Design, synthesis and antiproliferative activity studies of novel 1,2,3-triazole-dithiocarbamate-urea hybrids

Article abstract of DOI:10.1016/j.ejmech.2013.03.058

A series of novel 1,2,3-triazole-dithiocarbamate-urea hybrids were designed, synthesized and their antiproliferative activities against four selected human cancer cell lines were evaluated. The results showed that a number of the hybrids exhibited potent activity in selected human cancer cell lines. Among them, compounds 27 and 34 showed broad spectrum anticancer activity with IC₅₀ values ranging from 1.62 to 20.84 μM and 0.76 to 13.55 μM, respectively. Interestingly, compounds 27 and 34, being very potent against MGC-803 cells, exhibited no significant cytotoxicity against normal human embryonic kidney cells at up to 55 μM and 70 μM, respectively. Evidences of cell cycle arrest and apoptosis induction were obtained for the most effective compounds 27 and 34 by means of flow cytometry and microscopic techniques.

Full text of DOI:10.1016/j.ejmech.2013.03.058

Accepted Manuscript
Design, synthesis and antiproliferative activity studies of novel 1,2,3-triazole-
dithiocarbamate-urea hybrids
Ying-Chao Duan, Yi-Chao Zheng, Xiao-Chen Li, Meng-Meng Wang, Xian-Wei Ye,
Yuan-Yuan Guan, Gai-Zhi Liu, Jia-Xin Zheng, Hong-Min Liu
PII:
S0223-5234(13)00218-3
10.1016/j.ejmech.2013.03.058
EJMECH 6096
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 January 2013
Revised Date: 23 March 2013
Accepted Date: 26 March 2013
Please cite this article as: Y.-C. Duan, Y.-C. Zheng, X.-C. Li, M.-M. Wang, X.-W. Ye, Y.-Y. Guan, G.-
Z. Liu, J.-X. Zheng, H.-M. Liu, Design, synthesis and antiproliferative activity studies of novel 1,2,3-
triazole-dithiocarbamate-urea hybrids, European Journal of Medicinal Chemistry (2013), doi: 10.1016/
j.ejmech.2013.03.058.
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ACCEPTED MANUSCRIPT
Novel 1,2,3-triazole-dithiocarbamate-urea hybrids 34 exhibited potent anticancer
activity with IC50 values ranging from 0.76 to 13.55µM and was highly selective in its
cytotoxicity activity.

ACCEPTED MANUSCRIPT
Design, synthesis and antiproliferative activity studies of novel
1
,2,3-triazole-dithiocarbamate-urea hybrids
Ying-Chao Duan,Yi-Chao Zheng, Xiao-Chen Li, Meng-Meng Wang, Xian-Wei Ye, Yuan-Yuan Guan,
Gai-Zhi Liu, Jia-Xin Zheng, Hong-Min Liu
*
New Drug Research & Development Center, School of Pharmaceutical Sciences, Zhengzhou University, No. 100, Avenue
Kexue, Zhengzhou 450001, PR China
*Corresponding Author.Tel./ Fax: 86-371-67781739; e-mail: liuhm@zzu.edu.cn
Abstract: A series of novel 1,2,3-triazole-dithiocarbamate-urea hybrids were designed, synthesized
and their antiproliferative activities against four selected human cancer cell lines were evaluated. The
results showed that a number of the hybrids exhibited potent activity in selected human cancer cell
lines. Among them, compounds 27 and 34 showed broad spectrum anticancer activity with IC50
values ranging from 1.62 to 20.84ꢀM and 0.76 to 13.55ꢀM, respectively. Interestingly, compounds
2
7 and 34, being very potent against MGC-803 cells, exhibited no significant cytotoxicity against
normal human embryonic kidney cells at up to 55ꢀM and 70ꢀM, respectively. Evidences of cell
cycle arrest and apoptosis induction were obtained for the most effective compounds 27 and 34 by
means of flow cytometry and microscopic techniques.
Keywords: 1,2,3-triazole; dithiocarbamate; urea; hybrid; antiproliferative; apoptosis
1
. Introduction
Cancer, being one of the leading causes of death globally, causes almost 8 million people death each
year and poses a major socioeconomic hazard to humanity at large. Among all factors resulting in the
ultimate failure of cancer treatment, drug resistance is a significant player [1]. Development of new
molecules with novel mechanisms of action to fight cancer are urgently needed as most anticancer
drugs are ineffective due to drug resistance. Molecular hybridization which covalently combines two
or more drug pharmacophores into a single molecule is an effective tool to design highly active novel
entities [2]. These merged pharmacophores may act on multiple therapeutic targets and offer the
possibility of circumventing drug resistance. In addition, the hybrids may also minimize unwanted
side effects and allow for synergic action [3]. The molecular hybridization approach has already been
applied in developing novel antimalarial agents to overcome drug resistance [4].
1
,2,3-Triazoles have been a fruitful source of inspiration for medicinal chemists for many years due
to their synthetic accessibility by click chemistry as well as their numerous biological activities [5].
,2,3-Triazole is a versatile moiety found in a large variety of bioactive molecules, such as anti-fungal
1
[
6], anti-bacterial [7], anti-allergic [8], anti-HIV [9], anti-tubercular [10] and anti-inflammatory agents
11]. Recent research of 1,2,3-triazole's pharmacological effects became much more appealing and
[
1

ACCEPTED MANUSCRIPT
promising for anticancer agents design. 1,2,3-Triazoles conjugated with a wide range of moieties were
reported to exhibit potent anticancer activity [12]. N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)
arylamide was identified as a novel and proprietary small molecule scaffold for potential antitumor
agents by M.J. Miller group, and compound 1 exhibited an IC of 46 nM against MCF-7 cancer cell
5
0
line [13]. Compound 2, a 1,2,3-triazol-naphthalimide hybrid, showed IC values of 0.348 and 0.258
5
0
ꢀM against cell lines MCF-7 and SMMC-7721, respectively [14]. Carboxyamidotriazole (3) [15], a
1
,2,3-triazole-containing anticancer agent, is now available in the market (Fig. 1).
Dithiocarbamates are considered privileged scaffolds in drug discovery with a wide array of
biological activities. In the literature, dithiocarbamate derivatives have been described as anti-fungal
16], anti-bacterial [17], and carbonic anhydrase inhibitors [18]. In particular, their applications in the
[
treatment of cancer have been explored [19]. Brassinin (4), a phytoalexin in cruciferous plants, has
demonstrated cancer preventive activity [20]. Structural modifications of compound 4 led to the
synthesis of sulforamate (5), which was found to exhibit antitumor activity both in vitro and in vivo
[21]. Our group recently reported the synthesis of novel butenolide-containing dithiocarbamates (6),
and several compounds exhibited good anticancer activities (Fig. 2) [22].
Urea-based compounds are largely present in nature and have received special attention due to their
potent anticancer properties [23]. The urea derivatives, including ureas, arylureas, and thioureas,
represent one of the most useful classes of anticancer agents, with a wide range of activities against
various cancers [24] (Fig. 3).
In the course of our search for new anticancer agents, we recently reported the synthesis and
biological activities of a series of 1,2,3-triazole-dithiocarbamate hybrids with general structure 10,
several compounds showed excellent broad spectrum anticancer activity [25]. The study of new hybrid
systems in which 1,2,3-triazole, dithiocarbamate and urea are combined comprises an unexplored field
of research. These findings have encouraged us to investigate the potential synergistic effect of
1
,2,3-triazole, dithiocarbamate and urea scaffolds (Fig. 4). Herein, for the first time, the hybridization
of these three pharmacophores into novel scaffold and their ability to inhibit four selected human
tumor cell lines were reported.
2
. Results and discussion
2
.1. Chemistry
The general route for the synthesis of the target 1,2,3-triazole-dithiocarbamate-urea hybrids is
depicted in Scheme 1. Commercially available tert-butyl piperazine-1-carboxylate 11 [26] was reacted
with CS and propargyl bromide in the presence of Na PO ·12H O at room temperature to form
2
3
4
2
compound 12, which was subjected to click reaction with appropriately substituted benzyl azides to
afford compounds 13-18 with good yields. The substituted benzyl azides were readily synthesized
from the corresponding halides and sodium azide following literature procedures [27]. Compounds
1
3-18 were converted to compounds 19-24 by removing the protective group in a TFA/CH Cl2
2
solution. Without further purification, compounds 19-24 were transformed into the corresponding urea
derivatives 25-52 by treatment with triphosgene and various amines in one pot.
2
.2. Evaluation of biological activity
2

