Discovery of novel indole derivatives that inhibit NEDDylation and MAPK pathways against gastric cancer MGC803 cells

Article abstract of DOI:10.1016/j.bioorg.2021.104634

A series of novel indole derivatives were synthesized and evaluated for their antiproliferative activity against three selected cancer cell lines (MGC803, EC-109 and PC-3). Among these analogues, 2-(5-methoxy-1H-indol-1-yl)-N-(4-methoxybenzyl)-N-(3,4,5-trimethoxyphenyl)acetamide (V7) showed the best inhibitory activity against MGC803 cells with an IC₅₀ value of 1.59 μM. Cellular mechanisms elucidated that V7 inhibited colony formation, induced apoptosis and arrested cell cycle at G2/M phase. Importantly, indole analogue V7 inhibited NEDDylation pathway and MAPK pathway against MGC803 cells.

Full text of DOI:10.1016/j.bioorg.2021.104634

Bioorganic Chemistry 107 (2021) 104634
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Discovery of novel indole derivatives that inhibit NEDDylation and MAPK
pathways against gastric cancer MGC803 cells
,c,d
Dong-Jun Fua, Xin-Xin Cuib, Ting Zhub, Yan-Bing Zhangb, Yang-Yang Hue, Li-Rong Zhangb
,
,
c
,
,c,d,f,*
Sheng-Hui Wangb d, Sai-Yang Zhangb
a Modern Research Center for Traditional Chinese Medicine, School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100029, China
b School of Basic Medical Science, Zhengzhou University, Zhengzhou 450001, China
c The Academy of Medical Science, Zhengzhou University, Zhengzhou 450001, China
d Henan Institute of Advanced Technology, Zhengzhou University, Zhengzhou 450001, China
e Faculty of Science, The University of Melbourne, Victoria 3010, Australia
f State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210023, Jiangsu, 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 indole derivatives were synthesized and evaluated for their antiproliferative activity against
three selected cancer cell lines (MGC803, EC-109 and PC-3). Among these analogues, 2-(5-methoxy-1H-indol-1-
yl)-N-(4-methoxybenzyl)-N-(3,4,5-trimethoxyphenyl)acetamide (V7) showed the best inhibitory activity against
Indole
Apoptosis
NEDDylation pathway
MAPK pathway
MGC803 cells
MGC803 cells with an IC50 value of 1.59 μM. Cellular mechanisms elucidated that V7 inhibited colony formation,
induced apoptosis and arrested cell cycle at G2/M phase. Importantly, indole analogue V7 inhibited NEDDy-
lation pathway and MAPK pathway against MGC803 cells.
1. Introduction
FRET binding assay and inhibit cullin NEDDylation in the pulse-chase
NEDD8 transfer assay [12]. In addition, some NAE inhibitors has been
Neddylation,
a
3-step enzymatic cascade involving NEDD8-
reported, such as compounds 3–6, but while there still were some
shortages about potency and druglike physical properties in those
compounds [13]. Nucleoside analogue 7 as an NAE and UAE dual in-
hibitor inhibited conjugation of ubiquitin to intracellular proteins and
prevented the formation of cytoprotective aggresomes in A549 lung
cancer cells [14].
activating enzyme (NAE3/UBA3, E1), NEDD8-conjugating enzyme E2
(UBC12 or UBE2F) and substrate-specific E3s, regulates a variety of
biological processes by affecting the subcellular localization, stability,
conformation and function of target proteins [1–3]. Recently, accumu-
lated experimental data reported that the levels of Neddylation enzymes
(e.g. NEDD8 E1 and NAE1/UBA3) are higher in human cancers,
including gastric cancer, lung cancer, liver cancer, esophagus cancer and
colorectal cancer, when compared to adjacent normal tissues [4,5].
Thus, elevated status of NEDDylation modification might be an onco-
genic event during carcinogenesis, leading to degradation and ubiq-
uitylation of many tumor suppressor substrates (e.g. ATF4, c-myc and
p21) [6]. Scheme 1.
In continuation with our efforts toward the identification of NED-
Dylation inhibitors with anticancer potential, we attempt to discovery
structurally diverse scaffolds to design novel NEDDylation inhibitors.
Recently, our group has reported three NEDDylation inhibitors (Fig. 2):
(1) DC-2 specifically inhibited the interactions of DCN1 and UBE2M at
molecule and cellular levels, resulting in the decrease of cull-
in3 NEDDylation and accumulation of its substrate [15]; (2) WS-383
inhibited Cul3/1 Neddylation selectively over other cullins and also
induced accumulation of p21, p27, and NRF2 against MGC803 cells
[16]; (3) Flavokawain B inhibited NEDD8 conjugations to both Cullin1
and Ubc12 in prostate cancer cell lines [17].
Inhibition of protein Neddylation has emerged as a promising anti-
cancer strategy since the discovery of the NAE inhibitor MLN4924
(Fig. 1), and MLN4924 is currently in several phase I and II clinical trials
for cancer therapy [7–10]. However, as a result of its broad ablation of
Neddylation, MLN4924 has a series of toxicities [11]. Compound 2
could inhibit the DCN1-UBE2M protein–protein interaction in the TR-
Indoles as one of the most abundant heterocycles among biologically
active natural products and chemical agents possess interesting
* Corresponding author at: School of Basic Medical Science, Zhengzhou University, Zhengzhou 450001, China.
E-mail addresses: zzufdj@sina.com (D.-J. Fu), saiyangz@zzu.edu.cn (S.-Y. Zhang).
https://doi.org/10.1016/j.bioorg.2021.104634
Received 8 September 2020; Received in revised form 3 January 2021; Accepted 4 January 2021
Available online 8 January 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

