Design, synthesis, and biological evaluation of 3′,4′,5′-trimethoxy evodiamine derivatives as potential antitumor agents

Article abstract of DOI:10.1002/ddr.21806

A series of compounds bearing 3′,4′,5′-trimethoxy module into the core structure of evodiamine were designed and synthesized. The synthesized compounds were screened in vitro for their antitumor potential. MTT results showed that compounds 14a–14c and 14i–14j had significant effects, with compound 14h being the most prominent, with an IC₅₀ value of 3.3 ± 1.5?μM, which was lower than evodiamine and 5-Fu. Subsequent experiments further confirmed that compound 14h could inhibit cell proliferation and migration, and induce G2/M phase arrest to inhibit the proliferation of HGC-27 cells, which is consistent with the results of the cytotoxicity experiment. Besides, 14h could inhibit microtubule assembly and might kill tumor cells by inhibiting VEGF and glycolysis. All experimental results indicate that compound 14h might be a potential drug candidate for the treatment of gastric cancer and was worthy of further study.

Full text of DOI:10.1002/ddr.21806

Received: 30 November 2020
DOI: 10.1002/ddr.21806
Revised: 8 February 2021
Accepted: 8 February 2021
R E S E A R C H A R T I C L E
Design, synthesis, and biological evaluation of 30,40,50-
trimethoxy evodiamine derivatives as potential antitumor
agents
Yijiao Peng1,2 | Runde Xiong1,2 | Zhen Li2 | Junmei Peng2 | Zhi-Zhong Xie2
Xiao-Yong Lei2 | Dongxiu He1,2 | Guotao Tang1,2
|
1Institute of Pharmacy and Pharmacology,
Abstract
Hunan Province Cooperative Innovation
A series of compounds bearing 30,40,50-trimethoxy module into the core structure of
evodiamine were designed and synthesized. The synthesized compounds were
screened in vitro for their antitumor potential. MTT results showed that compounds
14a–14c and 14i–14j had significant effects, with compound 14h being the most
prominent, with an IC50 value of 3.3 1.5 μM, which was lower than evodiamine and
5-Fu. Subsequent experiments further confirmed that compound 14h could inhibit
cell proliferation and migration, and induce G2/M phase arrest to inhibit the prolifer-
ation of HGC-27 cells, which is consistent with the results of the cytotoxicity experi-
ment. Besides, 14h could inhibit microtubule assembly and might kill tumor cells by
inhibiting VEGF and glycolysis. All experimental results indicate that compound 14h
might be a potential drug candidate for the treatment of gastric cancer and was wor-
thy of further study.
Center for Molecular Target New Drug Study,
University of South China, Hengyang City,
China
2Hunan Provincial Key Laboratory of Tumor
Microenvironment Responsive Drug Research,
Hengyang City, Hunan Province, China
Correspondence
Guotao Tang and Dongxiu He, Hunan
Provincial Key Laboratory of Tumour
Microenvironment Responsive Drug Research
University of South China 28# Chang-Sheng
Road, Hengyang City, Hunan Province,
421001, People's Republic of China.
Email: tgtzq@163.com (G.-T.T.) and
hdx711106@126.com (D.-X.H.)
Funding information
Hunan Postgraduate Research and Innovation
Project, Grant/Award Number: CX20190751;
Hunan Province Cooperative Innovation
Center for Molecular Target New Drug Study,
The natural science foundation of hunan
province, Grant/Award Number: 2020JJ4534;
Hunan Provincial Key Laboratory of Tumor
Microenvironment Responsive Drug Research,
Grant/Award Number: 2019-56
K E Y W O R D S
evodiamine, microtubule assembly, trimethoxy benzene
1
|
INTRODUCTION
inhibitor (AI) and vascular disrupting agent (VDA). Both preclinical and
clinical studies have shown that the combination of AI and VDA can
The tumor microenvironment plays an important role in the develop-
ment and progression of tumors, and its main features include low pH,
hypoxia, angiogenesis, and abnormal glucose metabolism (Chen
et al., 2018; Yang, 2017). When tumors grow beyond a certain size,
overexuberant growth requires tumor blood vessels to provide suffi-
cient oxygen and nutrients. The important role of tumor blood vessels
in tumor development makes it one of the antitumor targets (Gaya &
Rustin, 2005; McKeage & Baguley, 2010; Porcu et al., 2014). Tumor
vascular targeted drugs can be divided into two types: angiogenesis
increase antitumor effectiveness (Siemann et al., 2017; Siemann &
Shi, 2004).
