Discovery of 1,2,4-triazine-based derivatives as novel neddylation inhibitors and anticancer activity studies against gastric cancer MGC-803 cells

Article abstract of DOI:10.1016/j.bmcl.2019.126791

Neddylation modification is often over-expressed in a variety of human tumor cells. Therefore, targeting neddylation pathway may represent a potential approach to the treatment of human tumors. Herein, we describe the discovery of a hit scaffold from our

Full text of DOI:10.1016/j.bmcl.2019.126791

Journal Pre-proofs
Discovery of 1,2,4-triazine-based derivatives as novel neddylation inhibitors
and anticancer activity studies against gastric cancer MGC-803 cells
Jian Song, Xin-Xin Cui, Bo-Wen Wu, Dong Li, Sheng-Hui Wang, Lei Shi, Ting
Zhu, Yan-Bing Zhang, Sai-Yang Zhang
PII:
S0960-894X(19)30756-5
DOI:
https://doi.org/10.1016/j.bmcl.2019.126791
BMCL 126791
Reference:
Bioorganic & Medicinal Chemistry Letters
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19 August 2019
28 October 2019
29 October 2019
Please cite this article as: Song, J., Cui, X-X., Wu, B-W., Li, D., Wang, S-H., Shi, L., Zhu, T., Zhang, Y-B., Zhang,
S-Y., Discovery of 1,2,4-triazine-based derivatives as novel neddylation inhibitors and anticancer activity studies
against gastric cancer MGC-803 cells, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/
10.1016/j.bmcl.2019.126791
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Discovery of 1,2,4-triazine-based derivatives as novel neddylation
inhibitors and anticancer activity studies against gastric cancer MGC-
803 cells
Jian Song a, b, c, 1, Xin-Xin Cui a, b, c, 1, Bo-Wen Wu a, c, Dong Li a, c, Sheng-Hui Wang a , Lei Shi a, c
,
Ting Zhu a, c, Yan-Bing Zhang a, c, Sai-Yang Zhang a, c, d,
*
a. School of Basic Medical Sciences, Institute of Drug Discovery & Development, Zhengzhou
University, Zhengzhou 450001, China
b. School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, China
c. Key Laboratory of Advanced Drug Preparation Technologies (Ministry of Education),
Zhengzhou University, Zhengzhou, 450001, China
d. Henan Institute of Advanced Technology, Zhengzhou University, Zhengzhou 450001, China
* Corresponding author: saiyangz@zzu.edu.cn (Sai-Yang Zhang)
1 These authors contributed equally to this work

Abstract:
Neddylation modification is often over-expressed in a variety of human tumor
cells. Therefore, targeting neddylation pathway may represent a potential approach to
the treatment of human tumors. Herein, we describe the discovery of a hit scaffold from
our in-house library and further structure-based optimizations. In this work, compound
V11 could block the neddylation and inhibit the activity of NAE (with an EC50 value
of 3.56 µM), and a dose-dependent reduction of the Ubc12-NEDD8 conjugations was
also observed. Molecular docking results suggest compound V11 could bind tightly to
NAE via hydrogen bonds and hydrophobic interactions. Compound V11 showed the
best antiproliferative ability with an IC50 value of 8.22 μM against gastric cancer MGC-
803 cells. Further anticancer activity studies suggested that compound V11 inhibited
MGC-803 cell growth, caused a cell cycle arrestment at G2/M phase and induced
apoptosis via extrinsic and intrinsic apoptosis pathways. All the findings suggest that
1,2,4-triazine scaffold might provide a novel scaffold for the further development of
neddylation inhibitors and compound V11 might be a potential neddylation inhibitor
with anticancer activity.
Keywords: 1,2,4-Triazine; Neddylation; MGC-803; UBC12-NEDD8 conjugation;
Neddylation is a novel type of posttranslational modification, in which the Neural
Precursor Cell-Expressed Developmentally Downregulated protein 8 (NEDD8) can be
conjugated to substrate proteins in a process and regulates their functions1-3. In
neddylation cascade, an E1 enzyme (NEDD8-activating enzyme (NAE; a heterodimer
of NAE1 and UBA3 subunits)) activates NEDD8 and then transfers it to an E2 enzyme
(NEDD8-conjugating E2 enzyme, also known as UBC12). Finally, the NEDD8 is
conjugated to cullin proteins to activate the cullin-RING ubiquitin E3 ligases (CRLs)
activity4-6(Fig.1). Neddylation regulates a wide range of biological processes such as
cell cycle, signal transduction and immune recognition1, 7-9. Neddylation modification
is highly activated in many cancer cells and promotes cancer development such as
gastric cancer10, lung cancer8, liver cancer11. Further studies suggest blocking the
neddylation modification has anti-tumor effects12-15. Therefore, targeting neddylation
pathway may represent a potential approach to the treatment of human cancers.

