both cell lines, pro-apoptosis proteins
Bid, Bim, Noxa were up regulated, the anti-apoptosis protein Bcl-2, Mcl-
1, XIAP were down regulated, while the Bax, Bak do not have sig-
nificant changes. Among which Noxa made the most tremendous
changes after the treatment in both cell lines. Next siRNA silencing was
performed to downregulate Noxa. In the Noxa silenced cell lines, the
apoptosis and PARP cleavage induced by compound 20 had been evi-
dently rescued (Fig. 6C and D). In conclusion, the apoptosis induced by
compound 20 is via intrinsic apoptosis pathway and depend on the
participation of Noxa.
A549
HepG2
KYSE30
5FU
12
13
15
16
17
–
22.08
8.46
5.98
7.57
4.32
> 80
0.05 4.22
0.04
1.32
8.09
0.04
0.05
0.02
1.46
0.03
0.04
0.11
0.03
0.07
0.09
2
1
2
1
3
0.04
> 80
20.71
34.05
18.41
31.38
10.42
17.67
0.04
0.06
0.05
0.08 3.61
0.05 13.73
0.08 23.72
0.14 21.74
0.09 7.92
0.11 12.76
0.07 6.55
a
Antiproliferative activity was assayed by exposure for 48 h. Data are pre-
sented as the means
experiments.
SDs from the dose-response curves of three independent
analogues 29 and 30 with substituents in the indoline ring exhibited
better antiproliferative activity results against Hela cells and A549 cells.
Replacing the indoline scaffold of 31 with 1,2,3,4-tetrahydroquinoline
ring of 32 led to a decrease of the antiproliferative activity against the
KYSE30 cells and an increase of the antiproliferative activity against the
A549 cells. All these results suggested that the indoline ring with sub-
stituents played a significant role in their activities (see Table 3).
3. Conclusions
2.3. Biology mechanism
A series of novel indoline derivatives were design, synthesized and
evaluated for their antiproliferative activity. Kyse30 was chosen to
determine the anti-cancer mechanisms, three other cancer cell lines
were used to appraise the broad-spectrum anti-cancer activity.
Compound 20 inhibits tumor growth against ESCC cell lines in vitro and
in vivo. Mechanism studies showed that compound 20 could arrest the
cells at M phase. In addition, compound 20 induced Noxa-dependent
apoptosis. These observations demonstrate that indoline 20 might be a
lead compound for the discovery of more Noxa mediated apoptosis
inducers against esophageal squamous cell carcinoma.
2.3.1. Compound 20 inhibited ESCC cell growth in vitro and in vivo
Indoline derivative compound 20 was chosen to do the further re-
search, and four ESCC cell lines (Kyse30, Kyse450, Kyse510, EC109)
were selected to test the inhibition activities against ESCCs of com-
pound 20 in vitro. Compound 20 treatment showed a strong inhibition
against the ESCCs in a concentration depend manner, the IC50 values
against cell lines were all less than 2 μM, presented in a dose-dependent
manner (Fig. 3A). Results in Fig. 3B showed that as the concentration
increased the cell colonies were evidently decreased by compound 20,
which illustrated compound 20 inhibited the colony formatting. To
verify the consistency of results in vitro and in vivo, mice xenograft
Kyse450 tumor model was established. Mice were treated for 30 days at
dose of 150 mg/kg/day. As shown in Fig. 3B and D, the tumors had
been significantly reduced by compound 20. These data confirmed that
the indoline 20 showed a high inhibition activity against ESCC cell lines
in vitro and in vivo.
4. Experimental section
4.1. General chemistry
All chemical agents were obtained from Bei Jing Yinuo Kai com-
pany. Melting points were taken on an X-5 micromelting apparatus
from Shang Hai Jing Ke company. 1H and 13C NMR spectra were re-
spectively determined with a 400 and 100 MHz spectrometer (Agilent,
Santa Clara, CA, USA). Mass spectra were recorded on Esquire 3000
mass spectrometer (Varian, Palo Alto, CA, USA). The spectra data of
compounds are provided in the supporting information.
2.3.2. Compound 20 induced G2/M phase arrest
To test the effect of compound 20 on the cell cycle arrestment, cells’
nucleus were stained with PI and analyzed by flow cytometry. As shown
in Fig. 4A and B, the percentage of cells at G2/M phase was markedly
elevated with the compound 20 treatment. For the Kyse30 and Kyse450
cell lines, 2.5 μM treatment up regulated G2/M percentage up to 4.9
times (Kyse30) and 6.1 times (Kyse450) comparing with control group.
Since the flow cytometry results cannot distinguish G2 or M ar-
restment, some G2/M phase related proteins were detected. In both
Kyse30 and Kyse450 cells Wee1, p-Wee1, CDK1, Cyclin B1 and p-
Histone H3 were up regulated as the concentration of compound 20
raises, which indicated the cell cycle arrestment caused by compound
20 is M phase arrestment (Fig. 4C).
4.2. General procedure for the synthesis of compounds 12–13
To
a solution of 5-(bromomethyl)-1,2,3-trimethoxybenzene 11
(6 mmol) in acetone (30 ml), K2CO3 (7 mmol) and arylamine (6 mmol)
were added. After the mixture was stirred at 60 °C for 5 h. The organic
layer was concentrated and then purified by column chromatography
with petroleum/ethyl acetate (9:1) to afford the target compound
12–13.
