Design and synthesis of novel 1,2,3-triazole-dithiocarbamate hybrids as potential anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2012.12.046

A series of novel 1,2,3-triazole-dithiocarbamate hybrids were designed, synthesized and evaluated for anticancer activity against four selected human tumor cell lines (MGC-803, MCF-7, PC-3, EC-109). Majority of the synthesized compounds exhibited moderate to potent activity against MGC-803 and MCF-7. Among them, compounds 3a and 3c showed excellent broad spectrum anticancer activity with IC₅₀ values ranging from 0.73 to 11.61 μM and 0.49-12.45 μM, respectively. Particularly, compound 3a was more potent than 5-fluorouracil against all tested human cancer cell lines. Flow cytometry analysis demonstrated that treatment of MGC-803 with 3c led to cell cycle arrest at G2/M phase accompanied by an increase in apoptotic cell death after 12 h.

Full text of DOI:10.1016/j.ejmech.2012.12.046

European Journal of Medicinal Chemistry 62 (2013) 11e19
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design and synthesis of novel 1,2,3-triazole-dithiocarbamate
hybrids as potential anticancer agents
Ying-Chao Duan, Yong-Cheng Ma, En Zhang, Xiao-Jing Shi, Meng-Meng Wang,
*
Xian-Wei Ye, Hong-Min Liu
New Drug Research & Development Center, School of Pharmaceutical Sciences, Zhengzhou University, No. 100, Avenue Kexue,
Zhengzhou 450001, PR China
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 1,2,3-triazole-dithiocarbamate hybrids were designed, synthesized and evaluated for
anticancer activity against four selected human tumor cell lines (MGC-803, MCF-7, PC-3, EC-109). Ma-
jority of the synthesized compounds exhibited moderate to potent activity against MGC-803 and MCF-7.
Among them, compounds 3a and 3c showed excellent broad spectrum anticancer activity with IC50
Received 31 October 2012
Received in revised form
25 December 2012
Accepted 29 December 2012
Available online 4 January 2013
values ranging from 0.73 to 11.61 mM and 0.49e12.45 mM, respectively. Particularly, compound 3a was
more potent than 5-fluorouracil against all tested human cancer cell lines. Flow cytometry analysis
demonstrated that treatment of MGC-803 with 3c led to cell cycle arrest at G2/M phase accompanied by
an increase in apoptotic cell death after 12 h.
Keywords:
1,2,3-triazole
Dithiocarbamate
Hybrid
Ó 2013 Elsevier Masson SAS. All rights reserved.
Anticancer
Cell cycle arrest
Apoptosis
1. Introduction
majority of the compounds proved to be more potent than etoposide
in selected human cancer cell lines [27]. A library of 1,2,3-triazole
1,2,3-Triazoles have occupied an important role not only in
organic chemistry but also in medicinal chemistry due to their easy
synthesis by click chemistry and attractive features as well as
numerous biological activities [1e3].1,2,3-Triazoles are highly stable
under basic and acid hydrolysis and reductive and oxidative condi-
tions, indicative of a high aromatic stabilization [4,5]. Moreover, this
heterocycle has a high dipole moment and is capable of hydrogen
bonding, which could be favorable in the binding of biomolecular
targets [6]. 1,2,3-Triazole is one of the key structural units found in
a large variety of bioactive molecules as anti-fungal [7], anti-
bacterial [8,9], anti-allergic [10], anti-HIV [11,12], anti-tubercular
[13,14] and anti-inflammatory agents [15]. Several 1,2,3-triazole-
containing drug molecules including tazobactam [16], cefatrizine
[4], carboxyamidotriazole [17] are now available in the market. In
recent years, people are increasingly focused on their anticancer
activity [18e26]. By combining 1,2,3-triazole with other pharmaco-
phores via click chemistry, a number of compounds with potent
antitumor activity were synthesized. A series of 1,2,3-triazole-
bearing podophyllotoxins were synthesized by H.M.S. Kumar,
analogs of combretastatin A-4 were prepared by Odlo, and one of
the triazole analogs displayed potent cytotoxic activity against sev-
eral cancer cell lines with IC50 values in the nanoMolar range [28]. A
family of 1,2,3-triazole-tethered
b-lactam-chalcones bifunctional
hybrids were designed and synthesized by V. Kumar, preliminary
studies showed that several compounds exhibited moderate to good
cytotoxic activity [29]. By incorporating the 1,2,3-triazole with ary-
lamides, M.J. Miller identified N-((1-benzyl-1H-1,2,3-triazol-4-yl)
methyl)arylamide as a novel and proprietary small molecule scaffold
for potential antitumor agents, and one of the compounds exhibited
an IC50 of 46 nM against MCF-7 cancer cell line [30].
On the other hand, dithiocarbamates have been attracting
considerable interest because of their diverse activities. In the lit-
erature, dithiocarbamate derivatives have been described as anti-
fungal [31], anti-bacterial [32] and carbonic anhydrases inhibitor
[33,34]. In particular, their applications in the treatment of cancer
have been exploring [35e43]. Our group recently reported the
synthesis of novel butenolide-containing dithiocarbamates, and
several compounds exhibited good anticancer activity [44,45].
While the pharmacological fight against cancer has made signifi-
cant progress in the last twenty years, novel molecules to fight this
disease are still urgently needed. Inspired by the biological importance
of 1,2,3-triazoles and dithiocarbamates as anticancer agents, we
* Corresponding author. Tel./fax: 86 371 67781739.
E-mail address: liuhm@zzu.edu.cn (H.-M. Liu).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.12.046

12
Y.-C. Duan et al. / European Journal of Medicinal Chemistry 62 (2013) 11e19
herein reported the synthesis of novel 1,2,3-triazole-dithiocarbamates
hybrids and their anticancer activity. The anticancer activity evalua-
tion results revealed that the 1,2,3-triazole-dithiocarbamate hybrids
exhibited potent anticancer activity.
From the screening results in Table 1, it was observed that com-
pounds 3aeh exhibited moderate to good anticancer activity against
MGC-803 and MCF-7. Two of the most active compounds are 3a and
3c, with IC50 values against the four tested human cancer cell lines
ranging from 0.73 to 11.61
mM and 0.49e12.45 mM, respectively.
Compound 3a was more cytotoxic than 5-fluorouracil against all
tested four human cancer cell lines, while 3c was less active than 5-
fluorouracil only against the EC-109 cells. Compound 3c proved to be
14-fold more potent than 5-fluorouracil in the case of MGC-803.
Starting from compound 3aeh, removing the tertiary butylox-
ycarbonyl group resulted in a dramatic drop of potency (4aeh).
2. Results and discussion
2.1. Chemistry
The synthetic route for 1,2,3-triazole-dithiocarbamate hybrids
3e6 is outlined in Scheme 1. Commercially available compound 1
reacting with CS2 and propargyl bromide in the presence of
Na3PO4$12H2O in one pot gave compound 2, which was further
reacted with appropriately substituted benzyl azides or aromatic
azides by click reaction to afford compounds 3aeh and 6aed with
good yields. The benzyl azides and aromatic azides were previously
obtained according to references [46,47]. Compounds 4aeh were
synthesized by removing the tertiary butyloxycarbonyl group of the
3aeh in a TFA/CH2Cl2 solution. Without further purification, cou-
pling 4aeh to carbobenzoxy chloride yielded compounds 5aeh.
The 1,2,3-triazole-dithiocarbamate hybrids bearing a coumarin
ring (9 and 13) were synthesized according to Scheme 2 and Scheme
3. Starting from m-hydroxy phenol (Scheme 2), compound 7 could
be obtained directly following literature procedures [48,49]. 7 was
converted to the azide derivative 8 via nucleophilic substitution by
using sodium azide in acetonitrile. Compound 9 was prepared by
Cu(I)-mediated Huisgen cycloaddition reaction of compound 2 with
the azide derivative 8. Compounds 13aeb were obtained from 4-
methylumbelliferone and 7-hydroxy coumarin, respectively
(Scheme 3). In the first step, 11aeb were prepared by alkylation of
phenolic group with 1,2-dibromoethane. Then, compounds 11ae
b reacted with sodium azide in acetone-water at reflux tempera-
ture to form compounds 12aeb, which were subjected to click re-
action with 2 to yield compounds 13aeb. All the synthesized
compounds were characterized by 1H NMR, 13C NMR, HRMS and IR.