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2
.2.1. Antiproliferative activity
All synthesized compounds were evaluated for their antiproliferative activity against four human
cancer cell lines, MGC-803 (human gastric cancer cell line), MCF-7 (human breast cancer cell line),
SMMC-7721(human hepatocellular carcinoma cell line), and EC-9706 (human esophageal cancer cell
line) using MTT assay method [28] and compared with the well-known anticancer drug 5-fluorouracil.
As shown in Table 1, the antiproliferative activity of the tested compounds was generally more
pronounced against MGC-803 and MCF-7 cells as compared with the other cell lines. With the
MGC-803 and MCF-7 cell, compounds 26-28 and 33-35 were more potent than 5-fluorouracil in the
single-digit micro molar range. Among them, the two most potent compounds were 27 and 34,
exhibiting IC50 values against the four tested human cancer cell lines ranging from 1.62 to 20.84ꢀM
and 0.76 to 13.55ꢀM, respectively. With the exception of EC-9706, compounds 27 and 34 were more
potent than 5-fluorouracil against three of the four cancer cell lines. Structure-activity relationship
(SAR) analysis showed that the steric hindrance of substituents on the N-atom played a critical role
for the antiproliferative activities. Compound 32, bearing hydrogen atoms on the N-atom,
demonstrated an IC50 of 9.33µM against MGC-803 which was significantly enhanced when the
hydrogen atom was replaced by an ethyl group (compound 33, IC = 2.17µM) or, even better, by an
5
0
isopropyl group (compound 34, IC = 0.76µM). However, changing the isopropyl group to bulkier
5
0
benzyl group (compound 36) led to a complete loss of activity with all four cell lines. The
antiproliferative activities of the hybrids were also influenced by the substituents on the phenyl ring.
The p-fluoro compound 34 proved to be nearly 10-fold more potent than fluorouracil in the case of
MGC-803, while p-methoxy compound 47 and m, p, m-trimethoxy compound 51 showed almost no
cytotoxicity against MGC-803. The replacement of p-fluoro atom (34) with p-chloro atom (39) or
p-methyl group (43) was also detrimental to the anticancer activity.
Compounds 27 and 34 were further examined for possible cytotoxicity against normal human
embryonic kidney cells (HEK293). As can be seen in Figure 5A, we found that compound 27, being
very potent against MGC-803 cells (1.62ꢀM), exhibited no significant cytotoxicity against HEK293
cells at up to 55ꢀM. Compound 34, with an IC 0f 0.76ꢀM against MGC-803 cells also exhibited no
5
0
significant cytotoxicity against HEK293 cells at up to 70ꢀM (Figure 5B). The result indicated that
compounds 27 and 34 were highly selective in their cytotoxicity activity.
2
.2.2. Apoptosis assay
Apoptosis is considered a major way that most of the anticancer drugs kill tumor cells. To test
whether the inhibition of cell growth of the hybrids was related to cell apoptosis, MGC-803 cells were
treated with compounds 27 or 34 at different concentrations (0, 0.25, 0.5, 1.0ꢀmol/L) for 12 h, and
changes in cell morphology were observed by fluorescence microscopy using Hoechst 33258 staining
[29]. As shown in Figure 6A and 6B, compounds 27 and 34 altered cell morphology with respect to
those of control cultures, and caused apoptosis characteristics including cellular nuclear shrinkage,
chromatin condensation and nuclear fragmentation.
We also performed a biparametric cytofluorimetric analysis using propidium iodide (PI) and
annexin-V-FITC to measure the apoptosis inducing activity of compound 34 in MGC-803 cells [30].
3

ACCEPTED MANUSCRIPT
After treatment with compound 34 for 12 h at different concentrations (0, 0.25, 0.5, 1.0ꢀmol/L),
MGC-803 cells were labeled with the two dyes, and the resulting red (PI) and green (FITC)
fluorescences were monitored by flow cytometry. As shown in Fig.6C, the apoptosis rates were
significantly increased from 2.8% (DMSO control) to 31.4%. The results showed that compound 34
caused a significant induction of apoptotic cells in a concentration-dependent manner.
2
.2.3. Cell cycle analysis
Because the anticancer efficacy of many current chemotherapeutic agents is correlated with their
ability to arrest the cell cycle in cancer cells. To determine whether the high anticancer effects of the
hybrids were caused by cell cycle accumulation at a certain phase, the effects of different
concentrations (0, 0.25, 0.5, 1.0ꢀmol/L) of compounds 27 and 34 on cell cycle progression were
examined with MGC-803 cell line [31]. After treatment with compound 27 for 12 h, it was observed
that the percentage of cells in G2/M phase were 18.14%, 21.86%, 50.35%, and 59.80%, respectively
(
Fig.7B), whereas after 24 h of exposure, the percentage of cells in G2/M phase were 15.21%, 32.93%,
8.71%, and 74.95%, respectively. After treatment with compound 34 for 12 h, the percentage of cells
6
in G2/M phase were increased from 16.76% to 68.86%, whereas after 24 h of exposure, the
percentage of cells in G2/M phase were increased from 16.04% to 79.02%. The results suggested that
compounds 27 and 34 caused an obvious increase in cell numbers in the G2/M phase with a
concomitant decrease of cells in the G1 and S phases in a concentration and time-dependent manner.
3
. Conclusions
In conclusion, we have discovered that a number of 1,2,3-triazole-dithiocarbamate-urea hybrids
displayed high activity against the proliferation of different human cancer cells in vitro. Two
promising compounds 27 and 34 exhibited broad spectrum anticancer activity in vitro and were more
potent than 5-fluorouracil against three human cancer cell lines. Moreover, compound 27 and 34 did
not affect the normal cells (HEK-293) at up to 55ꢀM and 70ꢀM, respectively, despite being very
potent against MGC-803 cells. The results of apoptosis assay and cell cycle analysis demonstrated that
2
7 and 34 could obviously inhibit the proliferation of MGC-803 cancer cells by inducing apoptosis
and arresting the cell cycle at G2/M phase. Collectively, the potent anticancer activity, synthetic
accessibility and high selectivity strongly encourage further optimization of 27 and 34 as leads to
develop more potent anticancer agents.
4
. Experimental section
4
.1. General
Reagents and solvents were purchased from commercial sources and were used without further
purification. Melting points were determined on an X-5 micromelting apparatus and are uncorrected.
1
13
H NMR and C NMR spectra were recorded on a Bruker 400 MHz and 100MHz spectrometer
respectively. IR spectra were recorded on a Nicolet iS10 infrared spectrometer. High resolution mass
spectra (HRMS) were recorded on a Waters Micromass Q-Tof Micro mass spectrometer.
4