D.-J. Fu et al.
Bioorganic Chemistry 107 (2021) 104634
pharmacological properties [18–20]. Importantly, various indoles were
designed and synthesized as potential anticancer candidates [21,22]. To
discovery more potent Neddylation inhibitors and anticancer agents
[23–32], we synthesized a series of novel indole derivatives based on the
anticacer indole scaffold. In this work, synthetic indoles were evaluated
for their antiproliferative activity against three cancer cell lines (EC-109,
PC-3 and MGC803). Among them, indole V7 was evaluated for its
apoptosis and deneddylation effects in gastric cancer MGC803 cells. Our
findings in this work might suggest that the indole would be a new
scaffold for the development of NEDDylation inhibitors.
1.59
μ
M against MGC803 cancer cells. The relationships between the
halogen substituents and the antiproliferative activities were 4-Br > 4-
Cl > 4-F. Compounds V6 ~ V10, V12 and V14 with electron-donating
groups on the phenyl ring A displayed moderate to potent inhibitory
effects against all cancer cells with IC50 values from 1.59 μM to 71.83
μ
M. Replacing the 4-OCH3 of V7 with 4-CH3 of V6 led to a decrease of
the anticancer activity in vitro against all cancer cells. However,
changing the 4-C(CH3)3 of V9 to 3-OCH3 of V12 led to a significant
improvement of the activity against MGC803, PC-3 and EC-109 cell
lines. These inhibitory results indicate that substituent groups on the
phenyl ring A exhibited an important effect for the anticancer activity in
vitro.
2. Results and discussion
In addition, the importance of 3,4,5-triOCH3 group on the phenyl
ring B for inhibitory activity was also explored. When the 3,4,5-triOCH3
group was replaced by 3,4-diOCH3, 4-OCH3, or hydrogen atom, the
antiproliferative activity was obviously decreased. Indole derivatives
2.1. Chemistry
The synthetic route of novel indole derivatives was shown in Scheme
1. Phenylamine derivatives I was subjected to nucleophilic substitution
reaction with benzyl chloride analogues II to afford secondary amine III
in the presence of potassium carbonate. The intermediate III was reacted
with chloroacetyl chloride to obtain the tertiary amide analogues IV in
the ethyl acetate solvent system. The target analogues V1 ~ V17 were
easily obtained at the reflux condition with 5-methoxy-1H-indole in the
presence of sodium hydroxide. The structure confirmation and NMR
analysis of indole derivatives V1 ~ V17 were described in the Sup-
porting Information.
V15 ~ V17 exhibited very weak activity with IC50 values of > 80 μM
against PC-3 and EC-109 cell lines.
2.3. Compound V7 selectively inhibited the viability against gastric cancer
cells
Based on the results of inhibitory activities, compound V7 was
selected to explore its cell viabilities against gastric cancer MGC803
cells, HGC-27 cells, SGC-7901 cells, and normal GES-1 cells. Among all
four cell lines, compound V7 displayed best antiproliferative activity
against MGC803 cells in a concentration dependent manner (Fig. 3A).
2.2. Antiproliferative activity evaluation
The viability of MGC803 cells after the 2 μmol/L treatment is approxi-
mately 28%, while the viability of GES-1 cells remains higher than 78%.
In addition, compound V7 inhibited the growth against MGC803 cells,
HGC-27 cells and SGC-7901 cells in a time-dependent manner (Fig. 3B).
Taking the results together, V7 selectively inhibited the viability against
gastric cancer cells.
In order to discovery potent anticancer agents and potential Ned-
dylation pathway inhibitors, we evaluated the anticancer activity in vitro
of all indole derivatives V1 ~ V17 against cancer cell lines (PC-3,
MGC803 and EC-109) using the MTT assay. In this paper, 5-fluorouracil
(5-Fu) was used as the control drug in the MTT assay. The anticancer
activity in vitro results against all three cancer cells for indole derivatives
V1 ~ V17 were shown in Table 1.
2.4. Compound V7 caused proliferation inhibition against MGC803 cells
To explore the effect of substituent groups on the phenyl ring A for
inhibitory activity, different electron-withdrawing groups and electron-
donating groups were introduced into the targeted compounds. All these
indole derivatives V1 ~ V14 exhibited the effective activity against
MGC803 cells and EC-109 cells. Especially, 2-(5-methoxy-1H-indol-1-
yl)-N-(4-methoxybenzyl)-N-(3,4,5-trimethoxyphenyl)acetamide (V7)
showed the most excellent antiproliferative activity with an IC50 value of
In order to evaluated for the proliferation ability of indole V7 against
MGC803 cells, the effects of cell morphology changes and colony for-
mation were also evaluated. MGC803 cell morphology changes after 48
h treatment were shown in Fig. 4A. As the concentration rised, cells
rounding up and cell debris were observed especially at high concen-
trations. We next checked the growth rate influenced by V7. As shown in
R1
R1
NH2
O
Cl
R1
NH
N
c
a
b
+
Cl
R2
R2
R2
IV
I
II
III
O
R1
O
N
N
R2
V1~V17
Scheme 1. Reagents and conditions: (a) K2CO3, acetone, reflux; (b) chloroacetyl chloride, ethyl acetate, rt; (c) NaOH, 5-methoxy-1H-indole, acetone, reflux.
2

D.-J. Fu et al.
Bioorganic Chemistry 107 (2021) 104634
the Fig. 4B and Fig. 4D, MGC803 cells were co-incubated with different
concentrations of V7 to determine the effects on formatting colonies.
After 7 days incubation, the cell colonies were obviously decreased by
compound V7. We next checked the growth effects influenced by V7. As
shown in the Fig. 4C, following the treatment with compound V7, the
viability of MGC803 cells was decreased in concentration-dependent
and time-dependent manners. All these results revealed that V7
inhibited the cell proliferation.
two different signaling pathways. The extrinsic apoptotic signal, which
responded mainly to extracellular stimuli, involves death receptors, and
the intrinsic apoptotic process, activated by modulators within the cell
itself, involves the mitochondria [34]. The complex interplay between
proapoptotic and antiapoptotic members of the Bcl-2 family plays a
crucial role in cellular fate determination [35].
As shown in Fig. 6, Bcl-2 family members highly involved in intrinsic
mitochondria apoptosis pathway were detected. The pro-apoptosis
protein Bax was upregulated, while the anti-apoptosis protein Bcl-2
and Bcl-xL were downregulated. The executers Caspase 3/7 were
evidently activated, and their substrate PARP was also activated by V7
in a concentration dependent manner. The important apoptosis inhibitor
cIAP1 was down-regulated. All these results suggested that V7 induced
cell apoptosis of MGC803 cells in an extrinsic and intrinsic combined
pathway.
2.5. 2.5 compound V7 induced the apoptosis against MGC803 cells
In order to explore the apoptotic effects, 4′,6-diamidino-2-phenyl-
indole (DAPI) staining was performed in MGC803 cells. Some remark-
able nuclear changes including nuclear fragmentation and condensation
were observed when MGC803 cells were treated with V7 (Fig. 5A).
MGC803 cells were analyzed after stained by Annexin V-FITC/PI to
determine the apoptosis percentages. As shown in Fig. 5B, apoptosis
effects of MGC803 cells was induced by compound V7. From the right
panel, it revealed the apoptosis percentages in MGC803 cells after
treated by V7. Treatment of MGC803 cells with compound V7 resulted
in a concentration-dependent increase (Fig. 5C), and the percentage of
apoptotic cells was up to 4.4%, 11.1%, and 65.2%, respectively,
compared to the control (3.2%).
2.7. Compound V7 arrested cell cycle at G2/M phase and induced DNA
damage
Cell cycle checkpoint mechanisms are crucial for the protection and
maintenance of genome integrity during tumorigenesis [36]. Cell cycle
progression is tightly controlled by cyclins and cyclin-dependent kinases
(CDKs), the sequential activation of which allows for the initiation of
and transition between different phases of the cell cycle [37]. Results in
Fig. 7A&B showed that V7 arrested cell cycle at G2/M phase
concentration-dependently. In addition, V7 decreased the expression
level of p-Histone H3 in MGC803 cells (Fig. 7C). The expression levels of
CDK1 and CyclinB1 in MGC803 cells were unchanged.
MGC803 cells were analyzed after stained by Annexin V-FITC/PI to
determine the apoptosis percentages. As shown in Fig. 5B, apoptosis
effects of MGC803 cells was induced by compound V7. From the right
panel, it revealed the apoptosis percentages in MGC803 cells after
treated by V7. Treatment of MGC803 cells with compound V7 resulted
in a concentration-dependent increase (Fig. 5C), and the percentage of
apoptotic cells was up to 4.4%, 11.1%, and 65.2%, respectively,
compared to the control (3.2%).
DNA repair can determine fate of a cell with treatment stress which
either resists over to survive or respond to toxic agents [38]. When the
repair mechanisms are unsuccessful, it may cause cellular senescence
(permanent cell cycle arrest), oncogenesis, normal embryonic develop-
ment, and maintenance of cell homeostasis [39]. In the comet assay, the
tail lengths of comet were lengthened by V7 (Fig. 7D). These results
indicated that V7 caused a strong DNA damage in MGC803 cells.
2.6. Compound V7 activated extrinsic and intrinsic apoptosis pathways
against MGC803 cells
Apoptosis is a very tightly programmed cell death with distinct
biochemical and genetic pathways that play a critical role in the ho-
meostasis of cancer cells [33]. The process of apoptosis is triggered by
NH2
O
O
O
S
NH2
O
HO
S
N
N
N
N
O
O
F
F
O
N
N
N
N
N
H
NH
F
O
N
N
N
Rh
N
O
NH
S
O
1(MLN4924)
4
2
3
OH
HO
OH
O
O
O
O
H
O
O
N
N
N
O
NH
HO
N
N
O
O
N
OH
O
H
H2N
O
OH
S
O
N
N
O
OH
HO
HO
5
6
7
Fig. 1. Structures of reported NEDDylation inhibitors in other groups.
3