Fig. 1 The neddylation pathway
Targeting neddylation pathway has emerged as a new approach to the treatment of
human cancers since the finding of NEDD8-activating enzyme (NAE) inhibitor
MLN492415. MLN4924 could covalently bind to NAE, block all CRLs neddylation and
cause the accumulation of CRLs substrates15, 16. MLN4924 has entered phase II clinical
trials at present and showed effective activity against both solid and hematological
human cancers17. However, as results of its broad ablation of neddylation, MLN4924
has a series of toxicities18. Recently, some other neddylation inhibitors have been
reported15, 19-27. Compound 1 was reported as a novel NAE inhibitor which inhibited
NEDD8 protein and subsequently elevated UBC12 levels20. Compound 2 inhibited
NAE/UAE activities, downregulated degradations of related substrates in AGS cells,
and promoted apoptosis in low micromole concentrations21. Compound 3 inhibited
cullins neddylation and the DCN1−UBE2M protein−protein interaction in TR-FRET
binding assay(IC50 = 62 nM)23(Fig.1). Compound 4 named WS-383 reversibly blocked
the DCN1-UBC12 interaction (IC50 = 11 nM), exhibited cellular target engagement to
DCN1 in MGC-803 cells and selectively inhibited Cul3/1 neddylation 24. Compound 5,
which was discovered from in-house structurally diverse molecular library (ca.1000
25
compounds), inhibited the interaction of DCN1 and UBE2M (IC50 = 15 nM)
.
Compound 5 inhibited the interaction of UBE2M and DCN1 at molecular and cellular
levels, resulting in the decrease of cullin3 neddylation and accumulation of its
substrates. Compound 6 is a predominant chalcone in the kava plants named
Flavokawain A26. Compound 6 inhibited NEDD8 conjugations to both Cullin1 and
Ubc12 in PC-3 cells and Ubc12 Neddylation in vitro assay (Fig.1).

CF3
O
CF3
NH
O
O
NH
N
N
O
N
S
N
N
N
N
N
NH
H
O
S
HN
O
H2N
O
OH
O
S
H2N
3
1
2
MLN4924
N
N
O
O
N
N
S
S
S
N
N
CN
N
S
N
N
O
OH
O
Cl
N
N
N
S
4
5
6
Fig. 2 Structures of reported neddylation inhibitors
In continuation with our efforts toward to the discovery of neddylation inhibitors
with potential anticancer activity, we attempt to discover structurally diverse
compounds as novel neddylation inhibitors. In this study, by screening our in-house
structurally diverse molecular library, we found 1,2,4-triazine scaffold might be a
potential scaffold for the inhibition of neddylation by western blotting and molecular
docking studies. What’s more, we have reported a series of 1,2,4-triazine derivatives as
potential anticancer agents28 and 1,3,5- triazine derivatives were selected out of 100
top-ranked compounds as potential NAE inhibitors by structure-based virtual
screening29. Thus, the hit 1,2,4-triazine scaffold (compound IV) was first selected and
its effects on the inhibition of neddylation were identified (Fig. 3A).
Initially, we predicted the binding mode of compound IV within the NAE binding
site using the reported NAE/ MLN4924 complex (PDB code 3GZN). As shown in Fig.
3A, the 1,2,4-triazine core ring of compound IV form two hydrogen bonds with the
positively charged side chain of Lys124. And compound IV, Lys124 and Asp102 make
up hydrogen bond networks, which could obviously increase the binding affinity of
compound IV. The 3,4,5-trimethoxyl-phenyl group is located in a hydrophobic pocket
surrounded by Ile75, Ile99, Met101, Ile148, Leu166, Ile170 and Ala171. Meanwhile,
the methoxy group form a hydrogen bond with the main chain of Ile148. However,
from the docking results, we could find that only a part of the binding pocket is occupied