Replacing the tertiary butyloxycarbonyl group with
a carbo-
benzoxy group caused a slight loss of the IC50 values (5aeh). The
substituents on benzyl azides had a profound influence on anti-
cancer activity, such as 3c (0.49
mM) as compared to 3f (22.83
mM)
against MGC-803, 3a (2.44 M) as compared to 3b (58.9
m
mM) against
EC-109. Compounds 6aed showed weak or no cytotoxicity against
all tested cell lines, suggesting that the length of azides may play an
important role in determining activity. The 1,2,3-triazole-dithio-
carbamate hybrids bearing a coumarin ring (9) also displayed good
anticancer activity against MGC-803 and MCF-7 but less than 3a and
3c bearing a benzene ring, while 13a-b showed weak anticancer
activity against all tested cell lines.
2.2.2. Apoptosis assay
Because compound 3c had a remarkable broad spectrum ac-
tivity against all tested human cancer cell lines and the best activity
against MGC-803 cell line, it was chosen to be further investigated
regarding its mechanism of action. In order to better characterize
the mode of cell death induced by compound 3c, we performed
a biparametric cytofluorimetric analysis using propidium iodide
(PI) and annexin-V-FITC in MGC-803 cells. After treatment with
compound 3c for 12 h at different concentrations (0, 0.25, 0.5,
1.0 mmol/L), MGC-803 cells were labeled with the two dyes, and the
resulting red (PI) and green (FITC) fluorescence was monitored by
flow cytometry. It can be observed from Fig. 1 that the apoptosis
rates were significantly increased from 3.7% (DMSO control) to
35.1%. The results showed that 3c markedly increased the cellular
apoptosis in a concentration-dependent manner.
2.2. Evaluation of biological activity
2.2.1. Anticancer activity
2.2.3. Cell cycle analysis
The IC50 values (concentration required to inhibit tumor cell
proliferation by 50%) for the synthesized compounds against four
human cancer cell lines including MGC-803 (human gastric cancer
cell line), MCF-7 (human breast cancer cell line), PC-3 (human
prostate cancer cell line), and EC-109 (human esophageal cancer
cell line) were determined using MTT assay method. The IC50 values
were listed in Table 1 and the well-known anticancer drug
5-fluorouracil was used as positive control.
Many anticancer drugs interact with cells leading to cell growth
arrest. To determine whether the high anticancer effects of the
hybrids were caused by cell cycle accumulated at a certain phase,
the effects of different concentrations of compound 3c on cell cycle
progression were examined with MGC-803 cell line. After treat-
ment with compound 3c at various concentrations (0, 0.25, 0.5,
1.0 mmol/L) for 12 h, it was observed that the percentage of cells in
Scheme 1. Synthesis of the 1,2,3-triazole-dithiocarbamate hybrids (3-6). Reagent and reaction conditions: (a) CS2, Na3PO4$12H2O, propargyl bromide, acetone, rt; (b) ArN3,
CuSO4$5H2O, Sodium ascorbate, THF-H2O (1:1), rt; (c) BnN3, CuSO4$5H2O, Sodium ascorbate, THF-H2O (1:1), rt; (d) CF3COOH, CH2Cl2, rt; (e) CbzCl, K2CO3, CH2Cl2, rt.

Y.-C. Duan et al. / European Journal of Medicinal Chemistry 62 (2013) 11e19
13
Scheme 2. Synthesis of the 1,2,3-triazole-dithiocarbamate hybrid (9). Reagent and conditions: (a) Con H2SO4, 0 ꢀC; (b) NaN3, CH3CN, reflux; (c) 2, CuSO4$5H2O, Sodium ascorbate,
THF-H2O (1:1), rt.
G2/M phase were 17.59%, 22.20%, 34.29%, and 64.10%, respectively
4.2. Procedure for the synthesis of tert butyl 4-((prop-2-ynylthio)
(Fig. 2 A), whereas after treatment of compound 3c (0, 0.25, 0.5,
carbonothioyl)piperazine-1-carboxylate (2)
1.0 mmol/L) for 24 h, the percentage of cells in G2/M phase were
15.12%, 38.75%, 68.10%, and 80.05%, respectively (Fig. 2 B). The re-
sults suggested that 3c caused a clear G2/M arrest pattern in
a concentration and time-dependent manner, with a concomitant
decrease of cells in other phases of the cell cycle.
CS2 (2.28 g, 30 mmol) was added drop wise to the solution of
1-Boc-piperazine (1.86 g, 10 mmol) and Na3PO4$12H2O (2.28 g,
6 mmol) in acetone (40 mL). The reaction mixture was stirred at
room temperature for 0.5 h. Then propargyl bromide (1.31 g,
11 mmol) was added to the mixture, the reaction mixture was
stirred at room temperature for another 0.5 h. Upon completion,
the reaction mixture was filtered and the filtrate was concentrated
under reduced pressure, the residue was dissolved in EtOAc
(50 mL), washed with water, brine, dried over anhydrous Na2SO4
and concentrated under vacuum to afford compound 2 (2.78 g,
3. Conclusions
In conclusion, a new class of 1,2,3-triazole-dithiocarbamate
hybrids were synthesized and screened for anticancer activity
against four human cancer cell lines. Compounds 3a and 3c
exhibited excellent broad spectrum anticancer activity in vitro,
especially compound 3a, it was more potent than 5-fluorouracil
against all tested human cancer cell lines. The results of apoptosis
assay and cell cycle analysis demonstrated that 3c could obviously
inhibit the proliferation of MGC-803 cancer cells by inducing
apoptosis and arresting the cell cycle at G2/M phase. These com-
pounds are currently being evaluated for their in vivo efficacy in
animal models. The 1,2,3-triazole-dithiocarbamate hybrids have
simple structures and are easy to synthesize. These findings have
encouraged us to continue the development and testing of novel
1,2,3-triazole-dithiocarbamate hybrids to conduct further studies
to investigate the structureeactivity relationship and elucidate the
detailed pharmacological mechanism(s).
yield 92.2%). white solid. Mp: 87e88 ꢀC. IR (KBr, cmꢁ1
2980, 1670, 1420, 1124, 931, 845, 767, 657, 544; 1H NMR (400 MHz,
Acetone-d6,
) n: 3215,
d
, ppm): 4.28 (br, 2H), 4.14 (d, 2H, J ¼ 2.7 Hz), 4.00 (br,
2H), 3.58 (br, 4H), 2.78 (t, 1H, J ¼ 2.7 Hz), 1.46 (s, 9H); HRMS (ESI)
calcd for C13H21N2O2S2 [M þ H]þ: 301.1044, found: 301.1046.
4.3. General procedure for the synthesis of compounds 3aeh, 6aed,
9 and 13aeb
In a round-bottom flask equipped with a magnetic stirred bar, 2
(1.51 g, 5 mmol), azide derivatives (5.5 mmol), CuSO4$5H2O (62 mg,
0.25 mmol), sodium ascorbate (100 mg, 0.5 mmol), THF (20 mL)
and H2O (20 mL) were added. The resulting mixture was stirred at
room temperature. The disappearance of compound 2 was moni-
tored by TLC. Upon completion, water (40 mL) was added and the
reaction mixture was extracted with EtOAc (3 ꢂ 40 mL). The
combined organic layer was washed with brine, dried over anhy-
drous Na2SO4 and concentrated under vacuum to afford the crude
product. The crude product was recrystallized from acetone to yield
the pure product.
4. Experimental section
4.1. General
Reagents and solvents were purchased from commercial sour-
ces and were used without further purification. Melting points
were determined on a X-5 micromelting apparatus and are un-
corrected. 1H NMR and 13C NMR spectra were recorded on a Bruker
400 MHz and 100 MHz spectrometer respectively. IR spectra were
recorded on a Nicolet iS10 infrared spectrometer. High resolution
mass spectra (HRMS) were recorded on a Waters Micromass Q-T of
Micromass spectrometer.
4.3.1. tert Butyl 4-(((1-(2-fluorobenzyl)-1H-1,2,3-triazol-4-yl)
methylthio)carbonothioyl)-piperazine-1-carboxylate (3a) ꢁ1
) n: 3447,
yield 79.0%. white solid. Mp: 109e110 ꢀC; IR (KBr, cm
2979, 1693, 1494, 1478, 1424, 1279, 1167, 1012, 986, 932, 791, 757,
695; 1H NMR (400 MHz, CDCl3,
, ppm): 7.66 (s, 1H), 7.09e7.38 (m,
4H), 5.55 (s, 2H), 4.69 (s, 2H), 4.29 (br, 2H), 3.91 (br, 3H), 3.54 (t, 4H,
d
Scheme 3. Synthesis of the 1,2,3-triazole-dithiocarbamate hybrids (13). Reagent and conditions: (a) 1,2-dibromoethane, K2CO3, CH3CN, reflux; (b) NaN3, Acetone-H2O (4:1), reflux;
(c) 2, CuSO4$5H2O, Sodium ascorbate, THF-H2O (1:1), rt.

