Design and synthesis of formononetin-dithiocarbamate hybrids that inhibit growth and migration of PC-3?cells via MAPK/Wnt signaling pathways

Article abstract of DOI:10.1016/j.ejmech.2016.12.027

A series of novel formononetin-dithiocarbamate derivatives were designed, synthesized and evaluated for antiproliferative activity against three selected cancer cell line (MGC-803, EC-109, PC-3). The first structure-activity relationship (SAR) for this formononetin-dithiocarbamate scaffold is explored in this report with evaluation of 14 variants of the structural class. Among these analogues, tert-butyl 4-(((3-((3-(4-methoxyphenyl)-4-oxo-4H-chromen-7-yl)oxy)propyl)thio)carbonothioyl)piperazine-1-carboxylate (8i) showed the best inhibitory activity against PC-3?cells (IC₅₀?=?1.97?μM). Cellular mechanism studies elucidated 8i arrests cell cycle at G1 phase and regulates the expression of G1 checkpoint-related proteins in concentration-dependent manners. Furthermore, 8i could inhibit cell growth via MAPK signaling pathway and inhibit migration via Wnt pathway in PC-3?cells.

Full text of DOI:10.1016/j.ejmech.2016.12.027

European Journal of Medicinal Chemistry 127 (2017) 87e99
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design and synthesis of formononetin-dithiocarbamate hybrids that
inhibit growth and migration of PC-3 cells via MAPK/Wnt signaling
pathways
,
,
,
Dong-Jun Fu a 1, Li Zhang a 1, Jian Song a 1, Ruo-Wang Mao a, Ruo-Han Zhao a,
Ying-Chao Liu a, Yu-Hui Hou a, Jia-Huan Li a, Jia-Jia Yang a, Cheng-Yun Jin a, Ping Li a,
Xiao-Lin Zi c, Hong-Min Liu a, Sai-Yang Zhang b *, Yan-Bing Zhang a
,
, **
a School of Pharmaceutical Sciences & Collaborative Innovation Center of New Drug Research and Safety Evaluation, Zhengzhou University, Zhengzhou
450001, China
b School of Basic Medical Sciences, Zhengzhou University, Zhengzhou 450001, China
c Pathology and Laboratory Medicine, University of California, Irvine, Orange, CA 92868, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel formononetin-dithiocarbamate derivatives were designed, synthesized and evaluated
for antiproliferative activity against three selected cancer cell line (MGC-803, EC-109, PC-3). The first
structure-activity relationship (SAR) for this formononetin-dithiocarbamate scaffold is explored in this
report with evaluation of 14 variants of the structural class. Among these analogues, tert-butyl 4-(((3-((3-
(4-methoxyphenyl)-4-oxo-4H-chromen-7-yl)oxy)propyl)thio)carbonothioyl)piperazine-1-carboxylate
Received 3 November 2016
Received in revised form
28 November 2016
Accepted 11 December 2016
Available online 14 December 2016
(8i) showed the best inhibitory activity against PC-3 cells (IC50 ¼ 1.97
mM). Cellular mechanism studies
elucidated 8i arrests cell cycle at G1 phase and regulates the expression of G1 checkpoint-related pro-
teins in concentration-dependent manners. Furthermore, 8i could inhibit cell growth via MAPK signaling
pathway and inhibit migration via Wnt pathway in PC-3 cells.
Keywords:
Formononetin
Dithiocarbamate
Growth
© 2016 Elsevier Masson SAS. All rights reserved.
Migration
1. Introduction
3.8
mM against HCT-116 cells (Fig. 1).
Dithiocarbamate, a privileged scaffold in drug discovery with a
Natural products have played prominent roles for drug discov-
ery and cancer therapy [1]. Formononetin 1, a bioactive isoflavone
found in red clover plant and widespread in the Leguminosae
family, was reported to have many potent pharmacological activ-
ities, including antioxidant, antiviral, antitumor, antihypertensive,
antibacterial, antiangiogenic effects and so on [2e6]. In recent
years, many formononetin analogues were designed as antitumor
agents and explored their biological mechanism against different
human cancer cell lines [7e9]. Its 7-phosphoramidate derivative 2
significantly induced early apoptosis in HepG-2 cells [10]. For-
mononetin nitrogen mustard derivative 3 could induce cell cycle
arrest at G2/M phase and cell apoptosis [11], with an IC50 value of
wide array of biological activities, including antifungal, antibacte-
rial, antitumor, inhibition of carbonic anhydrase activities and so on
[12e17]. Besides, the dithiocarbamate has always been used as a
linkage to combine different antiproliferative active scaffolds to
design new chemical entities. Our group have reported three series
of dithiocarbamates derivatives as potential antitumor agents: the
butenolide-containing dithiocarbamate 4 displayed an excellent
activity against Hela cells with an IC50 value of 0.77 mM [18]; the
novel dithiocarbamate-chalcone analogue 5 could change the
expression of apoptosis-related proteins and arrest the cell cycle at
G0/G1 phase against SK-N-SH cells [19]; triazoleꢀdithiocarbamate
based selective lysine specific demethylase 1 inactivator 6 inhibited
gastric cancer cell growth, invasion, and migration (Fig. 2) [20].
Molecular hybridization is a useful strategy of rational design of
new ligands based on the recognition of pharmacophoric subunits
in the molecular structure of two or more known bioactive de-
rivatives [21]. These above interesting findings and our continuous
quest to identify more potent anticancer agents, led to the
* Corresponding author.
** Corresponding author.
E-mail addresses: celeron16@163.com (S.-Y. Zhang), zhangyb@zzu.edu.cn
(Y.-B. Zhang).
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejmech.2016.12.027
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