ACCEPTED MANUSCRIPT
4
.2. Procedure for the synthesis of tert Butyl 4-((prop-2-ynylthio)carbonothioyl)piperazine-1-
carboxylate compound 12.
CS (4.56g, 60mmol) was added drop wise to the solution of tert-butyl piperazine-1-carboxylate 11
2
(
3.72g, 20mmol) and Na PO ·12H O (4.56g, 12mmol) in acetone (80mL). The reaction mixture was
3 4 2
stirred at room temperature for 0.5 h. Then propargyl bromide (2.32g, 22mmol) 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 (80mL), washed with water, brine, dried over anhydrous Na SO and concentrated
2
4
o
1
under vacuum to afford compound 2 (5.56g, yield 92.2%). white solid. Mp: 87-88 C. H NMR (400
MHz, Acetone-d , δ, ppm): 4.28 (br, 2H), 4.14 (d, 2H, J = 2.7 Hz), 4.00 (br, 2H), 3.58 (br, 4H), 2.78 (t,
6
1
3
1
2
H, J = 2.7 Hz), 1.46 (s, 9H); C NMR (100 MHz, CDCl , δ, ppm): 195.2, 154.4, 80.7, 78.2, 71.8,
3
+
8.4, 26.0; HRMS (ESI) calcd for C H N O S [M+H] : 301.1044, found: 301.1046.
1
3
21
2
2 2
4
.3. General procedure for the synthesis of compounds13-18
In a round-bottom flask equipped with a magnetic stirred bar, 12 (1.51g, 5mmol), azide derivatives
(
5.5mmol), CuSO ·5H O (62mg, 0.25mmol), sodium ascorbate (100mg, 0.5mmol), THF (20mL) and
4 2
H O (20mL) were added. The resulting mixture was stirred at room temperature. The disappearance of
2
compound 12 was monitored by TLC. Upon completion, water (40mL) was added and the reaction
mixture was extracted with EtOAc (3×40mL). The combined organic layer was washed with brine,
dried over anhydrous Na SO and concentrated under vacuum to afford the crude product. The crude
2
4
product was recrystallized from acetone to yield the pure product.
4
.3.1.tert Butyl 4-(((1-(2-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methylthio)carbonothioyl)-
o
1
piperazine-1-carboxylate (13): yield 79.0%. white solid. Mp: 109-110 C; H NMR (400 MHz, CDCl ,
3
δ, ppm): 7.66 (s, 1H), 7.09-7.38 (m, 4H), 5.55 (s, 2H), 4.69 (s, 2H), 4.29 (br, 2H), 3.91 (br, 3H), 3.54
1
3
(
t, 4H, J = 5.1 Hz), 1.47 (s, 9H); C NMR (100 MHz, CDCl , δ, ppm): 196.4, 161.7, 159.3, 154.4,
3
1
3
44.0, 130.9, 130.8, 130.5, 130.5, 124.8, 124.9, 123.0, 122.0, 121.8, 115.9, 115.7, 80.6, 47.7, 47.6,
+
1.8, 28.3; HRMS (ESI) calcd for C H FN O S [M+H] : 452.1590, found: 452.1598.
2
0
27
5
2 2
4
.3.2. tert Butyl 4-(((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methylthio)carbonothioyl)-
o
1
piperazine-1-carboxylate (14): yield 79.7%. white solid. Mp: 171-172 C; H NMR (400 MHz, CDCl ,
3
δ, ppm): 7.59 (s, 1H), 7.04-7.27 (m, 4H), 5.46 (s, 2H), 4.68 (s, 2H), 4.31 (br, 2H), 3.90 (br, 3H), 3.53
1
3
(
s, 4H), 1.47 (s, 9H); C NMR (100 MHz, CDCl , δ, ppm): 196.40, 164.07, 161.60, 154.40, 130.49,
3
1
30.45, 129.96, 129.87, 116.20, 115.98, 80.66, 53.42, 31.78, 28.34; HRMS (ESI) calcd for
+
C H FN O S [M+H] : 452.1590, found: 452.1588.
2
0
27
5
2 2
4
.3.3. tert Butyl 4-(((1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methylthio)carbonothioyl)-
o
1
piperazine-1-carboxylate (15): yield 85.5%. white solid. Mp: 177-178 C; H NMR (400 MHz, CDCl ,
3
δ, ppm): 7.59 (s, 1H), 7.35 (d, 2H, J = 8.4 Hz), 7.20 (d, 2H, J = 8.4 Hz), 5.45 (s, 2H), 4.68 (s, 2H),
1
3
4
.29 (br, 2H), 3.90 (br, 2H), 3.54 (t, 4H, J = 5.2 Hz), 1.47(s, 9H); C NMR (100 MHz, CDCl , δ,
3
5

ACCEPTED MANUSCRIPT
ppm):196.4, 154.4, 144.4, 134.8, 133.1, 129.4, 129.3, 122.8, 80.7, 53.4, 31.7, 28.4; HRMS (ESI) calcd
+
for C H ClN O S [M+H] : 468.1295, found: 468.1291.
2
0
27
5
2 2
4
.3.4. tert Butyl 4-(((1-(4-methylbenzyl)-1H-1,2,3-triazol-4-yl)methylthio)carbonothioyl)-
o
1
piperazine-1-arboxylate (16): yield 81.9%. white solid. Mp: 182-183 C; H NMR (400 MHz, CDCl ,
3
δ, ppm): 7.55 (s, 1H), 7.16-7.21 (m, 4H), 5.43 (s, 2H), 4.67 (s, 2H), 4.29 (br, 2H), 3.91 (br, 2H), 3.54
1
3
(
t, 4H, J = 5.2 Hz), 2.35 (s, 3H), 1.47 (s, 9H); C NMR (100 MHz, CDCl , δ, ppm): 196.5, 154.4,
3
1
38.7, 131.6, 129.8, 128.1, 80.6, 54.0, 31.9, 28.4, 21.2; HRMS (ESI) calcd for C H N O S
21 30 5 2 2
+
[M+H] :448.1841, found: 448.1840.
4
.3.5. tert Butyl 4-(((1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methylthio)carbonothioyl)-
o
1
piperazine-1-carboxylate (17): yield 85.8%. white solid. Mp: 129-130 C; H NMR (400 MHz, CDCl ,
3
δ, ppm): 7.54 (s, 1H), 7.21(d, 2H, J = 8.7 Hz), 6.88 (d, 2H, J = 8.7 Hz), 5.41 (s, 2H), 4.67 (s, 2H), 4.29
1
3
(
br, 2H), 3.90 (br, 2H), 3.80 (s, 3H), 3.51 (t, 4H, J = 5.2 Hz), 1.47 (s, 9H); C NMR (100 MHz, CDCl ,
3
δ, ppm): 196.5, 159.9, 154.4, 143.9, 129.6, 126.6, 122.5, 114.4, 80.6, 55.3, 53.7, 31.9, 28.4; HRMS
+
(
ESI) calcd for C H N O S [M+H] : 464.1790, found: 464.1794.
2
1
30
5
3 2
4
.3.6. tert Butyl 4-(((1-(3,4,5-trimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methylthio)carbonothioyl)-
o
1
piperazine-1-carboxylate (18): yield 83.4%. white solid. Mp: 134-135 C; H NMR (400 MHz, CDCl ,
3
δ, 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
5
.84 (s, 6H), 3.55 (t, 4H, J = 5.2 Hz), 1.48 (s, 9H); HRMS (ESI) calcd for C H N O S [M+H] :
2
3
34
5
2 2
24.2001, found: 524.2005.
4
.4. General procedure for the synthesis of compounds 19-24.
CF COOH (4.56g, 40mmol) was added to a solution of 13-18 (2mmol) in CH Cl (20mL) at 0 C.
o
3
2
2
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 CH Cl ,
2
2
washed with saturated NaHCO , brine, dried over anhydrous Na SO and concentrated under vacuum
3
2
4
to afford compounds 19-24, which were used in the next reaction without further purification.
4
.4.1. (1-(2-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl piperazine-1-carbodithioate (19):
o
1
yield 96.7%. white solid. Mp: 93-94 C; H NMR (400 MHz, CDCl , δ, ppm): 7.67 (s, 1H), 7.09-7.36
3
1
3
(m, 4H), 5.55 (s, 2H), 4.69 (s, 2H), 4.30 (br, 2H), 3.93 (br, 2H), 2.96 (t, 4H, J = 4.8 Hz); C NMR
(
100 MHz, CDCl , δ, ppm): 195.8, 161.7, 159.3, 144.3, 130.9, 130.8, 130.5, 130.5, 124.8, 124.8,
3
1
23.0, 122.0, 121.9, 115.9, 115.7, 47.7, 47.6, 45.6, 31.8, 30.9; HRMS (ESI) calcd for C H FN S
15 19 5 2
+
[M+H] : 352.1066, found: 352.1064.
4
.4.2. (1-(4-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl piperazine-1-carbodithioate (20):
o
1
yield 93.9%. white solid. Mp: 138-139 C; H NMR (400 MHz, CDCl , δ, ppm): 7.60 (s, 1H),
3
1
3
7
.04-7.27 (m, 4H), 5.45 (s, 2H), 4.69 (s, 2H), 4.31 (br, 2H), 3.89 (br, 2H), 2.93 (s, 4H); C NMR (100
MHz, CDCl , δ, ppm): 196.0, 164.0, 161.6, 144.3, 130.5, 130.4, 130.0, 129.9, 122.7, 116.2, 116.0,
3
6