14
Y.-C. Duan et al. / European Journal of Medicinal Chemistry 62 (2013) 11e19
Table 1
Inhibitory results of 1,2,3-triazole-dithiocarbamate hybrids against four human cancer cell lines.
Com.
R
IC50 (m
M)a
MGC-803
MCF-7
PC-3
EC-109
3a
3b
3c
3d
3e
3f
3g
3h
4a
4b
4c
4d
4e
4f
4g
4h
5a
5b
5c
5d
5e
5f
5g
5h
6a
6b
6c
o-F
p-F
o-Cl
p-Cl
p-CH3
p-OCH3
p, m-diCl
m, p, m-triOCH3
o-F
p-F
o-Cl
p-Cl
p-CH3
p-OCH3
p, m-diCl
m, p, m-triOCH3
o-F
0.73 ꢃ 0.11
1.93 ꢃ 0.13
0.49 ꢃ 0.07
9.79 ꢃ 1.41
3.49 ꢃ 0.43
22.83 ꢃ 2.21
33.66 ꢃ 2.38
27.34 ꢃ 1.97
19.35 ꢃ 2.45
32.46 ꢃ 1.39
45.19 ꢃ 4.47
ntb
5.67 ꢃ 0.91
3.34 ꢃ 0.40
6.09 ꢃ 0.97
2.96 ꢃ 0.30
19.65 ꢃ 2.11
4.96 ꢃ 0.78
19.09 ꢃ 1.91
22.65 ꢃ 1.63
33.16 ꢃ 2.08
49.89 ꢃ 2.17
38.86 ꢃ 3.77
ntb
11.61 ꢃ 1.59
>128
2.44 ꢃ 0.10
58.9 ꢃ 3.15
11.93 ꢃ 1.60
>128
47.86 ꢃ 2.97
36.84 ꢃ 2.66
>128
12.45 ꢃ 1.63
>128
17.25 ꢃ 1.98
>128
>128
>128
44.39 ꢃ 3.82
56.16 ꢃ 2.87
>128
>128
>128
>128
>128
>128
>128
>128
>128
26.96 ꢃ 2.30
32.03 ꢃ 2.45
56.79 þ 2.88
>128
32.56 ꢃ 5.40
58.27 ꢃ 2.94
29.41 ꢃ 4.28
>128
ntb
>128
64.27 ꢃ 3.42
53.16 ꢃ 2.84
19.76 ꢃ 2.41
ntb
ntb
>128
ntb
ntb
ntb
ntb
ntb
>128
5.06 ꢃ 0.80
17.61 ꢃ 1.92
15.50 ꢃ 1.04
31.30 ꢃ 2.38
9.37 ꢃ 1.36
23.80 ꢃ 2.16
7.14 ꢃ 1.08
10.86 þ 1.40
119.40 ꢃ 3.93
48.30 ꢃ 2.04
66.84 ꢃ 3.10
87.33 ꢃ 3.97
4.96 ꢃ 0.78
76.90 ꢃ 3.56
67.05 ꢃ 2.98
7.01 ꢃ 1.34
7.95 ꢃ 0.33
10.32 ꢃ 1.44
21.37 ꢃ 2.08
27.50 ꢃ 2.27
37.78 ꢃ 2.65
30.47 ꢃ 2.37
10.63 ꢃ 1.39
>128
89.59 ꢃ 4.91
74.16 ꢃ 4.45
80.38 ꢃ 3.11
93.05 ꢃ 4.77
10.44 ꢃ 2.34
90.38 ꢃ 4.05
79.80 ꢃ 3.09
7.54 ꢃ 0.7
68.29 ꢃ 3.13
p-F
o-Cl
p-Cl
>128
>128
>128
>128
>128
>128
>128
>128
>128
p-CH3
p-OCH3
p, m-diCl
m, p, m-triOCH3
o-F
p-CH3
m-CF3
p-OCH3
>128
113.60 ꢃ 3.80
69.32 ꢃ 2.62
>128
>128
>128
>128
3.34 ꢃ 0.86
>128
>128
6d
9
13a
13b
5-Fu
36.84 ꢃ 2.66
CH3
H
>128
>128
27.07 ꢃ 4.21
a
Inhibitory 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 ꢃ SDs of three independent experiments.
b
Not tested.
J ¼ 5.1 Hz), 1.47 (s, 9H); 13C NMR (100 MHz, CDCl3,
d
, ppm): 196.42,
4.3.4. tert Butyl 4-(((1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-yl)
161.72, 159.26, 154.41, 144.00, 130.89, 130.81, 130.51, 130.48, 124.82,
124.78, 122.96, 121.98, 121.84, 115.92, 115.71, 80.63, 47.66, 47.61,
31.84, 28.34; HRMS (ESI) calcd for C20H27FN5O2S2 [M þ H]þ:
452.1590, found: 452.1598.
methylthio)carbonothioyl)-piperazine-1-carboxylate (3d) ꢁ1
yield 85.5%. white solid. Mp: 177e178 ꢀC; IR (KBr, cm
) n: 3130,
2979, 2912, 1678, 1491, 1422, 1224, 1161, 1024, 994, 932, 777, 543,
499; 1H NMR (400 MHz, CDCl3,
d
, ppm): 7.59 (s, 1H), 7.35 (d, 2H,
J ¼ 8.4 Hz), 7.20 (d, 2H, J ¼ 8.4 Hz), 5.45 (s, 2H), 4.68 (s, 2H), 4.29 (br,
4.3.2. tert Butyl 4-(((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)
methylthio)carbonothioyl)-piperazine-1-carboxylate (3b)ꢁ1
2H), 3.90 (br, 2H), 3.54 (t, 4H, J ¼ 5.2 Hz), 1.47(s, 9H); 13C NMR
(100 MHz, CDCl3, d, ppm):196.39, 154.42, 144.35, 134.78, 133.11,
yield 81.5%. white solid. Mp: 171e172 ꢀC; IR (KBr, cm
)
n
: 3482,
129.36, 129.31, 122.79, 80.69, 53.40, 31.72, 28.35; HRMS (ESI) calcd
3139, 2974, 1690, 1470, 1420, 1281, 1167, 1051, 994, 824, 781, 541; 1H
NMR (400 MHz, CDCl3, , ppm): 7.59 (s, 1H), 7.04e7.27 (m, 4H), 5.46
(s, 2H), 4.68 (s, 2H), 4.31 (br, 2H), 3.90 (br, 3H), 3.53 (s, 4H), 1.47 (s,
9H); 13C NMR (100 MHz, CDCl3,
, ppm): 196.40, 164.07, 161.60,
for C20H27ClN5O2S2 [M þ H]þ: 468.1295, found: 468.1291.
d
4.3.5. tert Butyl 4-(((1-(4-methylbenzyl)-1H-1,2,3-triazol-4-yl)
methylthio)carbonothioyl)-piperazine-1-arboxylate (3e)
d
154.40, 130.49, 130.45, 129.96, 129.87, 116.20, 115.98, 80.66, 53.42,
31.78, 28.34; HRMS (ESI) calcd for C20H27FN5O2S2 [M þ H]þ:
452.1590, found: 452.1588.
yield 81.9%. white solid. Mp: 182e183 ꢀC; IR (KBr, cmꢁ1
)
n
: 3454,
2975, 1682, 1457, 1422, 1224, 1078, 994, 933, 867, 772, 524; 1H NMR
(400 MHz, CDCl3, , ppm): 7.55 (s, 1H), 7.16e7.21 (m, 4H), 5.43 (s,
2H), 4.67 (s, 2H), 4.29 (br, 2H), 3.91 (br, 2H), 3.54 (t, 4H, J ¼ 5.2 Hz),
2.35 (s, 3H), 1.47 (s, 9H); 13C NMR (100 MHz, CDCl3,
, ppm): 196.49,
d
4.3.3. tert Butyl 4-(((1-(2-chlorobenzyl)-1H-1,2,3-triazol-4-yl)
d
methylthio)carbonothioyl)-piperazine-1-carboxylate (3c)
154.41, 138.67, 131.57, 129.75, 128.09, 80.64, 54.01, 31.91, 28.35,
21.15; HRMS (ESI) calcd for C21H30N5O2S2 [M þ H]þ:448.1841,
found: 448.1840.