4

D.-J. Fu, et al.
BioorganicChemistry92(2019)103190
Table 2
Antiproliferative activity of the synthetic derivatives 18–27.
Compound
R1
IC50 (μM)a
Hela
A549
HepG2
KYSE30
5FU
18
–
–
22.08
4.66
0.05
4.22
2.74
0.04
0.05
1.32
1.35
0.04
0.06
1.46
1.54
0.03
0.05
3,4,5-trimethoxy
0.02
19
20
3,4,5-trimethoxy
3,4,5-trimethoxy
6.36
1.30
0.04
0.08
18.06
1.24
0.05
16.14
1.42
0.13
19.45
1.21
0.08
0.05
0.05
0.06
21
22
3,4,5-trimethoxy
3,4,5-trimethoxy
3.24
2.32
0.04
0.09
4.30
4.00
0.04
0.05
27.97
1.92
0.07
38.04
1.31
0.15
0.03
0.09
0.06
0.05
23
24
3,4,5-trimethoxy
3,4-dimethoxy
4.60
0.04
4.05
0.05
12.29
14.56
0.04
6.53
5.72
> 80
> 80
0.03
25
26
3,4-dimethoxy
3,4-dimethoxy
7.08
7.73
0.05
0.05
> 80
> 80
13.55
24.93
0.04
0.06
14.51
6.79
0.05
0.05
0.15
27
4-methoxy
5.83
0.07
> 80
12.96
0.06
7.37
a
Antiproliferative activity was assayed by exposure for 48 h. Data are presented as the means
SDs from the dose-response curves of three independent
experiments.
4.3. General procedure for the synthesis of compounds 15–27 and 29–32
(dt, J = 11.8, 6.1 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 153.50,
145.80, 136.73, 134.86, 129.04, 127.17, 122.47, 116.14, 111.30,
103.31, 60.86, 56.10, 55.67, 49.83, 28.20, 22.46. HRMS (ESI): Calcd.
Formyl chloride derivatives (7 mmol) was added to the solution of
various arylamines (7 mmol) in acetone (25 ml). After stirring at 60 °C
for 6 h, the reaction mixture was concentrated to remove acetone. The
residue was purified by column chromatography with petroleum/ethyl
acetate (9:1) to afford the target compound 15–27 and 29–32.
C
19H24NO3, [M+H]+ m/z: 314.1756, found: 314.1759.
4.3.2. 1-(3,4,5-Trimethoxybenzyl)indoline (13)
White solid, m.p.: 71–72 °C, yield: 75%. 1H NMR (400 MHz, CDCl3)
δ 7.15–7.02 (m, 2H), 6.69 (t, J = 7.3 Hz, 1H), 6.60 (s, 2H), 6.53 (d,
J = 7.8 Hz, 1H), 4.17 (s, 2H), 3.84 (d, J = 4.2 Hz, 9H), 3.31 (t,
J = 8.3 Hz, 2H), 2.98 (t, J = 8.3 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ
152.31, 151.51, 135.93, 133.24, 129.09, 126.29, 123.53, 117.00,
106.31, 103.57, 59.84, 55.10, 53.32, 52.80, 27.53. HRMS (ESI): Calcd.
4.3.1. 1-(3,4,5-Trimethoxybenzyl)-1,2,3,4-tetrahydroquinoline (12)
White solid, m.p.: 62–63 °C, yield: 80%. 1H NMR (400 MHz, CDCl3)
δ 7.04–6.93 (m, 2H), 6.65–6.52 (m, 2H), 6.50 (s, 2H), 4.39 (s, 2H), 3.82
(d, J = 10.5 Hz, 9H), 3.41–3.26 (m, 2H), 2.82 (t, J = 6.3 Hz, 2H), 2.01
5

D.-J. Fu, et al.
BioorganicChemistry92(2019)103190
Table 3
Antiproliferative activity of the synthetic derivatives 29–32.
Compound
IC50 (μM)a
Hela
A549
HepG2
KYSE30
5FU
29
–
22.08
3.87
0.05
4.22
7.32
0.04
0.04
1.32
0.04
0.08
1.46
8.56
0.03
0.08
0.12
20.85
30
9.11
0.04
6.65
0.04
7.34
0.07
24.61
0.13
31
32
> 80
> 80
> 80
11.05
2.12
5.75
0.04
0.03
7.86
0.10
0.06
30.97
0.10
a
Antiproliferative activity was assayed by exposure for 48 h. Data are presented as the means
SDs from the dose-response curves of three independent
experiments.
C18H22NO3, [M+H]+ m/z: 300.1600, found: 300.1607.
46.75, 33.85, 28.65, 25.27. HRMS (ESI): Calcd. C20H24NO4, [M+H]+
m/z: 342.1705, found: 342.1709.
