Design, synthesis and biological evaluation of tryptamine salicylic acid derivatives as potential antitumor agents

Article abstract of DOI:10.1039/c8md00484f

A series of tryptamine salicylic acid derivatives were synthesized and their antiproliferative activity against MGC-803, MCF-7, HepG2, A549 and HeLa cell lines was evaluated. The structure-activity relationship (SAR) study revealed that different substitutions of the C5 and C3′-C5′ positions have certain effects on the anti-proliferation activity. The growth assay revealed that N-[2-(5-bromo-1H-indol-3-yl)-ethyl]-2-hydroxy-3-methyl-benzamide (E20) showed the most potent and broad-spectrum anticancer inhibition of all the cell lines evaluated, and was only more potent than 5-Fu for the gastric cancer cell line. Preliminary studies indicated that compound E20 could inhibit colony formation and migration of MGC-803 cells. The flow cytometry (FCM) results showed that compound E20 arrested the cell cycle in the G2/M phase and induced apoptosis of MGC-803 cells in a concentration-dependent manner. In addition, the western blot results showed that E20 can down-regulate the expression of hexokinase 2. Our studies suggest that the framework of N-[2-(5-bromo-1H-indol-3-yl)-ethyl]-2-hydroxy-3-methyl-benzamide may be consider as a new type of chemical for designing effective anti-cancer drugs targeting gastric cancer cells.

Full text of DOI:10.1039/c8md00484f

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RESEARCH ARTICLE
Design, synthesis and biological evaluation of
tryptamine salicylic acid derivatives as potential
Cite this: DOI: 10.1039/c8md00484f
antitumor agents†
Runde Xiong,‡a Dongxiu He,‡a Xiangping Deng,a Juan Liu,a Xiaoyong Lei,a
a
Zhizhong Xie,a Xuan Cao,a Yanming Chen,b Junmei Penga and Guotao Tang
*
A series of tryptamine salicylic acid derivatives were synthesized and their antiproliferative activity against
MGC-803, MCF-7, HepG2, A549 and HeLa cell lines was evaluated. The structure–activity relationship (SAR)
study revealed that different substitutions of the C5 and C3′–C5′ positions have certain effects on the anti-
proliferation activity. The growth assay revealed that N-[2-(5-bromo-1H-indol-3-yl)-ethyl]-2-hydroxy-3-
methyl-benzamide (E20) showed the most potent and broad-spectrum anticancer inhibition of all the cell
lines evaluated, and was only more potent than 5-Fu for the gastric cancer cell line. Preliminary studies in-
dicated that compound E20 could inhibit colony formation and migration of MGC-803 cells. The flow cy-
tometry (FCM) results showed that compound E20 arrested the cell cycle in the G2/M phase and induced
apoptosis of MGC-803 cells in a concentration-dependent manner. In addition, the western blot results
showed that E20 can down-regulate the expression of hexokinase 2. Our studies suggest that the frame-
work of N-[2-(5-bromo-1H-indol-3-yl)-ethyl]-2-hydroxy-3-methyl-benzamide may be consider as a new
type of chemical for designing effective anti-cancer drugs targeting gastric cancer cells.
Received 27th September 2018,
Accepted 5th January 2019
DOI: 10.1039/c8md00484f
rsc.li/medchemcomm
burg effect) in human leiomyosarcoma,10 and others consid-
ered it to be an angiogenesis inhibitor which is used to com-
1. Introduction
Cancer is a common disease that does great harm to human
health. There is an exigent need for high efficiency and low
toxicity anticancer drugs that will improve the cure rate of
cancer. Melatonin (N-acetyl-5-methoxytryptamine) is a kind of
indole hormone which is secreted mainly from the pineal
gland. However, it is discovered in various other tissues such
as the gastrointestinal tract, retina, lens, skin, testes, lympho-
cytes and hematopoietic cells.1 As a ubiquitous molecule,
melatonin is involved in the regulation of many biological
functions2–5 and protection against a spectrum of cancers
such as breast cancer,6 gastric cancer7 and liver cancer.8
Moreover, the mechanisms which have been investigated
demonstrated that melatonin could induce cell apoptosis and
inhibit proliferation in gastric cancer cells,9 and western blot
analysis showed that melatonin could mediate the
upregulation of apoptotic proteins Bax/Bcl-2 and cleaved-
caspase 3 in gastric cancer cells.7 Besides, some researchers
reported that melatonin could inhibit aerobic glycolysis (War-
bat cancer.11 Melatonin showed broad antitumor activities
through various mechanisms. Previous studies have focused
on the anti-tumor activity of melatonin itself; however, there
were fewer studies on the anti-tumor activity of melatonin
derivatives.12–14 This has driven us to transform it into a new
anti-tumor agent.
Salicylic acid (SA) and acetylsalicylic acid (ASA) are well
known as anti-inflammatory drugs, but they also showed
anti-tumor activity,15 induction of apoptosis16,17 and modifi-
cation of tumor glucose utilization.18 Studies have discovered
a Institute of Pharmacy and Pharmacology, Hunan Province Cooperative
Innovation Center for Molecular Target New Drug Study, University of South
China, Hengyang, China. E-mail: tgtzq@163.com
b Jiuzhitang Co. Ltd, Changsha, China
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c8md00484f
Fig. 1 Design of novel tryptamine salicylic acid derivatives as anti-
‡ These authors contributed equally to this work.
cancer agents.
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that the anti-tumor activity of SA and ASA was produced by
acting on cyclooxygenase (COX)16,19 and 6-phosphofructo-1-
kinase (PFK).18 SA and ASA could be used as anti-tumor
agents.
antitumor activity, especially on MGC-803 cells. We summed
the IC50 values of each compound (the IC50 value greater than
100 is converted to 100) and made a histogram. It is shown
in Fig. 3 that different substituents on C5 had a great effect
on the antitumor activity of the compounds, and the differ-
ent substituents on C3′–C5′ also affected the antitumor activ-
ity. According to the histogram, the structure–activity rela-
tionship (SAR) of the tryptamine salicylic acid derivatives
E1–E36 was proposed as shown in Fig. 1. The non-
substituted compounds on C5 were inactive and the different
substitutions on C5 had different activity intensities. Bro-
mine and chlorine had the best activity at the C5 position,
followed by methoxy and methyl groups, and finally fluorine.
When the same substitution occurred at the C5 position of
the compounds, the comparison showed that the methyl sub-
stitution of C3′ was the best substitution.
Although both melatonin and acetylsalicylic acid have
antitumor activity, these two drugs show antitumor effects
only in the millimolar range.4,11 In order to obtain a novel
small molecular structure with better anti-tumor activity, mel-
atonin is combined with salicylic acid to form new chemo-
types, and is modified as shown in Fig. 1. In this study, a se-
ries of tryptamine salicylic acid derivatives have been synthe-
sized based on the above structure and evaluated for anti-
tumor activity in vitro. Since both have an inhibitory effect on
the glycolytic pathway, we investigated whether the most po-
tent drug synthesized (E20) has an effect on the expression of
three glycolysis-related proteins.
Among these compounds, compound E20 showed the
most potent inhibition of the tested cancer cells with IC50
values below 70 μM, but exhibited values greater than that
observed for 5-Fu at 50–70 μM. There was one exception – the
gastric cancer cell line, MGC-803, which exhibited an IC50 of
29 μM (cf. 58 μM for 5-Fu). Given the acceptable anti-
proliferative activity of compound E20 toward MGC-803 cells,
the possible toxicity against the normal human gastric epi-
thelial cell line GES-1 was also preliminarily examined. Com-
pound E20 showed IC50 values of 75 2 μM. It was obvious
that the toxicity of E20 to normal human gastric epithelial
cells was significantly lower than that to gastric cancer cells.
Because E20 showed certain selectivity between normal and
cancer cells, our studies envisioned that the framework of N-
[2-(5-bromo-1H-indol-3-yl)-ethyl]-2-hydroxy-3-methyl-benzamide
(E20) might be considered as a new type of chemical for de-
signing effective anti-cancer drugs targeting gastric cancer
cells. Further structural modifications based on such a scaf-
fold and mechanistic investigations are ongoing in our lab.
We used MOLSOFT online molecular computing service to
predict its molecular characteristics (HTTP://MLSOF.COM/).
2. Results and discussion
2.1 Chemistry
As shown in Scheme 1, all the compounds were adopted to
this synthetic pathway. As shown in Fig. 2, 36 compounds
were synthesized and purified by column chromatography.
These compounds were obtained in good yields after
depuration. All the compounds were characterized by nuclear
magnetic resonance-hydrogen spectrum, nuclear magnetic
resonance-carbon spectrum and mass spectrometry.
2.2 Antiproliferative activity and SAR discussion
All the compounds were evaluated for their antiproliferative
activity in five cell lines namely the human breast cancer cell
line (MCF-7), human lung cancer cell line (A549), human
liver cancer cell line (HepG2), human gastric cancer cell line
(MGC-803) and human cervical cancer cell line (HeLa) by the
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide) assay.20 5-Fu was used as a positive control. As shown
in Table 1, most of the synthesized compounds have good
Scheme 1 Reagents and conditions: (a) NaNO2, HCl, Na2SO3, reflux 2 h, yield 90–95%; (b) 4-Cl-1-OH-1-butanesulfonate, Na2HPO4, CH3CH2OH
reflux 6 h, yield 50–65%; (c) EDCI, HOBT, acetone, room temperature, yield 70–80%.