The synthesis of novel colchicine site tubulin inhibitors based on
trimethoxyphenyl has become a research hotspot (Belleri et al., 2005; Li
et al., 2018). Some microtubule targeting agents containing TMP struc-
tures have been found to inhibit microtubule polymerization and act as
effective tumor-VDAs against cancer. CA4P (Figure 1) (Liang
et al., 2016; Quan et al., 2008; Zhang et al., 2013) and its similar
OXi4503 (Bothwell et al., 2016), AVE8062 (Sessa et al., 2013), colchi-
cine analog ZD6126 (LoRusso et al., 2008), BNC-105 (Nowak
et al., 2013), CKD-516 (Lee et al., 2010) all have 3,4,5-trimethoxyphenyl
Yijiao Peng and Runde Xiong contributed equally to this study.
Drug Dev Res. 2021;1–12.
wileyonlinelibrary.com/journal/ddr
© 2021 Wiley Periodicals, LLC.
1

2
PENG ET AL.
FIGURE 1 Schematic diagram of the structure of some compounds containing trimethoxyphenyl
scaffold. As a pharmacophore of Tumor-VDAs, Trimethoxyphenyl is
retained as the necessary active part, and the bridge chain in the middle
can be modified with amide bond, sulfonamide bond, and so on
(Herdman et al., 2016; Nguyen et al., 2012; Yan et al., 2015).
VEGF-induced angiogenesis (Huang et al., 2015) indicating that
evodiamine may be a potent angiogenesis inhibitor. At the same time,
evodiamine can downregulate the expression of PhosphoFructoKi-
nase (PFK) protein, the key enzyme of glucose metabolism, and inhibit
the transcription of Hypoxia Inducible Factor (HIF) and AKT genes
related to the Warburg effect (Jiang, 2015).
Evodiamine is an alkaloid extracted from Evodia (Tan
&
Zhang, 2016), which has a wide range of biological activities such as
antiinflammatory (Liao et al., 2011), antiobesity (Bak et al., 2010),
thermoregulation, and vasodilation (Gavaraskar et al., 2015). In
recent years, studies on evodiamine have found that its antitumor
function was realized by inhibiting proliferation, blocking the cell
cycle, and inducing apoptosis. Evodiamine can inhibit the prolifera-
tion of a variety of tumor cells, such as colon cancer cells (Huang
et al., 2015; Zhao et al., 2015), human gastric cancer cells (Rasul
et al., 2012; Yang et al., 2014), human lung cancer cells (Hong
et al., 2014), human hepatic cells (Hu et al., 2017), etc. Evodiamine
can significantly block the percentage of tumor cells in the G2/M of
the cell cycle. Its mechanism is related to the regulation of the
expression of cyclin, CDKs, and cell cycle checkpoint kinases (Fang
et al., 2014). In addition, it can also block some tumor cells in the G1
phase by affecting the expression of G1 phase-related proteins and
activating the p53-p21-Rb pathway (Chien et al., 2014). In terms of
inducing apoptosis, evodiamine can increase the expression of death
receptors on the surface of tumor cells and promote the binding of
death receptors and ligands, thereby promoting the death signal
complex formation and inducing tumor cell apoptosis (Khan
et al., 2015). Evodiamine can also change the permeability of the
mitochondrial membrane to release cytochrome C and apoptosis-
inducing factors into the cytoplasm to induce tumor cell apoptosis
(Chen et al., 2016; Jaiswal et al., 2015; Shen et al., 2015).
To enhance the blocking effect of evodiamine on tumor vascula-
ture, we have proposed combination principles to modify the struc-
ture of evodiamine by introducing the trimethoxy benzene group into
the E ring or modify it on indole N atom. Herein, we have synthesized
a series of new 30,40,50-trimethoxy evodiamine derivatives and identi-
fied compound 14h as an applicable drug candidate for antitumor che-
motherapy of cancer.
2
|
MATERIALS AND METHODS
| Chemicals
2.1
2.1.1
|
Materials and reagents
The materials and reagents used in the experiment were purchased
through commercial channels and could be used directly without fur-
ther purification unless otherwise specified. Using thin-layer chroma-
tography (TLC) on a silica gel 60F254 plates, visualizing the results
under 254 or 365 nm ultraviolet light as a reference, the compounds were
separated using silica gel (200 mesh size) by column chromatography. The
melting point of the compound was determined with a Thermo Scientific
electrothermal digital melting point instrument (uncorrected). ESI mass
spectrometry data was obtained by Waters GCT mass spectrometer. The
1H NMR spectrum was measured by Bruker AV-400 spectrometer, and
the experiment was carried out in the designated solvent (TMS as internal
The most interesting thing is that evodiamine can down-regulate
the expression of β-catenin, which leads to a decline of vascular endo-
thelial growth factor (VEGF) at the transcriptional level and affects

PENG ET AL.
3
standard). The δ value (ppm) was used to indicate the chemical shift value,
and the Hz was used to indicate the coupling constant (J).
