Discovery of novel heteroarylmethylcarbamodithioates as potent anticancer agents: Synthesis, structure-activity relationship analysis and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2016.02.015

A series of new analogs based on the structure of lead compound 10 were designed, synthesized and evaluated for their in vitro anti-cancer activities against four selected human cancer cell lines (HL-60, Bel-7402, SK-BR-3 and MDA-MB-468). Several synthesized compounds exhibited improved anti-cancer activities comparing with lead compound 10. Among them, 1,3,4-oxadiazole analogs 17o showed highest bioactivity with IC₅₀ values of 1.23, 0.58 and 4.29 μM against Bel-7402, SK-BR-3 and MDA-MB-468 cells, respectively. It is noteworthy that 17o has potent anti-proliferation activity toward a panel of cancer cells with relatively less cytotoxicity to nonmalignant cells. The further mechanistic study showed that it induced apoptosis and cell cycle arrest through disrupting spindle assembly in mitotic progression, indicating these synthesized dithiocarbamates represented a novel series of anti-cancer compounds targeting mitosis.

Full text of DOI:10.1016/j.ejmech.2016.02.015

Accepted Manuscript
Discovery of novel heteroarylmethylcarbamodithioates as potent anticancer agents:
synthesis, structure-activity relationship analysis and biological evaluation
Ying-Bo Li, Xu Yan, Ri-Dong Li, Peng Liu, Shao-Qian Sun, Xin Wang, Jing-Rong Cui,
De-Min Zhou, Ze-Mei Ge, Run-Tao Li
PII:
S0223-5234(16)30085-X
10.1016/j.ejmech.2016.02.015
EJMECH 8366
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 September 2015
Revised Date: 11 December 2015
Accepted Date: 4 February 2016
Please cite this article as: Y.-B. Li, X. Yan, R.-D. Li, P. Liu, S.-Q. Sun, X. Wang, J.-R. Cui, D.-M.
Zhou, Z.-M. Ge, R.-T. Li, Discovery of novel heteroarylmethylcarbamodithioates as potent anticancer
agents: synthesis, structure-activity relationship analysis and biological evaluation, European Journal of
Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.02.015.
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ACCEPTED MANUSCRIPT
Graphical abstract
SK-BR-3 (Breast)
Bel-7402(Hepatoma) IC50 = 1.23±0.49 µM
PC-3ꢀProstateꢁ IC50 = 0.39±0.07 µM
HEK293TꢀNormalꢁ IC50 = 14.63±0.98 µM
IC50 = 0.58±0.05 µM