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D.-J. Fu et al. / European Journal of Medicinal Chemistry 127 (2017) 87e99
Fig. 1. Formononetin derivatives with anticancer activity.
Fig. 2. Dithiocarbamate derivatives with anticancer activity.
molecular hybridization of formononetin and dithiocarbamate to
integrate them in one molecular platform to generate a new hybrid
with an excellent antiproliferative activity (Fig. 3).
2.2. Antiproliferative activity
In continuation with our efforts toward the identification of
novel derivatives with anticancer potential, we evaluated the
antiproliferative activity of formononetin analogues 7aed and
formononetin-dithiocarbamate hybrids 8aej against several cancer
cell lines (PC-3, MGC-803 and EC-109) using the MTT assay. Due to
potentially similar mode of action between reported dithiocarba-
mate derivatives and well-known 5-fluorouracil (5-FU), 5-Fu was
used as the reference drug in the MTT assay [23]. Besides, for-
mononetin 1 also was a positive control.
As shown in Fig. 3, a molecular hybridization strategy based on
the structures of a natural formononetin 1 and a bioactive dithio-
carbamate analogue 6 yielded a scaffold which has four parts: (i) a
dithiocarbamate group with drug-like properties, (ii) a natural
formononetin scaffold as an antitumor pharmacophore fragment
(iii) a medium-chain alkoxyl group for lipophilicity, and (Ⅳ) various
aliphatic amine units attached with dithiocarbamate fragment. To
the best of our knowledge, there have been no literature reports
regarding formononetin-dithiocarbamate hybrids so far. These
findings have encouraged us to investigate the potential synergistic
effect of dithiocarbamate and formononetin scaffolds.
The antiproliferative activity results against all three cancer cells
for the candidate compounds were shown in Table 1. The replace-
ment of the bromine atom by the dithiocarbamate scaffold resulted
in
a powerful improvement of activity for formononetin-
dithiocarbamate derivatives compared with the corresponding
formononetin analogues (7aed). Especially, compound 8i showed
the inhibitory effect against PC-3 cell line with an IC50 value of
2. Results and discussion
2.1. Chemistry
1.97
mM (>30-fold and 15-fold more potent than 7b and 5-Fu,
respectively). This result suggests that dithiocarbamate moiety may
play a synergistic role in determining activity.
In order to complete the SAR study, a series of formononetin-
dithiocarbamate hybrids were prepared and evaluated for their
antiproliferative activity. With the exception of compounds 8aej,
all the compounds bearing a dithiocarbamate moiety exhibit
The synthetic route were shown in Scheme 1. Commercially
available formononetin 1 was subjected to etherification reaction
with 1,2-dibromoethane, 1,3-dibromopropane, 1,4-dibromobutane,
or 1,5-dibromopentane to afford 7aed in the presence of potassium
carbonate. The target analogues were easily obtained in high yields
with the mature reaction conditions developed by our group [22].
Fig. 3. Rational molecular hybridization strategy for target antiproliferative derivatives.

D.-J. Fu et al. / European Journal of Medicinal Chemistry 127 (2017) 87e99
89
Scheme 1. Reagents and conditions: (a) 1,2-dibromoethane, 1,3-dibromopropane, 1,4-dibromobutane, or 1,5-dibromopentane, K2CO3, THF, reflux, 70e83% yield; (b) CS2, substituted
amine, Na3PO4e12H2O, acetone, rt, 78e85% yield.
antiproliferative activity with IC50 values ranging from 1.97 to
53.95 M. To determine whether the amine unites have an effect on
transformation [24]. As shown in Fig. 6A and B, PC-3 cells treated
with 8i exhibited fewer and smaller colonies compared to the
m
the activity, compounds with a piperazine unit (8aed, 8g), a mor-
pholine unit (8e), and a pyrrolidine unit (8f) were synthesized.
Replacement of the pyrrolidine unit (8f) with a morpholine unit
(8e) led to a decrease of the activity. However, changing the pyr-
rolidine unit (8f) to a 1-methylpiperazine unit (8d) led to a signif-
icant improvement of the activity against all the tested cell lines. All
these results indicated that the piperazine unit may play an
important role for their inhibitory activity.
Furthermore, the importance of substituents on the piperazine
unit was investigated. Replacing the methyl (8d) group by benzyl
group (8a), ethyl (8b), pyridine (8g), or acetyl (8c) caused a
decrease of activity. Removing the tert-butyloxycarbonyl group was
clearly detrimental for the activity, such as compound 8i compared
to 8d. The modifications and SAR studies revealed that the tert-
butyloxycarbonyl group is important for their inhibitory activity.
To investigate whether the length of carbon linker between the
formononetin scaffold and the dithiocarbamate scaffold might
affect the antiproliferative activity, compounds possessing a tert-
butyl piperazine-1-carboxylate moiety (8hej) were synthesized.
With either extension or reduction of the carbon linker length by
one carbon, the antiproliferative activity was decreased (8i VS. 8h
or 8j). These results suggested that the linker between the for-
mononetin and the dithiocarbamate scaffold plays a significant role
in their activities.
control with a colony formation efficiency of 6% at 2.5 mM, which
indicated that compound 8i could significantly inhibit the prolif-
eration of PC-3 cells in a concentration-dependent manner.
2.5. Inhibition of MAPK signaling pathway
Mitogen-activated protein kinase (MAPK) signal transduction
pathways are among the most widespread mechanisms of
eukaryotic cell regulation and play important roles in cell growth
[25]. Some distinct groups of MAPKs have been characterized in
mammals: the extracellular signal-regulated kinases (ERK 1/2,
ERK3/4, ERK5, ERK 7/8), the Jun N-terminal kinases (JNK 1/2/3) and
the p38 MAPKs (p38a/b/g/d) [26]. According to the inhibitory re-
sults of cells growth, it was hypothesized that 8i treatment might
affect the MAPK signaling pathway of PC-3 cells. To confirm this
property, the expression of related MAPK signaling proteins (p-ERK,
p-c-Jun, c-Myc, p38, p-p38) were determined. As shown in Fig. 6C
and D, the protein levels of p-ERK, p-c-Jun, c-Myc, p38, and p-p38
were decreased in concentration-dependent manners after treat-
ment of compound 8i. The tests on MAPK signaling pathway
illustrated that compound 8i might inhibit growth of PC-3 cells via
inhibiting MAPK signaling pathway.
2.6. 8i induces G1 phase arrest and regulates the expression of G1-
related proteins
Compound 8i was further examined for possible cytotoxicity
against GES-1 (normal human gastric epithelial cell line). We found
that compound 8i exhibited no cytotoxicity against GES-1
Targeting the cell cycle of tumor cells has been recognized as a
promising strategy for cancer therapy [27]. In this study, compound
8i was chosen to investigate the effect of our designed derivatives
on the cell cycle of PC-3 cells. After treating PC-3 cells with com-
(>100
mM). The results indicated that compound 8i had good
selectivity between the selected cancer cell line (MGC-803) and a
normal cell line (GES-1). The detailed illustration for SAR of target
derivatives was shown in Fig. 4.
pound 8i at different concentrations (0 mM, 0.1 mM, 0.5 mM, 2.5 mM)
for 48 h, cells were then fixed and stained with PI for flow
cytometry analysis. As shown in Fig. 7, compound 8i concentration
dependently arrested cell cycle at G1 phase, accompanied with the
decrease of cells at G2 and S phase. Specifically, the percentage of
2.3. Cell viability test and cell growth curve
Based on the screening activity results of all synthetic de-
rivatives, the most potent formononetin-dithiocarbamate hybrid,
8i, was prioritized to perform further experiment for evaluating its
antiproliferative potential in PC-3 cells (Fig. 5). As shown in Fig. 5A,
following treatment with 8i, the viability of the PC-3 cells
decreased in a concentration-dependent manner. As shown in cell
growth curve (Fig. 5B), the PC-3 cells treated by 8i began to grow
slower than control after 6 h, and the difference of growth speed
became significant after 12 h. These results suggested that 8i could
significantly inhibit growth of PC-3 cells.
cells at G1 phase for the high concentration group (2.5
83.32%, about 30% higher than that of the control group.
mM) was
The G1-phase arrest induced by compound 8i was further
confirmed by investigation of the changes in G1 checkpoint-related
proteins, including cyclin D1 and CDK4 [28]. It is well known that
the levels of cyclin A and cyclin B1 decreased during G1 phase [29].
Our results as shown in Fig. 7C verified it. Cyclin D-Cdk4 complex
accumulation promotes G1-S transition in cell cycle [30]. Com-
pound 8i down regulated cyclin D1 and CDK4 concentration
dependently (Fig. 7C), which lead to cell arrested in G1 phase.
2.4. Clone assays
2.7. Compound 8i could not induce apoptosis of PC-3 cells
Based on the most excellent activity against PC-3 cells, com-
pound 8i was chosen to perform colony formation to investigate
whether 8i could inhibit PC-3 cells proliferation (Fig. 6A and B). The
assay measures the ability of PC-3 cells to grow and form foci,
which represents an indirect estimation of neoplastic
Based on the biological experiments above, compound 8i was
chosen to perform apoptosis assays to investigate whether 8i could
induce apoptosis of PC-3 cells (Fig. 8AeC). After treatment with
different concentrations of compound 8i (0, 0.1, 0.5 and 2.5 mM) for