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Fig. 2 The tryptamine salicylic acid derivatives with different substituents.
As shown in Table 2, compound E20 showed acceptable mo-
lecular properties and might have acceptable pharmacoki-
netic properties.
shown in Fig. 4B, the wounded area was almost completely
healed in the DMSO-treated control after 48 h, while com-
pound E20 inhibited the healing of the wounded area in a
concentration-dependent manner.
2.3 Colony formation assay of E20 on MGC-803 cells
2.5 Effect of compound E20 on apoptosis in MGC-803 cells
To prove the anti-proliferative activity of compound E20, the
colony formation assay was used to test the effect of com-
pound E20 on MGC-803 cells. Cells were treated with differ-
ent concentrations (10, 20 and 40 μM) of E20. As shown in
Fig. 4A, the treatment of E20 significantly reduced the num-
ber of viable cells in a concentration-dependent manner com-
pared to the DMSO-treated group, and the cells were
completely killed at 40 μM.
Flow cytometry was used to analyze the cells. MGC-803 cells
were treated with compound E20 and the Annexin V-FITC/
propidium iodide (PI) double staining assay (BD Pharmingen,
USA)23 was used. MGC-803 cells were treated with different
concentrations (10, 20, and 30 μM) of E20 for 48 h and the
control group was treated with 3‰ DMSO, then stained with
Annexin V-FITC and propidium iodide. The stained cells were
analyzed using a flow cytometer. As shown in Fig. 5, the per-
centage of apoptosis in the treated group was higher than
that in the control group, and compound E20 induced apo-
2.4 E20 can inhibit the migration of MGC-803 cells
The strong ability of migration of cancer cells plays an impor-
tant role in the mortality and prognosis of cancer pa-
tients;21,22 so we further studied the effect of E20 on cell mi-
gration activity using the wound healing assay. We scratched
the cells within each well with a 200 μL sterile pipette tip, re-
moved the floating cells by rinsing with PBS, and treated the
remaining cells with different concentrations of E20 for 48 h.
The control group was treated with 3‰ DMSO for 48 h. We
captured images of the cells with a microscopic camera sys-
tem at the time of treatment (0 h) and after 48 h to assess
the re-growth of the colony back into the damaged region. As
ptosis of MGC-803 cells in
manner.
a concentration-dependent
2.6 Effect of compound E20 on the cell cycle in MGC-803
cells
To demonstrate the effect of compound E20 on the cell cycle,
we used flow cytometry for analysis. MGC-803 cells were
treated with compound E20 (20, 40, and 60 μM) and the con-
trol group was treated with 3‰ DMSO, then mixed with
propidium iodide (PI) staining solution and RNase A. As
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Table 1 IC50 (μM) of analogues against different cell lines
IC50 SDa (μM)
vant anti-tumor targets, we performed western blot experi-
ments on the above three proteins. As shown in Fig. 7, com-
pound E20 had no effect on HIF-1 and PFK but down-
regulated the expression of HK2.
Compounds
MCF-7
A549
MGC-803
HepG2
HeLa
E1
E2
E3
E4
E5
E6
E7
E8
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
62
>100
90
89
>100
>100
64
>100
98
>100
93
>100
45
80
92
87
>100
>100
>100
>100
>100
>100
>100
>100
85
80
>100
75
>100
>100
63
80
80
74
78
35
56
37
34
31
35
45
38
38
47
42
61
37
29
48
43
63
80
63
52
60
62
53
80
59
43
59
56
55
62
58
3
4
3
5
5
5
5
5
3
6
6
5
3
4
4
2
1
3
3
6
5
6
4
1
4
3
3
6
3
3
1
2
3
1
2
1
82
2
>100
>100
>100
>100
3. Conclusion
In summary, a new class of tryptamine salicylic acid deriva-
tives was synthesized and evaluated against MGC-803, MCF-
7, A549, HepG2 and HeLa cell lines. The MTT assay revealed
that compound E20 was the most potent, showed broad-
spectrum anticancer inhibition against all the evaluated cell
lines, and was especially sensitive to the MGC-803 cell line.
Our preliminary observations indicated that compound E20,
when added to MGC-803 cells: (1) inhibited colony formation
and migration; (2) induced apoptosis in a concentration-
dependent manner; (3) inhibited their growth by blocking
the G2/M phase of the cell cycle; and (4) down-regulated the
expression of HK2. Our studies envisioned that the frame-
work of N-[2-(5-bromo-1H-indol-3-yl)-ethyl]-2-hydroxy-3-methyl-
benzamide (E20) might be considered as a new type of chemi-
cal for designing effective anti-cancer drugs targeting gastric
cancer cells.
75
70
73
69
80
75
93
62
60
2
4
2
3
2
4
4
3
2
60
48
63
46
62
63
3
81
56
64
64
80
85
2
3
1
6
1
4
2
3
1
2
3
6
E9
E10
E11
E12
E13
E14
E15
E16
E17
E18
E19
E20
E21
E22
E23
E24
E25
E26
E27
E28
E29
E30
E31
E32
E33
E34
E35
E36
5-Fu
1
5
>100
>100
50
75
68
89
80
59
36
61
63
63
62
1
5
61
3
3
1
4
3
4
3
1
3
3
4
>100
>100
6
93
4
79
89
80
63
80
98
73
2
>100
>100
3
2
1
5
5
4
5
79
52
61
71
80
5
5
2
3
2
3
6
6
5
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
67
4
>100
>100
4. Experimental section
4.1 Chemical synthesis
53
77
4
3
76
82
42
80
71
3
All chemicals (reagent grade) were purchased from Aladdin-
Reagent Co., Ltd (China) and used without further purifica-
tion unless otherwise stated. Melting points (uncorrected)
were determined on a Thermo Scientific electrothermal digi-
tal melting point apparatus. ESI mass spectra were obtained
on a Waters GCT mass spectrometer, and 1H NMR spectra
were measured on a Bruker AV-400 model spectrometer with
TMS and solvent signals slotted as internal standards. Chem-
ical shifts were reported in ppm (δ).
>100
>100
>100
>100
>100
>100
>100
3
3
3
5
97
4
2
1
>100
>100
>100
>100
3
>100
74
50
4
7
57
3
68
8
a
Data are the average of three independent assays; SD: standard
deviation.
4.1.1 Synthetic procedure for the synthesis of B1–B6. A
stirred solution of aniline (2.80 g, 0.03 mol) in 12% hydro-
chloric acid solution (27 mL) was cooled to 0–5 °C. Then a so-
dium nitrite solution (2.16 g, 0.031 mol) was added dropwise
and reacted for 1 h. After the reaction was completed, the so-
lution was added to an aqueous solution of sodium sulfite
(11.34 g, 0.09 mol) at room temperature and then heated to
80 °C. After 2 h, concentrated hydrochloric acid (6 mL) was
added and the reaction was stirred at 100 °C for 2 h. After
cooling to room temperature, phenylhydrazine hydrochloride
(B1) was precipitated from the solution. Compounds B2–B6
were prepared in a similar way. Yields: 90–95%.
4.1.2 Synthetic procedure for the synthesis of C1–C6. To a
solution of B1 (2.90 g, 0.02 mol) in 30% ethanol solution
were added 4-chloro-1-hydroxy-1-butanesulfonate sodium
(4.21 g, 0.02 mol) and Na2HPO4 (0.14 g, 0.001 mol), the mix-
ture was stirred at 70 °C for 6 h. After evaporating the etha-
nol, the mixture was washed with dichloromethane and
chloroform, then the pH was adjusted to 7 with saturated so-
dium bicarbonate and the mixture was cooled to 4 °C, during
shown in Fig. 6, the percentage of cells in the G0/G1, S and
G2/M phase was obtained with flow cytometry. Compound
E20 caused accumulation of cells in the G2/M phase of the
cell cycle and the shift to this phase was stronger with in-
creasing concentration of E20.
2.7 E20 inhibited the expression of HK2
It has been reported that the anti-tumor effects of melatonin
and salicylic acid are related to glycolysis-related proteins,
and representative proteins are hypoxia-inducible factor-1α
(HIF-1α), hexokinase
2 (HK2) and phosphofructokinase
(PFK).24 Compound E20 was a modified structure based on
the combination of melatonin and salicylic acid. According
to the combination principles, it is likely that E20 also
inhibited glycolysis by inhibiting the above-related proteins.
To further investigate whether the anti-proliferative effect of
compound E20 on gastric cancer cells is related to the rele-
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Fig. 3 The ordinate axis represents the sum of the IC50 values of the five cell lines and the abscissa axis represents different compounds. We can
more intuitively observe the structure–activity relationship of tryptamine salicylic acid derivatives.
