Discovery of tertiary amide derivatives incorporating benzothiazole moiety as anti-gastric cancer agents in vitro via inhibiting tubulin polymerization and activating the Hippo signaling pathway

Article abstract of DOI:10.1016/j.ejmech.2020.112618

On the basis and continuation of our previous studies on anti-tubulin and anti-gastric cancer agents, novel tertiary amide derivatives incorporating benzothiazole moiety were synthesized and the antiproliferative activity was studied in vitro. Preliminary structure activity relationships (SARs) were explored according to the in vitro antiproliferative activity results. Some of compounds could significantly inhibit the proliferation of three cancer cells (HCT-116, MGC-803 and PC-3 cells) and compound F10 exhibited excellent antiproliferative activity against HCT-116 cells (IC₅₀ = 0.182 μM), MGC-803 cells (IC₅₀ = 0.035 μM), PC-3 cells(IC₅₀ = 2.11 μM) and SGC-7901 cells (IC₅₀ = 0.049 μM). Compound F10 effectively inhibited tubulin polymerization (IC₅₀ = 1.9 μM) and bound to colchicine binding site of tubulin. Molecular docking results suggested compound F10 could bind tightly into the colchicine binding site of β-tubulin. Moreover, compound F10 could regulate the Hippo/YAP signaling pathway. Compound F10 activated Hippo signaling pathway from its very beginning MST1/2, as the result of Hippo cascade activation YAP were inhibited. And then it led to a decrease of c-Myc and Bcl-2 expression. Further molecular experiments showed that compound F10 arrested at G2/M phase, inhibited cell colony formatting and induced extrinsic and intrinsic apoptosis in MGC-803 and SGC-7901 cells. Collectively, compound F10 was the first to be reported as a new anticancer agent in vitro via inhibiting tubulin polymerization and activating the Hippo signaling pathway.

Full text of DOI:10.1016/j.ejmech.2020.112618

European Journal of Medicinal Chemistry 203 (2020) 112618
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Discovery of tertiary amide derivatives incorporating benzothiazole
moiety as anti-gastric cancer agents in vitro via inhibiting tubulin
polymerization and activating the Hippo signaling pathway
,
b
,
,
b
,
e
,
,
,
, b
Jian Song a 1, Qiu-Lei Gao a
1, Bo-Wen Wu a b, Ting Zhu a b, Xin-Xin Cui a
,
Cheng-Jun Jin a b, Shu-Yu Wang a b, Sheng-Hui Wang a b, Dong-Jun Fu d, Hong-Min Liu a
,
,
,
,
, b
Sai-Yang Zhang a
*, Yan-Bing Zhang a **, Yong-Chun Li a
,
b,
c
,
,
b,
, b, ***
a Institute of Drug Discovery & Development, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, 450001, China
b School of Pharmaceutical Sciences, Key Laboratory of Advanced Drug Preparation Technologies (Ministry of Education), Zhengzhou University, Zhengzhou,
450001, China
c Henan Institute of Advanced Technology, Zhengzhou University, Zhengzhou, 450001, China
d Modern Research Center for Traditional Chinese Medicine, School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, 100029,
China
e School of Mechanics and Safety Engineering, ZhengZhou University, China
a r t i c l e i n f o
a b s t r a c t
Article history:
On the basis and continuation of our previous studies on anti-tubulin and anti-gastric cancer agents,
novel tertiary amide derivatives incorporating benzothiazole moiety were synthesized and the anti-
proliferative activity was studied in vitro. Preliminary structure activity relationships (SARs) were
explored according to the in vitro antiproliferative activity results. Some of compounds could significantly
inhibit the proliferation of three cancer cells (HCT-116, MGC-803 and PC-3 cells) and compound F10
Received 27 April 2020
Received in revised form
23 June 2020
Accepted 23 June 2020
Available online 13 July 2020
exhibited excellent antiproliferative activity against HCT-116 cells (IC50 ¼ 0.182
(IC50 ¼ 0.035 M), PC-3 cells(IC50 ¼ 2.11 M) and SGC-7901 cells (IC50 ¼ 0.049
effectively inhibited tubulin polymerization (IC50 ¼ 1.9 M) and bound to colchicine binding site of
tubulin. Molecular docking results suggested compound F10 could bind tightly into the colchicine
binding site of -tubulin. Moreover, compound F10 could regulate the Hippo/YAP signaling pathway.
m
M), MGC-803 cells
m
m
mM). Compound F10
Keywords:
Gastric cancer
Tertiary amide
Benzothiazole
m
b
Compound F10 activated Hippo signaling pathway from its very beginning MST1/2, as the result of Hippo
cascade activation YAP were inhibited. And then it led to a decrease of c-Myc and Bcl-2 expression.
Further molecular experiments showed that compound F10 arrested at G2/M phase, inhibited cell colony
formatting and induced extrinsic and intrinsic apoptosis in MGC-803 and SGC-7901 cells. Collectively,
compound F10 was the first to be reported as a new anticancer agent in vitro via inhibiting tubulin
polymerization and activating the Hippo signaling pathway.
Colchicine binding site tubulin inhibitor
Hippo signaling pathway
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
Gastric cancer (GC) is one of the most aggressive and common
cancers in the world. In 2018, there were over 1000000 new cases
* Corresponding author. Henan Institute of Advanced Technology, Zhengzhou
University, Zhengzhou 450001, China.
** Corresponding author. School of Pharmaceutical Sciences, Key Laboratory of
Advanced Drug Preparation Technologies (Ministry of Education), Zhengzhou
University, Zhengzhou 450001, China.
diagnosed worldwide and an estimated 783,000 people died of
gastric cancer [1]. Although cancer diagnosis technology and anti-
cancer agents have been evolving steadily, gastric cancer remains
one of the most lethal malignant tumors among China [2e4].
Therefore, it is urgent to develop novel agents for the treatment of
gastric cancer.
*** Corresponding author. Institute of Drug Discovery & Development, School of
Basic Medical Sciences, Zhengzhou University, Zhengzhou 450001, China.
E-mail addresses: saiyangz@zzu.edu.cn (S.-Y. Zhang), zhangyb@zzu.edu.cn
(Y.-B. Zhang), ycli78@zzu.edu.cn (Y.-C. Li).
Microtubule (composed of a-tubulin and b-tubulin heterodimers)
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.ejmech.2020.112618
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