The solution of 3,4-dihydro-β-carbene (3, 0.17 g, 1 mmol) and 4 (0.25 g,
1 mmol) in CH2Cl2 (20 ml) was stirred at room temperature for 24 h. The
crude product was purified by a silica gel column (CH2Cl2). Compound 6a
(0.38 g, 1 mmol) was added IBX (0.41 g, 1.5 mmol) in DMSO (20 ml) solu-
tion and stirred at room temperature for 2 h. Then added water (40 ml)
and extracted with ethyl acetate (3 × 30 ml). The composite organic phase
was removed under reduced pressure. The crude product was purified by
silica gel column (CH2Cl2) to get 7a–b.
2.1.2
|
Synthesis methods of compound 6a–b and
7a–b
The solution of tryptamine (1, 1.60 g, 0.01 mol) in ethyl formate (20 ml)
was heated to 60ꢀC, stirred for 6 h, and the compound 2 was obtained
after the ethyl formate was removed under reduced pressure. POCl3
(1 ml) was added to the solution of compound 2 in CH2Cl2 (30 ml) and
stirred at 0–5ꢀC for 6 h. The solvent was then removed under reduced
pressure. The crude product was purified by the silica gel column (CH2Cl2:
The compounds are listed in Table 1.
The 1H NMR data of compounds 6a–6b, and 7a–7b were sup-
plied as experiment data (Scheme 1).
CH3OH
=
10:1) to obtain 3. The solution of 2-amino-3,-
2.1.3
|
Synthesis methods of compound 14a–n
4,5-trimethoxybenzoic acid (4, 0.23 g, 1 mmol) and acetyl chloride (0.8 ml)
in ethyl chloroformate (15 ml) was heated to 95ꢀC and stirred for 2 h.
Cool to room temperature and precipitate compound 5 from the solution.
2-Oxybenzoic acid (12, 0.69 g, 5 mmol) was added with thionyl chlo-
ride (1.18 g, 10 mmol) in CH2Cl2 (25 ml) solution and stirred at 40ꢀC
for 1 h. The solvent was then removed under reduced pressure. The
residue was added to the solution of intermediate 3 (0.85 g, 5 mmol)
in CH2Cl2 (40 ml) and stirred at room temperature for 24 h. After the
reaction, the solvent was removed under reduced pressure, and the
crude product was purified by a silica gel column (CH2Cl2). The inter-
mediate 13 was obtained. NAH (0.05 g, 2 mmol) was added to the
solution of compound 13 (0.29 g, 1 mmol) in DMF (20 ml) and stirred
at 0ꢀC for 20 min. Then 3,4,5-trimethoxybenzoyl chloride (0.23 g,
1 mmol) was added and stirred at 0–5ꢀC for 24 h. Then add water
(40 ml) and precipitate the crude from the solution. The crude product
was purified by silica gel column (CH2Cl2) for 14a.
TABLE 1 Synthesis of E-ring substituted trimethoxy evodiamine
derivatives (6a, 6b, 7a, and 7b)
Compound
R
X
The preparation method of compound 14b–n was similar to the
above method.
6a
6b
7a
7b
–H
NH
=N
NH
=N
–H
The compounds are listed in Table 2.
The 1H NMR and ESI-MS spectra data of compounds 14a–14n
–CH3O
–CH3O
were supplied as experiment data (Scheme 2).
SCHEME 1 Synthetic route of 6a-b, 7a-b

4
PENG ET AL.
2.2
|
Cell culture
normal cell (GES-1) cytotoxicity. Added the cell suspension to a 96-well
plate and waited for the cells to adhere to the wall. After 48 h of incuba-
tion with different concentrations of drugs as required, 20 μl MTT was
added to each well. After the culture continued for 4 h, the liquid was
aspirated and 150 μl DMSO was added. Shaked in the dark for 10 min,
and used a Wellscan MK-2 microplate reader to measure the OD value at
a wavelength of 490 nm. GraphPad Prism 8.0 software was used to pro-
cess and analyze the experimental data. All experiments were performed
three times independently (Supporting Information).
The Bel-7402, A549, HCT-116, MCF-7, HGC-27, and GES-1 cells
used in the experiment were purchased from the Typical Culture Col-
lection Center of the Chinese Academy of Sciences in Shanghai,
China. The culture medium used for culturing these cells is configured
according to the relevant culture instructions. All cells were cultured
in a humidified incubator at 37ꢀC and 5% CO2.
