Synthesis, molecular modeling and biological evaluation of dithiocarbamates as novel antitubulin agents

Article abstract of DOI:10.1016/j.bmc.2010.04.091

A series of novel dithiocarbamate compounds with the chalcone scaffold have been designed and synthesized, and their biological activities were also evaluated as potential antiproliferation and antitubulin polymerization inhibitors. Compound 2n showed the most potent biological activity in vitro, which inhibited the growth of MCF-7 cells with IC₅₀ of 0.04 ± 0.01 μM and the polymerization of tubulin with IC₅₀ of 6.8 ± 0.6 μM. To understand the tubulin-inhibitor interaction and the selectivity of the most active compound towards tubulin, molecular modeling studies were performed to dock compound 2n into the colchicine binding site, which suggested probable inhibition mechanism.

Full text of DOI:10.1016/j.bmc.2010.04.091

Bioorganic & Medicinal Chemistry 18 (2010) 4310–4316
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, molecular modeling and biological evaluation of dithiocarbamates
as novel antitubulin agents
*
Yong Qian, Gao-Yuan Ma, Ying Yang, Kui Cheng, Qing-Zhong Zheng, Wen-Jun Mao, Lei Shi, Jing Zhao ,
*
Hai-Liang Zhu
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel dithiocarbamate compounds with the chalcone scaffold have been designed and synthe-
sized, and their biological activities were also evaluated as potential antiproliferation and antitubulin
polymerization inhibitors. Compound 2n showed the most potent biological activity in vitro, which
Received 23 February 2010
Revised 24 April 2010
Accepted 27 April 2010
Available online 20 May 2010
inhibited the growth of MCF-7 cells with IC50 of 0.04 0.01
lM and the polymerization of tubulin with
IC50 of 6.8 0.6 M. To understand the tubulin–inhibitor interaction and the selectivity of the most active
l
compound towards tubulin, molecular modeling studies were performed to dock compound 2n into the
colchicine binding site, which suggested probable inhibition mechanism.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Dithiocarbmate
Chalcone
Molecular modeling
Antitubulin polymerization
1. Introduction
suppressive, antiprotozoan activity, antimalarial,10–16 antimitotic,
and antiproliferative.3,11,12,17–19
Microtubules are key cytoskeletal filaments and are involved in
numerous cellular functions, including cell motility, vesicle trans-
port and cell division. Microtubules are in dynamic equilibrium
with tubulin dimers.1 Disruption of the dynamic equilibrium will
lead to cell cycle arrest or cell apoptosis. Given their significant role
in the growth and function of cells, microtubules are among the
most important molecular targets for cancer chemotherapeutic
agents.
Recently a number of small molecules were discovered to bind
to tubulins, interfering with the polymerization or depolymeriza-
tion of microtubules and then inducing cell cycle arrest, resulting
in cell death.2–5 Fore example, Combretastatin A-4 (CSA-4), which
was isolated from the South African tree combretum cattrum, was
found to inhibit the polymerization of tubulin by binding to the
colchicine site.6,7 Colchicine is the first drug that is well known
to bind tubulin, and the colchicine binding site has been character-
ized recently.8 The development of ketones as tubulin binding
agents such as chalcones was reviewed.9
On the other hand, dithiocarbamate (DTC) derivatives are well
known as rubber additives, vulcanizing agents, fungicides,20 anti-
microbial agents,21 antitumor drugs,22 and prophylactic or thera-
peutic agents for metal toxicity.23–25 Recently, Yang et al. have
described a series of chromone derivatives bearing dithiocarba-
mate moieties which displayed apoptosis-inducing effects on tu-
mor cell lines.26
Herein we report the synthesis and bioactivities of a series of
new substituted dithiocarbamate compounds based on intermedi-
ate chalcones. Their antiproliferative and inhibition of tubulin
polymerization activities were evaluated. Molecular modeling
studies were consequently performed to understand tubulin–
inhibitor interaction. The docking results confirmed that the com-
bination of static and hydrophobic interactions and hydrogen
bonding may contribute to the potent biological activities.
2. Results and discussion
2.1. Chemistry
Chalcones (1,3-diarylpropenones) are an important pharmaco-
phore of natural products and the synthetic precursors to various
flavonoids and isoflavonoids.
activities of chalcones have been reported, including anticancer,
anti-inflammatory, immunomodulatory, antibacterial, immuno-
A variety of pharmacological
The synthesis of compounds 2a–2t is outlined in Scheme 1.