Table 2 Molecular properties of E20a
Compound
MW
HBA
HBD
MolLogP
MolPSA (A2)
MV (A3)
Desirable value
E20
<500
372.05
<10
2
<5
3
<5
4.05
<140
49.84
—
318.79
a
MW: molecular weight; HBA: number of hydrogen bond acceptors; HBD: number of hydrogen bond donors; MolLogP: log p value predicted
by Molsoft; MolPSA: polar surface area; MV: molecular volume.
which C1 was precipitated from the solution. After filtration,
the product was used in the next step without purification.
Compounds C2–C6 were prepared in a similar way. Yields:
50–65%.
4.1.3 Synthetic procedure for the synthesis of E1–E36. To a
solution of salicylic acid (0.14 g, 0.001 mol) in acetone (15.0
ml) were added EDCI (0.573 g, 0.003 mol) and HOBt (0.405 g,
0.003 mol) at room temperature. After 4 h, compound E1 was
added and the reaction mixture was stirred at room tempera-
ture for 24 h. The crude product purified by silica gel column
chromatography (petroleum ether/EtOAc = 2 : 1) to obtain the
final product.
2-Hydroxy-N-[2-(1H-indol-3-yl)-ethyl]-benzamide
(E1).
1
Yellow-white powder, 80.53% yield, mp 154–156 °C; H NMR
(400 MHz, CDCl3) δ 12.41 (s, 1H), 8.20 (s, 1H,), 7.62 (s, 1H),
7.39 (t, J = 7.6 Hz, 1H), 7.33 (d, J = 8.5 Hz, 1H), 7.29 (s, 1H),
7.21 (s, 1H), 7.19 (s, 1H), 7.12 (s, 1H), 7.01 (d, J = 8.3 Hz, 1H),
6.81 (t, J = 7.3 Hz, 1H), 6.44 (s, 1H), 3.79 (d, J = 5.7 Hz, 2H),
3.08 (t, J = 6.2 Hz, 2H); 13C NMR (101 MHz, acetone) δ 170.66
(s), 160.25 (s), 135.26 (s), 134.35 (s), 127.97 (s), 127.35 (s),
126.60 (s), 123.90 (s), 122.88 (s), 122.67 (s), 118.13 (s), 117.56
(s), 113.81 (s), 111.86 (s), 111.02 (s), 40.33 (s), 25.08 (s). MS
(ESI): 280.32 (C17H16N2O2, [M + H+]).
2-Hydroxy-N-[2-(1H-indol-3-yl)-ethyl]-3-methyl-benzamide
(E2). White powder, 80.53% yield, mp 165–167 °C; 1H NMR
(400 MHz, CDCl3) δ 12.39 (s, 1H), 8.22 (s, 1H), 7.61 (s, 1H),
Fig. 4 (A) E20 inhibited the growth and migration of MGC-803 cells.
Representative images of MGC-803 cell colonies after treatment with
E20 at different concentrations for 9 days. (B) Wound healing assay.
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Fig. 5 Apoptotic effect of E20 on MGC-803 cells after treatment for 48 h. The lower left quadrant represents live cells, the lower right is for
early/primary apoptotic cells, the upper right is for late/secondary apoptotic cells, and the upper left represents cells damaged during the
procedure.
Fig. 6 Effect of E20 on cell cycle progression in MGC-803 cells. (A) Cells were treated with different concentrations of E20 for 24 h, then the cell
cycle distribution was analysed by flow cytometry. (B) Graph reporting percentile cell-cycle phase distributions. G0/G1, S and G2/M phase distribu-
tions were determined by FlowJo analysis.
7.33 (d, J = 8.5 Hz, 1H), 7.29 (s, 1H), 7.20 (d, J = 7.9 Hz, 1H),
7.11 (s, 1H), 7.08 (d, J = 7.8 Hz, 1H), 6.82 (s, 1H), 6.62 (d, J =
7.3 Hz, 1H), 6.37 (s, 1H), 3.77 (d, J = 5.1 Hz, 2H), 3.07 (d, J =
5.9 Hz, 2H), 2.33 (s, 3H); 13C NMR (101 MHz, acetone) δ
170.69 (s), 160.25 (s), 136.88 (s), 134.40 (s), 127.69 (s), 126.63
(s), 123.91 (s), 122.58 (s), 121.32 (s), 118.64 (s), 118.43 (s),
117.59 (s), 113.79 (s), 112.33 (s), 111.34 (s), 40.22 (s), 25.13
(s), 14.92 (s). MS (ESI): 294.35 (C18H18N2O2, [M + H+]).
2-Hydroxy-N-[2-(1H-indol-3-yl)-ethyl]-4-methyl-benzamide
(E3). White powder, 82.63% yield, mp 164–167 °C. 1H NMR
(400 MHz, CDCl3) δ 12.72 (s, 1H), 8.02 (s, 1H), 7.43 (s, 1H),
7.31 (d, J = 8.3 Hz, 1H), 7.29 (s, 1H), 7.25 (d, J = 7.2 Hz, 1H),
7.10 (s, 1H), 7.07 (s, 1H), 6.97 (d, J = 7.9 Hz, 1H), 6.67 (t, J =
7.6 Hz, 1H), 6.42 (s, 1H), 3.83–3.73 (m, 2H), 3.10 (t, J = 6.3
Hz, 2H), 2.46 (s, 3H); 13C NMR (101 MHz, acetone) δ 170.68
(s), 160.26 (s), 136.88 (s), 134.39 (s), 127.69 (s), 126.63 (s),
123.91 (s), 122.58 (s), 121.31 (s), 118.62 (s), 118.42 (s), 117.58
(s), 113.79 (s), 112.32 (s), 111.33 (s), 40.21 (s), 25.13 (s), 14.90
(s). MS (ESI): 294.35 (C18H18N2O2, [M + H+]).
4-Chloro-2-hydroxy-N-[2-(1H-indol-3-yl)-ethyl]-benzamide
(E4). White powder, 84.53% yield, mp 158–160 °C. 1H NMR
(400 MHz, CDCl3) δ 12.55 (s, 1H), 8.12 (s, 1H), 7.51 (s, 1H),
7.22 (d, J = 8.6 Hz, 1H), 7.18 (s, 1H), 7.16 (d, J = 7.3 Hz, 1H),
7.09 (d, J = 8.6 Hz, 1H), 7.01 (s, 1H), 6.94 (d, J = 7.9 Hz, 1H),
6.60 (t, J = 7.6 Hz, 1H), 6.32 (s, 1H), 3.67 (q, J = 6.0 Hz, 2H),
2.96 (t, J = 6.5 Hz, 2H); 13C NMR (101 MHz, acetone) δ 169.51
(s), 162.72 (s), 138.59 (s), 136.88 (s), 128.04 (s), 127.66 (s),
122.61 (s), 121.32 (s), 118.74–118.28 (m), 117.56 (s), 113.60
(s), 112.20 (s), 111.35 (s), 40.27 (s), 25.01 (s). MS (ESI): 314.77
(C17H15ClN2O2, [M + H+]).
5-Chloro-2-hydroxy-N-[2-(1H-indol-3-yl)-ethyl]-benzamide
(E5). White powder, 74.56% yield, mp 170–173 °C. 1H NMR
(400 MHz, CDCl3) δ 12.35 (s, 1H), 8.20 (s, 1H), 7.60 (s, 1H),
7.46 (d, J = 8.8 Hz, 1H), 7.33 (d, J = 8.6 Hz, 1H), 7.28 (s, 1H),
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Fig. 7 Western blot analysis of E20 in MGC3-803 cells. Cells were cultured in the presence of different concentrations of E20 (μM) for 24 h. (A)
The expression of HIF-1α, HK2 and PFK proteins. The expression of HIF-1α required the addition of CoCl2 (100 μM) to produce hypoxic conditions.
Representative photographs from three independent experiments are shown. (B) E20 only inhibited the expression of HK2, but had no obvious ef-
fect on PFK and HIF-1α. Western blot results of the HK2 protein were selected for statistical analysis. Quantification of the western blot result was
conducted by calculating the ratio of the value to the control group. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control.
7.25(d, J = 7.3 Hz, 1H), 7.21 (d, J = 8.6 Hz, 1H), 7.12 (s, 1H),
6.90 (d, J = 8.8 Hz, 1H), 6.38 (s, 1H), 3.77 (q, J = 6.4 Hz, 2H),
3.07 (t, J = 6.6 Hz, 2H). 13C NMR (101 MHz, acetone) δ 169.02
(s), 160.50 (s), 136.87 (s), 133.47 (s), 127.66 (s), 126.18 (s),
122.58 (d, J = 8.3 Hz), 121.31 (s), 119.57 (s), 118.63 (s), 118.37
(s), 115.83 (s), 112.18 (s), 111.34 (s), 40.35 (s), 24.96 (s). MS
(ESI): 314.77 (C17H15ClN2O2, [M + H+]).