2.3
|
Cytotoxicity test
2.4
|
Cell migration experiment
Detected 18 kinds of trimethoxyphenyl evodiamine derivatives on five
tumor cells (Bel-7402, A549, HCT-116, MCF-7, and HGC-27) and a
The scratch test was carried out in a six-well plate. After the cells
were evenly filled in the six-well plate, used a 10 μl pipette tip to draw
straight “wounds,” washed the suspended cells with phosphate buff-
ered solution (PBS), and then added different concentrations of drugs
to continue incubate for 48 h in an incubator. Observed and photo-
graphed the width of the “wound” at 0 and 48 h under the micro-
scope. The migration rate calculated by the formula was used as the
basis for the drug's ability to inhibit cell migration.
TABLE 2 Synthesis of B-ring substituted trimethoxy evodiamine
derivatives (14a–14n)
2.5
|
Colony experiment
A single-cell suspension was spread on six-well plates and incubated
in an incubator for 24 h. Cells were cultured with different concentra-
tions of compound 14h (0, 0.75, 1.5, and 3 μmol/L) for 9 days, and the
medium was changed every 3 days. Then the cells were fixed with 4%
paraformaldehyde for 15 min. The colonies were washed with PBS
and stained with 0.1% crystal violet for 15 min. After washed away
excess crystal violet in PBS, observed the colony distribution in the
six-well plate, and recorded the experimental results by taking
pictures.
Compound
14a
R
R1
–H
–H
14b
14c
–H
1–CH3
2–CH3
2–Cl
–H
14d
14e
–H
–H
3–Cl
14f
–H
3–Br
2–NO2
–H
14g
–H
14h
14i
–CH3O
–CH3O
–CH3O
–CH3O
–CH3O
–CH3O
–CH3O
2.6
|
Cell morphology analysis
1–CH3
2–CH3
2–Cl
14j
Hoechst 33258 can label living cells. The cell culture in the experi-
ment was similar to the colony experiment, incubating cells (12 h) and
drug action (48 h) were in a six-well plate. Washed twice with PBS
and fixed with 4% paraformaldehyde for 15 min. Then stained with
1 ml Hoechst 33258 solution in the dark for 10 min at 37ꢀC. After
14k
14l
3–Cl
14m
14n
3–Br
2–NO2
SCHEME 2 Synthetic route of trimethoxyphenyl evodiamine derivative 14a-n

PENG ET AL.
5
washed with PBS, the cells were observed with a fluorescence micro-
scope (Olympus, Japan), and the detection wavelength was 350 nm.
conditions, HGC-27 cells were incubated in different concentrations
(0, 0.75, 1.5, and 3 μmol/L) for compound 14h in triplicate for 48 h.
Lyse the HGC-27 cells after 48 h of drug treatment in the six-well
plate with lysis solution (phenylmethylsulfonyl fluoride [PMSF]:
RIPA = 1:100). Aspirated the centrifuged supernatant and took 5 μg
to detect protein concentration with the BCA kit. An equal amount
(30 μg) of protein was separated by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (8% gel, SDS-PAGE) and trans-
ferred to PVDF membrane. Blocked with 5% skim milk at room
2.7
|
Cell cycle and apoptosis
| Apoptosis detection assay
2.7.1
HGC-27 cells were transplanted into six-well plates overnight, then
different concentrations of compound 14h dissolved in DMSO were
added in triplicate for 48 h incubation and the control group was
treated with 3‰ DMSO. After that, the cells were collected by
trypsinization and washed twice with PBS. Then the cells resuspended
in 400 μl of 1× binding buffer, 5 μl of Annexin V-FITC, and 5 μl of PI
(BD Pharmingen, The United States). Finally, the stained cells were
incubated at 25ꢀC for 15 min in the dark and analyzed by flow
cytometry.
temperature for
2 h, and incubated the membrane with the
corresponding antibody at 4ꢀC overnight. TBST washes away excess
antibody and then incubated with an anti-rabbit horseradish
peroxidase-conjugated secondary antibody (VEGF [1:2000], Hexoki-
nase [HK] [1:2000], pyruvate kinase [PK] [1:1000], and PFK [1:1000])
at room temperature for 1 h. Enhanced chemiluminescence system
(Tanon6200, China) for visual detection of proteins. Each experiment
was performed three times independently.
2.7.2
|
Cell cycle analysis
2.10
|
Immunofluorescence
HGC-27 cells were transplanted into six-well plates overnight, then
different concentrations of compound 14h dissolved in DMSO
were added in triplicate for 48 h incubation and the control group
was treated with 3‰ DMSO. After that, the cells were washed with
PBS and immobilized with a 70% ethanol solution for 12 h at 4ꢀC.
Then the cells were mixed with RNase A and propidium iodide
staining solution (Beyotime, China). Finally, the stained cells were
incubated at 25ꢀC for 15 min in the dark and analyzed by flow
cytometry.