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D.-J. Fu et al. / European Journal of Medicinal Chemistry 127 (2017) 87e99
Table 1
Antiproliferative activity of formononetin-dithiocarbamate derivatives.
Compound
na
IC50 (m
M)b
MGC-803
EC-109
PC-3
7a
7b
7c
7d
8a
e
e
e
e
0
1
2
3
1
>100
55.05 3.98
39.67 1.14
66.31 0.75
59.10 0.81
25.27 0.59
41.89 0.53
62.61 0.80
55.62 0.89
44.76 1.82
14.92 0.09
64.87 0.82
52.72 1.07
>100
43.81 1.43
8b
8c
1
1
53.95 3.91
39.42 1.87
36.91 0.67
24.68 1.36
7.08 0.74
4.31 0.98
8d
8e
8f
1
1
1
1
21.93 2.00
49.62 2.07
39.97 1.84
42.02 1.39
24.24 1.16
34.04 0.95
30.79 0.73
38.28 0.91
2.21 0.97
27.36 1.29
19.36 2.03
17.39 0.27
8g
8h
8i
0
1
2
11.61 0.46
6.07 0.88
31.27 1.15
4.09 0.83
3.54 1.47
8.84 1.27
3.64 0.90
1.97 0.01
4.61 1.21
8j
1
5-FU
e
e
e
e
>100
7.21 1.04
>100
10.30 0.83
57.01 1.04
29.31 1.87
a
"n" represents the number of eCH2e.
b
Antiproliferative activity was assayed by exposure for 48 h to substances and expressed as concentration required to inhibit tumor cell proliferation by 50% (IC50). Data are
presented as the means SDs from the dose-response curves of three independent experiments.
48 h, PC-3 cells were stained with PI and Annexin V-FITC, and then
analyzed by the flow cytometry. As illustrated in Fig. 8A and B, the
percentage of apoptotic cells was not changed obviously from the
control group. Then, we performed to examine the expression of