ACCEPTED MANUSCRIPT
+
5
3.4, 45.2, 31.8; HRMS (ESI) calcd for C H FN S [M+H] : 352.1066, found: 352.1063.
1
5
19
5 2
4
.4.3. (1-(4-Chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl piperazine-1-carbodithioate (21):
o
1
yield 95.1%. white solid. Mp: 79-80 C; H NMR (400 MHz, CDCl , δ, ppm): 7.61 (s, 1H), 7.35 (d, 2H,
3
J = 8.1 Hz), 7.20 (d, 2H, J = 8.1 Hz), 5.45 (s, 2H), 4.69 (s, 2H), 4.31 (br, 2H), 3.90 (br, 2H), 2.94 (s,
1
3
4
3
H); C NMR (100 MHz, CDCl , δ, ppm): 195.6, 144.5, 134.7, 133.2, 129.4, 129.3, 122.8, 53.4, 45.6,
3
+
1.7; HRMS (ESI) calcd for C H ClN S [M+H] : 368.0770, found: 368.0767.
1
5
19
5 2
4
.4.4. 1-(4-Methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl piperazine-1-carbodithioate (22):
o
1
yield 94.0%. white solid. Mp: 74-75 C; H NMR (400 MHz, CDCl , δ, ppm): 7.56 (s, 1H), 7.14-7.27
3
1
3
(
(
(
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); C NMR
100 MHz, CDCl , δ, ppm): 195.8, 138.6, 131.6, 129.7, 128.1, 122.7, 54.0, 31.8, 29.7, 21.2; HRMS
3
+
ESI) calcd for C H N S [M+H] :348.1317, found: 348.1319.
1
6
22
5 2
4
.4.5. (1-(4-Methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl piperazine-1-carbodithioate (23):
o
1
yield 92.7%. white solid. Mp: 95-96 C; H NMR (400 MHz, CDCl , δ, ppm): 7.55 (s, 1H), 7.24 (d, 2H,
3
J = 8.7 Hz), 6.91 (d, 2H, J = 8.7 Hz), 5.42 (s, 2H), 4.68 (s, 2H), 4.31 (br, 2H), 3.95 (br, 2H), 3.80 (s,
1
3
3
1
3
H), 2.96 (t, 4H, J = 4.6 Hz); C NMR (100 MHz, CDCl , δ, ppm): 195.8, 159.9, 144.1, 129.6, 126.6,
3
+
22.5, 114.5, 55.3, 53.7, 45.6, 31.8; HRMS (ESI) calcd for C H N OS [M+H] :364.1266, found:
1
6
22
5
2
64.1263.
4
.4.6. (1-(3,4,5-trimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl piperazine-1-carbodithioate (24):
o
1
yield 96.0%. white solid. Mp: 120-121 C; H NMR (400 MHz, CDCl , δ, ppm): 7.64 (s, 1H), 6.47 (s,
3
2
H), 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);
1
3
C NMR (100 MHz, CDCl , δ, ppm): 195.6, 153.7, 144.4, 138.2, 130.2, 122.8, 105.2, 60.9, 56.2, 54.4,
3
+
4
5.7, 31.7; HRMS (ESI) calcd for C H N O S [M+H] : 424.1477, found: 424.1472.
1
8
26
5
3 2
4
.5. General procedure for the synthesis of compounds 25-52.
To a solution of triphosgene (0.7mmol) in CH Cl (10mL) under nitrogen, Et N (2.1mmol) was
2
2
3
o
added drop wise, the reaction mixture was stirred at 0 C for 20 minutes, then a solution of 19-24
2mmol) in CH Cl (10mL) were added drop wise to the mixture, the reaction mixture was stirred at
(
2
2
room temperature for another 1 h, then various amines were added to the mixture, the mixture were
allowed to stand at room temperature for 1 to 4 h. The reaction mixture was quenched by the addition
of diluted HCl (1N, 50 mL), the organic layer was washed with saturated NaHCO , brine, dried over
3
anhydrous Na SO and concentrated under vacuum to give the crude product. The crude product were
2
4
recrystallized from acetone to yield the pure product 25-53 as white solids.
4
.5.1. (1-(2-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl 4-carbamoylpiperazine-1-carbodithioate (25):
o
1
yield 78.4%. white solid. Mp: 143-144 C; H NMR (400 MHz, DMSO-d , δ, ppm): 8.12 (s, 1H),
6
7
.20-7.42 (m, 4H), 6.13 (br, 2H), 5.64 (s, 2H), 4.58 (s, 2H), 4.21 (br, 2H), 3.87 (br, 2H), 3.44 (t, 4H, J
1
3
=
5.2 Hz); C NMR (100 MHz, DMSO-d , δ, ppm): 194.79, 161.77, 159.31, 158.23, 142.65, 131.26,
6
7

ACCEPTED MANUSCRIPT
1
31.23, 131.19, 125.34, 125.30, 124.46, 123.35, 123.21, 116.20, 115.99, 47.33, 47.29, 43.00, 31.85;
+
HRMS (ESI) calcd for C H FN OS [M+Na] : 417.0943, found: 417.0943.
1
6
19
6
2
4
.5.2.(1-(2-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl4-(ethylcarbamoyl)piperazine-1-carbodithioate
o
1
(26): yield 74.9%. white solid. Mp: 185-186 C; H NMR (400 MHz, CDCl , δ, ppm): 7.68 (s, 1H),
3
7
.11-7.37 (m, 4H), 5.56 (s, 2H), 4.70 (s, 2H), 4.55 (br, 1H), 4.35 (br, 2H), 3.96 (br, 2H), 3.56 (t, 4H, J
1
3
=
5.4 Hz), 3.31 (q, 2H, J = 7.1 Hz), 1.18 (t, 3H, J = 7.1 Hz); C NMR (100 MHz, CDCl , δ, ppm):
3
1
1
96.37, 161.73, 159.27, 157.24, 143.94, 130.95, 130.87, 130.53, 130.50, 124.84, 124.81, 123.02,
21.92, 121.78, 115.95, 115.74, 47.71, 47.67, 42.54, 35.83, 31.77, 15.52; HRMS (ESI) calcd for
+
C H FN NaOS [M+Na] : 445.1256, found: 445.1258.
1
8
23
6
2
4
.5.3. (1-(2-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl4-(isopropylcarbamoyl)piperazine-1-
o
1
carbodithioate (27): yield 80.9%. white solid. Mp: 159-160 C; H NMR (400 MHz, CDCl , δ, ppm):
3
7
.66 (s, 1H), 7.10-7.37 (m, 4H), 5.54 (s, 2H), 4.68 (s, 2H), 4.26 (br, 3H), 3.95-4.01 (m, 3H), 3.51 (s,
1
3
4
H), 1.17 (d, 6H, J = 6.4 Hz); C NMR (100 MHz, CDCl , δ, ppm): 196.42, 161.73, 159.27, 156.56,
3
1
43.96, 130.94, 130.86, 130.54, 130.51, 124.85, 124.81, 123.01, 121.93, 121.79, 115.95, 115.74,
+
4
7.70, 47.66, 42.81, 31.76, 29.70, 23.42; HRMS (ESI) calcd for C H FN OS [M+H] : 437.1594,
1
9
26
6
2
found: 437.1592.
4
.5.4 (1-(2-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl4-(ethyl(methyl)carbamoyl)piperazine-1-
o
1
carbodithioate (28): yield 83.7%. white solid. Mp: 144-145 C; H NMR (400 MHz, CDCl , δ, ppm):
3
7
4
.68 (s, 1H), 7.09-7.38 (m, 4H), 5.55 (s, 2H), 4.68 (s, 2H), 4.34 (br, 2H), 3.95 (br, 2H), 3.36 (t, 4H, J =
1
3
.8 Hz), 3.27 (q, 2H, J = 7.1 Hz), 2.84 (s, 3H), 1.18 (t, 3H, J = 7.1 Hz ); C NMR (100 MHz, CDCl ,
3
δ, ppm): 196.37, 161.73, 159.27, 157.24, 143.94, 130.95, 130.87, 130.53, 130.50, 124.84, 124.81,
1
23.02, 121.92, 121.78, 115.95, 115.74, 53.5, 46.3, 44.8, 35.3, 31.7, 12.6; HRMS (ESI) calcd for
+
C H FN OS [M+H] : 437.1594, found: 437.1595.
1
9
26
6
2
4
.5.5. (1-(2-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl4-(tert-butylcarbamoyl)piperazine-1-
o
1
carbodithioate (29):yield 73.6%. white solid. Mp: 74-75 C; H NMR (400 MHz, CDCl , δ, ppm): 7.68
3
(
s, 1H), 7.10-7.40 (m, 4H), 5.57 (s, 2H), 4.71 (s, 2H), 4.28 (br, 3H), 3.96 (br, 2H), 3.52 (t, 4H, J = 5.0
1
3
Hz), 1.38 (s, 9H); C NMR (100 MHz, CDCl , δ, ppm): 196.43, 161.73, 159.27, 156.35, 144.01,
3
1
4
30.93, 130.84, 130.54, 130.50, 124.84, 124.80, 122.97, 121.96, 121.81, 115.94, 115.73, 51.13, 47.68,
+
7.63, 31.77, 29.39; HRMS (ESI) calcd for C H FN OS [M+H] : 451.1570, found: 451.1572.
2
0
28
6
2
4
.5.6. (1-(2-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl4-(cyclohexylcarbamoyl)piperazine-1-
o
1
carbodithioate (30): yield 71.7%. white solid. Mp: 124-125 C; H NMR (400 MHz, CDCl , δ, ppm):
3
7
1
1
1
.70 (s, 1H), 7.10-7.40 (m, 4H), 5.57 (s, 2H), 4.72 (s, 2H), 4.37 (br, 3H), 3.97 (br, 2H), 3.63-3.71 (m,
1
3
H), 3.55 (t, 4H, J = 5.1 Hz), 1.07-2.00 (m, 10H); C NMR (100 MHz, CDCl , δ, ppm): 196.42,
3
61.74, 159.27, 156.49, 143.95, 130.96, 130.88, 130.55, 130.52, 124.85, 124.82, 123.05, 121.89,
21.75, 115.96, 115.75, 49.64, 47.75, 47.70, 33.93, 31.71, 29.70, 25.60, 25.04; HRMS (ESI) calcd for
+
C H FN NaOS [M+Na] : 499.1726, found: 499.1724.
2
2
29
6
2
8