2
J. Song et al. / European Journal of Medicinal Chemistry 203 (2020) 112618
is a component of the cytoskeleton and plays an important role in
cellular shape maintenance, mitosis, signal transduction and material
transportation [5e7]. Because of its significant role in the mitosis and
growth of eukaryotic cells, microtubule system has been regarded as
an attractive and successful molecular target for the treatment of
cancers as chemotherapeutic agents [8e13] such as paclitaxel [11],
vincristine [12] and vinblastine [13]. But the marketed tubulin in-
hibitors used in clinical treatment (e.g. paclitaxel/taxol, vincristine,
vinblastine) bind to the taxanes or vinca alkaloids binding sites in
tubulin [14,15]. Unfortunately, they general have poor in water solu-
bility, toxic, side effects, and drug resistance [10,16,17]. Therefore,
polymerization inhibitors targeting tubulin colchicine binding site
havereceivedextraordinaryattentionintherecentyears, importantly
tubulin inhibitors targeting colchicine binding site could largely
overcome the above drawbacks and have more therapeutic advan-
tages over other binding sites tubulin inhibitors [18e21].
gastric cancer [45]. What’s more, it also has been found to func-
tionally interact with many other cellular pathways and act as a
central node in the regulation of cell division in various cancers
including gastric cancer [46e48]. Activation of Hippo pathway
would result in phosphorylation of YAP. The phosphor-YAP is
inactivated and would be degraded via Ubiquitin-proteasome sys-
tem. Herein, targeting the Hippo pathway with small molecule
inhibitors may represent a potential approach to treat cancers with
high YAP activity. However, only rare small molecule activators of
Hippo pathway have been reported.
In this work, as the continuation of our studies on colchicine
binding site tubulin inhibitors and anti-gastric cancer agents, novel
tertiary amide derivatives incorporating benzothiazole moiety
were designed via opening ring of b-lactam and explored its anti-
proliferative activity (Fig. 2). We screened the most potent anti-
proliferative activity compound F10 through in vitro tubulin
polymerization assay and Hippo related protein expression in
MGC-803 and SGC-7901 cells. Fortunately, compound F10 is a novel
colchicine binding site tubulin inhibitor and could activate the
Hippo signaling pathway. Therefore, we first report novel tertiary
amide derivatives incorporating benzothiazole moiety as new anti-
gastric cancer agents in vitro via inhibiting tubulin polymerization
and activating the Hippo signaling pathway.
To date, various tubulin inhibitors that target colchicine binding
site have been developed [22e27] and some of them have entered
clinical trials [28e32] such as CA-4P [29], ZD6126 [30] and ABT-751
[31]. In addition, our group have reported several series colchicine
binding site anti-tubulin agents against gastric cancers [33e35]. For
example,
b
-lactam-azide analogue 1 played excellent anticancer
M) and was identified
M)31
activity against MGC-803 cells (IC50 ¼ 0.106
m
as colchicine binding site tubulin inhibitor (IC50 ¼ 2.26
m
.
What’s more, benzothiazole moiety has been reported as a poten-
tial antitumor fragment to design colchicine binding site tubulin
inhibitors [36e38] such as compound 2 [38] and 3 [36]with IC50
2. Chemistry
The chemical synthesis of compounds incorporating benzo-
thiazole moiety is outlined in Scheme 1. Substituted benzaldehyde
derivatives A1-A10 reacted with 3,4,5-trimethoxyaniline (B) to
afford compounds C1-C10 in EtOH. Compounds C1-C10 then reac-
ted with NaBH4 in the presence of benzoic acid in DCM to give
compounds D1-D10. Then compounds D1-D10 reacted with
chloroacetyl chloride to give compounds E1-E11 in DMF. Finally,
compounds E1-E11 was reacted with substituted benzothiazoles to
afford the target compounds F1-F14 in the presence of K2CO3 in
acetonitrile. Furthermore, compound B reacted with substituted
benzyl chlorides G1-G6 to afford compounds H1-H6 in the pres-
ence of K2CO3 in DMF. Next, compounds H1-H6 reacted with
values of 1.67 mM and 2.01 mM, respectively (Fig. 1).
Hippo is a highly conserved signaling pathway which plays an
crucial role in the occurrence and growth of most tumors by
regulating cell proliferation and apoptosis [39e41]. The activation
of Hippo signaling pathway can affect the genesis, proliferation,
drug resistance and metastasis of tumor stem cells [42]. In mam-
mals, Hippo signaling pathway consists of protein kinases MST1/2,
SAV1, LATS1/2, MOB1, and downstream effectors YAP, TAZ and
TEADs. Activated YAP and TEADs together express oncogenes like c-
Myc and Bcl-2 and would contribute to the development of tumors,
such as colorectal cancer [42], lung cancer [43], breast cancer [44],
Fig. 1. Structures of colchicine binding site tubulin inhibitors.

J. Song et al. / European Journal of Medicinal Chemistry 203 (2020) 112618
3
Fig. 2. Design of tertiary amide derivative F10 in this work.
Scheme 1. Synthesis of target compounds F1-F14 and J1-J6. Reagents and conditions: a) EtOH, 80 ꢀC, 6 h; b) Benzoic acid, NaBH4, anhydrous CH2Cl2, rt, 8 h; c) chloroacetyl chloride,
DMF, rt. d) substituted benzothiazoles, acetonitrile, K2CO3, reflux, 6 h; e) substituted benzyl chlorides, DMF, K2CO3, rt, 7 h.
chloroacetyl chloride in DMF to obtain tertiary amide derivatives
I1-I6. Tertiary amide derivatives I1-I6 then reacted with 2-
mercaptobenzothiazole in the presence of K2CO3 in acetonitrile to
afford the target compounds J1-J6. Characterization of compounds
F1-F14 and J1-J6 was carried out by means of NMR and HREI-mass
spectra.
colchicine as control drugs. The antiproliferative activity results are
depicted in https://www.sciencedirect.com/science/article/pii/
S0223523419306440 - tbl1 Table 1 and https://www.science
direct.com/science/article/pii/S0223523419306440 - tbl2 Table 2.
The structure-activity relationships of synthesized compounds F1-
F14 and J1-J6 are preliminarily summarized (Fig. 3).
The in vitro antiproliferative efficacy of compounds F1-F14 are
listed in Table 1. In this series of compounds, we primarily explored
the importance of the substituent groups on phenyl ring (A ring) and
benzothiazolemoiety(Bring)inantiproliferativeactivity. Asshownin
https://www.sciencedirect.com/science/article/pii/S022352341830
9280 - tbl1 Table 1, most of synthetic compounds displayed moderate
to excellent antiproliferative activity. The in vitro antiproliferative
efficacy of the compounds varies with its substituent groups on the
ringA. CompoundsF1-F2andF7-F14inhibitedMGC-803cellsgrowth
3. Results and discussion
3.1. Antiproliferative activity and structure activity relationships
The antiproliferative activity of synthesized compounds F1-F14
and J1-J6 was evaluated against MGC-803 cell line (human gastric
cancer), HCT-116 cell line (human colon cancer) and PC-3 cell line
(human prostate cancer) using MTT assay and 5-fluorouracil and

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J. Song et al. / European Journal of Medicinal Chemistry 203 (2020) 112618
Table 1
Antiproliferative activity of compounds F1-F14 against MGC-803, HCT-116 and PC-3 cells.
Compound
R1
R2
R3
IC50 (m
mol/L)a
MGC-803
HCT-116
PC-3
F1
F2
F3
F4
F5
F6
F7
F8
-F
-Br
H
H
H
H
H
-OCH3
H
H
H
H
H
H
H
H
e
H
H
H
H
H
H
H
H
0.416 0.021
0.852 0.72
6.36 0.89
25.2 1.64
34.1 2.13
1.83 0.26
4.55 1.35
17.1 2.42
>80
20.1 2.02
16.0 1.38
34.3 2.04
>80
>80
>80
26.5 2.68
14.4 1.72
7.51 2.58
2.11 0.76
12.3 1.84
11.2 1.65
8.21 1.18
4.11 0.84
18.4 1.73
0.338 0.014
0.033 0.004
-CH3
-CF3
-OCH3
-OCH3
NO2
-N3
-NH2
-OH
-OH
-OH
-OH
-OH
e
33.9 2.49
65.3 2.54
1.28 0.11
2.86 1.69
0.491 0.025
0.182 0.012
0.852 0.098
0.429 0.063
3.38 1.25
0.352 0.043
13.8 1.96
0.129 0.020
0.042 0.006
45.7 1.83
0.682 0.052
0.187 0.027
0.084 0.017
0.035 0.006
0.493 0.033
0.085 0.012
0.372 0.028
0.071 0.009
6.82 1.17
F9
H
H
F10
F11
F12
F13
F14
5-FU
Colchicine
CA-4P
4-CH3
5-Cl
5-Br
6-OCH3
e
e
e
e
0.107 0.010
0.020 0.002
e
e
e
a
Antiproliferative activity was assayed by exposure for 48 h.
Fig. 3. Summary of the structure-activity relationships.
with IC50 values less than 1
116 cells with IC50 values less than 5
m
M and inhibited the growth of HCT-
9280 - tbl3 Table 1). Compounds F11- F14 retained potent in vitro
antiproliferative efficacy on MGC-803 cells with IC50 values less than
mM. But most of compounds
exhibited weaker antiproliferative activity against PC-3 cells
compared to MGC-803 cells and HCT-116 cells. Only compounds F9,
F10 and F13 showed the potent antiproliferative activity of PC-3 cells
0.5 mM, and HCT-116 cells with IC50 values lessthan4.0 mM. Compared
compound F10 with compoundsF11-F14, substituted benzothiazoles
of ring B might slightly impair antiproliferative activity than without
substituted groups of ring B against tested cancer cells.
With compound F10 in hand, we then performed further SARs
studies (Table 2). In this series of target compounds, we explored
the importance of the 3-hydroxyl group of A ring. These anti-
proliferative efficacy results suggested that 4-methoxy group on
ring A had significant effect on antiproliferative activity. When the
4-methoxy group of ring A was replaced by other groups (4-CH3, 4-
F, 4-Cl, 4-Br, and 4-H), compounds J1-J5 exhibited poorer activity
with IC50 values less than 10
3-hydroxyl group and 4-methoxy group on the ring A exhibited most
active activity with IC50 values of 0.035 M (MGC-803 cells), 0.182
(HCT-116 cells) and 2.11 M (PC-3 cells). Compounds F1, F2, F4 and F7
mM. To our delight, compound F10 with
m
mM
m
showed improved efficacy against MGC-803 cells with electron-
withdrawing groups on the ring A compared to compounds F3, F5
and F6 with electron-donating groups on the ring A. However,
introduction of 3-OH, 3-NH2, and 3-N3 groups on the ring A could
significantly enhance the antiproliferative activity compared with
other compounds against MGC-803 cells with IC50 values less than
(almost IC50 values > 40
mM) compared with compound F10.
However, compound J6 with a 4-methoxy group on ring A showed
1.0
mM. These inhibitory results suggested that substituent groups on
decent activity against MGC-803 and HCT-116 cells with IC50 values
the phenyl ringA exhibiteda significanteffect for the antiproliferative
efficacy.
Based on compound F10 and the SAR analysis, the effects of
substituted benzothiazoles (ring B) on activity were further explored
(https://www.sciencedirect.com/science/article/pii/S022352341830
of 0.163 mM and 0.426 mM, which were lower than compound F10
with 3-hydroxyl group and 4-methoxy groups of A ring. The in vitro
antiproliferative efficacy results indicated the 4-methoxy group on
the ring A plays a significant role in antiproliferative activity and
introduction of 3-OH and 3-NH2 groups can significantly enhance