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Discovery of novel heteroarylmethylcarbamodithioates as potent
anticancer agents: synthesis, structure-activity relationship analysis
and biological evaluation
Ying-Bo Li a, Xu Yan a, Ri-Dong Li, Peng Liu, Shao-Qian Sun, Xin Wang, Jing-Rong
Cui, De-Min Zhou*, Ze-Mei Ge*, Run-Tao Li
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University,
Beijing 100191, PR China
a These authors contributed equally.
Abstract
A series of new analogs based on the structure of lead compound 10 were
designed, synthesized and evaluated for their in vitro anti-cancer activities against
four selected human cancer cell lines (HL-60, Bel-7402, SK-BR-3 and
MDA-MB-468). Several synthesized compounds exhibited improved anti-cancer
activities comparing with lead compound 10. Among them, 1,3,4-oxadiazole
analogues 17o showed highest bioactivity with IC50 values of 1.23, 0.58 and 4.29 ꢀM
against Bel-7402, SK-BR-3 and MDA-MB-468 cells, respectively. It is noteworthy
that 17o has potent anti-proliferation activity toward a panel of cancer cells with
relatively less cytotoxicity to nonmalignant cells. The further mechanistic study
showed that it induced apoptosis and cell cycle arrest through disrupting spindle
assembly in mitotic progression, indicating these synthesized dithiocarbamates
represented a novel series of anti-cancer compounds targeting mitosis.
Keywords:
Dithiocarbamate; 1,3,4-Oxadiazole; Anti-tumor activity; Structure-activity
relationship; Cell cycle arrest;
1. Introduction
Despite that the substantial progress has been made in cancer therapies, cancer is
still a major health problem which concerns the medical community throughout the
world. This is due to the involvement of multiple signaling pathway disruption and
the incidence of resistance to current therapeutic agents [1]. Hence, it is still urgent to
discover anti-cancer agents with new scaffolds. Cumulative data suggested that
dithiocarbamate represents a privileged scaffold in medicinal chemistry. This kind of
compounds showed a broad spectrum of biological activities [2-12]. Particularly, their
anti-cancer activities have attracted much more attention from medicinal chemists in
*Corresponding author. Tel./fax: +86 10 82801504.
E-mail address: zmge@bjmu.edu.cn (Z.-M. Ge), deminzhou@bjmu.edu.cn (D.-M. Zhou)
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recent years [13-28]. For examples, Zahran et al. reported Thalidomide derivatives
bearing dithiocarbamate groups (1) displayed anti-tumor activity against Ehrlich
ascites carcinoma cell line [14]. Thomas et al. revealed dithiocarbamate derivatives (2)
as HDAC inhibitors to inhibit tumor proliferation[16]. By combining the
dithiocarbamate with 2,4-diaminoquinazoline, Cao's group synthesized a series of
compounds (3) which displayed potent anti-proliferative activity against human
cancer cell lines [20]. Manga. V et al. demonstrated that the Michael additional
compounds of chalcone with dithiocarbamate (4) exhibited significant anti-mitotic
activities [22]. Sharma et al. reported that alkylphospholipid-dithiocarbamate hybrids
(5) were a kind of promising agents against endocrine related cancers, acting via
modulation of Akt-pathway[23]. More recently, Liu's group published a novel type of
1,2,3-triazole-dithiocarbamate hybrids (6), which acted as selective lysine specific
demethylase 1 (LSD1) inactivators [26] (Fig. 1).
Fig.1. Representative dithiocarbamate derivatives with anti-cancer activity
In view of the anti-cancer activities of dithiocarbamtes, intense efforts have been
made in our group to develop novel anti-cancer agents with the dithiocarbamte
scaffold. As shown in Fig.2, several compounds with anti-cancer efficiency have been
identified (7-10) [29-38]. Particularly, compound 10 showed potent anti-proliferation
activity against different tumor cell lines with IC50 low to the ꢀM range, making it a
valuable lead compound for anti-cancer agent. However, there was still gap in
potency and drug-like properties between compound 10 and launching anti-cancer
drugs. In the present paper, the systemic SAR study of compound 10 was carried out
in two aspects (region A and region B) based on its structure (Fig. 3). The novel
compound 17o was identified as the most potent compound, which shows much better
efficiency than the lead compound 10.
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Fig. 2. Anti-cancer dithiocarbamate derivatives developed by our group.
Fig. 3. Optimization strategy of lead compound 10.
2. Results and discussion
2.1. Optimization of region A in lead compound 10
For the structural optimization of region A, a variety of N-containing
heterocycles were selected to replace the pyridine of compound 10 to examine the
effect of different heterocycles on the bioactivity (11a-11j, Scheme 1). Target
compounds 11a-11j were synthesized from the reaction of heterocyclic methylamines
12 with carbon disulfide and 3-bromopropionitrile 13 using our developed one-pot
procedure [39-40]. The heterocyclic methylamines 12 were commercially available or
prepared according to literature procedures [41-44].
Compd.
11a
R
Compd.
11e
R
Compd.
11i
R
11b
11c
11d
11f
11g
11h
11j
Scheme 1. Synthesis of compounds 11a-11j. Reagents and conditions: (a) TEA, H2O, rt, 2h.
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The synthesized compounds 11a-11j were evaluated for their anti-cancer
activities against human tumor cell lines: HL-60 (leukemia) and Bel-7402 (hepatoma)
ꢀ
4219; Arch.
referring to the results of our previous research.(Bioorg. Med. Chem. Lett. 2006, 16, 4214
Pharm. Chem. Life Sci. 2011, 11, 320–332) As shown in Table 1, pyrazole (11a, IC50 = 6.05 ꢀM; 11c,
IC50 = 4.17 ꢀM), oxazole (11d, IC50 = 7.88 ꢀM) and pyrimidine (11h, IC50 = 5.03 ꢀM)
derivatives exhibited good activities against HL-60 cell line with IC50 value less than
10 ꢀM. Compound 11c also showed potent anti-cancer activity against Bel-7402 cell
line with IC50 value of 4.26 ꢀM. However, none of the tested compounds was superior
to lead compound 10, which inhibited the proliferation of HL-60 and Bel-7402 cell
lines with IC50 values of 0.74 ꢀM and 1.03 ꢀM, respectively. These results
demonstrate that pyridine is the most favored group in region A for the anti-tumor
activities among the selected heterocycles.
Table 1.
Anti-cancer activities of compounds 11a-11j against HL-60 and Bel-7402 cancer cell lines (IC50, ꢀM).
Compd.
10
R
HL-60
Bel-7402
0.74±0.10
1.03±0.50
6.05±0.59
24.94±3.58
11a
108.60±19.18
4.17±0.08
295.60±22.68
4.26±1.87
11b
11c
7.88±0.47
14.20±3.92
11.77±27.53
282.50±48.45
17.95±4.33
25.66±3.44
18.65±3.98
226.70±32.55
11d
11e
11f
11g
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5.03±0.45
22.39±4.91
11h
5.18±1.23
96.39±9.92
11i
11j
82.80±5.49
327.30±157.96
2.2. Optimization of region B in lead compound 10
Based on the optimization results of region A, the cyano group in region B was
modified with the pyridine substitution at A. According to bioisosterism principles,
the cyano group was replaced with ketone, hydroxyl, ester, amide, sulfonyl hydroxide,
phosphate, borate and several five-member heterocycles, respectively (17a-17p,
Scheme 2). All target compounds were prepared using the similar synthetic procedure
of 11 from the reaction of pyridin-3-ylmethanamine 18 with carbon disulfide, and
corresponding halides 19 or Michael acceptors 20. 19 and 20 were commercially
available or prepared in according to literature procedures. Such as, the synthetic
routes for the corresponding halides 19m, 19o and 19p were depicted in Scheme 3.
19m was synthesized by the cyclization of 2-aminophenol and 3-bromopropanimidate
hydrochloride 22 which was obtained by 3-bromopropionitrile 21 treated with HCl
gas. Benzoylhydrazine 23 reacted with 3-bromopropionic acid under the catalysis of
POCl3 affording the compound 19o. Benzonitrile 24 was treated with hydroxylamine
hydrochloride to afford intermediate 25, and subsequent coupling with
3-chloropropionyl chloride and intra-molecular condensation gave 19p.
Compd.
17a
R
Compd.
17g
R
Compd.
17m
R
O
17b
17c
17d
17h
17i
17j
17n
17o
17p
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17e
17f
17k
17l
Scheme 2. Synthesis of compounds 17a-17p. Reagents and conditions: (a) TEA, H2O, rt, 2h.
Scheme 3. Synthesis of compounds 19m, 19o and 19p. Reagents and conditions: (a) HCl gas, MeOH; (b)
2-aminophenol, ethanol, reflux; (c) 3-bromopropionic acid, POCl3, toluene, reflux; (d) NH2OH HCl, TEA, EtOH,
80 ºC; (e) 3-chloropropionyl chloride, CH2Cl2, r.t.; (f) K2CO3, 1,4-dioxane, reflux, 6 h.
Our latest research results show that lead compound 10 has good activities
ꢀ
96
against not only liver cancer cell, but also breast cancer cell. Cancer Letters 2013, 340, 88
Therefore, anti-cancer activities of target compounds 17a-17p were evaluated in three
human cancer cell lines, i.e. hepatoma cells Bel-7402, breast cancer cells SK-BR-3
and MDA-MB-468. Compound 10 was used as a positive control. The results in
Table 2 showed that most compounds exhibited potent anti-cancer activities against
selected cancer cell lines with IC50 less than 10 ꢀM. Among them, derivatives
(17a-17i) without the heterocyclic substitution at B showed quite or weaker
anticancer activity against Bel-7402 cells than positive control compound 10 (IC50 =
3.29 ꢀM). However, the replacements of cyano group by ketone (17a, IC50 = 1.12 ꢀM),
hydroxyl (17b, IC50 = 1.32 ꢀM), or amide (17e, IC50 = 1.16 ꢀM) enhanced the
inhibitory activities against SK-BR-3 cells by 2 fold. Compound 17a also exhibited
more potent efficiency against MDA-MB-468 cells (IC50 = 4.22 ꢀM) than compound
10 (IC50 = 9.53 ꢀM). On the contrary, the other non-heterocyclic replacements (17c,
17d, 17g, 17i) led to a slight reduction in bioactivity. N-benzyl amide (17f) and
phosphate (17h) derivatives lost the inhibitory activities completely (IC50 > 50 ꢀM).
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Different heterocyclic groups in region B showed great impact on the anti-cancer
activities except tetrazole (17j), which barely showed anti-cancer effect against all the
selected cell lines. Compounds 17l-17p were more potent against SK-BR-3 than lead
compound 10 (IC50 = 2.84 ꢀM) with IC50 in the range of 0.58-1.69 ꢀM, indicating
N-containing five-member heterocyclic groups favored the inhibitory activities
against SK-BR-3 cells. Replacing of cyano group with benzoxazole (17m, IC50 = 4.30
ꢀM), and 1,3,4-oxadiazole (17o, IC50 = 4.29 ꢀM) also improved the bioactivity
against MDA-MB-468 cells. On the other hand, only 1,3,4-oxadiazole derivative 17o
(IC50 = 1.23 ꢀM) had significantly improved inhibitory activity against Bel-7402 cells.
Remarkably, this compound also showed the highest inhibitory activity against
SK-BR-3 and MDA-MB-468 cells with IC50 value of 0.58 ꢀM and 4.29 ꢀM,
respectively. Because oxadiazoles widely exist in drug molecules [25-28], the
compound 17o was chosen for further optimization.
Table 2.
Anti-cancer activities of compounds 17a-17p against Bel-7402, SK-BR-3, and MDA-MB-468 cancer cell lines
(IC50, ꢀM).
S
R
N
S
H
N
Compd.
10
R
Bel-7402
SK-BR-3
MDA-MB-468
CN
3.29±0.72
2.84±0.28
9.53±0.11
3.43±0.40
3.65±1.37
7.42±1.50
6.35±1.55
3.33±2.29
>100
1.12±0.09
1.32±0.18
7.97±0.39
4.46±0.54
1.16±0.11
>100
4.22±0.38
6.61±0.34
9.32±0.63
12.79±1.61
4.63±1.19
>100
17a
17b
17c
17d
17e
17f
O
6.59±0.72
6.77±0.28
11.06±0.82
17g
17h
50.83±39.25
51.01±10.45
60.37±198.90
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4.27±0.81
6.82±1.90
6.62±0.76
17i
17j
O
B
O
57.67±9.35
>100
>100
3.56±0.39
9.30±2.27
1.39±0.10
1.69±0.10
9.71±1.14
17k
17l
11.53±0.71
2.88±5.85
2.66±0.99
0.82±0.06
0.98±0.29
4.30±0.97
17m
17n
11.61±0.57
1.23±0.49
2.80±0.47
0.58±0.05
1.29±0.28
4.29±1.13
17o
17p
10.06±2.08
2.3. Optimization of compound 17o
Subsquently, we turned our attention to the modification of 2-phenyl ring of
1,3,4-oxadiazole of compound 17o. Different substituents were introduced to intact
phenyl group (27a-27e), or the phenyl group was replaced by different heterocyclic
groups (27f-27o). Target compounds 27a-27o were prepared using the similar
synthetic procedure of 17o, with replacement of the starting material by
corresponding hydrazides 28 with substituted aromatic and heterocyclic groups
(Scheme 4). The anti-cancer activities of these compounds were also evaluated
towards Bel-7402, SK-BR-3 and MDA-MB-468 cancer cell lines, comparing with
compound 17o (Table 3).
Compd.
27a
R
Compd.
27f
R
Compd.
27k
R
27b
27g
27l
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27c
27d
27e
27h
27m
27n
27o
27i
27j
Scheme 4. Synthesis of compounds 27a-27o. Reagents and conditions: (a) 3-Bromopropionic acid, POCl3, toluene,
reflux; (b) TEA, H2O, rt, 2h.
As shown in Table 3, all the 4-substituted phenyl derivatives, either
electron-donating group (MeO in 27b, IC50 = 1.54 ꢀM) or electron-withdrawing group
(CF3 in 27c, IC50 = 1.22 ꢀM; Cl in 27d, IC50 = 1.26 ꢀM), exhibit higher activity
against Bel-7402 than compound 17o (IC50 = 1.58 ꢀM) except compound 27e (R =
4-NO2Ph, IC50 = 1.84 ꢀM). However, 2-fluoro-phenyl substituted derivatives (27a)
led to significant decrease of the activities not only for Bel-7402, but also for
SK-BR-3 and MDA-MB-468. Comparing with 17o, only 4-chloro-phenyl derivative
27d and pyrrole derivative 27f showed a little better activity against Bel-7402 and
MDA-MB-468, respectively. Results in Table 3 also showed that most of heterocyclic
derivatives exhibited comparable anti-cancer activities with 17o. Only 2-isoxazole
derivatives 27j showed dramatically decrease of the activity against MDA-MB-468,
and the activities of 2-benzothiophene derivatives 27o significantly decreased against
all three tested models. These results suggested that there is still more optimization
space for R.
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Table 3.
Anti-cancer activities of compounds 27a-27o against Bel-7402, SK-BR-3, MDA-MB-468 cell lines (IC50, ꢀM).
Compd.
17o
R
Bel-7402
SK-BR-3
MDA-MB-468
1.58±0.20
0.79±0.07
2.77±0.13
5.54±1.44
4.69±1.53
3.42±1.91
27a
1.54±0.10
1.22±0.17
1.26±0.18
1.84±0.63
1.17±0.67
1.59±1.33
1.23±0.21
1.34±0.57
1.68±0.71
1.37±0.14
1.43±0.16
1.79±1.09
1.95±1.43
6.89±0.89
2.96±1.19
2.40±0.22
0.86±0.03
1.54±0.03
0.82±0.06
0.81±0.08
1.03±0.34
1.07±0.13
1.73±0.29
0.80±0.10
1.29±0.25
0.85±0.21
0.79±0.17
5.32±0.65
5.22±0.60
5.49±0.31
1.98±0.18
4.60±0.50
2.04±0.06
3.32±0.56
3.26±1.10
3.41±0.48
6.14±1.15
3.16±0.50
1.71±2.27
1.69±0.15
3.50±0.54
6.77±0.71
27b
27c
27d
27e
27f
27g
27h
27i
27j
27k
27l
N
27m
27n
27o
2.4. Anti-cancer activity of 17o on a panel of cancer cell lines and two nonmalignant
cell lines
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Compounds 17o that demonstrated the most potent cytotoxic activities against
three cancer cell lines was subjected to further evaluation in a panel of 12 tumor cell
lines with different tissue origins, including lung cancer (H1299, H460, H522),
hepatoma (Bel-7402), breast (MDA-MB-468, SK-BR-3), colorectal (COLO205,
HCT-8, HCT-116, SW620), and prostate (DU145, PC3) cell lines. Human hepatic cell
line LO2 and human embryonic kidney 293 cells (HEK293T) were used as
nonmalignant cell control. As shown in Table.4, 17o inhibited the viability of the
tested cancer cells with an IC50 value ranging from 0.39 ꢀM to 7.91 ꢀM. Meanwhile,
17o showed relatively less cytotoxicity against nonmalignant cells LO2 and
HEK293T with IC50 value of 9.66 ꢀM and 14.63 ꢀM respectively, indicating its
selectivity to cancer cells. Among these cancer cells, PC-3 cells from human prostate
cancer (IC50 value =0.39 ꢀM) and Bel-7402 cells from hepatoma cancer (IC50 value
=1.23 ꢀM) revealed relatively higher sensitivity to 17o, and therefore were used for
subsequent experiment.
Table 4.
Anti-cancer activity of compound 17o on a panel of malignant cell lines and two nonmalignant cell lines.
Cell line a
H1299
H460
IC50 b (ꢀM)
1.89±0.30
6.07±0.76
2.16±0.34
1.23±0.49
3.34±0.28
Cell line a
MDA-MB-468
SK-BR-3
COLO205
HCT-8
IC50 b (ꢀM)
4.30±1.13
0.58±0.05
7.92±3.19
5.98±0.74
0.39±0.07
Cell line a
HCT-116
SW620
IC50 b(ꢀM)
8.21±0.10
3.80±0.33
9.67±0.87
14.63±0.98
H522
LO2
Bel-7402
DU145
HEK293T
PC-3
a H1299, H460, H522: lung cancer; Bel-7402: hepatoma; MDA-MB-468, SK-BR-3: breast carcinoma; COLO205,
HCT-8, HCT-116, SW620: colorectal carcinoma; DU145, PC-3: prostate carcinoma; LO2: human hepatic cell line;
HEK293T: human embryonic kidney cell line.
bDose-response curves were determined at least at five concentrations. The IC50 values are the concentrations
needed to inhibit cell growth by 50% as determined from these curves.
2.5. Effects of 17o in cell apoptosis
In order to identify whether 17o-induced cell death through apoptosis, Hoechst
33342 staining was used to evaluate nuclei condensation and Annexin V-FITC/PI
staining assay was used to evaluate phosphatidylserine (PS) translocation. Hoechst
staining results in Fig.4A showed that treatment with 17o induced chromatin
condensation, which is commonly accepted as a marker of apoptosis. Another
characteristic of apoptosis is PS translocation to the outer surface of plasma
membrane. In Annexin V-FITC/PI dual staining assay, apoptotic cells which presented
transmembrane PS were labeled by Annexin V-FITC. Meanwhile, PI, which is
impermeant to live cells and apoptotic cells, was used to stain dead cells. Therefore,
Annexin V+ and PI- staining indicates early apoptotic cells, whereas Annexin V+ and
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PI+ staining indicate late apoptotic cells. As shown in Fig.4B, treatments of 17o for
48 h dose-dependently increased both early and late apoptotic cells in PC-3 and
Bel-7402 cell lines.
Fig.4. 17o induced apoptosis in PC-3 and Bel-7402 cancer cells. PC-3 and Bel-7402 cells were exposed to
different concentrations of 17o for 48 h respectively, and then cells were analyzed by (A) Hoechst 33342 staining
or (B) Annexin V/PI staining assays.
2.6. 17o induced mitotic arrest
Flow cytometry analysis was used to determine the effect of 17o on cell cycle
distribution. As shown in Fig.5A, a typical G2/M phase arrest was observed in
response to the treatment of 17o in PC-3 and Bel-7402 cells. In order to further
determine whether 17o induced a G2 or M phase cell cycle arrest, two specific mitotic
molecular markers, MPM-2 and phosphorylated histone H3 (Pi-H3), were examined
by Western blot assay. The results in Fig.5B showed that 17o induced potent increase
in these two markers whose tendency was similar to the well-known mitotic inhibitor
taxol, indicating 17o induced a specific mitotic arrest. Moreover, the
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immunofluorescence results (Fig.6) showed that 17o induced abnormal mitosis of
cancer cells which was similar to the behavior of the mitotic inhibitor Taxol. After
treatment with 17o or Taxol for 6 h, cells were fixed and costained for DNA, β-tubulin
and γ-tubulin. As shown in Fig.6, normal mitotic cells showed intact and stretched
microtubule and two centrioles per centrosome, which were respectively evidenced by
β-tubulin and γ-tubulin staining. In contrast, 17o-treated cells exhibited
supernumerary centrosomes and multipolar spindles formation, a similar behavior of
Taxol. These data suggested that 17o -induced mitotic arrest was through disruption of
spindle assembly.
Fig.5. 17o induced a specific mitotic arrest. (A) PC-3 and Bel-7402 cancer cells were treated with 17o (0.5 - 4 ꢀM)
for 24 h, then subjected to cell cycle analysis. Representative flow cytometric histograms and statistic analysis
were shown. (B) PC-3 cells were treated with 17o (0.5 - 4 ꢀM) for 24 h, then protein were subjected to Western
blot assay. Taxol (TAX, 40 nM) were used as positive control.
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Fig.6. 17o disrupted of spindle assembly in cell mitosis. PC-3 cells were exposed to 17o (2 ꢀM) or taxol (TAX, 40
nM) for 6 h, and then subjected to Immunofluorescence assay to observe effects of agents on the distribution of
β-tubulin and γ-tubulin. The scale bar is 15 ꢀm.
3. Conclusion
In summary, a series of novel dithiocarbamates were designed and synthesized to
explore the structural activity relationship of lead compound 10. Several compounds
inhibited the growth of cancer cells with lower IC50 values than those of lead
compound 10. Among them, 17o showed potent anti-proliferation activity toward a
panel of cancer cells with relatively less cytotoxicity to nonmalignant cells. It induced
apoptosis and mitotic arrest through disrupting spindle assembly in mitotic
progression, indicating these synthesized dithiocarbamates represented a novel series
of anti-cancer compounds targeting mitosis. Analysis of structure activity relationship
(SAR) indicated that: (a) Among the selected heterocycles in region A, pyridine is
most favored for the anti-cancer activities; (b) Both non-heterocyclic and heterocyclic
bioisosteres showed great impact on the anti-cancer activities of compounds, in which
5-phenyl-1,3,4-oxadiazole is the most favored substituent; (c) Introduction of
methoxyl, trifluoromethyl, chloro into para position of the benzene ring in the
structure of 5-phenyl-1,3,4-oxadiazole is favored to the cytotoxic activity against
Bel-7402 cells; Also, chloro group at para position enhance the inhibitory activities
against MDA-MB-468 cells; (d) Replacement of the benzene ring with some
heterocycles, such as pyrroleand pyrazine, improved activities against both Bel-7402
and MDA-MB-468 cells. These results provide valuable information for the further
investigation of this type of potent anti-tumor compounds.
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4. Experiment
4.1. General
All reagents and solvents were purchased from commercial sources and were
used without further purification. Melting points were determined on X4 microscope
1
and are uncorrected. H NMR and 13C NMR spectra were recorded on Bruker
AVANCEꢁ 400 MHz and 100 MHz spectrometer respectively. Elemental analyses
were performed on a Vario EL ꢁ (Germany) instrument.
4.2. General procedure for the synthesis of compounds 11a-11d, 11h-11j.
To a mixture of corresponding heterocyclic methylamine 12 (1 mmol), TEA (100
mg, 1 mmol) in water (10 mL), CS2 (91 mg, 1.2 mmol) was added dropwise. After
stirring for 5 min, 3-bromopropionitrile 13 (134 mg, 1 mmol) was added. The reaction
mixture was stirred at room temperature for 2 h, then extracted with ethyl acetate (30
mL×3). The organic phase was combined and dried over Na2SO4, concentrated under
vacuum and purified by column chromatography on silica gel or recrystallization
(ethyl acetate/ petroleum ether) to afford compound 11a-10d, 11h-11j.
4.2.1. 2-Cyanoethyl((5-methyl-1-phenyl-1H-pyrazol-4-yl)methyl)carbamodithioate
(11a)
Yield 79%. White solid. MP: 135-138 ºC. 1H NMR (400 MHz, CDCl3): δ = 2.31
(s, 3H), 2.85-2.88 (t, J = 6.9 Hz, 2H), 3.51-3.54 (t, J = 6.9 Hz, 2H), 4.75 (d, J = 4.8
13
Hz, 2H), 7.40-7.61 (m, 6H). C NMR (100 MHz, CDCl3): δ = 10.77, 18.52, 30.57,
41.92, 114.39, 118.18, 125.01, 128.12, 129.22, 137.82, 139.54, 140.15, 194.68. Anal.
Cald for C15H16N4S2: C, 56.93; H, 5.10; N, 17.71; Found: C, 57.05; H, 5.30; N, 17.54.
4.2.2. 2-Cyanoethyl (thiazol-5-ylmethyl) carbamodithioate (11b)
1
Yield 78%. White solid. MP: 76-78 ºC. H NMR (400 MHz, CDCl3): δ =
2.84-2.86 (t, J = 6.9 Hz, 2H), 3.51-3.54 (t, J = 6.9 Hz, 2H), 5.23 (d, J = 5.2 Hz, 2H),
13
7.37 (d, J = 3.2 Hz, 1H), 7.79 (d, J = 3.6 Hz, 1H), 9.30 (br s, 1H). C NMR (100
MHz, CDCl3): δ = 18.44, 30.63, 47.14, 118.21, 120.45, 142.08, 164.45, 196.26. Anal.
Cald for C8H9N3S3: C, 41.83; H, 4.68; N, 16.26; Found: C, 42.03; H, 4.24; N, 16.23.
4.2.3. 2-Cyanoethyl ((1,3-dimethyl-1H-pyrazol-5-yl)methyl) carbamodithioate (11c)
1
Yield 82%. White solid. MP: 112-114 ºC. H NMR (400 MHz, CDCl3): δ = 2.09
(s, 3H), 2.84-2.92 (t, J = 6.9 Hz, 2H), 3.45-3.48 (t, J = 6.9 Hz, 2H), 3.69 (s, 3H), 4.81
(d, J = 5.2 Hz, 2H), 5.99 (s, 1H), 10.54 (br s, 1H). 13C NMR (100 MHz, CDCl3): δ =
13.63, 18.23, 30.14, 36.46, 41.52, 106.12, 119.64, 138.36, 146.04, 195.63. Anal. Cald
for C10H14N4S2: C, 47.22; H, 5.55; N, 22.03; Found: C, 47.39; H, 5.52; N, 21.69.
4.2.4. 2-Cyanoethyl ((4-methyl-2-phenyloxazol-5-yl)methyl) carbamodithioate (11d)
Yield 85%. White solid. MP: 221-224 ºC. 1H NMR (400 MHz, CDCl3): δ = 2.28
(s, 3H), 2.87-2.90 (t, J = 6.9 Hz, 2H), 3.54-3.57 (t, J = 6.9 Hz, 2H), 4.95 (d, J = 4.8
Hz, 2H), 7.46-7.48 (m, 4H), 7.98-8.00 (m, 2H). 13C NMR (100 MHz, CDCl3): δ =
11.62, 18.42, 30.72, 40.97, 118.08, 126.32, 128.84, 130.62, 136.28, 140.73, 161.00,
15