yield 78.7%. white solid. Mp: 105e106 ꢀC; IR (KBr, cmꢁ1
)
n
: 3447,
2975, 1691, 1460, 1423, 1219, 1167, 1039, 935, 788, 746; 1H NMR
(400 MHz, CDCl3,
d
, ppm): 7.70 (s, 1H), 7.46 (dd, 1H, J1 ¼ 1.4 Hz,
J2 ¼ 7.8 Hz), 7.23e7.33 (m, 2H), 7.18 (dd, 1H, J1 ¼1.6 Hz, J2 ¼ 7.4 Hz),
4.3.6. tert Butyl 4-(((1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)
5.62 (s, 2H), 4.70 (s, 2H), 4.28 (br, 2H), 3.93 (br, 2H), 3.54 (t, 4H,
methylthio)carbonothioyl)-piperazine-1-carboxylate (3f)
J ¼ 5.2 Hz), 1.47 (s, 9H); 13C NMR (100 MHz, Acetone-d6,
d
, ppm):
yield 85.8%. white solid. Mp: 129e130 ꢀC; IR (KBr, cmꢁ1
)
n
: 3502,
3125, 2975, 1686, 1542, 1453, 1281, 1173, 1016, 982, 932, 775, 698;
1H NMR (400 MHz, CDCl3,
, ppm): 7.54 (s, 1H), 7.21(d, 2H,
J ¼ 8.7 Hz), 6.88 (d, 2H, J ¼ 8.7 Hz), 5.41 (s, 2H), 4.67 (s, 2H), 4.29 (br,
195.68, 154.33, 143.01, 133.30, 133.16, 130.60, 130.31, 129.74, 127.71,
123.79, 79.78, 50.99, 31.63, 27.63; HRMS (ESI) calcd for
C20H27ClN5O2S2 [M þ H]þ: 468.1295, found: 468.1292.
d

Y.-C. Duan et al. / European Journal of Medicinal Chemistry 62 (2013) 11e19
15
163.64, 161.17, 154.40, 133.32, 122.56, 122.48, 116.78, 116.55, 80.67,
31.56, 28.35; HRMS (ESI) calcd for C19H25FN5O2S2 [M þ H]þ:
438.1434, found: 438.1437.
4.3.10. tert Butyl 4-(((1-p-tolyl-1H-1,2,3-triazol-4-yl)methylthio)
carbonothioyl)piperazine-1-carboxylate (6b)
yield 78.4%. white solid. Mp: 130e131 ꢀC; IR (KBr, cmꢁ1
)
n
: 3140,
2975, 1686, 1598, 1497, 1366, 1223, 1124, 1019, 995, 934, 836, 770,
519; 1H NMR (400 MHz, CDCl3,
, ppm): 8.08 (s, 1H), 7.59 (d, 2H,
d
J ¼ 8.4 Hz), 7.30 (d, 2H, J ¼ 8.4 Hz), 4.79 (s, 2H), 4.32 (br, 2H), 3.93
(br, 2H), 3.56 (t, 4H, J ¼ 5.2 Hz), 2.41 (s, 3H), 1.47 (s, 9H); 13C NMR
(100 MHz, DMSO-d6, d, ppm): 196.39, 154.40, 138.81, 134.73, 130.17,
121.09, 120.43, 80.62, 31.71, 28.35, 21.09; HRMS (ESI) calcd for
C20H28N5O2S2 [M þ H]þ: 434.1684, found: 434.1681.
Fig. 1. Apoptosis effect on human MGC-803 cell line induced by compound 3c.
Apoptotic cells were detected with Annexin V/PI double staining after incubation with
compounds 3c (0, 0.25, 0.5, 1.0 mmol/L) for 12 h. The lower left quadrants represent live
cells, the lower right quadrants are for early/primary apoptotic cells, upper right
quadrants are for late/secondary apoptotic cells, while the upper left quadrants rep-
resent cells damaged during the procedure. The experiments were performed three
times, and a representative experiment is shown.
2H), 3.90 (br, 2H), 3.80 (s, 3H), 3.51 (t, 4H, J ¼ 5.2 Hz), 1.47 (s, 9H);
13C NMR (100 MHz, CDCl3,
d, ppm): 196.47, 159.89, 154.40, 143.86,
129.62, 126.59, 122.52, 114.44, 80.61, 55.34, 53.70, 31.90, 28.35;
HRMS (ESI) calcd for C21H30N5O3S2 [M þ H]þ: 464.1790, found:
464.1794.
4.3.7. tert Butyl 4-(((1-(3,4-dichlorobenzyl)-1H-1,2,3-triazol-4-yl)
methylthio)carbonothioyl)-piperazine-1-carboxylate (3g) ꢁ1
yield 90.2%. white solid. Mp: 153e154 ꢀC; IR (KBr, cm
) n: 3129,
2984, 1693, 1470, 1347, 1159, 1131, 990, 963, 798, 740, 698, 542; 1H
NMR (400 MHz, CDCl3,
d
, ppm): 7.64 (s, 1H), 7.43 (d, 1H, J ¼ 8.2 Hz),
7.35 (d, 1H, J ¼ 2.0 Hz), 7.08 (dd, 1H, J1 ¼ 2.0 Hz, J2 ¼ 8.2 Hz), 5.44 (s,
2H), 4.70 (s, 2H), 4.30 (br, 2H), 3.91 (br, 2H), 3.52 (t, 4H, J ¼ 5.1 Hz),
1.47 (s, 9H); 13C NMR (100 MHz, CDCl3,
d, ppm): 196.29, 154.39,
144.59, 134.77, 133.26, 133.08, 131.08, 129.86, 127.18, 122.94, 80.65,
52.82, 31.65, 28.34; HRMS (ESI) calcd for C20H26Cl2N5O2S2
[M þ H]þ: 502.0905, found: 502.0900.
4.3.8. tert Butyl 4-(((1-(3,4,5-trimethoxybenzyl)-1H-1,2,3-triazol-
4-yl)methylthio)carbonothioyl)-piperazine-1-carboxylate ꢁ(31h)
yield 83.4%. white solid. Mp: 134e135 ꢀC; IR (KBr, cm
) n: 3148,
2970, 1688, 1464, 1424, 1384, 1242, 1127, 1011, 933, 843, 774, 738,
615; 1H NMR (400 MHz, CDCl3,
d, ppm): 7.63 (s, 1H), 6.48 (s, 2H),
5.41 (s, 2H), 4.70 (s, 2H), 4.30 (br, 2H), 3.91 (br, 2H), 3.85 (s, 3H), 3.84
(s, 6H), 3.55 (t, 4H, J ¼ 5.2 Hz), 1.48 (s, 9H); HRMS (ESI) calcd for
C23H34N5O2S2 [M þ H]þ: 524.2001, found: 524.2005.
4.3.9. tert Butyl 4-(((1-(4-fluorophenyl)-1H-1,2,3-triazol-4-yl)
methylthio)carbonothioyl)-piperazine-1-carboxylate (6a) ꢁ1
yield 77.5%. white solid. Mp: 178e179 ꢀC; IR (KBr, cm
) n: 3447,
Fig. 2. Effect of compound 3c on the cell cycle distribution of MGC-803 cells. Cells
were treated with different concentrations (0, 0.25, 0.5, 1.0 mmol/L) for 12 h or 24 h.
Then the cells were fixed and stained with PI to analyze DNA content by flow
cytometry. (A) incubated for 12 h; (B) incubated for 24 h. The experiments were
performed three times, and a representative experiment is shown.
3086, 2983, 1694, 1514, 1456, 1419, 1224, 1165, 1012, 992, 939, 838,
746, 553; 1H NMR (400 MHz, CDCl3,
d, ppm): 8.09 (s, 1H), 7.18e7.71
(m, 4H), 4.79 (s, 2H), 4.32 (br, 2H), 3.93 (br, 2H), 3.56 (t, 4H,
J ¼ 5.2 Hz), 1.47 (s, 9H); 13C NMR (100 MHz, CDCl3,
d, ppm): 196.30,

16
Y.-C. Duan et al. / European Journal of Medicinal Chemistry 62 (2013) 11e19
4.3.11. tert Butyl 4-(((1-(3-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-
4-yl)methylthio)-carbonothioyl) piperazine-1-carboxylate (6c)
4.4. General procedure for the synthesis of compounds 4a-h
yield 87.6%. white solid. Mp: 109e110 ꢀC; IR (KBr, cmꢁ1
)
n
: 3142,
2983, 1687, 1598, 1483, 1417, 1325, 1283, 1224, 1166, 1071, 1037, 993,
937, 807, 772, 698, 540; 1H NMR (400 MHz, CDCl3,
, ppm): 8.21 (s,
CF3COOH (4.56 g, 40 mmol) was added to a solution of 3
(2 mmol) in CH2Cl2 (20 mL) at 0 ꢀC. The reaction mixture was
warmed to room temperature and stirred at the same temperature.