4.3.3. (3,4-Dihydroquinolin-1(2H)-yl)(3,4,5-trimethoxyphenyl)
methanone (15)
4.3.6. (5-Methoxy-1H-indol-1-yl)(3,4,5-trimethoxyphenyl)methanone
(18)
White solid, m.p.: 102–103 °C, yield: 60%. 1H NMR (400 MHz,
CDCl3) δ 7.06 (d, J = 7.3 Hz, 1H), 6.91 (t, J = 7.3 Hz, 1H), 6.82 (t,
J = 7.5 Hz, 1H), 6.66 (d, J = 7.8 Hz, 1H), 6.50 (s, 2H), 3.82 (t,
J = 6.5 Hz, 2H), 3.74 (s, 3H), 3.58 (s, 6H), 2.75 (t, J = 6.5 Hz, 2H),
2.06–1.85 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 168.60, 151.69,
138.80, 138.51, 130.65, 129.96, 127.16, 124.85, 124.34, 123.59,
105.48, 59.84, 55.01, 43.53, 25.94, 23.22. HRMS (ESI): Calcd.
Yellow liquid, yield: 65%. 1H NMR (400 MHz, CDCl3) δ 8.18 (d,
J = 9.0 Hz, 1H), 7.26 (d, J = 3.7 Hz, 1H), 6.98 (d, J = 2.5 Hz, 1H), 6.91
(dd, J = 9.1, 2.5 Hz, 1H), 6.89 (s, 2H), 6.47 (d, J = 3.7 Hz, 1H), 3.86 (s,
3H), 3.81 (s, 6H), 3.79 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 166.84,
155.71, 152.17, 140.21, 130.74, 129.74, 128.52, 127.18, 116.09,
112.38, 107.43, 105.79, 102.59, 59.98, 55.36, 54.67. HRMS (ESI):
Calcd. C19H20NO5, [M+H]+ m/z: 342.1341, found: 342.1347.
C
19H22NO4, [M+H]+ m/z: 328.1549, found: 328.1552.
4.3.4. Indolin-1-yl(3,4,5-trimethoxyphenyl)methanone (16)
4.3.7. (2-(Trifluoromethyl)-10H-phenothiazin-10-yl)(3,4,5-
White solid, m.p.: 105–106 °C, yield: 90%. 1H NMR (400 MHz,
CDCl3) δ 7.27 (s, 1H), 7.22 (d, J = 7.4 Hz, 1H), 7.12 (s, 1H), 7.02 (t,
J = 7.1 Hz, 1H), 6.78 (s, 2H), 4.12 (t, J = 8.0 Hz, 2H), 3.88 (d,
J = 14.8 Hz, 9H), 3.13 (t, J = 8.3 Hz, 2H). 13C NMR (100 MHz, CDCl3)
δ 168.58, 153.38, 142.51, 139.78, 132.42, 132.20, 127.24, 124.96,
123.95, 123.89, 104.55, 60.98, 56.29, 50.45, 28.06. HRMS (ESI): Calcd.
trimethoxyphenyl)methanone (19)
White solid, m.p.: 159–160 °C, yield: 43%. 1H NMR (400 MHz,
DMSO) δ 7.91 (s, 1H), 7.83 (d, J = 8.2 Hz, 1H), 7.72–7.55 (m, 2H), 7.38
(dd, J = 6.4, 2.7 Hz, 1H), 7.35–7.21 (m, 2H), 6.60 (s, 2H), 3.63 (s, 3H),
3.59 (s, 6H). 13C NMR (100 MHz, DMSO) δ 167.34, 152.30, 139.36,
138.98, 138.53, 136.90, 130.19, 129.11, 128.51, 127.76, 127.66,
127.36, 127.12, 127.05, 125.05, 123.54, 123.23, 122.34, 106.25,
C
18H20NO4, [M+H]+ m/z: 314.1392, found: 314.1398.
60.07, 55.78. HRMS (ESI): Calcd.
462.0987, found: 462.0989.
C
23H19F3NO4S, [M+H]+ m/z:
4.3.5. (2,3,4,5-tetrahydro-1H-benzo[b]azepin-1-yl)(3,4,5-
trimethoxyphenyl)methanone (17)
White solid, m.p.: 144–145 °C, yield: 50%. 1H NMR (400 MHz,
CDCl3) δ 7.16 (d, J = 7.5 Hz, 1H), 7.02 (t, J = 7.4 Hz, 1H), 6.89 (t,
J = 7.5 Hz, 1H), 6.60 (d, J = 7.7 Hz, 1H), 6.38 (s, 2H), 4.95 (d,
J = 13.2 Hz, 1H), 3.69 (s, 3H), 3.52 (s, 6H), 2.97 (t, J = 13.0 Hz, 1H),
2.82 (dd, J = 13.6, 4.4 Hz, 1H), 2.71 (t, J = 10.1 Hz, 1H), 2.02 (d,
J = 11.5 Hz, 1H), 1.89 (dd, J = 11.8, 7.5 Hz, 2H), 1.45 (d, J = 11.5 Hz,
1H). 13C NMR (100 MHz, CDCl3) δ 167.16, 151.25, 143.46, 138.14,
138.01, 129.72, 128.78, 127.15, 126.31, 126.20, 105.23, 59.78, 54.85,
4.3.8. (5-Bromoindolin-1-yl)(3,4,5-trimethoxyphenyl)methanone (20)
White solid, m.p.: 126–127 °C, yield: 31%. 1H NMR (400 MHz,
DMSO) δ 7.89 (s, 1H), 7.48 (s, 1H), 7.37 (d, J = 7.9 Hz, 1H), 6.90 (s,
2H), 4.06 (t, J = 8.4 Hz, 2H), 3.82 (d, J = 8.0 Hz, 6H), 3.73 (d,
J = 4.9 Hz, 3H), 3.10 (t, J = 8.3 Hz, 2H). 13C NMR (100 MHz, DMSO) δ
168.29, 153.28, 142.63, 139.31, 136.21, 132.46, 130.03, 128.30,
115.66, 107.00, 105.02, 60.57, 56.58. HRMS (ESI): Calcd.
