Synthesis, biological evaluation and molecular modeling of 1,3,4-thiadiazol-2-amide derivatives as novel antitubulin agents

Article abstract of DOI:10.1016/j.bmc.2014.05.017

A series of 1,3,4-thiadiazol-2-amide derivatives (6a-w) were designed and synthesized as potential inhibitors of tubulin polymerization and as anticancer agents. The in vitro anticancer activities of these compounds were evaluated against three cancer cel

Full text of DOI:10.1016/j.bmc.2014.05.017

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, biological evaluation and molecular modeling
of 1,3,4-thiadiazol-2-amide derivatives as novel antitubulin agents
Yu-Jing Li , Ya-Juan Qin , Jigar A. Makawana, Yan-Ting Wang, Yan-Qing Zhang, Ya-Liang Zhang,
⇑
⇑
Meng-Ru Yang, Ai-Qin Jiang , Hai-Liang Zhu
State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210093, PR China
School of Medicine, Nanjing University, Nanjing 210093, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 1,3,4-thiadiazol-2-amide derivatives (6a–w) were designed and synthesized as potential
inhibitors of tubulin polymerization and as anticancer agents. The in vitro anticancer activities of these
compounds were evaluated against three cancer cell lines by the MTT method. Among all the designed
compounds, compound 6f exhibited the most potent anticancer activity against A549, MCF-7 and HepG2
Received 13 April 2014
Revised 9 May 2014
Accepted 10 May 2014
Available online xxxx
cancer cell lines with IC50 values of 0.03
ited significant tubulin polymerization inhibitory activity (IC50 = 1.73
l
M, 0.06
l
M and 0.05
l
M, respectively. Compound 6f also exhib-
M), which was superior to the
l
Keywords:
positive control. The obtained results, along with a 3D-QSAR study and molecular docking that were used
for investigating the probable binding mode, could provide an important basis for further optimization of
compound 6f as a novel anticancer agent.
1,3,4-Thiadiazol-2-amide derivatives
Tubulin polymerization inhibitors
Anticancer activity
Ó 2014 Elsevier Ltd. All rights reserved.
Molecular docking
3D-QSAR
1. Introduction
tubulin by binding to the colchicine binding site resulting in
strong cytotoxicity against multiple human tumor.12 Last decade,
Microtubules play a key role in organizing the spatial distribu-
tion of organelles throughout interphase and of chromosomes
during cell division. The mitotic spindle, constituted of microtu-
extensive studies have been conducted to examine the structure–
activity relationship (SAR) of CA-4 and its analogues. The trimeth-
oxy substitutions on the A ring and the cis-olefin configuration at
the bridge have been reported as prerequisites for potent cytotox-
icity, while the B ring is tolerant of structural modifications.13,14
The trimethoxy substitutions on the A ring appear to be critical
for efficient binding to tubulin and are thought to make an additive
contribution to the strength of binding of colchicine to tubulin,
serving as an anchor that maintains the whole molecule in the
proper orientation within the binding locus.15–17cis-Olefin configu-
ration at the bridge of CA-4 has been reported as prerequisites for
potent cytotoxicity. Some alternative nonheterocyclic bridges
(e.g., amide derivatives) and heterocyclic bridges (e.g., thiazoles)
were used to modify olefin configuration.18–20
1,3,4-Thiadiazoles are five-membered ring systems that have
gained prominence by exhibiting a wide variety of biological
activities as well as producing useful intermediates in several
organic preparations. 1,3,4-Thiadiazole derivatives have been
reported to be anticancer,21 antimicrobial,22 anti-tubercular,23
anti-inflammatory24 and analgesic.23 The action of 1,3,4-thiadiaz-
oles connected with the apoptotic mechanisms and angiogenesis,
which is a crucial step in the tumorigenesis seems to be very
promising in anticancer therapy.25 Besides, amide derivatives were
associated with broad spectrum of biological activities including
antitumor properties and many tubulin binding agents containing
bules generated by noncovalent polymerization of
a-tubulin and
b-tubulin heterodimers has become an attractive and successful
target to treat many types of malignancies.1,2 Drugs that acts on
microtubules are well researched and of wide structural heteroge-
neity.3 Many of these agents work by inhibiting the polymerization
of tubulin into microtubules, thereby arresting cells in the G2/M
phase of the cell cycle, inhibiting cell division and leading to cell
death.4–6 Generally, there are three ligand binding sites in tubulin
a
/b-heterodimer: paclitaxel binding site, vinblastine binding site7,8
and colchicine binding site.8,9 Natural products such as Colchicine
(Fig. 1) and Combretastatin A-4 (CA-4, Fig. 1) exhibited potent
tubulin polymerization inhibitory ability by binding to colchicine
binding site and their distinctive structural features started
numerous projects in the development of antimitotic agents.10,11
CA-4 isolated from the bark of the South African tree
Combretum caffrum can strongly inhibit the polymerization of
⇑
Corresponding authors at: State Key Laboratory of Pharmaceutical Biotechnol-
ogy, School of Life Sciences, Nanjing University, Nanjing 210093, PR China. Tel.: +86
25 8359 2572; fax: +86 25 8359 2672.
E-mail addresses: jianaq@nju.edu.cn (A.-Q. Jiang), zhuhl@nju.edu.cn (H.-L. Zhu).
These two authors equally contributed to this paper.
http://dx.doi.org/10.1016/j.bmc.2014.05.017
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
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Y.-J. Li et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
H3CO
O
NHCOCH3
H3CO
O
O
OCH3
O
OH
OCH3
OCH3
Colchicine (1)
CA-4 (2)
OCH3
OCH3
H3CO
H3CO
H3CO
H3CO
S
N
NH
N
O
O
IC50=21-71 nM
IC50=133-288 nM
H3CO
H3CO
extracted
H3CO
H3CO
OCH3
OCH3
HN
N
OCH3
H3CO
H3CO
N
O
N
N
LYS B:352
N
IC50=6-24 nM
IC50=7-23 nM
N
N
H3CO
linker
NH
S
O
H3CO
OCH3
CYS B:241
SER A:140
Tubulin inhibihors
O
N
OCH3
OCH3
N
NH
N
N
H3CO
H3CO
S
extracted
S
NH
or
NH
S
N
O
O
H3CO
IC50=45-70 nM
IC50=280 nM
Figure 1. Chemical structures of antimitotic agents and lead tubulin inhibitors.
the amide moieties are now first-line clinical drugs or currently in
clinical trials for the treatment of solid tumors.26–28 Moreover, a
series of 1,3,4-thiadiazol-2-amide derivatives have been well designed
and found to be differentiating inducers of human cancer cells.29
These previous researches encouraged us to integrate
3,4,5-trimethylphenyl group with 1,3,4-thiadiazol-2-amide to
screen new 1,3,4-thiadiazol-2-amide derivatives as potential
antitubulin agents (Fig. 1). The two combined substructures might
exhibit synergistic effect in anticancer activities. A series of novel
thiadiazol derivatives were recently reported as potent anticancer
agents targeting tubulin in our group and some of them had
demonstrated potent antitumor activity.19 In order to extend
our research, here we describe the synthesis of these target
compounds and the ensuing structure–activity relationship (SAR)
N
N
R1
NH2
R1
COOH
S
R2
R3
R2
R3
S
i
H2N
N
H
NH2
R4
R4
3a-e
4
N
N
N
N
R5
R1
R1
R2
R6
R7
COOH
NH2
NH
S
S
ii
R2
R3
R8
R7
R3
O
R8
5
R5
R4
R4
R6
6a-w
4
Scheme 1. Synthesis of 1,3,4-thiadiazol-2-amide derivatives (6a–w). Reagents and conditions: (i) POCl3, 75 °C, 1 h; 50% KOH; (ii) EDCl, HOBt, dichloromethane, reflux 24 h.