J. Song et al. / European Journal of Medicinal Chemistry 203 (2020) 112618
5
Table 2
3.3. Compound F10 inhibited tubulin polymerization in vitro
Antiproliferative activity of compounds J1-J6 against MGC-803, HCT-116 and PC-
3 cells.
Since this series of compounds were designed as tubulin
polymerization inhibitor, the inhibiting polymerization activity of
tubulin was first to be verified. The in vitro tubulin polymeriza-
tion inhibition activity was evaluated for compound F10 and
colchicine (as a positive control group) at different concentra-
tions. As shown in Fig. 5D, the results suggested that compound
F10 and colchicine produced a dose-dependent inhibition of
tubulin polymerization and gave IC50 values 1.9 mM and 7.5 mM,
Compound
R4
IC50 (m
mol/L)a
respectively. As shown in Fig. 5B, these results are consistent
with the observed higher potency of compound F10 in anti-
proliferative activity and tubulin polymerization than that of
MGC-803
HCT-116
PC-3
J1
J2
J3
J4
J5
J6
H
-F
-Cl
-Br
-CH3
-OCH3
e
44.3 2.59
>80
>80
48.1 5.18
54.2 12.4
0.163 0.032
6.82 1.17
0.107 0.010
0.020 0.002
54.5 1.94
59.8 1.93
60.3 1.34
63.8 1.96
>80
0.426 0.022
13.8 1.96
0.129 0.020
0.042 0.006
54.1 2.14
50.82 1.86
>80
>80
>80
8.46 1.78
18.4 1.73
0.338 0.014
0.033 0.004
colchicine. EBI (N,N0-ethylenebis(iodoacetamide)) can alkylate
b-
tubulin when its colchicine binding site is free. Compound F10
down regulated tubulin adduct in a dose-dependent manner
(Fig. 5C), which indicated compound F10 could competitively
bind to the colchicine binding site of b-tubulin. 2 gastric cancer
5-FU
Colchicine
CA-4P
cell lines were stained and observed using microscope after
treated with compound F10, the mid and high dose group of
compound F10 prevented the polymerization of tubulin from
formatting microtubule which is a major component of the
e
e
a
Antiproliferative activity was assayed by exposure for 48 h.
cytoskeleton and actor of mitosis. b-tubulin did not form a fila-
mentous structure, instead they appeared dotted. Multiple nu-
cleus was observed as the result of failed in mitosis (Fig. 5A).
These results indicated that compound F10 could as a novel anti-
tubulin polymerization agent targeting colchicine binding site,
which lead to tubulin polymerization inhibition intracellular and
extracellular.
the in vitro antiproliferative activity.
Based on above inhibitory activity results and SARs analysis, we
conclude that 4-methoxy group on the ring A plays a significant
role in antiproliferative activity and introduction of 3-OH, 3-NH2,
and 3-N3 groups could significantly enhance the antiproliferative
activity. The antiproliferative efficacy of target compounds varies
with its substituent groups on the ring A. Substituted benzothia-
zoles of B ring may slightly impair antiproliferative activity against
tested cancer cells compared with compound F10. The SARs of
target compounds were summarized in Fig. 3.
3.4. Molecular docking study
To explore the binding mechanism of compound F10 with
tubulin, a molecular docking study was performed of compound
F10 into the colchicine binding pocket of b-tubulin (PDB ID: 6O61).
The docking result showed that compound F10 could well bind into
the colchicine binding site of tubulin (Fig. 6). The 3,4,5-
trimethoxyphenyl group could form an unconventional hydrogen
bond with the side chain of Cys239, and the hydrogen bond dis-
tance was 1.9 Å. The 3-hydroxyl-4- methoxyphenyl could form two
hydrogen bonds with the main chain of Leu246 and the side chain
of Asn101. The hydrogen bonds distances were 1.6 Å and 2.0 Å,
respectively. These two groups were also had hydrophobic inter-
action with Leu240, Leu246 and Lys252. The benzothiazole group
was located in a hydrophobic pocket consist of Tyr200, Val236,
Leu240, Leu250, Leu253 and Ile316. Meanwhile, the carbonyl group
formed a hydrogen bond with the side chain of Asn256 in the
distance of 1.9 Å. These interactions made compound F10 tightly
bind in the colchicine binding site and exhibit a strong polymeri-
zation inhibiting activity against tubulin.
3.2. Compound F10 selectively inhibits gastric cancer cells
The most active compound F10 was selected to do the mecha-
nism studies according to above inhibitory activity results. SGC-
7901 and GES-1 cells were used to detect the selective inhibition
of compound F10. The 48 h IC50 values of MGC-803, SGC-7901 and
GES-1 cells were 0.035 mM, 0.049 mM and 0.249 mM, respectively
(Fig. 4B). As shown in Fig. 4A, compound F10 dose-dependently
inhibited MGC-803 and SGC-7901 cells. With high dose treatment
of compound F10, the inhibition rates of MGC-803 and SGC-
7901 cells respectively were 72% and 63% while its inhibition rate of
GES-1 were under 20%. Results in Fig. 4C&D showed compound F10
inhibited MGC-803 and SGC-7901 cells growth in dose and time-
dependent manners. These results suggested compound F10
selectively inhibited gastric cancer cells and showed much a lower
toxicity against normal gastric cell lines GES-1. Therefore, Com-
pound F10 was selected to do the further study for its high inhi-
bition activity against MGC-803 and SGC-7901 cells.
Moreover, we detected the effects of compound F10 on drug
resistance at cell levels. As shown in Fig. 4E, paclitaxel (PTX)
resistant MGC-803 cells were less sensitive to PTX than regular
MGC-803 cells. The IC50 of PTX against MGC-803 was 8.11 nM,
while IC50 against MGC-803/PTX was 88.68 nM. Therefore, the PTX
resistance Index of MGC-803/PTX is 10.93. With pre-incubation of
compound F10 (15 nM, 48 h inhibition rate against MGC-803/PTX
<15%) for 24 h, the IC50 of PTX against MGC-803/PTX was
reduced from 88.68 nM to 45.22 nM (Fig. 4F). This result illustrated
that compound F10 could rescue the PTX resistant on MGC-
803 cells.
3.5. Compound F10 inhibited proliferation of gastric cancer cells
Tubulin polymerization inhibition could lead to proliferation
inhibition, cell cycle arrestment at G2/M phase to be specific. As
shown in Fig. 7A, compound F10 dose-dependently arrested 2
gastric cancer cells at G2/M phase. The percentage of G2/M cells
with high dose treatment both 2 gastric cell lines rose to about 2
folds that of control group. Cell cycle arrestment would be
embodied in activity of cell formatting colonies. With 7 days
treatment with compound F10, cells’ activities formatting colony
were obviously inhibited (Fig. 7B). G2/M phase related proteins
were detected next. As expected, M phase marker p-Histone H3Ser10
was up regulated and G2 phase related proteins CyclinB1 and CDK1
were down regulated (Fig. 7C).

