Discovery of novel chalcone-dithiocarbamates as ROS-mediated apoptosis inducers by inhibiting catalase

Article abstract of DOI:10.1016/j.bioorg.2019.01.023

Novel chalcone-dithiocarbamate hybrids were designed, synthesized and evaluated for antiproliferative activity against selected cancer cell lines (MGC803, MCF7, and PC3). Among these analogues, (E)-2-oxo-2-((4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenyl)am

Full text of DOI:10.1016/j.bioorg.2019.01.023

Accepted Manuscript
Discovery of novel chalcone-dithiocarbamates as ROS-mediated apoptosis in-
ducers by inhibiting catalase
Dong-Jun Fu, Jia-Huan Li, Jia-Jia Yang, Ping Li, Simeng Liu, Zhong-Rui Li,
Sai-Yang Zhang
PII:
S0045-2068(18)31351-8
DOI:
https://doi.org/10.1016/j.bioorg.2019.01.023
YBIOO 2727
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 November 2018
9 January 2019
12 January 2019
Please cite this article as: D-J. Fu, J-H. Li, J-J. Yang, P. Li, S. Liu, Z-R. Li, S-Y. Zhang, Discovery of novel chalcone-
dithiocarbamates as ROS-mediated apoptosis inducers by inhibiting catalase, Bioorganic Chemistry (2019), doi:
https://doi.org/10.1016/j.bioorg.2019.01.023
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Discovery of novel chalcone-dithiocarbamates as ROS-mediated
apoptosis inducers by inhibiting catalase
Dong-Jun Fua, b, d, 1, Jia-Huan Lib, 1, Jia-Jia Yangb, 1, Ping Lib, Simeng Liuc, Zhong-Rui
Lib, Sai-Yang Zhanga, b*
aSchool of Basic Medical Science, Zhengzhou University, Zhengzhou, 450001, China
bSchool of Pharmaceutical Sciences & Collaborative Innovation Center of New Drug Research
and Safety Evaluation, Zhengzhou University, Zhengzhou 450001, China
cDepartment of Gastroenterology, the Fifth Affiliated Hospital of Zhengzhou University,
Zhengzhou 450052, China
dDepartment of Urology, University of California, Irvine, Orange, CA 92868, USA
1These authors contributed equally to this work.
*Corresponding author, E-mail addresses: saiyangz@zzu.edu.cn (S.-Y. Zhang)
Abstract: Novel chalcone-dithiocarbamate hybrids were designed, synthesized and
evaluated for antiproliferative activity against selected cancer cell lines (MGC803,
MCF7,
and
PC3).
Among
these
analogues,
(E)-2-oxo-2-((4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenyl)amino)ethyl-4-(2-hydrox
yethyl)piperazine-1-carbodithioate (12d) showed the best inhibitory activity against
PC3 cells (IC50 = 1.05 uM). Cellular mechanism studies elucidated 12d could inhibit
colony formation, arrest cell cycle at G2/M phase and induce DNA damage against
PC3 cells. Compound 12d also induced mitochondrial apoptosis by caspase activation,
MMP decrease, ROS production and catalase (CAT) inhibition. Importantly, 12d
inhibited epithelial-mesenchymal transition (EMT) process by regulating
EMT-related proteins (E-cadherin, N-cadherin, Vimentin, MMP2, MMP9). These
results indicated that 12d is a promising lead compound and deserves further
investigation for prevention and treatment of human prostate cancer.
Keywords: Chalcone-dithiocarbamate; Catalase; DNA damage; Apoptosis;
Epithelial-mesenchymal transition.
1. Introduction
Chalcone derivatives have broad spectrum of biological activities including
antimicrobial, anticancer, antiviral, antioxidant, anti-inflammatory, anticonvulsant,
and antimalarial activities [1-3]. Especially, chalcones exhibited potent anticancer
activity against cancer cells and the antitumor activity may be attributed to induction
of apoptosis, blocking cell cycle progression, inhibition of angiogenesis and so on [4,
5]. Chalcone analogue 1 (Fig.1) displayed the potent antiproliferative activity against
A549 cell lines with an IC50 value of 6.06 μM [6]. Chalcone 2 induced cell cycle
blockage in G1 phase of cell cycle and inhibition of S phase in PC3 cells [7].
Chalcone 3 exhibited the inhibitory effect against MDA-MB-231 cells and HCT116
cells with IC50 values of 3.42 μM and 5.29 μM, respectively [8]. Chalcone 4 showed
well cytotoxic activity against MCF7 and PC3 cell lines with IC50 values of 3.88 ±
1.03 μM and 3.15 ± 0.81 μM, respectively [9]. All these findings revealed that
chalcone skeleton contaning a 3,4,5-trimethoxyphenyl ring might be a potential
antiproliferative fragement in anticancer drugs design.

Fig.1. Antitumor chalcone derivatives
Dithiocarbamates (DTCs) have received considerable attention due to their
potent anticancer
activity
[10-12].
Tert-butyl
4-(((3-((3-(4-methoxyphenyl)-4-oxo-4H-chromen-7-yl)oxy)propyl)thio)carbonothioyl
)piperazine-1-carboxylate 5 (Fig.2) reported in our group displayed the potent
inhibition of cell growth via MAPK signaling pathway and inhibit migration via Wnt
pathway in PC3 cells [13]. Dual dithiocarbamate 6 showed the IC50 values of 54 nM
and 23 nM against HepG2 and MCF7 cell lines, respectively [14].
(1,3-Benzodioxol-5-ylmethyl)-2-[4-(4-nitrophenyl)-1-piperazinylthiocarbamoylthio]a
cetamide 7 exhibited notable inhibitory effect on C6 cells with an IC50 value of
23.33 ± 7.63 μg/mL
[15].
N-((2-methyl-4(3H)-quinazolinon-6-yl)methyl)dithiocarbamate 8 could arrested A549
cells at G2/M phase [16].
Fig.2. Antitumor dithiocarbamate derivatives
On the basis of these findings, in the present investigation we reported the synthesis