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Y.-J. Li et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
Table 1
Table 2
Structure of compounds 6a–w
Crystallographical and experimental data for compound 6u
N
N
Compound
6u
R1
R2
NH
R5
S
Empirical formula
Formula weight
Crystal system
Space group
a (Å)
b (Å)
c (Å)
a (°)
C
21H20BrN3O5S
R8
R7
506.37
Triclinic
P-1
11.0460(11)
11.0650(12)
18.0719(18)
90.00
98.172(3)
90.00
2186.4(4)
20
2.354
1.86–25.14
1460
20364/3901
3901/0/288
9.693
R3
O
R4
R6
Compounds R1 R2
R3
R4
R5
R6
R7
R8
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
Br OCH3 OCH3 OCH3 Br
Br OCH3 OCH3 OCH3
Br OCH3 OCH3 OCH3 OCH3
Br OCH3 OCH3 OCH3
Br OCH3 OCH3 OCH3
Br OCH3 OCH3 OCH3
OCH3 OCH3 OCH3
OCH3 OCH3 OCH3
H
OCH3
H
OCH3 OCH3
H
Br
H
Cl
H
F
H
b (°)
c (°)
V (Å)
H
H
OCH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Z
Dcalcd/g cmÀ3
Br OCH3 OCH3 OCH3 Br
H
H
Br
H
Cl
H
F
h range (°)
F(000)
Reflections collected/unique
Data/restraints/parameters
Absorption coefficient (mmÀ1
)
Br OCH3 OCH3 OCH3
Br OCH3 OCH3 OCH3
Br OCH3 OCH3 OCH3
Br OCH3 OCH3 OCH3
Br OCH3 OCH3 OCH3
Br OCH3 OCH3 OCH3
Br OCH3 OCH3 OCH3
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
Br
Br
H
R1; wR2 [I > 2r(I)]
0.0539/0.1179
0.0924/0.1350
1.020
H
OH
R1; wR2 (all data)
GOF
H
H
H
H
H
H
H
H
H
OCH3 OCH3 OCH3
OCH3 OCH3 OCH3
OCH3 OCH3 OCH3
OCH3 OCH3 OCH3
OCH3 OCH3
OCH3 OCH3
OCH3 OCH3
OCH3 OCH3 OCH3
OCH3
OH
OCH3 OCH3 OCH3
H
OCH3 OCH3 OCH3
OCH3 OCH3 OCH3
H
H
H
H
H
2-amino-1,3,4-thiadiazoles and different substituted carboxylic
acids was performed by using 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide hydrochloride (EDCl) and N-hydroxybenzotriazole
(HOBt) in anhydrous methylene dichloride under refluxed condi-
tion to give the desired compounds 6a–w (Table 1). The synthesis
of compound 3a is shown in Scheme 2. 2-Bromo-3,4,5-trimethoxy
cinnamic acid was synthesized from 3,4,5-trimethoxy benzalde-
hyde by N-bromosuccinimide followed by treatment with malonic
acid and piperidine to yield the target product. All of the synthetic
compounds 6a–w gave satisfactory elementary analytical and spec-
troscopic data. 1H NMR, 13C NMR and ESI-MS spectra were consis-
tent with the assigned structures.
6s
6t
6u
6v
6w
H
H
H
H
H
H
H
OCH3
H
OCH3
Br OCH3 OCH3 OCH3
H
H
studies. Thus, once the 5-(3,4,5-trimethoxystyryl)-1,3,4-thiadiazol-
2-amide scaffold was identified as the minimum structural
requirement for activity in this new series of compounds, our strat-
egy for further development of active antimitotic agents was to
perform modifications on the phenyl ring and in the 2-position
of the 3,4,5-trimethylphenyl group. Molecular docking of some of
these inhibitors bound to tubulin revealed that introducing –Br
in the 2-position of the 3,4,5-trimethylphenyl group could
decrease the CDOCK-interaction-energy. We therefore designed a
series of 5-(2-bromo-3,4,5-trimethoxystyryl)-1,3,4-thiadiazol-
2-amide derivatives to improve potency and selectivity against
tubulin.
2.2. Crystal structure of compound 6u
Among these compounds, the crystal structure of compound 6u
was determined by X-ray diffraction analysis. The crystal data
presented in Table 2 and Figure 2 gave perspective views of
compound 6u with the atomic labeling system.
2.3. Biological activity
2. Results and discussion
2.1. Chemistry
2.3.1. In vitro antiproliferative activities
The cytotoxicity of a series of new 1,3,4-thiadiazol-2-amide
derivatives was evaluated against a panel of three different human
cancer cell lines including MCF-7 (human breast cancer cell line),
A549 (human lung cancer cells) and HepG2 (human hepatocellular
liver carcinoma cells) after a continuous exposure of 24 h. The
results are summarized in Table 3.
According to the data presented in Table 3, all the compounds
exhibited significant cytotoxicity with an IC50 values of 0–0.21 lM
in A549 cell line. In all detected cell lines, compound 6f was found
The synthetic route of the 1,3,4-thiadiazol-2-amide derivatives
6a–w is outlined in Scheme 1. These compounds were synthesized
from 2-amino-1,3,4-thiadiazoles
carboxylic acids 5. Firstly, the different substituted cinnamic acids
3a–e were treated with thiosemicarbazide in presence of phos-
phoryl chloride to yield 2-amino-1,3,4-thiadiazoles. Secondly, the
coupling reaction between the obtained different substituted
4 and different substituted
COOH
Br
Br
COOH
H2C
H3CO
NBS
i
H3CO
H3CO
CHO
H3CO
H3CO
CHO
COOH
ii
H3CO
H3CO
OCH3
OCH3
1
2
3a
Scheme 2. Synthesis of 2-bromo-3,4,5-trimethoxy cinnamic acid (3a). Reagents and conditions: (i) Bromosuccinimide, CHCl3, 90 °C, 3 h. (ii) Piperidine, pyridine, 80–90 °C,
12 h.
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Y.-J. Li et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
of –Cl > –Br > –F in HepG2 cell line, while the p-substituted showed
the similar order. Compounds (6h, IC50 = 0.16 M) with
m-substituted group showed slightly more potent activities than
l
those of p-substituted (6i, IC50 = 0.20 lM), while o-substituted
compound has the lowest activities.
2.3.2. Inhibition of tubulin polymerization
To validate whether the above anti-proliferative effect was
produced by interaction between tubulin and the synthesized
compounds, the synthesized compounds were evaluated for their
abilities to inhibit the tubulin polymerization. As expected, com-
pound 6f displayed the most potent anti-tubulin polymerization
activity and the most of the activity data suggested that the IC50
values of these compounds showed a similar tendency as their
relevant IC50 values in the anti-proliferative assay. Hence, a further
study comparing the anti-proliferative activity of the top 10 com-
pounds (6a, 6b, 6d, 6f, 6h, 6j, 6l, 6n, 6p and 6w) against A549 cell
line with the tubulin polymerization inhibitory activity was per-
formed and the result indicated that there was a moderate correla-
tion between tubulin polymerization inhibition and the inhibition
of cancer cellular proliferation as evidenced in Figure 3 with an R2
value of 0.947. Therefore, we could conclude that the synthesized
molecule inhibitors can inhibit the tubulin polymerization and
the anti-proliferative effect was produced partly by interaction
between tubulin and the compounds.