C
18H19BrNO4, [M+H]+ m/z: 392.0497, found: 392.0499.
6

D.-J. Fu, et al.
BioorganicChemistry92(2019)103190
120
100
80
60
40
20
0
A
120
100
80
60
40
20
0
Kyse450
Kyse30
3
0
5
5
5
5
0
0 0
4
0
8
O
0
.
2
1
3
2
6
2
.
1
1
2
3
0
5
5
5
.
2
5
0
0
0
0
S
O
0
1
3
2
2
.
1
2
4
8
.
D
. Concentration (
)
S
M
6
.
.
1
D
M
Concentration (
Kyse510
)
120
100
80
60
40
20
0
120
100
80
60
40
20
0
Ec109
3
0
5
1
5
5
.
2
5
0
0 0
4
0
8
O
3
0
5
5
5
.
2
5
0
0
0
0
8
1
3
2
2
.
1
2
O
S
1
3
2
2
.
1
1
2
4
6
.
S
.
6
.
M
.
0
M
0
D
D
Concentration (
)
B
Kyse30
Kyse450
Kyse510
Ec109
400
300
200
100
0
400
300
200
100
0
160
120
80
40
0
250
200
150
100
50
0
5
0
0
3
(µM)
1
(µM)
(µM)
3
1
(µM)
3
1
O
5
0
0
5
0
0
O
2
5
5
0
0
3
O
S
1
O
2
5
3
.
0
S
2
5
2
5
1
S
1
2
.
3
.
0
1
2
.
.
S
2
3
.
0
1
.
2 3
.
0
.
M
.
.
.
M
0
M
0
M
0
D
0
D
D
D
Concentration
Concentration
Concentration
Concentration
DMSO
0.125 µM
0.313 µM
DMSO
0.125 µM
0.313 µM
DMSO
0.125 µM
0.313 µM
DMSO
0.125 µM
0.313 µM
0.250 µM
0.250 µM
0.250 µM
0.250 µM
P=0.047
2.0
1.5
1.0
0.5
0.0
D
C
1 cm
Control #20
Fig. 3. A. Cell viabilities of four ESCC cell lines after 48 h compound 20 treatment; B. 4 ESCC cell lines colonies formed after 7 days compound 20 treatment; C&D.
Tumors and tumors’ weight after 30 days compound 20 treatment.
4.3.9. (2-Methylindolin-1-yl)(3,4,5-trimethoxyphenyl)methanone (21)
White solid, m.p.: 102–103 °C, yield: 23%. 1H NMR (400 MHz,
CDCl3) δ 7.13 (d, J = 7.1 Hz, 1H), 6.92 (dt, J = 15.5, 7.7 Hz, 3H), 6.66
(s, 2H), 4.77–4.63 (m, 1H), 3.81 (s, 3H), 3.75 (s, 6H), 3.34 (dd,
J = 15.7, 8.8 Hz, 1H), 2.56 (dd, J = 15.7, 1.1 Hz, 1H), 1.21 (d,
J = 6.3 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 168.31, 153.39, 141.29,
139.71, 132.18, 131.51, 127.01, 125.48, 123.78, 116.78, 104.67,
60.96, 57.13, 56.28, 35.84, 20.84. HRMS (ESI): Calcd. C19H22NO4, [M
+H]+ m/z: 328.1549, found: 328.1554.
(s, 3H), 3.64 (s, 6H), 2.77 (t, J = 6.6 Hz, 2H), 2.21 (s, 3H), 1.92 (p,
J = 6.5 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 168.53, 151.66, 138.62,
135.89, 133.24, 130.38, 130.17, 127.61, 125.58, 124.11, 105.35,
59.89, 55.00, 43.56, 25.88, 23.24, 19.77. HRMS (ESI): Calcd.
C
20H24NO4, [M+H]+ m/z: 342.1705, found: 342.1708.
4.3.11. (7-Nitro-3,4-dihydroquinolin-1(2H)-yl)(3,4,5-trimethoxyphenyl)
methanone (23)
White solid, m.p.: 129–130 °C, yield: 21%. 1H NMR (400 MHz,
DMSO) δ 8.21 (s, 1H), 7.94 (dd, J = 8.4, 2.4 Hz, 1H), 7.54 (d,
J = 8.5 Hz, 1H), 6.85 (s, 2H), 3.83–3.80 (m, 2H), 3.79 (d, J = 5.1 Hz,
6H), 3.74 (s, 3H), 3.01 (t, J = 6.6 Hz, 2H), 2.09–1.93 (m, 2H). 13C NMR
(100 MHz, DMSO) δ 170.24, 153.24, 145.51, 139.65, 139.61, 138.86,
131.47, 130.50, 119.52, 118.71, 105.97, 60.60, 56.55, 46.44, 27.15,
4.3.10. (6-Methyl-3,4-dihydroquinolin-1(2H)-yl)(3,4,5-
trimethoxyphenyl)methanone (22)
White solid, m.p.: 92–93 °C, yield: 37%. 1H NMR (400 MHz, DMSO)
δ 7.01 (s, 1H), 6.77 (s, 2H), 6.62 (s, 2H), 3.71 (t, J = 6.4 Hz, 2H), 3.67
7

D.-J. Fu, et al.