ACCEPTED MANUSCRIPT
173.36, 195.77. Anal. Cald for C8H9N3S3: C, 56.76; H, 4.76; N, 13.24; Found: C,
57.01; H, 4.81; N, 12.94.
4.2.5. 2-Cyanoethyl ((2-morpholinopyrimidin-5-yl)methyl)carbamodithioate (11h)
1
Yield 83%. White solid. MP: 151-154 ºC. H NMR (400 MHz, CDCl3): δ =
2.87-2.91 (t, J = 6.9 Hz, 2H), 3.45-3.64 (t, J = 6.9 Hz, 2H), 3.64-3.68 (m, 8H),
4.62-4.63 (d, J = 5.2 Hz, 2H), 8.37 (s, 1H), 10.50 (bs, 1H). 13C NMR (100 MHz,
CDCl3): δ = 18.23, 30.07, 44.43, 44.55, 66.42, 119.20, 119.66, 158.55, 161.23, 195.24.
Anal. Cald for C13H17N5OS2: C, 48.27; H, 5.30; N, 21.65; Found: C, 48.27; H, 5.33;
N, 21.38.
4.2.6. 2-Cyanoethyl (quinolin-3-ylmethyl)carbamodithioate (11i)
Yield 72%. White solid. MP: 120-122 ºC. 1HNMR(CDCl3,400 MHz): δ 9.14 (m,
1H), 8.78 (s, 1H), 8.05 (m, 2H), 7.70 (m, 2H), 7.52 (t, 1H, J = 5.7 Hz), 5.07(d, 2H, J
13
=5.6 Hz), 3.53 (t, 2H, J= 6.8 Hz), 2.89 (t, 2H, J = 6.8 Hz). C NMR(CDCl3, 100
MHz): δ = 196.24, 150.69, 147.27, 135.66, 129.87, 129.09, 128.75, 127.85, 127.23,
127.16, 118. 36, 48.50, 30.51, 18.50. HRMS (ESI+) m/z calcd for C14H14N3S2 (M +
H)+, 288.0623, found 288.0620.
4.2.7. 2-cyanoethyl ((4-methyl-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazin-7-yl)methyl)
carbamodithioate (11j)
1
Yield 79%. White solid. MP: 117-118 ºC. HNMR (CDCl3, 400 MHz): δ = 8.63
(s, 1H), 7.50 (sꢂ1H), 6.73 (d, 2H, J = 2.0 Hz), 4.57 (d, 2H, J = 4.0 Hz), 4.15 (t, 2H, J
= 3.6 Hz), 3.45 (t, 2H, J = 6.8 Hz), 3.37 (t, 2H, J = 3.6 Hz), 3.02 (s, 3H), 2.81 (t, 2H, J
13
= 6.8 Hz). CNMR (CDCl3,100 MHz): δ = 194.91, 147.72, 139.47, 121.18, 120.33,
118.36, 63.95, 48.83, 47.84, 36.02, 30.38, 18.51. Anal. Calcd for C13H16N4OS2: C,
50.63; H, 5.23; N, 18.17. Found: C, 50.90; H, 5.23; N,18.19.
4.3. Synthesis of 2-cyanoethyl ((5-phenyl-1,3,4-oxadiazol-2-yl)methyl) carbamodi-
thioate (11e)
A mixture of benzoylhydrazine (136 mg, 10 mmol) and 2-choloro-acetic acid (96
mg, 12 mmol) in glacial acetic acid (30 mL) was refluxed for 6 h. After cooling to
room temperature, the solvent was removed under vacuum and the residue was
purified by flash column to afford 2-(chloromethyl)-5-phenyl-1,3,4-oxadiazole as a
white solid.
Under N2 atmosphere, a solution of 2-(chloromethyl)-5-phenyl-1,3,4-oxadiazole
(92 mg, 0.5 mmol) in DMF (20 mL) was added NaN3 (84 mg, 1.5mmol) portion-wise.
After stirring at room temperature for 6 h, the reaction was quenched by adding water,
and then extracted with ethyl acetate (15 mL×3). The organic phase was combined
and
dried
over
Na2SO4,
concentrated
under
vacuum
to
afford
2-(azidomethyl)-5-phenyl-1,3,4-oxadiazole as a yellow oil. This crude product was
used in next step without purification.
A mixture of 2-(azidomethyl)-5-phenyl-1,3,4-oxadiazole (43 mg, 0.25 mmol) and
Pd/C (5 mg) in methanol (15 mL) was stirred under H2 atmosphere (4 atm) at room
16