Upon completion, the reaction mixture was concentrated under
vacuum, the residue was dissolved in CH2Cl2, washed with satu-
rated NaHCO3, brine, dried over anhydrous Na2SO4 and con-
centrated under vacuum to afford compounds 4, which were used
in the next reaction without further purification.
d
1H), 8.01 (s, 1H), 7.96 (d, 1H, J ¼ 7.6 Hz), 7.66e7.73 (m, 2H), 4.82 (s,
2H), 4.34 (br, 2H), 3.95 (br, 2H), 3.57 (t, 4H, J ¼ 5.2 Hz), 1.48 (s, 9H);
13C NMR (100 MHz, CDCl3,
d, ppm): 196.13, 154.39, 145.29, 137.34,
132.87, 132.54, 132.21, 131.88, 130.51, 127.38, 125.37, 125.34, 125.30,
125.26, 124.67, 123.56, 121.96, 121.10, 119.25, 117.49, 117.46, 117.42,
117.38, 80.67, 31.45, 28.33; HRMS (ESI) calcd for HRMS (ESI) calcd
for C20H25F3N5O2S2 [M þ H]þ: 488.1402, found: 488.1398.
4.4.1. (1-(2-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl piperazine-
1-carbodithioate (4a)
4.3.12. tert Butyl 4-(((1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)
yield 96.7%. white solid. Mp: 93e94 ꢀC; IR (KBr, cmꢁ1
)
n
: 3211,
2908,1492, 1469, 1387, 1261, 1222, 1121, 1021, 983, 852, 753, 735; 1H
NMR (400 MHz, CDCl3, , ppm): 7.67 (s, 1H), 7.09e7.36 (m, 4H), 5.55
methylthio)carbonothioyl)-piperazine-1-carboxylate (6d) ꢁ1
yield 83.7%. white solid. Mp: 133e134 ꢀC; IR (KBr, cm
)
n
: 3146,
d
2976, 1686, 1518, 1421, 1254, 1161, 1125, 1041, 995, 935, 810, 770,
(s, 2H), 4.69 (s, 2H), 4.30 (br, 2H), 3.93 (br, 2H), 2.96 (t, 4H,
694, 532; 1H NMR (400 MHz, CDCl3,
d, ppm): 8.04 (s, 1H), 7.61 (d,
J ¼ 4.8 Hz); 13C NMR (100 MHz, CDCl3,
d, ppm): 195.81, 161.72,
2H, J ¼ 9.0 Hz), 7.01 (d, 2H, J ¼ 9.0 Hz), 4.79 (s, 2H), 4.32 (br, 2H),
159.26, 144.27, 130.88, 130.80, 130.50, 130.47, 124.82, 124.79, 122.98,
122.00, 121.86, 115.93, 115.72, 47.66, 47.61, 45.58, 31.76, 30.93;
HRMS (ESI) calcd for C15H19FN5S2 [M þ H]þ: 352.1066, found:
352.1064.
3.94 (br, 2H), 3.86 (s, 3H), 3.56 (t, 4H, J ¼ 5.3 Hz), 1.47 (s, 9H); 13C
NMR (100 MHz, CDCl3, d, ppm): 196.43, 159.80, 154.41, 144.33,
130.48, 122.17, 121.24, 114.72, 80.64, 55.63, 31.72, 28.35; HRMS (ESI)
calcd for C20H28N5O3S2 [M þ H]þ: 450.1634, found: 450.1638.
4.4.2. (1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl piperazine-
1-carbodithioate (4b)
4.3.13. tert Butyl 4-(((1-((7-hydroxy-2-oxo-2H-chromen-4-yl)
methyl)-1H-1,2,3-triazol-4-yl)-methylthio)carbonothioyl)
piperazine-1-carboxylate (9)
yield 93.9%. white solid. Mp: 138e139 ꢀC; IR (KBr, cmꢁ1
)
n
: 3152,
2912, 1601, 1508, 1425, 1243, 1215, 1141, 1136, 1031, 987, 843, 775,
729, 528; 1H NMR (400 MHz, CDCl3,
, ppm): 7.60 (s, 1H), 7.04e7.27
(m, 4H), 5.45 (s, 2H), 4.69 (s, 2H), 4.31 (br, 2H), 3.89 (br, 2H), 2.93 (s,
4H); 13C NMR (100 MHz, CDCl3,
, ppm): 196.03, 164.07, 161.60,
yield 91.3%. Yellow white solid. Mp: 219e220 ꢀC; IR (KBr, cmꢁ1
)
n:
d
3161, 2972, 2852, 1693, 1562, 1423, 1280, 1152, 1007, 932, 797, 689,
504; 1H NMR (400 MHz, DMSO-d6,
d, ppm): 10.73 (s, 1H), 8.24 (s, 1H),
d
7.68 (d, 1H, J ¼ 9.0 Hz), 6.82 (d,1H, J ¼ 9.0 Hz), 6.77 (s, 1H), 5.88 (s, 2H),
144.33, 130.47, 130.44, 129.97, 129.88, 122.74, 116.21, 116.00, 53.41,
45.15, 31.76; HRMS (ESI) calcd for C15H19FN5S2 [M þ H]þ: 352.1066,
found: 352.1063.
5.54 (s, 1H), 4.63 (s, 2H), 4.23 (br, 2H), 3.92 (br, 2H), 3.45 (t, 4H,
J ¼ 5.2 Hz),1.42 (s, 9H); 13C NMR (100 MHz, DMSO-d6,
d, ppm): 194.86,
162.14, 160.39, 155.57, 154.15, 150.99, 143.22, 126.54, 125.42, 113.65,
109.84, 109.66, 103.01, 79.87, 67.48, 49.64, 31.77, 28.49, 25.59; HRMS
(ESI) calcd for C23H27N5NaO2S2 [M þ Na]þ: 540.1351, found: 540.1353.
4.4.3. (1-(2-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl piperazine-
1-carbodithioate (4c)
yield 94.8%. white solid. Mp: 98e99 ꢀC; IR (KBr, cmꢁ1
)
n
: 3143,
2927, 1468, 1407, 1388, 1250, 1211, 1139, 1105, 1014, 953, 806, 774,
531; 1H NMR (400 MHz, CDCl3,
, ppm): 7.68 (s, 1H), 7.43 (dd, 1H,
4.3.14. tert Butyl 4-(((1-(2-(4-methyl-2-oxo-2H-chromen-7-yloxy)
ethyl)-1H-1,2,3-triazol-4-yl)-methylthio)carbonothioyl)piperazine-
1-carboxylate (13a)
d
J1 ¼ 1.4 Hz, J2 ¼ 7.8 Hz), 7.24e7.33 (m, 2H), 7.18 (dd, 1H, J1 ¼ 1.6 Hz,
yield 89.6%. white solid. Mp: 137e138 ꢀC; IR (KBr, cmꢁ1
)
n
: 2954,
2104, 1708, 1613, 1508, 1392, 1273, 1071, 916, 847, 535; 1H NMR
(400 MHz, DMSO-d6,
J2 ¼ 7.4 Hz), 5.61 (s, 2H), 4.64 (s, 2H), 4.45 (br, 4H), 3.34 (s, 4H); 13
C
NMR (100 MHz, CDCl3, d, ppm): 197.25, 143.28, 133.50, 132.25,
d, ppm): 8.16 (s, 1H), 7.68 (d, 1H, J ¼ 8.8 Hz),
130.34,130.19,129.97,127.65,123.43, 51.57, 43.19, 32.18; HRMS (ESI)
6.99 (d, 1H, J ¼ 2.4 Hz), 6.95 (dd, 1H, J1 ¼ 2.4 Hz, J2 ¼ 8.8 Hz), 6.22 (s,
1H), 4.80 (t, 2H, J ¼ 4.8 Hz), 4.58 (s, 2H), 4.52 (t, 2H, J ¼ 4.8 Hz), 4.23
(br, 2H), 3.90 (br, 2H), 3.44 (s, 4H), 2.40 (s, 3H), 1.41 (s, 9H); 13C NMR
calcd for C15H19ClN5S2 [M þ H]þ: 368.0770, found: 368.0772.