First, a series of chalcones were prepared by the Claisen–Schmidt
condensation between ketones and aldehydes in the presence of
KOH in MeOH in 80–85% yield.11 The desired dithiocarbamates
2a–2t could be obtained by one-pot reaction of diethylamine,
carbon disulfide and intermediate chalcones (yield: 85–90%).27
* Corresponding authors. Tel.: +86 25 8359 2572; fax: +86 25 8359 2672 (H.-L.Z.).
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.091

Y. Qian et al. / Bioorg. Med. Chem. 18 (2010) 4310–4316
4311
Scheme 1. Synthesis of dithiocarbamates 2a–2t. Reagents and conditions: (a) KOH, MeOH, rt; (b) CS2, dithylamine, CH2Cl2.
(Table 1) All the target compounds were characterized by 1H NMR,
Elemental analysis, and mass spectrum. Furthermore, the crystal
structure of compound 2l was determined by single crystal X-ray
diffraction analysis in Figure 1.
2.2. Bioactivity
All synthesized dithiocarbamate compounds 2a–2t were evalu-
ated for their ability to antiproliferative activities against MCF-7
human breast carcinoma cells. The results were summarized in Ta-
ble 2. A number of dithiocarbamate compounds bearing the same
dithiocarbamate (DTC) moiety showed remarkable effects on anti-
proliferative activities. The compounds with para halogen substi-
Table 1
Structure of dithiocarbamates 2a–2t
Figure 1. Crystal structure diagrams of compound 2l. Molecule structure diagram
with displacement ellipsoids being at the 30% probability level and H atoms are
shown as small spheres of arbitrary radii.
tuted (2d, 2e, 2f) exhibited significant antiproliferative activity
and the potency order is F < Cl < Br. The results demonstrated that
a hydrophobic and electron-withdrawing halogen group may have
slightly improved antiproliferative activity and other electron-
donating substituted (2b, 2c) had minimal effects relative to those
of 2d. Compounds (2d, 2e) with p-substituted group showed
slightly more potent activities than those of o-substituted (2g,
2h) and m-substituted (2i). Compound 2j in which A-ring was
substituted by methyl group showed poor activity, suggesting that
aromaticity of the A-ring was critical for the potent activity. Com-
pounds (2n, 2k, 2m) with chloro-substitution or methoxy-substi-
tution showed stronger antitumor activity compared to
compound with a p-bromo group substituted in the A ring such
as 2l, suggesting that the enhancing order of the substituent on
A-ring is: 4-Br < 4-OMe = 3, 4-Cl < 4-Cl. Interestingly, a similar phe-
nomenon was also observed with substituents of p-Br (2o) and p-
OMe (2q) on A-ring, having the same p-OMe substituent on B-ring,
Compound
R1
R2
R3
R4
R5
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
2r
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OMe
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
NO2
H
H
H
H
H
H
H
H
H
H
H
H
OMe
Me
Br
Cl
F
H
H
H
H
H
H
H
H
OMe
F
OMe
Me
H
H
A=CH3
OMe
Br
Cl
Cl
Br
Br
OMe
OMe
OMe
OMe
H
H
Cl
H
H
H
H
H
H
H
H
Cl
H
and showed IC50 values of 5 0.6 and 0.8 0.05
lM, respectively.