D.-J. Fu et al. / European Journal of Medicinal Chemistry 127 (2017) 87e99
91
Fig. 4. Summary of SAR of target derivatives.
inhibition was concentration-independent.
The Wnt signaling pathway plays a critical role in numerous
cellular processes including cell proliferation, tissue homeostasis,
and cell migration [31e33]. Specifically once activated,
disassembles from complex comprised of Axin, APC and GSK3
b
-Catenin
and
b
translocates into nuclear [34]. Then it interacts with TCF4 which
promotes the downstream target gene transcription [35]. Accord-
ing to the transwell migration assay above, we next tested whether
compound 8i could influence Wnt signaling pathway in PC-3 cells.
Western blotting analysis of related Wnt pathway proteins (b-cat-
enin, Axin, TCF-4) were performed to confirm this property. As
shown in Fig. 9C and D, treatment of PC-3 cells with 8i resulted in
the increase expression of Axin and the decrease expression of b-
catenin and TCF-4 in concentration-dependent manners. These
results indicated that compound 8i might inhibit migration of PC-
3 cells via Wnt signaling pathway.
3. Conclusions
Following our previous work, we designed a series of new
formononetin-dithiocarbamate derivatives based on the natural
formononetin skeleton by molecular hybridization strategy. All
hybrids possessed moderate to good growth inhibition against the
tested cancer cells. Particularly, compound 8i exhibited excellent
growth inhibition against PC-3 cells with an IC50 value of 1.97 mM.
Fig. 5. (A) The effect of 8i in reducing cell viabilities of PC-3 cells measured by MTT
assay. The cells were treated with the indicated concentrations of 8i for 48 h. The
columns of each index have * p < 0.01 vs. untreated group; (B) Growth curve of PC-
3 cells. 2.5 ꢁ 104 cells were seeded in 24-well plate. After 24 h, they were treated with
The preliminary SAR illustrated that dithiocarbamate as a reported
antitumor scaffold could play potential synergistic effect for natural
formononetin skeleton. These hybrids in this work might serve as
bioactive fragments and lead compounds for developing more
potent cytotoxic agents.
8i (2.5 mM) for various time. The cell growth curve was plotted with culture time as the
X axis and the fold of cell numbers gained from MTT assay as the Y axis.
The mechanism investigation showed that compound 8i
inhibited the colony formation, halted cell cycle progression at the
G1 phase, altered the expression of cell cycle-related proteins, and
induced the inhibition of MAPK signaling pathway. These results
investigated that formononetin-dithiocarbamate derivative 8i
could inhibit the growth of PC-3 cells. In addition to the mechanism
study, we also explored the effect on migration. 8i inhibited
migration of PC-3 cells in a concentration-dependent manner
involved to Wnt signaling pathway. More mechanism studies are
underway and will be reported in due course.
key protein (Bax) related to apoptosis. As shown in Fig. 8C, the
expression of Bax protein did not increase after treatment of PC-
3 cells with compound 8i. All these results in Fig. 8 illustrated that
compound 8i could not induce apoptosis of PC-3 cells.
2.8. 8i inhibits migration involved to Wnt signaling pathway on PC-
3 cells
From above studies, we can conclude that compound 8i
inhibited growth of PC-3 cells. We next investigated whether
compound 8i could inhibit migration of cancer cells. After incuba-
4. Experimental section
tion at different concentrations (0, 0.1, 0.5, 2.5
m
M) for 48 h, PC-
4.1. General
3 cells were stained with crystal violet, and migrated cells were
detected and numbered using the high content screening system.
As shown in Fig. 9A and B, compound 8i inhibited migration of PC-
Reagents and solvents were used without special treatment.
Melting points were determined on an X-5 micromelting apparatus
and are uncorrected. 1HNMR and 13CNMR spectra were recorded on
3 cells even at low concentration (0.1
mM). Intriguingly, the

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D.-J. Fu et al. / European Journal of Medicinal Chemistry 127 (2017) 87e99
Fig. 6. (A) Representative images of PC-3 cells colonies after treatment with various concentrations for a week; (B) Quantitative analysis of the colony formation efficiency. The
results shown were representative of three independent experiments. **: p < 0.01 verse control. (c) Inhibition of MAPK signaling pathway; (D) Statistical analysis of MAPK pathway
protein expression levels. The results shown were representative of three independent experiments. *: p < 0.05 verse control, **: p < 0.01 verse control.
a Bruker 400 MHz and 100 MHz spectrometer, respectively. High
resolution mass spectra (HRMS) of all derivatives were recorded on
a Waters Micromass Q-T of Micromass spectrometer by electro-
spray ionization (ESI).
(0.75 mmol,1.5 eq) was added dropwise. The mixture was stirred at
60 ꢂC. Upon completion, EtOAc and H2O were added. The aqueous
layer was extracted with EtOAc for several times; the combined
organic layers were washed with H2O for several times to remove
the THF, and then washed with brine, dried over MgSO4 and
evaporated to give the products. The residue was purified with
column chromatography (hexane: EtOAc ¼ 6:1) to obtain analogue
7a ~ d.
4.2. General procedure for the synthesis of compounds 7a ~ d
To a solution of formononetin (0.5 mmol, 1.0 eq) in THF (5 mL)
was added K2CO3 (0.75 mmol, 1.5 eq) at room temperature, the
mixture was stirred for about 30 min, and then the bromide

D.-J. Fu et al. / European Journal of Medicinal Chemistry 127 (2017) 87e99
93
Fig. 7. (A) Effects of 8i on PC-3 cell cycle progress for 48 h; (B) Quantitative analysis of the percentage of cell cycle phase; (C) Effects of 8i on G1 regulatory protein. PC-3 cells were
treated for 48 h with the indicated concentration of 8i. The cells were harvested and lysed for the detection of cyclin A, cyclin B1, CDK4, cyclin D1; (D) Staticstical analysis of G1
regulatory protein expression levels. The results shown were representative of three independent experiments. *: p < 0.05 verse control, **: p < 0.01 verse control.