4.4.4. (1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl piperazine-
(100 MHz, DMSO-d6,
d
, ppm): 195.02, 170.79, 161.33, 160.65, 155.74,
1-carbodithioate (4d)
154.14, 144.70, 142.49, 130.02, 124.84, 124.50, 113.29, 101.97, 79.86,
67.25, 60.22, 49.26, 31.93, 31.15, 28.48, 21.23; HRMS (ESI) calcd for
C25H32N5O5S2 [M þ H]þ: 546.1845, found: 546.1846.
yield 95.1%. white solid. Mp: 79e80 ꢀC; IR (KBr, cmꢁ1
)
n
: 3138,
2921, 1472, 1407, 1386, 1258, 1226, 1139, 1118, 1014, 980, 806, 774,
501; 1H NMR (400 MHz, CDCl3,
, ppm): 7.61 (s, 1H), 7.35 (d, 2H,
J ¼ 8.1 Hz), 7.20 (d, 2H, J ¼ 8.1 Hz), 5.45 (s, 2H), 4.69 (s, 2H), 4.31 (br,
2H), 3.90 (br, 2H), 2.94 (s, 4H); 13C NMR (100 MHz, CDCl3,
, ppm):
195.63, 144.54, 134.71, 133.17, 129.35, 129.27, 122.81, 53.36, 45.61,
31.65; HRMS (ESI) calcd for C15H19ClN5S2 [M þ H]þ: 368.0770,
found: 368.0767.
d
4.3.15. tert Butyl 4-(((1-(2-(2-oxo-2H-chromen-7-yloxy)ethyl)-1H-
1,2,3-triazol-4-yl)methylthio)-carbonothioyl)piperazine-1-
carboxylate (13b)
d
yield 94.1%. white solid. Mp: 135e136 ꢀC; IR (KBr, cmꢁ1
)
n
: 2977,
1735, 1686, 1612, 1460, 1420, 1365, 1226, 1126, 994, 937, 832, 758;
1H NMR (400 MHz, CDCl3,
, ppm): 7.88 (s, 1H), 7.64 (d, 1H,
d
4.4.5. 1-(4-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl piperazine-
J ¼ 9.5 Hz), 7.39 (d, 1H, J ¼ 8.5 Hz), 6.84 (dd, 1H, J1 ¼ 2.0 Hz,
J2 ¼ 8.5 Hz), 6.79 (d, 1H, J ¼ 2.0 Hz), 6.29 (d, 1H, J ¼ 9.5 Hz), 4.78 (t,
2H, J ¼ 5.0 Hz), 4.70 (s, 2H), 4.43 (t, 2H, J ¼ 5.0 Hz), 4.32 (br, 2H), 3.91
1-carbodithioate (4e)
yield 94.0%. white solid. Mp: 74e75 ꢀC; IR (KBr, cmꢁ1
)
n
: 3267,
2919, 1514, 1474, 1420, 1385, 1258, 1137, 1021, 979, 803, 772, 525; 1H
NMR (400 MHz, CDCl3, , ppm): 7.56 (s, 1H), 7.14e7.27 (m, 4H), 5.43
(s, 2H), 4.67 (s, 2H), 4.31 (br, 2H), 3.92 (br, 2H), 2.96 (br, 4H), 2.35 (s,
3H); 13C NMR (100 MHz, CDCl3,
, ppm): 195.82, 138.61, 131.59,
(br, 2H), 3.53 (s, 4H), 1.47 (s, 9H); 13C NMR (100 MHz, DMSO-d6,
d
,
d
ppm): 195.01, 170.80, 161.24, 160.52, 155.09, 154.14, 153.79, 126.99,
124.85, 114.00, 112.90, 111.89, 101.96, 79.86, 67.23, 60.22, 49.27,
31.93, 31.15, 28.48; HRMS (ESI) calcd for C24H30N5O5S2 [M þ H]þ:
532.1688, found: 532.1686.
d
129.74, 128.08, 122.72, 53.96, 31.80, 29.68, 21.17; HRMS (ESI) calcd
for C16H22N5S2 [M þ H]þ:348.1317, found: 348.1319.

Y.-C. Duan et al. / European Journal of Medicinal Chemistry 62 (2013) 11e19
17
4.4.6. (1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl
4.5.3. Benzyl 4-(((1-(2-chlorobenzyl)-1H-1,2,3-triazol-4-yl)
methylthio)carbonothioyl)piperazine-1-carboxylate (5c)
yield 71.7%. white solid. Mp: 91e92 ꢀC; IR (KBr, cmꢁ1
piperazine-1-carbodithioate (4f)
yield 92.7%. white solid. Mp: 95e96 ꢀC; IR (KBr, cmꢁ1
)
n
: 3279,
)
n
:3457,
2916, 1610, 1514, 1420, 1230, 1178, 1125, 1025, 992, 901, 838, 784,
2974, 1692, 1511, 1474, 1425, 1360, 1130, 1094, 976, 785, 727, 692,
694, 554; 1H NMR (400 MHz, CDCl3,
d, ppm): 7.55 (s, 1H), 7.24 (d,
523; 1H NMR (400 MHz, CDCl3,
d
, ppm): 7.67 (s, 1H), 7.13e7.43 (m,
2H, J ¼ 8.7 Hz), 6.91 (d, 2H, J ¼ 8.7 Hz), 5.42 (s, 2H), 4.68 (s, 2H), 4.31
9H), 5.61 (s, 2H), 5.15 (s, 2H), 4.69 (s, 2H), 4.28 (br, 2H), 3.95 (br, 2H),
(br, 2H), 3.95 (br, 2H), 3.80 (s, 3H), 2.96 (t, 4H, J ¼ 4.6 Hz); 13C NMR
3.61 (t, 4H, J ¼ 5.3 Hz); 13C NMR (100 MHz, CDCl3,
d, ppm): 196.60,
(100 MHz, CDCl3,
d, ppm): 195.75, 159.89, 144.12, 129.61, 126.61,
155.05, 143.86, 136.20, 133.42, 132.46, 130.20, 129.90, 128.61, 128.31,
128.10, 127.58, 126.98, 123.20, 67.65, 51.40, 43.04, 31.89; HRMS (ESI)
calcd for C23H25ClN5O2S2 [M þ H]þ: 502.1138, found: 502.1137.
122.53, 114.45, 55.34, 53.69, 45.63, 31.79; HRMS (ESI) calcd for
C16H22N5OS2 [M þ H]þ:364.1266, found: 364.1263.
4.4.7. (1-(3,4-dichlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl
4.5.4. Benzyl 4-(((1-(4-methylbenzyl)-1H-1,2,3-triazol-4-yl)
piperazine-1-carbodithioate (4g)
methylthio)carbonothioyl)piperazine-1-carboxylate (5e)
yield 95.5%. white solid. Mp: 122e123 ꢀC; IR (KBr, cmꢁ1
)
n
: 3238,
2914, 1472, 1418, 1256, 1224, 1047, 978, 776, 739, 537; 1H NMR
(400 MHz, CDCl3,
yield 81.8%. white solid. Mp: 99e100 ꢀC; IR (KBr, cmꢁ1
)
n
: 3141,
2983, 1686, 1598, 1417, 1325, 1224, 1165, 1037, 993, 936, 858, 806,
771, 698, 540; 1H NMR (400 MHz, CDCl3,
, ppm): 7.55 (s, 1H), 7.14e
d
, ppm): 7.65 (s, 1H), 7.45 (d, 1H, J ¼ 8.3 Hz), 7.35
d
(d, 1H, J ¼ 2.0 Hz), 7.10 (dd, 1H, J1 ¼ 2.0 Hz, J2 ¼ 8.3 Hz), 5.44 (s, 2H),
7.37 (m, 9H), 5.44 (s, 2H), 5.16 (s, 2H), 4.67 (s, 2H), 4.29 (br, 2H), 3.96
4.70 (s, 2H), 4.31 (br, 2H), 3.92 (br, 2H), 2.95 (s, 4H); 13C NMR
(br, 2H), 3.59 (t, 4H, J ¼ 5.1 Hz), 2.35 (s, 3H); 13C NMR (100 MHz,
(100 MHz, CDCl3,
d, ppm): 195.62, 144.87, 134.79, 133.27, 133.07,
CDCl3, d, ppm): 196.67, 155.04, 138.68, 136.21, 131.56, 129.76, 128.61,
131.09, 129.86, 127.17, 122.92, 52.81, 45.63, 31.56; HRMS (ESI) calcd
128.31, 128.10, 122.73, 67.64, 54.02, 43.03, 31.95, 21.17; HRMS (ESI)
for C15H18Cl2N5S2 [M þ H]þ:402.0381, found: 402.0379.
calcd for C24H28N5O2S2 [M þ H]þ: 482.1684, found: 482.1683.