5-Bromo-2-hydroxy-N-[2-(1H-indol-3-yl)-ethyl]-benzamide
(E6). White powder, 75.56% yield, mp 174–177 °C. 1H NMR
(400 MHz, CDCl3) δ 12.40 (s, 1H), 8.15 (s, 1H), 7.67 (d, J = 7.8
Hz, 1H), 7.46 (s, 1H), 7.44 (s, 1H), 7.27 (s, 1H), 7.23 (s, 1H),
7.20 (d, J = 7.4 Hz, 1H), 7.12 (s, 1H), 6.90 (d, J = 8.8 Hz, 1H),
6.36 (s, 1H), 3.92–3.68 (m, 2H), 3.13 (t, J = 6.5 Hz, 2H). 13C
NMR (101 MHz, DMSO) δ 167.83 (s), 159.51 (s), 136.72 (s),
136.46 (s), 130.57 (s), 127.63 (s), 123.22 (s), 121.43 (s), 120.21
(s), 118.71 (d, J = 6.4 Hz), 117.83 (s), 111.92 (d, J = 7.2 Hz),
110.14 (s), 25.23 (s). MS (ESI): 359.22 (C17H15BrN2O2, [M +
H+]).
135.25 (s), 134.39 (s), 128.86 (s), 126.62 (s), 124.53 (s), 123.95
(d, J = 14.7 Hz), 121.31 (s), 117.85 (s), 117.60 (s), 113.75 (s),
112.72 (s), 112.30 (s), 40.09 (s), 24.90 (s), 14.87 (s). MS (ESI):
328.79 (C18H17ClN2O2, [M + H+]).
N-[2-(5-Chloro-1H-indol-3-yl)-ethyl]-2-hydroxy-4-methyl-
benzamide (E9). Brown powder, 80.46% yield, mp 174–176 °C.
1H NMR (400 MHz, DMSO) δ 12.60 (s, 1H), 11.08 (s, 1H), 8.87
(t, J = 5.6 Hz, 1H), 7.77 (d, J = 1.8 Hz, 1H), 7.75 (d, J = 8.4 Hz,
1H), 7.31 (d, J = 8.6 Hz, 1H), 7.26 (d, J = 2.2 Hz, 1H), 7.20 (dd,
J = 8.6, 1.9 Hz, 1H), 6.70 (s, 1H), 6.73 (s, 1H), 3.51 (dd, J =
13.3, 7.1 Hz, 2H), 2.98 (t, J = 7.3 Hz, 2H), 2.29 (s, 3H); 13C
NMR (101 MHz, acetone) δ 170.27 (s), 161.93 (s), 144.57 (s),
135.25 (s), 128.86 (s), 126.30 (s), 124.51 (s), 124.03 (s), 121.31
(s), 119.35 (s), 117.91 (d, J = 9.1 Hz), 112.72 (s),112.21 (d, J =
26.4 Hz), 39.99 (s), 24.94 (s), 20.61 (s). MS (ESI): 328.79
(C18H17ClN2O2, [M + H+]).
4-Chloro-N-[2-(5-chloro-1H-indol-3-yl)-ethyl]-2-hydroxy-
benzamide (E10). White powder, 78.47% yield, mp 140–144
1
N-[2-(5-Chloro-1H-indol-3-yl)-ethyl]-2-hydroxy-benzamide
°C. H NMR (400 MHz, DMSO) δ 12.91 (s, 1H), 11.05 (s, 1H),
1
(E7). White-brown powder, 78.46% yield, mp 132–137 °C. H
8.96 (t, J = 5.4 Hz, 1H′), 7.85 (d, J = 8.4 Hz, 1H), 7.61 (d, J =
1.7 Hz, 1H), 7.36 (d, J = 8.6 Hz, 1H), 7.27 (d, J = 2.0 Hz, 1H),
7.06 (dd, J = 8.6, 2.0 Hz, 1H), 6.99 (dd, J = 3.3, 2.0 Hz, 1H),
6.97–6.95 (m, 1H), 3.55 (dd, J = 13.2, 6.9 Hz, 2H), 2.95 (t, J =
7.3 Hz, 2H); 13C NMR (101 MHz, DMSO) δ 168.35 (s), 161.16
(s), 137.98 (s), 135.16 (s), 129.93 (s), 128.84 (s), 125.20 (s),
123.52 (s), 121.34 (s), 119.29 (s), 118.05 (s), 117.46 (s), 115.04
(s), 113.40 (s), 112.04 (s), 25.05 (s). MS (ESI): 349.21
(C17H14Cl2N2O2, [M + H+]).
NMR (400 MHz, CDCl3) δ 12.41 (s, 1H), 8.18 (s, 1H), 7.62 (d, J
= 1.9 Hz, 1H), 7.40 (ddd, J = 8.6, 7.3, 1.5 Hz, 1H), 7.33 (d, J =
8.6 Hz, 1H), 7.22–7.17 (m, 2H), 7.13 (d, J = 2.2 Hz, 1H), 7.00
(dd, J = 8.4, 1.0 Hz, 1H), 6.83–6.78 (m, 1H), 6.41 (s, 1H), 3.79
(dd, J = 12.6, 6.6 Hz, 2H), 3.08 (t, J = 6.7 Hz, 2H); 13C NMR
(101 MHz, acetone) δ 170.23 (s), 161.86 (s), 135.25 (s), 133.75
(s), 128.85 (s), 126.47 (s), 124.53 (s), 124.03 (s), 121.31 (s),
118.27 (s), 117.83 (d, J = 4.6 Hz), 114.67 (s), 112.72 (s), 112.29
(s), 40.07 (s), 24.88 (s). MS (ESI): 314.77 (C17H15ClN2O2, [M +
H+]).
N-[2-(5-Chloro-1H-indol-3-yl)-ethyl]-2-hydroxy-3-methyl-
benzamide (E8). Brown powder, 80.46% yield, mp 174–176 °C.
1H NMR (400 MHz, CDCl3) δ 12.65 (s, 1H), 8.15 (s, 1H), 7.62
(d, J = 2.0 Hz, 1H), 7.35–7.32 (m, 1H), 7.26 (d, J = 7.3 Hz, 1H),
7.20 (dd, J = 8.6, 2.0 Hz, 1H), 7.13 (d, J = 2.3 Hz, 1H), 7.05–
7.01 (m, 1H), 6.71 (dd, J = 9.5, 5.8 Hz, 1H), 6.39 (s, 1H), 3.78
(dd, J = 12.6, 6.6 Hz, 2H), 3.08 (q, J = 6.4 Hz, 2H), 2.29 (s,
3H). 13C NMR (101 MHz, acetone) δ 170.70 (s), 160.23 (s),
5-Chloro-N-[2-(5-chloro-1H-indol-3-yl)-ethyl]-2-hydroxy-
benzamide (E11). White powder, 75.47% yield, mp 164–165
1
°C. H NMR (400 MHz, DMSO) δ 12.61 (s, 1H), 11.05 (s, 1H),
8.99 (t, J = 5.5 Hz, 1H), 7.92 (d, J = 2.6 Hz, 1H), 7.61 (d, J = 2.0
Hz, 1H), 7.43 (dd, J = 8.8, 2.6 Hz, 1H), 7.36 (d, J = 8.6 Hz,
1H), 7.28 (d, J = 2.2 Hz, 1), 7.06 (dd, J = 8.6, 2.0 Hz, 1H), 6.94
(d, J = 8.8 Hz, 1H), 3.59–3.51 (m, 2H), 2.95 (t, J = 7.2 Hz, 2H).
13C NMR (101 MHz, DMSO) δ 167.88 (s), 159.03 (s), 135.16
(s), 133.65 (s), 128.85 (s), 127.69 (s), 125.23 (s), 123.53 (s),
122.76 (s), 121.34 (s), 119.76 (s), 118.05 (s), 117.29 (s), 113.40
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(s), 112.04 (s), 25.00 (s). MS (ESI): 349.21 (C17H14Cl2N2O2, [M
+ H+]).
(s), 138.56 (s), 135.25 (s), 127.99 (d, J = 9.4 Hz), 127.37 (s),
122.81 (d, J = 18.3 Hz), 118.53 (s), 118.10 (s), 117.54 (s),
113.60 (s), 111.73 (s), 111.05 (s), 40.40 (s), 24.97 (s), 20.73 (s).
MS (ESI): 328.79 (C18H17ClN2O2, [M + H+]).
5-Bromo-N-[2-(5-chloro-1H-indol-3-yl)-ethyl]-2-hydroxy-
benzamide (E12). White powder, 75.47% yield, mp 182–185
1
°C. H NMR (400 MHz, DMSO) δ 12.66 (s, 1H), 11.07 (s, 1H),
5-Chloro-2-hydroxy-N-[2-(5-methyl-1H-indol-3-yl)-ethyl]-
benzamide (E17). Brown powder, 70.38% yield, mp 162–164
9.01 (t, J = 5.5 Hz, 1H), 8.04 (d, J = 2.4 Hz, 1H), 7.61 (d, J = 1.8
Hz, 1H), 7.54 (dd, J = 8.8, 2.4 Hz, 1H), 7.36 (d, J = 8.6 Hz,
1H), 7.28 (d, J = 2.1 Hz, 1H), 7.07 (dd, J = 8.6, 2.0 Hz, 1H),
6.89 (d, J = 8.8 Hz, 1H), 3.56 (dd, J = 13.1, 7.0 Hz, 2H), 2.96 (t,
J = 7.2 Hz, 2H). 13C NMR (101 MHz, acetone) δ 168.97 (s),
160.90 (s), 136.35 (s), 135.24 (s), 129.10 (s), 128.83 (s), 124.58
(s), 124.07 (s), 121.33 (s), 120.00 (s), 117.83 (s), 116.40 (s),
112.73 (s), 112.19 (s), 109.52 (s), 40.25 (s), 24.73 (s). MS (ESI):
393.66 (C17H14BrClN2O2, [M + H+]).