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J. Song et al. / European Journal of Medicinal Chemistry 203 (2020) 112618
Fig. 4. Compound F10 selectively inhibited MGC-803 and SGC-7901 cells. Cells were incubated with 20, 40 and 60 nM F10 for 48 h. A. Cell viabilities of 3 gastric cell lines; B. The
48 h IC50 values of 3 gastric cell lines; C&D. Growth curves of 2 gastric cancer cell lines. E. Cell viabilities of MGC-803 and MGC-803/PTX cells. Cells were treated with PTX for 48 h. F.
Cell viabilities of MGC-803/PTX. Cells were pre-incubated with or without 15 nM compound F10 for 24 h, then treated with PTX for 48 h.
To sum up, compound F10 arrested MGC-803 and SGC-
7901 cells at G2/M phase and inhibited cell colony formatting
resulting in proliferation inhibition in MGC-803 and SGC-7901 cells.
3.6. Compound F10 activated Hippo signaling pathway
Hippo signaling pathway plays an important role in tissue
regeneration, organ size control, and cancer. In this work,

J. Song et al. / European Journal of Medicinal Chemistry 203 (2020) 112618
7
Fig. 5. Compound F10 inhibited tubulin polymerization in vitro. A. b-tubulin (green) nucleus (blue) in MGC-803 and SGC-7901 cells. Cells were incubated with 20, 40 and 60 nM F10
for 48 h; B. Tubulin polymerization extracellular, concentrations of Paclitaxel, Colchicine and F10 were 3 mmol/L; C. EBI competition assay on MGC-803 and SGC-7901 cells; D. Effects
of F10 and colchicine on tubulin polymerization in vitro.
compound F10 could effectively activated Hippo pathway and
then lead to down-regulation of activated YAP. As shown in
Fig. 7A, compound F10 promoted phosphorylation of MST1/2
which was the upstream of Hippo. Increased p-MST1/2 promoted
phosphorylation of its downstream protein LATS. p-LATS then
caused inactivation (phosphorylation) of YAP protein. Activated
YAP together with transcription factor TEAD express downstream
proteins that contribute to surviving and proliferation. Besides
inactivating YAP, compound F10 also down regulated TEAD
(Fig. 8A). c-Myc and Bcl-2 are 2 proteins regulated by YAP-TEAD

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J. Song et al. / European Journal of Medicinal Chemistry 203 (2020) 112618
Fig. 6. The proposed binding mode of compound F10 with tubulin (PDB ID: 6O61).
Fig. 7. Compound F10 inhibited cell proliferation. A. Cell cycle analysis of 2 gastric cancer cell lines, cells were incubated with 20, 40 and 60 nM F10 for 24 h; B. Colony formatting
analysis, cells were incubated with 20, 40 and 60 nM F10 for 7 days; C. Cell cycle related proteins in 2 cancer cell lines were detected by western blotting, cells were incubated with
20,40 and 60 nM F10 for 48 h.
complex. Among them c-Myc is a proliferation and anti-apoptosis
protein, Bcl-2 is an anti-apoptosis protein. The decrease of YAP
and TEAD led to low expression of c-Myc and Bcl-2 (Fig. 8A). This
2 decreases induced by compound F10 were obviously rescued
(Fig. 8C). The cell viability inhibitions caused by compound F10
were also reversed when YAP silenced in both MGC-803 cell lines
and SGC-7901 cell lines (Fig. 8D).
cascade above was involved by compound F10 in
a dose-
dependent manner. Next siRNA silencing was performed to
downregulate YAP. The siYAP-1557 was the most effective one
(Fig. 8B). For YAP silenced MGC-803 cell lines, the c-Myc and Bcl-
In conclusion, compound F10 activated Hippo signaling
pathway from its very beginning MST1/2, as the result of Hippo
cascade YAP were inhibited. Which led to a decrease of c-Myc and

J. Song et al. / European Journal of Medicinal Chemistry 203 (2020) 112618
9
Fig. 8. Compound F10 activated Hippo pathway. A. Hippo related proteins in 2 cancer cell lines, cells were incubated with 20,40 and 60 nM F10 for 48 h; B. Effects of 3 siYAP on
silencing YAP; C. Bcl-2 and c-Myc expression levels, MGC-803 cells were treated with siYAP-1557 and compound F10 (60 nmol/L) for 48 h; D. Cell viabilities of 2 cancer cells, cells
were treated with siYAP-1557 and compound F10 (60 nmol/L) for 48 h.
Fig. 9. Compound F10 regulated apoptosis related proteins in 2 gastric cancer cell lines, cells incubated with 20, 40 and 60 nM F10 for 48 h.

10
J. Song et al. / European Journal of Medicinal Chemistry 203 (2020) 112618
Fig. 10. Compound F10 induced cell apoptosis in both 2 cell lines, cells were incubated with 20, 40 and 60 nM F10 for 48 h in A, B and C. A. Depolarize of Mitochondrial membrane
potential; B. Cell apoptosis analysis; C. Cell nucleus’ morphological changes; D. Live-Dead cell stain, cells are divided into living cells (green) and dead cells (red), cells were t
incubated with 20, 40 and 60 nM F10 for 24 h. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

J. Song et al. / European Journal of Medicinal Chemistry 203 (2020) 112618
11
Fig. 11. Model of a mechanism by which compound F10 inhibits tubulin polymerization and activates Hippo signaling pathway to inhibit gastric cancers.
Bcl-2 expression, the target genes of YAP. It was proved the down
regulation of c-Myc, Bcl-2 led the inhibition of cell viabilities caused
by compound F10 were depend on its activity activating Hippo
pathway.
4. Conclusion
In summary, novel tertiary amide derivatives incorporating
benzothiazole moiety were explored via opening ring of -lactam
b
derivatives. In vitro Antiproliferative activity of compounds was
evaluated for three human cancer cells. Some of them displayed
potent antiproliferative efficacy and compound F10 showed
excellent antiproliferative activity against MGC-803 cells
3.7. Compound F10 induced cell apoptosis
Down regulation of c-Myc, Bcl-2 were the results of activation of
Hippo induced by compound F10. The protein of c-Myc is a pro-
liferation and anti-apoptosis protein. It has been reported that c-
Myc negative regulates pro-apoptotic protein DR5 (Death Receptor
5), which can induce an extrinsic apoptosis. Bcl-2 is a well-known
anti-apoptotic protein. The decrease of Bcl-2 could lead to an
intrinsic apoptosis. As shown in Fig. 9, with the compound F10
treatment on 2 gastric cancer cells, DR5 was up regulated. FADD,
protein with DD domain which can be recruited by DR5 was up
regulated. Their downstream t-Bid (activated Bid) was also up
regulated. Anti-apoptosis proteins in Bcl-2 family Bcl-xL, Mcl-1
were decreased. Pro-apoptosis proteins in Bcl-2 family Noxa, Bak
and Bax were increased (Fig. 9). Mitochondrial membrane potential
depolarization is an important event of intrinsic apoptosis Bcl-2
family regulated. There occurs an obvious depolarize of mito-
chondrial membrane potential after compound F10 treatment
(Fig. 10A). XIAP and c-IAP1, 2 Inhibitor of Apoptosis Proteins, were
down regulated by compound F10.2 Caspase family members
Caspase3 and Caspase9 were evidently activated (cleaved). Their
substrate PARP was also cleaved (Fig. 9). At cell level, cells were
induced apoptosis by compound F10. Compound F10 rose
apoptosis percentages to 73% (MGC-803 cells) and 66% (SGC-
7901 cells) from 7% to 4% (Fig. 10B). Apoptosis-like changes, like
concentration and fragmentation, occurred in nucleus of 2 gastric
cancer cells (Fig.10C). With the concentration of F10 rises, more and
more cells were induced dead (Fig. 10D).
(IC50 ¼ 0.035
m
M), HCT-116 cells (IC50 ¼ 0.182
m
M), PC-3 cells
(IC50 ¼ 2.11
m
M) and SGC-7901 cells (IC50 ¼ 0.049
mM), respectively.
In addition, the SARs of the synthesized compounds were studied
and summarized.
In further study, the results of in vitro tubulin polymerization
inhibitory assay and EBI alkylate assay suggested compound F10
effectively inhibited tubulin polymerization binding to colchicine
site with an IC50 value of 1.9 mM. And also, compound F10 exhibited
a strong activity against tubulin polymerization intracellular and
extracellular. Molecular docking results suggested compound F10
could bind tightly into the colchicine binding site of tubulin.
Further molecular experiments indicated that compound F10
arrested MGC-803 and SGC-7901 cells at G2/M phase and inhibited
cell colony formatting (Fig. 11). On the other hand, compound F10
activated Hippo signaling pathway to inactive the YAP protein.
Then down regulated Bcl-2 and c-Myc depend on YAP inactivation.
Then compound F10 induced extrinsic and intrinsic apoptosis of 2
gastric cancer cells (Fig. 11). As the result of proliferation inhibition
and apoptosis inducement compound F10 selectively inhibited
gastric cancer cells.
Here, compound F10 was the first to be reported as a new
anticancer agent in vitro via inhibiting
b-tubulin polymerization
and activating the Hippo signaling pathway.
5. General
The results show that compound regulated apoptosis related
proteins then induced extrinsic and intrinsic apoptosis of gastric
cancer cells.
In this work, commercial materials were purchased from re-
agent companies (Beijing Innochem Science &Technology Co. Ltd