ACCEPTED MANUSCRIPT
temperature overnight. The resulting mixture was filtrated through Celite, the filtrate
was concentrated to provide crude (5-phenyl-1,3,4-oxadiazol-2-yl)methanamine as a
yellow oil.
Following the same procedure for the synthesis of 11a, target compound 11e was
obtained using (5-phenyl-1,3,4-oxadiazol-2-yl)methanamine as amine. Yield 54%.
1
White solid. MP: 148-152 ºC. H NMR (400 MHz, CDCl3): δ = 2.87-2.90 (t, J = 6.9
Hz, 2H), 3.55-3.59 (t, J = 6.9 Hz, 2H), 5.40 (d, J = 5.6 Hz, 2H), 7.49-7.52 (m, 3H),
7.96-7.98 (m, 2H), 8.59 (s, 1H). 13C NMR (100 MHz, CDCl3): δ = 18.04, 30.35, 41.24,
119.57, 123.58, 126.95, 129.98, 132.60, 163.00, 164.67, 197.62. HRMS (ESI+) m/z
calcd for C13H12N4OS2 (M + H)+, 305.0452, found 305.0538.
4.4. Synthesis of 2-cyanoethyl ((5-phenyl-1,3,4-thiadiazol-2-yl)methyl) carbamodi-
thioate (11f)
To a solution of benzoylhydrazine (136 mg, 10 mmol) in ethyl acetate (20 mL),
2-chloroacetyl chloride (134 mg, 12 mmol) was added dropwise. After refluxing for 3
h, the mixture was cooled to room temperature and concentrated to afford
N'-(2-chloroacetyl)benzohydrazide as a white solid. This crude product was used
without purification.
Under N2 atmosphere, a solution of N'-(2-chloroacetyl)benzohydrazide (106 mg,
5 mmol) and Lawesson’s reagent (241 mg, 6 mmol) in toluene (100 mL) was stirred at
115 ºC for 4 h. After cooling to room temperature, the mixture was concentrated and
purified by a flash column to afford crude 2-(chloromethyl)-5-phenyl-1,3,4-
thiadiazole as a pale green solid.
Following the same procedure for the synthesis of 11e, target compound 11f was
obtained. Yield 26%. White solid. MP: 131-134 ºC. 1H NMR (400 MHz, CDCl3): δ =
2.89-2.92 (t, J = 6.9 Hz, 2H), 3.46-3.49 (t, J = 6.9 Hz, 2H), 5.12 (d, J = 5.6 Hz, 2H),
7.58-7.66 (m, 3H), 7.96-7.98 (m, 2H). 13C NMR (100 MHz, CDCl3): δ = 18.04, 30.35,
41.24, 119.57, 123.58, 126.95, 129.98, 132.60, 164.66, 172.49, 197.61. Anal. Cald for
C13H12N4S3: C, 48.72,; H, 3.77; N, 17.48; Found: C, 49.04; H, 4.11; N, 17.14.
4.5. Synthesis of 2-cyanoethyl((1-methyl-1H-pyrrol-2-yl)methyl)carbamodithioate
(11g)
A mixture of 1H-pyrrole-2-carbaldehyde 14 (190 mg, 2 mmol) and K3PO4 (420
mg, 2 mmol) in DMF (20 mL) was stirred at room temperatrure for 15 min, and then
CH3I (570mg, 4 mmol) was added dropwise. The reaction mixture was stirred at room
temperature for another 8 h, then, water was added, extracted with ethyl acetate (15
mL×3). The organic phase was combined and dried over Na2SO4, concentrated under
vacuum to afford crude 1-methyl-1H-pyrrole-2-carbaldehyde 15 as a colorless oil.
A mixture of crude 1-methyl-1H-pyrrole-2-carbaldehyde 15 (200 mg, 1.8 mmol),
hydroxylamine hydrochloride (283 mg, 4 mmol), sodium acetate (500 mg, 6 mmol) in
ethanol (20 mL) was refluxed for 2 h. After cooling to room temperature, the mixture
was
concentrated
and
purified
by
a
flash
column
to
afford
1-methyl-1H-pyrrole-2-carbaldehyde oxime 16 as white solid. This crude product was
used without purification.
17

ACCEPTED MANUSCRIPT
A mixture of 1-methyl-1H-pyrrole-2-carbaldehyde oxime (120 mg, 0.9 mmol)
and fresh made Raney-Ni (400 mg) in ethanol (15 mL) was stirred under H2
atmosphere (4 atm) at room temperature overnight. The resulting mixture was filtered,
the filtrate was concentrated to provide crude (1-methyl-1H-pyrrol-2-yl)methanamine
12g as a yellow oil.
Following the same procedure for the synthesis of 11a, target compound 11g was
1
obtained using 12g as amine. Yield 47%. White solid. MP: 91-93 ºC. H NMR (400
MHz, CDCl3): δ = 2.85-2.97 (t, J = 6.9 Hz, 2H), 3.51-3.54 (t, J = 6.9 Hz, 2H), 3.60 (s,
3H), 4.87-4.89 (d, J = 4.4 Hz, 2H), 6.09-6.11 (m, 1H), 6.19 (s, 1H), 6.68 (s, 1H). 13C
NMR (100 MHz, CDCl3): δ = 18.50, 30.50, 33.98, 43.24, 110.25, 118.18, 123.84,
126.20, 128.86, 130.96, 194.41. HRMS (ESI+) m/z calcd for C10H13N3S2 (M + H)+,
240.0550, found 240.0481.
4.6. General procedure for the synthesis of compounds 17a-17i
To a mixture of pyridin-3-ylmethanamine 18 (108 mg, 1mmol), TEA (100 mg, 1
mmol) in water (10 mL), CS2 (91 mg, 1.2 mmol) was added dropwise. After stirring
for 5 min, the corresponding halide 19 (1mmol) or Michael acceptor 20 (1mmol) was
added. The reaction mixture was stirred at room temperature for 2 h, then extracted
with ethyl acetate (30 mL×3). The organic phase was combined and dried over
Na2SO4, concentrated under vacuum and purified by column chromatography on
silica gel or recrystallization (ethyl acetate/ petroleum ether) to afford the
corresponding compound 17.
4.6.1. 3-Oxobutyl (pyridin-3-ylmethyl)carbamodithioate (17a)
4-Bromobutan-2-one was used as halide. Yield 26%. White solid. MP: 135-138
ºC. H NMR (400 MHz, DMSO-d6): δ = 2.10 (s, 3H), 2.84-2.88 (t, J = 6.9 Hz, 2H),
1
3.30-3.37 (t, J = 6.9 Hz, 2H), 4.83-4.85 (d, J = 5.2 Hz, 2H), 7.26-7.29 (m, 1H),
7.70-7.72 (m, 1H), 8.49-8.51 (m, 2H), 10.45 (bs, 1H); 13C NMR (100 MHz,
DMSO-d6): δ = 28.73, 30.10, 42.98, 47.55, 123.97, 133.41, 135.96, 148.89, 149.54,
197.76, 207.16; Anal. Cald for C11H14N2OS2: C, 51.94; H, 5.55; N, 11.01; Found: C,
51.88; H, 5.50; N, 10.88.
4.6.2. 2-Hydroxyethyl (pyridin-3-ylmethyl)carbamodithioate (17b)
3-Bromopropan-1-ol was used as halide. Yield 70%. White solid. MP: 104-105
ºC. H NMR (400 MHz, MeOD): δ = 3.40-3.43 (t, J = 6.8 Hz, 2H), 3.73-3.76 (t, J =
1
6.8 Hz, 2H), 4.93 (s, 2H), 7.36-7.39 (m, 1H), 7.79-7.81 (m, 1H), 8.41-8.51 (m, 2H);
13C NMR (100 MHz, MeOD): δ = 36.87, 47.07, 60.59, 123.81, 133.97, 136.55,
147.57, 148.35, 198.63; Anal. Cald for C9H12N2OS2: C, 47.34; H, 5.30; N, 12.27;
Found: C, 47.51; H, 5.33; N, 12.30.
4.6.3. 3-{[(Pyridin-3-ylmethyl)carbamothioyl]thio}propanoic acid (17c)
3-Bromopropanoic acid was used as halide. Yield 70%. White solid. MP:
104-107 ºC. 1H NMR (400 MHz, MeOD): δ = 2.56-2.60 (t, J = 6.8 Hz, 2H), 3.32-3.35
(t, J = 6.8 Hz, 2H), 4.78 (s, 2H), 7.27-7.30 (m, 1H), 7.70-7.72 (m, 1H), 8.30-8.38 (m,
18