D.-J. Fu et al.
Bioorganic Chemistry 107 (2021) 104634
2.8. Compound V7 inhibited the MAPK pathway
Table 1
Antiproliferative activity of the indole derivatives V1
~
V17.
Mitogen-activated protein kinase (MAPK) pathway is a critical
pathway for human cancer cell survival [40]. There are three indepen-
dent MAPK pathways composed of three signaling families closely
related to cell proliferation: the ERK family, the P38 family and the JNK
family [41]. It has been originally shown that ERKs are important for cell
survival, whereas JNKs and p38-MAPKs were deemed stress responsive
and thus involved in apoptosis [42,43].
O
R1
A
O
N
N
B
R2
V1~V17
Biomarker of each pathway was chosen to determine the effect of V7
on MAPK. As shown in the Fig. 8, p-c-Jun was not regulated by V7, while
p-ERK and p-P38 have been down-regulated. Among them, p-ERK has a
most significant change, so we detected the up-stream proteins nextly.
Results showed that c-Raf, p-c-Raf, p-MEK1/2 and p-ERK1/2 were
down-regulated while Ras remained the same. All these results indicated
that compound V7 inhibited the MAPK pathway in MGC803 cells.
Co-incubating with V7, proteasome inhibitor MG132 did not recover
the down-regulation of c-Raf and p-c-Raf (Fig. 9A). Moreover, com-
pound V7 cannot promote the degradation of c-Raf and p-c-Raf
(Fig. 9B). Results here suggested the down-regulation of p-c-Raf by V7 is
not the result of promoting the degradation of p-c-Raf. V7 inhibited the
activity of Ras to have an influence on the phosphorylation of c-Raf.
Taking these together, V7 may inhibit the expression of c-Raf, then
inhibit the activation of MEK1/2 and ERK1/2 resulting in the cell pro-
liferation inhibition.
Compound
R1
R2
IC50
(
μ
M)a
EC-109
47.57 ±
2.52
PC-3
MGC803
22.59 ±
1.74
V1
H
3,4,5-
>80
triOCH3
3,4,5-
V2
4-F
47.77 ±
3.50
>80
43.47 ±
1.11
triOCH3
3,4,5-
V3
4-Cl
46.82 ±
2.89
34.53 ±
1.52
51.52 ±
1.12
triOCH3
3,4,5-
V4
4-Br
3-F
29.07 ±
0.84
21.23 ±
1.48
25.09 ±
1.64
triOCH3
3,4,5-
V5
64.61 ±
3.08
>80
20.69 ±
1.87
triOCH3
3,4,5-
V6
4-CH3
4-OCH3
3-CH3
21.00 ±
0.44
18.04 ±
0.84
7.62 ±
0.04
triOCH3
3,4,5-
V7
14.52 ±
0.29
3.56 ±
0.08
1.59 ±
0.01
triOCH3
3,4,5-
V8
39.40 ±
0.64
43.83 ±
2.55
64.67 ±
2.59
triOCH3
3,4,5-
V9
4-C
58.25 ±
1.16
8.36 ±
0.22
34.55 ±
0.77
(CH3)3
2-CH3
triOCH3
3,4,5-
2.9. Compound V7 inhibited NEDDylation
V10
V11
V12
V13
V14
V15
71.83 ±
4.67
39.03 ±
1.11
18.56 ±
0.86
triOCH3
3,4,5-
3-Cl
37.45 ±
0.83
38.09 ±
0.77
66.93 ±
2.37
To discovery a novel NEDDylation inhibitor, the effects of V7 on
NEDDylation were detected. In an in vitro NEDD8 conjugation assay, V7
inhibited the conjugation of Ubc12 and NEDD8 formatting Ubc12-
NEDD8, the band of Ubc12-NEDD8 is getting smaller evidently with
the effect of 2803 (Fig. 10A).
triOCH3
3,4,5-
3-OCH3
3-CH2Cl
7.58 ±
0.65
7.01 ±
0.17
19.04 ±
1.95
triOCH3
3,4,5-
52.08 ±
0.51
18.94 ±
0.47
36.57 ±
0.56
triOCH3
3,4,5-
3,5-
37.45 ±
0.83
38.09 ±
0.77
66.93 ±
2.37
Inner the MGC803 cells, V7 also inhibited the NEDDylation of
NEDDylation E1 (NAE1) and two members of NEDDylation E3 com-
plexes (Cullin 1 and Cullin 3). In Fig. 10B, the NEDDylation of each
protein (with one NEDD8 conjugated, the NEDDylation band will be 9
kDa higher than the original proteins) were significantly inhibited by
V7. To see the overall effect of V7 on NEDDylation, the whole protein
lysate was used to do the western blotting with anti-NEDD8. As shown in
the Fig. 10C, V7 inhibited the general tendency of NEDDylation inner
MGC803 cells. Nextly, we checked the two important NEDDylation
substrates involved in cell death and proliferation. Both Death Receptor
5 (DR5) and P21 were clearly accumulated as a result of NEDDylation
inhibition (Fig. 10E). To sum up, V7 inhibited NEDDylation in a con-
centration dependent manner against MGC803 cells.
diOCH3
4-OCH3
triOCH3
H
>80
>80
19.43 ±
0.82
V16
V17
4-OCH3
4-OCH3
4-OCH3
3,4-
>80
>80
>80
>80
>80
>80
diOCH3
–
5-Fu
–
15.53 ±
12.87 ±
8.22 ±
0.27
1.10
0.56
a
Antiproliferative activity was assayed by exposure for 48 h.
resolution: 3.00 Å). We could find that compound V7 could be well
docked in the active cavity of NAE (Fig. 10D). Its 4-methoxyl-phenyl
group was located in a hydrophobic pocket consist of Ile75, Met101,
Lys147, Ile148, Leu166 and Ala171. Meanwhile, the 4-methoxyl-phenyl
group had a hydrogen bond with the backbone of Ile148. The 3,4,5-tri-
methoxyl-phenyl group could have hydrophobic interaction with
Met101, Ala170, Ile309 and Ile310, and form a hydrogen bond with
The biological tests have proved that compound V7 interacted with
NAE1 regulatory subunit to inhibit UBC12 NEDDylation in MGC-803
cells. Based on this, a docking study was performed to explore the
detailed interactions between compound V7 and NAE (PDB code: 3GZN,
Neddylation inhibitors reported by our group:
N
S
N
N
N
OH
O
O
N
S
N
N
S
N
CN
N
Cl
N
N
O
S
S
N
Flavokawain B
DC-2
WS-383
Fig. 2. NEDDylation inhibitors reported by our group.
4