12
J. Song et al. / European Journal of Medicinal Chemistry 203 (2020) 112618
þ
and Zhengzhou HeQi Compony). We used an X-5 micromelting
apparatus (Hanon Compony) to determine the melting points. NMR
spectra was recorded on a Bruker spectrometer. HRMS spectra was
recorded on a Waters Micromass spectrometer by ESI.
51.98, 36.94, 15.92. HR-MS (ESI): Calcd. C27H28N2O5S2, [MþH]
525.1512, found: 525.1508.
2-(benzo[d]thiazol-2-ylthio)-N-(4-methoxy-3-(tri-
fluoromethyl)benzyl)-N-(3,4,5-trimethoxyphenyl)
acetamide(F4).
5.1. Synthesis of compounds C1-C10, D1-D10 and E1-E12
Yield, 57%, M.p. 159e160 ꢀC, White solid. 1H NMR (400 MHz,
DMSO‑d6)
d
7.99 (d, J ¼ 7.9 Hz, 1H), 7.72 (d, J ¼ 8.0 Hz, 1H), 7.48 (dd,
A solution of various aromatic aldehydes A1-A10 (1.0 mmol, 1.0
eq) and 3,4,5-trimethoxyaniline (1.0 mmol, 1.0 eq) were added into
EtOH (20 ml) at 80 ꢀC for 6 h. When the reactions were complete,
organic phases were collected to give crude the products C1-C10
without further purified with column chromatography.
J ¼ 18.4, 10.1 Hz, 3H), 7.36 (t, J ¼ 7.6 Hz, 1H), 7.15 (d, J ¼ 8.5 Hz, 1H),
6.66 (s, 2H), 4.89 (s, 2H), 4.25 (s, 2H), 3.86 (s, 3H), 3.68 (d,
J ¼ 20.7 Hz, 9H). 13C NMR (100 MHz, DMSO‑d6)
d 166.37, 165.98,
156.23, 153.08, 152.40, 137.08, 136.60, 134.63, 134.36, 129.15, 126.89,
126.85, 126.28, 124.44, 121.80, 120.84, 116.32, 112.69, 106.07, 60.01,
56.10, 55.93, 51.52, 36.86. HR-MS (ESI): Calcd. C27H25F3N2O5S2,
[MþH]þ m/z: 579.1230, found: 579.1224.
A solution of compounds C1-C10 (1.0 mmol, 1.0 eq), benzoic acid
(1.0 mmol, 1.0 eq) and NaBH4 (1.0 mmol, 1.0 eq) were added into
DCM (20 ml) at 25 ꢀC for 8 h. When the reactions were complete,
organic phases were collected to obtain crude products and then
were purified by column chromatography to obtain compounds
D1-D10.
A solution of compounds D1-D10 (1.0 mmol, 1.0 eq) and acyl
chloride derivatives (1.0 mmol, 1.0 eq) were added into DMF (8 ml)
at 25 ꢀC for 6e8 h. Then using EA (20 ml) extracted aqueous layers
for three times. Collected organic layers were washed by saturated
salt water, dried over magnesium sulfate anhydrous and evapo-
rated to get crude products. Crude products were purified by col-
umn chromatography to obtain compounds E1-E11.
2-(benzo[d]thiazol-2-ylthio)-N-(3,4-dimethoxybenzyl)-N-
(3,4,5-trimethoxyphenyl) acetamide(F5).
Yield, 49%, M.p. 121e123 ꢀC, White solid.1H NMR (400 MHz,
DMSO‑d6)
d
7.59 (dd, J ¼ 17.1, 7.3 Hz, 2H), 7.34e7.28 (m, 2H), 6.84 (d,
J ¼ 8.1 Hz, 1H), 6.80e6.74 (m, 2H), 6.67 (s, 2H), 4.82 (s, 2H), 4.27 (s,
2H), 3.71 (s, 9H), 3.67 (s, 3H), 3.64 (s, 3H).13C NMR (100 MHz,
DMSO‑d6)
d 166.08, 163.98, 152.98, 151.14, 148.46, 148.05, 141.10,
136.99, 136.60, 129.39, 124.61, 124.26, 120.75, 118.06, 112.13, 111.44,
110.10, 106.10, 60.02, 55.94, 55.44, 55.34, 52.34, 36.39. HR-MS (ESI):
Calcd. C27H28N2O6S2, [MþH] þ m/z: 563.1281, found: 563.1244.
2-(benzo[d]thiazol-2-ylthio)-N-(3,4,5-trimethoxybenzyl)-N-
(3,4,5-trimethoxyphenyl) acetamide(F6).
5.2. Synthesis of compounds F1-F14
Yield, 44%, M.p. 149e150 ꢀC, White solid.1H NMR (400 MHz,
DMSO‑d6)
d
7.99 (d, J ¼ 7.9 Hz, 1H), 7.72 (d, J ¼ 8.1 Hz, 1H), 7.44 (t,
A solution of compounds E1-E11 (0.5 mmol, 1.0 eq), substituted
benzothiazoles (0.6 mmol, 1.2 eq) and K2CO3 (0.75 mmol, 1.5 eq)
were added into acetonitrile (10 ml) at 80 ꢀC for 6 h. Upon
completion, organic phase was collected to obtain crude products
and then were purified with column chromatography to give
compounds F1-F14.
J ¼ 7.6 Hz, 1H), 7.36 (t, J ¼ 7.6 Hz, 1H), 6.69 (s, 2H), 6.50 (s, 2H), 4.83
(s, 2H), 4.29 (s, 2H), 3.72 (s, 6H), 3.64 (d, J ¼ 11.6 Hz, 12H).13C NMR
(100 MHz, DMSO‑d6) d 166.35, 166.01, 152.98, 152.65, 152.41, 137.02,
136.74, 136.64, 134.59, 132.84, 126.33, 124.47, 121.78, 120.83, 106.09,
105.67, 60.02, 59.97, 55.97, 55.75, 52.60, 36.99. HR-MS (ESI): Calcd.
C
28H30N2O7S2, [MþH] þ m/z: 571.1567, found: 571.1562.