and antiproliferative evaluation of a new series of chalcone-based dithiocarbamate
derivatives as anticancer agents against selected cancer cell lines (MGC803, MCF7,
and PC3) [17-22]. In this work, molecular hybridization strategy based on the
incorporation of the chalcone and the dithiocarbamate into a single molecule has the
potential to improve the antiproliferative efficacy. Importantly, the synthesized
chalcone-dithiocarbamate hybrids were identified as ROS-mediated apoptosis
inducers.
2. Results and discussion
2.1. Chemistry
The synthetic routes towards chalcone analogues (10a~10c and 12a~12m) were
shown in Scheme 1. Commercially available 9 was reacted with acetophenone
derivatives to form chalcones 10a~10c by the claisen-schmidt condensation, which
was subjected to the acylation reaction to afford 11. The target chalcone hybrids
12a~12m were easily obtained with the mature reaction conditions developed by our
group [23-25]. The purity of all biologically evaluated compounds was determined to
be >95% by reverse phase high performance liquid chromatography (HPLC) analysis.
Scheme 1. Reagents and conditions: A: (a) acetophenone derivatives, NaOH, EtOH,
reflux; (b) 2-chloroacetyl chloride, K2CO3, Acetone, reflux; (c) CS2, substituted
piperazine derivatives, Na3PO4.12H2O, acetone, rt.
2.2. Antiproliferative activity
In continuation with our efforts toward the identification of novel derivatives with
anticancer potential [26], we evaluated the antiproliferative activity of chalcone

analogues (10a~10c and 12a~12m) against several cancer cell lines (MGC803, MCF7,
and PC3) using the MTT assay. Due to potentially similar mode of action between
reported dithiocarbamate derivatives and well-known 5-fluorouracil (5-FU), 5-Fu was
used as the reference drug in the MTT assay [24].
From the antiproliferative results of compounds 10a~10c in Table 1, chalcones
without dithiocarbamate group displayed very weak activity against all cancer cell
lines with IC50 values of > 40μM. However, chalcone-dithiocarbamate hybrid 12a
showed moderate to potent activity with IC50 values from 2.40μM to 15.88 μM
against all cancer cell lines. Based on this finding, it revealed that the dithiocarbamate
group might exhibit an important role for the antiproliferative activity of chalcones.
Table 1. Antiproliferative activity of chalcones 10a~10c and hybrid 12a
IC50 (μM)
R
Compound
MGC803
> 40
PC3
> 40
MCF7
> 40
NH2
10a
10b
10c
H
> 40
> 40
> 40
CH3
> 40
> 40
> 40
-
-
3.57±0.08
2.40±0.06
15.88±1.10
12a
5-FU
12.41±0.94 29.31±1.87 21.20±3.60
aAntiproliferative activity was assayed by exposure for 72 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.
In order to complete the structure activity relationships, a series of
chalcone-dithiocarbamate hybrid were prepared and evaluated for their
antiproliferative activity (MGC803, MCF7, and PC3). As shown in Table 2,
chalcone-dithiocarbamate hybrid 12b~12i exhibited the antiproliferative activity with
IC50 values ranging from 1.05 to 37.25 μM. For these chalcone-dithiocarbamate
hybrids 12b~12i, the importance of substituents on the piperazine unit was also
investigated in Table 2. Replacing the acetyl group (12b) by t-butyloxycarboryl group
(12e) caused a decrease of activity. When the ethyl group attaching piperazine unit of
compound 12c was replaced by a hydroxyethyl group of compound 12d, the
antiproliferative activity was improved against MGC803 and PC3 cancer cell lines. In
addition, chalcone-dithiocarbamate hybrids with 4-methoxy phenyl ring (12f) and

pyrimidine ring (12g) attaching piperazine unit displayed weak acitivty against all
cancer cell lines. All these modifications and structure activity relationship studies
revealed that the substituents on the piperazine unit is important for their inhibitory
activity.
Table 2. Antiproliferative activity of chalcone-dithiocarbamate hybrid 12b~12i
R
IC50(μM)
Compound
MGC803
2.48±0.02
1.99±0.23
1.74±0.16
20.56±0.18
PC3
MCF7
1.12±0.02
1.09±0.04
1.05±0.02
15.60±1.31
12.25±0.31
17.70±0.90
17.90±1.70
26.72±1.23
28.25±0.87
31.08±0.59
7.35±1.14
11.23±0.33
21.20±3.60
12b
12c
12d
12e
12f
31.41±1.42 27.08±1.46
22.82±0.78 27.25±0.09
22.20±0.64 17.34±0.76
12g
12h
12i
37.25±0.14
4.19±2.20
5-FU
-
12.41±0.94 29.31±1.87
aAntiproliferative activity was assayed by exposure for 72 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.
To investigate the effect of piperazine unit, we also explored the antiproliferative
activity of chalcone-dithiocarbamate hybrid 12j~12m (Table 3). Compounds with
morpholine (12j), thiomorpholine-1,1-dioxide (12k), piperidine (12l) and pyrrolidine
(12m) were synthesized and evaluated for their antiproliferative activity against all
these cell lines. Among them, compound 12j with morpholine unit exhibited the most
potent antiproliferative activity with an IC50 value of 1.28 μM against PC3 cells. We
found that the N-heterocycle was important for the activity showing an over 12-fold
activity loss, when the morpholine group (12j) was replaced with the pyrrolidine

(12m).
Table 3. Antiproliferative activity of chalcone-dithiocarbamate hybrid 12j~12m
IC50(μM)
PC3
Compound
MGC803
8.71±0.45
11.21±0.87
5.36±0.19
MCF7
1.28±0.08
4.25±0.47
1.88±0.07
9.52±0.62
3.98±0.69
17.25±0.15
18.25±0.87
21.20±3.60
12j
12k
12l
11.24±0.24 15.39±0.34
12m
5-FU
-
12.41±0.94 29.31±1.87
aAntiproliferative activity was assayed by exposure for 72 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.
2.3. 12d Inhibits prostate cell proliferation and reduces colony
formation
Among all these targeted hybrids, compound 12d showed the best anticancer
activity in vitro with an IC50 value of 1.05 μM against PC3 cell lines. Thus, we
selected 12d to explore its detailed antiproliferative mechanisms against PC3 cells.
PC3 cell line showed the obvious inhibition with the treatment of compound 12d in
time dependent and concentration dependent manners (Fig.3.A). We next studied the
ability of PC3 cell line to form colonies on 6-well cell culture plates in the presence of
compound 12d (0.5 μM and 2 μM). The number of colonies was reduced in a
concentration dependent manner, as shown in Fig.3.B. At the highest concentration of
12d (2 μM), colony formation was reduced over 90% as compared to the untreated
controls.