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D.-J. Fu et al. / European Journal of Medicinal Chemistry 127 (2017) 87e99
Fig. 8. Compound 8i could not induce apoptosis of PC-3 cells. (A) Apoptosis analysis with PI staining after 48 h in PC-3 cells; (B) Quantitative analysis of the percentage of early
apoptosis and late apoptosis; (C) Western blot analysis of Bax protein.
4.2.1. 7-(2-bromoethoxy)-3-(4-methoxyphenyl)-4H-chromen-4-
one (7a)
J ¼ 6.6 Hz, 2H), 2.31 (p, J ¼ 6.2 Hz, 2H). 13C NMR (100 MHz, DMSO)
d 174.59, 162.71, 159.00, 157.37, 153.46, 130.04, 127.01, 124.04,
123.36, 117.71, 114.93, 113.60, 101.17, 66.30, 55.13, 31.56, 30.94. HR-
MS (ESI): Calcd.
C
19H18BrO4, [MþH]þm/z: 389.0380, found:
389.0388. IR: 3437, 2930, 1623, 1609, 1513, 1446, 541 cmꢀ1
.
4.2.3. 7-(4-bromobutoxy)-3-(4-methoxyphenyl)-4H-chromen-4-
one (7c)
Yield 79%. White solid. Mp: 109e111 ꢂC. 1H NMR (400 MHz,
DMSO)
d
8.50 (s, 1H), 8.12 (d, J ¼ 8.9 Hz, 1H), 7.60 (d, J ¼ 8.7 Hz, 2H),
7.28 (d, J ¼ 2.3 Hz, 1H), 7.19 (dd, J ¼ 8.9, 2.3 Hz, 1H), 7.06 (d,
J ¼ 8.7 Hz, 2H), 4.72e4.43 (m, 2H), 4.02e3.89 (m, 2H), 3.86 (s, 3H).
13C NMR (100 MHz, DMSO)
d 162.73, 157.82, 154.34, 154.05, 130.45,
Yield 75%. White solid. Mp: 109e111 ꢂC. 1H NMR (400 MHz,
127.41, 124.53, 123.81, 118.43, 115.39, 113.95, 109.48, 101.79, 69.01,
55.63, 31.46. HR-MS (ESI): Calcd.
C
18H16BrO4, [MþH]þm/z:
DMSO)
d
8.47 (s, 1H), 8.09 (d, J ¼ 8.9 Hz, 1H), 7.59 (d, J ¼ 8.7 Hz, 2H),
7.22 (d, J ¼ 2.2 Hz, 1H), 7.14 (dd, J ¼ 8.9, 2.2 Hz, 1H), 7.05 (d,
J ¼ 8.7 Hz, 2H), 4.23 (t, J ¼ 6.2 Hz, 2H), 3.85 (s, 3H), 3.69 (t, J ¼ 6.5 Hz,
2H), 2.18e2.00 (m, 2H), 2.01e1.80 (m, 2H). 13C NMR (100 MHz,
375.0239, found: 375.0232. IR: 3007, 2953, 1609, 1576, 1498, 1446,
544 cmꢀ1
.
DMSO)
d 175.11, 163.43, 159.48, 157.90, 153.95, 130.55, 127.43,
4.2.2. 7-(3-bromopropoxy)-3-(4-methoxyphenyl)-4H-chromen-4-
124.54, 123.83, 118.03, 115.51, 114.09, 101.54, 68.10, 55.62, 35.23,
one (7b)
29.43, 27.58. HR-MS (ESI): Calcd.
C
20H20BrO4, [MþH]þm/z:
403.0548, found: 403.0545. IR: 2963, 1633, 1608, 1515, 1446,
696 cmꢀ1
.
4.2.4. 7-((5-bromopentyl)oxy)-3-(4-methoxyphenyl)-4H-chromen-
4-one (7d)
Yield 70%. White solid. Mp: 131e132 ꢂC. 1H NMR (400 MHz,
DMSO)
d
8.42 (d, J ¼ 1.2 Hz, 1H), 8.04 (d, J ¼ 8.9 Hz, 1H), 7.59e7.45
(m, 2H), 7.19 (d, J ¼ 2.0 Hz, 1H), 7.10 (dd, J ¼ 8.9, 2.4 Hz, 1H),
7.04e6.89 (m, 2H), 4.25 (t, J ¼ 6.0 Hz, 2H), 3.79 (s, 3H), 3.70 (t,

D.-J. Fu et al. / European Journal of Medicinal Chemistry 127 (2017) 87e99
95
Fig. 9. Migration inhibition induced by compound 8i. (A) Anti-migration effect of compound 8i on PC-3 cells at concentrations of 0, 0.1, 0.5 and 2.5
mM via transwell migration
assay; (B) Quantitative analysis of migration rate; (C) Inhibition of Wnt signaling pathway; (D) Staticstical analysis of Wnt pathway protein expression levels. The results shown
were representative of three independent experiments. **: p < 0.01 verse control.
Yield 83%. White solid. Mp: 110e111 ꢂC. 1H NMR (400 MHz,
DMSO)
4.3. General procedure for the synthesis of compounds 8a ~ j
d
8.46 (s, 1H), 8.07 (d, J ¼ 8.9 Hz, 1H), 7.58 (d, J ¼ 8.7 Hz, 2H),
7.19 (d, J ¼ 2.2 Hz, 1H), 7.12 (dd, J ¼ 8.9, 2.3 Hz, 1H), 7.04 (d,
J ¼ 8.8 Hz, 2H), 4.18 (t, J ¼ 6.4 Hz, 2H), 3.84 (s, 3H), 3.62 (t, J ¼ 6.7 Hz,
2H), 2.03e1.89 (m, 2H), 1.89e1.79 (m, 2H), 1.61 (dt, J ¼ 15.1, 7.6 Hz,
CS2 (2 eq) was added to the mixture of substituted amine (0.1 g,
1 eq) and Na3PO4$12H2O (0.5 eq) in acetone (5 mL). The mixture
was stirred at room temperature for 0.5 h. Then product 7a ~ d (1
eq) was added to the mixture cautiously, the mixture was
continued to stir at room temperature for another 0.5 h. Upon
completion, the solvent was removed under reduced pressure, the
residue was extracted with dichloromethane, washed with water,
brine, dried with anhydrous Na2SO4 and concentrated under
reduced pressure. The residue was purified with column chroma-
tography (hexane: EtOAc ¼ 7:1) to obtain analogue 8a ~ j.
2H). 13C NMR (100 MHz, DMSO)
d 175.10, 163.52, 159.47, 157.90,
153.91, 130.53, 127.40, 124.55, 123.82, 117.98, 115.50, 114.08, 101.47,
68.79, 55.62, 35.51, 32.37, 28.00, 24.68. HR-MS (ESI): Calcd.
C
21H22BrO4, [MþH]þm/z: 417.0707, found: 417.0701. IR: 3450, 2962,
1632, 1610, 1576, 1515, 1446, 542 cmꢀ1
.