To investigate the effect of compound 14h on tubulin, immunofluores-
cence was used to study the effect of synthetic compounds on cellular
tubulin. In short, HGC-27 cells were treated with DMSO or different
concentrations of compounds 14h (0.75, 1.5, and 3 μmol/L) for 48 h,
and the dead cells were washed with PBS and fixed with 5% parafor-
maldehyde (PFA) at room temperature for 15 min, then Block for
30 min in PBS containing 1% bovine serine albumin (BSA). At room
temperature, cells were incubated with Cy3 labeled goat anti-mouse
IgG (1:250; Service Bio, CN) in a wet box for 1 h. Finally, DAPI (1:1000)
stained the nuclei in the dark for 15 min, and the samples were
mounted with an antifluorescence quencher. Sent the detection mech-
anism to test with a confocal laser microscope (Nikon C2 in Japan).
2.8
|
Determination of lactic acid content
HGC-27 cells were prepared as single-cell suspensions and cultured in
six-well plates for 24 h (37ꢀC, 5% CO2). Replaced with fresh culture
medium containing different concentrations of compound 14h
(0, 0.75, 1.5, and 3 μmol/L) and continue culturing for 48 h. The drug-
free medium containing 3‰ DMSO was used as the control group.
The working solution and cells in the lactic acid kit (Nanjing Jiancheng
Bioengineering Institute, Nanjing, China) were vortexed and mixed
and incubated at 37ꢀC for 10 min. The reaction was terminated under
the action of a terminator. The Wellscan MK-2 microplate reader
measures the absorbance at 530 nm. Calculate the LD level according
to the relevant formula. All experiments were repeated three times
independently.
2.11
|
Drug likeness
This study used the descriptor calculation module of MOE 2019. The
ligand property calculation function of MOE 2019 can calculate
molecular descriptors for all series of compounds (Table 3).
3
|
RESULTS AND DISCUSSION
| Chemistry
3.1
A series of evodiamine derivatives were synthesized by splicing
trimethoxyphenyl on the B ring or E ring of evodiamine (Figure 2).
During the synthesis process, it was found that the yield of the product
with a methoxy group at the 10-position was significantly higher than
that of the unsubstituted product at the 10-position, and the yield of
the compound with a halogen atom was also higher than that of the
2.9
|
Western blotting
The expression of the selected protein under the action of compound
14h was analyzed by Western blotting. In a nutshell, under normoxic

6
PENG ET AL.
TABLE 3 Physicochemical
descriptors of compounds 6a–b, 7a–b,
and 14a–n
Compound
6a
LogP
2.64
3.39
2.50
3.25
5.50
5.72
6.0
Lip_acc
Lip_don
LogD (pH = 7)
2.64
3.39
2.50
3.25
5.50
5.72
6.0
LogS
TPSA
75.82
76.15
81.29
85.38
79.23
79.23
79.23
79.23
79.23
79.23
125.05
88.46
88.46
88.46
88.46
88.46
88.46
134.28
Weight
379.42
377.40
409.44
407.43
484.51
498.53
498.53
518.95
518.95
563.40
529.50
514.53
528.56
528.56
548.98
548.98
593.43
559.53
4
5
5
6
6
6
6
6
6
6
6
7
7
7
7
7
7
7
2
1
2
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
−4.58
−5.23
−4.62
−5.27
−7.23
−7.56
−7.67
−8.01
−7.99
−8.17
−7.85
−7.37
−7.60
−7.71
−8.05
−8.03
−8.22
−7.89
6b
7a
7b
14a
14b
14c
14d
14e
14f
6.21
6.18
6.35
5.62
5.37
5.59
5.87
6.08
6.05
6.22
5.49
6.21
6.18
6.35
5.62
5.37
5.59
5.87
6.08
6.05
6.22
5.49
14g
14h
14i
14j
14k
14l
14m
14n
FIGURE 2 The design and
synthesis of trimethoxyphenyl
evocarpine derivatives
methyl and nitro groups. The compound obtained through spectral data
analysis was consistent with the expected design compound.
different data we detected might be related to the modification of the
structure of the E ring and the B ring, especially the B ring compounds
were more sensitive to HGC-27 cells. Among them, 14a–c and 14i–j had
significant effects, 14h (IC50 = 3.3 1.5 μM) was the most prominent,
even lower than evodiamine and 5-Fu, so compound 14h was selected
for further study. In addition to its good antiproliferative activity against
cancer cells, the toxicity of the compound to noncancer cells reflected
its safety to a certain extent and was also an important indicator. All
compounds were tested with GES-1 cells and did not show relatively
high cytotoxicity. The results illustrated that these compounds were
selective for the attack of cancer cells.
3.2
|
In vitro antitumor cell proliferation
experiment
In the experiment, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT) assay was mainly used to detect the cytotoxicity of the
drug. Evodiamine derivatives had good antiproliferative activity against
five types of cancer cells, as shown in the data recorded in Table 4. The

PENG ET AL.