by compound IV. The non-binding sub-pocket provided us a way to optimize our
inhibitors based on the compound IV and the structure of sub-pocket (Fig. 3B). Then,
the overall effects of compound IV on neddylation were next investigated. The results
showed that the formation of Cullin1-NEDD8 was dose-dependently inhibited in MGC-
803 cells (Fig. 3C). Cullin1 is a substrate protein of neddylation. Therefore, all the
findings suggested that compound IV might serve as a novel scaffold and might block
the neddylation in MGC-803 cells.
Fig 3. (A) Structure of hit 1,2,4-triazine scaffold (compound IV). (B) Docking results of compound
IV with NAE (PDB code: 3GZN). Compound IV is shown in yellow stick model, and key residues
of NAE are shown in cyan stick models. The binding pocket is shown in an orange surface. The
nitrogen, oxygen, sulfur and chlorine atoms are shown in blue, red, yellow and green, respectively.
Hydrogen bonds are shown as black dashed lines. (C) Compound IV inhibited neddylation in MGC-
803 cells.
Based on above results, a series of novel 1,2,4-triazine derivatives were
synthesized based on 1,2,4-triazine scaffold by incorporating bioactive substructures
such as aromatic groups and indole groups for better efficacy. What’s more, various
indoles were designed and synthesized as potential anticancer agents30-32. Among them,
compound V11 was investigated the impact of V11 on neddylation activity (Fig. 4).

O
H
O
O
Cl
N
N
N
N
N
N
O
Structure-based
discovery Strategy
H
H
Our in-house structurally
diverse molecular library
Screening out
O
O
Cl
N
N
N
Compound V11
N
Cl
inhibited NAE activity with an
H
EC50 value of 3.56 mol/L
μ
Hit scaffold IV
inhibited neddylation in MGC-803 cells at 20 mol/L
μ
Fig. 4 1,2,4-triazine-based derivatives as novel neddylation inhibitors
The synthetic route of 1,2,4-triazine-based derivatives is shown in Scheme-1.
Starting from commercially available compound 1,2,4-triazine-3,5(2H,4H)-dione (I),
the electrophilic bromine was introduced in the position 6 of the triazinic ring at room
temperature to obtain the compound II. In the presence of N, N-diethylaniline,
phosphorus pentachloride and phosphoryl trichloride, compound II was converted to
compound III at 130oC. The first chlorine atom in the position 5 of intermediate
compound III was displaced by 3,4,5-trimethoxyaniline in the presence of TEA in THF
at 65oC to afford a key compound IV. Finally, compound IV reacted with substituted
arylamines in the presence of camphor sulfonic acid (CSA) in isopropanol to give the
target compounds V1-V12. Finally, all the target compounds were fully characterized
by NMR and HRMS.

O
N
N
Br
O
O
O
Cl
N
N
Cl
Cl
N
N
HN
HN
c
N
N
a
b
O
N
H
O
O
N
H
N
Cl
Cl
H
I
II
III
IV
O
O
Cl
N
N
N
d
R
O
N
N
H
H
V1-V12
Br
Cl
R=
R=
V3
V6
R=
R=
R=
V2
V1
V4
O
R=
R=
R=
V5
V8
O
O
O
O
O
NH2
R=
V9
R=
V7
O
O
H
N
N
O
R=
V12
R=
V11
V10
Scheme 1. (a) Br2, H2O. b) PCl5, N, N-diethyl aniline, POCl3, 130oC. (c) 3,4,5-trimethoxyaniline,
Et3N, THF, 65oC. (d) arylamines, CSA, isopropanol, reflux.
The antiproliferative activity of all the target compounds was evaluated against
three human cancer cell lines (MGC-803, PC-3, and EC-109) using MTT assay method
and the well-known anticancer drug 5-fluorouracil as reference drug (Table 1).
As shown in Table 1, most of compounds exerted moderate to potent cytotoxic
activity against three human cancer cell lines. Particularly, compound V5 and V11
exhibited the better growth inhibition against the tested cell lines (MGC-803, PC-3 and
MCF-7) with IC50 values of 8.75 µM, 9.75 μM, 12.57 μM and 8.22 µM, 9.28 μM, 9.64
μM, respectively. The potency of the compounds varies with respect to substitutions on
the simple phenyl ring. Comparing V1-V2 with V3-V7, compounds with electron-
donating groups at phenyl ring exhibited weaker cytotoxic activity than compounds
with electron-withdrawing groups and compound V5 without substituent group against
MGC-803 cells. The relationships between the substituent groups and the