BioorganicChemistry92(2019)103190
A
B
Kyse30
Kyse450
DMSO
0.313 µM
DMSO
0.313 µM
1.25 µM
2.5 µM
1.25 µM
2.5 µM
Kyse30
Kyse450
**
**
90
80
70
60
50
40
30
20
10
0
90
80
70
60
50
40
30
20
10
0
G0/G1
S
G2/M
G0/G1
S
G2/M
2.5 µM
DMSO
0.313 µM
1.25 µM
C
Kyse30
0 0.313 1.25 2.5
Kyse450
0 0.313 1.25 2.5
(µM)
Compd. 20
Wee1
p-Wee1 (Ser642)
Cyclin B1
p-Histone H3 (Ser10)
GAPDH
Fig. 4. A&B. Two cell lines were treated with compound 20 as shown in this figure for 48 h, and cell cycle phases’ percentage were analyzed; C. The change of cell
cycle related proteins in cells after 48 h treatment by compound 20.
22.97. HRMS (ESI): Calcd. C19H21N2O6, [M+H]+ m/z: 373.1400,
1.99 (p, J = 6.6 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 168.84, 149.64,
147.30, 138.84, 130.63, 127.24, 127.18, 124.80, 124.42, 123.36,
121.45, 111.19, 109.03, 54.85, 54.75, 43.40, 25.98, 23.25. HRMS
(ESI): Calcd. C18H20NO3, [M+H]+ m/z: 298.1443, found: 298.1447.
found: 373.1406.
4.3.12. (3,4-Dihydroquinolin-1(2H)-yl)(3,4-dimethoxyphenyl)methanone
(24)
White solid, m.p.: 117–118 °C, yield: 30%. 1H NMR (400 MHz,
CDCl3) δ 7.08 (d, J = 7.4 Hz, 1H), 6.92 (t, J = 7.4 Hz, 1H), 6.88–6.84
(m, 2H), 6.81 (t, J = 7.7 Hz, 1H), 6.63 (dd, J = 8.3, 4.9 Hz, 2H), 3.84 (t,
J = 6.6 Hz, 2H), 3.79 (s, 3H), 3.65 (s, 3H), 2.76 (t, J = 6.6 Hz, 2H),
4.3.13. (3,4-Dimethoxyphenyl)(2-methylindolin-1-yl)methanone (25)
White solid, m.p.: 130–131 °C, yield: 27%. 1H NMR (400 MHz,
CDCl3) δ 7.13 (d, J = 7.0 Hz, 1H), 7.08–6.93 (m, 3H), 6.91 (dd,
J = 16.4, 9.1 Hz, 2H), 6.80 (d, J = 8.2 Hz, 1H), 4.87–4.59 (m, 1H), 3.86
8

D.-J. Fu, et al.
BioorganicChemistry92(2019)103190
A
DMSO
0.313 µM
1.25 µM
2.5 µM
60
50
40
30
20
10
0
Kyse30
DMSO
Kyse450
0.313 µM
2.5 µM
1.25 µM
Annexin V-FITC
B
DMSO
0.313 µM
1.25 µM
2.5 µM
50
40
30
20
10
0
Kyse30
DMSO
Kyse450
0.313 µM
2.5 µM
FITC-DEVD-FMK
Kyse30
1.25 µM
C
Kyse450
0 0.313 1.25 2.5
(µM)
Compd. 20
0 0.313 1.25 2.5
c-CASP9
c-CASP3
c-PARP
GAPDH
Fig. 5. A. The activity of compound 20 to induce apoptosis after 72 h treatment; Representative images were shown (left panel). The early apoptotic (Annexin V-FITC
positive) and late apoptotic (Annexin V-FITC positive, PI positive) were quantified as apoptotic cells. The percentage of apoptotic cells were shown in right panel. B.
The activity of compound 20 to activate Caspase-3 after 72 h treatment; Representative images were shown (left panel). The percentage of cells with active caspase 3
was shown in right panel. C. The change of cleaved-Caspase 9, cleaved-Caspase-3, cleaved PARP in cells after 72 h treatment by compound 20.
(s, 3H), 3.80 (s, 3H), 3.34 (dd, J = 15.3, 8.9 Hz, 1H), 2.57 (dd,
J = 15.7, 1.4 Hz, 1H), 1.21 (d, J = 6.3 Hz, 3H). 13C NMR (100 MHz,
CDCl3) δ 167.54, 149.71, 147.93, 140.65, 130.52, 128.27, 125.92,
124.44, 122.50, 119.56, 115.70, 109.82, 109.58, 56.27, 55.01, 54.96,
34.87, 19.68. HRMS (ESI): Calcd. C18H20NO3, [M+H]+ m/z: 298.1443,
found: 298.1446.