1
°C. H NMR (400 MHz, acetone) δ 12.93 (s, 1H), 9.91 (s, 1H),
8.38 (s, 1H), 7.78 (s, 1H), 7.42 (s, 1H), 7.40 (s, 1H), 7.28 (d, J =
8.2 Hz, 1H), 7.18 (s, 1H,), 6.95 (d, J = 3.8 Hz, 1H), 6.93 (s,
1H), 3.74 (q, J = 6.5 Hz, 2H), 3.08 (t, J = 7.2 Hz, 2H), 2.39 (s,
3H); 13C NMR (101 MHz, acetone) δ 169.01 (s), 160.50 (s),
135.24 (s), 133.45 (s), 127.96 (s), 127.40 (s), 126.19 (s), 122.73
(t, J = 18.4 Hz), 119.56 (s), 118.11 (s), 115.85 (s), 111.77 (s),
111.05 (s), 40.54 (s), 24.93 (s), 20.73 (s). MS (ESI): 328.79
(C18H17ClN2O2, [M + H+]).
2-Hydroxy-N-[2-(5-methyl-1H-indol-3-yl)-ethyl]-benzamide
1
(E13). Brown powder, 78.46% yield, mp 156–158 °C. H NMR
5-Bromo-2-hydroxy-N-[2-(5-methyl-1H-indol-3-yl)-ethyl]-
benzamide (E18). Brown powder, 72.34% yield, mp 161–163
(400 MHz, acetone) δ 12.78 (s, 1H), 9.74 (s, 1H), 8.11 (s, 1H),
7.58 (d, J = 8.0 Hz, 1H), 7.26 (s, 1H), 7.23 (s, 1H), 7.12 (d, J =
8.2 Hz, 1H), 7.02 (s, 1H), 6.81–6.76 (m, 1H), 6.75 (s, 1H), 6.68
(t, J = 7.6 Hz, 1H), 3.58 (dd, J = 13.5, 6.7 Hz, 2H), 2.92 (t, J =
7.2 Hz, 2H), 2.23 (s, 3H). 13C NMR (101 MHz, acetone) δ
170.68 (s), 160.26 (s), 136.88 (s), 134.39 (s), 127.69 (s), 126.63
(s), 123.91 (s), 122.58 (s), 121.31 (s), 118.62 (s), 118.42 (s),
117.58 (s), 113.79 (s), 112.32 (s), 111.33 (s), 40.21 (s), 25.13
(s), 14.90 (s). MS (ESI): 294.35 (C18H18N2O2, [M + H+]).
1
°C. H NMR (400 MHz, acetone) δ 12.97 (s, 1H), 9.91 (s, 1H),
8.40 (s, 1H), 7.90 (s, 1H), 7.54 (d, J = 8.8 Hz, 1H), 7.40 (s, 1H),
7.28 (d, J = 8.3 Hz, 1H), 7.17 (s, 1H), 6.95 (d, J = 8.2 Hz, 1H),
6.89 (d, J = 8.8 Hz, 1H), 3.74 (q, J = 6.5 Hz, 2H), 3.08 (t, J = 7.1
Hz, 2H), 2.39 (s, 3H); 13C NMR (101 MHz, acetone) δ 170.21
(s), 161.89 (s), 135.26 (s), 133.73 (s), 127.97 (s), 127.37 (s),
126.52 (s), 122.90 (s), 122.70 (s), 118.20 (d, J = 10.9 Hz),
117.81 (s), 114.73 (s), 111.85 (s), 111.05 (s), 40.33 (s), 25.09
(s), 20.75 (s). MS (ESI): 373.24 (C18H17BrN2O2, [M + H+]).
2-Hydroxy-3-methyl-N-[2-(5-methyl-1H-indol-3-yl)-ethyl]-
benzamide (E14). Brown powder, 78.46% yield, mp 142–144
N-[2-(5-Bromo-1H-indol-3-yl)-ethyl]-2-hydroxy
benzamide
1
1
°C. H NMR (400 MHz, DMSO) δ 12.75 (s, 1H), 10.67(s, 1H),
(E19). Brown powder, 79.38% yield, mp 124–126 °C. H NMR
(400 MHz, DMSO) δ 12.63 (d, J = 4.5 Hz, 1H), 11.06 (s, 1H),
8.94 (t, J = 5.6 Hz, 1H), 7.83 (dd, J = 7.9, 1.3 Hz, 1H), 7.77 (d,
J = 1.8 Hz, 1H), 7.42–7.37 (m, 1H), 7.32 (d, J = 8.6 Hz, 1H),
7.26 (d, J = 2.3 Hz, 1H), 7.18 (dd, J = 8.6, 1.9 Hz, 1H), 6.91 (s,
1H), 6.89–6.86 (m, 1H), 3.56 (dd, J = 13.2, 7.1 Hz, 2H), 3.01–
2.90 (m, 2H). 13C NMR (101 MHz, DMSO) δ 169.38 (s), 160.57
(s), 135.39 (s), 134.08 (s), 129.59 (s), 128.08 (s), 125.02 (s),
123.85 (s), 121.10 (s), 119.00 (s), 117.84 (s), 115.69 (s), 113.88
(s), 112.04 (s), 111.47 (s), 25.13 (s). MS (ESI): 359.22
(C17H15BrN2O2, [M + H+]).
8.88 (t, J = 5.6 Hz, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.35 (s, 1H),
7.23 (d, J = 8.2 Hz, 1H), 7.13 (d, J = 2.2 Hz, 1H), 6.85 (dd, J =
8.3, 1.3 Hz, 1H), 6.71 (d, J = 6.1 Hz, 1H), 6.70 (s, 1H), 3.55
(dd, J = 13.3, 7.2 Hz, 2H), 2.94 (t, J = 7.4 Hz, 2H), 2.36 (s, 3H),
2.28 (s, 3H); 13C NMR (101 MHz, acetone) δ 170.66 (s), 160.25
(s), 135.26 (s), 134.35 (s), 127.97 (s), 127.35 (s), 126.60 (s),
123.90 (s), 122.88 (s), 122.67 (s), 118.13 (s), 117.56 (s), 113.81
(s), 111.86 (s), 111.02 (s), 40.33 (s), 25.08 (s), 20.73 (s), 14.88
(s). MS (ESI): 308.37 (C19H20N2O2, [M + H+]).
2-Hydroxy-4-methyl-N-[2-(5-methyl-1H-indol-3-yl)-ethyl]-
benzamide (E15). Brown powder, 79.78% yield, mp 142–144
N-[2-(5-Bromo-1H-indol-3-yl)-ethyl]-2-hydroxy-3-methyl-
benzamide (E20). Brown powder, 80.23% yield, mp 183–185
1
°C. H NMR (400 MHz, DMSO) δ 12.73 (s, 1H), 10.69 (s, 1H),
1
8.86 (t, J = 5.6 Hz, 1H), 7.73 (d, J = 8.0 Hz, 1H), 7.34 (s, 1H),
7.23 (d, J = 8.2 Hz, 1H), 7.13 (d, J = 2.2 Hz, 1H), 6.89 (dd, J =
8.3, 1.3 Hz, 1H), 6.71 (d, J = 6.1 Hz, 1H), 6.70 (s, 1H), 3.55
(dd, J = 13.3, 7.2 Hz, 2H), 2.94 (t, J = 7.4 Hz, 2H), 2.36 (s, 3H),
2.28 (s, 3H). 13C NMR (101 MHz, DMSO) δ 169.56 (s), 160.82
(s), 144.48 (s), 135.11 (s), 127.87 (d, J = 10.7 Hz), 127.09 (s),
123.25 (s), 123.00 (s), 119.97 (s), 118.38 (s), 117.98 (s), 112.93
(s), 111.58 (d, J = 3.9 Hz), 25.42 (s), 21.63 (d, J = 17.3 Hz). MS
(ESI): 308.37 (C19H20N2O2, [M + H+]).
°C. H NMR (400 MHz, DMSO) δ 13.27 (s, 1H), 11.06 (s, 1H),
8.99 (t, J = 5.6 Hz, 1H), 7.76 (d, J = 1.8 Hz, 1H), 7.65 (d, J = 7.2
Hz, 1H), 7.32 (d, J = 8.6 Hz, 1H), 7.29 (s, 1H), 7.26 (d, J = 2.3
Hz, 1H), 7.18 (dd, J = 8.6, 1.9 Hz, 1H), 6.78 (t, J = 7.7 Hz, 1H),
3.55 (dd, J = 13.2, 7.1 Hz, 2H), 2.96 (t, J = 7.3 Hz, 2H), 2.16 (s,
3H). 13C NMR (101 MHz, DMSO) δ 170.57 (s), 159.83 (s),
135.37 (s), 134.89 (s), 129.59 (s), 126.43 (s), 124.96 (d, J = 11.6
Hz), 123.84 (s), 121.07 (s), 118.13 (s), 113.93 (d, J = 9.2 Hz),
112.01 (s), 111.47 (s), 25.05 (s), 15.93 (s). MS (ESI): 373.24
(C18H17BrN2O2, [M + H+]).