7
TABLE 4 IC50 value of compounds
6a–b, 7a–b, and 14a–n
IC50 SDa (μM)
Compound
6a
Bel-7402
>100
A549
HCT-116
>100
MCF-7
>100
HGC-27
GES-1
>100
36.3 1.8
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
25.6 2.2
>100
>100
>100
6b
35.4 2.3
>100
25.3 1.2
>100
64.3 3.3
>100
>100
38.2 3.1
>100
7a
>100
7b
>100
98.2 2.3
17.3 2.1
85.6 3.2
13.5 1.5
>100
>100
>100
88.2 1.3
6.1 2.5
7.3 1.3
6.2 2.6
92.9 1.3
58.7 2.0
68.0 3.2
90.2 1.5
3.3 1.5
7.3 2.1
6.7 1.4
90.2 2.0
49.3 1.3
10.7 4.3
85.0 1.3
22.6 3.2
18.9 2.7
14a
36.5 1.2
>100
85.2 2.3
>100
80.8 2.0
92.3 1.3
95.7 3.3
>100
14b
14c
33.3 2.3
>100
95.2 2.3
>100
14d
14e
>100
>100
>100
>100
14f
>100
>100
>100
>100
14g
>100
>100
>100
>100
14h
14i
28.1 1.3
8.2 3.3
>100
8.5 2.1
29.3 1.3
11.9 2.3
>100
80.8 2.3
91.2 1.5
>100
72.3 1.5
90.2 3.3
85.3 1.6
>100
14j
14k
>100
>100
14l
>100
>100
>100
>100
14m
14n
Evodiamine
5-Fu
>100
>100
>100
>100
>100
>100
>100
>100
38.1 1.3
36.9 1.2
>100
>100
>100
61.7 5.4
32.6 2.1
57.9 3.7
aData are the average of three independent assays; SD, standard deviation.
FIGURE 3 Detection of the ability of drugs to inhibit cell migration. Compound 14h inhibited the migration of HGC-27 cells. (a) The width of
the “wound” after 0 and 48 h was observed and photographed under a microscope; (b) HGC-27 cell mobility was negatively correlated with
compound 14h. Error basis showed the SD, “**” p < .01, compared with the control group
3.3
|
Cell migration and colony formation
intuitively see the inhibitory effect was concentration-dependent.
Besides, The colonies stained with crystal violet reflected the effects of
different concentrations of drugs in a six-well plate. Statistical results
(Figure 4) showed that the cell proliferation of the drug group was more
or less inhibited compared with the control group, and the high concen-
tration group was almost completely suppressed. Reflected the good
antiproliferative activity against HGC-27 cells in compound 14h.
The scratch experiment explored the effect of compound 14h on the
migration of HGC-27 cells. As shown in Figure 3, the scratch area of
the control group healed almost completely after 48 h, while the
healing rate of the scratch in the drug-treated group was significantly
slowed down (Figure 3(a)). The migration rate (Figure 3(b)) could

8
PENG ET AL.
FIGURE 4 Compound 14h
inhibited the proliferation of HGC-27
cells
FIGURE 5 Cell morphology
analysis. Hoechst staining was
used to observe the effect of
HGC-27 cells treated with
compound 14h on apoptosis.
(a) Cell chromatogram under the
fluorescence microscope.
(b) There was a positive
correlation between the total
amount of apoptosis and the
concentration of compound 14h.
Error basis showed the SD, “**”
p < .01, compared with the
control group
FIGURE 6 Apoptosis analysis. Effect of compound 14h on apoptosis of HGC-27 cells after 48 h treatment. (a) Flow Jo software was used to
calculate the number of apoptotic cells. Q1, Q2, Q3, and Q4, respectively, represent cell fragments, early apoptotic cells, late apoptotic cells, and
normal living cells. (b) There was a positive correlation between the total amount of apoptosis and the concentration of compound 14h. Error
basis showed the SD, “**” p < .01, compared with the control group
3.4
|
Apoptosis and cycle experiment
cells with Annexin V-FITC and PI showed changes in the proportion
of apoptosis. Compared with the control group (4.53%), the apoptosis
rates of drug action were 6.66%, 13.63%, and 17.88% (Figure 6),
which showed a certain concentration dependence. These results
proved that compound 14h can induce apoptosis of HGC-27 cells,
After 48 h of drug action, the results (Figure 5) showed that HGC-27
cells showed chromatin condensation and nuclear fragmentation,
marking cell apoptosis. Flow cytometry analysis of the combination of

PENG ET AL.