ACCEPTED MANUSCRIPT
13
2H); C NMR (100 MHz, MeOD): δ = 29.52, 33.80, 46.80, 123.94, 134.27, 136.96,
147.12, 147.91, 174.11, 198.52; HRMS (ESI+) m/z calcd for C10H12N2O2S2 (M + H)+,
257.0340, found 257.0413.
4.6.4. Butyl 3-(((pyridin-3-ylmethyl)carbamothioyl)thio)propanoate (17d)
n-Butyl acrylate was used as Michael acceptor. Yield 70%. White solid. MP:
1
54-56 ºC. H NMR (400 MHz, CDCl3): δ = 0.86-0.90 (t, J = 7.2 Hz, 3H), 1.30-1.35
(m, 2H), 1.54-1.58 (m, 2H), 2.72-2.76 (t, J = 6.8 Hz, 2H), 3.46-3.50 (t, J = 6.8 Hz,
2H), 4.02-4.05 (t, J = 6.8 Hz, 2H), 4.90 (s, 2H), 7.20-7.23 (m, 1H), 7.66-7.68 (m, 1H),
8.38-8.39 (m, 2H), 9.10 (bs, H); 13C NMR (100 MHz, CDCl3): δ = 13.68, 19.07,
30.14, 30.54, 34.37, 48.03, 64.73, 123.72, 132.65, 136.30, 148.68, 149.14, 172.11,
198.17; Anal. Cald for C14H20N2O2S2: C, 53.82; H, 6.45; N, 8.97; Found: C, 53.82; H,
6.44; N, 8.99.
4.6.5. 3-Amino-3-oxopropyl (pyridin-3-ylmethyl)carbamodithioate (17e)
Acrylamide was used as Michael acceptor. Yield 84%. White solid. MP: 138-139
ºC. 1H NMR (400 MHz, DMSO-d6): δ = 2.42-2.49 (t, J = 6.8 Hz, 2H), 3.31-3.35 (t, J
= 6.8 Hz, 2H), 4.82-4.83 (d, J = 4.8 Hz, 2H), 6.87 (bs, 1H), 7.33-7.36 (m, 2H),
7.66-7.68 (m, 1H), 8.45-8.49 (m, 2H), 10.44 (bs, 1H); 13C NMR (100 MHz,
DMSO-d6): δ = 30.45, 35.03, 47.50, 123.94, 133.42, 135.91, 148.86, 149.53, 172.69,
197.88; Anal. Cald for C10H13N3OS2: C, 47.03; H, 5.13; N, 16.46; Found: C, 47.17; H,
5.21; N, 16.36.
4.6.6. 3-(Benzylamino)-3-oxopropyl (pyridin-3-ylmethyl)carbamodithioate (17f)
N-benzylacrylamide was used as Michael acceptor. Yield 72%. White solid. MP:
1
120-122 ºC. H NMR (400 MHz, DMSO-d6): δ = 2.57-2.60 (t, J = 6.8 Hz, 2H),
3.42-3.45 (t, J = 6.8 Hz, 2H), 4.28-4.29 (d, J = 5.6 Hz, 2H), 4.86-4.87 (d, J = 5.2 Hz,
2H), 7.22-7.37 (m, 6H), 7.69-7.71 (m, 1H), 8.43-8.54 (m, 3H), 10.49 (bs, 1H). 13C
NMR (100 MHz, DMSO-d6): δ = 30.66, 35.31, 42.59, 47.55, 123.96, 127.21, 127.68,
128.74, 133.43, 135.95, 139.85, 148.88, 149.56, 170.65, 197.77; Anal. Cald for
C17H19N3OS2: C, 59.10; H, 5.54; N, 12.16; Found: C, 59.31; H, 5.60; N, 12.04.
4.6.7. 3-{[(Pyridin-3-ylmethyl)carbamothioyl]thio}propane-1-sulfonic acid (17g)
3-Bromopropane-1-sulfonic acid was used as halide. Yield 78%. White solid.
1
MP: >250 ºC. H NMR (400 MHz, D2O): δ = 1.85-1.93 (m, 2H), 2.77-2.81 (m, 2H),
3.13-3.16 (m, 2H), 4.72(s, 2H), 7.20-7.23 (m, 1H), 7.59-7.61 (m, 1H), 8.15-8.37 (m,
2H); 13C NMR (100 MHz, D2O): δ = 24.21, 33.01, 47.48, 49.58, 124.35, 133.06,
136.99, 147.65, 147.85, 199.25; HRMS (ESI+) m/z calcd for C10H14N2O3S3 (M + H)+,
307.0167, found 307.0235.
4.6.8. 2-(Diethoxyphosphoryl)ethyl (pyridin-3-ylmethyl)carbamodithioate (17h)
Diethyl (2-bromoethyl)phosphonate was used as halide. Yield 84%. White solid.
1
MP: 66-68 ºC. H NMR (400 MHz, CDCl3): δ = 1.21-1.25 (t, J = 6.8 Hz, 6H),
2.08-2.17 (m 2H), 3.31-3.37 (m 2H), 3.98-4.02 (q, J = 7.6 Hz, 4H), 4.85(s, 2H),
19

ACCEPTED MANUSCRIPT
7.12-7.17 (m, 1H), 7.62-7.64 (m, 1H), 8.32-8.43 (m, 2H), 9.90 (bs, 1H); 13C NMR
(100 MHz, CDCl3): δ = 16.30, 16.38, 25.61, 26.95, 28.21, 47.87, 61.92, 61.99, 123.51,
132.87, 136.11, 148.46, 149.25, 197.42; Anal. Cald for C13H21N2O3PS2: C, 44.81; H,
6.08; N, 8.04; Found: C, 44.63; H, 6.21; N, 7.85.
4.6.9. 3-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)propyl(pyridin-3-ylmethyl)
carbamodithioate (17i)
2-(2-Bromoethyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane was used as halide.
1
Yield 71%. White solid. MP: 93-95 ºC. H NMR (400 MHz, DMSO-d6): δ =
0.74-0.78 (t, J = 7.6 Hz, 2H), 1.61-1.65 (m, 2H), 3.14-3.18 (t, J = 7.6 Hz, 2H),
4.83-4.85 (d, J = 5.6 Hz, 2H), 7.35-7.38 (m, 1H), 7.67-7.69 (m, 1H), 8.47-8.51 (m,
2H), 10.40 (bs, 1H); 13C NMR (100 MHz, DMSO-d6): δ = 24.20, 25.12, 36.82, 47.45,
83.24, 123.95, 133.45, 135.91, 135.91, 148.84, 149.05, 197.94; Anal. Cald for
C16H25BN2O2S2: C, 54.54; H, 7.15; N, 7.95; Found: C, 54.29; H, 6.93; N, 7.98.
4.7. Synthesis of 2-(1H-tetrazol-5-yl)ethyl (pyridin-3-ylmethyl)carbamodithioate (17j)
A mixture of 3-bromoethyl cyanide (0.27 g, 2 mmol), sodium azide (0.16 g, 2.5
mmol), and ammonium choloride (0.13 g, 2.5 mmol) in dry DMF (20 mL) was stirred
at 100 ºC for 6 h. After cooling to room temperature, the mixture was poured into ice
water, then extracted with ethyl acetate (20 mL×3). The organic phase was combined
and dried over Na2SO4, concentrated under vacuum and purified by flash column
chromatography to afford 5-(2-bromoethyl)-1H-tetrazole without further purification.
Following the same procedure for the synthesis of 17a, target compound 17j was
obtained using 5-(2-bromoethyl)-1H-tetrazole as halide. Yield 49%. White solid. MP:
121-124 ºC. 1H NMR (400 MHz, MeOD): δ = 3.32-3.43 (t, J = 6.8 Hz, 2H), 3.69-3.72
(t, J = 6.8 Hz, 2H), 4.94(s, 2H), 7.41-7.44 (m, 1H), 7.82-7.84 (m, 1H), 8.45-8.53 (m,
13
2H); C NMR (100 MHz, MeOD): δ = 23.77, 31.81, 47.01, 123.88, 134.01, 136.77,
147.40, 148.19, 155.64, 197.56; HRMS (ESI+) m/z calcd for C10H12N6S2 (M + H)+,
281.0565, found 281.0640.
4.8. Synthesis of 2-(benzo[d]thiazol-2-yl)ethyl (pyridin-3-ylmethyl) carbamodithioate
(17k)
To a mixture of 2-mercaptoaniline (0.25 g, 2 mmol) and TEA (0.2 g, 2 mmol) in
ethyl acetate (20 mL), 3-bromoethyl cyanide (0.27 g, 2 mmol) was added dropwis.
After stirring at room temperature for 6h, the resulting mixture was concentrated to
afford crude 2-(2-bromoethyl)benzo[d]thiazole. This crude product was used without
purification.
Following the same procedure for the synthesis of 17a, target compound 17k was
obtained using 2-(2-bromoethyl)benzo[d]thiazole as halide. Yield 19%. White solid.
1
MP: 96-99 ºC. H NMR (400 MHz, DMSO-d6): δ = 3.60-3.72 (m, 4H) 4.85(s, 2H),
7.32-7.69 (m, 4H), 8.48-8.53 (m, 2H), 10.61 (bs, 1H); 13C NMR (100 MHz,
DMSO-d6): δ = 32.04, 34.12, 47.78, 110.70, 118.78, 123.99, 124.80, 125.10, 133.24,
141.72, 148.95, 149.56, 151.77, 164.16, 196.49; HRMS (ESI+) m/z calcd for
C16H15N3S3 (M + H)+, 346.0428, found 346.0522.
20