Compound 2s, with a p-OMe group in the A-ring and an o-Cl group
in the B-ring, revealed the similar activity to 2n containing p-Cl
2s
2t
Cl

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Y. Qian et al. / Bioorg. Med. Chem. 18 (2010) 4310–4316
Table 2
To gain more understanding on compound 2n’s potency, we
proceeded to examine the interaction of the compound 2n with
tubulin (PDB code: 1SA0). The molecular docking was performed
by simulation of compound 2n in the colchicine binding site of
tubulin. All docking runs were applied the Lamarckian genetic
algorithm of Auto-Dock 4.0.28 The calculated free energies of bind-
ing were used as the parameter for the selection of the cluster of
docking posed to be evaluated, in which the binding mode of the
lowest energy structure located in the top docking cluster. The se-
lected pose of 2n had an estimated free energy of binding of
ꢀ10.76 kcal/mol (free energy of binding of control compounds col-
chicine and CSA-4 is ꢀ8.86 kcal/mol and ꢀ7.62 kcal/mol, respec-
tively). The structure of lowest energy in the top docking cluster
was showed in Figure 2A. The docking results demonstrated that
2n interacted with the colchicine binding site of tubulin via hydro-
phobic interactions and binding is stabilized by one hydrogen bond
(SER178: angle Oꢁ ꢁ ꢁH–O = 168.022°, distance = 2.111 Å). These res-
idues (CYS241, SER178) were also found to be involved in the bind-
ing of other tubulin binding agents.29 As depicted in Figure 2B, a, c
and d depicted the 3D model of the interaction between 2n, colchi-
cine, CSA-4 and the colchicine binding site. Comparing this model,
the hydrophobic pockets of colchicine binding site were occupied
by compounds, and the difference was the arrangement on the en-
zyme surface. In particular, b depicted the molecular surface model
of compound 2n interaction with the site, showing well binding
affinity to the target. In the 2n binding model, more details re-
vealed that there are some key roles of the interaction of 2n and
tubulin (Fig. 2A). The chlorophenyl moiety of A-ring occupied a
pocket bounded by ALA180, LEU255, and LYS352. It should be
noted that the static interaction between Cl group and LYS352
might play a role. The phenyl moiety of B ring was embedded in
a hydrophobic pocket constructed by the side chains of residues
of CYS241, LEU248 and LEU255. Moreover, the binding of com-
pound 2n to tubulin was stabilized by hydrogen bond with
SER178. Overall, the results suggested that 2n interacted well with
tubulin, similar to colchicine and CSA-4.3,30,31
Inhibition of tubulin polymerizaton and inhibition (IC50) of MCF-7 cells proliferation
by compound 2a–2t
Compound
MCF-7a IC50 SD (
17
4.6 0.3
20
0.08 0.03
0.12 0.02
0.7 0.08
l
M)
Tubulinb IC50 SD (
130 40
lM)
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
2r
3
45
2
8
220 10
12
15
80
4
3
7
16
4
150 14
4.5 0.12
52
90
5
8
9
23
0.7
3
250 20
62 40
65 15
60 10
6.8 0.6
4.5 0.7
5
0.15
4.5 0.2
0.04 0.01
5
1
0.6
0.25
52
18
84
7
4
6
0.8 0.05
17
0.07 0.01
16
4
180 20
10
2s
2t
2
4
140 30
3.2 0.4
2.2 0.2
Colchicine
CSA-4
0.017 0.01
0.013 0.003
a
Inhibition the growth of MCF-7 human breast carcinoma cells.
Inhibition of tubulin polymerization.
b
substituent on A-ring. However, compound 2t displayed a poor
activity.
To examine whether the compounds interact with tubulin and
inhibit tubulin polymerization in vitro, we performed the tubulin
assembly assay. As shown in Table 2, compounds 2d, 2e, 2n, 2s
showed strong inhibitory effect and their 50% tubulin polymeriza-
tion inhibition about 12
4
l
M, 15
3 lM, 6.8 0.6 lM,
10 M, respectively. Compound 2n displayed the most potent
2
l
anti-tubulin polymerization activity (Table 2). The result con-
firmed that the antiproliferative effect was mediated by direct
interaction of compounds with tubulin.
Compound 2n was also performed cell cycle analysis using flow
cytometry. Cell cycle analysis (Fig. 3) showed that the compound
2n strongly induced G2/M arrest in MCF-7 cells, and the effect
was observed in a dose-independent manner after treatment for
24 h increasing amounts of the compound. About 39.5% of the cells
were arrested in the G2/M phase after treatment with 250 nm 2n
for 24 h. These findings indicated a continuing impairment of cell
division and confirmed compound 2n was a potent antitubulin
agent.
3. Conclusion
In the present work, we synthesized and evaluated a series of
novel antitubulin polymerization inhibitors containing a dithiocar-
bamate moiety. These chalcone-type compounds exhibited potent
tubulin polymerization inhibitory activity and antiproliferative
activity against MCF-7 human breast carcinoma cells. Compound
2n demonstrated the most potent activity which inhibited the
growth of MCF-7 cells with IC50 of 0.04 0.01 lM and inhibited
Figure 2A. compound 2n (carbon atom are green) docked into the tubulin pocket. Both side chains of important active site amino acids and hydrogen bonds are shown: Ser-
178 (angle Oꢁ ꢁ ꢁH–O = 168.022°, distance = 2.111 Å).