D.-J. Fu et al.
Bioorganic Chemistry 107 (2021) 104634
MGC803
A
B
100%
50%
0%
SGC-7901
HGC-27
GES-1
MGC803
0.5 μM
1 μM
2 μM
0
12
24
36
48
Time Interval (Hours)
HGC-27
SGC-7901
100%
50%
0%
100%
50%
0%
0.5 μM
0.5 μM
1 μM
2 μM
1 μM
2 μM
0
12
24
36
48
0
12
24
36
48
Time Interval (Hours)
Time Interval (Hours)
Fig. 3. A. Cell viabilities of different cell lines with the treatment of V7 for 48 h treatment; B. Cell viabilities of MGC803 cells, HGC-27 cells and SGC-7901 cells with
the treatment of V7 for different hours. The results shown were representative of three independent experiments. *: p < 0.05 verse control. **: p < 0.01 verse control.
A
2 μmol/L
0 μmol/L
1 μmol/L
0.5 μmol/L
2 μmol/L
0 μmol/L
1 μmol/L
0.5 μmol/L
B
Fig. 4. A. Cell morphology changes of MGC803 cells after 48 h treatment; B. Representative images of MGC803 cells colonies after treatment for a week; C. Growth
curve of MGC803 cells; D. Statistical analysis of colony formation rates. The results shown were representative of three independent experiments. **: p < 0.01 verse
control. ****: p < 0.0001 verse control.
Serl68. Moreover, the 5-methoxy-indole group has hydrophobic inter-
action with surrounding residues Leu80, Lys124, Asp167, Thr203 and
Ile310. The 5-methoxy-indole group could also form hydrogen bonds
with Leu80 and Lys124. From the docking result, we could find com-
pound V7 could bind to NAE via hydrogen bonds and hydrophobic in-
teractions and therefore inhibit NAE as well as NEDD8 pathway, which
could explain the good anticancer activity of compound V7.
3. Conclusion
A series of indole derivatives were synthesized and evaluated for
their antiproliferative activity against PC-3, MGC803 and EC-109 cells.
Among all these analogues, compound V7 possessed the best anti-
proliferative ability with an IC50 value of 1.59 μM against MGC803 cells.
Compound V7 could inhibit MGC803 cell growth selectively and
5

D.-J. Fu et al.
Bioorganic Chemistry 107 (2021) 104634
A
2 μmol/L
0 μmol/L
1 μmol/L
0.5 μmol/L
2 μmol/L
0 μmol/L
1 μmol/L
0.5 μmol/L
B
Annexin V-FITC
D
C
80
60
40
20
0
Early apoptosis Late apoptosis
Cell rates (%)
Control 0.5 μmol/L
1 μmol/L
3.5±2.01
7.6±1.23
11.1±1.78
2 μmol/L
48.1±3.24
17.1±2.11
65.2±2.89
Early apoptosis 1.5±0.12
2.0±1.02
2.4±1.71
4.4±1.87
Late apoptosis
Total
1.7±0.27
3.2±0.32
Control
0.5 μmol/L
1 μmol/L
2 μmol/L
Fig. 5. A. MGC803 cells were stained by DAPI after 48 h treatment, red arrows were drawn to point out the nuclear condensation; B. MGC803 cells were stained by
Annexin V-FITC/PI and analyzed by flow cytometry after 48 h treatment; C&D. Apoptotic rates of MGC803 cells. (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
arrest cell cycle at G2/M phase. Importantly, indole V7 inhibited
NEDDylation and MAPK pathways against MGC803 cells, which led to
activated extrinsic and intrinsic apoptosis pathways (Fig. 11). From the
results of docking analysis, compound V7 could bind to NEDD8 acti-
vating enzyme (NAE) via hydrogen bonds and hydrophobic interactions.
It suggested that the indole derivative V7 as a NEDDylation inhibitor
might be a promising lead compound.
Supporting information.
N-benzyl-2-(5-methoxy-1H-indol-1-yl)-N-(3,4,5-trimethox-
yphenyl)acetamide (V1)
White solid, m.p.:114 ~ 115 ◦C, yield: 45%. 1H NMR (400 MHz,
DMSO‑d6) δ 7.34 – 7.29 (m, 2H), 7.26 (dd, J = 10.3, 7.3 Hz, 3H), 7.17 (d,
J = 8.9 Hz, 1H), 7.13 (t, J = 3.3 Hz, 1H), 7.04 (d, J = 2.4 Hz, 1H), 6.76
(dd, J = 8.8, 2.4 Hz, 1H), 6.57 (s, 2H), 6.32 (d, J = 2.9 Hz, 1H), 4.85 (t, J
= 8.7 Hz, 4H), 3.75 (s, 3H), 3.69 (d, J = 6.4 Hz, 6H), 3.66 (s, 3H). 13
C
4. Materials and methods
NMR (100 MHz, DMSO‑d6) δ 167.30, 153.48, 153.05, 137.33, 137.01,
136.13, 131.57, 130.07, 128.39, 128.28, 128.23, 127.18, 110.94,
110.39, 105.88, 102.04, 100.42, 59.97, 55.92, 55.31, 52.48, 48.06.
HRMS (ESI) (m/z) [M + H]+ calcd for C27H29N2O5, 461.2076; found,
461.2078.
4.1. Chemistry part
All reagents and solvents used were of analytical grade and were
purchased from Zhengzhou Research Biotechnology Co., Ltd. 1H NMR
and 13C NMR spectra were recorded on a Bruker 400 and 100 MHz
spectrometer with TMS as internal standard in DMSO‑d6. High-
resolution mass spectra (HRMS) were recorded on a Waters Micromass
QT of Micromass spectrometer by electrospray ionization (ESI).
N-(4-fluorobenzyl)-2-(5-methoxy-1H-indol-1-yl)-N-(3,4,5-trime-
thoxyphenyl)acetamide (V2)
White solid, m.p.:107 ~ 108 ◦C, yield: 85%. 1H NMR (400 MHz,
DMSO‑d6) δ 7.27 (dd, J = 8.2, 5.8 Hz, 2H), 7.19 – 7.10 (m, 4H), 7.03 (d,
J = 2.3 Hz, 1H), 6.75 (dd, J = 8.8, 2.4 Hz, 1H), 6.57 (s, 2H), 6.31 (d, J =
2.9 Hz, 1H), 4.84 (d, J = 8.8 Hz, 4H), 3.75 (s, 3H), 3.71 (s, 6H), 3.66 (s,
3H). 13C NMR (100 MHz, DMSO‑d6) δ 167.31, 162.60, 160.18, 153.48,
153.10, 137.05, 136.00, 133.57, 133.54, 131.58, 130.45, 130.37,
130.06, 128.38, 115.08, 114.87, 110.94, 110.40, 105.90, 102.03,
100.41, 59.97, 55.96, 55.31, 51.72, 48.05. HRMS (ESI) (m/z) [M + H]+
calcd for C27H28FN2O5, 479.1982; found, 479.1987.
General procedure for the synthesis of compounds V1 ~ V17:
Compound IV was synthesized according to our previous reference [23].
5-Methoxy-1H-indole (10 mmol) and sodium hydroxide (10 mmol) was
added to the solution of crude product IV (10 mmol) in acetone (15 mL).
After stirring at 60 ◦C for 6 h, the reaction mixturewas concentrated to
remove then treated with a solution of ethyl acetate (20 mL) and H2O
(20 mL). The organic layer was washed with aqueous NaHCO3, water,
and brine, dried over anhydrous Na2SO4, and then concentrated. The
residue was purified by column chromatography with petroleum/ethyl
acetate (10:1) as an eluent to afford the target compounds V1 ~ V17.
The NMR analysis of all indole derivatives was showed in the
N-(4-chlorobenzyl)-2-(5-methoxy-1H-indol-1-yl)-N-(3,4,5-trime-
thoxyphenyl)acetamide (V3)
White solid, m.p.:113 ~ 114 ◦C, yield: 72%. 1H NMR (400 MHz,
DMSO‑d6) δ 7.37 (d, J = 8.4 Hz, 2H), 7.26 (d, J = 8.3 Hz, 2H), 7.16 (d, J
= 8.9 Hz, 1H), 7.10 (d, J = 2.9 Hz, 1H), 7.02 (d, J = 2.3 Hz, 1H), 6.74
6