4-Chloro-2-hydroxy-N-[2-(5-methyl-1H-indol-3-yl)-ethyl]-
benzamide (E16). Brown powder, 75.38% yield, mp 152–154
N-[2-(5-Bromo-1H-indol-3-yl)-ethyl]-2-hydroxy-4-methyl-
benzamide (E21). Brown powder, 75.23% yield, mp 135–137
1
°C. H NMR (400 MHz, acetone) δ 13.27 (s, 1H), 9.90 (s, 1H),
1
8.34 (s, 1H), 7.75 (d, J = 8.5 Hz, 1H), 7.40 (s, 1H), 7.28 (d, J =
8.2 Hz, 1H), 7.17 (s, 1H), 6.96 (s, 1H), 6.94 (s, 1H), 6.87 (d, J =
8.5 Hz, 1H), 3.73 (q, J = 6.5 Hz, 2H), 3.07 (t, J = 7.2 Hz, 2H),
2.39 (s, 3H); 13C NMR (101 MHz, acetone) δ 169.50 (s), 162.73
°C. H NMR (400 MHz, DMSO) δ 12.68 (s, 1H), 11.06 (s, 1H),
8.85 (t, J = 5.6 Hz, 1H), 7.77 (d, J = 1.8 Hz, 1H), 7.72 (d, J = 8.4
Hz, 1H), 7.32 (d, J = 8.6 Hz, 1H), 7.26 (d, J = 2.2 Hz, 1H), 7.18
(dd, J = 8.6, 1.9 Hz, 1H), 6.72 (s, 1H), 6.70 (s, 1H), 3.54 (dd, J
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= 13.3, 7.1 Hz, 2H), 2.95 (t, J = 7.3 Hz, 2H), 2.27 (s, 3H). 13C
NMR (101 MHz, DMSO) δ 169.56 (s), 160.78 (s), 144.51 (s),
135.38 (s), 129.59 (s), 127.80 (s), 124.99 (s), 123.85 (s), 121.10
(s), 120.01 (s), 117.99 (s), 113.88 (s), 112.90 (s), 112.06 (s),
111.47 (s), 25.16 (s), 21.55 (s). MS (ESI): 373.24
(C18H17BrN2O2, [M + H+]).
N-(2-(5-Fluoro-1H-indol-3-yl)ethyl)-2-hydroxy-3-methyl-
benzamide (E26). White powder, 79.43% yield, mp 147–149
1
°C. H NMR (400 MHz, DMSO) δ 13.30 (s, 1H), 10.94 (s, 1H),
8.99 (t, J = 5.6 Hz, 1H), 7.67 (dd, J = 5.9, 2.1 Hz, 1H), 7.35 (s,
1H), 7.34 (d, J = 3.9 Hz, 1H), 7.32–7.30 (m, 1H), 7.28 (d, J =
2.3 Hz, 1H), 6.91 (td, J = 9.3, 2.5 Hz, 1H), 6.78 (t, J = 7.7 Hz,
1H), 3.56 (dd, J = 13.3, 7.2 Hz, 2H), 2.95 (t, J = 7.4 Hz, 2H),
2.16 (s, 3H). 13C NMR (101 MHz, DMSO) δ 170.59 (s), 159.85
(s), 134.89 (s), 133.36 (s), 126.44 (s), 125.37 (s), 124.90 (s),
118.11 (s), 112.78 (d, J = 9.8 Hz), 112.38 (d, J = 4.8 Hz), 109.63
(s), 109.37 (s), 103.48 (s), 103.25 (s), 25.19 (s), 15.92 (s). MS
(ESI): 312.34 (C18H17FN2O2, [M + H+]).
N-[2-(5-Bromo-1H-indol-3-yl)-ethyl]-4-chloro-2-hydroxy-
benzamide (E22). Brown powder, 72.23% yield, mp 143–144
1
°C. H NMR (400 MHz, DMSO) δ 12.92 (s, 1H), 11.06 (s, 1H),
8.96 (t, J = 5.6 Hz, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.75 (d, J = 1.9
Hz, 1H), 7.32 (d, J = 8.6 Hz, 1H), 7.26 (d, J = 2.3 Hz, 1H), 7.17
(dd, J = 8.6, 1.9 Hz, 1H), 6.99 (d, J = 1.5 Hz, 1H), 6.96 (d, J =
2.1 Hz, 1H), 3.55 (dd, J = 13.1, 7.1 Hz, 2H), 2.95 (t, J = 7.3 Hz,
2H). 13C NMR (101 MHz, DMSO) δ 168.39 (s), 161.20 (s),
137.99 (s), 135.39 (s), 129.91 (s), 129.57 (s), 125.05 (s), 123.85
(s), 121.10 (s), 119.29 (s), 117.47 (s), 115.01 (s), 113.88 (s),
111.98 (s), 111.48 (s), 25.04 (s). MS (ESI): 393.66
(C17H14BrClN2O2, [M + H+]).
N-[2-(5-Bromo-1H-indol-3-yl)-ethyl]-5-chloro-2-hydroxy-
benzamide (E23). Brown powder, 70.23% yield, mp 161–164
°C. 1H NMR (400 MHz, DMSO) δ 12.61 (s, 1H), 11.06 (s,
1H), 8.99 (t, J = 5.5 Hz, 1H), 7.91 (d, J = 2.6 Hz, 1H),
7.75 (d, J = 1.8 Hz, 1H), 7.43 (dd, J = 8.8, 2.6 Hz, 1H),
7.32 (d, J = 8.6 Hz, 1H), 7.27 (d, J = 2.2 Hz, 1H), 7.18
(dd, J = 8.6, 1.9 Hz, 1H), 6.94 (d, J = 8.8 Hz, 1H), 3.55
(dd, J = 13.1, 7.1 Hz, 2H), 2.95 (t, J = 7.2 Hz, 2H). 13C
NMR (101 MHz, DMSO) δ 167.89 (s), 159.04 (s), 135.39
(s), 133.65 (s), 129.57 (s), 127.69 (s), 125.08 (s), 123.86 (s),
122.77 (s), 121.09 (s), 119.77 (s), 117.28 (s), 113.89 (s),
111.98 (s), 111.49 (s), 24.98 (s). MS (ESI): 393.66
(C17H14BrClN2O2, [M + H+]).
5-Bromo-N-[2-(5-bromo-1H-indol-3-yl)-ethyl]-2-hydroxy-
benzamide (E24). White powder, 71.23% yield, mp 152–154
°C. 1H NMR (400 MHz, DMSO) δ 12.63 (d, J = 4.9 Hz, 1H),
11.06 (s, 1H), 8.99 (t, J = 5.5 Hz, 1H), 8.03 (d, J = 2.5 Hz, 1H),
7.75 (d, J = 1.8 Hz, 1H), 7.54 (dd, J = 8.8, 2.5 Hz, 1H), 7.32 (d,
J = 8.6 Hz, 1H), 7.26 (d, J = 2.2 Hz, 1H), 7.18 (dd, J = 8.6, 1.9
Hz, 1H), 6.89 (d, J = 8.8 Hz, 1H), 3.55 (dd, J = 13.1, 7.1 Hz,
2H), 2.95 (t, J = 7.2 Hz, 2H). 13C NMR (101 MHz, DMSO) δ
167.84 (s), 159.47 (s), 136.46 (s), 135.39 (s), 130.56 (s), 129.57
(s), 125.07 (s), 123.86 (s), 121.09 (s), 120.20 (s), 117.83 (s),
113.89 (s), 111.98 (s), 111.49 (s), 110.20 (s), 24.98 (s). MS
(ESI): 438.11 (C17H14Br2N2O2, [M + H+]).
N-(2-(5-Fluoro-1H-indol-3-yl)ethyl)-2-hydroxy-4-methyl-
benzamide (E27). White powder, 79.43% yield, mp 144–148
1
°C. H NMR (400 MHz, DMSO) δ 12.69 (s, 1H), 10.94 (s, 1H),
8.86 (t, J = 5.6 Hz, 1H), 7.72 (d, J = 8.0 Hz, 1H), 7.34 (dd, J =
5.1, 3.6 Hz, 1H), 7.32 (d, J = 2.2 Hz, 1H), 7.27 (d, J = 2.2 Hz,
1H), 6.91 (td, J = 9.2, 2.5 Hz, 1H), 6.72 (s, 1H), 6.70 (s, 1H),
3.55 (dd, J = 13.3, 7.2 Hz, 2H), 2.94 (t, J = 7.4 Hz, 2H), 2.27 (s,
3H). 13C NMR (101 MHz, DMSO) δ 169.55 (s), 160.77 (s),
144.51 (s), 133.36 (s), 127.81 (s), 125.36 (s), 120.00 (s), 117.99
(s), 112.77 (d, J = 9.9 Hz), 112.43 (d, J = 4.8 Hz), 109.63 (s),
109.37 (s), 103.51 (s), 103.28 (s), 25.28 (s), 21.54 (s). MS (ESI):
312.34 (C18H17FN2O2, [M + H+]).