Y. Qian et al. / Bioorg. Med. Chem. 18 (2010) 4310–4316
4313
the polymerization of tubulin with IC50 of 6.8 0.6
compound CSA-4, showed antiproliferation activity against MCF-
l
M (control
7 cells (IC50 = 0.013 0.003
activity (IC50 = 2.2 0.2
l
M) and antitubulin polymerization
l
M), respectively). Molecular docking
Figure 2B. Comparison of colchicine, CSA-4 and compounds 2n binding model with tubulin complex. (a) 3D model of the interaction between compound 2n and the
colchicine binding site. The protein is represented by molecular surface. 2n is depicted by sticks and balls. (b) 3D model of interaction between compound 2n and the
colchicine binding site. The protein is represented by molecular surface. 2n is depicted by molecular surface and sticks and balls. (c) 3D model of the interaction between
colchicine and the colchicine binding site. The protein is represented by molecular surface. colchicine is depicted by sticks and balls. (d) 3D model of the interaction between
CSA-4 and the colchicine binding site. The protein is represented by molecular surface. CSA-4 is depicted by sticks and balls.
Figure 3. Cell cycle analysis of MCF-7 cells treated with compound 2n using flow cytometry. Cells were harvested after treatment with 2n for 24 h and subjected to cell cycle
analysis. The percentage of cells in each cell cycle phase was indicated.

4314
Y. Qian et al. / Bioorg. Med. Chem. 18 (2010) 4310–4316
was performed to study the inhibitor–tubulin protein interactions.
Analysis of the compound 2n’s binding conformation in the colchi-
cine binding site demonstrated that a combination of several inter-
actions with the protein residues led to the antitubulin
polymerization and cytotoxicity. We are presently plan to utilize
the molecular modeling studies to design and synthesize more po-
tent tubulin polymerization inhibitors.
J = 7.68 Hz, 2H); 7.30 (d, J = 8.04 Hz, 2H); 7.38–7.43 (m, 2H);
7.49–7.54 (m, 1H); 7.95–7.99 (m, 2H). ESI-MS: 392.1 (C21H26NOS2,
[M+H]+). Anal. Calcd for C21H25NOS2: C, 67.88; H, 6.78; N, 3.77.
Found: C, 68.12; H, 6.98; N, 4.08.
4.3.4. 1-(4-Bromophenyl)-3-oxo-3-phenylpropyl diethylcar-
bamodithioate (2d)
1H NMR (300 MHz, CDCl3): d 1.25–1.33 (m, 6H); 3.66–4.08 (m,
6H); 5.67–5.72 (dd, J = 4.2, 9.9 Hz, 1H); 7.30–7.33 (m, 2H); 7.40–
7.47 (m, 4H); 7.53–7.58 (m, 1H); 7.95–7.97 (m, 2H). ESI-MS:
436.0 (C20H23BrNOS2, [M+H]+). Anal. Calcd for C20H22BrNOS2: C,
55.04; H, 5.08; N, 3.21. Found: C, 55.37; H, 5.27; N, 3.43.
4. Experiments
4.1. Materials and measurements
All chemicals and reagents used in current study were of analyt-
ical grade. All the 1H NMR spectra were recorded on a Bruker DRX
500 or DPX 300 model Spectrometer in CDCl3 and chemical shifts
was reported in ppm (d). ESI-MS spectra were recorded on a Mar-
iner System 5304 Mass spectrometer. Elemental analyses were
performed on a CHN-O-Rapid instrument. TLC was performed on
the glass-backed silica gel sheets (Silica Gel 60 Å GF254) and visu-
alized in UV light (254 nm). Column chromatography was per-
formed using silica gel (200–300 mesh) eluting with ethyl
acetate and petroleum ether.
4.3.5. 1-(4-Chlorophenyl)-3-oxo-3-phenylpropyl diethylcar-
bamodithioate (2e)
1H NMR (300 MHz, CDCl3): d 1.23–1.29 (m, 6H); 3.68–4.03 (m,
6H); 5.67–5.72 (dd, J = 4.3, 10.0 Hz, 1H); 7.23 (s, 1H); 7.35–7.59
(m, 6H); 7.94–8.00 (m, 2H). ESI-MS: 392.1 (C20H23ClNOS2,
[M+H]+). Anal. Calcd for C20H22ClNOS2: C, 61.28; H, 5.66; N, 3.57.
Found: C, 61.55; H, 5.87; N, 3.86.