6.81 (dd, J = 11.2, 4.2 Hz, 1H), 6.73–6.65 (m, 2H), 6.62 (d, J = 8.0 Hz,
1H), 3.82 (dd, J = 8.0, 5.2 Hz, 2H), 3.71 (s, 3H), 2.76 (t, J = 6.6 Hz,
2H), 1.97 (p, J = 6.6 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 168.94,
160.06, 138.80, 130.52, 129.75, 127.30, 127.27, 124.73, 124.39,
123.28, 112.24, 54.24, 43.38, 25.96, 23.21. HRMS (ESI): Calcd.
C
17H18NO2, [M+H]+ m/z: 268.1338, found: 268.1339.
4.3.14. (3,4-Dimethoxyphenyl)(6-methyl-3,4-dihydroquinolin-1(2H)-yl)
methanone (26)
4.3.16. (2-Methylindolin-1-yl)(thiophen-2-yl)methanone (29)
Yellow liquid, yield: 72%. 1H NMR (400 MHz, DMSO) δ 8.00–7.72
(m, 2H), 7.71 (d, J = 3.7 Hz, 1H), 7.32 (d, J = 7.3 Hz, 1H), 7.25–7.13
(m, 2H), 7.08 (t, J = 7.4 Hz, 1H), 5.15–4.91 (m, 1H), 3.48 (d,
White solid, m.p.: 109–110 °C, yield: 43%. 1H NMR (400 MHz,
CDCl3) δ 6.95–6.80 (m, 3H), 6.68–6.57 (m, 2H), 6.50 (d, J = 8.2 Hz,
1H), 3.82 (t, J = 6.6 Hz, 2H), 3.79 (s, 3H), 3.66 (s, 3H), 2.72 (t,
J = 6.6 Hz, 2H), 2.18 (s, 3H), 1.96 (p, J = 6.6 Hz, 2H). 13C NMR
(100 MHz, CDCl3) δ 169.73, 150.56, 148.31, 137.28, 133.99, 131.38,
128.69, 128.47, 126.55, 125.19, 122.38, 112.22, 110.05, 55.87, 55.78,
44.43, 26.93, 24.29, 20.81. HRMS (ESI): Calcd. C19H22NO3, [M+H]+
m/z: 312.1600, found: 312.1605.
J = 8.7 Hz, 1H), 2.71 (d, J = 15.9 Hz, 1H), 1.19 (d, J = 6.4 Hz, 3H). 13
C
NMR (100 MHz, DMSO) δ 161.31, 142.18, 139.22, 131.80, 131.16,
129.89, 128.25, 127.46, 125.90, 124.64, 117.71, 57.05, 36.37, 21.73.
HRMS (ESI): Calcd. C14H13NOS, [M+H]+ m/z: 243.0718, found:
243.0719.
4.3.17. (5-Bromoindolin-1-yl)(thiophen-2-yl)methanone (30)
4.3.15. (3,4-Dihydroquinolin-1(2H)-yl)(4-methoxyphenyl)methanone
(27)
White solid, m.p.: 118–119 °C, yield: 32%. 1H NMR (400 MHz,
DMSO) δ 7.97 (d, J = 8.5 Hz, 1H), 7.88 (dd, J = 5.0, 0.9 Hz, 1H),
7.81–7.65 (m, 1H), 7.49 (s, 1H), 7.38 (d, J = 8.6 Hz, 1H), 7.22 (dd,
J = 5.0, 3.8 Hz, 1H), 4.43 (t, J = 8.4 Hz, 2H), 3.21 (t, J = 8.3 Hz, 2H).
White solid, m.p.: 84–85 °C, yield: 54%. 1H NMR (400 MHz, CDCl3)
δ 7.25 (d, J = 8.8 Hz, 2H), 7.07 (d, J = 7.2 Hz, 1H), 6.95–6.86 (m, 1H),
9

D.-J. Fu, et al.
BioorganicChemistry92(2019)103190
Fig. 6. A. Mitochondrial membrane po-
tential change of both cells after 24 h
compound 20 treatment; Representative
images were shown in left panel;
Percentage of cells that had lost mi-
tochondrial membrane potential is
shown in right panel. B. The change of
mitochondrial related proteins in cells
after 72 h treatment by compound 20; C
&D. Downregulation of Noxa reduced
A
DMSO
0.313 µM
1.25 µM
2.5 µM
60
50
40
30
20
10
0
Kyse30
DMSO
1.25 µM
Kyse450
0.313 µM
2.5 µM
compound
20-induced
apoptosis.
Kyse30 and Kyse450 cells were trans-
fected with control siRNA or Noxa
siRNA and then treated with DMSO or
1.25 μmol/L compound 20 for 48 h.
Apoptosis induction was quantified by
Annexin V-FITC/PI double-staining
analysis. Knockdown efficiency and
cleaved PARP were assessed by western
blot assay.