D.-J. Fu et al.
Bioorganic Chemistry 107 (2021) 104634
0
0.5
1
2 μmol/L
PARP
cIAP1
cleaved-Parp
1.5
1.0
0.5
200
150
100
50
***
cleaved-PARP
*
**
**
cIAP1
***
Bax
0.0
1.5
0
0
0.5 μM 1 μM 2 μM
0
0.5 μM 1 μM 2 μM
Noxa
Bcl-xl
15
10
5
Noxa
Bcl2
****
1.0
0.5
0.0
**
****
Bcl-xl
0
0
0.5 μM 1 μM 2 μM
0
0.5 μM 1 μM 2 μM
cleaved-Caspase3
cleaved-Caspase8
cleaved-Caspase8
cleaved-Caspase3
80
60
40
20
0
40
30
20
10
0
****
****
Actin
***
0.5 μM 1 μM 2 μM
**
0.5 μM 1 μM 2 μM
***
*
0
0
Fig. 6. The effects of V7 on apoptosis proteins, MGC803 cells were treated with V7 for 48 h. The results shown were representative of three independent exper-
iments. *: p < 0.05 verse control. **: p < 0.01 verse control. ***: p < 0.001 verse control. ****: p < 0.0001 verse control.
(dd, J = 8.8, 2.4 Hz, 1H), 6.59 (s, 2H), 6.30 (d, J = 2.9 Hz, 1H), 4.84 (d, J
= 8.7 Hz, 4H), 3.74 (s, 3H), 3.70 (d, J = 13.2 Hz, 6H), 3.65 (s, 3H). 13C
NMR (100 MHz, DMSO‑d6) δ 167.40, 153.47, 153.10, 137.04, 136.36,
136.03, 131.79, 131.57, 130.18, 130.07, 128.36, 128.20, 110.93,
110.41, 105.86, 102.03, 100.41, 59.98, 56.00, 55.32, 51.83, 48.030.
HRMS (ESI) (m/z) [M + H]+ calcd for C27H28ClN2O5, 495.1687; found,
495.1689.
trimethoxyphenyl)acetamide (V6)
White solid, m.p.:96 ~ 97 ◦C, yield: 62%. 1H NMR (400 MHz,
DMSO‑d6) δ 7.15 (d, J = 8.9 Hz, 1H), 7.11 (s, 5H), 7.03 (d, J = 2.4 Hz,
1H), 6.75 (dd, J = 8.8, 2.4 Hz, 1H), 6.56 (s, 2H), 6.30 (d, J = 2.9 Hz, 1H),
4.84 (s, 2H), 4.79 (s, 2H), 3.75 (s, 3H), 3.69 (d, J = 8.9 Hz, 6H), 3.65 (s,
3H), 2.27 (s, 3H). 13C NMR (100 MHz, DMSO‑d6) δ 167.19, 153.46,
153.02, 136.97, 136.29, 136.16, 134.27, 131.56, 130.08, 128.76,
128.55, 128.37, 128.28, 110.92, 110.38, 105.92, 102.03, 100.38,
100.16, 59.98, 55.95, 55.32, 52.20, 48.04, 20.65. HRMS (ESI) (m/z) [M
+ H]+ calcd for C28H31N2O5, 475.2233; found, 475.2237.
N-(4-bromobenzyl)-2-(5-methoxy-1H-indol-1-yl)-N-(3,4,5-trime-
thoxyphenyl)acetamide (V4)
White solid, m.p.:119 ~ 121 ◦C, yield: 77%. 1H NMR (400 MHz,
DMSO‑d6) δ 7.50 (d, J = 8.3 Hz, 2H), 7.19 (dd, J = 15.3, 8.6 Hz, 3H),
7.11 (d, J = 2.9 Hz, 1H), 7.03 (d, J = 2.3 Hz, 1H), 6.75 (dd, J = 8.8, 2.4
Hz, 1H), 6.60 (s, 2H), 6.31 (d, J = 2.9 Hz, 1H), 4.85 (d, J = 8.6 Hz, 2H),
4.82 (s, 2H), 3.75 (s, 3H), 3.72 (s, 6H), 3.66 (s, 3H). 13C NMR (100 MHz,
DMSO‑d6) δ 167.41, 153.49, 153.12, 137.07, 136.78, 136.04, 131.58,
131.12, 130.54, 130.06, 128.37, 120.32, 110.95, 110.41, 105.87,
102.04, 100.42, 59.98, 56.00, 55.32, 51.92, 48.04. HRMS (ESI) (m/z)
[M + H]+ calcd for C27H28BrN2O5, 539.1182; found, 539.1187.
N-(3-fluorobenzyl)-2-(5-methoxy-1H-indol-1-yl)-N-(3,4,5-trime-
thoxyphenyl)acetamide (V5)
2-(5-Methoxy-1H-indol-1-yl)-N-(4-methoxybenzyl)-N-(3,4,5-tri-
methoxyphenyl)acetamide (V7)
White solid, m.p.:103 ~ 104 ◦C, yield: 50%. 1H NMR (400 MHz,
DMSO‑d6) δ 7.17 – 7.09 (m, 4H), 7.03 (d, J = 2.3 Hz, 1H), 6.86 (d, J =
8.5 Hz, 2H), 6.74 (dt, J = 11.9, 6.0 Hz, 1H), 6.52 (s, 2H), 6.30 (d, J = 3.0
Hz, 1H), 4.80 (d, J = 13.3 Hz, 2H), 4.76 (s, 2H), 3.74 (s, 3H), 3.71 (d, J =
8.6 Hz, 9H), 3.65 (s, 3H). 13C NMR (100 MHz, DMSO‑d6) δ 178.96,
168.05, 162.86, 156.28, 152.78, 149.30, 139.04, 136.65, 133.86,
129.86, 129.24, 128.61, 125.30, 124.70, 124.13, 120.40, 34.84, 30.79.
HRMS (ESI) (m/z) [M + H]+ calcd for C28H31N2O6, 491.2182; found,
491.2189.
White solid, m.p.:114 ~ 115 ◦C, yield: 45%. 1H NMR (400 MHz,
DMSO‑d6) δ 7.38 – 7.31 (m, 1H), 7.18 (d, J = 8.9 Hz, 1H), 7.11 (dd, J =
8.1, 4.2 Hz, 2H), 7.08 (d, J = 6.5 Hz, 2H), 7.03 (d, J = 2.3 Hz, 1H), 6.75
(dd, J = 8.8, 2.4 Hz, 1H), 6.63 (s, 2H), 6.31 (d, J = 2.9 Hz, 1H), 4.87 (d, J
= 6.8 Hz, 4H), 3.75 (s, 3H), 3.72 (s, 6H), 3.66 (s, 3H). 13C NMR (100
MHz, DMSO‑d6) δ 167.52, 163.32, 160.90, 153.48, 153.11, 140.32,
140.25, 137.06, 136.08, 131.58, 130.21, 130.13, 130.08, 128.38,
124.24, 114.94, 114.73, 114.06, 113.85, 110.93, 110.43, 105.85,
102.03, 100.42, 59.97, 55.99, 55.31, 52.03, 48.05. HRMS (ESI) (m/z)
[M + H]+ calcd for C27H28FN2O5, 479.1982; found, 479.1989.
2-(5-Methoxy-1H-indol-1-yl)-N-(4-methylbenzyl)-N-(3,4,5-
2-(5-Methoxy-1H-indol-1-yl)-N-(3-methylbenzyl)-N-(3,4,5-trime-
thoxyphenyl)acetamide (V8)
White solid, m.p.:115 ~ 116 ◦C, yield: 72%. 1H NMR (400 MHz,
DMSO‑d6) δ 7.21 – 7.14 (m, 2H), 7.05 (dt, J = 12.0, 10.6 Hz, 5H), 6.75
(dd, J = 8.8, 2.2 Hz, 1H), 6.56 (s, 2H), 6.30 (d, J = 2.8 Hz, 1H), 4.86 (s,
2H), 4.80 (s, 2H), 3.75 (s, 3H), 3.70 (s, 6H), 3.65 (s, 3H), 2.27 (s, 3H).
13C NMR (100 MHz, DMSO‑d6) δ 167.25, 153.49, 153.02, 137.32,
137.25, 137.02, 136.20, 131.54, 130.09, 128.83, 128.40, 128.11,
127.77, 125.35, 110.93, 110.36, 105.91, 102.08, 100.38, 59.98, 55.94,
55.34, 52.42, 48.12, 20.90. HRMS (ESI) (m/z) [M + H]+ calcd for
7