4.3.6. 1-(4-Fluorophenyl)-3-oxo-3-phenylpropyl diethylcar-
bamodithioate (2f)
1H NMR (300 MHz, CDCl3): d 1.26–1.28 (m, 6H); 3.65–4.09 (m,
6H); 5.67–5.72 (dd, J = 4.4, 10.2 Hz, 1H); 6.93–6.99 (t, J = 8.61 Hz,
2H); 7.37–7.56 (m, 5H); 7.94–7.97 (m, 2H). ESI-MS: 376.1
(C20H23FNOS2, [M+H]+). Anal. Calcd for C20H22FNOS2: C, 63.97; H,
5.91; N, 3.73. Found: C, 64.33; H, 6.13; N, 3.99.
4.2. General procedure for synthesis of chalcones
To a stirred solution of acetophenone derivatives or acetone
(1 mmol) and a benzaldehyde derivatives (1 mmol) in MeOH
(30 mL) was added 6 M KOH (4 mL) and the reaction mixture
was stirred until the solids formed. The products were filtrated
and washed carefully with ice water and cool MeOH; the resulting
chalcones were purified by crystallization from MeOH in
refrigerator.
4.3.7. 1-(2-Methoxyphenyl)-3-oxo-3-phenylpropyl diethylcar-
bamodithioate (2g)
1H NMR (300 MHz, CDCl3): d 1.16–1.20 (m, 6H); 3.62–4.08 (m,
6H); 3.74 (s, 3H); 5.91–5.96 (dd, J = 4.7, 9.9 Hz, 1H); 6.75–6.82
(m, 2H); 7.09–7.18 (m, 1H); 7.31–7.46 (m, 4H); 7.89–7.92 (m,
2H). ESI-MS: 388.1 (C21H26NO2S2, [M+H]+). Anal. Calcd for
C21H25NO2S2: C, 65.08; H, 6.50; N, 3.61. Found: C, 65.39, H, 6.72;
N, 3.92.
4.3. General procedure for the preparation of dithiocarbamates
The CS2 (2.5 mmol) and chalcones (2 mmol) were dissolved in
CH2Cl2 (2 mL) and the solution was cooled to 0 °C in an ice bath.27
Amine (2.25 mmol) was slowly added and the reaction mixture
was stirred at 0 °C for 30 min. Then, the solution was warmed to
room temperature and stirred for another 24 h. After the end of
the reaction, the solvents were removed in vacuum and the residue
was purified by column chromatography on silica gel (ethyl ace-
tate–petroleum ether, 1/10–1/20) affording the compound dithio-
carbamates (2a–2t). (Scheme 1).
4.3.8. 1-(2-Chlorophenyl)-3-oxo-3-phenylpropyl diethylcar-
bamodithioate (2h)
1H NMR (300 MHz, CDCl3): d 1.24–1.29 (m, 6H); 3.66–4.03 (m,
6H); 5.69–5.73 (dd, J = 4.3, 10.0 Hz, 1H); 7.19–7.56 (m, 7H);
7.95–7.98 (m, 2H). ESI-MS: 392.1 (C20H23ClNOS2, [M+H]+). Anal.
Calcd for C20H22ClNOS2: C, 61.28; H, 5.66; N, 3.57. Found: C,
61.57; H, 5.86; N, 3.87.
4.3.1. 3-Oxo-1,3-diphenylpropyl diethylcarbamodithioate (2a)
1H NMR (300 MHz, CDCl3): d 1.24–1.29 (m, 6H); 3.67–4.19 (m,
6H); 6.08–6.13 (dd, J = 4.7, 9.7 Hz, 1H); 7.13–7.21 (m, 2H); 7.32–
7.56 (m, 6H); 7.96–8.00 (m, 2H). ESI-MS: 358.1 (C20H23NOS2,
[M+H]+). Anal. Calcd for C20H23NOS2: C, 67.19; H, 6.48; N, 3.92.
Found: C, 67.58; H, 6.61; N, 4.28.
4.3.9. 1-(3-Nitrophenyl)-3-oxo-3-phenylpropyl diethylcar-
bamodithioate (2i)
1H NMR (300 MHz, CDCl3): d 1.23–1.29 (m, 6H); 3.64–3.92 (m,
6H); 5.68–5.72 (dd, J = 4.4, 10.2 Hz, 1H); 7.43–7.88 (m, 7H);
7.98–8.12 (m, 2H). ESI-MS: 403.1 (C21H22N2O3S2, [M+H]+). Anal.
Calcd for C21H21N2O3S2: C, 59.68; H, 5.51; N, 6.96. Found: C,
59.93; H, 5.66; N, 7.28.