Figure 2. Crystal structure diagram of compound 6u. H atoms are shown as small
spheres of arbitrary radii.
Table 3
In vitro cell growth inhibitory effects of 1,3,4-thiadiazol-2-amide derivatives (6a–w)
against human tumor cell lines
Compounds
IC50 SD (lM)
A549a
MCF-7a
HepG2a
Tubulinb
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
0.13 0.02
0.14 0.01
0.17 0.03
0.06 0.01
0.15 0.02
0.03 0.01
0.21 0.05
0.08 0.03
0.16 0.04
0.07 0.01
0.14 0.02
0.08 0.02
0.19 0.03
0.12 0.05
0.15 0.02
0.13 0.04
0.16 0.06
0.19 0.03
0.16 0.04
0.17 0.02
0.18 0.05
0.19 0.01
0.11 0.04
0.10 0.02
0.47 0.20
0.51 0.12
0.60 0.21
0.17 0.04
0.56 0.14
0.06 0.03
0.37 0.11
0.26 0.12
0.32 0.10
0.23 0.13
0.28 0.12
0.34 0.09
0.38 0.05
0.36 0.10
0.48 0.19
0.48 0.10
0.49 0.14
0.46 0.07
0.56 0.11
0.54 0.12
0.55 0.21
0.59 0.20
0.29 0.04
0.31 0.06
0.20 0.02
0.21 0.06
0.23 0.03
0.12 0.04
0.22 0.10
0.05 0.01
0.25 0.13
0.16 0.04
0.20 0.05
0.15 0.02
0.18 0.07
0.17 0.10
0.24 0.08
0.22 0.04
0.27 0.12
0.25 0.04
0.28 0.08
0.26 0.09
0.22 0.06
0.26 0.09
0.21 0.10
0.21 0.11
0.18 0.04
0.16 0.02
16.79 2.12
16.95 1.33
17.91 1.56
3.08 0.32
18.79 2.42
1.73 0.28
19.45 1.02
6.02 0.45
15.09 1.01
5.68 0.53
11.81 1.35
7.08 0.32
15.63 0.91
13.44 0.55
22.5 1.52
15.5 1.46
23.94 1.63
23.67 1.21
28.06 1.15
29.44 1.16
28.58 1.09
30.36 1.21
9.83 0.30
2.1 0.12
2.3.3. Compound 6f induced apoptosis
In order to test whether the inhibition of cell growth of A549
was related to cell apoptosis, we performed a biparametric
cytofluorimetric analysis using propidium iodide (PI) and
annexin-V-FITC, which stain DNA and phosphatidylserine (PS) res-
idues, respectively.
A549 cells were treated with compound 6f at 0.01
and 0.10 M for 24 h, respectively, and DMSO served as a control.
The results showed that the percentage of cell apoptosis was 4.01%
at 0 M for compound 6f, and cell apoptosis was increased to
7.96%, 12.7% and 22.0% at 0.01 M, 0.05 M and 0.10 M, respec-
lM, 0.05 lM
l
l
l
l
l
6s
6t
6u
6v
6w
CA-4
tively, (Fig. 4). Thus, compound 6f could cause cell apoptosis like
other tubulin-binding agents.
2.3.4. Analysis of cell cycle effects
The effect of 6f on cell cycle progression was examined by flow
cytometry in A549 cells (Fig. 5). The compound 6f caused a
a
Inhibition of the growth of tumor cell lines.
Inhibition of tubulin polymerization.
b
to be the most active among all compounds exhibiting IC50 values of
0.03 M, 0.06 M and 0.05 M against A549, MCF-7 and HepG2 cell
l
l
l
lines, respectively. Of the 23 tested compounds, 6d, 6f, 6h, 6j and 6l
possessed the highest overall potency with IC50 values of 0.03–
0.34 lM against all the three typical cancer cell lines.
Meanwhile, we could draw a conclusion that the activity of the
tested compounds may be correlated to the variation and modifica-
tions of the structure. The importance of substituents on the phenyl
ring was demonstrated for activity and selectivity against the
different cancer cell lines. Comparing with 6w, the 3,4-dimethoxy
substituent of phenyl ring (6f) has a higher activity. Structure–
activity relationships in these new compounds demonstrated that
phenyl ring substituent containing 3-OCH3 (6d), 3-Br (6h) and
3-Cl (6j) resulted in enhancement of antiproliferative activity
against three cell lines, while the 3-F in phenyl ring (6l) resulted
in enhanced activity in two cell lines (A549 and HepG2 cells).
However, antitumor activities of the remaining compounds were
decreased.
Figure 3. Correlation between the anti-proliferative activity against A549 and the
tubulin polymerization inhibitory activity, R2 = 0.947, which indicated that there
In the halogen-substituted compounds, the compounds with
m-substituted (6h, 6j, 6l) (IC50 = 0.16
lM, 0.15 lM, 0.17 lM)
was
a moderate correlation between tubulin polymerization inhibition and
exhibited significant antiproliferative activities in the order
inhibition of cellular proliferation.
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5
Figure 4. Representative flow cytometric histograms of apoptotic A549 cells after 24 h treatment with 6f. The cells were harvested and labeled with Annexin-V-FITC and PI
and then analyzed by flow cytometry.
significant G2/M arrest in a concentration-dependent manner with
a rise in G2/M cells occurring at a concentration as low as 0.01 M.
At higher concentration of 0.10–1 M up to 89.26% of the cells
were arrested in G2/M. There was a concomitant decrease of cells
in the other phases of the cell cycle (G1 and S) as shown in Figure 5.
These findings confirmed a continuing impairment of cell division
and proved that compound 6f was a potent antitubulin agent.
hydrophobic interactions and binding was stabilized by two
hydrogen bonds and one -cation interaction. The oxygen atom
l
p
l
of one of the methoxyl groups on A-ring of 6f formed one hydrogen
bond with the amino hydrogen of Cys B:241 (bond length:
Cys241 N–H. . .O = 3.00) and the oxygen atom of one of the meth-
oxyl groups on B-ring of 6f formed another one with the amino
hydrogen of Ser A:140 (bond length: Ser140 N–H. . .O = 3.09). The
enzyme surface model (Fig. 6a) revealed that the molecule was
well embedded in the active pocket. In addition, a benzene ring
2.4. Computational
formed one
p–p interaction with Lys352. This molecular docking
2.4.1. Molecular docking
results along with the biological assay data suggested that com-
Molecular docking is an application wherein molecular model-
ing techniques are used to predict how protein receptors interact
with small molecules. Then, molecular docking of all the synthe-
sized compounds into active binding site of tubulin was performed
to simulate a binding mode derived from tubulin (PDB code: 1SA0).
All docking runs were applied LigandFit Dock protocol of Discovery
Studio 3.5.