ACCEPTED MANUSCRIPT
4
.5.7. (1-(2-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl4-(benzylcarbamoyl)piperazine-1-
o
1
carbodithioate (31): yield 75.5%. white solid. Mp: 131-132 C; H NMR (400 MHz, CDCl , δ, ppm):
3
7
3
1
.65 (s, 1H), 7.08-7.37 (m, 9H), 5.54 (s, 2H), 4.81 (br, 1H), 4.67 (s, 2H), 4.43 (s, 2H), 4.32 (br, 2H),
1
3
.94 (br, 2H), 3.56 (t, 4H, J = 5.2 Hz); C NMR (100 MHz, CDCl , δ, ppm): 196.4, 161.7, 159.3,
3
57.2, 143.9, 139.1, 131.0, 130.9, 130.5, 130.5, 128.7, 127.8, 127.5, 124.9, 124.8, 123.0, 121.9, 121.8,
+
116.0, 115.8, 47.7, 47.7, 45.0, 42.7, 31.8; HRMS (ESI) calcd for C H FN NaOS [M+Na] :
2
3
25
6
2
5
07.1413, found: 507.1416.
4
.5.8. (1-(4-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl 4-carbamoylpiperazine-1-carbodithioate (32):
o
1
yield 72.5%. white solid. Mp: 163-164 C; H NMR (400 MHz, Acetone-d , δ, ppm): 7.98 (s, 1H),
6
7
.13-7.46 (m, 4H), 5.61 (s, 2H), 5.54 (br, 1H), 4.62 (s, 2H), 4.30 (br, 2H), 3.98 (br, 2H), 3.57 (t, 4H, J
1
3
=
5.5 Hz); C NMR (100 MHz, CDCl , δ, ppm): 194.8, 163.6, 161.1, 158.2, 142.8, 132.8, 132.7,
3
+
1
4
30.8, 130.8, 124.2, 116.2, 116.0, 52.4, 43.0, 31.9; HRMS (ESI) calcd for C H FN NaOS [M+Na] :
1
6
19
6
2
17.0943, found: 417.0946.
4
.5.9.(1-(4-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl4-(ethylcarbamoyl)piperazine-1-carbodithioate
o
1
(33): yield 79.7%. white solid. Mp: 193-194 C; H NMR (400 MHz, CDCl , δ, ppm): 7.58 (s, 1H),
3
7
3
1
.03-7.27 (m, 4H), 5.46 (s, 2H), 4.68 (s, 2H), 4.34 (br, 3H), 3.95 (br, 2H), 3.54 (t, 4H, J = 5.0 Hz),
1
3
.32 (q, 2H, J = 7.2 Hz), 1.17 (t, 3H, J = 7.2 Hz ); C NMR (100 MHz, CDCl , δ, ppm): 196.4, 164.1,
3
61.6, 157.2, 144.2, 130.4, 130.4, 130.0, 129.9, 122.7, 116.2, 116.0, 53.4, 42.6, 35.9, 31.7, 15.5;
+
HRMS (ESI) calcd for C H FN NaOS [M+Na] : 445.1256, found: 445.1255.
1
8
23
6
2
4
.5.10. (1-(4-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl 4-(isopropylcarbamoyl)piperazine-1-
o
1
carbodithioate (34): yield 76.1%. white solid. Mp: 182-183 C; H NMR (400 MHz, Acetone-d , δ,
6
ppm): 7.98 (s, 1H), 7.13-7.46 (m, 4H), 5.69 (d, 1H, J = 6.6 Hz), 5.61 (s, 2H), 4.61 (s, 2H), 4.26 (br,
1
3
2
H), 3.85-3.91 (m, 3H), 3.54 (t, 4H, J = 5.0 Hz), 1.12 (d, 6H, J = 6.6 Hz); C NMR (100 MHz, CDCl ,
3
δ, ppm): 196.4, 164.1, 161.6, 156.6, 144.2, 130.5, 130.4, 130.0, 129.9, 122.7, 116.2, 116.0, 53.4, 42.8,
+
3
1.7, 23.4; HRMS (ESI) calcd for C H FN NaOS [M+Na] : 459.1413, found: 459.1414.
1
9
25
6
2
4
.5.11. (1-(4-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl4-(ethyl(methyl)carbamoyl)piperazine-1-
o
1
carbodithioate (35): yield 81.4%. white solid. Mp: 85-86 C; H NMR (400 MHz, CDCl , δ, ppm):
3
7
4
.61 (s, 1H), 7.05-7.28 (m, 4H), 5.47 (s, 2H), 4.70 (s, 2H), 4.34 (br, 2H), 3.95 (br, 2H), 3.35 (t, 4H, J =
1
3
.8 Hz), 3.27 (q, 2H, J = 7.1 Hz), 2.85 (s, 3H), 1.18 (t, 3H, J = 7.1 Hz); C NMR (100 MHz, CDCl , δ,
3
ppm): 196.2, 164.0, 163.9, 161.6, 130.5, 130.5, 130.0, 129.9, 122.7, 116.2, 116.0, 53.4, 46.2, 44.8,
+
3
5.3, 31.7, 12.6; HRMS (ESI) calcd for C H FN OS [M+H] : 437.1594, found:437.1591.
1
9
26
6
2
4
.5.12. (1-(4-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl4-(cyclohexylcarbamoyl)piperazine-1-
o
1
carbodithioate (36): yield 77.0%. white solid. Mp: 196-197 C; H NMR (400 MHz, CDCl , δ, ppm):
3
7
1
1
.60 (s, 1H), 7.05-7.28 (m, 4H), 5.47 (s, 2H), 4.69 (s, 2H), 4.33 (br, 3H), 3.95 (br, 2H), 3.60-3.71 (m,
1
3
H), 3.53 (s, 4H), 1.07-2.00 (m, 10H); C NMR (100 MHz, CDCl , δ, ppm): 196.4, 164.1, 161.6,
3
56.5, 144.2, 130.4, 130.4, 130.0, 129.9, 122.7, 116.2, 116.0, 53.4, 49.7, 33.9, 31.7, 29.7, 25.6, 25.0;
9