4.3.2. 1-(4-Methoxyphenyl)-3-oxo-3-phenylpropyl diethyl-
carbamodithioate (2b)
1H NMR (300 MHz, CDCl3): d 1.26–1.27 (m, 6H); 3.65–3.74 (m,
3H); 3.76 (s, 3H); 4.01–4.19 (m, 3H); 5.62–5.67 (dd, J = 4.2,
10.4 Hz, 1H); 6.80–6.83 (m, 2H); 7.32–7.35 (m, 2H); 7.40–7.45
(m, 2H); 7.51–7.56 (m, 1H); 7.97–8.00 (m, 2H). ESI-MS: 388.1
(C21H26NO2S2, [M+H]+). Anal. Calcd for C21H25NO2S2: C, 65.08; H,
6.50; N, 3.61. Found: C, 65.44; H, 6.72; N, 3.94.
4.3.10. 3-Oxo-1-phenylbutyl diethylcarbamodithioate (2j)
1H NMR (300 MHz, CDCl3): d 1.22–1.28 (m, 6H); 2.14 (s, 3H);
3.61–4.24 (m, 6H); 5.61–5.67 (m, 1H); 7.18–7.42 (m, 3H); 7.38–
7.51 (m, 2H). ESI-MS: 296.1 (C15H22NOS2, [M+H]+). Anal. Calcd
for C15H21NOS2: C, 60.98; H, 7.16; N, 4.74. Found: C, 61.33; H,
7.37; N, 4.96.
4.3.11. 3-(4-Methoxyphenyl)-3-oxo-1-phenylpropyl diethylcar-
bamodithioate (2k)
4.3.3. 3-Oxo-3-phenyl-1-p-tolylpropyl diethylcarbamodithioate
(2c)
1H NMR (300 MHz, CDCl3): d 1.25–1.28 (m, 6H); 3.63–3.71 (m,
3H); 3.84 (s, 3H); 4.00–4.11 (m, 3H); 5.68–5.73 (dd, J = 4.4,
10.0 Hz, 1H); 6.87–6.92 (m, 2H); 7.18–7.43 (m, 5H); 7.94–8.06
1H NMR (300 MHz, CDCl3): d 1.22–1.34 (m, 6H); 2.88 (s, 3H);
3.67–4.17 (m, 6H); 5.63–5.68 (dd, J = 4.2, 10.2 Hz, 1H); 7.08 (d,

Y. Qian et al. / Bioorg. Med. Chem. 18 (2010) 4310–4316
4315
(m, 2H). ESI-MS: 388.1 (C21H26NO2S2, [M+H]+). Anal. Calcd for
C21H25NO2S2: C, 65.08; H, 6.50; N, 3.61. Found: C, 65.41; H, 6.78;
N, 3.98.
C21H24ClNO2S2: C, 59.77; H, 5.73; N, 3.32. Found: C, 59.95; H,
5.92; N, 3.51.
4.3.20. 1-(4-Chlorophenyl)-3-(4-methoxyphenyl)-3-oxopropyl
diethylcarbamodithioate (2t)
4.3.12. 3-(4-Bromophenyl)-3-oxo-1-phenylpropyl diethylcar-
bamodithioate (2l)
1H NMR (300 MHz, CDCl3): d 1.23–1.27 (m, 6H); 3.63–3.70 (m,
2H); 3.85 (s, 3H); 3.88–4.05 (m, 4H); 5.67–5.72 (dd, J = 4.4,
10.1 Hz, 1H); 6.87–6.91 (m, 2H); 7.32–7.58 (m, 4H); 7.92–7.97
(m, 2H). ESI-MS: 422.1 (C21H25ClNO2S2, [M+H]+). Anal. Calcd for
C21H24ClNO2S2: C, 59.77; H, 5.73; N, 3.32. Found: C, 59.97; H,
5.94; N, 3.52.
1H NMR (300 MHz, CDCl3): d 1.22–1.29 (m, 6H); 3.62–4.15 (m,
6H); 5.62–5.67 (dd, J = 4.4, 10.3 Hz, 1H); 7.19–7.31 (m, 3H);
7.37–7.45 (m, 2H); 7.54–7.59 (m, 2H); 7.77–7.90 (m, 2H). ESI-
MS: 436.0 (C20H23BrNOS2, [M+H]+). Anal. Calcd for C20H22BrNOS2:
C, 55.04; H, 5.08; N, 3.21. Found: C, 55.35; H, 5.32; N, 3.54.