The docking calculation of the synthesized compounds was
showed in Table 4. The interaction energy between compounds
6a–w and tubulin showed the corresponding results with tubulin
polymerization inhibitory activity. Among the synthesized
compounds, compound 6f showed the lowest interaction energy.
The binding modes of compound 6f and tubulin were depicted in
Figure 6. The amino acid residue which had interaction with
tubulin was labeled in Figure 6c. In the binding mode, compound
6f was nicely bound to the colchicine binding site of tubulin via
pound 6f was a potential inhibitor of tubulin.
2.4.1.1. 3D-QSAR model. In order to obtain a systematic SAR pro-
file on 1,3,4-thiadiazol-2-amide derivatives as antitumor agents
and to explore the more potent and selective tubulin inhibitors,
3D-QSAR model was built to choose activity conformation of the
designed molecular and reasonably evaluated the designed
molecules by using the corresponding pIC50 values which were
converted from the obtained IC50 (lM) values of tubulin inhibition
(the way of this transformation was derived from an online
calculator developed by an indian medicinal chemistry lab
(http://www.sanjeevslab.org/tools-IC50.html)) and performed by
built-in QSAR software of DS 3.5 (Discovery Studio 3.5, Accelrys,
Co. Ltd). The training and test sets were divided by the random
diverse molecules method of DS 3.5, in which the training set
accounted for 83% of all the molecules while the test set was set
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Y.-J. Li et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Figure 5. Effects of compound 6f on cell cycle progression of A549 cells were determined by flow cytometry analysis. A549 cells were treated with different concentrations of
6f for 48 h. The percentage of cells in each cycle phase was indicated.
Table 4
expected to increase activity. Similarly, steric map indicated areas
where steric bulk was predicted to increase (green) or decrease
(yellow) activity. It was widely acceptable that a better inhibitor
based on the 3D-QSAR model should have strong Van der Waals
attraction in the green areas and a polar group in the blue electro-
static potential areas (which were dominant close to the skeleton).
As expected, those potent compounds (6d, 6f, 6h and so on) not
only could circumvent the red subregion or the unfavorable yellow
steric subregion but also can get more close to the favorable blue
and green spaces. Thus, this promising model would provide a
guideline to design and optimize more effective tubulin inhibitors
and pave the way for us in the further study.
The docking calculation of 1,3,4-thiadiazol-2-amide derivatives (6a–w)
Compounds
CDOCKER
Compounds
CDOCKER
interaction energy
interaction energy
D
D
Gb(kcal/mol)
Gb(kcal/mol)
6a
6b
6c
6d
6e
6f
6g
6h
6i
À46.5067
À45.4588
À44.5666
À63.0058
À43.8867
À65.3216
À43.0038
À58.2294
À47.0025
À60.1096
À49.0023
À55.3425
6m
6n
6o
6p
6q
6r
6s
6t
6u
6v
6w
À46.8223
À48.5668
À42.5589
À47.0013
À40.8819
À41.3388
À40.5534
À40.1226
À39.2298
À38.3395
À50.9166
6j
6k
6l
3. Conclusion
In this paper, a series of novel compounds (6a–w) bearing
thiadiazol skeleton and amide moiety has been synthesized and
evaluated as tubulin polymerization inhibitors. These compounds
exhibited potent tubulin polymerization inhibitory activities and
antiproliferative activities against A549, MCF-7 and HepG2 cells.
Compound 6f showed the most potent antiproliferative activity
to 17%. The training set composes 19 agents and 4 agents were
consisted of the relative test set. The graphical relationship of
observed and predicted values has illustrated in Figure 7. In which
the plot of the observed IC50 versus the predicted values showed
that this model could be used in prediction of activity for 1,3,4-thia-
diazol-2-amide derivatives.
Also the molecules aligned with the iso-surfaces of the 3D-QSAR
model coefficients on electrostatic potential grids (Fig. 8a) and Van
der Waals grids (Fig. 8b) were listed. Electrostatic map indicated
red contours around regions where high electron density (negative
charge) was expected to increase activity, and blue contours repre-
sent areas where low electron density (partial positive charge) was
with an IC50 value of 0.03
A549, MCF-7 and HepG2 cell lines, respectively, as well as tubulin
polymerization inhibitory activity with an IC50 of 1.73 M. Further-
lM, 0.06 lM and 0.05 lM against
l
more, we also showed that 6f was a potent inducer of apoptosis in
A549 cells and it had cellular effects typical for microtubule inter-
acting agents, causing accumulation of cells in the G2/M phase of
the cell cycle. Molecular docking and 3D QSAR were further
performed to study the inhibitor-tubulin protein interactions.
According to the analysis of the binding model of compound 6f
with tubulin, several interactions were observed with the protein
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Y.-J. Li et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
7
Figure 7. The predicted versus experimental pIC50 values for the inhibition of
tubulin (PDB code: 1SA0).
4. Experimental section
4.1. Materials and measurements
All reagents and solvents were of analysis or synthesis grade. 1H
NMR and 13C NMR-spectra were recorded on a Bruker DPX300
model Spectrometer at 300 MHz, respectively, in DMSO-d6 and
chemical shifts were reported in ppm (d). TLC was performed on
the glass backed silica gel sheets (Silica Gel 60 GF254) and visual-
ized in UV light (254 nm and 365 nm). Column chromatography
was performed using silica gel (100–200 mesh) eluting with ethyl
acetate and petroleum ether. Melting points (uncorrected) were
measured on a X-4 MP apparatus (Taike Corp, Beijing, China).
4.2. Synthesis
4.2.1. General procedure for Synthesis of 2-bromo-3,4,5-
trimethoxybenzaldehyde (2)
To a solution of 3,4,5-trimethoxy benzaldehyde 1 (5.0 g, 25.4
mmol) in 50 mL chloroform (CHCl3) was added N-bromosuccini-
mide (5.44 g, 30.5 mmol). The solution was heated at reflux
temperature for 3 h. After the reaction was completed by TLC, the
reaction mixture was brought to room temperature, and the
solution was washed with water. The combined extracts were dried
with anhydrous Na2SO4 and concentrated. The crude bromobenzal-
dehyde was recrystallized from Et2O to get pure white solid with
97% yield.
4.2.2. General procedure for synthesis of 2-bromo-3,4,5-
trimethoxy cinnamic acid (3a)
Figure 6. (a) The surface model structure of compound 6f binding model with
tubulin complex. (b) Compound 6f (colored by atom: carbons: light blue; nitrogen:
blue; oxygen: red; sulfur: yellow; Bromine: dark red) is bond into tubulin (entry
1SA0 in the Protein Data Bank). The dotted lines show the hydrogen bonds and the
yellow line show the p-cation interactions. (c) 2D Ligand interaction diagram of
compound 6f with tubulin using Discovery Studio program with the essential
amino acid residues at the binding site are tagged in circles. The purple circles show
the amino acids which participate in hydrogen bonding, electrostatic or polar
interactions and the green circles show the amino acids which participate in the
Van der Waals interactions.
A mixture of aromatic aldehydes 2 (10 mmol), malonic acid
(12 mmol), piperidine (12 mmol) was dissolved in pyridine and
stirred on 80–90 °C for 12 h. The pyridine was removed under
vacuum. The reaction mixture was poured into ice water. The pre-
cipitate was filtered and washed with 10% HCl and dried under
vacuum to afford the 2-bromo-3,4,5-trimethoxy cinnamic acid 3a.