A
B
Control
2.0μM
0.5μM
Fig.3. A: Cell viability of PC3 cell line with compound 12d treatment; B: Colony
formation assay with 12d (0.5 μM and 2 μM) at 10 days.
2.4. 12d Arrests cell cycle at G2/M phase and regulates the
expression of related proteins
Cell cycle analysis of compound 12d was done to detect cell distribution in each
phase of cell cycle. From data obtained in (Fig.4.A and B), cell number at G2/M
phase with the treatment of 5 μM 12d changed to 67.36% compared with control,
indicating compound 12d could arrest cell cycle at G2/M phase against PC3 cell lines.
Cyclin-dependent kinases (CDKs) play a key role in the control of the G2/M
transition of the cell cycle in prostate cancers [27]. To explore the effect of compound
12d for cell cycle related proteins, the western blot assay was proceeded. As shown in
Fig.4.C, compound 12d upregulated the expression of P21 and decreased the
expression of CDK1 and CyclinB1.

A
Control
1.25μM
2.5μM
5.0μM
DNA content
B
C
0
1.25 2.5
5
(μM)
Concentrations (uM)
Cell number(%)
P21
G1
S
G2/M
0
56.70±1.18
56.62±1.34
45.37±0.75
22.06±0.33
24.20±1.34
23.44±1.04
19.09±2.10
8.30±1.83
18.36±2.71
18.73±0.64
34.49±2.53
67.36±3.07
CDK1
CyclinB1
β-Actin
1.25
2.5
5.0
Fig.4. A: Cell cycle analysis of PC3 cells with treatment of 12d (0, 1.25 μM, 2.5 μM
and 5 μM) after 48 hours treatment; B: Cell number of cycle phase; C: The expression
level of G2/M phase related proteins (P21, CDK1 and CyclinB1) after 48 hours
treatment.
2.5. 12d Inhibited epithelial-mesenchymal transition process
The epithelial-mesenchymal transition (EMT) is a highly conserved cellular
program that allows polarized, immotile epithelial cells to convert to motile
mesenchymal cells [28]. Epithelial-mesenchymal transition could inhibit tubulin
tyrosine ligase and promote microtubule stability, resulting in tubulin detyrosination
and microtentacles formation for the attachment endothelial cell [29]. As shown in
Fig.5. A, the purple part of pictures is the migrated cell, and the quantity of migrated
cells are decreased with the treatment of 12d. The transwell results were also shown
in Fig.5. B, the migration rate of migrated PC3 cells with the treatment of 12d
(control, 0.25 and 0.5 μM) were 100%, 50% and 31%, respectively. Then, we
detected the expression level of epithelial-mesenchymal transition markers
(E-cadherenin, N-cadherenin, MMP9, MMP2, and Vementin). From the results of
Fig.5. C, 12d could inhibit epithelial-mesenchymal transition process on PC3 cells by
upregulation of E-cadherenin and downregulation of N-cadherenin, actived-MMP9,
actived-MMP2, and Vementin.

Fig.5. A: Transwell test of compound 12d at 48 hours on PC3 cells, the purple part of
pictures is the migrated cell. B: Migration rate (%) of PC3 cells with the treatment of
12d (0, 0.25 and 0.5 μM). C: Western blot analysis of EMT-related proteins at 48
hours. ***p < 0.001 compared with the control.
2.6. 12d Induced DNA damage
DNA damage response constantly monitors DNA integrity and, in the presence of
any type of DNA damage, activates transient cell cycle arrest and repair of DNA to
ensure maintenance of genomic stability and cell viability [30, 31]. Based on the
blockage of cell cycle by compound 12d, we assume that compound 12d might
induced DNA danmage. The comet assay is a versatile and sensitive method for
measuring single- and double-strand breaks in DNA [30]. To confirm whether the
potent compound 12d generated the action to DNA, we quantified DNA damage by
the comet assay in PC3 cells treated with 12d at different concentrations (control,
1.25 μM, 2.5 μM and 5 μM). As shown in Fig.6, the length of DNA tails
was increased compared with control at a concentration dependent manner, indicating
that compound 12d displayed potent DNA-damaging ability.

Control
1.25μM
5.0μM
2.5μM
Fig.6. Representative images of nuclei in the comet assay. PC3 cells were treated with
compound 12d at 48 hours.
2.7. 12d Induced a decrease in the mitochondrial membrane
potential.
The loss of mitochondrial membrane potential (ΔΨm) displayed a key role during
drug-induced apoptosis [31]. The maintenance of mitochondrial membrane potential
(ΔΨm) is very important for bioenergetic function and mitochondrial
integrity [32]. As shown in Fig.7.A, red fluorescence decreased with the increase of
concentrations treatment and bright green fluorescence increased with the increase of
concentrations, indicating that 12d can induce a decrease in the mitochondrial
membrane potential against PC3 cells. In addition, the percentage of cells at 5 μM
was 20.9%, significantly higher than that of the control group (2.2%), also
demonstrating that compound 12d caused the mitochondrial membrane potential
decrease in a concentration-dependent manner.