4-Chloro-N-(2-(5-fluoro-1H-indol-3-yl)ethyl)-2-hydroxy-
benzamide (E28). White powder, 75.54% yield, mp 175–180
1
°C. H NMR (400 MHz, DMSO) δ 12.93 (s, 1H), 10.94 (s, 1H),
8.97 (t, J = 5.5 Hz, 1H), 7.87 (d, J = 8.4 Hz, 1H), 7.36–7.33 (m,
1H), 7.32–7.30 (m, 1H), 7.27 (d, J = 2.2 Hz, 1H), 6.99 (dd, J =
3.7, 2.0 Hz, 1H), 6.96 (d, J = 2.1 Hz, 1H), 6.91 (td, J = 9.2, 2.5
Hz, 1H), 3.57 (dd, J = 13.2, 7.2 Hz, 2H), 2.94 (t, J = 7.3 Hz,
2H). 13C NMR (101 MHz, DMSO) δ 168.37 (s), 161.20 (s),
137.99 (s), 133.37 (s), 129.92 (s), 125.41 (s), 119.28 (s), 117.47
(s), 112.78 (d, J = 9.8 Hz), 112.33 (d, J = 4.8 Hz), 109.64 (s),
109.39 (s), 103.51 (s), 103.28 (s), 25.17 (s). MS (ESI): 332.76
(C17H14ClFN2O2, [M + H+]).
5-Chloro-N-(2-(5-fluoro-1H-indol-3-yl)ethyl)-2-hydroxy-
benzamide (E29). White powder, 70.54% yield, mp 170–172
1
°C. H NMR (400 MHz, DMSO) δ 12.61 (s, 1H), 10.95 (s, 1H),
8.99 (t, J = 5.4 Hz, 1H), 7.93 (d, J = 2.6 Hz, 1H), 7.43 (dd, J =
8.8, 2.6 Hz, 1H), 7.34 (t, J = 4.3 Hz, 1H), 7.32 (d, J = 3.6 Hz,
1H), 7.28 (d, J = 2.2 Hz, 1H), 6.96–6.92 (m, 1H), 6.90 (dd, J =
9.1, 2.4 Hz, 1H), 3.57 (dd, J = 13.2, 7.1 Hz, 2H), 2.94 (t, J = 7.3
Hz, 2H). 13C NMR (101 MHz, DMSO) δ 167.86 (s), 159.02 (s),
133.64 (s), 133.37 (s), 127.71 (s), 125.43 (s), 119.76 (s), 117.32
(s), 112.79 (d, J = 9.8 Hz), 112.31 (d, J = 4.7 Hz), 109.65 (s),
109.39 (s), 103.50 (s), 103.28 (s), 25.11 (s). MS (ESI): 332.76
(C17H14ClFN2O2, [M + H+]).
N-(2-(5-Fluoro-1H-indol-3-yl)ethyl)-2-hydroxybenzamide
1
(E25). White powder, 78.23% yield, mp 117–120 °C. H NMR
(400 MHz, DMSO) δ 12.65 (s, 1H), 10.94 (s, 1H), 8.94 (t, J =
5.5 Hz, 1H), 7.84 (dd, J = 7.9, 1.5 Hz, 1H), 7.42–7.37 (m, 1H),
7.35 (d, J = 4.1 Hz, 1H), 7.34–7.31 (m, 1H), 7.28 (d, J = 2.2 Hz,
1H), 6.94 (d, J = 2.5 Hz, 1H), 6.91 (s, 1H), 6.89 (d, J = 1.5 Hz,
1H), 3.57 (dd, J = 13.3, 7.2 Hz, 2H), 2.95 (t, J = 7.4 Hz, 2H).
13C NMR (101 MHz, DMSO) δ 169.38 (s), 160.56 (s), 158.29
(s), 156.00 (s), 134.08 (s), 133.37 (s), 128.32–127.77 (m),
125.38 (s), 118.98 (s), 117.85 (s), 115.71 (s), 112.78 (d, J = 9.8
Hz), 112.41 (d, J = 4.8 Hz), 109.64 (s), 109.38 (s), 103.52 (s),
103.29 (s), 25.26 (s). MS (ESI): 298.11 (C17H15FN2O2, [M +
H+]).
5-Bromo-N-(2-(5-fluoro-1H-indol-3-yl)ethyl)-2-hydroxy-
benzamide (E30). White powder, 71.54% yield, mp 176–179
1
°C. H NMR (400 MHz, DMSO) δ 12.64 (s, 1H), 10.95 (s, 1H),
8.99 (t, J = 5.5 Hz, 1H), 8.04 (d, J = 2.5 Hz, 1H), 7.54 (dd, J =
8.8, 2.5 Hz, 1H), 7.36–7.33 (m, 1H), 7.32–7.30 (m, 1H), 7.28
(d, J = 2.2 Hz, 1H), 6.92–6.90 (m, 1H), 6.90–6.87 (m, 1H), 3.56
(dd, J = 13.1, 7.1 Hz, 2H), 2.94 (t, J = 7.3 Hz, 2H). 13C NMR
(101 MHz, DMSO) δ 167.81 (s), 159.45 (s), 136.45 (s), 133.37
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(s), 130.58 (s), 125.43 (s), 120.20 (s), 117.86 (s), 112.79 (d, J =
9.7 Hz), 112.31 (d, J = 4.8 Hz), 109.65 (s), 109.39 (s), 103.50
(s), 103.27 (s), 25.11 (s). MS (ESI): 377.21 (C17H14BrFN2O2, [M
+ H+]).
Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 168.82 (s), 160.11 (s),
154.32 (s), 133.99 (s), 131.54 (s), 127.59 (s), 124.99 (s), 123.24
(s), 122.90 (s), 120.07 (s), 115.22 (s), 112.76 (s), 112.25 (s),
100.23 (s), 55.83 (s), 40.21 (s), 29.73 (s), 24.99 (s). MS (ESI):
344.79 (C18H17ClN2O3, [M + H+]).
5-Bromo-2-hydroxy-N-(2-(5-methoxy-1H-indol-3-yl)ethyl)-
benzamide (E36). White powder, 68.34% yield, mp 139–142
°C. 1H NMR (400 MHz, CDCl3) δ 12.43 (s, 1H), 8.09 (d, J =
15.9 Hz, 1H), 7.45 (dd, J = 8.8, 2.3 Hz, 1H), 7.32 (d, J = 8.8 Hz,
1H), 7.28 (s, 1H), 7.23 (d, J = 2.3 Hz, 1H), 7.06 (d, J = 2.3 Hz,
1H), 6.93 (dd, J = 8.8, 2.4 Hz, 1H), 6.89 (d, J = 8.8 Hz, 1H),
6.41 (s, 1H), 3.84 (s, 3H), 3.77 (q, J = 6.4 Hz, 2H), 3.09 (t, J =
6.6 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 168.73 (s), 160.56
(s), 154.31 (s), 136.81 (s), 131.53 (s), 127.98 (s), 127.59 (s),
122.90 (s), 120.48 (s), 115.86 (s), 112.78 (s), 112.25 (s), 110.14
(s), 100.20 (s), 55.85 (s), 40.25 (s), 24.98 (s). MS (ESI): 389.24
(C18H17BrN2O3, [M + H+]).
2-Hydroxy-N-(2-(5-methoxy-1H-indol-3-yl)ethyl)benzamide
1
(E31). White powder, 70.34% yield, mp 125–127 °C. H NMR
(400 MHz, CDCl3) δ 12.40 (s, 1H), 8.07 (s, 1H), 7.32 (d, J = 8.8
Hz, 2H), 7.29 (s, 1H), 7.10 (d, J = 2.6 Hz, 2H), 6.96–6.93 (m,
1H), 6.93–6.91 (m, 1H), 6.41 (s, 1H), 3.84 (s, 3H), 3.78 (dd, J =
12.4, 6.3 Hz, 2H), 3.09 (t, J = 6.5 Hz, 2H). 13C NMR (101 MHz,
CDCl3) δ 169.91 (s), 161.57 (s), 154.23 (s), 134.16 (s), 131.54
(s), 127.65 (s), 125.31 (s), 122.93 (s), 118.59 (d, J = 2.8 Hz),
114.28 (s), 112.76 (s), 112.48 (s), 112.19 (s), 100.26 (s), 55.81
(s), 40.04 (s), 25.11 (s). MS (ESI): 310.35 (C18H18N2O3, [M +
H+]).