JC-1 monomers
Kyse450
B
Kyse30
Kyse450
Kyse30
(µM)
0 0.313 1.25 2.5 0 0.313 1.25 2.5
Compd. 20 0 0.313 1.25 2.5 0 0.313 1.25 2.5
Bax
Bcl-xl
Bcl-2
Bak
Bid
Mcl-1
XIAP
Bim
GAPDH
Noxa
C
D
Kyse30
Kyse450
Kyse30
Kyse450
siControl
+
-
-
-
-
+
-
-
-
-
+
-
+
-
-
+
-
+
-
+
-
-
+
+
+
-
-
-
-
+
-
-
-
-
+
-
+
-
-
+
-
-
-
+
+
**
30
20
10
0
30
20
10
**
+
-
+
siNoxa-1
siNoxa-2
Compd. 20
Noxa
0
c-PARP
Control #20
Control #20
siNoxa-2 GAPDH
siControl
siNoxa-1
13C NMR (100 MHz, DMSO) δ 161.21, 142.99, 139.62, 135.92, 132.07,
130.92, 130.11, 128.42, 128.22, 118.99, 115.93, 50.80, 28.40. HRMS
(ESI): Calcd. C13H11BrNOS, [M+H]+ m/z: 307.9745, found: 307.9748.
were cultured in an atmosphere with 5% CO2 at 37 °C, using RPMI-1640
with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 0.1 mg/ml
streptomycin as culture medium.
4.3.18. Indolin-1-yl(thiophen-2-yl)methanone (31)
White solid, m.p.: 95–96 °C, yield: 14%. 1H NMR (400 MHz, DMSO)
δ 8.03 (d, J = 7.8 Hz, 1H), 7.87 (d, J = 5.0 Hz, 1H), 7.75 (d, J = 3.7 Hz,
1H), 7.29 (d, J = 7.3 Hz, 1H), 7.21 (dt, J = 13.0, 6.3 Hz, 2H), 7.06 (t,
J = 7.4 Hz, 1H), 4.41 (t, J = 8.3 Hz, 2H), 3.20 (t, J = 8.2 Hz, 2H). 13C
NMR (100 MHz, CDCl3) δ 160.48, 142.06, 138.43, 130.81, 129.10,
128.75, 126.46, 126.16, 123.64, 123.21, 116.74, 49.62, 27.66. HRMS
(ESI): Calcd. C13H12NOS, [M+H]+ m/z: 230.0640, found: 230.0647.
4.5. CCK8 assay
Cells were seeded in 96-well plates at a density of 3 × 103 per well.
After 24 h incubation, they were treated with different concentrations
of compound as shown in the Figures and Tables. After 48 h, 5 μL CCK8
solution was added to each well, and co-incubated for 4 h at 37 °C. The
absorbance was determined at 450 nm. Data are calculated with
GraphPad Prism5 software.
4.3.19. (3,4-Dihydroquinolin-1(2H)-yl)(thiophen-2-yl)methanone (32)
White solid, m.p.: 73–74 °C, yield: 55%. 1H NMR (400 MHz, DMSO)
δ 7.72 (dd, J = 5.0, 1.1 Hz, 1H), 7.24 (d, J = 7.2 Hz, 1H), 7.11–7.03 (m,
2H), 7.03–6.96 (m, 3H), 3.81 (t, J = 6.5 Hz, 2H), 2.77 (t, J = 6.6 Hz,
2H), 1.94 (p, J = 6.6 Hz, 2H). 13C NMR (100 MHz, DMSO) δ 163.17,
139.39, 138.78, 132.69, 131.37, 131.09, 128.88, 127.49, 126.09,
125.52, 125.39, 45.22, 26.54, 24.30. HRMS (ESI): Calcd. C14H14NOS,
[M+H]+ m/z: 244.0796, found: 244.0798.
4.6. Colony formation assay
Kyse30, Kyse450, Kyse510 and EC109 cells were seeded in a 6-well
plate at a density of 5 × 102 per well and incubated at 37 °C in 5% CO2
for 24 h, then they were treated with different concentrations of com-
pound 20. After 7 days, the culture medium was removed, and the cells
were washed with PBS and fix with 4% paraformaldehyde. Then stain
the cells with 0.1% crystal violet. The images were captured with
camera. Colonies comprising 50 cells or more were counted.
4.4. Cell culture
Hela, A549, HepG2, Kyse30, Kyse450, Kyse510, EC109 cell lines
10

D.-J. Fu, et al.
BioorganicChemistry92(2019)103190
4.7. Cell cycle distribution assay
4.13. Subcutaneous transplantation tumor model of human esophageal
cancer and treatment
Kyse30 and Kyse450 cells were seeded in 6-well culture plate. After
24 h incubation, cells were treated with certain concentration of com-
pound 20 for 24 h. Then cells were harvested and fixed at 4 °C with 70%
ethanol. The fixed cells were washed twice and resuspended using PBS
with 10 mg/mL RNaseA and 50 mg/mL PI incubated for 30 min in the
dark. Samples were analyzed for DNA content with flow cytometry then
(Becton, Dickinson and Company, NJ). Data were analyzed with ModFit
LT software (Verity).