4.2.3. General procedure for synthesis of 2-amino-1,3,4-
thiadiazoles (4)
A stirring mixture of substituted Cinnamic acid (10 mmol),
N-aminothiourea (10 mmol) and POCl3 (10 mL) was heated at
75 °C for 1 h. After cooling down to room temperature, the mixture
was quenched with water and refluxed for 1 h. After cooling, the
residues in the colchicine binding site might play a crucial role in
its antitubulin polymerization and antiproliferative activities. The
research of these well designed compounds might be helpful for
the design and synthesis of tubulin polymerization inhibitors with
stronger activities.
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8
Y.-J. Li et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Figure 8. 3D-QSAR of 1,3,4-thiadiazol-2-amide derivatives for tubulin (PDB code: 1SA0). (a) Isosurface of the 3D-QSAR model coefficients on electrostatic potential grids. The
blue triangle mesh represents positive electrostatic potential and the red area represents negative electrostatic potential. (b) Isosurface of the 3D-QSAR model coefficients on
Van der Waals grids. The green triangle mesh representation indicates positive coefficients; the yellow triangle mesh indicates negative coefficients.
mixture was basified to pH 8 by dropwise addition of 50% KOH
solution under stirring. The precipitate was filtered and recrystal-
lized from ethanol to gain compound 4.
(ESI): 506.03 [M+H]+. Anal. Calcd for C21H20BrN3O5S: C, 49.81; H,
3.98; N, 8.30. Found: C, 49.82; H, 3.97; N, 8.29.
4.3.4. (E)-N-(5-(2-Bromo-3,4,5-trimethoxystyryl)-1,3,4-
4.2.4. General procedure for synthesis of target compounds
(6a–w)
thiadiazol-2-yl)-3-methoxybenzamide (6d)
White powders, yield 59.2%. Mp: 226–227 °C. 1H NMR
(DMSO-d6, 300 MHz) d: 13.17 (s, 1H), 7.71 (s, 2H), 7.67 (d, J
= 1.53 Hz, 2H), 7.49 (t, J = 6.12 Hz, 1H), 7.43 (s, 1H), 7.24 (d, J
= 7.47 Hz, 1H), 3.93(s, 3H), 3.87 (s, 3H), 3.83 (s, 3H), 3.82 (s, 3H).
13C NMR (DMSO-d6, 300 MHz) d: 55.88, 56.74, 61.23, 61.28,
106.70, 111.25, 113.40, 119.80, 121.21, 121.24, 130.32, 130.64,
133.14, 134.05, 144.00, 150.82, 152.22, 153.33, 159.73, 160.71,
165.22. MS (ESI): 506.03 [M+H]+. Anal. Calcd for C21H20BrN3O5S:
C, 49.81; H, 3.98; N, 8.30. Found: C, 49.80; H, 3.97; N, 8.31.
Compounds 6a–w were synthesized by coupling of substituted
2-amino-1,3,4-thiadiazoles with substituted carboxylic acids using
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
(EDCl) and N-hydroxybenzotriazole (HOBt) as catalysts. The mix-
ture was refluxed in anhydrous CH2Cl2 for 24 h. The products were
extracted with ethyl acetate. The extract was washed successively
with 10% HCl, saturated NaHCO3 and water. Then dried over anhy-
drous Na2SO4, filtered and evaporated. The residue was purified by
column chromatography using petroleum ether and ethyl acetate
(5:1).
4.3.5. (E)-N-(5-(2-Bromo-3,4,5-trimethoxystyryl)-1,3,4-
thiadiazol-2-yl)-4-methoxybenzamide (6e)
4.3. Spectral properties of 1,3,4-thiadiazol-2-amide derivatives
Yellow powders, yield 57.2%. Mp: 243–244 °C. 1H NMR
(DMSO-d6, 300 MHz) d: 12.98 (s, 1H), 8.16 (s, 1H), 8.14 (s, 1H),
7.65 (d, J = 2.07 Hz, 2H), 7.42 (s, 1H), 7.12 (s, 1H), 7.10 (s, 1H),
3.93(s, 3H), 3.87 (s, 3H), 3.83 (s, 3H), 3.82 (s, 3H). MS (ESI):
506.03 [M+H]+. Anal. Calcd for C21H20BrN3O5S: C, 49.81; H, 3.98;
N, 8.30. Found: C, 49.82; H, 3.97; N, 8.31.
4.3.1. (E)-2-Bromo-N-(5-(2-bromo-3,4,5-trimethoxystyryl)-
1,3,4-thiadiazol-2-yl)-3,4,5-trimethoxybenzamide (6a)
White powders, yield 40.6%. Mp: 179–180 °C. 1H NMR
(DMSO-d6, 300 MHz) d: 13.17 (s, 1H), 7.68 (d, J = 5.4 Hz, 2H), 7.44
(s, 1H), 7.24 (s, 1H), 3.94 (s, 3H), 3.88 (s, 3H), 3.85 (s, 3H), 3.84
(s, 3H), 3.84(s, 3H), 3.82 (s, 3H). MS (ESI): 643.96 [M+H]+. Anal.
Calcd for C23H23Br2N3O7S: C, 42.81; H, 3.59; N, 6.51. Found: C,
42.80; H, 3.60; N, 6.52.
4.3.6. (E)-N-(5-(2-Bromo-3,4,5-trimethoxystyryl)-1,3,4-
thiadiazol-2-yl)-3,4-dimethoxybenzamide (6f)
White powders, yield 54.9%. Mp: 262–263 °C. 1H NMR (DMSO-
d6, 300 MHz) d: 13.01 (s, 1H), 7.82 (d, J = 6.36 Hz, 1H), 7.78 (s, 1H),
7.65 (s, 2H), 7.42 (s, 1H), 7.14 (d, J = 6.39 Hz, 1H), 3.93(s, 3H), 3.88
(s, 3H), 3.87 (s, 3H), 3.83 (s, 3H), 3.82(s, 3H). 13C NMR (DMSO-d6,
300 MHz) d: 56.16, 56.22, 56.81, 61.26, 61.30, 105.60, 106.81,
111.23, 111.66, 121.39, 122.99, 127.50, 130.73, 133.82, 144.01,
148.88, 150.87, 152.21, 153.29, 153.38, 158.91, 164.40. MS (ESI):
536.04 [M+H]+. Anal. Calcd for C22H22BrN3O6S: C, 49.26; H, 4.13;
N, 7.83. Found: C, 49.27; H, 4.12; N, 7.82.
4.3.2. (E)-N-(5-(2-Bromo-3,4,5-trimethoxystyryl)-1,3,4-
thiadiazol-2-yl)-3,4,5-trimethoxybenzamide (6b)
Light yellow powders, yield 45.5%. Mp: 232–233 °C. 1H NMR
(DMSO-d6, 300 MHz) d: 13.15 (s, 1H), 7.67 (s, 2H), 7.53 (s, 2H),
7.43 (s, 1H), 3.93(s, 3H), 3.90 (s, 6H), 3.83 (s, 3H), 3.82 (s, 3H),
3.77(s, 3H). MS (ESI): 567.05 [M+H]+. Anal. Calcd for C23H24BrN3O7
S: C, 48.77; H, 4.27; N, 7.42. Found: C, 48.78; H, 4.26; N, 7.43.