1.25μM
2.5μM
Control
5.0μM
A
B
5.0μM
Control
1.25μM
2.5μM
20.9%
17.5%
2.3%
2.2%
JC-1 monomer
Fig. 7. A: PC3 cells were treated with 12d for 48 hours. The samples were stained
with JC-1 (magnification, 400X); B: The treated samples were analyzed after JC-1
staining.
2.8. 12d Induced apoptosis against PC3 cells
In this work, the apoptotic analysis of 12d agsinst PC3 cells was performed with
annexin V-FITC/PI double staining and quantitated by flow cytometry. From the
results of Fig. 8. A and B, the apoptotic population on PC3 cells increased to 27.9%
(1.25 μM), 50.7% (2.5 μM), and 55.9% (5 μM), respectively, compared to control. All
these datas indicated that compound 12d could induce cell apoptosis against PC3 cell
line.
Bcl-2 family proteins are important apoptosis regulators and overexpression of the
prominent pro-survival Bcl-2 family members like Bcl-xl and Bcl-2 is a common
feature responsible for deregulation of apoptosis in cancer cells [33]. We can see from
the result in Fig. 8. C that compound 12d dramatically increased the level of Bad, and
reduced the level of Bcl-2, Bid, and Bcl-xl. These results demonstrated that
compound 12d regulated Bcl-2 family proteins against PC3 cell line as a novel
apoptosis inducer.

A
5.0μM
Control
2.5μM
1.25μM
Annexin V-FITC
B
C
Bcl-2
Bid
Cell Rate
Control 1.25μΜ 2.5μΜ 5.0μΜ
14.1% 15.2%
Late apoptosis 3.7% 20.0% 36.6% 40.7%
Total 7.1% 27.9% 50.7% 55.9%
Early apoptosis 3.40% 7.9%
Bcl-xl
Bad
β-Actin
Fig. 8. A: The apoptosis analysis of 12d treated cells after Annexin V-FITC/PI
staining; B: Quantitative analysis of cell apoptosis; C: Western blot analysis of
apoptosis-related proteins at 48 hours.
2.9. 12d Induced the high ROS generation and inhibited the
catalase (CAT) activity
Reactive oxygen species (ROS) as regulators for cell apoptosis were appreciated to
function as signaling molecules to affect cancer cells [34]. In this work, we tried to
identify ROS effect in the course of 12d induced-apoptosis. Therefore, the
intracellular ROS generation in PC3 cells was marked by measuring the DCFH-DA
fluorescence intensity. As shown in Fig. 9. A and B, imaging analysis revealed
that 12d strongly generated ROS in PC3 cells in a concentration dependent manner.
Therefore, all these results suggested that compound 12d could trigger the
accumulation of intracellular ROS in PC3 cells.
Catalase (CAT) as an enzyme degrading hydrogen peroxide could be used as a
therapeutic agent for cancer, but its successful application will depend on the
distribution of the enzyme to the sites where ROS are generated [35]. Because 12d
generated high ROS accumulation, it was selected to explore the effect targeted to
CAT enzyme. With the increase of concentrations, the activity of CAT was decreased
( Fig. 9. C). These results indicate that compound 12d could induce the ROS level
increase by inhibiting the activity of catalase.

1.25μM
5.0μM
Control
2.5μM
A
C
B
**
**
Fig. 9. A: PC3 cells were stained by DCFH-DA after 24 hours treatment to detect
intracellular ROS level; B: Quantitative analysis of fluorescence; C: Effect of 12d on
inhibiting CAT activity. **p < 0.01 compared with the control.
2.10. Molecular docking studies
Molecular docking was performed to investigate binding modalities of the
synthesized chalcone derivative 12d and the catalase protein. In this paper, we chosed
the PDB code：1DGG based on the previous report [36]. As shown in Fig. 10, two
carbonyl groups of 12d formed two hydrogen bonds with the residues Tyr215 and
Arg203. The nitrogen atom of amino formed a hydrogen bond with the residue Val450.
Importantly, two sulphur atoms of the dithiocarbamate unit formed two hydrogen
bonds with the residues Arg203 and Gln458, respectively, indicating the important
role of dithiocarbamate for inhibitory activity against PC3 cells. In addition, the
3,4,5-trimethoxyphenyl ring of derivative 12d formed hydrophobic interactions with
the residues Pro304, Val450, Phe446 and Phe198.
A
B
Fig. 10. A: Protein surface map was used to show the binding of 12d and CAT, where,
blue is the hydrophobic surface of CAT, yellow is the 12d.; B: Low-energy binding
conformation of 12d bound to CAT. 12d (green), hydrogen bonds (purple) and protein
residues (pink) were as shown.