ACCEPTED MANUSCRIPT
4.9. Synthesis 2-(1H-benzo[d]imidazol-2-yl)ethyl (pyridin-3-ylmethyl) carbamodithio-
ate (17l)
A mixture of 1,2-diaminobenzene (0.22 g, 2 mmol) and 3-bromopropionic acid
(0.61g, 4 mmol) in concentrated HCl (15 mL) was refluxed for 6 h. After cooling to
room temperature, the mixture was adjusted to pH = 7 with sodium bicarbonate, then
extacted with ethyl acetate (20 mL×3). The organic layer was combined and
concentrated to afford crude 2-(2-bromoethyl)-1H-benzo[d]imidazole as a yellow oil.
This crude product was used without purification.
Following the same procedure for the synthesis of 17a, target compound 17l was
obtained using 2-(2-bromoethyl)-1H-benzo[d]imidazole as halide. Yield 33%. White
solid. MP: 149-151 ºC. 1H NMR (400 MHz, DMSO-d6): δ = 3.20-3.24 (t, J = 6.8 Hz,
2H), 3.67-3.71 (t, J = 6.8 Hz, 2H), 4.85 (d, J = 5.2 Hz, 2H), 7.13-7.15 (m, 2H),
7.35-7.38 (m, 1H), 7.48-7.50 (m, 2H), 7.68-7.70 (m, 1H), 8.47-8.52 (m, 2H), 10.54
13
(bs, 1H); C NMR (100 MHz, DMSO-d6): δ = 23.71, 29.00, 47.56, 122.02, 123.98,
148.88, 149.52, 153.44, 197.41; Anal. Cald for C16H16N4S2: C, 58.51; H, 4.91; N,
17.06; Found: C, 58.64; H, 4.58; N, 16.69.
4.10. Synthesis of 2-(benzo[d]oxazol-2-yl)ethyl (pyridin-3-ylmethyl) carbamodithioate
(17m)
To a solution of 3-bromoethyl cyanide 21 (2.66 g, 20 mmol) and methanol (1.28
g, 40 mmol) in ethyl ether (100 mL), dry HCl gas was introduced for 6 h at room
temperature. The resulting white solid was filtrate and dried to afford methyl
3-bromopropanimidate hydrochloride 22 (2.08g, 12.6 mmol), yiled 63%.
A mixture of methyl 3-bromopropanimidate hydrochloride 22 (0.16 g, 1 mmol)
and 2-aminophenol (0.15g, 1.3 mmol) in ethanol (20 mL) was heated at 80 ºC for 6 h.
The mixture was filtrated and the filtrate was concentrated to afford crude
2-(2-bromoethyl)benzo[d]oxazole 19m as yellow oil. This crude product was used
without purification.
Following the same procedure for the synthesis of 21a, target compound 17m
was obtained using 2-(2-bromoethyl)benzo[d]oxazole 19m as halide. Yield 57%.
1
White solid. MP: 116-117 ºC. H NMR (400 MHz, CDCl3): δ = 3.38-3.41 (t, J = 6.8
Hz, 2H), 3.80-3.84 (t, J = 6.8 Hz, 2H), 4.95 (s, 2H), 7.25-7.33 (m, 3H), 7.48-7.70 (m,
3H), 8.17 (bs, 1H), 8.50-8.53 (m, 2H); 13C NMR (100 MHz, CDCl3): δ = 28.96, 31.81,
48.16, 110.46, 119.63, 123.68, 124.29, 124.84, 136.05, 149.19, 149.37, 150.86,
164.88, 197.75; Anal. Cald for C16H15N3OS2: C, 58.33; H, 4.59; N, 12.76; Found: C,
58.08; H, 4.69; N, 12.50.
4.11. Synthesis of 2-(2H-benzo[d][1,2,3]triazol-2-yl)ethyl(pyridin-3-ylmethyl)
carbamodithioate (17n)
A mixture of 1,2,3-benzotriazole (1.19 g, 10 mmol), 1,2-dibromoethane (1.86 g,
10 mmol), KOH (1.12 g, 20 mmol), and TBAI (0.37 g, 1 mmol) in DMF (40 mL) was
heated at 80 ºC overnight. After cooling to room temperature, the mixture was added
water (40 mL), then extracted with ethyl acetate (20 mL×3). The organic layer was
21

ACCEPTED MANUSCRIPT
combined and concentrated to afford crude 2-(2-bromoethyl)-2H-benzo[d][1,2,3]
triazole as a white solid. This crude product was used without purification.
Following the same procedure for the synthesis of 17a, target compound 17n was
obtained using 2-(2-bromoethyl)-2H-benzo[d][1,2,3]triazole as halide. Yield 63%.
1
White solid. MP: 147-148 ºC. H NMR (400 MHz, DMSO-d6): δ = 3.78-3.81 (t, J =
6.8 Hz, 2H), 3.82-3.84 (t, J = 6.8 Hz, 2H), 4.95 (m, 2H), 7.34-7.55 (m, 4H), 7.64-7.83
(m, 2H), 8.48-8.50 (m, 2H), 10.58 (bs, 1H); 13C NMR (100 MHz, DMSO-d6): δ =
34.09, 46.97, 47.77, 110.99, 119.59, 123.95, 124.43, 127.74, 133.18, 135.93, 148.95,
196.41; HRMS (ESI+) m/z calcd for C15H15N5S2 (M + H)+, 330.0769, found
330.0847.
4.12. Synthesis of 2-(5-phenyl-1,3,4-oxadiazol-2-yl)ethyl(pyridin-3-ylmethyl)
carbamodithioate (17o)
A mixture of benzoylhydrazine 23 (0.68 g, 5 mmol), 3-bromopropionic acid (1.2g,
7.5 mmol), POCl3 (4 mL) in toluene (20 mL) was stirred at 100 ºC for 2 h. After
cooling to room temperature, the mixture was poured into ice water slowly, and
extracted with ethyl acetate (20 mL×3). The organic layer was combined, dried over
Na2SO4, and concentrated to afford crude 2-(2-bromoethyl)-5-phenyl-1,3,4-
oxadiazole 19o as a yellow solid in 41% of yield. This crude product was used
without purification.
Following the same procedure for the synthesis of 17a, target compound 17o was
obtained using crude 2-(2-bromoethyl)-5-phenyl-1,3,4-oxadiazole 19o as halide. Yield
62%. White solid. MP: 134-136 ºC. 1H NMR (400 MHz, CDCl3): δ 3.35-3.38 (t, J =
6.8 Hz, 2H), 3.76-3.79 (t, J = 6.8 Hz, 2H), 4.95 (d, J = 5.2 Hz, 2H), 7.19-7.21 (m, 1H),
7.45-7.51 (m, 3H), 7.68-7.70 (m, 1H), 7.98-8.00 (m, 2H), 8.39-8.49 (m, 2H), 9.11 (bs,
13
1H). C NMR (100 MHz, CDCl3): δ = 26.08, 31.43, 48.18, 123.62, 126.85, 129.04,
131.75, 132.45, 136.24, 148.85, 149.40, 165.02, 165.07, 197.39. Anal. Cald for
C17H16N4OS2: C, 57.28; H, 4.52; N, 15.72; Found: C, 57.32; H, 4.64; N, 15.66.
4.13. Synthesis of 2-(5-phenyl-1,2,4-oxadiazol-3-yl)ethyl(pyridin-3-ylmethyl)
carbamodithioate (17p)
A mixture of benzonitrile 24 (1.03 g, 10 mmol), TEA (4.0 g, 40 mmol), and
hydroxylammonium chloride (1.39 g, 20 mmol) in ethanol (60 mL) was heated at 80
ºC for 12 h. After cooling to room temperature and concentrating, water and ethyl
acetate was added, the organic layer was separated and concentrated to afford crude
N-hydroxybenzimidamide 25 as a yellow oil. This crude product was used without
purification.
To a mixture of N-hydroxybenzimidamide 25 (0.63 g, 5 mmol) and TEA (0.5 g, 5
mmol) in dichloromethane (30 mL), 3-chloropropionyl chloride (0.63 g, 5 mmol) was
added dropwise. After stirring for another 2 h, water (20 mL) was added and the
organic layer was dried over Na2SO4, concentrated to afford crude
N-((3-chloropropanoyl)oxy)benzimidamide 26 as a yellow oil. This crude product was
used without purification.
A mixture of N-((3-chloropropanoyl)oxy)benzimidamide 26 (0.23 g, 1 mmol) and
22

ACCEPTED MANUSCRIPT
K2CO3 (0.28 g, 2 mmol) in 1,4-dioxane (10 mL) was refluxed for 6 h. After
concentrating, water (20 mL) and ethyl acetate (20 mL) was added, the organic layer
was
dried
over
Na2SO4,
concentrated
to
afford
crude
5-(2-chloroethyl)-3-phenyl-1,2,4-oxadiazole 19p as a yellow oil. This crude product
was used without purification.
Following the same procedure for the synthesis of 17a, target compound 17p was
obtained using crude 5-(2-chloroethyl)-3-phenyl-1,2,4-oxadiazole 19p as halide. Yield
43%. White solid. MP: 118-120 ºC. 1H NMR (400 MHz, CDCl3): δ = 3.40-3.43 (t, J =
6.8 Hz, 2H), 3.80-3.84 (t, J = 6.8 Hz, 2H), 4.95 (m, 2H), 7.25-7.28 (m, 1H), 7.45-7.51
13
(m, 3H), 7.68-7.70 (m, 1H), 8.06-8.08 (m, 2H), 8.53 (m, 2H); C NMR (100 MHz,
CDCl3): δ = 27.09, 31.65, 48.26, 123.68, 127.44, 128.83, 131.19, 136.01, 149.42,
168.36, 177.74, 197.32; Anal. Cald for C17H16N4OS2: C, 57.28; H, 4.52; N, 15.72;
Found: C, 57.28; H, 4.68; N, 15.24.
4.14. General procedure for the synthesis of compounds 27a-27o.
Following the procedure for the synthesis of compound 17o, compounds 27a-27o
were prepared from corresponding aromatic or heterocyclic hydrazine.
4.14.1. 2-(5-(2-Fluorophenyl)-1,3,4-oxadiazol-2-yl)ethyl (pyridin-3-ylmethyl)car-
bamodithioate (27a)
1
Yield 67%. White solid. MP: 108-110 ºC. H NMR (400 MHz, DMSO-d6): δ =
3.35-3.38 (t, J = 6.8 Hz, 2H), 3.67-3.71 (t, J = 6.8 Hz, 2H), 4.85 (d, J = 5.2 Hz, 2H),
7.36-8.00 (m, 6H), 8.47-8.52 (m, 2H), 10.62 (bs, 1H); 13C NMR (100 MHz,
DMSO-d6): δ = 25.88, 31.06, 47.70, 110.00, 117.76, 124.03, 125.81, 130.03, 133.31,
134.55, 134.63, 136.10, 148.80, 149.41, 161.14, 165.80, 196.84; Anal. Cald for
C17H15FN4OS2: C, 54.53; H, 4.04; N, 14.96; Found: C, 54.26; H, 4.11; N, 14.69.
4.14.2. 2-(5-(4-Methoxyphenyl)-1,3,4-oxadiazol-2-yl)ethyl (pyridin-3-ylmethyl)car-
bamodithioate (27b)
1
Yield 70%. White solid. MP: 149-151 ºC. H NMR (400 MHz, DMSO-d6): δ =
3.30-3.33 (t, J = 6.8 Hz, 2H), 3.67-3.71 (t, J = 6.8 Hz, 2H), 3.85 (s, 3H), 4.85 (d, J =
5.2 Hz, 2H), 7.13-7.37 (m, 3H), 7.67-7.69 (m, 1H), 7.90-7.93 (m, 2H), 8.47-8.52 (m,
2H), 10.59 (bs, 1H); 13C NMR (100 MHz, DMSO-d6): δ = 25.88, 31.10, 47.70, 55.98,
115.32, 116.27, 123.98, 128.73, 133.26, 135.96, 148.92, 149.53, 162.40, 164.46,
164.99, 196.85; Anal. Cald for C18H18N4O2S2: C, 55.94; H, 4.69; N, 14.50; Found: C,
56.20; H, 4.71; N, 14.94.
4.14.3. 2-(5-(4-(Trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)ethyl (pyridin-3-yl
methyl)carbamodithioate (27c)
1
Yield 65%. White solid. MP: 145-146 ºC. H NMR (400 MHz, DMSO-d6): δ
3.36-3.39 (t, J = 6.8 Hz, 2H), 3.69-3.73 (t, J = 6.8 Hz, 2H), 4.85 (d, J = 5.2 Hz, 2H),
7.33-7.37 (m, 1H), 7.67-7.69 (m, 1H), 7.90-7.93 (m, 2H), 8.18-8.20 (m, 2H),
13
8.47-8.51 (m, 2H), 10.60 (bs, 1H); C NMR (100 MHz, DMSO-d6): δ 26.00, 31.01,
47.72, 123.95, 126.91, 127.78, 133.23, 135.94, 148.92, 149.54, 163.57, 166.26,
196.83; Anal. Cald for C18H15F3N4OS2: C, 50.93; H, 3.56; N, 13.20; Found: C, 50.81;
23