4.3.3. (E)-N-(5-(2-Bromo-3,4,5-trimethoxystyryl)-1,3,4-
thiadiazol-2-yl)-2-methoxybenzamide (6c)
4.3.7. (E)-2-Bromo-N-(5-(2-bromo-3,4,5-trimethoxystyryl)-
1,3,4-thiadiazol-2-yl)benzamide (6g)
White powders, yield 56.3%. Mp: 204–205 °C. 1H NMR
(DMSO-d6, 300 MHz) d: 12.43 (s, 1H), 7.70 (t, J = 6.12 Hz, 2H),
7.58–7.65 (m, 2H), 7.44 (s, 1H), 7.24 (d, J = 6.24 Hz, 1H), 7.11
(t, J = 5.55 Hz, 1H), 3.93(s, 6H), 3.83 (s, 3H), 3.82 (s, 3H). MS
White powders, yield 40.2%. Mp: 218–219 °C. 1H NMR
(DMSO-d6, 300 MHz) d: 13.30 (s, 1H), 7.80 (d, J = 6.42 Hz, 1H),
7.70 (s, 1H), 7.67 (s, 1H), 7.48–7.57 (m, 2H), 7.45 (s, 1H),
3.94(s, 3H), 3.84 (s, 3H), 3.82 (s, 3H). MS (ESI): 553.93 [M+H]+. Anal.
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Y.-J. Li et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
9
Calcd for C20H17Br2N3O4S: C, 43.26; H, 3.09; N, 7.57. Found: C,
4.3.14. (E)-N-(5-(2-Bromo-3,4,5-trimethoxystyryl)-1,3,4-
43.25; H, 3.10; N, 7.56.
thiadiazol-2-yl)-3-hydroxybenzamide (6n)
White powders, yield 69.5%. Mp: 312–313 °C. 1H NMR (DMSO-
d6, 300 MHz) d: 13.06 (s, 1H), 9.90 (s, 1H), 7.65 (d, J = 2.82 Hz, 2H),
7.60 (d, J = 6.15 Hz, 1H), 7.49 (s, 1H), 7.42 (s, 1H), 7.37 (t, J
= 5.94 Hz, 1H), 7.06 (d, J = 7.44 Hz, 1H), 3.93 (s, 3H), 3.83 (s,3H),
3.82 (s,3H). MS (ESI): 492.02 [M+H]+. Anal. Calcd for C20H18BrN3O5
S: C, 48.79; H, 3.69; N, 8.53. Found: C, 48.80; H, 3.68; N, 8.54.
4.3.8. (E)-3-Bromo-N-(5-(2-bromo-3,4,5-trimethoxystyryl)-
1,3,4-thiadiazol-2-yl)benzamide (6h)
White powders, yield 47.2%. Mp: 279–280 °C. 1H NMR (DMSO-
d6, 300 MHz) d: 13.29 (s, 1H), 8.34 (s, 1H), 8.12 (d, J = 5.94 Hz, 1H),
7.89 (d, J = 5.97 Hz, 1H), 7.67 (d, J = 2.16 Hz, 2H), 7.55 (t, J = 5.91 Hz,
1H), 7.43 (s, 1H), 3.93(s, 3H), 3.83 (s, 3H), 3.82 (s, 3H). 13C NMR
(DMSO-d6, 300 MHz) d: 56.10, 61.24, 61.29, 105.60, 106.40,
116.90, 123.20, 126.55, 129.56, 129.89, 130.51, 133.53, 134.86,
136.05, 144.02, 150.11, 152.59, 153.14, 159.22, 165.00. MS (ESI):
553.93 [M+H]+. Anal. Calcd for C20H17Br2N3O4S: C, 43.26; H, 3.09;
N, 7.57. Found: C, 43.27; H, 3.10; N, 7.56.
4.3.15. (E)-3-Methoxy-N-(5-(3,4,5-trimethoxystyryl)-1,3,4-
thiadiazol-2-yl)benzamide (6o)
Yellow powders, yield 69.5%. Mp: 190–191 °C. 1H NMR
(DMSO-d6, 300 MHz) d: 13.08 (s, 1H), 7.71 (s, 2H), 7.59 (d, J
= 12.3 Hz, 1H), 7.43–7.51 (m, 2H), 7.24 (d, J = 6.27 Hz, 1H), 7.10
(s, 2H), 3.87 (s, 3H), 3.85 (s,6H), 3.70 (s, 3H). MS (ESI): 428.12
[M+H]+. Anal. Calcd for C21H21N3O5S: C, 59.00; H, 4.95; N, 9.83.
Found: C, 59.01; H, 4.94; N, 9.82.
4.3.9. (E)-4-Bromo-N-(5-(2-bromo-3,4,5-trimethoxystyryl)-
1,3,4-thiadiazol-2-yl)benzamide (6i)
White powders, yield 50.2%. Mp: 295–296 °C. 1H NMR (DMSO-
d6, 300 MHz) d: 13.28 (s, 1H), 8.08 (s, 1H), 8.06 (s, 1H), 7.81 (s, 1H),
7.79 (s, 1H), 7.66 (d, J = 1.62 Hz, 2H), 7.42 (s, 1H), 3.93(s, 3H), 3.83
(s, 3H), 3.82 (s, 3H). MS (ESI): 553.93 [M+H]+. Anal. Calcd for C20
H17Br2N3O4S: C, 43.26; H, 3.09; N, 7.57. Found: C, 43.27; H,
3.08 N, 7.56.
4.3.16. (E)-3,4-Dimethoxy-N-(5-(3,4,5-trimethoxystyryl)-1,3,4-
thiadiazol-2-yl)benzamide (6p)
White powders, yield 40.9%. Mp: 234–235 °C. 1H NMR
(DMSO-d6, 300 MHz) d: 12.93 (s, 1H), 7.80 (t, J = 74.7 Hz, 2H),
7.58 (d, J = 12.3 Hz, 1H), 7.44 (d, J = 12.27 Hz, 1H), 7.14 (d, J
= 6.42 Hz, 1H), 7.10(s, 2H), 3.89 (s, 3H), 3.87 (s, 3H), 3.86 (s, 6H),
3.70 (s, 3H). MS (ESI): 458.13 [M+H]+. Anal. Calcd for C22H23N3O6S:
C, 57.76; H, 5.07; N, 9.18. Found: C, 57.77; H, 5.06; N, 9.19.
4.3.10. (E)-N-(5-(2-Bromo-3,4,5-trimethoxystyryl)-1,3,4-
thiadiazol-2-yl)-3-chlorobenzamide (6j)
White powders, yield 59.2%. Mp: 293–294 °C.1H NMR (DMSO-
d6, 300 MHz) d: 13.28 (s, 1H), 8.20 (s, 1H), 8.08 (d, J = 5.88 Hz,
1H), 7.76 (d, J = 5.94 Hz, 1H), 7.68 (d, J = 2.04 Hz, 2H), 7.62
(t, J = 5.94 Hz, 1H), 7.44 (s, 1H), 3.93(s, 3H), 3.83 (s, 3H), 3.82 (s,
3H). 13C NMR (DMSO-d6, 300 MHz) d: 56.11, 61.20, 61.25, 104.89,
106.26, 116.55, 125.45, 127.33, 129.88, 130.12, 132.21, 133.40,
134.50, 135.82, 144.03, 50.28, 151.99, 152.50, 159.00, 165.32. MS
(ESI): 509.98 [M+H]+. Anal. Calcd for C20H17BrClN3O4S: C, 47.03;
H, 3.35; N, 8.23. Found: C, 47.02; H, 3.34; N, 8.24.