4.3.13. 3-(3,4-Dichlorophenyl)-3-oxo-1-phenylpropyl diethyl-
carbamodithioate (2m)
4.4. Crystal structure determination
1H NMR (300 MHz, CDCl3): d 1.25–1.32 (m, 6H); 3.60–4.05 (m,
6H); 5.59–5.65 (dd, J = 4.4, 10.4 Hz, 1H); 7.20–8.10 (m, 8H). ESI-
MS: 425.0 (C20H22Cl2NOS2, [M+H]+). Anal. Calcd for C20H21Cl2NOS2:
C, 56.33; H, 4.96; N, 3.28. Found: C, 56.79; H, 5.25; N, 3.44.
Crystal structure determination of compound 2l were carried
out on a Nonius CAD4 diffractometer equipped with graphite-
0
monochromated Mo Ka (k = 0.71073 ÅA) radiation. The structure
was solved by direct methods and refined on F2 by full-matrix
least-squares methods using SHELX-97.32 All the non-hydrogen
atoms were refined anisotropically. All the hydrogen atoms were
placed in calculated positions and were assigned fixed isotropic
thermal parameters at 1.2 times the equivalent isotropic U of the
atoms to which they are attached and allowed to ride on their
respective parent atoms. The contributions of these hydrogen
atoms were included in the structure-factors calculations. The
crystal data, data collection, and refinement parameter for the
compound 2l is listed in Table 3.
4.3.14. 3-(4-Chlorophenyl)-3-oxo-1-phenylpropyl diethyl-
carbamodithioate (2n)
1H NMR (300 MHz, CDCl3): d 1.24–1.28 (m, 6H); 2.97–4.01 (m,
6H); 5.63–5.68 (dd, J = 4.4, 10.2 Hz, 1H); 7.19–7.98 (m, 9H). ESI-
MS: 392.1 (C20H23ClNOS2, [M+H]+). Anal. Calcd for C20H22ClNOS2:
C, 61.28; H, 5.66; N, 3.56. Found: C, 61.54; H, 5.87; N, 3.87.
4.3.15. 3-(4-Bromophenyl)-1-(4-methoxyphenyl)-3-oxopropyl
diethylcarbamodithioate (2o)
1H NMR (300 MHz, CDCl3): d 1.22–1.27 (m, 6H); 3.51–3.73 (m,
3H); 3.75 (s, 3H); 3.85–4.02 (m, 3H); 5.56–5.61 (dd, J = 4.4,
10.6 Hz, 1H); 6.77–6.83 (m, 1H); 7.25–7.32 (m, 1H); 7.53–7.89
(m, 6H). ESI-MS: 466.0 (C21H25BrNO2S2, [M+H]+). Anal. Calcd for
C21H24BrNO2S2: C, 54.07; H, 5.19; N, 3.00. Found: C, 54.38; H,
5.28; N, 3.17.
4.5. Anti-proliferation assay
The antiproliferative activity of the prepared compounds
against MCF-7 human breast carcinoma cells was evaluated as de-
scribed elsewhere11 with some modifications. Target tumor cell
line was grown to log phase in RPMI 1640 medium supplemented
with 10% fetal bovine serum. After diluting to 2 ꢂ 104 cells mLꢀ1
4.3.16. 3-(4-Bromophenyl)-1-(4-fluorophenyl)-3-oxopropyl
diethylcarbamodithioate (2p)
with the complete medium, 100 lL of the obtained cell suspension
was added to each well of 96-well culture plates. The subsequent
incubation was permitted at 37 °C, 5% CO2 atmosphere for 24 h be-
fore the cytotoxicity assessments. Tested samples at pre-set con-
centrations were added to 6 wells with colchicine and CSA-4 co-
1H NMR (300 MHz, CDCl3): d 1.25–1.32 (m, 6H); 3.66–4.07 (m,
6H); 5.62–5.67 (dd, J = 4.2, 10.3 Hz, 1H); 6.94–6.99 (m, 2H);
7.35–7.39 (m, 2H); 7.56–7.66 (m, 2H); 7.82–7.85 (m, 2H). ESI-
MS: 454.0 (C20H22BrFNOS2, [M+H]+). Anal. Calcd for C20H21BrF-
NOS2: C, 52.86; H, 4.66; N, 3.08. Found: C, 52.97; H, 4.73; N, 3.31.
assayed as positive reference. After 48 h exposure period, 40 lL
of PBS containing 2.5 mg mLꢀ1 of MTT (3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide)) was added to each well.