96
D.-J. Fu et al. / European Journal of Medicinal Chemistry 127 (2017) 87e99
4.3.1. 3-((3-(4-methoxyphenyl)-4-oxo-4H-chromen-7-yl)oxy)
propyl 4-benzylpiperazine-1-carbodithioate (8a)
3.64e3.55 (m, 4H), 3.44 (t, J ¼ 7.2 Hz, 2H), 2.24e2.11 (m, 2H), 2.03
(s, 3H). 13C NMR (100 MHz, DMSO)
195.33, 174.61, 168.68, 162.81,
d
158.99, 157.38, 153.47, 130.05, 126.97, 124.04, 123.35, 117.62, 115.05,
113.61, 101.13, 67.15, 55.15, 44.41, 32.69, 27.81, 21.21. HR-MS (ESI):
Calcd. C26H29N2O5S2, [MþH]þm/z: 513.1510, found: 513.1518. IR:
3439, 2922, 1648, 1633, 1609, 1567, 1515, 1444, 1252, 1026 cmꢀ1
.
4.3.4. 3-((3-(4-methoxyphenyl)-4-oxo-4H-chromen-7-yl)oxy)
propyl 4-methylpiperazine-1-carbodithioate (8d)
Yield 83%. White solid. Mp: 124e125 ꢂC. 1H NMR (400 MHz,
DMSO)
d
8.42 (s, 1H), 8.03 (d, J ¼ 8.9 Hz, 1H), 7.53 (d, J ¼ 8.7 Hz, 2H),
7.38e7.29 (m, 4H), 7.29e7.22 (m, 1H), 7.17 (d, J ¼ 2.2 Hz, 1H), 7.09
(dd, J ¼ 8.9, 2.3 Hz, 1H), 7.00 (d, J ¼ 8.8 Hz, 2H), 4.22 (t, J ¼ 6.1 Hz,
4H), 3.92 (s, 2H), 3.79 (s, 3H), 3.51 (s, 2H), 3.42 (t, J ¼ 7.2 Hz, 2H),
2.45 (s, 4H), 2.22e2.09 (m, 2H). 13C NMR (100 MHz, DMSO)
d 194.74,
174.60, 162.81, 158.99, 157.38, 153.45, 137.53, 130.04, 128.91, 128.22,
127.09, 126.96, 124.05, 123.35, 117.63, 115.02, 113.60, 101.12, 67.14,
61.27, 55.13, 51.93, 32.72, 27.86. HR-MS (ESI): Calcd. C31H33N2O4S2,
[MþH]þm/z: 561.1885, found: 561.1882. IR: 3445, 2935, 1628, 1609,
1566, 1514, 1445, 1251 cmꢀ1
.
Yield 82%. Yellow solid. Mp: 167e169 ꢂC. 1H NMR (400 MHz,
DMSO)
d
8.43 (s, 1H), 8.04 (d, J ¼ 8.9 Hz, 1H), 7.53 (d, J ¼ 8.7 Hz, 2H),
4.3.2. 3-((3-(4-methoxyphenyl)-4-oxo-4H-chromen-7-yl)oxy)
propyl 4-ethylpiperazine-1-carbodithioate (8b)
7.18 (d, J ¼ 2.3 Hz, 1H), 7.10 (dd, J ¼ 8.9, 2.3 Hz, 1H), 7.00 (d,
J ¼ 8.8 Hz, 2H), 4.22 (t, J ¼ 6.1 Hz, 4H), 3.92 (s, 2H), 3.79 (s, 3H), 3.42
(t, J ¼ 7.2 Hz, 2H), 2.44e2.32 (m, 4H), 2.20 (s, 3H), 2.14 (dd, J ¼ 13.7,
7.0 Hz, 2H). 13C NMR (100 MHz, DMSO)
d 194.86, 174.60, 162.81,
158.99, 157.38, 153.47, 130.04, 126.96, 124.04, 123.35, 117.62, 115.03,
113.60, 101.12, 67.15, 55.13, 53.94, 45.03, 32.71, 27.86. HR-MS (ESI):
Calcd. C25H29N2O4S2, [MþH]þm/z: 485.1574, found: 485.1569. IR:
3442, 2940, 1625, 1595, 1576, 1513, 1442, 1294, 1249, 1198, 1181,
1031 cmꢀ1
.
4.3.5. 3-((3-(4-methoxyphenyl)-4-oxo-4H-chromen-7-yl)oxy)
propyl morpholine-4-carbodithioate (8e)
Yield 85%. Yellow solid. Mp: 167e168 ꢂC. 1H NMR (400 MHz,
DMSO)
d
8.43 (s, 1H), 8.03 (d, J ¼ 8.9 Hz, 1H), 7.53 (d, J ¼ 8.8 Hz, 2H),
7.18 (d, J ¼ 2.3 Hz, 1H), 7.09 (dd, J ¼ 8.9, 2.3 Hz, 1H), 7.00 (d,
J ¼ 8.8 Hz, 2H), 4.22 (t, J ¼ 6.1 Hz, 4H), 3.91 (s, 2H), 3.79 (s, 3H), 3.42
(t, J ¼ 7.2 Hz, 2H), 2.47e2.39 (m, 4H), 2.36 (q, J ¼ 7.2 Hz, 2H),
2.21e2.10 (m, 2H), 1.01 (t, J ¼ 7.2 Hz, 3H). 13C NMR (100 MHz,
DMSO)
d 194.69, 174.60, 162.82, 158.99, 157.38, 153.46, 130.05,
126.96, 124.05, 123.35, 117.62, 115.03, 113.61, 101.13, 67.15, 55.14,
51.71, 50.99, 32.69, 27.87, 11.85. HR-MS (ESI): Calcd. C26H31N2O4S2,
[MþH]þm/z: 499.1729, found: 499.1725. IR: 3423, 2969, 1625, 1594,
1576, 1513, 1467, 1253, 1181, 1031 cmꢀ1
.
4.3.3. 3-((3-(4-methoxyphenyl)-4-oxo-4H-chromen-7-yl)oxy)
propyl 4-acetylpiperazine-1-carbodithioate (8c)
Yield 79%. White solid. Mp: 156e158 ꢂC. 1H NMR (400 MHz,
DMSO)
d
8.43 (s, 1H), 8.04 (d, J ¼ 8.9 Hz, 1H), 7.53 (d, J ¼ 8.8 Hz, 2H),
7.18 (d, J ¼ 2.3 Hz, 1H), 7.10 (dd, J ¼ 8.9, 2.3 Hz, 1H), 7.00 (d,
J ¼ 8.8 Hz, 2H), 4.23 (t, J ¼ 6.1 Hz, 4H), 3.94 (s, 2H), 3.79 (s, 3H),
3.72e3.60 (m, 4H), 3.44 (t, J ¼ 7.2 Hz, 2H), 2.23e2.11 (m, 2H). 13
C
NMR (100 MHz, DMSO)
d 195.39, 174.60, 162.81, 158.99, 157.38,
153.47, 130.05, 126.97, 124.04, 123.35, 117.62, 115.03, 113.60, 101.12,
67.14, 65.56, 55.13, 32.56, 27.84. HR-MS (ESI): Calcd. C24H26NO5S2,
[MþH]þm/z: 472.1257, found: 472.1252. IR: 3442, 3077, 1638, 1624,
1609, 1575, 1514, 1441, 1250, 1199, 1177, 1138, 1116 cmꢀ1
.
Yield 78%. White solid. Mp: 174e176 ꢂC. 1H NMR (400 MHz,
DMSO)
d
8.43 (s, 1H), 8.04 (d, J ¼ 8.9 Hz, 1H), 7.53 (d, J ¼ 8.7 Hz, 2H),
4.3.6. 3-((3-(4-methoxyphenyl)-4-oxo-4H-chromen-7-yl)oxy)
propyl pyrrolidine-1-carbodithioate (8f)
7.18 (d, J ¼ 2.2 Hz, 1H), 7.10 (dd, J ¼ 8.9, 2.3 Hz, 1H), 7.00 (d,
J ¼ 8.8 Hz, 2H), 4.23 (t, J ¼ 6.1 Hz, 4H), 3.97 (s, 2H), 3.79 (s, 3H),