D.-J. Fu et al.
Bioorganic Chemistry 107 (2021) 104634
A
2 μmol/L
1 μmol/L
0 μmol/L
0.5 μmol/L
PI
C
B
0
0.5
1
2 μmol/L
CDK1
CyclinB1
p-Histone H3
Actin
D
2 μmol/L
1 μmol/L
0 μmol/L
0.5 μmol/L
Fig. 7. A&B. MGC803 cells were stained by PI and cell cycle percentages were analyzed after 48 h treatment; C. The expression levels of CDK1, CyclinB1 and p-
Histone H3 in MGC803 cells affected by V7; D. The comet assay.
C28H31N2O5, 475.2233; found, 475.2236.
7.07 (m, 3H), 7.04 (d, J = 2.3 Hz, 1H), 6.75 (dd, J = 8.8, 2.4 Hz, 1H),
6.62 (d, J = 9.0 Hz, 2H), 6.31 (d, J = 2.5 Hz, 1H), 4.93 – 4.80 (m, 4H),
3.75 (s, 3H), 3.72 (s, 6H), 3.66 (s, 3H). 13C NMR (100 MHz, DMSO‑d6) δ
167.52, 163.32, 160.90, 153.48, 153.10, 140.31, 140.24, 139.85,
137.07, 136.08, 136.00, 132.88, 131.58, 131.55, 130.21, 130.12,
130.08, 128.38, 128.10, 127.17, 126.96, 124.24, 114.94, 114.72,
114.06, 113.85, 110.93, 110.42, 105.85, 102.04, 100.42, 59.98, 55.99,
55.32, 52.02, 51.93, 48.04. HRMS (ESI) (m/z) [M + H]+ calcd for
N-(4-(tert-butyl)benzyl)-2-(5-methoxy-1H-indol-1-yl)-N-(3,4,5-
trimethoxyphenyl)acetamide (V9)
White solid, m.p.:46 ~ 47 ◦C, yield:45%. 1H NMR (400 MHz,
DMSO‑d6) δ 7.37 – 7.28 (m, 2H), 7.25 – 7.05 (m, 4H), 7.04 (d, J = 2.2 Hz,
1H), 6.75 (dt, J = 9.4, 4.7 Hz, 1H), 6.43 (d, J = 68.3 Hz, 2H), 6.32 (t, J =
6.8 Hz, 1H), 4.83 (d, J = 12.2 Hz, 2H), 4.80 (s, 2H), 3.75 (s, 3H), 3.74 –
3.67 (m, 6H), 3.66 (s, 3H), 1.26 (s, 9H). 13C NMR (100 MHz, DMSO‑d6) δ
167.13, 153.49, 153.22, 153.01, 149.63, 137.04, 136.09, 134.41,
131.59, 131.00, 130.07, 128.39, 128.19, 127.97, 125.65, 124.92,
111.90, 111.07, 110.93, 110.40, 106.01, 102.06, 101.64, 100.77,
100.39, 59.98, 55.87, 55.33, 55.22, 52.15, 48.06, 34.14, 31.08. HRMS
(ESI) (m/z) [M + H]+ calcd for C31H37N2O5, 517.2702; found,
517.2705.
C27H28ClN2O5, 495.1687; found, 495.1689.
2-(5-Methoxy-1H-indol-1-yl)-N-(3-methoxybenzyl)-N-(3,4,5-tri-
methoxyphenyl)acetamide (V12)
White solid, m.p.:97 ~ 98 ◦C, yield: 47% 1H NMR (400 MHz,
DMSO‑d6) δ 7.23 (d, J = 6.5 Hz, 1H), 7.15 (dd, J = 14.5, 9.0 Hz, 2H),
7.04 (s, 1H), 6.78 (dd, J = 16.6, 11.2 Hz, 4H), 6.59 (d, J = 5.0 Hz, 2H),
6.31 (s, 1H), 4.85 (d, J = 11.9 Hz, 2H), 4.82 (s, 2H), 3.82 – 3.72 (m, 3H),
3.68 (dd, J = 19.0, 3.8 Hz, 12H). 13C NMR (100 MHz, DMSO‑d6) δ
167.30, 159.20, 153.48, 153.04, 138.92, 137.05, 136.19, 131.57,
130.07, 129.29, 128.39, 120.45, 113.75, 112.68, 110.92, 110.38,
105.92, 102.06, 100.40, 59.98, 55.96, 55.33, 54.93, 52.42, 48.07.
HRMS (ESI) (m/z) [M + H]+ calcd for C28H31N2O6, 491.2182; found,
491.2187.
2-(5-Methoxy-1H-indol-1-yl)-N-(2-methylbenzyl)-N-(3,4,5-trime-
thoxyphenyl)acetamide (V10)
White solid, m.p.:108 ~ 110 ◦C, yield:70%. 1H NMR (400 MHz,
DMSO‑d6) δ 7.19 – 7.10 (m, 6H), 7.03 (d, J = 2.3 Hz, 1H), 6.75 (dd, J =
8.8, 2.3 Hz, 1H), 6.55 (s, 2H), 6.31 (d, J = 2.9 Hz, 1H), 4.86 (d, J = 6.0
Hz, 4H), 3.75 (s, 3H), 3.68 (s, 6H), 3.64 (s, 3H), 2.15 (s, 3H). 13C NMR
(100 MHz, DMSO‑d6) δ 166.99, 153.47, 152.97, 137.05, 136.19, 135.74,
134.95, 131.58, 130.09, 129.91, 129.15, 128.38, 127.21, 125.63,
110.92, 110.38, 106.08, 102.06, 100.39, 60.01, 55.96, 55.34, 49.93,
48.06, 18.66. HRMS (ESI) (m/z) [M + H]+ calcd for C28H31N2O5,
475.2233; found, 475.2238.
N-(3-(chloromethyl)benzyl)-2-(5-methoxy-1H-indol-1-yl)-N-
(3,4,5-trimethoxyphenyl)acetamide (V13)
White solid, m.p.:71 ~ 72 ◦C, yield: 64%. 1H NMR (400 MHz,
DMSO‑d6) δ 7.33 (d, J = 4.3 Hz, 2H), 7.30 (s, 1H), 7.21 (s, 1H), 7.16 (d, J
= 8.8 Hz, 1H), 7.12 (d, J = 2.5 Hz, 1H), 7.04 (d, J = 2.1 Hz, 1H), 6.76
(dd, J = 8.8, 2.2 Hz, 1H), 6.57 (s, 2H), 6.31 (d, J = 2.7 Hz, 1H), 4.87 (s,
4H), 4.73 (s, 2H), 3.75 (s, 3H), 3.70 (s, 6H), 3.65 (s, 3H). 13C NMR (100
MHz, DMSO‑d6) δ 167.36, 153.50, 153.08, 137.82, 137.70, 137.04,
N-(3-chlorobenzyl)-2-(5-methoxy-1H-indol-1-yl)-N-(3,4,5-trime-
thoxyphenyl)acetamide (V11)
White solid, m.p.:109 ~ 110 ◦C, yield: 80%. 1H NMR (400 MHz,
DMSO‑d6) δ 7.47 – 7.21 (m, 2H), 7.18 (dd, J = 8.8, 5.6 Hz, 1H), 7.14 –
8