antiproliferative activities were 4-H > 4-Br > 4-Cl > 3-OCH3>4-OCH3 > 4-CH3 > 3,4,5-
triOCH3. Moreover, compound V11 with the indole group exhibited the best activity
against three human cancer cell lines. Importantly, MGC-803 cells were more sensitive
to most of the compounds, including compound V11, than PC-3 cells and EC-109 cells.
Therefore, compound V11 was chosen to investigate its impact of neddylation activity
on MGC-803 cells.
Table 1. The cytotoxicity results of 1,2,4-triazine compounds (V1-V12) against human cancer
cell lines (MGC-803, PC-3, and EC-109).
IC50(μM) a
Compounds
MGC-803
PC-3
EC-109
12.59±0.53
32.61±2.64
9.61±0.22
V1
V2
V3
V4
V5
11.35±1.02
19.79±0.51
18.95±2.71
8.75±0.79
8.99±1.32
14.26±0.51
13.47±0.62
9.75±1.46
41.57±1.91
27.34±2.06
22.34±1.06
12.57±1.23
V6
11.26±0.31
16.46±0.82
24.31±2.67
13.01±0.68
15.22±1.77
53.62±3.23
8.22±0.283
28.13±1.42
9.79±0.17
15.94±5.23
>50
V7
V8
34.31±3.88
38.63±1.37
32.15±2.56
58.45±3.42
9.28±1.14
36.34±1.76
42.38±2.13
46.64±1.25
9.64±1.47
42.12±1.84
21.21±3.61
V9
V10
V11
V12
48.48±2.42
20.42±1.83
5-FU

aAntiproliferative activity was assayed by exposure for 48 hours.
The impacts of compound V11 on neddylation activity were investigated. First, a
dose–response experiment was performed using neddylation kit in vitro which
measured the formation of the Ubc12-NEDD8 conjugation products. The inhibition of
NAE would decrease the levels of Ubc12-NEDD8 conjugation, therefore the results
could be used to evaluate the impacts of compound V11 on the NAE activity15. As
shown in Fig. 5A, the formation of Ubc12-NEDD8 was almost suppressed in the
presence of compound V11 at 5 μM. MLN4924 also significantly inhibited Ubc12-
NEDD8 conjugation at 10 μM (Fig. 5A). Therefore, compound V11 inhibits
neddylation (enzyme-based assay).
The ability of compound V11 to inhibit Ubc12-NEDD8 formation in MGC-803
cells was next evaluated (Fig. 5B). MGC-803 cells were incubated with compound V11
for 24 hours. According to the results, a dose-dependent inhibition of Ubc12–NEDD8
formation was observed upon treatment with compound V11. Furthermore, the ability
of the compound V11 to inhibit the levels of Ubc12-NEDD8 conjugation in MGC-803
cells was assessed by Western blotting. The formation of Ubc12-NEDD8 was
obviously suppressed at 5μM and nearly complete inhibited at 10μM in the presence of
compound V11(Fig. 5B). The results showed that compound V11 decreased Ubc12-
NEDD8 conjugation in MGC-803 cells in a dose-dependent manner with an EC50 value
of 3.56 μM (Fig. 5G). What’s more, the formation of NAE1-NEDD8 and Cullin3-
NEDD8 were almost suppressed in the presence of compound V11 at 10 μM. These
results suggested that compound V11 blocked the neddylation, in which NAE activated
NEDD8 and transferred it to Ubc12 and Cullin3.
The neddylation of Cullin1 (another E3 enzyme) was decreased in a time-
dependent manner (Fig. 5D). The overall effects on neddylation of compound IV and
V11 were next investigated. The results indicated that both compound IV and
compound V11 inhibited neddylation in MGC-803 cells (the smears of neddylation
protein binds were decreased by compounds IV and V11) (Fig. 5C). Furthermore,