D.-J. Fu et al. / European Journal of Medicinal Chemistry 127 (2017) 87e99
97
J ¼ 8.6 Hz, 2H), 4.38 (t, J ¼ 6.2 Hz, 2H), 4.26 (s, 2H), 3.97 (s, 2H), 3.80
(s, 3H), 3.77 (t, J ¼ 6.2 Hz, 2H), 3.48 (s, 4H), 1.42 (s, 9H). 13C NMR
(100 MHz, DMSO)
d 195.25, 190.44, 175.10, 162.88, 159.52, 157.85,
154.03, 130.55, 127.56, 124.33, 123.87, 118.19, 115.46, 114.10, 101.78,
79.89, 75.53, 67.15, 55.63, 35.36, 28.49. HR-MS (ESI): Calcd.
C
28H33N2O6S2, [MþH]þm/z: 557.1788, found: 557.1780. IR: 3440,
2972, 1701, 1627, 1513, 1443, 1289, 1251, 1179 cmꢀ1
.
4.3.9. tert-butyl 4-(((3-((3-(4-methoxyphenyl)-4-oxo-4H-
chromen-7-yl)oxy)propyl)thio)carbonothioyl)piperazine-1-
carboxylate (8i)
Yield 81%. White solid. Mp: 159e161 ꢂC. 1H NMR (400 MHz,
DMSO)
d
8.43 (s, 1H), 8.04 (d, J ¼ 8.9 Hz, 1H), 7.53 (d, J ¼ 8.8 Hz, 2H),
7.18 (d, J ¼ 2.3 Hz, 1H), 7.09 (dd, J ¼ 8.9, 2.3 Hz, 1H), 7.00 (d,
J ¼ 8.8 Hz, 2H), 4.22 (t, J ¼ 6.2 Hz, 2H), 3.79 (s, 3H), 3.76 (d,
J ¼ 6.9 Hz, 2H), 3.62 (t, J ¼ 6.8 Hz, 2H), 3.40 (t, J ¼ 7.2 Hz, 2H),
2.23e2.10 (m, 2H), 2.07e1.96 (m, 2H), 1.91 (p, J ¼ 6.7 Hz, 2H). 13
C
NMR (100 MHz, DMSO)
d 190.61, 174.60, 162.83, 158.99, 157.39,
153.47, 130.05, 126.96, 124.05, 123.35, 117.62, 115.04, 113.60, 101.11,
67.15, 55.13, 54.93, 50.53, 32.03, 28.03, 25.53, 23.71. HR-MS (ESI):
Calcd. C24H26NO4S2, [MþH]þm/z: 456.1306, found: 456.1303. IR:
3445, 2848, 1623, 1609, 1574, 1513, 1443, 1290, 1265, 1201, 1177,
Yield 83%. White solid. Mp: 165e167 ꢂC. 1H NMR (400 MHz,
1031 cmꢀ1
.
DMSO)
d
8.42 (s, 1H), 8.03 (d, J ¼ 8.9 Hz, 1H), 7.53 (d, J ¼ 8.8 Hz, 2H),
7.17 (d, J ¼ 2.3 Hz, 1H), 7.09 (dd, J ¼ 8.9, 2.3 Hz, 1H), 7.00 (d,
J ¼ 8.8 Hz, 2H), 4.22 (t, J ¼ 6.1 Hz, 4H), 3.95 (s, 2H), 3.79 (s, 3H), 3.44
(dd, J ¼ 15.1, 7.3 Hz, 6H), 2.22e2.10 (m, 2H), 1.42 (s, 9H). 13C NMR
4.3.7. 3-((3-(4-methoxyphenyl)-4-oxo-4H-chromen-7-yl)oxy)
propyl 4-(pyridin-2-yl)piperazine-1-carbodithioate (8g)
(100 MHz, DMSO)
d 195.33, 174.59, 162.80, 158.98, 157.37, 153.68,
153.44, 130.04, 126.95, 124.04, 123.34, 117.62, 115.02, 113.59, 101.10,
79.35, 67.14, 55.13, 32.69, 27.99, 27.81, 18.53. HR-MS (ESI): Calcd.
C
29H35N2O6S2, [MþH]þm/z: 571.1940, found: 571.1937. IR: 3440,
2931,1690,1608,1566,1513,1444,1419,1250,1223,1178 1031 cmꢀ1
.
4.3.10. tert-butyl 4-(((4-((3-(4-methoxyphenyl)-4-oxo-4H-
chromen-7-yl)oxy)butyl)thio)carbonothioyl)piperazine-1-
carboxylate (8j)
Yield 84%. White solid. Mp: 178e180 ꢂC. 1H NMR (400 MHz,
DMSO)
d
8.42 (s, 1H), 8.13 (dd, J ¼ 4.9, 1.3 Hz, 1H), 8.04 (d, J ¼ 8.9 Hz,
1H), 7.60e7.55 (m, 1H), 7.55e7.48 (m, 2H), 7.19 (d, J ¼ 2.3 Hz, 1H),
7.10 (dd, J ¼ 8.9, 2.3 Hz, 1H), 7.00 (d, J ¼ 8.8 Hz, 2H), 6.81 (d,
J ¼ 8.6 Hz, 1H), 6.68 (dd, J ¼ 7.0, 5.0 Hz, 1H), 4.34 (s, 2H), 4.24 (t,
J ¼ 6.1 Hz, 2H), 4.06 (s, 2H), 3.79 (s, 3H), 3.66 (s, 4H), 3.46 (t,
J ¼ 7.1 Hz, 2H), 2.26e2.08 (m, 2H). 13C NMR (100 MHz, DMSO)
d
194.93, 174.52, 162.74, 158.91, 158.07, 157.30, 153.38, 147.47, 137.58,
Yield 85%. White solid. Mp: 143e144 ꢂC. 1H NMR (400 MHz,
129.97, 126.88, 124.42, 123.27, 117.54, 114.97, 113.52, 113.18, 106.86,
101.04, 67.07, 55.05, 43.51, 32.57, 27.76. HR-MS (ESI): Calcd.
DMSO)
d
8.41 (s, 1H), 8.02 (d, J ¼ 8.9 Hz, 1H), 7.53 (d, J ¼ 8.7 Hz, 2H),
C
29H30N3O4S2, [MþH]þm/z: 548.1684, found: 548.1678. IR: 3441,
7.15 (d, J ¼ 2.2 Hz, 1H), 7.07 (dd, J ¼ 8.9, 2.2 Hz, 1H), 7.00 (d,
J ¼ 8.8 Hz, 2H), 4.23 (s, 2H), 4.16 (t, J ¼ 5.8 Hz, 2H), 3.94 (s, 2H),
3.51e3.42 (m, 4H), 1.95e1.76 (m, 4H), 1.42 (s, 9H). 13C NMR
2915, 1639, 1627, 1599, 1567, 1514, 1442, 1251, 1224, 1035 cmꢀ1
.
4.3.8. tert-butyl 4-(((2-((3-(4-methoxyphenyl)-4-oxo-4H-
chromen-7-yl)oxy)ethyl)thio)carbonothioyl)piperazine-1-
carboxylate (8h)
(100 MHz, DMSO)
d 196.13, 175.10, 163.47, 159.48, 157.89, 154.18,
153.92, 130.54, 127.41, 124.55, 123.83, 118.01, 115.51, 114.09, 101.51,
79.86, 68.48, 55.62, 36.24, 28.49, 28.09, 25.55. HR-MS (ESI): Calcd.
C
30H37N2O6S2, [MþH]þm/z: 585.2096, found: 585.2093. IR: 3435,
2931, 1707, 1630, 1514, 1445, 1406, 1252, 1264, 1168 1027 cmꢀ1
.
4.4. Antiproliferative activity assays
MGC-803 cells (human gastric cancer), EC-109 (human esoph-
agus cancer), PC-3 (human prostate Cancer) and GES-1 (human
gastric epithelial cell) were cultured by RPMI 1640 medium with
10% FBS and 100 U/ml penicillin and 0.1 mg/ml streptomycin in the
37 ꢂC in an atmosphere containing 5% CO2. After 24 h of incubation,
the culture medium was removed and fresh medium containing
various concentrations of the candidate compounds was added to
Yield 79%. White solid. Mp: 140e142 ꢂC. 1H NMR (400 MHz,
DMSO)
d
8.43 (s, 1H), 8.04 (d, J ¼ 8.9 Hz, 1H), 7.53 (d, J ¼ 8.6 Hz, 2H),
7.26 (d, J ¼ 1.8 Hz, 1H), 7.11 (dd, J ¼ 9.0, 2.0 Hz, 1H), 7.00 (d,