ACCEPTED MANUSCRIPT
H, 4.01; N, 12.91.
4.14.4. 2-(5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl)ethyl(pyridin-3-ylmethyl)
carbamodithioate (27d)
1
Yield 64%. White solid. MP: 126-127 ºC. H NMR (400 MHz, DMSO-d6) δ =
3.34 (t, J = 6.9 Hz, 2H), 3.70 (t, J = 6.9 Hz, 2H), 4.85 (d, J = 5.5 Hz, 2H), 7.36 (dd, J
= 7.7, 4.8 Hz, 1H), 7.45 (t, J = 8.9 Hz, 2H), 7.69 (d, J = 7.8 Hz, 1H), 8.07 – 8.01 (m,
2H), 8.48 (dd, J = 4.7, 1.4 Hz, 1H), 8.52 (d, J = 1.6 Hz, 1H),10.59 (t, J = 5.3 Hz, 1H);
13C NMR (100 MHz, DMSO-d6) δ = 25.92, 31.06, 47.72, 117.01, 117.24, 120.57,
123.95, 129.54, 129.63, 133.24, 135.93, 148.93, 149.55, 163.82, 165.60, 196.84; Anal.
Cald for C17H15ClN4OS2: C, 52.23; H, 3.87; N, 14.33; Found: C, 52.61; H, 4.00; N,
14.21.
4.14.5. 2-(5-(4-Nitrophenyl)-1,3,4-oxadiazol-2-yl)ethyl(pyridin-3-ylmethyl)carbamo-
dithioate (27e)
1
Yield 37%. White solid. MP: 157-158 ºC. H NMR (400 MHz, DMSO-d6) δ =
3.39 (t, J = 6.6 Hz, 2H), 3.72 (t, J = 6.6 Hz, 2H), 4.84 (d, J = 5.1 Hz, 2H), 7.43 – 7.32
(m, 1H), 7.69 (d, J = 7.3 Hz, 1H), 8.24 – 8.19 (m, 2H), 8.40 (t, J = 7.7 Hz, 2H), 8.48
(dd, J = 15.4, 11.6 Hz, 2H), 10.62 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ = 26.04,
31.00, 47.71, 123.98, 125.06, 128.18, 128.24, 129.36, 133.27, 136.06, 148.82, 149.44,
163.26, 166.57, 196.80; HRMSꢃCalcd for C17H16N5O3S2 (M++H) : 402.06891,
Foundꢃ402.06957.
4.14.6. 2-(5-(1H-Pyrrol-3-yl)-1,3,4-oxadiazol-2-yl)ethyl (pyridin-3-ylmethyl)carba-
modithioate (27f)
1
Yield 73%. White solid. MP: 167-168 ºC. H NMR (400 MHz, DMSO-d6) δ =
3.28 (t, J = 6.9 Hz, 2H), 3.66 (t, J = 6.9 Hz, 2H), 4.86 (d, J = 5.5 Hz, 2H), 6.25 (dd, J
= 5.8, 2.4 Hz, 1H), 6.78 (d, J = 3.5 Hz, 1H), 7.07 (dd, J = 3.9, 2.6 Hz, 1H), 7.37 (dd, J
= 7.8, 4.8 Hz, 1H), 7.69 (d, J = 7.9 Hz, 1H), 8.48 (dd, J = 4.7, 1.3 Hz, 1H), 8.53 (d, J
= 1.6 Hz, 1H), 10.60 (t, J = 5.5 Hz, 1H), 12.16 (s, 1H); 13C NMR (100 MHz,
DMSO-d6) δ = 25.73, 31.14, 47.71, 110.32, 112.25, 115.79, 123.90, 123.99, 133.26,
135.98, 148.93, 149.55, 159.77, 163.61, 196.83; HRMSꢃCalcd for C15H16N5OS2
ꢄM++Hꢅꢃ346.07908ꢂFoundꢃ346.07884.
4.14.7. 2-(5-(Furan-2-yl)-1,3,4-oxadiazol-2-yl)ethyl (pyridin-3-ylmethyl)carbamo-
dithioate (27g)
1
Yield 85%. White solid. MP: 155-156 ºC. H NMR (400 MHz, DMSO-d6) δ =
3.32 (t, J = 6.9 Hz, 2H), 3.67 (t, J = 6.9 Hz, 2H), 4.85 (d, J = 5.5 Hz, 2H), 6.79 (dd, J
= 3.5, 1.7 Hz, 1H), 7.31 (d, J = 3.5 Hz, 1H), 7.37 (dd, J = 7.7, 4.8 Hz, 1H), 7.68 (d, J
= 7.8 Hz, 1H), 8.04 (d, J = 1.0 Hz, 1H), 8.48 (dd, J = 4.7, 1.2 Hz, 1H), 8.52 (d, J = 1.5
Hz, 1H), 10.58 (t, J = 5.3 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ = 25.76, 31.05,
47.72, 113.01, 114.73, 123.96, 133.23, 135.93, 139.10, 147.28, 148.93, 149.55,
157.52, 164.85, 196.79; HRMS: Calcd for C15H15N4O2S2 (M++H): 347.06309, Found:
347.06367.
24

ACCEPTED MANUSCRIPT
4.14.8. 2-(5-(Thiophen-3-yl)-1,3,4-oxadiazol-2-yl)ethyl (pyridin-3-ylmethyl)carbamo-
dithioate (27h)
1
Yield 89%. White solid. MP: 141-142 ºC. H NMR (400 MHz, DMSO-d6) δ =
3.32 (t, J = 6.8 Hz, 2H), 3.68 (t, J = 6.7 Hz, 2H), 4.85 (d, J = 5.2 Hz, 2H), 7.40 – 7.33
(m, 1H), 7.60 (d, J = 4.9 Hz, 1H), 7.68 (d, J = 7.6 Hz, 1H), 8.31 (s, 1H), 7.81 (s, 1H),
8.48 (d, J = 4.5 Hz, 1H), 8.52 (s, 1H), 10.60 (s, 1H); 13C NMR (100 MHz, DMSO-d6)
δ = 196.81, 164.85, 161.48, 149.54, 148.93, 135.95, 133.25, 129.48, 128.93, 125.99,
124.97, 123.98, 47.71, 31.08, 25.84. HRMSꢃCalcd for C15H15N4OS3 (M++H):
363.04025, Found: 363.03961.
4.14.9. 2-(5-(Thiophen-2-yl)-1,3,4-oxadiazol-2-yl)ethyl (pyridin-3-ylmethyl)carbamo-
dithioate (27i)
1
Yield 92%. White solid. MP: 145-145 ºC. H NMR (400 MHz, DMSO-d6) δ =
3.32 (t, J = 6.9 Hz, 2H), 3.68 (t, J = 6.9 Hz, 2H), 4.85 (d, J = 5.6 Hz, 2H), 7.28 (dd, J
= 5.0, 3.7 Hz, 1H), 7.38 – 7.34 (m, 1H), 7.71 – 7.66 (m, 1H), 7.78 (dt, J = 6.4, 3.2 Hz,
1H), 7.92 (dd, J = 5.0, 1.2 Hz, 1H), 8.48 (dd, J = 4.7, 1.5 Hz, 1H), 8.52 (d, J = 1.7 Hz,
13
1H), 10.59 (t, J = 5.4 Hz, 1H); C NMR (100 MHz, DMSO-d6); δ = 25.84, 31.07,
47.73, 123.96, 124.83, 129.15, 130.51, 131.81, 133.24, 135.94, 148.93, 149.55,
160.87, 165.00, 196.82. HRMS: Calcd for C15H15N4OS3 (M++H): 363.04025ꢂFound:
363.04064.
4.14.10. 2-(5-(5-methylisoxazol-3-yl)-1,3,4-oxadiazol-2-yl)ethyl (pyridin-3-ylmethyl)
carbamodithioate (27j)
1
Yield 75%. White solid. MP: 149-150 ºC. H NMR (400 MHz, v) δ = 2.49 (s,
3H); 3.33 (t, J = 6.3 Hz, 2H), 3.63 (t, J = 6.4 Hz, 2H), 4.80 (d, J = 4.7 Hz, 2H), 7.37 –
7.28 (m, 1H), 6.84 (s, 1H), 7.64 (d, J = 7.2 Hz, 1H), 8.43 (d, J = 3.4 Hz, 1H), 8.47 (s,
1H),10.56 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ = 12.27, 25.90, 30.96, 47.74,
101.59, 123.96, 133.22, 135.95, 148.92, 149.55, 150.44, 157.39, 166.61, 172.78,
196.74. HRMSꢃCalcd for C15H16N5O2S2 (M++H): 362.07399, Found: 362.07377.
4.14.11. 2-(5-(Pyridin-3-yl)-1,3,4-oxadiazol-2-yl)ethyl (pyridin-3-ylmethyl)carbamo-
dithioate (27k)
1
Yield 75%. White solid. MP: 151-152 ºC. H NMR (400 MHz, DMSO-d6): δ =
3.39 (t, J = 6.6 Hz, 2H), 3.74 (t, J = 6.7 Hz, 2H), 4.87 (d, J = 5.2 Hz, 2H), 7.37 (dd, J
= 7.4, 4.8 Hz, 1H), 7.64 (dd, J = 7.7, 4.8 Hz, 1H), 7.71 (d, J = 7.5 Hz, 1H), 8.35 (d, J
= 7.8 Hz, 1H), 8.50 (d, J = 3.7 Hz, 1H), 8.54 (s, 1H), 8.81 (d, J = 3.7 Hz, 1H), 9.17 (s,
1H), 10.64 (s, 1H); 13C NMR (100 MHz, DMSO-d6): δ = 25.98, 31.04, 47.74, 120.44,
123.95, 124.78, 133.23, 134.48, 135.97, 147.57, 148.90, 149.53, 152.87, 162.81,
166.02, 196.82; HRMSꢃCalcd for C16H16N5OS2 (M++H): 358.07908ꢂ Found:
358.07826.
4.14.12. 2-(5-(Pyrazin-2-yl)-1,3,4-oxadiazol-2-yl)ethyl (pyridin-3-ylmethyl)carbamo-
dithioate (271)
25