9
FIGURE 7 Cell cycle analysis. The effect of compound 14h on the cell cycle distribution of HGC-27. (a) Flow cytometry analysis showed that
the distribution of the HGC-27 cell cycle was affected after 14h treatment for 48 h. The percentage of different stages of the cell cycle was
calculated by flow software. (b) The percentage of G1/G0, G2/M, and S phase was plotted by origin8 software. “*” p < .05, “**” p < .01
TABLE 5 Determination of lactate content in HGC-27 cells
concentration decreased the most. Phosphofructose kinase and hexo-
kinase played a key role in the glycolysis pathway. If the drug could
inhibit the expression of these two enzymes, it might be possible to
inhibit glycolysis by blocking the energy supply of the cells, thereby
starving the tumor cells. To verify this conjecture, we analyzed the
compound 14h by immunoblotting. Western blot analysis
(WB) results (Figure 8) showed that compound 14h had a certain
degree of inhibitory effect on the expression of VEGF, HK, PK, and
PFK. Compared with the control group, the protein expression in the
drug-treated group decreased significantly, indicating that these pro-
teins could be down-regulated at compound 14h. This suggested that
compound 14h might kill tumors by inhibiting VEGF and glycolysis.
treated with different concentrations for 14h comparison with control
group: “**” p<.01
Group
Control
14h
Concentration(μmol/L)
Lactate content(mmol/L)
13.251 0.005**
9.660 0.005**
—
0.75
1.5
3
8.732 0.006**
7.428 0.009**
and its antiproliferative effect was closely related to their strong
proapoptotic activity.
To investigate the effect of compound 14h on the cycle of HGC-
27 cells, we used flow cytometry to detect the cells after 48 h of drug
action. The cell cycle stage had a significant change (Figure 7), in
which the cell cycle distribution changes the most when the drug con-
centration is 3 μM. The number of cells in the G2/M phase increased
to 66.39%, the number of cells in the G0/G1 phase decreased signifi-
cantly to 3.04%, and there was no significant difference in the per-
centage of cells in the s phase (Figure 7(b)). Therefore, compound 14h
inhibited the proliferation of HGC-27 cells by inducing G2/M phase
arrest, which was consistent with the cytotoxicity test results.
3.6
|
Immunofluorescence and molecular docking
simulation
The control group's tubulin spread out around, showing a network struc-
ture, but the cellular tubulin of the drug action gathered around the cell
periphery (Figure 9), indicating that the aggregation of microtubules and
the formation of the spindle were disturbed, hindering the mitosis of the
cells leading to abnormal spindles. It was suggested that compound 14h
could inhibit the tubulin assembly of HGC-27 cells.
3.5
|
Western blot analysis of the effect on
glycolysis
3.7
|
Drug likeness
Lactic acid is closely related to intracellular energy metabolism. Com-
pared with the control group (Table 5), the lactic acid content of dif-
ferent concentrations of drugs decreased after 24 h, and the highest
The log P, Lipinski's acceptors (lip-acc), Lipinski's donors (lip-don), log
D (pH = 7), molecular weight, and total polar specific surface area

10
PENG ET AL.
FIGURE 8 Western blot data analysis. The expression of VEGF, PFKM, PKM2, and HK-2 in HGC-27 cells was influenced by the compound
14h. The Western blot results were quantitatively analyzed by calculating the ratio of the experimental group to the control group.
Representative photos of four independent experiments are shown. Error basis showed the SD, “**” p < .01, compared with the control group
FIGURE
9
Immunofluorescence
experiment results. The effect of
compound 14h (0, 0.75, 1.5, and
3 μmol/L) on tubulin. HGC-27
cells were treated with solvent
(0.1% DMSO, negative control)
and 14h (0, 0.75, 1.5, and 3 μmol/
L) for 48 h. Then cells were
stained with Cy3 binding anti-
β-tubulin antibody and DAPI.
Tubulin was shown in red and
DNA was shown in blue
(TPSA) were calculated to test the pharmaceutical properties of the
synthesized trimethoxyphenyl evodiamine derivatives (Aziz, Saeed,
Khan, Afridi, & Jabeen, 2020a; Aziz, Saeed, Khan, Afridi, Jabeen,
Ashfaq Ur, & Hashim, 2020b; Lipinski et al., 2001). These descriptors
have predictive power in evaluating the likelihood of a drug being
made into a drug. Drug-like molecules must have log P (the logarithm
of the octanol/water partition coefficient) ≤5, total polar specific
surface area (TPSA) <140 Å2, molecular weight ≤500, number of
hydrogen bond donors ≤5, and hydrogen bond acceptor ≤10 as per
Lipinski's RO5. The compounds conform to the drug-like principle to
provide a basis for its preparation. The detailed results of the drug
similarity of the 18 compounds are listed in Table 3. According to
Lipinski's rule, the bioavailability of 14h may not be as expected, and
more experiments are needed to explore and modify.

PENG ET AL.