ACCEPTED MANUSCRIPT
+
HRMS (ESI) calcd for C H FN NaOS [M+Na] : 499.1726, found: 499.1725.
2
2
29
6
2
4
.5.13. (1-(4-Chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl4-carbamoylpiperazine-1-carbodithioate (37):
o
1
yield 82.3%. white solid. Mp: 97-98 C; H NMR (400 MHz, Acetone-d , δ, ppm): 8.02 (s, 1H),
6
7
.40-7.46 (m, 4H), 5.65 (s, 2H), 5.57 (br,1H), 4.65 (s, 2H), 4.32 (br, 2H), 3.99 (br, 2H), 3.59 (t, 4H, J
1
3
=
5.2 Hz); C NMR (100 MHz, CDCl , δ, ppm): 194.8, 158.2, 142.8, 135.5, 133.3, 130.4, 129.2,
3
+
1
4
4
24.4, 52.4, 43.0, 31.9; HRMS (ESI) calcd for C H ClN NaOS [M+Na] : 433.0648, found:
1
6
19
6
2
33.0646.
.5.14. (1-(4-Chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl 4-(ethylcarbamoyl)piperazine-1-
o
1
carbodithioate (38): yield 75.5%. white solid. Mp: 195-196 C; H NMR (400 MHz, CDCl , δ, ppm):
3
7
3
.60 (s, 1H), 7.36 (d, 2H, J = 8.0 Hz), 7.21 (d, 2H, J = 8.0 Hz), 5.46 (s, 2H), 4.69 (s, 2H), 4.32 (br, 3H),
1
3
.95 (br, 2H), 3.53 (s, 4H), 3.32 (q, 2H, J = 7.2 Hz), 1.17 (t, 3H, J = 7.2 Hz); C NMR (100 MHz,
CDCl , δ, ppm): 196.5, 157.2, 144.3, 134.8, 133.1, 129.4, 129.3, 122.9, 53.5, 42.6, 35.9, 31.7, 15.6;
3
+
HRMS (ESI) calcd for C H ClN OS [M+H] : 439.1142, found: 439.1144.
1
8
24
6
2
4
.5.15. (1-(4-Chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl4-(isopropylcarbamoyl)piperazine-1-
o
1
carbodithioate (39): yield 76.9%. white solid. Mp: 202-203 C; H NMR (400 MHz, CDCl , δ, ppm):
3
7
4
.59 (s, 1H), 7.36 (d, 2H, J = 8.4 Hz), 7.21 (d, 2H, J = 8.4 Hz), 5.46 (s, 2H), 4.68 (s, 2H), 4.34 (br, 2H),
1
3
.21 (d, 1H, J = 6.4 Hz), 3.93-4.01 (m, 3H), 3.53 (s, 4H), 1.17 (d, 6H, J = 6.4 Hz); C NMR (100
MHz, CDCl , δ, ppm): 196.4, 156.6, 144.3, 134.8, 133.1, 129.4, 129.3, 122.8, 53.4, 42.8, 31.7, 23.4;
3
+
HRMS (ESI) calcd for C H ClN OS [M+H] : 453.1298, found: 453.1298.
1
9
26
6
2
4
.5.16. (1-(4-Chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl4-(ethyl(methyl)carbamoyl)piperazine-1-
o
1
carbodithioate (40): yield 81.0%. white solid. Mp: 112-113 C; H NMR (400 MHz, CDCl , δ, ppm):
3
7
.61 (s, 1H), 7.36 (d, 2H, J = 8.4 Hz), 7.22 (d, 2H, J = 8.4 Hz), 5.47 (s, 2H), 4.70 (s, 2H), 4.34 (br, 2H),
.95 (br, 2H), 3.50 (t, 4H, J = 5.0 Hz), 3.27 (q, 2H, J = 7.1 Hz), 2.84 (s, 3H), 1.18 (t, 3H, J = 7.1 Hz);
3
1
3
C NMR (100 MHz, CDCl , δ, ppm): 196.2, 163.9, 144.3, 134.7, 133.2, 129.4, 129.3, 122.8, 53.3,
3
+
5
0.9, 49.6, 46.2, 44.8, 35.3, 31.7, 12.6; HRMS (ESI) calcd for C H ClN OS [M+H] : 453.1298,
1
9
26
6
2
found: 453.1299.
4
.5.17.(1-(4-Methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl4-carbamoylpiperazine-1-carbodithioate (41):
o
1
Yield 72.9%. white solid. Mp:179-180 C. H NMR (400 MHz, CDCl , δ, ppm): 7.57 (s, 1H),
3
7
.17-7.21 (m, 4H), 5.46 (s, 2H), 4.69 (s, 2H), 4.67 (br, 2H), 4.37 (br, 2H), 4.00 (br, 2H), 3.59 (t, 4H, J
1
3
=
5.4 Hz), 2.37 (s, 3H); C NMR (100 MHz, CDCl , δ, ppm): 194.8, 158.2, 142.7, 138.0, 133.5, 129.8,
3
+
1
28.5, 124.1, 53.0, 43.0, 31.9, 21.2; HRMS (ESI) calcd for C H N NaOS [M+Na] : 413.1194,
1
7
22
6
2
found: 413.1192.
4
.5.18. (1-(4-Methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl 4-(ethylcarbamoyl)piperazine-1-
o
1
carbodithioate (42): yield 73.0%. white solid. Mp:123-124 C. H NMR (400 MHz, CDCl , δ, ppm):
3
7
5
.56 (s, 1H), 7.15-7.19 (m, 4H), 5.45 (s, 2H), 4.68 (s, 2H), 4.35 (br, 3H), 3.92 (br, 2H), 3.53 (t, 4H, J =
1
3
.1 Hz), 3.32 (q, 2H, J = 7.2 Hz), 2.35 (s, 3H), 1.17 (t, 3H, J = 7.2 Hz); C NMR (100 MHz, CDCl , δ,
3
1
0