4.3.17. (E)-3,4,5-Trimethoxy-N-(5-(3,4,5-trimethoxystyryl)-
1,3,4-thiadiazol-2-yl)benzamide (6q)
White powders, yield 39.5%. Mp: 205–206 °C. 1H NMR
(DMSO-d6, 300 MHz) d: 13.05 (s, 1H), 7.57 (d, J = 12.3 Hz, 1H),
7.51 (s, 2H), 7.43 (d, J = 9.3 Hz, 1H), 7.09 (s, 2H), 3.89 (s, 6H),
3.84 (s, 6H), 3.76 (s, 3H), 3.69 (s, 3H). MS (ESI): 488.14 [M+H]+.
Anal. Calcd for C23H25N3O7S: C, 56.66; H, 5.17; N, 8.62. Found: C,
56.65; H, 5.16; N, 8.63.
4.3.11. (E)-N-(5-(2-Bromo-3,4,5-trimethoxystyryl)-1,3,4-
thiadiazol-2-yl)-4-chlorobenzamide (6k)
4.3.18. (E)-3-Hydroxy-N-(5-(3,4,5-trimethoxystyryl)-1,3,4-
thiadiazol-2-yl)benzamide (6r)
White powders, yield 44.5%. Mp: 285–286 °C. 1H NMR (DMSO-
d6, 300 MHz) d: 13.26 (s, 1H), 8.16 (s, 1H), 8.14 (s, 1H), 7.62–7.71
(m, 4H), 7.43 (s, 1H), 3.93(s, 3H), 3.83 (s, 3H), 3.82 (s, 3H). MS
(ESI): 509.98 [M+H]+. Anal. Calcd for C20H17BrClN3O4S: C, 47.03;
H, 3.35; N, 8.23. Found: C, 47.04; H, 3.34; N, 8.24.
White powders, yield 69.5%. Mp: 281–282 °C. 1H NMR (DMSO-
d6, 300 MHz) d: 12.98 (s, 1H), 9.89 (s, 1H), 7.58 (t, J = 7.11 Hz, 2H),
7.47 (d, J = 3.57 Hz, 2H), 7.37 (t, J = 5.94 Hz, 1H), 7.10 (s, 2H), 7.06
(d, J = 7.50 Hz, 1H), 3.95 (s, 6H), 3.70 (s,3H). MS (ESI): 414.10
[M+H]+. Anal. Calcd for C20H19N3O5S: C, 58.10; H, 4.63; N, 10.16.
Found: C, 58.11; H, 4.64; N, 10.15.
4.3.12. (E)-N-(5-(2-Bromo-3,4,5-trimethoxystyryl)-1,3,4-
thiadiazol-2-yl)-3-fluorobenzamide (6l)
4.3.19. (E)-N-(5-(3,4-Dimethoxystyryl)-1,3,4-thiadiazol-2-yl)-
3,4,5-trimethoxybenzamide (6s)
White powders, yield 48.2%. Mp: 273–274 °C. 1H NMR
(DMSO-d6, 300 MHz) d: 13.28 (s, 1H), 7.98 (t, J = 6.0 Hz, 2H), 7.67
(d, J = 1.59 Hz, 3H), 7.53–7.57 (m, 1H), 7.43 (s, 1H), 3.93(s, 3H),
3.83 (s, 3H), 3.82 (s, 3H). 13C NMR (DMSO-d6, 300 MHz) d: 56.15,
61.22, 61.27, 105.22, 106.35, 116.60, 124.26, 127.00, 129.78,
130.00, 131.98, 133.45, 134.67, 135.99, 144.02, 150.20, 152.00,
153.11, 159.12, 165.13. MS (ESI): 494.01 [M+H]+. Anal. Calcd for
White powders, yield 40.6%. Mp: 203–204 °C. 1H NMR
(DMSO-d6, 300 MHz) d: 13.02 (s, 1H), 7.52 (s, 2H), 7.46 (s, 1H),
7.44 (s, 1H), 7.39 (d, J = 1.23 Hz, 1H), 7.24–7.26 (m, 1H), 7.00 (d, J
= 6.3 Hz, 1H), 3.90 (s, 6H), 3.85 (s, 3H), 3.81 (s, 3H), 3.77 (s, 3H).
MS (ESI): 458.13 [M+H]+. Anal. Calcd for C22H23N3O6S: C, 57.76;
H, 5.07; N, 9.18. Found: C, 57.75; H, 5.06; N, 9.19.
C20H17BrFN3O4S: C, 48.59; H, 3.47; N, 8.50. Found: C, 48.58; H,
3.48; N, 8.51.
4.3.20. (E)-N-(5-(3,4-Dimethoxystyryl)-1,3,4-thiadiazol-2-yl)-2-
methoxybenzamide (6t)
4.3.13. (E)-N-(5-(2-Bromo-3,4,5-trimethoxystyryl)-1,3,4-
thiadiazol-2-yl)-4-fluorobenzamide (6m)
White powders, yield 42.5%. Mp: 207–208 °C. 1H NMR
(DMSO-d6, 300 MHz) d: 12.31 (s, 1H), 7.70–7.73 (m, 1H), 7.60 (t, J
= 6.57 Hz, 1H), 7.47 (s, 1H), 7.45 (s, 1H), 7.40 (d, J = 1.53 Hz, 1H),
7.23–7.27 (m, 2H), 7.12 (t, J = 5.55 Hz, 1H), 7.00 (d, J = 6.30 Hz,
1H), 3.94 (s, 3H), 3.84 (s, 3H), 3.81 (s, 3H). MS (ESI): 398.11
[M+H]+. Anal. Calcd for C20H19N3O4S: C, 60.44; H, 4.82; N, 10.57.
Found: C, 60.43; H, 4.83; N, 10.56.
White powders, yield 46.8%. Mp: 277–278 °C. 1H NMR
(DMSO-d6, 300 MHz) d: 13.19 (s, 1H), 8.21–8.24 (m, 2H), 7.66
(d, J = 1.77 Hz, 2H), 7.43 (t, J = 6.66 Hz, 3H), 3.93(s, 3H), 3.83 (s, 3H),
3.82 (s, 3H). MS (ESI): 494.01 [M+H]+. Anal. Calcd for C20H17BrFN3O4S:
C, 48.59; H, 3.47; N, 8.50. Found: C, 48.60; H, 3.48; N, 8.51.
Please cite this article in press as: Li, Y.-J.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.017

10
Y.-J. Li et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
4.3.21. (E)-2-Bromo-3,4,5-trimethoxy-N-(5-(4-methoxystyryl)-
1,3,4-thiadiazol-2-yl)benzamide (6u)
4.6. Tubulin polymerization assay
White powders, yield 40.5%. Mp: 256–257 °C. 1H NMR
(DMSO-d6, 300 MHz) d: 13.06 (s, 1H), 7.70 (s, 1H), 7.68 (s, 1H),
Bovine brain tubulin was purified as described previously.31 To
evaluate the effect of the compounds on tubulin assembly in vitro,
varying concentrations of compounds were preincubated with
7.48 (d,
J = 12.33 Hz, 1H), 7.41 (d, J = 12.33 Hz, 1H), 7.23
(s, 1H), 7.01 (s, 1H), 6.99 (s, 1H), 3.87 (s, 3H), 3.84 (s,3H), 3.84
(s,3H), 3.81 (s, 3H). MS (ESI): 506.03 [M+H]+. Anal. Calcd for C21H20
BrN3O5S: C, 49.81; H, 3.98; N, 8.30. Found: C, 49.80; H, 3.97; N, 8.31.