4.3.17. 1,3-Bis (4-methoxyphenyl) -3-oxopropyl diethylcar-
bamodithioate (2q)
Table 3
1H NMR (300 MHz, CDCl3): d 1.20–1.26 (m, 6H); 3.51–3.75 (m,
3H); 3.77 (s, 3H); 3.81 (s, 3H); 3.85–3.99 (m, 3H); 5.54–5.60 (dd,
J = 4.4, 10.3 Hz, 1H); 6.78–6.89 (m, 4H); 7.25–7.82 (m, 4H). ESI-
MS: 418.1 (C22H28NO3S2, [M+H]+). Anal. Calcd for C22H27NO3S2: C,
63.28; H, 6.52; N, 3.35. Found: C, 63.53; H, 6.64; N, 3.57.
Crystallographical and experimental data for compound 2l
Compounds
2l
Empirical formula
Formula weight
Crystal system
Space group
a (Å)
C20H22BrNOS2
436.42
Monoclinic
P2(1)/n
7.5824(6)
15.8153(14)
16.9183(15)
90.00
94.548(4)
90.00
2022.4(3)
4
1.433
2.42–25.00
896
10,445/3549 [Rint = 0.0287]
3549/0/228
2.247
4.3.18. 3-(4-Methoxyphenyl)-3-oxo-1-p-tolylpropyl diethylcar-
bamodithioate (2r)
b (Å)
c (Å)
1H NMR (300 MHz, CDCl3): d 1.12–1.25 (m, 6H); 2.39 (s, 3H);
3.60–3.68 (m, 2H); 3.84 (s, 3H); 3.88–4.02 (m, 4H); 5.62–5.67
(dd, J = 4.4, 10.3 Hz, 1H); 6.88–6.99 (m, 2H); 7.20–7.31 (m, 2H);
7.44–7.55 (m, 2H); 7.94–8.05 (m, 2H). ESI-MS: 402.1 (C22H28NO2S2,
[M+H]+). Anal. Calcd for C22H27NO2S2: C, 65.80; H, 6.78; N, 3.49.
Found: C, 65.93; H, 6.86; N, 3.78.
a
(°)
b (°)
(°)
c
V (Å)
Z
Dcalcd/g cmꢀ3
h range (°)
F(0 0 0)
Reflections collected/unique
Data/restraints/parameters
Absorption coefficient (mmꢀ1
R1; wR2 [I > 2
R1; wR2 (all data)
GOF
4.3.19. 1-(2Chlorophenyl)-3-(4-methoxyphenyl)-3-oxopropyl
diethylcarbamodithioate (2s)
1H NMR (300 MHz, CDCl3): d 1.24–1.32 (m, 6H); 3.69–4.07 (m,
6H); 3.84 (s, 3H); 6.07–6.12 (dd, J = 4.9, 9.7 Hz, 1H); 6.82–6.97
(m, 2H); 7.13–7.20 (m, 2H); 7.30–7.52 (m, 2H); 7.97–8.05 (m,
2H). ESI-MS: 422.1 (C21H25ClNO2S2, [M+H]+). Anal. Calcd for
)
r(I)]
0.0315/0.0636
0.0531/0.0709
1.020

4316
Y. Qian et al. / Bioorg. Med. Chem. 18 (2010) 4310–4316
4 h later, 100
l
L extraction solution (10% SDS–5% isobutyl alcohol–
charge from The Director, CCDC, 12 Union Road, Cambridge CB2
1EZ, UK (fax: +44 1223 336 033; e-mail: deposit@ccdc.cam.ac.uk
or www: http://www.ccdc.cam.ac.uk).
0.01 M HCl) was added. After an overnight incubation at 37 °C, the
optical density was measured at a wavelength of 570 nm on an ELI-
SA microplate reader. In all experiments three replicate wells were
used for each drug concentration. Each assay was carried out at
least three times. The results were summarized in Table 2.
Acknowledgement
This work was supported by the Jiangsu National Science Foun-
dation (No. BK2009239) and by a grant (Project 30772627) from
National Natural Science Foundation of China.
4.6. Effects on tubulin polymerization
Bovine brain tubulin was purified as described previously.33 To
evaluate the effect of the compounds on tubulin assembly
References and notes
in vitro,34 varying concentrations were preincubated with 10
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Crystallographic data for the structural analysis have been
deposited with the Cambridge Crystallographic Data Centre Nos.
750890 for 2l. A copy of this information can be obtained free of