11
Bothwell, K. D., Folaron, M., & Seshadri, M. (2016). Preclinical activity of
the vascular disrupting agent OXi4503 against head and neck cancer.
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Trends in Pharmacological Sciences, 39(1), 59–74. https://doi.org/10.
1016/j.tips.2017.10.009
4
|
CONCLUSION
By modifying the structure of ethylenediamine, a series of com-
pounds containing 3,4,5-trimethoxyphenyl scaffolds were obtained,
and their activities were tested in vitro. From the MTT results, we
found that compound 14h had high cytotoxicity to five kinds of can-
cer cells, especially HGC-27 cells. Subsequent experiments further
confirmed that compound 14h could inhibit cell proliferation and
migration, and induce G2/M phase arrest to inhibit the proliferation
of HGC-27 cells, which is consistent with the results of the cytotox-
icity experiment. Besides, 14h could inhibit microtubule assembly
and might kill tumor cells by inhibiting VEGF and glycolysis. In sum-
mary, compound 14h might be a potential drug candidate for the
treatment of gastric cancer, but its mechanism was still unclear and
deserves further study.
Chen, T. C., Chien, C. C., Wu, M. S., & Chen, Y. C. (2016). Evodiamine from
Evodia rutaecarpa induces apoptosis via activation of JNK and PERK
in human ovarian cancer cells. Phytomedicine, 23(1), 68–78. https://
doi.org/10.1016/j.phymed.2015.12.003
Chien, C. C., Wu, M. S., Shen, S. C., Ko, C. H., Chen, C. H., Yang, L. L., &
Chen, Y. C. (2014). Activation of JNK contributes to evodiamine-
induced apoptosis and G2/M arrest in human colorectal carcinoma
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Fang, C., Zhang, J., Qi, D., Fan, X., Luo, J., Liu, L., & Tan, Q. (2014).
Evodiamine induces G2/M arrest and apoptosis via mitochondrial and
endoplasmic reticulum pathways in H446 and H1688 human small-cell
lung cancer cells. PLoS One, 9(12), e115204. https://doi.org/10.1371/
journal.pone.0115204
ACKNOWLEDGMENTS
Gavaraskar, K., Dhulap, S., & Hirwani, R. R. (2015). Therapeutic and cos-
metic applications of evodiamine and its derivatives: A patent review.
Fitoterapia, 106, 22–35. https://doi.org/10.1016/j.fitote.2015.07.019
Gaya, A. M., & Rustin, G. J. (2005). Vascular disrupting agents: A new class
of drug in cancer therapy. Clinical Oncology (Royal College of Radiolo-
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Herdman, C. A., Strecker, T. E., Tanpure, R. P., Chen, Z., Winters, A.,
Gerberich, J., Liu, L., Hamel, E., Mason, R. P., Chaplin, D. J.,
Trawick, M. L., & Pinney, K. G. (2016). Synthesis and biological evalua-
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19.1.7
This work was financially supported by Hunan Provincial Key Labora-
tory of Tumor Microenvironment Responsive Drug Research
(Approval number: 2019-56), Hunan Province Cooperative Innovation
Center for Molecular Target New Drug Study, The Natural Science
Foundation of Hunan Province (Approval number: 2020JJ4534), and
Hunan Postgraduate Research and Innovation Project (Approval num-
ber: CX20190751).
CONFLICT OF INTEREST
The authors declare no conflicts of interest.
DATA AVAILABILITY STATEMENT
All data included in this study are available upon request by contact
with the corresponding author.
Hu, C. Y., Wu, H. T., Su, Y. C., Lin, C. H., Chang, C. J., & Wu, C. L. (2017).
Evodiamine exerts an anti-hepatocellular carcinoma activity through a
WWOX-dependent pathway. Molecules, 22(7), 1–8. https://doi.org/
10.3390/molecules22071175
ORCID
Zhi-Zhong Xie
Guotao Tang
https://orcid.org/0000-0003-3365-0379
https://orcid.org/0000-0001-9673-4727
Huang, J., Chen, Z. H., Ren, C. M., Wang, D. X., Yuan, S. X., Wu, Q. X.,
Chen, Q. Z., Zeng, Y. H., Shao, Y., Li, Y., Wu, K., Yu, Y., Sun, W. J., &
He, B. C. (2015). Antiproliferation effect of evodiamine in human colon
cancer cells is associated with IGF-1/HIF-1alpha downregulation.
Oncology Reports, 34(6), 3203–3211. https://doi.org/10.3892/or.
2015.4309
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How to cite this article: Peng Y, Xiong R, Li Z, et al. Design,
synthesis, and biological evaluation of 30,40,50-trimethoxy
evodiamine derivatives as potential antitumor agents. Drug
Dev Res. 2021;1–12. https://doi.org/10.1002/ddr.21806