2-(benzo[d]thiazol-2-ylthio)-N-(3-fluoro-4-methoxybenzyl)-
N-(3,4,5-trimethoxyphenyl) acetamide(F1).
2-(benzo[d]thiazol-2-ylthio)-N-(4-methoxy-3-nitrobenzyl)-
N-(3,4,5-trimethoxyphenyl) acetamide(F7).
Yield, 49%, M.p. 118e119 ꢀC, White solid.1H NMR (400 MHz,
Yield, 61%, M.p. 147e148 ꢀC, White solid. 1H NMR (400 MHz,
DMSO‑d6)
J ¼ 7.6 Hz,1H), 7.36 (t, J ¼ 7.6 Hz,1H), 7.13e6.96 (m, 3H), 6.69 (s, 2H),
4.83 (s, 2H), 4.26 (s, 2H), 3.80 (s, 3H), 3.71 (s, 6H), 3.66 (s, 3H). 13
NMR (100 MHz, DMSO‑d6) 166.21, 153.05, 152.40, 149.94, 146.23,
d
8.00 (d, J ¼ 7.9 Hz, 1H), 7.75 (d, J ¼ 8.0 Hz, 1H), 7.46 (t,
DMSO‑d6)
d
7.99 (d, J ¼ 7.7 Hz, 1H), 7.78e7.70 (m, 2H), 7.53 (d,
J ¼ 8.3 Hz, 1H), 7.45 (t, J ¼ 7.1 Hz, 1H), 7.35 (t, J ¼ 7.5 Hz, 1H), 7.24 (d,
C
J ¼ 8.7 Hz, 1H), 6.73 (s, 2H), 4.91 (s, 2H), 4.27 (s, 2H), 3.89 (s, 3H),
d
3.73 (s, 6H), 3.66 (s, 3H). 13C NMR (100 MHz, DMSO‑d6)
d 166.56,
137.04, 136.75, 134.65, 130.16, 130.11, 126.30, 124.53, 124.50, 124.45,
121.83, 115.65, 113.46, 106.00, 60.01, 56.01þ, 55.92, 51.66, 36.83. HR-
MS (ESI): Calcd. C26H25FN2O5S2, [MþH] m/z: 529.1262, found:
529.1256.
165.97, 153.13, 152.38, 151.05, 138.90, 137.07, 136.58, 134.62, 134.30,
129.64, 126.31, 124.55, 124.45, 121.79, 120.80, 114.06, 106.02, 60.01,
56.61, 56.00, 51.21, 36.84. HR-MS (ESI): Calcd. C26H25N3O7S2,
[MþH]þ m/z: 556.1207, found: 556.1201.
2-(benzo[d]thiazol-2-ylthio)-N-(3-bromo-4-
N-(3-azido-4-methoxybenzyl)-2-(benzo[d]thiazol-2-ylthio)-
N-(3,4,5-trimethoxyphenyl) acetamide(F8).
methoxybenzyl)-N-(3,4,5-trimethoxyphenyl) acetamide(F2).
Yield, 47%, M.p. 136e137 ꢀC, White solid.1H NMR (400 MHz,
Yield, 55%, M.p. 121e122 ꢀC, White solid. 1H NMR (400 MHz,
DMSO‑d6)
d
7.99 (d, J ¼ 8.0 Hz, 1H), 7.75 (d, J ¼ 8.0 Hz, 1H), 7.46 (d,
DMSO‑d6)
d
7.99 (d, J ¼ 7.8 Hz, 1H), 7.73 (d, J ¼ 8.0 Hz, 1H), 7.46 (t,
J ¼ 9.0 Hz, 2H), 7.36 (t, J ¼ 7.6 Hz,1H), 7.21 (d, J ¼ 8.0 Hz,1H), 6.99 (d,
J ¼ 8.4 Hz, 1H), 6.68 (s, 2H), 4.83 (s, 2H), 4.26 (s, 2H), 3.81 (s, 3H),
J ¼ 7.5 Hz, 1H), 7.36 (t, J ¼ 7.4 Hz, 1H), 7.04e6.89 (m, 3H), 6.68 (s,
2H), 4.81 (s, 2H), 4.25 (s, 2H), 3.81 (s, 3H), 3.72 (s, 6H), 3.66 (s,
3.72 (s, 6H), 3.66 (s, 3H).13C NMR (100 MHz, DMSO‑d6)
d
166.34,
3H).13C NMR (100 MHz, DMSO‑d6)
d 166.33, 165.99, 153.05, 152.39,
166.00, 154.54, 153.05, 152.40, 137.04, 136.64, 134.64, 132.81, 130.87,
129.07, 126.30, 124.44, 121.80, 120.91, 112.28, 110.19, 106.07, 60.03,
56.14, 56.00, 51.34, 36.88. HR-MS (ESI): Calcd. C26H25BrN2O5S2,
[MþH] þ m/z: 589.0461, found: 589.0455.
151.14, 137.04, 136.79, 134.67, 130.38, 126.84, 126.24, 126.16, 124.43,
121.80, 120.85, 120.59, 112.41, 106.02, 60.02, 56.01, 51.70, 36.80. HR-
þ
MS (ESI): Calcd. C26H25N5O5S2, [MþH]
m/z:552.1370, found:
552.1365.
2-(benzo[d]thiazol-2-ylthio)-N-(4-methoxy-3-
N-(3-amino-4-methoxybenzyl)-2-(benzo[d]thiazol-2-
ylthio)-N-(3,4,5-trimethoxyphenyl) acetamide(F9).
methylbenzyl)-N-(3,4,5-trimethoxyphenyl) acetamide(F3).
Yield, 61%, M.p. 136e138 ꢀC, White solid. 1H NMR (400 MHz,
Yield, 63%, M.p. 149e150 ꢀC, White solid.1H NMR (400 MHz,
DMSO‑d6)
d
7.99 (d, J ¼ 7.9 Hz, 1H), 7.73 (d, J ¼ 8.0 Hz, 1H), 7.45 (t,
DMSO‑d6)
d
8.00 (d, J ¼ 7.9 Hz, 1H), 7.77 (d, J ¼ 8.0 Hz, 1H), 7.46 (t,
J ¼ 7.5 Hz, 1H), 7.35 (t, J ¼ 7.5 Hz, 1H), 7.01 (s, 2H), 6.79 (d, J ¼ 8.7 Hz,
J ¼ 7.5 Hz,1H), 7.36 (t, J ¼ 7.5 Hz,1H), 6.71e6.59 (m, 3H), 6.55 (s,1H),
6.39 (d, J ¼ 7.7 Hz, 1H), 4.68 (d, J ¼ 19.2 Hz, 4H), 4.24 (s, 2H), 3.71 (d,
1H), 6.65 (s, 2H), 4.80 (s, 2H), 4.25 (s, 2H), 3.74 (s, 3H), 3.70 (s, 6H),
3.66 (s, 3H).13C NMR (100 MHz, DMSO‑d6)
d
166.11, 166.05, 156.51,
J ¼ 8.1 Hz, 9H), 3.66 (s, 3H).13C NMR (100 MHz, DMSO‑d6)
d 166.05,
152.98, 152.43, 137.00, 136.85, 134.66, 130.51, 128.68, 127.07, 126.28,
125.23, 124.42, 121.79, 120.87, 109.81, 106.11, 60.01, 55.96, 55.14,
165.94,152.92,152.42,145.62,137.28,136.96,136.85,134.63,129.44,
126.32, 124.44, 121.79, 120.93, 116.35, 113.99, 110.15, 106.09, 60.02,