ACCEPTED MANUSCRIPT
ppm): 196.5, 156.6, 138.7, 129.8, 128.1, 122.7, 53.8, 44.8, 35.9, 31.8, 21.3, 15.5; HRMS (ESI) calcd
+
for C H N NaOS [M+Na] : 441.1507, found: 441.1504.
1
9
26
6
2
4
.5.19. (1-(4-Methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl 4-(isopropylcarbamoyl)piperazine-1-
o
1
carbodithioate (43): yield 83.1%. white solid. Mp: 198-199 C; H NMR (400 MHz, CDCl , δ, ppm):
3
7
3
.55 (s, 1H), 7.15-7.19 (m, 4H), 5.44 (s, 2H), 4.67 (s, 2H), 4.33 (br, 2H), 4.21 (br, 1H), 3.92-3.97 (m,
1
3
H), 3.52 (t, 4H, J = 5.3 Hz), 2.35 (s, 3H), 1.17 (d, 6H, J = 6.5 Hz); C NMR (100 MHz, CDCl , δ,
3
ppm): 196.5, 156.6, 138.7, 129.8, 128.1, 122.7, 54.0, 42.8, 31.8, 23.4, 21.2; HRMS (ESI) calcd for
+
C H N NaOS [M+Na] : 455.1664, found: 455.1666.
2
0
28
6
2
4
.5.20. (1-(4-Methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl 4-(ethyl(methyl)carbamoyl)piperazine-1-
o
1
carbodithioate (44): yield 73.7%. white solid. Mp: 110-111 C. H NMR (400 MHz, CDCl , δ, ppm):
3
7
5
.56 (s, 1H), 7.14-7.19 (m, 4H), 5.44 (s, 2H), 4.68 (s, 2H), 4.31 (br, 2H), 3.94 (br, 2H), 3.34 (t, 4H, J =
1
3
.1 Hz), 3.26 (q, 2H, J = 7.1 Hz), 2.83 (s, 3H), 2.34 (s, 3H), 1.17 (t, 3H, J = 7.1 Hz); C NMR (100
MHz, CDCl , δ, ppm): 196.3, 163.9, 138.7, 131.6, 129.8, 128.1, 54.0, 46.1, 44.8, 35.4, 31.8, 21.2, 12.6;
3
+
HRMS (ESI) calcd for C H N OS [M+H] : 433.1844, found: 433.1843.
2
0
29
6
2
4
.5.21.(1-(4-Methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl4-carbamoylpiperazine-1-carbodithioate
o
1
(45): yield 80.4%. white solid. Mp: 189-190 C; H NMR (400 MHz, DMSO-d , δ, ppm): 8.08 (s, 1H),
6
7
3
1
.30 (d, 2H, J = 8.4 Hz), 6.94 (d, 2H, J = 8.4 Hz), 6.12 (s, 2H), 5.48 (s, 2H), 4.57 (s, 2H), 4.21 (br, 2H),
1
3
.87 (br, 2H), 3.74 (s, 3H), 3.43 (t, 4H, J = 5.2 Hz); C NMR (100 MHz, DMSO-d , δ, ppm): 194.8,
6
59.6, 158.2, 142.6, 130.1, 128.4, 124.0, 114.6, 55.6, 52.8, 43.0, 31.9; HRMS (ESI) calcd for
+
C H N NaO S [M+Na] : 429.1143, found: 429.1142.
1
7
22
6
2 2
4
.5.22.(1-(4-Methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl4-(ethylcarbamoyl)piperazine-1-
o
1
carbodithioate (46): yield 71.9%. white solid. Mp: 179-180 C; H NMR (400 MHz, CDCl , δ, ppm):
3
7
3
.54 (s, 1H), 7.23 (d, 2H, J = 8.6 Hz), 6.90 (d, 2H, J = 8.6 Hz), 5.41 (s, 2H), 4.67 (s, 2H), 4.35 (br, 3H),
.94 (br, 2H), 3.81 (s, 3H), 3.54 (t, 4H, J = 5.3 Hz), 3.32 (q, 2H, J = 7.2 Hz), 1.17 (t, 3H, J = 7.2 Hz);
1
3
C NMR (100 MHz, CDCl , δ, ppm): 196.5, 159.9, 157.2, 143.8, 129.7, 126.5, 122.5, 114.5, 55.4,
3
+
5
3.8, 35.9, 31.8, 15.5; HRMS (ESI) calcd for C H N NaO S [M+Na] : 457.1456, found: 457.1458.
1
9
26
6
2 2
4
.5.23.(1-(4-Methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl4-(isopropylcarbamoyl)piperazine-1-
o
1
carbodithioate (47): yield 83.2%. white solid. Mp: 190-191 C; H NMR (400 MHz, CDCl , δ, ppm):
3
7
4
.56 (s, 1H), 7.25 (d, 2H, J = 8.6 Hz), 6.92 (d, 2H, J = 8.6 Hz), 5.44 (s, 2H), 4.69 (s, 2H), 4.36 (br, 2H),
1
3
.23 (br, 1H), 3.92-4.03 (m, 3H), 3.83 (s, 3H), 3.54 (t, 4H, J = 5.1 Hz), 1.19 (d, 6H, J = 6.5 Hz); C
NMR (100 MHz, CDCl , δ, ppm): 196.5, 159.9, 156.6, 143.8, 129.7, 126.5, 122.6, 114.5, 55.4, 53.8,
3
+
4
2.8, 31.8, 29.7, 23.4; HRMS (ESI) calcd for C H N NaO S [M+Na] : 471.1613, found:471.1616.
2
0
28
6
2 2
4
.5.24. (1-(4-Methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl 4-(ethyl(methyl)carbamoyl)piperazine-1-
o
1
carbodithioate (48): yield 87.0%. white solid. Mp: 82-83 C; H NMR (400 MHz, CDCl , δ, ppm):
3
7
.56 (s, 1H), 7.24 (d, 2H, J = 8.6 Hz), 6.91 (d, 2H, J = 8.6 Hz), 5.42 (s, 2H), 4.86 (s, 2H), 4.34 (br, 2H),
11

ACCEPTED MANUSCRIPT
3
.94 (br, 2H), 3.81(s, 3H), 3.34 (s, 4H), 3.27 (q, 2H, J = 7.1 Hz), 2.84 (s, 3H), 1.18 (t, 3H, J = 7.1 Hz);
1
3
C NMR (100 MHz, CDCl , δ, ppm): 196.3, 163.9, 159.9, 143.9, 129.6, 126.6, 122.5, 114.5, 55.4,
3
+
5
4
3.7, 46.1, 44.8, 35.4, 31.8, 12.6; HRMS (ESI) calcd for C H N NaO S [M+Na] : 471.1613, found:
2
0
28
6
2 2
71.1615.
4
.5.25. (1-(3,4,5-Trimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl 4-carbamoylpiperazine-1-
o
1
carbodithioate (49): yield 79.4%. white solid. Mp: 142-143 C; H NMR (400 MHz, CDCl , δ, ppm):
3
7
3
1
4
.62 (s, 1H), 6.48 (s, 2H), 5.41 (s, 2H), 4.94 (br, 2H), 4.68 (s, 2H), 4.33 (br, 2H), 3.97 (br, 2H), 3.83 (s,
1
3
H), 3.82 (s, 6H), 3.57 (s, 4H) ; C NMR (100 MHz, CDCl , δ, ppm): 194.8, 158.2, 153.4, 142.7,
3
+
37.7, 131.8, 124.3, 106.1, 60.4, 56.4, 53.5, 43.0, 31.9; HRMS (ESI) calcd for C H N O S [M+H] :
1
9
27
6
4 2
67.1535, found: 467.1537.
4
.5.26. (1-(3,4,5-Trimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl 4-(ethylcarbamoyl)piperazine-1-
o
1
carbodithioate (50): yield 81.7%. white solid. Mp: 154-155 C; H NMR (400 MHz, CDCl , δ, ppm):
3
7
3
.62 (s, 1H), 6.48 (s, 2H), 5.41 (s, 2H), 4.69 (s, 2H), 4.50 (br, 1H), 4.34 (br, 2H), 3.95 (br, 2H), 3.84 (s,
1
3
H), 3.83 (s, 6H), 3.52 (s, 4H), 3.31 (q, 2H, J = 7.2 Hz), 1.17 (t, 3H, J = 7.2 Hz); C NMR (100 MHz,
CDCl , δ, ppm): 196.3, 157.3, 153.7, 144.0, 138.2, 130.1, 122.9, 105.2, 60.9, 56.2, 54.4, 42.5, 35.8,
3
+
3
1.7, 15.5; HRMS (ESI) calcd for C H N O S [M+H] : 495.1848, found: 495.1849.
2
1
31
6
4 2
4
.5.27. (1-(3,4,5-Trimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl4-(isopropylcarbamoyl)piperazine-1-
o
1
carbodithioate (51): yield 73.5%. white solid. Mp: 170-171 C; H NMR (400 MHz, CDCl , δ, ppm):
3
7
3
1
.61 (s, 1H), 6.47 (s, 2H), 5.40 (s, 2H), 4.69 (s, 2H), 4.33 (br, 3H), 3.93-4.02 (m, 3H), 3.84 (s, 3H),
1
3
.83 (s, 6H), 3.51 (s, 4H), 1.17 (d, 6H, J = 6.5 Hz); C NMR (100 MHz, CDCl , δ, ppm): 196.4, 156.5,
3
53.7, 144.1, 138.3, 130.0, 122.9, 105.2, 60.9, 56.3, 54.5, 42.8, 31.7, 23.4; HRMS (ESI) calcd for
+
C H N NaO S [M+Na] : 531.1824, found: 531.1822.
2
2
32
6
4 2
4
.5.28.(1-(3,4,5-Trimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl4-(ethyl(methyl)carbamoyl)piperazine
o
1
-1-carbodithioate (52): yield 86.2%. white solid. Mp:111-112 C; H NMR (400 MHz, CDCl , δ, ppm):
3
7
6
.62 (s, 1H), 6.47 (s, 2H), 5.41 (s, 2H), 4.70 (s, 2H), 4.32 (br, 2H), 3.94 (br, 2H), 3.84 (s, 3H), 3.83 (s,
1
3
H), 3.34 (t, 4H, J = 5.2 Hz), 3.25 (q, 2H, J = 7.1 Hz), 2.83 (s, 3H), 1.17 (t, 3H, J = 7.1 Hz); C NMR
(
100 MHz, CDCl , δ, ppm): 196.3, 163.9, 153.7, 144.2, 138.2, 130.1, 122.8, 105.2, 60.9, 56.2, 54.4,
3
+
4
5
6.2, 44.8, 35.3, 31.7, 12.6; HRMS (ESI) calcd for C H N O S [M+H] : 509.2005, found:
2
2
33
6
4 2
09.2006.
.6. Antiproliferative activity assays
4
3
Exponentially growing cells were seeded into 96-well plates at a concentration of 5×10 cells per
o
well. After 24 h incubation at 37 C, the culture medium was removed and replaced with fresh medium
containing the candidate compounds in different concentrations. The cells were incubated for another
7
3
2 h. Afterwards, 20 ꢀL of MTT solution (5mg/mL) was added to all wells and incubated for 4 h at
o
7 C. Discarded the suspension and added 150 ꢀL of dimethyl sulfoxide (DMSO) to each well and
1
2