compound V11 had a stronger activity than compound IV, which was consistent with
the cell viability inhibition results (Table. 1). According to these results, compound
V11 inhibited the NAE activity and blocked neddylation in MGC-803 cells, leading to
a corresponding decrease in the abundance of E3s. As a result of neddylation inhibition,
cell cycle related protein P21 was increased (Fig. 5E), and some pro-apoptosis proteins
such as ATF4, CHOP, DR5, Noxa were obviously accumulated (Fig. 5F) which
indicated compound V11 might have potential anticancer activity.
Fig.5 Compound V11 inhibited neddylation. (A) Compound V11 inhibited NAE activities in
enzyme-based assay. (B, C&D) Compound V11 inhibited neddylation in MGC-803 cells. (E&F)
Level of cell cycle or apoptosis related proteins. MGC-803 cells were treated treated with different
concentration of compound V11 for 24 hours. (G) The quantification of Ubc12-NEDD8 formation
was determined by densitometry of triplicate experiments.
The inhibited effects of compound V11 on proliferation of gastric cancer cell lines

MGC-803 were evaluated. As shown in Fig. 6A, the cell viability was decreased in a
dose-dependent manner by a 48 hours treatment with compound V11, with an IC50
value of 8.22 μmol/L. Then, the growth curve of MGC-803 cells was drawn (Fig. 6B),
and it showed that the compound V11 inhibited the growth of MGC-803 cells within
48 hours. The treated group increased to 1.3 times while the untreated group grew to
approximate 3.7 times. The anti-proliferation activity of compound V11 was evaluated
by colony formation assay. Within a 7 days treatment, the low dose treatment group
evidently inhibited the cell colony formation (Fig. 6C). These results suggested that the
compound V11 inhibited the proliferation of MGC-803 cells in a dose and time-
dependent manner.
Fig. 6 (A) Cell viability of MGC-803 cells. MGC-803 cells were treated with different
concentrations of compound V11 for 48 hours. (B) Cell growth curve of MGC-803 cells. MGC-803
cells were treated with compound V11 for different times, MGC-803 cells were treated with DMSO
as control. (C) Colony formatting analysis. MGC-803 cells were treated with different
concentrations of compound V11 for 7 days.
To determine the effects of compound V11 on cell cycle, the flow cytometry assay
was performed. As described in Fig. 7A&B, the results showed that the MGC-803 cells
were arrested in G2/M phase after a 24 hours treatment, the percentage of G2/M cells
rose to twice that of the untreated group approximately. Apart from of P21, some other

proteins involved in cell cycle regulation such as CyclinB1 and CDK1 play important
roles in G2-M phase transformation. As shown in Fig. 7C, CyclinB1 and CDK1 were
significantly down-regulated by compound V11. Therefore, compound V11 regulated
the cell cycle related proteins P21, CyclinB1 and CDK1, leading to a G2/M phase
arrestment.
Fig. 7 (A&B) Cell cycle analysis. MGC-803 cells were treated with different concentrations of
compound V11 for 24 hours. (C) Level of cell cycle related proteins. MGC803 cells were treated
with different concentrations of compound V11 for 24 hours.
The accumulations of ATF4, CHOP, DR5 and Noxa may lead to cell apoptosis.
Hence, the apoptosis induction of compound V11 against MGC-803 cells was next
investigated. As shown in Fig. 8 A&B, the compound V11 treatment group up-
regulated the apoptosis rate to about 60%. The results in Fig. 8C showed that the
apoptosis related proteins Bax, XIAP, Caspase9, Caspase3 and PARP were evidently
regulated. The pro-apoptosis protein Bax was up-regulated, while the anti-apoptosis
protein XIAP was down-regulated. Caspase9, Caspase3 and PARP were cleaved by the
compound V11. All these proteins together with ATF4, CHOP, DR5 and Noxa are
involved in extrinsic and intrinsic apoptosis pathways. These results suggested the
compound V11 induced apoptosis via extrinsic and intrinsic apoptosis pathways.