2-Hydroxy-N-(2-(5-methoxy-1H-indol-3-yl)ethyl)-3-methyl-
benzamide (E32). White powder, 69.34% yield, mp 125–127
°C. 1H NMR (400 MHz, CDCl3) δ 12.74 (s, 1H), 8.04 (s, 1H),
7.32 (s, 1H), 7.29 (d, J = 3.6 Hz, 2H), 7.25 (d, J = 7.3 Hz, 1H),
6.98 (d, J = 7.8 Hz, 1H), 6.91 (dd, J = 8.8, 2.3 Hz, 1H), 6.68 (t,
J = 7.6 Hz, 1H), 6.44 (s, 1H), 3.81 (s, 3H), 3.80–3.76 (m, 2H),
3.09 (t, J = 6.5 Hz, 2H), 2.28 (s, 3H). 13C NMR (101 MHz,
CDCl3) δ 170.32 (s), 160.00 (s), 154.23 (s), 134.84 (s), 131.52
(s), 127.64 (s), 122.83 (d, J = 13.8 Hz), 117.87 (s), 113.44 (s),
112.77 (s), 112.54 (s), 112.16 (s), 100.27 (s), 55.81 (s), 40.01
(s), 25.14 (s), 15.82 (s). MS (ESI): 324.37 (C19H20N2O3, [M +
H+]).
2-Hydroxy-N-(2-(5-methoxy-1H-indol-3-yl)ethyl)-4-methyl-
benzamide (E33). White powder, 68.04% yield, mp 125–127
°C. 1H NMR (400 MHz, CDCl3) δ 12.48 (s, 1H), 8.08 (s, 1H),
7.29 (d, J = 1.7 Hz, 1H), 7.06 (s, 2H), 7.01 (d, J = 8.1 Hz, 1H),
6.91 (dd, J = 8.8, 2.4 Hz, 1H), 6.81 (s, 1H), 6.58 (d, J = 8.1 Hz,
1H), 6.41 (s, 1H), 3.81 (s, 3H), 3.79–3.75 (m, 2H), 3.08 (t, J =
6.5 Hz, 2H), 2.32 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 169.94
(s), 161.55 (s), 154.21 (s), 145.21 (s), 131.53 (s), 127.67 (s),
125.15 (s), 122.92 (s), 119.77 (s), 118.69 (s), 112.64 (d, J = 19.5
Hz), 112.17 (s), 111.66 (s), 100.26 (s), 55.80 (s), 39.98 (s),
25.15 (s), 21.61 (s). MS (ESI): 324.37 (C19H20N2O3, [M + H+]).
4-Chloro-2-hydroxy-N-(2-(5-methoxy-1H-indol-3-yl)ethyl)-
benzamide (E34). White powder, 68.04% yield, mp 128–132
°C. 1H NMR (400 MHz, CDCl3) δ 12.70 (s, 1H), 8.09 (s, 1H),
7.31 (s, 1H), 7.29 (d, J = 2.6 Hz, 1H), 7.07–7.03 (m, 2H), 7.02–
6.99 (m, 1H), 6.92 (dd, J = 8.8, 2.4 Hz, 1H), 6.74 (dd, J = 8.5,
2.0 Hz, 1H), 6.41 (s, 1H), 3.82 (s, 3H), 3.79–3.75 (m, 2H), 3.08
(t, J = 6.5 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 169.25 (s),
162.33 (s), 154.24 (s), 139.66 (s), 131.55 (s), 127.62 (s), 126.31
(s), 122.95 (s), 119.06 (s), 118.60 (s), 112.78 (d, J = 9.7 Hz),
112.28 (d, J = 8.2 Hz), 100.27 (s), 55.84 (s), 40.11 (s), 25.01 (s).
MS (ESI): 344.79 (C18H17ClN2O3, [M + H+]).
4.2 Biological evaluation
4.2.1 Cell culture. The human lung adenocarcinoma epi-
thelial cell line (A549), human cervix adenocarcinoma cell
line (HeLa), human gastric cancer cell line (MGC-803), hu-
man hepatoma cell line (HepG-2) and human breast cancer
cell line (MCF-7) were purchased from the Type Culture Col-
lection of the Chinese Academy of Sciences, Shanghai, China.
Cells were grown in Dulbecco's Modified Eagle's Medium
(DMEM, Gibco, NY, USA) mixed with 10% fetal bovine serum
(Gibco, NY, USA), and all the cells were cultured in a 5% CO2
incubator at 37 °C.
4.2.2 MTT. Cells were transplanted into 96-well plates at a
density of 6000 cells per well and placed in the incubator for
24 h. For each assay, E20 at five different concentrations
(128, 64, 32, 16, and 8 μmol L−1) and one control (3‰
DMSO) were added to six different wells and placed in the in-
cubator for 48 h. After 48 h, MTT solution (20 μL) was added
to each well and the plate was placed in the incubator for 4
h. Formazan was dissolved in 150 μL of DMSO. The absor-
bance (optical density, OD) was monitored on a Wellscan
MK-2 microplate reader (LabSystems) at 490 nm. The IC50
values of each compound were calculated by Graph Pad Pro
5.0. The assay was performed three times.
4.2.3 Cell migration assay. MGC-803 cells were trans-
planted into 6-well plates at a density of 1 × 105 cells per well
for 24 h. We scratched the cell culture with a 200 μL sterile
pipette tip, then removed the floating cells with phosphate-
buffered saline (PBS) and treated the cell culture with differ-
ent concentrations of E20 and the control group (3‰ DMSO)
for 48 h. We captured images of the cells with a microscopic
camera system at the time of treatment (0 h) and after 48 h
to assess the re-growth of the colony back into the damaged
region. The assay was performed three times.
5-Chloro-2-hydroxy-N-(2-(5-methoxy-1H-indol-3-yl)ethyl)-
benzamide (E35). White powder, 68.22% yield, mp 131–135
°C. 1H NMR (400 MHz, CDCl3) δ 12.51 (s, 1H), 8.08 (s, 1H),
7.41–7.35 (m, 1H), 7.32 (s, 1H), 7.29 (d, J = 3.5 Hz, 1H), 7.13
(dd, J = 8.0, 1.2 Hz, 1H), 7.00 (d, J = 7.9 Hz, 1H), 6.91 (dd, J =
8.8, 2.4 Hz, 1H), 6.78 (dd, J = 11.2, 4.0 Hz, 1H), 6.47 (s, 1H),
3.81 (d, J = 4.1 Hz, 3H), 3.78 (d, J = 6.3 Hz, 2H), 3.09 (t, J = 6.5
4.2.4 Analysis of cellular apoptosis. MGC-803 cells (1 × 105
cells per well) were transplanted into 6-well plates and incu-
bated for 24 h. Then the cells were treated with different con-
centrations of E20 for 24 h and the control group was treated
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with 3‰ DMSO. The cells were collected by trypsin digestion
and washed twice at 4 °C with PBS. Then after centrifugation
at 1200 rpm and removal of the supernatants, the cells were
mixed with 400 μL of 1× binding buffer, 5 μL of Annexin
V-FITC and 5 μL of propidium iodide (PI) (BD Pharmingen,
USA), and incubated at 25 °C for 15 min in the dark. The
stained cells were analyzed by flow cytometry. The assay was
performed three times.
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4.2.5 Colony formation assay. MGC-803 cells (1000 cells
per well) were placed in 6-well plates and placed in the incu-
bator for 24 h. They were then treated with different concen-
trations of E20 and left in the incubator for 9 d, where the
culture medium was changed every 3 d. At the end of the ex-
periment the culture medium was removed, and the cells
washed twice with PBS, immobilized with 4% paraformalde-
hyde for 20 min, stained with crystal violet for 15 min and
washed with PBS. The assay was performed three times.
4.2.6 Cell cycle analysis. MGC-803 cells (1 × 105 cells per
well) were incubated in 6-well plates for 24 h and treated with
different concentrations of E20 (20, 40, and 60 μmol L−1) for
24 h and the control group was treated with 3‰ DMSO. After
this time period, the culture medium was removed and the
cells were washed twice with PBS, transferred to a centrifuge
tube and centrifuged (1200 rpm). The cells were then washed
at 4 °C with PBS and immobilized with 70% ethanol solution
for 12 h at 4 °C. Then the cells were mixed with RNase A and
propidium iodide staining solution (Beyotime, China). Flow
cytometry was used to analyze the cells. The assay was
performed three times.
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4.2.7 Western blot. MGC-803 cells (1 × 105 cells per well)
were placed in 6-well plates for 24 h and treated with differ-
ent concentrations of E20 (10, 20, and 40 μmol L−1) for 24 h.
The expression of HIF-1α was investigated under CoCl2 treat-
ment (100 μM) and serum starvation (0.1% FBS)25 in order to
create the hypoxia environment. Protein expression was ana-
lyzed by western blotting as described elsewhere26 (Image
J2x). The assay was performed three times.
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Conflicts of interest
The authors declare that there are no conflicts of interest.
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Acknowledgements
This work was financially supported by Hunan Province Cooper-
ative Innovation Center for Molecular Target New Drug Study
(0223-0002-0002000-49), Innovation Platform Open Foundation
of the Education, Department of Hunan province (contract grant
number 16K079, 17K082) and Graduate Student Science Foun-
dation of the University of South China (2018490).
24 C. Granchi and F. Minutolo, ChemMedChem, 2012, 7,
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