3. Conclusion
We designed and synthesized of a series of novel chalcone-dithiocarbamate hybrids
and evaluated their antiproliferative activity against cancer cell lines (MGC803,
MCF7, and PC3). Among all these hybrids, 12d showed the best inhibitory activity
against PC3 cells with an IC50 of 1.05 μM. Preliminary biological mechanisms
demonsrated that 12d could reduce colony formation, induce DNA damage and arrest
cell cycle at G2/M phase by the upregualtion of P21 and downregualtion of CDK1
and CyclinB1 in a concentration manner. By the scarification test and transwell test,
12d inhibited the epithelial-mesenchymal transition process by adjusting the
expression level of EMT related markers (E-cadherenin, N-cadherenin, MMP9,
MMP2, and Vementin). Importantly, 12d could cause PC3 cells apoptosis by inducing
the mitochondrial membrane potential decrease and changing the expression level of
BCl-2 family proteins. In addition, 12d as a novel apoptosis inducer can also trigger
the accumulation of intracellular ROS in PC3 cells by inhibiting the activity of
catalase enzyme. In summary, compound 12d could be a lead candidate for its further
application in treatment of prostate cancer.
4. Experimental section
4.1. General
Commercial reagents and solvents were purchased from Sigma Aldrich and
1
MedChemExpress. 13CNMR and HNMR spectra were recorded on a Bruker 100
MHz and 400 MHz spectrometer, respectively. High resolution mass spectra (HRMS)
of all derivatives were recorded on a Waters Micromass Q-T of Micromass
spectrometer by electrospray ionization (ESI). The purity of all biologically evaluated
compounds was determined to be >95% by reverse phase high performance liquid
chromatography (HPLC) analysis.
4.2
General procedure for the synthesis of compound 11
To a solution of (E)-1-(4-aminophenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one
10a (0.5 mmol, 1.0 eq) in acetone (5 mL) was added K2CO3 (0.75 mmol, 1.5 eq) at
reflux condition, and then the 2-chloroacetyl chloride (0.75 mmol, 1.5 eq) was added
dropwise. Upon completion, EtOAc and H2O were added. The organic layers were
washed with H2O for three times, and then washed with brine, dried over MgSO4 and
evaporated to give the products. The residue was purified with column
chromatography (hexane : EtOAc = 8:1) to obtain analogue 11.
4.3 General procedure for the synthesis of compounds 12a~12l
(E)-2-chloro-N-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenyl)acetamide (0.1 g, 1
eq) , CS2 (2 eq), secondary amine (1eq) and Na3PO4.12H2O (0.5 eq) were added in
acetone (5 ml). The mixture was stirred at room temperature for 6 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
chromatography (hexane : EtOAc = 10:1) to obtain analogue 12a~12l.
4.3.1(E)-2-chloro-N-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenyl)
acetamide (11)
1
Yellow solid; M.p.: 179~180oC; Yield: 30%. H NMR (400 MHz, DMSO-d6) δ 10.68
(s, 1H), 8.20 (d, J = 8.7 Hz, 2H), 7.90 (d, J = 15.5 Hz, 1H), 7.81 (d, J = 8.7 Hz, 2H),
7.69 (d, J = 15.5 Hz, 1H), 7.24 (s, 2H), 4.33 (s, 2H), 3.87 (s, 6H), 3.72 (s, 3H). 13C
NMR (100 MHz, DMSO-d6) δ 187.97, 165.70, 153.58, 144.51, 143.29, 140.15,
133.39, 130.77, 130.43, 121.53, 119.19, 106.96, 60.61, 56.60, 44.11. HR-MS
(ESI):Calcd. C20H21ClNO5, [M+H]+m/z: 390.1108, found: 390.1112.
4.3.2(E)-2-oxo-2-((4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenyl)am
ino)ethyl 4-methylpiperazine-1-carbodithioate (12a)
1
Yellow solid; M.p.: 176~177oC; Yield: 28%. H NMR (400 MHz, DMSO-d6) δ 10.70
(s, 1H), 8.18 (d, J = 8.7 Hz, 2H), 7.90 (d, J = 15.5 Hz, 1H), 7.80 (d, J = 8.7 Hz, 2H),
7.69 (d, J = 15.5 Hz, 1H), 7.23 (s, 2H), 4.33 (s, 2H), 4.20 (d, J = 9.2 Hz, 2H), 3.96 (s,
2H), 3.87 (s, 6H), 3.72 (s, 3H), 2.42 (s, 4H), 2.22 (s, 3H). 13C NMR (100 MHz,
DMSO-d6) δ 194.42, 187.40, 165.98, 153.07, 143.89, 143.35, 139.61, 132.44, 130.30,
129.92, 121.06, 118.35, 106.43, 60.10, 56.10, 53.87, 44.96, 41.31, 40.11. HR-MS
(ESI):Calcd. C26H32N3O5S2, [M+H]+m/z: 530.1783, found: 530.1787.
4.3.3(E)-2-oxo-2-((4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenyl)am
ino)ethyl 4-acetylpiperazine-1-carbodithioate (12b)
1
Yellow solid; M.p.: 207~208oC; Yield: 27%. H NMR (400 MHz, DMSO-d6) δ 10.71
(s, 1H), 8.19 (d, J = 8.7 Hz, 2H), 7.90 (d, J = 15.5 Hz, 1H), 7.80 (d, J = 8.7 Hz, 2H),
7.69 (d, J = 15.5 Hz, 1H), 7.23 (s, 2H), 4.35 (s, 2H), 4.32 – 4.17 (m, 2H), 4.02 (dd, J
= 13.7, 6.5 Hz, 2H), 3.87 (s, 6H), 3.72 (s, 3H), 3.63 (d, J = 7.0 Hz, 4H), 2.04 (s, 3H).
13C NMR (100 MHz, DMSO-d6) δ 194.81, 187.41, 168.67, 165.92, 153.07, 143.90,
143.32, 139.61, 132.46, 130.30, 129.93, 121.05, 118.36, 106.43, 60.10, 56.10, 44.40,
41.26, 30.65, 21.20. HR-MS (ESI):Calcd. C27H32N3O6S2, [M+H]+m/z: 558.1733,
found: 558.1737.
4.3.4(E)-2-oxo-2-((4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenyl)am
ino)ethyl 4-ethylpiperazine-1-carbodithioate (12c)
1
Yellow solid; M.p.: 162~163oC; Yield: 36%. H NMR (400 MHz, DMSO-d6) δ 10.69
(s, 1H), 8.18 (d, J = 8.7 Hz, 2H), 7.90 (d, J = 15.5 Hz, 1H), 7.80 (d, J = 8.6 Hz, 2H),
7.69 (d, J = 15.5 Hz, 1H), 7.23 (s, 2H), 4.32 (s, 2H), 4.21 (s, 2H), 3.95 (s, 2H), 3.87 (s,
6H), 3.72 (s, 3H), 2.46 (s, 4H), 2.37 (q, J = 7.1 Hz, 2H), 1.02 (t, J = 7.1 Hz, 3H). 13C
NMR (100 MHz, DMSO-d6) δ 194.22, 187.40, 166.00, 153.07, 143.90, 143.34,
139.61, 132.44, 130.30, 129.92, 121.05, 118.35, 106.43, 60.10, 56.10, 55.81, 51.67,
50.99, 41.27, 11.82. HR-MS (ESI):Calcd. C27H34N3O5S2, [M+H]+m/z: 544.1940,