A subcutaneous transplantation tumor model of human esophageal
cancer was established using Kyse450 esophageal cancer cells. The
tumor-bearing mice were randomized into 2 groups and subcutaneous
injection with normal saline or compound 20 (150 mg/kg) continuously
for 30 days (compound 20 was firstly resolved with the mixture of ethyl
alcohol and castor oil, and then diluted in normal saline). Tumor size
was determined by caliper measurement. The tumor volume was cal-
culated using the ellipsoid volume formula (Length × Width2/2).
Tumor tissues were harvested, photographed, and weighed. Animal
experiments were performed in accordance with animal protocols ap-
proved by the Institutional Animal Care and Use Committee of
Zhengzhou University.
4.8. Cell apoptosis assay
Kyse30 and Kyse450 cells were seeded in 6-well culture plate and
treated with certain concentration of compound 20 as shown in Figures
for 72 h. Cells were harvested and suspended in binding buffer con-
taining Annexin V-FITC (0.5 mg/mL) and PI (0.5 mg/mL) then. After
that, samples were incubated for 25 min in dark and analyzed with flow
cytometry (Becton, Dickinson and Company, NJ). The activities of
caspase-3 were quantitated by CaspGLOW Fluorescein Active Caspase-3
Staining Kit (BioVision, Inc. Milpitas, California) according to the
manufacturer's instructions.
4.14. Statistical analysis
Data from three independent experiments are presented as
mean
SD. IC50 values and Student’s t-tests were calculated by
GraphPad Prism5 software. ** Respectively represent p < 0.01.
4.15. Abbreviations
4.9. Activity assays of caspase-3
Structure-activity relationships
Esophageal squamous cell carcinoma
Histone deacetylase 6
SARs
ESCC
HDAC6
HSP90
A549
HCT116
HL60
MKN45
H460
HT29
PERK
Hela
1.5 × 105 (Kyse30) or 1 × 105 (Kyse450) cells were seeded per well
in 6-well plates. After 24 h, the cells were maintained with the indicated
concentration of compound 20 for 72 h. The cells were collected and
washed by PBS, and then resuspended with wash buffer supplied with
the kit (Bio Vision, Inc. Milpitas, California).1µL of FITC-DEVD-FMK
was added into tubes above-mentioned. And the cells were incubated
for 1 h at 37℃. Then cells were centrifuged at 3000 rpm for 5 min and
washed. Finally, the cells were resuspended in 0.5 ml of wash buffer on
ice, then analyzed by flow cytometry (Becton Dickinson FACScan;
Becton-Dickinson, San Jose, CA, USA).
Heat shock protein 90
Human lung carcinoma cell line
Human colon cancer cell line
Human leukemia cell line
Human gastric carcinoma cell
Human lung carcinoma cell line
Human colon cancer cell line
Protein kinase R-like endoplasmic reticulum kinase
Human cervical cancer cell line
Human liver cancer cell line
Human esophagus cancer cell line
5-Fluorouracil
HepG2
KYSE30
5Fu
N, N′-ethylenebis (iodoacetamide)
Cyclin-dependent kinase 1
EBI
4.10. Western blot analysis
CDK1
PARP
MMP
Poly ADP-ribose polymerase
Mitochondrial membrane potential
B-cell lymphoma-2
Treated and untreated cells were harvested and lysed. Cell extracts
were denatured by loading buffer and resolved by SDS-PAGE and
transferred to PVDF membrane. The membranes were incubated with
appropriate primary antibodies at 4 °C for 10 h after blocking with 5%
skimmed milk. After conjugated with secondary antibodies, the detec-
tion of proteins was carried out with an ECL kit.
Bcl-2
X-linked inhibitor of apoptosis protein
Myeloid cell leukemia-1
XIAP
MCl-1
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (Project No. 81703541, 81001102, 81101894,
81672421), China Postdoctoral Science Foundation, Natural Science
Foundation of Henan Province (Grant No. 162300410302),
Outstanding Young Talent Research Fund of Zhengzhou University
4.11. Measurement of mitochondrial membrane potential (MMP, ΔΨ)
JC-1 probe was used as a measure of loss of MMP. Cells were seeded
at 1.5 × 105 into 6-well plates and treated with certain concentrations
of compound 20 for 24 h. Then JC-1 (2.5 μg/ml), were added and co-
incubate with an equal volume of cell suspension at 37 °C for 15 min.
Then, cells were washed with PBS. Then samples were analyzed with
flow cytometry (Becton, Dickinson and Company, NJ).
(Grant No. 51999223, 32210449) and Program for Science
&
Technology Innovation Talents in Universities of Henan Province
(Grant No. 18HASTIT046).
Appendix A. Supplementary material
4.12. Gene silencing using small interfering RNA (siRNA)
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2019.103190.
Kyse30 and Kyse450 cells were seeded at a density of 5 × 105 cells
into 60-mm dishes and transfected with siRNA oligonucleotides (final
concentration: 100 nmol/L), synthesized by GenePharma (Shanghai,
China) using Lipofectamine 2000 (Cat. No. 11668019, Thermo Fisher
Scientific). Cells were transfected with siRNA for 48 h and further
treated with 1.25 μmol/L compound 20 for 48 h. The sequences of the
siRNA are as follows: siNOXA-1: GUAAUUAUUGACACAUUUC;
siNOXA-2: GGUGCACGUUUCAUCAAUUUG; siControl: UUCUCCG
AACGUGUCACGU.
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