D.-J. Fu et al.
Bioorganic Chemistry 107 (2021) 104634
A
0
0.5
1
2 μmol/L
p-c-Raf
c-Raf
1.5
1.0
0.5
0.0
1.5
1.0
0.5
0.0
Ras
c-Raf
***
p-c-Raf
***
****
p-MEK1/2
ERK1/2
p-ERK1/2
Actin
0
0.5 μM 1 μM 2 μM
0
0.5 μM 1 μM 2 μM
p-MEK1/2
p-ERK1/2
1.5
1.0
0.5
0.0
1.5
1.0
0.5
0
0.5
1
2 μmol/L
****
B
***
0.5 μM 1 μM 2 μM
p-P38
0.0
1.5
0
0.5 μM 1 μM 2 μM
0
p-c-Jun
p-JNK
p-P38
Actin
1.0
0.5
0.0
**
**
***
0
0.5 μM 1 μM 2 μM
Fig. 8. A&B: The expression levels of V7 on MAPK pathway proteins, MGC803 cells were treated with V7 for 48 h. The results shown were representative of three
independent experiments. **: p < 0.01 verse control. ***: p < 0.001 verse control. ****: p < 0.0001 verse control.
A
B
V7 (2μmol/L)
−
−
+
−
+
+
V7 (2μmol/L)
−
−
+
−
+
+
+
+
+
+
+
CHX (50μg/mL) +
MG132(5μmol/L)
−
+
0
12 24
0
12 24 hours
c-raf
c-raf
p-c-raf
actin
p-c-raf
actin
Fig. 9. A: MGC803 cells were treated by V7 with/without MG132 (a proteasome inhibitor) for 24 h; B: MGC803 cells were treated by V7 with/without CHX
(cycloheximide) for different time.
136.07, 131.54, 130.08, 128.63, 128.54, 128.41, 128.32, 127.70,
110.94, 110.36, 105.84, 102.09, 100.41, 59.96, 55.93, 55.34, 52.23,
48.11, 46.06. HRMS (ESI) (m/z) [M + H]+ calcd for C28H30ClN2O5,
509.1843; found, 509.1847.
131.56, 130.06, 128.40, 110.91, 110.36, 106.11, 105.91, 102.07,
100.40, 98.98, 59.98, 59.72, 55.97, 55.33, 55.08, 52.50, 48.10. HRMS
(ESI) (m/z) [M + H]+ calcd for C29H33N2O7, 521.2288; found,
521.2289.
N-(3,5-dimethoxybenzyl)-2-(5-methoxy-1H-indol-1-yl)-N-(3,4,5-
trimethoxyphenyl)acetamide (V14)
2-(5-Methoxy-1H-indol-1-yl)-N-(4-methoxybenzyl)-N-phenyl-
acetamide (V15)
Yellow solid, m.p.:46–47 ◦C, yield:50%. 1H NMR (400 MHz,
DMSO‑d6) δ 7.15 (d, J = 8.9 Hz, 1H), 7.11 (d, J = 2.8 Hz, 1H), 7.03 (d, J
= 2.3 Hz, 1H), 6.74 (dd, J = 8.8, 2.4 Hz, 1H), 6.61 (s, 2H), 6.39 (s, 3H),
6.31 (d, J = 2.9 Hz, 1H), 4.88 (s, 2H), 4.78 (s, 2H), 3.75 (s, 3H), 3.72 (s,
6H), 3.68 (d, J = 6.4 Hz, 6H), 3.66 (s, 3H). 13C NMR (100 MHz,
DMSO‑d6) δ 167.34, 160.36, 153.49, 153.04, 139.71, 137.07, 136.24,
Yellow solid, m.p.:44 ~ 45 ◦C, yield:55% 1H NMR (400 MHz, DMSO)
δ 7.46 (t, J = 7.2 Hz, 2H), 7.39 (d, J = 6.7 Hz, 1H), 7.33 (d, J = 7.4 Hz,
2H), 7.16 (d, J = 2.5 Hz, 1H), 7.11 (t, J = 7.4 Hz, 3H), 7.04 (d, J = 2.3
Hz, 1H), 6.84 (d, J = 8.4 Hz, 2H), 6.76 (dd, J = 8.8, 2.4 Hz, 1H), 6.32 (d,
J = 2.9 Hz, 1H), 4.80 (d, J = 13.9 Hz, 2H), 4.72 (s, 2H), 3.75 (s, 3H), 3.72
(s, 3H). 13C NMR (100 MHz, DMSO) δ 166.91, 158.41, 153.49, 140.68,
9

D.-J. Fu et al.
Bioorganic Chemistry 107 (2021) 104634
A
0
0.5
1
2 μmol/L
B
0
20
200 μmol/L
UBC12-NEDD8
NAE1-NEDD8
NAE1
Ubc12-NEDD8
Ubc12
0
0.5
1
2 μmol/L
C
100 kDa
60 kDa
Cullin1-NEDD8
Cullin1
Cullin3-NEDD8
Cullin3
NEDD8
Actin
Actin
E
0
0.5
1
2 μmol/L
D
DR5
P21
Actin
NAE1-NEDD8
Cullin1-NEDD8
Ubc12-NEDD8
1.5
1.0
0.5
0.0
1.5
1.5
1.0
0.5
0.0
F
1.0
0.5
0.0
*
**
***
***
***
0
0.5 μM 1 μM 2 μM
0
0.5 μM 1 μM 2 μM
0
0.5 μM 1 μM 2 μM
DR5
P21
Cullin3-NEDD8
6
6
1.5
1.0
0.5
0.0
***
*
4
2
0
4
2
0
*
**
**
0
0.5 μM 1 μM 2 μM
0
0.5 μM 1 μM 2 μM
0
0.5 μM 1 μM 2 μM
Fig. 10. A. An in vitro NEDD8 conjugation assay out of cells; B&C. The effect of V7 on NEDDylation, MGC803 cells were treated with V7 for 48 h; D. Docking results
of compound V7 that interacted with NAE (PDB code: 3GZN); E. The expression levels of NEDDylation substrates (DR5 and P21) by compound V7; F. Statistical
analysis of proteins expression in MGC803 cells. The results shown were representative of three independent experiments. *: p < 0.05 verse control. **: p < 0.01
verse control. ***: p < 0.001 verse control.
10