4.4.8. (1-(3,4,5-trimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl
4.5.5. Benzyl 4-(((1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)
piperazine-1-carbodithioate (4h)
methylthio)carbonothioyl)piperazine-1-carboxylate (5f)
yield 96.0%. white solid. Mp: 120e121 ꢀC; IR (KBr, cmꢁ1
)
n
: 3331,
2945, 1509, 1425, 1418, 1361, 1328, 1119, 1044, 979, 838, 784, 726, 536;
1H NMR (400 MHz, CDCl3,
, ppm): 7.64 (s, 1H), 6.47 (s, 2H), 5.40 (s,
2H), 4.69 (s, 2H), 4.30 (br, 2H), 3.91 (br, 2H), 3.83 (s, 3H), 3.82 (s, 6H),
2.93 (s, 4H); 13C NMR (100 MHz, CDCl3,
, ppm): 195.63,153.66,144.42,
yield 75.5%. white solid. Mp: 113e114 ꢀC; IR (KBr, cmꢁ1
)
n
: 3351,
3143, 2952, 1678, 1513, 1425, 1360, 1130, 1095, 986, 780, 727, 692,
542; 1H NMR (400 MHz, CDCl3,
, ppm): 7.53 (s, 1H), 7.31e7.39 (m,
d
d
5H), 7.22 (d, 2H, J ¼ 8.6 Hz), 6.89 (d, 2H, J ¼ 8.6 Hz), 5.40 (s, 2H), 5.15
d
(s, 2H), 4.66 (s, 2H), 4.28 (br, 2H), 3.89 (br, 2H), 3.80 (s, 3H), 3.61 (t,
138.23, 130.15, 122.83, 105.16, 60.85, 56.23, 54.35, 45.72, 31.68; HRMS
4H, J ¼ 5.3 Hz); 13C NMR (100 MHz, CDCl3,
d, ppm): 196.63, 159.90,
(ESI) calcd for C18H26N5O3S2 [M þ H]þ: 424.1477, found: 424.1472.
155.03, 143.75, 136.22, 129.64, 128.60, 128.29, 128.08, 126.61,
122.54, 114.45, 67.61, 55.35, 53.69, 43.02, 31.97; HRMS (ESI) calcd
for C24H28N5O3S2 [M þ H]þ: 498.1634, found: 498.1639.
4.5. General procedure for the synthesis of compounds 5a-h
A mixture of 4 (2 mmol), K2CO3 (0.28 g, 2 mmol) and CbzCl
(0.38 g, 2.2 mol) in CH2Cl2 (20 mL) was stirred at room temperature.
Upon completion, K2CO3 was removed by filtration and the solvent
was diluted with CH2Cl2, washed with water, brine, dried over
anhydrous Na2SO4 and concentrated under vacuum to give the
crude product, which were recrystallized from acetone to provide
compound 5 as white solids.
4.5.6. Benzyl 4-(((1-(3,4-dichlorobenzyl)-1H-1,2,3-triazol-4-yl)
methylthio)carbonothioyl)-piperazine-1-carboxylate (5g) ꢁ1
yield 86.5%. white solid. Mp: 106e107 ꢀC; IR (KBr, cm
) n: 3138,
2910, 1693, 1474, 1425, 1365, 1219, 1132, 1031, 996, 933, 785, 731,
694, 548; 1H NMR (400 MHz, CDCl3,
d
, ppm): 7.63 (s,1H), 7.45 (d,1H,
J ¼ 8.3 Hz), 7.33e7.37 (m, 6H), 7.09 (dd, 1H, J1 ¼ 2.0 Hz, J2 ¼ 8.3 Hz),
5.43 (s, 2H), 5.15 (s, 2H), 4.69 (s, 2H), 4.29 (br, 2H), 3.95 (br, 2H), 3.62
(t, 4H, J ¼ 5.2 Hz); 13C NMR (100 MHz, CDCl3,
d, ppm): 196.45,155.02,
4.5.1. Benzyl 4-(((1-(2-fluorobenzyl)-1H-1,2,3-triazol-4-yl)
methylthio)carbonothioyl)piperazine-1-carboxylate (5a)
144.45,136.20,134.82,133.22,133.04,131.08,129.88,128.60,128.30,
128.08, 127.21, 122.98, 67.63, 52.79, 43.01, 31.74; HRMS (ESI) calcd
for C23H24Cl2N5O2S2 [M þ H]þ: 536.0748, found: 536.0749.
yield 74.6%. white solid. Mp: 138e139 ꢀC; IR ( KBr, cmꢁ1
)
n
: 3446,
3131, 1686, 1492, 1465, 1422, 1360, 1211, 1095, 791, 761, 693, 584; 1H
NMR (400 MHz, CDCl3, , ppm): 7.66 (s, 1H), 7.08e7.37 (m, 9H), 5.54
d
4.5.7. Benzyl 4-(((1-(3,4,5-trimethoxybenzyl)-1H-1,2,3-triazol-4-yl)
(s, 2H), 5.15 (s, 2H), 4.69 (s, 2H), 4.30 (br, 2H), 3.95 (br, 2H), 3.61 (t,
methylthio)carbonothioyl)-piperazine-1-carboxylate (5h)
4H, J ¼ 5.2 Hz); 13C NMR (100 MHz, CDCl3,
d, ppm): 196.61, 161.73,
yield 82.0%. white solid. Mp: 134e135 ꢀC. IR (KBr, cmꢁ1
)
n
: 3141,
2911, 1713, 1594, 1506, 1459, 1431, 1385, 1218, 1094, 951, 695, 575;
1H NMR (400 MHz, CDCl3,
, ppm): 7.63 (s, 1H), 7.35e7.37 (m, 5H),
159.27, 155.04, 143.97, 136.21, 130.94, 130.85, 130.54, 130.51, 128.61,
128.30, 128.09, 124.84, 124.80, 123.04, 121.93, 121.79, 115.94, 115.73,
67.64, 47.72, 47.68, 43.02, 31.85; HRMS (ESI) calcd for
C23H25FN5O2S2 [M þ H]þ: 486.1434, found: 486.1432.
d
6.47 (s, 2H), 5.40 (s, 2H), 5.15 (s, 2H), 4.68 (s, 2H), 4.28 (br, 2H), 3.96
(br, 2H), 3.83 (s, 3H), 3 0.82 (s, 6H), 3.62 (t, 4H, J ¼ 5.0 Hz); 13C NMR
(100 MHz, CDCl3, d, ppm): 196.60, 155.02, 153.69, 144.02, 138.30,
4.5.2. Benzyl 4-(((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)
methylthio)carbonothioyl)piperazine-1-carboxylate (5b)
136.19, 130.08, 128.60, 128.30, 128.09, 122.82, 105.23, 67.64, 60.85,
56.25, 54.38, 43.01, 31.86; HRMS (ESI) calcd for C26H32N5O5S2
[M þ H]þ: 558.1845, found: 558.1841.
yield 90.2%. white solid. Mp: 114e115 ꢀC; IR (KBr, cmꢁ1
)
n
: 3446,
3056, 1697, 1511, 1474, 1425, 1360, 1130, 1094, 976, 785, 727, 692,
523; 1H NMR (400 MHz, CDCl3,
, ppm): 7.58 (s, 1H), 7.03e7.36 (m,
d
4.6. Procedure for the synthesis of 4-(azidomethyl)-7-hydroxy-2H-
9H), 5.45 (s, 2H), 5.15 (s, 2H), 4.67 (s, 2H), 4.29 (br, 2H), 3.94 (br, 2H),
chromen-2-one compound 8
3.62 (t, 4H, J ¼ 5.0 Hz); 13C NMR (100 MHz, CDCl3,
d, ppm): 196.53,
164.04, 161.57, 155.02, 136.21, 130.54, 130.51, 129.98, 129.90, 128.60,
128.30, 128.08, 122.84, 116.19, 115.97, 67.62, 53.37, 43.01, 31.85;
HRMS (ESI) calcd for C23H24FN5NaO2S2 [M þ Na]þ: 508.1253, found:
508.1250.
To a magnetically stirred solution of compound 7 (0.63 g,
3 mmol) in CH3CN (10 mL), sodium azide (0.59 g, 9 mmol) was
added carefully and the reaction mixture was refluxed for 10 h.
Upon completion, the reaction mixture was concentrated under

18
Y.-C. Duan et al. / European Journal of Medicinal Chemistry 62 (2013) 11e19
vacuum, the residue was dissolved in EtOAc (30 mL) and washed
with water, brine, dried over anhydrous Na2SO4 and concentrated
under vacuum to give compound 8 (0.60 g, yield 92.0%), which was
used in the next reaction without further purification. yellow solid.