ACCEPTED MANUSCRIPT
shaked 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 (IC ) was determined
5
0
from the dose-response curves according to the inhibition ratio for each concentration.
4
.7. Flow cytometric analysis of cell cycle distribution
For flow cytometric analysis of DNA content, 5×10 MGC-803 cells in exponential growth were
5
treated with different concentrations 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 analysed using the FlowJo software (Tree Star, Inc., Ashland, OR, USA)
4
.8. Morphologic analysis of cellular apoptosis.
MGC-803 cells were seeded in 24-well plates at a density of 8 ×10 cells/well, and incubated with
4 with different concentrations. After 12 h incubation, the cells were digested and collected. Then the
3
3
cells were fixed and stained with DNA fluorochrome Hoechst 33258 for 20 min. After washing with
phosphate buffered saline (PBS), the morphological features of apoptosis (including cellular nucleus
shrinkage, chromatin condensation, intense fluorescence and nuclear fragmentation) were monitored
by fluorescence microscopy (Nikon, Japan).
4
.9. Flow cytometric analysis of cellular apoptosis
MGC-803 cells were plated in 6-well plates (5.0×10 cells/mL) and incubated at 37 C for 12 h.
4
0
Exponentially growing cells were then incubated for 12 h with complete medium (blank) or with the
compound 34. 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.
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 Doctor Stations, Ministry of Education
(Project No. 20114101120013).
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1
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Fig. 1. 1,2,3-Triazole derivatives with anticancer activity.
Fig. 2. Dithiocarbamate derivatives with anticancer activity.
Fig.3. Urea derivatives with anticancer activity.
Fig. 4. Structures of 1,2,3-triazole-dithiocarbamate-urea hybrids.
Fig.5. A: Selectivity of compound 27 towards MGC-803 cells over normal cells HEK293; B: Selectivity of compound
3
4 towards MGC-803 cells over normal cells HEK293
Fig.6. A: Effect of compound 27 on morphology of MGC-803 cells; B: Effect of compound 34 on morphology of
MGC-803 cells; C: Representative flow cytometric histograms of apoptotic MGC-803 cells after 12 h treatment with
3
4 at different concentrations.
Fig.7. A: Effect of compound 27 on cell cycle in MGC-803 cells. a, b, c, d incubated for 12 h; e, f, g, h incubated for
4 h; B: Effect of compound 34 on cell cycle in MGC-803 cells. a, b, c, d incubated for 12 h; e, f, g, h incubated for 24
2
h.
Scheme 1. Synthesis of the 1,2,3-triazole-dithiocarbamate-urea hybrids. Reagent and reaction conditions: (a) CS2,
Na PO ·12H O, propargyl bromide, acetone, rt; (b) BnN , CuSO ·5H O, Sodium ascorbate, THF-H O (1:1), rt; (c)
3
4
2
3
4
2
2
o
CF COOH, CH Cl , rt; (d) BTC, Et N, R R NH, CH Cl , 0 C-rt.
3
2
2
3
4
5
2
2
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Table 1
Antiproliferative activity of 1,2,3-triazole-dithiocarbamate-urea hybrids
a
Com.
R
3
R
4
R
5
NH
IC50 (µM)
MGC-803
MCF-7
SMMC-7721
EC-9706
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
o-F
NH
2
11.30±1.64
2.83±0.18
1.62±0.12
5.86±2.19
40.7±2.25
36.51±2.60
13.27±1.67
9.33±1.04
2.17±0.22
0.76±0.03
1.55±0.14
>128
>128
>128
43.11±3.14
23.71±2.43
20.84±2.19
15.55±1.67
87.84±1.78
84.94±3.37
38.10±2.61
62.74±3.53
27.79±2.53
12.19±1.34
14.28±1.45
>128
o-F
CH
3
CH
CHNH
CH NCH
2
NH
5.76±1.21
1.86±0.41
7.21±0.46
18.80±1.37
84.96±3.37
38.10±2.53
22.05±2.38
3.66±0.51
1.66±0.53
2.15±0.61
>128
>128
o-F
(CH
3
)
2
7.13±0.36
19.65±2.44
>128
o-F
CH
3
2
3
o-F
(CH
3
6
)
3
CNH
o-F
C
6
H
NH
>128
o-F
PhCH
NH
2
>128
p-F
2
>128
p-F
CH
3
CH
2
NH
44.36±2.21
5.97±1.02
23.68±3.14
>128
p-F
(CH ) CHNH
3 2
p-F
CH
3
CH
2
NCH
3
p-F
C
6
H
6
NH
p-Cl
p-Cl
p-Cl
p-Cl
p-CH
p-CH
p-CH
p-CH
NH
2
>128
>128
>128
>128
CH
3
CH
2
NH
35.35±2.72
19.87±1.07
4.32±0.64
29.85±2.73
19.76±2.45
16.55±1.97
8.27±1.02
>128
>128
>128
>128
(CH
3
)
2
CHNH
>128
>128
25.60±2.35
17.68±1.99
>128
CH
NH
3
CH
2
NCH
3
14.73±1.82
>128
38.23±2.73
>128
3
3
3
3
2
CH
3
CH
2
NH
>128
>128
>128
(CH
3
)
2
CHNH
4.63±0.70
40.1±2.86
>128
>128
>128
CH
3
CH
2
NCH
3
>128
70.59±3.43
>128
p-OCH
p-OCH
p-OCH
p-OCH
3
3
3
3
NH
2
>128
CH
3
CH
2
NH
>128
97.57±1.99
19.58±2.10
21.41±1.01
>128
>128
>128
(CH
3
)
2
CHNH
99.23±3.68
87.89±3.79
>128
>128
>128
CH
NH
3
CH
2
NCH
3
>128
>128
m,p, m-triOCH
m,p, m-triOCH
m,p, m-triOCH
m, p,m-triOCH
3
3
3
3
2
>128
>128
CH
3
CH
2
NH
101.3±3.52
>128
>128
>128
>128
(CH
3
)
2
CHNH
>128
>128
>128
CH
3
CH NCH
2
3
85.73±3.23
7.14±1.26
68.47±3.03
7.33±0.54
>128
>128
-Fu
26.71±2.66
0.32±0.25
a
Antiproliferative activity was assayed by exposure for 72 hours 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.

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