Fig. 8 (A&B) Cell apoptosis analysis. MGC-803 cells were treated with different concentrations of
compound V11 for 48 hours. (C) Level of cell apoptosis related proteins. MGC-803 cells were
treated with different concentrations of compound V11 for 48 hours.
Based on the above experiments, 1,2,4-triazine derivative V11 showed the
inhibition of neddylation and inhibited the NAE activity. Because compound V11
might be a novel neddylation inhibitor, molecular docking study was carried out using
AutoDock Tools 1.5.6 as an automated tool to perform docking and predict the possible
binding mode of compound V11 with NAE (PDB: 3GZN).
The docking study was performed between NAE and most potent compound V11
(Fig. 9). We could find that compound V11 share a similar binding conformation with
compound IV, and its 1,2,4-triazine core ring and 3,4,5-trimethoxyl-phenyl group have
the same interactions with NAE. Moreover, the nitrogen atom linker could form a
hydrogen bond with the main chain of Gly165. The electron rich indole ring could have
strong electrostatic interactions with surrounding residues Gly79, Arg111, Gln112 and
Asn317. In addition, the indole ring could also have hydrophobic interactions with
Leu80, Thr203, Ile310 and Ala312. These additional interactions between NAE and
compound V11 might make compound V11 bind more tightly to NAE than compound
IV and also could explain that compound V11 have a better inhibitory activity of
neddylation.

Fig. 9 Docking results of compound V11 interact with NAE (PDB code: 3GZN). Compound V11
is shown in yellow stick model, and key residues of NAE are shown in cyan stick models. The
nitrogen, oxygen, sulfur and chlorine atoms are shown in blue, red, yellow and green, respectively.
The hydrogen bonds are shown as black dash lines.
In conclusion, 1,2,4-triazine scaffold was chosen as a hit scaffold via screening
our in-house structurally diverse molecular library further structure-based
optimizations. Molecular docking results and the overall effects of compound IV on
neddylation suggest that compound IV might serve as a novel scaffold and might block
the neddylation in MGC-803 cells. Therefore, a series of novel 1,2,4-triazine
derivatives were synthesized based on 1,2,4-triazine scaffold by incorporating bioactive
substructures such as aromatic groups and indole groups and evaluated for their
cytotoxic activity against MGC-803 cells, PC-3 cells and EC-109 cells using MTT
assay. The compound V11 was chosen to investigated its impacts of neddylation
activity in vitro. Compound V11 could block neddylation and inhibit the activity of
NAE (with an EC50 of 3.56 µM), leading to the decrease of UBC12-NEDD8
conjugation. The formation of Cullin3-NEDD8 and Cullin1-neddylation were also
inhibited. According to molecular docking results, we could find compound V11 would
bind tightly to NAE via hydrogen bonds and hydrophobic interactions. Compound V11
possessed the best antiproliferative ability with an IC50 value of 8.22μM against MGC-

803 cells. Anticancer activity studies suggested that compound V11 inhibited MGC-
803 cells growth, caused a cell cycle arrestment at G2/M phase and induced apoptosis
via extrinsic and intrinsic apoptosis pathways. All the findings suggest that 1,2,4-
triazine scaffold might be as a hit scaffold for the development of neddylation inhibitors
and compound V11 might be as a novel NAE inhibitor with potential anticancer activity
against MGC-803 cells.
Acknowledgements
This work was supported by the National Natural Sciences Foundations of China
(No. 81703541, 81673322) and China Postdoctoral Science Foundation (No.
2018M632812).
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Graphical Abstract
Highlights:
 1,2,4-triazine scaffold was screened out from in-house structurally diverse
molecular library.
 Novel 1,2,4-triazine derivatives as neddylation inhibitors were discovered.
 Compound V11 could block the neddylation in MGC-803 cells and inhibited NAE
activity.

Among them, compound V11 exerted the most excellent anti-proliferative
activities against MGC-803 cells.
 Compound V11 caused cell cycle arrest at G2/M phase and induced cell apoptosis.
Declaration of Interest Statement
The authors declare that they have no conflicts of interest.