J2 ¼ 8.6 Hz), 6.83 (d, 1H, J ¼ 2.4 Hz), 6.29 (d, 1H, J ¼ 9.5 Hz), 4.22 (t,
2H, J ¼ 4.9 Hz), 3.67 (t, 2H, J ¼ 4.9 Hz).13C NMR (100 MHz, CDCl3,
d,
ppm): 161.31, 161.00, 155.79, 143.29, 128.95, 113.57, 113.05, 112.95,
101.47, 67.45, 49.92; HRMS (ESI) calcd for C11H10N3O3 [M þ H]þ:
232.0722, found:232.0720.
Mp: 139e140 ꢀC; 1H NMR (400 MHz, DMSO-d6,
d, ppm): 4.79 (s,
2H), 6.39 (s, 1H), 6.82 (d, 1H, J ¼ 8.8 Hz), 6.75 (s, 1H), 7.57 (d, 1H,
J ¼ 8.8 Hz), 10.69 (s, 1H); HRMS (ESI) calcd for C10H8N3O3 [M þ H]þ:
218.0566, found: 218.0566.
4.9. Anticancer activity assays
Exponentially growing cells were seeded into 96-well plates at
a concentration of 5 ꢂ 103 cells per well. After 24 h incubation at
37 ꢀC, the culture medium was removed and replaced with fresh
medium containing the candidate compounds in different con-
centrations. The cells were incubated for another 72 h. Afterward,
4.7. General procedure for the synthesis of compounds 11a-b
K2CO3 (1.66 g,12 mmol) and 1,2-dibromoethane (1.12 g, 6 mmol)
were added to a solution of 10a or 10b (2 mmol) in CH3CN (20 mL).
The reaction mixture was refluxed for 2 h. Upon completion, K2CO3
was removed by filtration and the solvent was concentrated under
vacuum, the residue was dissolved in CH2Cl2, washed with water,
brine, dried over anhydrous Na2SO4 and concentrated under vac-
uum to give compounds 11a-b, which were used in the next reac-
tion without further purification.
20
m
L of MTT solution (5 mg/mL) was added to all wells and incu-
bated for 4 h at 37 ꢀC. Discarded the suspension and added 150
mL of
dimethyl sulfoxide (DMSO) to each well and shook the plates to
dissolve the dark blue crystals (formazan); the absorbance was
measured using a microplate reader at a wavelength of 570 nm.
Each concentration was analyzed in triplicate and the experiment
was repeated three times. The average 50% inhibitory concentration
(IC50) was determined from the doseeresponse curves according to
the inhibition ratio for each concentration.
4.7.1. 7-(2-bromoethoxy)-4-methyl-2H-chromen-2-one (11a)
yield 94.5%. white solid. Mp: 104e105 ꢀC; IR(KBr, cmꢁ1
)
n
: 3073,
2956, 1713, 1619, 1511, 1460, 1288, 1265, 1174, 1012, 986, 887, 845,
572, 520; 1H NMR (400 MHz, CDCl3,
d
, ppm): 7.51 (d, 1H, J ¼ 8.8 Hz),
4.10. Flow cytometric analysis of cell cycle distribution
6.89 (dd, 1H, J1 ¼ 2.5 Hz, J2 ¼ 8.8 Hz), 6.79 (d, 1H, J ¼ 2.5 Hz), 6.14 (s,
1H), 4.36 (t, 2H, J ¼ 6.0 Hz), 3.69 (t, 2H, J ¼ 6.0 Hz), 2.40 (s, 3H); 13C
For flow cytometric analysis of DNA content, 5 ꢂ 105 MGC-803
cells in exponential growth were treated with different concen-
trations of the test compounds for 12 or 24 h. After an incubation
period, the cells were collected, centrifuged and fixed with icecold
ethanol (70%). The cells were then treated with buffer containing
RNAse A and 0.1% Triton X-100 and then stained with PI. Samples
were analyzed on Accuri C6 flow cytometer (Becton, Dickinson).
Data obtained from the flow cytometer was analyzed using the
FlowJo software (Tree Star, Inc., Ashland, OR, USA).
NMR (100 MHz, CDCl3,
d, ppm): 161.07, 161.01, 155.12, 152.48,
125.77, 114.11, 112.48, 112.29, 101.69, 68.18, 28.59, 18.67; HRMS (ESI)
calcd for C12H12BrO3 [M þ H]þ: 282.9970, found: 282.9973.
4.7.2. 7-(2-bromoethoxy)-2H-chromen-2-one (11b)
yield 93.7%. white solid. Mp: 176e177 ꢀC; IR (KBr, cmꢁ1
)
n
: 3056,
1697,1511,1474,1425,1360,1130,1094, 976, 785, 727, 692, 523; 1HNMR
(400 MHz, actone-d6,
d, ppm): 7.91 (d, 1H, J ¼ 9.5 Hz), 7.61 (d, 1H,
J ¼ 8.6 Hz), 6.97 (dd,1H, J1 ¼2.4 Hz, J2 ¼ 8.6 Hz), 6.93 (d,1H, J ¼ 2.4 Hz),
6.24 (d, 1H, J ¼ 9.5 Hz), 4.51 (t, 2H, J ¼ 5.4 Hz), 3.84 (t, 2H, J ¼ 5.4 Hz);
HRMS (ESI) calcd for C11H10BrO3 [M þ H]þ: 268.9813, found: 268.9814.
4.11. Analysis of cellular apoptosis
MGC-803 cells were plated in 6-well plates (5.0 ꢂ 104 cells/mL)
and incubated at 37 ꢀC for 12 h. Exponentially growing cells were
then incubated for 12 h with complete medium (blank) or with the
compound 3c. Cells were then harvested and the Annexin-V-FITC/PI
apoptosis kit (Biovision) was used according to the manufacturer’s
instructions to detect apoptotic cells. Ten thousand events were
collected for each sample and analyzed by Accuri C6 flow cytometer.
4.8. General procedure for the synthesis of compounds 12a-b
To a magnetically stirred solution of compound 11a or 11b
(3 mmol) in acetone (32 mL), a solution of sodium azide (0.39 g,
6 mmol) in water (8 mL) was added drop wise and the reaction
mixture was refluxed for 8 h. Upon completion, the reaction mix-
ture was concentrated under vacuum to remove acetone, the res-
idue was extracted with EtOAc (3 ꢂ 30 mL), the combined organic
layer was washed with brine, dried over anhydrous Na2SO4 and
concentrated under vacuum to get 12a-b, which were used in the
next reaction without further purification.
Acknowledgments
We are grateful for the financial support from the National
Natural Science Foundation of China (Project No. 81172937,
U1204206), China Postdoctoral Science Foundation funded project
(Project No. 20100480857, 201104402) and New Teachers’ Fund for
4.8.1. 7-(2-azidoethoxy)-4-methyl-2H-chromen-2-one (12a)
yield 77.9%. white solid. Mp: 101e102 ꢀC; IR (KBr, cmꢁ1
)
n
: 2954,
2875, 2104,1708,1613,1508,1424,1369,1273,1154,1071, 988, 916, 847,
796, 535; 1H NMR (400 MHz, CDCl3,
Doctor
Stations,
Ministry of
Education
(Project
No.
20114101120013).
d
, ppm): 7.52 (d, 1H, J ¼ 8.8 Hz),
6.90 (dd,1H, J1 ¼2.5 Hz, J2 ¼ 8.8 Hz), 6.82 (d,1H, J ¼ 2.5 Hz), 6.16 (s,1H),
4.21 (t, 2H, J ¼ 4.8 Hz), 3.66 (t, 2H, J ¼ 4.8 Hz), 2.40(s, 3H); 13C NMR
Appendix A. Supplementary data
(100 MHz, CDCl3, d, ppm): 161.13, 161.10, 155.18, 152.45, 125.74, 114.11,
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2012.12.046.
112.64, 112.34, 101.46, 67.40, 49.94, 18.68; HRMS (ESI) calcd for
C12H12N3O3 [M þ H]þ: 246.0879, found: 246.0875.
References
4.8.2. 7-(2-azidoethoxy)-2H-chromen-2-one (12b)
yield 76.5%. white solid. Mp: 154e155 ꢀC; IR (KBr, cmꢁ1
)
n
: 3075,
2124, 1732, 1608, 1507, 1405, 1390, 1125, 1053, 995, 914, 892, 834,
749, 616, 522; NMR (400 MHz, actone-d6, , ppm): 7.66 (d, 1H,
J ¼ 9.5 Hz), 7.41 (d, 1H, J ¼ 8.6 Hz), 6.90 (dd, 1H, J1 ¼ 2.4 Hz,
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