D.-J. Fu et al.
Bioorganic Chemistry 107 (2021) 104634
N8
Ras
Raf
Comp. V7
E1
N8 E1
P
P
P
E2
MEK1/2
ERK1/2
CRL
N8 E2
N8
Proliferation
Apoptosis
P21 ↑
DR5 ↑
cleaved-Caspase 8
Caspase 8
Caspase 3
cleaved-Caspase 3
Noxa ↑, Bcl-xl ↓
Fig. 11. V7 inhibits NEDDylation and MAPK pathways against gastric cancer MGC803 cells.
131.59, 130.32, 129.68, 129.47, 129.01, 128.45, 128.31, 113.67,
111.00, 110.22, 102.14, 100.35, 55.33, 54.96, 51.88, 47.85. HRMS
(ESI) (m/z) [M + H]+ calcd for C25H25N2O3, 401.1865; found,
401.1869.
4.2. Cell culture and MTT assay
◦
Cell lines were cultured at 37 C in an atmosphere containing 5%
CO2, RPMI-1640 medium with 10% fetal bovine serum, 100 U/ml
penicillin and 0.1 mg/ml streptomycin was used as culture medium
(Servicebio, Wuhan, China). Cells were seeded at a density of 5 × 103
per well in 96-well plates for 24 h and treated with compound for 48 h.
2-(5-Methoxy-1H-indol-1-yl)-N-(4-methoxybenzyl)-N-(4-methox-
yphenyl)acetamide (V16)
Yellow solid, m.p.:48 ~ 49 ◦C, yield:75%. 1H NMR (400 MHz,
DMSO‑d6) δ 7.23 (d, J = 8.8 Hz, 2H), 7.16 (d, J = 3.0 Hz, 1H), 7.10 (dd,
J = 8.4, 6.4 Hz, 3H), 7.04 (d, J = 2.3 Hz, 1H), 6.99 (t, J = 7.7 Hz, 2H),
6.85 (d, J = 8.6 Hz, 2H), 6.76 (dd, J = 8.8, 2.4 Hz, 1H), 6.32 (d, J = 2.9
Hz, 1H), 4.76 (s, 2H), 4.65 (d, J = 35.1 Hz, 2H), 3.76 (d, J = 4.5 Hz, 6H),
3.74 – 3.71 (m, 3H). 13C NMR (100 MHz, DMSO‑d6) δ 167.16, 158.70,
158.40, 153.47, 133.18, 131.60, 130.33, 129.55, 129.50, 129.12,
128.87, 128.44, 128.29, 128.17, 114.78, 114.55, 113.65, 110.97,
110.21, 102.12, 100.31, 55.33, 55.28, 54.95, 51.96, 47.75. HRMS (ESI)
(m/z) [M + H]+ calcd for C26H27N2O4, 431.1971; found, 431.1975.
N-(3,4-dimethoxyphenyl)-2-(5-methoxy-1H-indol-1-yl)-N-(4-
methoxybenzyl)acetamide (V17)
Then, 20 μL MTT solution (Servicebio, Wuhan, China) was added to each
well, and incubated for 4 h at 37 ◦C. 150
μL DMSO was added to each
well to dissolve the formazan after removing the supernatant liquid, the
absorbance was determined at 570 nm.
4.3. Colony formatting assay
MGC803 cells were seeded in a 6-well plate and incubated in 5% CO2
at 37 ◦C for 24 h, then treated with different concentration of compound.
After 7 days, remove the culture medium, wash the cells twice with PBS,
fix cells with 4% paraformaldehyde and stain with 0.1% crystal violet.
The cells’ images were captured with microscope (Nikon, Japan).
White solid, m.p.:49 ~ 50 ◦C, yield:55%. 1H NMR (400 MHz,
DMSO‑d6) δ 7.12 (dd, J = 15.0, 5.4 Hz, 4H), 7.04 (d, J = 2.3 Hz, 1H),
6.96 (d, J = 8.5 Hz, 1H), 6.86 (s, 2H), 6.84 (s, 1H), 6.81 – 6.72 (m, 2H),
6.31 (d, J = 2.9 Hz, 1H), 4.75 (d, J = 7.8 Hz, 4H), 3.76 (d, J = 3.4 Hz,
6H), 3.72 (d, J = 3.5 Hz, 6H). 13C NMR (101 MHz, DMSO‑d6) δ 167.17,
158.43, 153.47, 149.08, 148.42, 133.22, 131.60, 130.27, 129.69,
129.26, 128.42, 120.47, 113.62, 112.03, 111.73, 110.93, 110.28,
102.11, 100.29, 55.58, 55.52, 55.34, 54.98, 51.87, 47.81. HRMS (ESI)
(m/z) [M + H]+ calcd for C27H29N2O5, 461.2076; found, 461.2079.
4.4. Cell cycle distribution assay
MGC803 cells were seeded in 6-well culture plate and treated with
compound for 48 h. Then cells were harvested and fixed with 70%
◦
ethanol for 8 h at 4 C. The fixed cells were washed and resuspended
with PBS containing 50 mg/mL and PI 10 mg/mL RNaseA (Servicebio,
Wuhan, China). Then cell suspension was incubated for 30 min in a dark
11

D.-J. Fu et al.
Bioorganic Chemistry 107 (2021) 104634
place. After that, samples were analyzed for DNA content with flow
cytometry. (Becton, Dickinson and Company, NJ).
Acknowledgment
This research was funded by the National Natural Sciences Founda-
tions of China (No. 81703541 & U2004123 for Sai-Yang Zhang and No.
81673322 for Yan-Bing Zhang), China Postdoctoral Science Foundation
(No. 2018M632812 for Sai-Yang Zhang), Henan Association of Science
and Technology (No. 2020HYTP056 for Sai-Yang Zhang, China), Science
and Technology Department of Henan Province (No. 20202310144, for
Sai-Yang Zhang, China), the open fund of state key laboratory of Phar-
maceutical Biotechnology, Nan-jing University, China (Grant no. KF-
GN-202104). The manuscript was also supported by China Post-
doctoral Science Foundation (Grant no. 2020M670239).
4.5. Cell apoptosis assay
MGC803 cells were seeded in 6-well culture plate and treated with
compound for 48 h. Then cells were harvested and resuspended in
binding buffer containing Annexin V-FITC (0.5 mg/mL) and PI (0.5 mg/
mL) and incubated for 30 min in a dark place. After that, samples were
analyzed with flow cytometry (Becton, Dickinson and Company, NJ).
4.6. Comet assay
MGC803 cells were treated with different concentration of com-
pound for 48 h. Then the cells were harvested and covered with comet
slides. The slides were immersed in the lysis buffer at 4 ◦C for 2 h. After
lysed, Comet slides were immersed in cold electrophoresis buffer for 25
min to equilibrate the slides. Then, electrophoresis was conducted at 25
V, 300 mA, for 30 min. Then the slides coated with drops of neutrali-
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104634.
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