J. Song et al. / European Journal of Medicinal Chemistry 203 (2020) 112618
13
55.95, 55.26, 52.35, 36.99. HR-MS (ESI): Calcd. C26H26N2O6S2,
[MþH] þ m/z:526.1465, found: 526.1460.
were purified by column chromatography to obtain compounds
H1-H6.
2-(benzo[d]thiazol-2-ylthio)-N-(3-hydroxy-4-
A solution of compounds H1-H6 (1.0 mmol, 1.0 eq) and
chloroacetic chloride (1.0 mmol,1.0 eq) were added into DMF (8 ml)
at 20 ꢀC for 6e8 h. When the reactions were complete, 20 ml H2O
and 20 ml ethyl acetate (three times) were added into aqueous
layers. Combined organic layers were washed by H2O and brine
successively, dried over magnesium sulfate anhydrous and then
evaporated to get crude products. The crude products were purified
by column chromatography to obtain compounds I1-I6.
methoxybenzyl)-N-(3,4,5-trimethoxyphenyl) acetamide(F10).
Yield, 52%, M.p. 140e141 ꢀC, White solid.1H NMR (400 MHz,
DMSO‑d6)
d
8.90 (s, 1H), 7.99 (d, J ¼ 7.9 Hz, 1H), 7.77 (d, J ¼ 8.0 Hz,
1H), 7.46 (t, J ¼ 7.5 Hz, 1H), 7.36 (t, J ¼ 7.5 Hz, 1H), 6.79 (d, J ¼ 8.2 Hz,
1H), 6.70 (s, 1H), 6.61 (d, J ¼ 8.1 Hz, 3H), 4.74 (s, 2H), 4.24 (s, 2H),
3.73 (s, 3H), 3.70 (s, 6H), 3.66 (s, 3H).13C NMR (100 MHz, DMSO‑d6)
d
166.07, 166.02, 152.95, 152.40, 146.88, 146.21, 136.97, 136.78,
134.62, 129.70, 126.32, 124.46, 121.77, 120.93, 119.30, 115.79, 111.86,
106.05, 60.03, 55.95, 55.58, 52.11, 36.86. HR-MS (ESI): Calcd.
C
5.4. Synthesis of compounds J1-J6
26H26N2O6S2, [MþH] þ m/z: 527.1305, found: 527.1300.
N-(3-hydroxy-4-methoxybenzyl)-2-((4-methylbenzo[d]thia-
A solution of compounds I1-I6 (0.5 mmol, 1.0 eq), 2-
zol-2-yl)thio)-N-(3,4,5-trimethoxyphenyl)acetamide(F11).
mercaptobenzothiazole (0.6 mmol, 1.2 eq) and K2CO3 (0.75 mmol,
1.5 eq) were added into acetonitrile (10 ml) at 80 ꢀC for 6 h. Upon
completion, the organic phases were collected and dried under a
vacuumtogivecrudethe productsand thenwerepurified bycolumn
chromatography to give compounds J1-J16.
Yield, 42%, M.p. 150e151 ꢀC, White solid.1H NMR (400 MHz,
DMSO‑d6)
d
8.90 (s, 1H), 7.78 (d, J ¼ 6.4 Hz, 1H), 7.24 (d, J ¼ 6.7 Hz,
2H), 6.78 (d, J ¼ 8.0 Hz, 1H), 6.70 (s, 1H), 6.57 (s, 3H), 4.74 (s, 2H),
4.29 (s, 2H), 3.71 (d, J ¼ 11.9 Hz, 9H), 3.64 (s, 3H).13C NMR (100 MHz,
DMSO‑d6)
d
166.02, 164.58, 152.99, 151.54, 146.92, 146.27, 137.01,
2-(benzo[d]thiazol-2-ylthio)-N-benzyl-N-(3,4,5-
136.76, 134.40, 130.46, 129.76, 126.72, 124.37, 119.36, 119.09, 115.85,
111.87, 105.96, 59.98, 55.95, 55.59, 52.00, 37.20, 17.62. HR-MS (ESI):
Calcd. C27H28N2O6S2, [MþNa] þ m/z: 563.1281, found: 563.1276.
2-((5-chlorobenzo[d]thiazol-2-yl)thio)-N-(3-hydroxy-4-
methoxybenzyl)-N-(3,4,5-trimethoxyphenyl)acetamide(F12).
Yield, 59%, M.p. 142e144 ꢀC, White solid.1H NMR (400 MHz,
trimethoxyphenyl) acetamide (J1).
Yield, 45%, M.p. 151e153 ꢀC, White solid. 1H NMR (400 MHz,
CDCl3)
(m, 2H), 7.18 (d, J ¼ 1.5 Hz, 4H), 6.25 (s, 2H), 4.83 (s, 2H), 4.00 (s, 2H),
3.74 (s, 3H), 3.62 (s, 6H). 13C NMR (100 MHz, CDCl3)
166.00,
d
7.65 (dd, J ¼ 14.2, 8.0 Hz, 2H), 7.33e7.28 (m, 1H), 7.24e7.19
d
164.62, 152.62, 151.85, 136.99, 136.18, 135.89, 134.55, 128.19, 127.42,
126.59, 124.93, 123.33, 120.28, 120.09, 104.80, 59.90, 55.15, 52.58,
35.62. HR-MS (ESI): Calcd. C25H24N2O4S2, [MþH] þm/z: 481.1250,
found: 481.1257.
DMSO‑d6)
d
8.91 (s, 1H), 8.14 (s, 1H), 7.74 (d, J ¼ 8.6 Hz, 1H), 7.48 (d,
J ¼ 8.6 Hz,1H), 6.80 (d, J ¼ 8.1 Hz,1H), 6.71 (s,1H), 6.61 (d, J ¼ 7.8 Hz,
3H), 4.75 (s, 2H), 4.25 (s, 2H), 3.74 (s, 3H), 3.71 (s, 6H), 3.67 (s,
3H).13C NMR (100 MHz, DMSO‑d6)
d
167.42, 165.93, 152.97, 151.24,
2-(benzo[d]thiazol-2-ylthio)-N-(4-fluorobenzyl)-N-(3,4,5-
trimethoxyphenyl) acetamide(J2).
146.91, 146.25, 137.01, 136.75, 136.21, 129.72, 128.80, 126.68, 121.95,
121.47, 119.32, 115.84, 111.86, 106.06, 60.02, 55.96, 55.58þ, 52.14,
Yield, 60%, M.p. 145e146 ꢀC, White solid.1H NMR (400 MHz,
37.08. HR-MS (ESI): Calcd.
583.0735, found: 583.0729.
C26H25ClN2O6S2, [MþNa]
m/z:
CDCl3)
d
7.67 (d, J ¼ 7.9 Hz,1H), 7.60 (d, J ¼ 8.1 Hz,1H), 7.35e7.28 (m,
1H), 7.25e7.21 (m, 1H), 7.15 (dd, J ¼ 8.5, 5.5 Hz, 2H), 6.87 (t,
J ¼ 8.7 Hz, 2H), 6.27 (s, 2H), 4.79 (s, 2H), 3.98 (s, 2H), 3.75 (s, 3H),
2-((5-bromobenzo[d]thiazol-2-yl)thio)-N-(3-hydroxy-4-
methoxybenzyl)-N-(3,4,5-trimethoxyphenyl)acetamide(F13).
3.65 (s, 6H).13C NMR (101 MHz, CDCl3)
d 166.04, 164.53, 152.70,
Yield, 46%, M.p. 136e137 ꢀC, White solid.1H NMR (400 MHz,
137.06, 135.82, 134.54, 132.04, 129.95, 129.87, 124.95, 123.37, 120.18,
DMSO‑d6)
d
8.90 (s, 1H), 7.99e7.91 (m, 2H), 7.52 (d, J ¼ 8.5 Hz, 1H),
120.12, 114.33, 114.12, 104.72, 59.91, 55.18, 51.88, 35.49. HR-MS
þ
6.79 (d, J ¼ 8.1 Hz, 1H), 6.68 (s, 1H), 6.61 (s, 3H), 4.74 (s, 2H), 4.23 (s,
(ESI): Calcd.
499.1151.
C
25H23FN2O4S2, [MþH]
m/z: 499.1156, found:
2H), 3.73 (s, 3H), 3.70 (s, 6H), 3.66 (s, 3H).13C NMR (100 MHz,
DMSO‑d6)
d
168.80, 165.93, 153.61, 152.94, 146.90, 146.22, 136.98,
2-(benzo[d]thiazol-2-ylthio)-N-(4-chlorobenzyl)-N-(3,4,5-
trimethoxyphenyl) acetamide(J3).
136.76, 133.94, 129.73, 127.04, 123.57, 123.29, 119.25, 119.14, 115.88,
111.80, 106.07, 60.02, 55.93, 55.59, 52.11, 37.02. HR-MS (ESI): Calcd.
Yield, 62%, M.p. 129e131 ꢀC, White solid. 1H NMR (400 MHz,
C
26H25BrN2O6S2, [MþNa]þ m/z: 605.0410, found: 605.0405.
CDCl3)
d
7.67 (d, J ¼ 7.9 Hz, 1H), 7.57 (d, J ¼ 8.0 Hz, 1H), 7.35e7.30
N-(3-hydroxy-4-methoxybenzyl)-2-((6-methoxybenzo[d]
(m, 1H), 7.25e7.21 (m, 1H), 7.14 (q, J ¼ 8.7 Hz, 4H), 6.29 (s, 2H), 4.79
thiazol-2-yl)thio)-N-(3,4,5-trimethoxyphenyl)acetamide(F14).
(s, 2H), 3.98 (s, 2H), 3.75 (s, 3H), 3.66 (s, 6H). 13C NMR (100 MHz,
Yield, 68%, M.p. 142e144 ꢀC, White solid.1H NMR (400 MHz,
CDCl3) d 166.13, 164.48, 152.74, 151.77, 137.10, 135.84, 134.70, 134.55,
DMSO‑d6)
d
8.90 (s, 1H), 7.66 (d, J ¼ 8.9 Hz, 1H), 7.58 (s, 1H), 7.04 (d,
132.50, 129.58, 127.56, 124.98, 123.39, 120.18, 120.14, 104.70, 59.92,
þ
J ¼ 8.9 Hz, 1H), 6.79 (d, J ¼ 8.1 Hz, 1H), 6.70 (s, 1H), 6.60 (s, 3H), 4.73
55.22, 52.02, 35.42. HR-MS (ESI): Calcd. C25H23ClN2O4S2, [MþH]
(s, 2H), 4.19 (s, 2H), 3.80 (s, 3H), 3.73 (s, 3H), 3.69 (s, 6H), 3.66 (s,
m/z: 515.0861, found: 515.0865.
3H).13C NMR (100 MHz, DMSO‑d6)
d
166.13, 162.54, 156.64, 152.94,
2-(benzo[d]thiazol-2-ylthio)-N-(4-bromobenzyl)-N-(3,4,5-
trimethoxyphenyl) acetamide(J4).
146.90, 146.89, 146.24, 136.97, 136.81, 136.12, 129.74, 121.47, 119.30,
115.82, 114.99, 111.86, 106.05, 104.87, 60.02, 55.94, 55.63,þ55.57,
Yield, 49%, M.p. 134e135 ꢀC, White solid.1H NMR (400 MHz,
52.09, 36.95. HR-MS (ESI): Calcd. C27H28N2O7S2, [MþNa]
m/z:
CDCl3)
d
7.68e7.65 (m, 1H), 7.56 (d, J ¼ 7.8 Hz, 1H), 7.36e7.28 (m,
579.1230, found: 579.1224.
3H), 7.24e7.20 (m, 1H), 7.07 (d, J ¼ 8.4 Hz, 2H), 6.29 (s, 2H), 4.77 (s,
2H), 3.97 (s, 2H), 3.75 (s, 3H), 3.65 (s, 6H).13C NMR (100 MHz, CDCl3)
5.3. Synthesis of compounds H1-H6 and I1-I10
d 161.86, 160.43, 148.52, 147.34, 132.89, 131.59, 130.97, 130.21,
126.30, 125.71, 120.82, 119.23, 116.40, 115.92, 100.48, 55.70,þ51.01,
47.87, 31.26. HR-MS (ESI): Calcd. C25H23BrN2O4S2, [MþH] m/z:
559.0355, found: 559.0360.
A solution of benzyl chloride derivatives G1-G6 (1.0 mmol, 1.0
eq), 3,4,5-trimethoxyaniline B (1.0 mmol, 1.0 eq) and K2CO3 were
added into DMF (20 ml) at 25 ꢀC for 6 h. When the reactions were
complete, 20 ml H2O and 20 ml ethyl acetate (three times) were
added into aqueous layers. Collected organic layers were washed by
H2O and brine successively, dried over magnesium sulfate anhy-
drous and then evaporated to get crude products. Crude products
2-(benzo[d]thiazol-2-ylthio)-N-(4-methylbenzyl)-N-(3,4,5-
trimethoxyphenyl) acetamide(J5).
Yield, 52%, M.p. 150e153 ꢀC, White solid.1H NMR (400 MHz,
CDCl3)
d
7.64 (dd, J ¼ 16.8, 8.0 Hz, 2H), 7.33e7.28 (m, 1H), 7.24e7.20
(m, 1H), 7.06 (d, J ¼ 8.0 Hz, 2H), 6.99 (d, J ¼ 7.9 Hz, 2H), 6.26 (s, 2H),