found: 544.1948.
4.3.5(E)-2-oxo-2-((4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenyl)am
ino)ethyl-4-(2-hydroxyethyl)piperazine-1-carbodithioate (12d)
1
Yellow solid; M.p.: 168~169oC; Yield: 38%. H NMR (400 MHz, DMSO-d6) δ 10.67
(s, 1H), 8.18 (d, J = 8.8 Hz, 2H), 7.90 (d, J = 15.5 Hz, 1H), 7.80 (d, J = 8.8 Hz, 2H),
7.69 (d, J = 15.5 Hz, 1H), 7.23 (s, 2H), 4.49 (t, J = 5.2 Hz, 1H), 4.33 (s, 2H), 4.21 (s,
2H), 3.95 (s, 2H), 3.87 (s, 6H), 3.72 (s, 3H), 3.53 (q, J = 5.7 Hz, 2H), 2.54 (s, 4H),
13
2.45 (t, J = 6.0 Hz, 2H). C NMR (100 MHz, DMSO-d6) δ 194.67, 187.91, 166.51,
153.58, 144.40, 143.84, 140.12, 132.95, 130.80, 130.43, 121.56, 118.86, 106.94,
60.61, 59.94, 58.94, 56.60, 56.32, 52.96, 41.69. HR-MS (ESI):Calcd. C27H34N3O6S2,
[M+H]+m/z: 560.1889, found: 560.1893.
4.3.6Tert-butyl-(E)-4-(((2-oxo-2-((4-(3-(3,4,5-trimethoxyphenyl)acry
loyl)phenyl)amino)ethyl)thio)carbonothioyl)piperazine-1-carboxylat
e(12e)
1
Yellow solid; M.p.: 207~209oC; Yield: 56%. H NMR (400 MHz, DMSO-d6) δ 10.69
(s, 1H), 8.18 (d, J = 8.8 Hz, 2H), 7.90 (d, J = 15.5 Hz, 1H), 7.79 (d, J = 8.8 Hz, 2H),
7.68 (d, J = 15.5 Hz, 1H), 7.23 (s, 2H), 4.32 (d, J = 14.3 Hz, 2H), 4.22 (s, 2H), 4.03
(dd, J = 12.6, 5.5 Hz, 2H), 3.87 (s, 6H), 3.72 (s, 3H), 3.49 (s, 4H), 1.43 (s, 9H). 13C
NMR (100 MHz, DMSO-d6) δ 194.83, 187.42, 165.93, 153.67, 153.07, 143.90,
143.32, 139.61, 132.45, 130.29, 129.92, 121.05, 118.36, 106.43, 79.40, 60.11, 56.10,
41.26, 27.99. HR-MS (ESI):Calcd. C30H38N3O7S2, [M+H]+m/z: 616.2151, found:
616.2155.
4.3.7(E)-2-oxo-2-((4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenyl)am
ino)ethyl- 4-(4-methoxyphenyl)piperazine-1-carbodithioate (12f)
1
Yellow solid; M.p.: 150~152oC; Yield: 74%. H NMR (400 MHz, DMSO-d6) δ 10.70
(s, 1H), 8.18 (d, J = 8.8 Hz, 2H), 7.90 (d, J = 15.5 Hz, 1H), 7.80 (d, J = 8.8 Hz, 2H),
7.69 (d, J = 15.5 Hz, 1H), 7.23 (s, 2H), 6.94 (d, J = 9.1 Hz, 2H), 6.85 (d, J = 9.1 Hz,
2H), 4.35 (s, 4H), 4.12 (s, 2H), 3.87 (s, 6H), 3.71 (d, J = 9.8 Hz, 6H), 3.16 (s, 4H). 13C
NMR (100 MHz, DMSO-d6) δ 194.47, 187.42, 165.98, 153.39, 153.08, 144.37,
143.91, 143.34, 139.61, 132.45, 130.30, 129.93, 121.06, 118.37, 117.89, 114.33,
106.43, 60.11, 56.11, 55.16, 49.33, 41.28. HR-MS (ESI):Calcd. C32H36N3O6S2,
[M+H]+m/z: 622.2046, found: 622.2049.
4.3.8(E)-2-oxo-2-((4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenyl)am
ino)ethyl -4-(pyrimidin-2-yl)piperazine-1-carbodithioate (12g)
1
Yellow solid; M.p.: 176~178oC; Yield: 80%. H NMR (400 MHz, DMSO-d6) δ 10.71
(s, 1H), 8.42 (s, 1H), 8.41 (s, 1H), 8.18 (d, J = 8.8 Hz, 2H), 7.90 (d, J = 15.5 Hz, 1H),
7.81 (d, J = 8.8 Hz, 2H), 7.69 (d, J = 15.5 Hz, 1H), 7.23 (s, 2H), 6.70 (s, 1H), 4.51 (s,
13
2H), 4.37 (s, 2H), 4.11 (s, 2H), 3.90 (s, 4H), 3.87 (s, 6H), 3.72 (s, 3H). C NMR (100
MHz, DMSO-d6) δ 210.20, 194.64, 187.41, 165.96, 160.80, 157.98, 153.08, 143.89,