98
D.-J. Fu et al. / European Journal of Medicinal Chemistry 127 (2017) 87e99
each well [23]. The cells were then incubated for 48 h, MTT assays
were performed and cell viability was assessed at 570 nm by a
microplate reader (Biotech, China). All experiments were per-
formed three times.
experiments. SPSS version 10.0 (SPSS, Inc., Chicago, IL, USA) was
used to calculate IC50 values and one-way analysis of variance
(ANOVA). *P < 0.05 and **P < 0.01 were considered to indicate as
statistical significant difference.
4.5. Clonogenicity assay
Acknowledgment
PC-3 cells were seeded in a 6-well plate and incubated for 24 h,
then the media were replaced with fresh media containing
different concentrations of compound 8i. After a week of treatment,
the cells were washed twice with PBS, fixed with 4% para-
formaldehyde, and colonies were visualized using 0.1% crystal vi-
olet staining. The cells were imaged, and the number of colonies
were quantified by Image J software (Developed by National In-
stitutes of Health).
This work was supported by the National Natural Sciences
Foundations of China (No. 81673322, 81273393, 81430085,
21372206, and 81172937), Ph.D. Educational Award from Ministry
of Education (No. 20134101130001).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.12.027.
4.6. Cell cycle distribution assay
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RNaseA and 50 mg/mL PI for 20 min in dark. Samples were then
analyzed for DNA content by flow cytometry (Becton, Dickinson
and Company, NJ.).
mM of compound 8i for 48 h. Then cells were collected and
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