14
J. Song et al. / European Journal of Medicinal Chemistry 203 (2020) 112618
4.78 (s, 2H), 4.00 (s, 2H), 3.75 (s, 3H), 3.63 (s, 6H), 2.25 (s, 3H).13
NMR (100 MHz, CDCl3) 165.94, 164.76, 152.62, 151.75, 137.02,
C
in MOE 2015.10 and was prepared by energy minimization and
conformational search. The ligands were docked into the colchicine
binding site of tubulin and 20 poses were exported for the next
analysis.
d
136.23, 135.97, 134.49, 133.12, 128.19, 128.04, 124.93, 123.35, 120.30,
120.07, 104.89, 59.90, 55.17, 52.37, 35.70, 20.11. HR-MS (ESI): Calcd.
C
26H26N2O4S2, [MþH] þ m/z:495.1407, found: 495.1411.
2-(benzo[d]thiazol-2-ylthio)-N-(4-methoxybenzyl)-N-(3,4,5-
5.10. Western blot analysis
trimethoxy phenyl) acetamide(J6).
Yield, 52%, M.p. 132e133 ꢀC, White solid. 1H NMR (400 MHz,
Gastric cancer cells were seeded in dishes and treated with
compound F10 or DMSO. After 48 h treatment, gastric cancers were
collected and then lysed. The lysates of each group were electro-
phoretic separated in SDS-PAGE after denatured. Proteins were
then transferred to PVDF membranes from the gel. After blocked,
the membranes were incubated for 1st antibody conjugation. Then
membranes were washed and incubated with 2nd antibody. The
target proteins were detected after washed.
CDCl3)
d
7.64 (dd, J ¼ 17.5, 8.0 Hz, 2H), 7.30 (t, J ¼ 7.6 Hz, 1H), 7.22 (d,
J ¼ 7.5 Hz, 1H), 7.09 (d, J ¼ 8.3 Hz, 2H), 6.71 (d, J ¼ 8.3 Hz, 2H), 6.25
(s, 2H), 4.76 (s, 2H), 3.99 (s, 2H), 3.75 (s, 3H), 3.71 (s, 3H), 3.64 (s,
6H).13C NMR (100 MHz, CDCl3)
d 165.84, 164.77, 158.10, 152.62,
151.72, 136.98, 135.92, 134.48, 129.59, 128.35, 124.93, 123.36, 120.27,
120.09, 112.72, 104.87, 59.91, 55.20, 54.26, 52.02, 35.73. HR-MS
(ESI): Calcd. C26H26N2O5S2, [MþH] þm/z:511.1356, found: 511.1360.
5.5. Cell culture
5.11. Gene silencing using small interfering RNA (siRNA)
Cells were cultured in RPMI-1640 medium supplemented with
10% fetal bovine serum (FBS), 100 U/ml penicillin and 0.1 mg/ml
streptomycin. All the cells were incubated at 37 ꢀC and 5% CO2.
Gastric cancer cells were seeded 5 ꢂ 105 cells per dish into 60-
mm dishes and transfected with siRNA oligonucleotides (100 nmol/
L), purchased from GenePharma (Shanghai, China) using Lipofect-
amine 2000 (Cat. No. 11668019, Thermo Fisher Scientific). Cells
were transfected with siRNA for 48 h and further treated with
compound for 48 h. The sequences of the siRNA are as follows:
5.6. MTT assay
5,000 cells were seeded into 96-well cell culture plates. After
24 h, cells were treated with synthesized compounds. And then,
MTT reagent was added 20 mL per well after 48 h treatment with
5.12. General methods
synthesized compounds. Cells were then incubated for 4 h at 37 ꢀC.
Formazan was then dissolved with DMSO. Absorbencies of for-
mazan solution at 490 nm were determined. SPSS version 10.0 was
used for 50% inhibitory concentration (IC50) calculation.
Colony formation assay, cell apoptosis assay, cell cycle distri-
bution assay, flow cytometry analysis, and immunostaining assay
were proceeded according to our group reported work in European
journal of medicinal chemistry [49] and Bioorganic & Medicinal
Chemistry Letters [50].
5.7. In vitro tubulin polymerization assay
Pig brain microtubule protein was isolated by three cycles of
temperature-dependent assembly/disassembly in PIPES (pH 6.5,
100 mM), MgSO4 (1.0 mM), EGTA (2.0 mM), GTP (1.0 mM) and 2-
mercaptoethanol (1.0 mM). In the first cycle of polymerization,
glycerol and phenylmethylsulfonyl fluoride were added to 4 M and
0.2 mM, respectively. Homogeneous tubulin was prepared from
microtubule protein by phosphocellulose (P11) chromatography.
The purified proteins were stored in aliquots at ꢁ70 ꢀC.
Tubulin protein was mixed with compound F10 and colchicine
in PEM buffer containing GTP (1 mM) and glycerol (5%). Microtu-
bule polymerization was monitored by light scattering at 340 nm
using a SPECTRA MAX 190(Molecular Device) spectrophotometer at
37 ꢀC.
5.13. Statistical analysis
The data of three independent experiments were expressed as
mean SD and calculated by SPSS version 17.0.
Competing financial interests
There is no conflict of interest about this article to declare.
Declaration of competing interest
There is no conflict of interest about this article to declare.
5.8. EBI alkylate assay
Acknowledgement
5 ꢂ 105 gastric cancer cells were seeded into 6-well cell culture
plates. After 24 h, cells were treated with compound F10, colchicine
and DMSO. After 2 h treatment, cells were then treated with
100 mmol/L EBI for another 2 h. At last, cells were harvested for the
This work was supported by the National Key Research Program
of Proteins (No. 2016YFA0501800 for Hong min Liu, China), Na-
tional Natural Science Foundation of China of China (No. 81703541
for Sai-Yang Zhang and No. 81673322 for Yan-Bing Zhang), China
Postdoctoral Science Foundation (No. 2018M632812 for Sai-Yang
Zhang), the Henan Scientific Innovation Talent Team, Department
for Education (No. 19ITSTHN001 for Wen Zhao, China), Henan As-
sociation of Science and Technology (2020HYTP056) and Science
and Technology Department of Henan Province (202102310144).
determine of
b-tubulin and b-tubulin adduct (alkylated b-tubulin)
with anti- -tubulin antibody.
b
5.9. Molecular docking study
The molecular docking study was performed using MOE
2015.10. The crystal structure of Tubulin (PDB ID: 6O61) was
retrieved from RCSB Protein Data Bank, and then was prepared by
adding hydrogen atoms, removing water molecules and repairing
the missing side chains. The protonation states of protein residues
were calculated in the pKa at 7. The ligand compound F10 was built
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112618.

J. Song et al. / European Journal of Medicinal Chemistry 203 (2020) 112618
evaluation and cocrystal structures with tubulin of chiral
15
b
-lactam bridged
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