143.33, 139.63, 132.46, 130.30, 129.92, 121.06, 118.37, 110.62, 106.44, 68.66, 60.11,
56.62, 56.11, 42.46. HR-MS (ESI):Calcd. C29H32N5O5S2, [M+H]+m/z: 594.1845,
found: 594.1849.
4.3.9(E)-2-oxo-2-((4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenyl)am
ino)ethyl -4-butylpiperazine-1-carbodithioate (12h)
1
Yellow solid; M.p.: 130~132oC; Yield: 82%. H NMR (400 MHz, DMSO-d6) δ 10.69
(s, 1H), 8.19 (d, J = 8.8 Hz, 2H), 7.91 (d, J = 15.5 Hz, 1H), 7.81 (d, J = 8.8 Hz, 2H),
7.70 (d, J = 15.5 Hz, 1H), 7.24 (s, 2H), 4.33 (s, 2H), 4.21 (s, 2H), 3.95 (s, 2H), 3.88 (s,
6H), 3.73 (s, 3H), 2.45 (s, 4H), 2.33 – 2.25 (m, 2H), 1.45-1.38 (m, 2H), 1.34-1.25 (m,
2H), 0.88 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 194.14, 187.40,
165.98, 153.08, 143.88, 143.34, 139.64, 132.46, 130.30, 129.90, 121.06, 118.36,
106.44, 60.10, 56.84, 56.10, 52.15, 41.30, 28.36, 19.99, 13.84. HR-MS (ESI):Calcd.
C29H38N3O5S2, [M+H]+m/z: 572.2253, found: 572.2258.
4.3.10(E)-2-oxo-2-((4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenyl)a
mino)ethyl -4-(methylsulfonyl)piperazine-1-carbodithioate (12i)
1
Yellow solid; M.p.: 183~185oC; Yield: 62%. H NMR (400 MHz, DMSO-d6) δ 10.70
(s, 1H), 8.18 (d, J = 8.8 Hz, 2H), 7.90 (d, J = 15.5 Hz, 1H), 7.79 (d, J = 8.8 Hz, 2H),
7.68 (d, J = 15.5 Hz, 1H), 7.23 (s, 2H), 4.35 (s, 2H), 4.32 (s, 2H), 4.13 (s, 2H), 3.87 (s,
6H), 3.72 (s, 3H), 3.29 (s, 4H), 2.95 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ
195.35, 187.42, 165.84, 153.08, 143.90, 143.31, 139.64, 132.47, 130.29, 129.93,
121.07, 118.36, 106.46, 60.11, 56.12, 44.87, 41.41, 34.40. HR-MS (ESI):Calcd.
C26H32N3O7S3, [M+H]+m/z: 594.1402, found: 594.1407.
4.3.11(E)-2-oxo-2-((4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenyl)a
mino)ethyl- morpholine-4-carbodithioate (12j)
1
Yellow solid; M.p.: 176~178oC; Yield: 60%. H NMR (400 MHz, DMSO-d6) δ 10.70
(s, 1H), 8.18 (d, J = 8.6 Hz, 2H), 7.90 (d, J = 15.5 Hz, 1H), 7.80 (d, J = 8.6 Hz, 2H),
7.69 (d, J = 15.5 Hz, 1H), 7.23 (s, 2H), 4.35 (s, 2H), 4.21 (s, 2H), 4.08 – 3.94 (m, 2H),
3.87 (s, 6H), 3.72 (s, 3H), 3.70 (s, 4H). 13C NMR (100 MHz, DMSO-d6) δ 194.90,
187.45, 165.96, 153.07, 143.91, 143.31, 139.61, 132.45, 130.29, 129.92, 121.06,
118.38, 106.41, 65.53, 60.11, 56.10, 55.63, 41.09. HR-MS (ESI):Calcd. C25H29N2O6S2,
[M+H]+m/z: 517.1467, found: 517.1469.
4.3.12(E)-2-oxo-2-((4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenyl)a
mino)ethyl -thiomorpholine-4-carbodithioate 1,1-dioxide (12k)
1
Yellow solid; M.p.: 179~181oC; Yield: 74%. H NMR (400 MHz, DMSO-d6) δ 10.74
(s, 1H), 8.20 (d, J = 8.7 Hz, 2H), 7.91 (d, J = 15.5 Hz, 1H), 7.81 (d, J = 8.7 Hz, 2H),
7.70 (d, J = 15.5 Hz, 1H), 7.24 (s, 2H), 4.57 (s, 4H), 4.37 (s, 2H), 3.88 (s, 6H), 3.73 (s,
13
3H), 3.38 (s, 4H). C NMR (100 MHz, DMSO-d6) δ 197.10, 187.44, 165.62, 153.07,
143.91, 143.23, 139.64, 132.54, 130.29, 129.93, 121.06, 118.43, 106.44, 60.11, 56.10,
50.54, 42.24, 29.56. HR-MS (ESI):Calcd. C25H29N2O7S3, [M+H]+m/z: 565.1137,

found: 565.1139.
4.3.13(E)-2-oxo-2-((4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenyl)a
mino)ethyl piperidine-1-carbodithioate (12l)
Yellow solid; M.p.: 168~170oC; Yield: 79%. 1H NMR (400 MHz, DMSO-d6) δ 10.67
(s, 1H), 8.18 (d, J = 8.8 Hz, 2H), 7.90 (d, J = 15.5 Hz, 1H), 7.80 (d, J = 8.8 Hz, 2H),
7.69 (d, J = 15.5 Hz, 1H), 7.23 (s, 2H), 4.31 (s, 2H), 4.21 (s, 2H), 3.94 (s, 2H), 3.87 (s,
6H), 3.72 (s, 3H), 1.70 – 1.54 (m, 6H). 13C NMR (100 MHz, DMSO-d6) δ 192.97,
187.45, 166.10, 153.09, 143.87, 143.34, 139.69, 132.47, 130.30, 129.89, 121.12,
118.39, 106.48, 60.11, 56.13, 52.58, 51.08, 41.39, 25.80, 25.13, 23.44. HR-MS
(ESI):Calcd. C26H31N2O5S2, [M+H]+m/z: 515.1674, found: 515.1679.
4.3.14(E)-2-oxo-2-((4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenyl)a
mino)ethylpyrrolidine-1-carbodithioate (12m)
Yellow solid; M.p.: 190~192oC; Yield: 84%. 1H NMR (400 MHz, DMSO-d6) δ 10.65
(s, 1H), 8.18 (d, J = 8.8 Hz, 2H), 7.90 (d, J = 15.5 Hz, 1H), 7.79 (d, J = 8.8 Hz, 2H),
7.68 (d, J = 15.5 Hz, 1H), 7.23 (s, 2H), 4.31 (s, 2H), 3.87 (s, 6H), 3.77 (t, J = 6.9 Hz,
2H), 3.72 (s, 3H), 3.71 – 3.66 (m, 2H), 2.10 – 2.01 (m, 2H), 1.94 (dd, J = 13.4, 6.5 Hz,
2H). 13C NMR (100 MHz, DMSO-d6) δ 190.09, 187.45, 166.11, 153.09, 143.87,
143.32, 139.69, 132.47, 130.30, 129.89, 121.11, 118.39, 106.49, 60.12, 56.14, 55.21,
50.61, 40.85, 25.67, 23.78. HR-MS (ESI):Calcd. C25H29N2O5S2, [M+H]+m/z:
501.1518, found: 501.1524.
4.4 Cell culture and MTT assay
MGC803 (human gastric cancer), PC3(human prostate cancer), and MCF7(human
breast cancer) were cultured in an atmosphere containing 5% CO2 at 37 °C, with
RPMI-1640 medium with 10% fetal bovine serum, 100 U/ml penicillin and 0.1 mg/ml
streptomycin.
Cells were seeded at a density of 5 x 103 per well in 96-well plates for 72 hours.
Then, 20 μL MTT solution was added to each well, and incubated for 4 hours at 37 °C.
150 μL DMSO was added to each well to dissolve the formazan after removing the
liquid, the absorbance was determined at 570 nm.
4.5 Clonogenicity assay
PC3 cells were seeded in a 6-well plate and incubated in 5% CO2 at 37 °C for 24 h,
then treated with the targeted compound 12d. After 10 days, remove the culture
medium, wash the cells twice with PBS, fix with 4% paraformaldehyde and stain with
0.1% crystal violet. The cells` image were captured, and the number of colonies were
quantified by Image J software (National Institutes of Health).
4.6 Cell cycle distribution assay
PC3 cells were seeded in 6-well culture plate and treated with compound 12d for 48
hours. Then cells were harvested and fixed with 70% ethanol at 4 °C for 8 hours. The
fixed cells were washed and resuspended with PBS containing 10 mg/mL RNaseA