ACCEPTED MANUSCRIPT
1
Yield 88%. White solid. MP: 143-144 ºC. H NMR (400 MHz, DMSO-d6) δ =
3.41 (t, J = 6.8 Hz, 2H), 3.71 (t, J = 6.8 Hz, 2H), 4.85 (d, J = 5.3 Hz, 2H), 7.37 (dd, J
= 7.3, 4.9 Hz, 1H), 7.69 (d, J = 7.6 Hz, 1H), 8.48 (d, J = 3.6 Hz, 1H), 8.52 (s, 1H),
8.88 (d, J = 5.8 Hz, 2H), 9.35 (s, 1H), 10.60 (s, 1H); 13C NMR (100 MHz, DMSO-d6)
δ = 26.00, 31.03, 47.73, 123.97, 133.23, 135.96, 139.51, 143.96, 145.49, 147.43,
148.92, 149.54, 162.55, 166.87, 196.77; HRMS: Calcd for C15H16N6OS2 (M++H):
359.07433, Found: 359.07482.
4.14.13. 2-(5-(Quinolin-6-yl)-1,3,4-oxadiazol-2-yl)ethyl (pyridin-3-ylmethyl)carbamo-
dithioate (27m)
1
Yield 54%. White solid. MP: 165-166 ºC. H NMR (400 MHz, DMSO-d6): δ =
3.40 (t, J = 6.8 Hz, 2H), 3.74 (t, J = 6.7 Hz, 2H), 4.85 (s, 2H), 7.35 (dd, J = 7.6, 4.8
Hz, 1H), 7.73 – 7.61 (m, 2H), 8.18 (d, J = 8.8 Hz, 1H), 8.29 (d, J = 8.8 Hz, 1H), 8.47
(d, J = 4.4 Hz, 1H), 8.52 (s, 1H), 8.58 (d, J = 8.2 Hz, 1H), 8.65 (s, 1H), 9.01 (d, J =
3.6 Hz, 1H), 10.63 (s, 1H); 13C NMR (100 MHz, DMSO-d6): δ = 26.02, 31.09, 47.73,
121.70, 123.11, 123.95, 126.87, 127.61, 128.17, 130.82, 133.24, 135.95, 137.48,
148.92, 149.07, 149.55, 152.92, 164.29, 165.93, 196.81. HRMS: Calcd for
C20H18N5OS2 (M++H): 408.09473, Found: 408.09520.
4.14.14. 2-(5-(quinolin-2-yl)-1,3,4-oxadiazol-2-yl)ethyl (pyridin-3-ylmethyl)carbamo-
dithioate (27n)
1
Yield 81%. White solid. MP: 139-140 ºC. H NMR (400 MHz, DMSO-d6) δ =
3.44 (t, J = 6.8 Hz, 2H), 3.73 (t, J = 6.8 Hz, 2H), 4.86 (d, J = 5.3 Hz, 2H), 7.41 – 7.34
(m, 1H), 7.71 (d, J = 7.6 Hz, 1H), 7.75 (t, J = 7.5 Hz, 1H), 7.90 (t, J = 7.5 Hz, 1H),
8.12 (d, J = 7.8 Hz, 1H), 8.18 (d, J = 8.4 Hz, 1H), 8.27 (d, J = 8.5 Hz, 1H), 8.48 (d, J
13
= 4.4 Hz, 1H), 8.53 (s, 1H), 8.63 (d, J = 8.5 Hz, 1H), 10.63 (s, 1H); C NMR (100
MHz, DMSO-d6) δ = 26.07, 31.12, 47.74, 119.93, 123.98, 128.68, 128.78, 128.85,
129.77, 131.37, 133.27, 136.02, 138.52, 143.36, 147.62, 148.88, 149.52, 164.39,
166.84, 196.81; HRMS: Calcd for C20H18N5OS2 (M++H): 408.09473, Found:
408.09504.
4.14.15. 2-(5-(benzo[b]thiophen-2-yl)-1,3,4-oxadiazol-2-yl)ethyl (pyridin-3-ylmethyl)
carbamodithioate (27o)
1
Yield 53%. White solid. MP: 249-250 ºC. H NMR (400 MHz, DMSO-d6) δ =
3.40 – 3.31 (m, 2H), 3.70 (t, J = 6.9 Hz, 2H), 4.85 (s, 2H), 7.36 (dd, J = 7.8, 4.8 Hz,
1H), 7.52 (dd, J = 7.2, 5.6 Hz, 2H), 7.71 – 7.67 (m, 1H), 8.04 (dd, J = 6.9, 1.8 Hz, 1H),
8.12 (d, J = 7.8 Hz, 1H), 8.47 (dd, J = 4.7, 1.5 Hz, 1H), 8.52 (d, J = 1.7 Hz, 1H),
10.66 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ = 25.96, 31.03, 47.71, 123.35,
123.93, 124.67, 125.74, 125.89, 127.35, 133.29, 135.96, 139.28, 140.39, 148.88,
149.57, 160.98, 165.71, 196.67; HRMS: Calcd for C19H17N4OS3 (M++H): 413.05590,
Found: 413.05648.
4.15. Cytotoxic activity analysis
Human cancer cell lines excepted SW620 and PC-3 were cultivated at 37˚C and
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5% CO2 in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS),
100 U/ml penicillin and 100 ꢀg/ml streptomycin. Human prostate cancer cells PC-3
were cultivated at 37˚C and 5% CO2 in F-12K medium supplemented with 10% FBS,
100 U/ml penicillin and 100 ꢀg/ml streptomycin. SW620 cells were cultivated at 37˚C
and 100% air in Leibovitz’s L-15 medium supplemented with 10% FBS, 100 U/ml
penicillin and 100 ꢀg/ml streptomycin. Cytotoxic effects of target compounds were
determined by Alamar blue assay. Cells were seeded into 96-well plates and cultured
overnight. After treatment by different concentrations of compounds for 72 h, cells
were incubated with 100 ꢀl medium containing 10% Alamar blue solution for 2-4 h. A
multipliable counter (Tecan Infinite M200 PRO, Swiss) was used to measure
fluorescence at 530 nm excitation and 590 nm emission.
4.16. Cell cycle analysis
Cells were cultured in medium containing 0.5% FBS overnight for
synchronization, and then exposed to different concentration of compound for 24 h.
Cells were collected and fixed in cold 70% ethanol at -20ꢆ overnight. Cells were
then washed with PBS and stained by propidium iodide (PI) staining solution (50
ꢀg/ml of PI and 100 ꢀg/ml of DNAse-free RNase), followed by immediate analysis
using flow cytometry. The results were analyzed with Mod Fit LT 4.0.4 (Verity Soft
House, USA).
4.17. Cell nuclear morphology
Hoechst 33342 staining was used to observe chromosome condensation and
nuclear morphology of cancer cells. After treatment, cells were washed with PBS and
fixed in 4% paraformaldehyde for 15 min. Cells were incubated in PBS containing
Hoechst 33342 (10 ꢀg/ml) for 10 min at room temperature. After staining, cells were
visualized and imaged under UV illumination using a fluorescence microscop
(Olympus, IX81).
4.18. Annexin V-FITC/PI staining assay
Cell apoptosis was determined using Annexin V-FITC/PI Apoptosis Detection Kit
(BD Pharmingen) in according to manufacturer’s instruction. Briefly, after treatment,
both non-adherent and adherent cells were collected and re-suspended in Binding
Buffer supplemented with 5 ꢀl FITC Annexin V and 10 ꢀl PI. Cells were gently
mixed and incubated for 15 min in the dark. After staning, 400 ꢀl Binding Buffer was
added to each tube. The apoptotic cells were immediately analyzed using flow
cytometry. Annexin V-FITC positive/PI negative cells were considered as early
apoptotic cells. All Annexin V-FITC staining positive cells were considered as total
apoptotic cells.
4.19. Western blot analysis
Cells were collected and lysed using RIPA lysis buffer containing 1% PMSF and
1% protease inhibitor cocktail (Roche). Proteins were separated by SDS-PAGE and
transferred to PVDF membrane. The PVDF membrane was blocked using 5% non-fat
27

ACCEPTED MANUSCRIPT
milk for 1 h, and then incubated with the primary antibodies against MPM-2
(Millipore, 1:1000), Pi-H3 (CST, 1:600), or β-actin (Beyotime company, 1:1000)
overnight at 4ꢆ. Horseradish peroxidase-conjugated antibody was used as secondary
antibody. The immunecomplexes were visualized using SuperSignal substrate reagent
(Pierce).
4.20. Immunofluorescence assay
Cells were grown on confocal dish and treated by 10p or Taxol for 6 h. After
gently wash by PBS, cells were fixed in 4% paraformaldehyde overnight at 4ꢆ. Then,
fixed cells were blocked, incubated in blocking buffer supplemented with primary
antibody against β-tubulin (CST, 1:100) and γ-tubulin (CST, 1:100) overnight at 4ꢆ,
washed three times in PBS, and incubated with Alexa Fluor® 488 conjugated- and
Alexa Fluor® 647-conjugated secondary antibodies for an additional hour. DAPI was
used to visualize the nuclei. Stained specimens were obtained with a TCS-SP5 laser
scanning confocal microscope (Leica).
Acknowledgements
The authors thank the National Natural Science Foundation of China (Grand Nos.
21172011 and 81301881) for financial support.
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