and 50 mg/mL PI incubated for 25 minutes in dark. After that, samples were analyzed
for DNA content with flow cytometry. (Becton, Dickinson and Company, NJ)
4.7 Cell apoptosis assay
PC3 cells were seeded in 6-well culture plate and treated with compound 12d for 48
hours. Then cells were harvested and suspended in binding buffer containing Annexin
V-FITC (0.5 mg/mL) and PI (0.5 mg/mL) and incubated for 25 min in dark. After that,
samples were analyzed with flow cytometry (Becton, Dickinson and Company, NJ)
4.8 Migration assay
100 μL RPMI-1640 medium containing 1% fetal bovine serum, different
concentrations of compound 12d and 1x104 cells were added to each trans-well upper
chamber. 500 μL medium with 20% fetal bovine serum was used as chemoattractant
in the lower chamber. After 48 hours incubation, both chambers were washed with
PBS three times. Then stain the cells with crystal violet and analyze the image with
image J software (National Institutes of Health).
4.9 Catalase specific activity
Human catalase activity of treated and untreated PC3 cells was detected using a
catalase analysis kit (Beyotime Biotechnology, China). Briefly, working solution was
heated in 37 °C 10 minutes previously, then add the treated and untreated PC3 cells
lysate and determine the samples at 520 nm.
4.10 Measurement of loss of mitochondrial membrane potential
(MMP, ΔΨ)
JC-1 probe was used to measure the loss of MMP. Cells were seeded at a density of
1.5 x 105 each well in 6-well plates, and treated with compound 12d for 48 hours. 2.5
μg/ml JC-1were added and incubated at 37 °C for 10 minutes. Then cells were
analyzed with fluorescence microscope and flow cytometry after another two times
wash.
4.11 Measurement of ROS
2,7-Dichlorodihydro fluorescent diacetate (DCFH-DA) was used to measure the
levels of intracellular reactive oxygen species (ROS). Cells were seeded into a 6-well
plate, and treated with compound 12d. Following the treatment, cells were incubated
with 20mM DCFH-DA for 30 minutes at 37 °C in dark. Then cells were analyzed
with fluorescence microscope after another two times wash.
4.12 Western blot analysis
Treated and untreated cells were harvested and lysed. Protein lysates were denatured
and resolved by SDS-PAGE and transferred to nitrocellulose membrane. The
membranes were incubated with appropriate antibodies at 4 °C for 8 hours after
blocking with 5% skimmed milk. After conjugated with secondary antibodies, the

detection of proteins was carried out with an ECL kit.
4.13 Molecular docking
The Molecular Operating Environment software was used for docking studies. The
X-ray crystal structure of the Catalase (CAT) was retrieved from the Protein Data
Bank (PDB ID:1DGG) and prepared using the default parameters. Hydrogen atoms
and the partial charges for all atoms were added and water molecules were deleted.
Energy-minimized was done to the CAT using Amber 10: EHT forcefield. With
MOE-site Finder the CAT docking site was generated. Amber 10: EHT forcefield was
also used for the 3D structures of compound building and energy-minimize. The
London dG and Triangle Matcher placement method was used for docking.
GBVI/WSA dG scoring function was used for conformation optimization to evaluate
the final docking conformation. The docking results retain the top 30 in scoring.
4.14 Comet assay
PC3 cells were treated with different concentration of compound 12d for 48 hours.
Then the cells were harvested and covered with comet slides. The slides were
immersed in the lysis buffer for 2 hours at 4 °C. After lyses, Comet slides were
immersed in freshly and cold electrophoresis buffer for 25min to equilibrate the slides.
After equilibrating, electrophoresis was conducted at 25 V, 300 mA, for 30 min. Then
the slides coated with drops of neutralization buffer for 5 min, repeated three times.
The slides were stained with 1.0 μg/ml DAPI, and observed with a fluorescent
microscope.
4.15 Statistical analysis
Data from three independent experiments are presented as mean ± SD. IC50 values
and Student’s t-tests were calculated by SPSS version 10.0 (SPSS, Inc., Chicago, IL,
USA). **and *** respectively represent p < 0.01 and p < 0.001.
Acknowledgment
This work was supported by the National Natural Sciences Foundations of China (No.
81703541 and 81673322), and project funded by China Postdoctoral Science
Foundation. This work was supported by MEDCHEMEXPRESS. Thanks to MCE
Award for Scientists Promoting Biology and Medicine Research.
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Highlights
.
.
.
.
Chalcone-dithiocarbamates were novel apoptosis inducers.
12d arrested cell cycle at G2/M phase and induced DNA damage.
12d induced ROS-mediated apoptosis by catalase inhibition.
12d inhibited epithelial-mesenchymal transition process.

Graphic abstract
Apoptosis
G2/M
G2/M arrest
ΔΨm
DNA damage
Inhibition of catalase
ROS