10 lM bovine brain tubulin in glutamate buffer at 30 °C and then
cooled to 0 °C.32,33 After the addition of 0.4 mM GTP, the mixtures
were transferred to 0 °C cuvettes in a recording spectrophotometer
and warmed up to 30 °C. Then, the assembly of tubulin was
observed turbidimetrically at 350 nm. The IC50 was defined as
the compound concentration that inhibited the extent of assembly
by 50% after 20 min incubation.
4.3.22. (E)-2-Bromo-3,4,5-trimethoxy-N-(5-(3-methoxystyryl)-
1,3,4-thiadiazol-2-yl)benzamide (6v)
White powders, yield 39.5%. Mp: 194–195 °C. 1H NMR (DMSO-
d6, 300 MHz) d: 13.11 (s, 1H), 7.59 (s, 1H), 7.52 (s, 1H), 7.30–7.36
(m, 3H), 7.24 (s, 1H), 6.95 (d, J = 5.97 Hz, 1H), 3.88 (s, 3H), 3.85
(s,3H), 3.84 (s,3H), 3.82 (s, 3H). MS (ESI): 506.03 [M+H]+. Anal.
Calcd for C21H20BrN3O5S: C, 49.81; H, 3.98; N, 8.30. Found: C,
49.82; H, 3.97; N, 8.31.
4.7. Annexin-V Assay
Surface exposure of PS on apoptotic cells was measured by flow
cytometry with a Coulter Cytomics FC500 (Beckman Coulter) by
adding annexin-V-FITC to cells according to the manufacturer’s
instructions (Annexin-V Fluos, Roche Diagnostic). Simultaneously,
the cells were stained with PI.
4.3.23. (E)-N-(5-(2-Bromo-3,4,5-trimethoxystyryl)-1,3,4-
thiadiazol-2-yl)benzamide (6w)
White powders, yield 45.9%. Mp: 237–238 °C. 1H NMR (DMSO-
d6, 300 MHz) d: 13.12 (s, 1H), 8.07 (d, J = 5.64 Hz, 2H), 7.60 (s, 3H),
7.52 (t, J = 5.61 Hz, 2H) 7.36 (s, 1H), 3.86 (s, 3H), 8.07 (d, J = 4.05 Hz,
6H). 13C NMR (DMSO-d6, 300 MHz) d: 56.02, 61.14, 61.20, 104.92,
106.22, 116.75, 126.88, 127.45, 128.88, 130.10, 132.12, 133.26,
134.81, 135.45, 144.30, 152.00, 152.12, 152.33, 158.26, 165.32.
MS (ESI): 476.34 [M+H]+. Anal. Calcd for C20H18BrN3O4S: C,
50.43; H, 3.81; N, 8.82. Found: C, 50.42; H, 3.80; N, 8.83.
4.8. Flow Cytometric Analysis of Cell Cycle Distribution
For flow cytometric analysis of DNA content, 5 Â 105 A549 cells
in exponential growth were treated with different concentrations
of 6f for 48 h. After the incubation, the cells were collected, centri-
fuged, and fixed with ice-cold ethanol (70%). The cells were treated
with lysis buffer containing RNase A and 0.1% Triton X-100 and
stained with PI. Samples were analyzed on a Cytomic FC500 flow
cytometer (Beckman Coulter). DNA histograms were analyzed
using ModFit for Windows.
4.4. Crystal structure determination
Crystal structure determination of compound 6u was carried
out on a Nonius CAD4 diffractometer equipped with graphite-
monochromated Mo Ka (k = 0.71073 Å) 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 parameters for the
compound 6u are listed in Table 2.
4.9. Experimental protocol of docking study
Molecular docking of compound 6f into the three dimensional
X-ray structure of tubulin (PDB code: 1SA0) was carried out using
the Discovery Studio (version 3.5) as implemented through
the graphical user interface DS-CDOCKER protocol. The three-
dimensional structure of the compound was constructed using
Chem. 3D ultra 12.0 software [Chemical Structure Drawing
Standard; Cambridge Soft corporation, USA (2010)], then it was
energetically minimized by using MMFF94 with 5000 iterations
and minimumRMS gradient of 0.10. The crystal structure of protein
complex was retrieved from the RCSB Protein Data Bank (http://
www.rcsb.org/pdb/home/home.do). All bound waters and ligands
were eliminated from the protein and the polar hydrogenwas
added to the proteins. Molecular docking of all twenty compound-
swas then carried out using the Discovery Stutio (version 3.5) as
implemented through the graphical user interface CDOCKER proto-
col. CDOCKER is an implementation of a CHARMm based molecular
docking tool using a rigid receptor.
4.5. Antiproliferation assay
The antiproliferative activities of the prepared compounds
against A549, MCF-7 and HepG2 cells were evaluated as described
elsewhere with some modifications.30 Generally, target tumor cell
lines were grown to log phase in RPMI 1640 medium supple-
mented with 10% fetal bovine serum,100 mg/mL streptomycinand
100 IU/mL penicillin. After diluted to 2 Â 104 cells mLÀ1 with the
4.9.1. 3D-QSAR
complete medium, 100
l
L of the obtained cell suspension was
Ligand-based 3D-QSAR approach was performed by QSAR soft-
ware of DS 3.5 (Discovery Studio 3.5, Accelrys, Co. Ltd). The train-
ing sets were composed of inhibitors with the corresponding pIC50
added to each well of 96-well culture plates. Following this, the
subsequent incubation was then permitted at 37 °C, 5% CO2 atmo-
sphere for 24 h before the cytotoxicity assessments.
values which were converted from the obtained IC50 (lM), and test
Tested samples at pre-set concentrations were added to six
wells with CA-4 as positive references. After 48 h exposure period,
sets comprised compounds of data sets. All the definition of the
descriptors can be seen in the ‘Help’ of DS 3.5 software and they
were calculated by QSAR protocol of DS 3.5. The alignment confor-
mation of each molecule was the one with lowest interaction
energy in the docked results of CDOCKER. The predictive ability
of 3D-QSAR modeling can be evaluated based on the cross-
validated correlation coefficient, which qualifies the predictive
ability of the models. Scrambled test (Y scrambling) was performed
to investigate the risk of chance correlations. The inhibitory
40
2-yl)-2,5-diphenyltetrazolium bromide)) was added to each well.
Four hours later, 100 L DMSO was added and the optical density
l
L PBS containing 2.5 mg mLÀ1 MTT (3-(4,5-dimethylthiazol-
l
was measured at a wavelength of 490 nm on an ELISA microplate
reader. In all experiments three replicate wells were used for each
drug concentration. Each assay was carried out for at least three
times. The results were summarized in Table 3.
Please cite this article in press as: Li, Y.-J.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.017

Y.-J. Li et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
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Acknowledgments
The work was financed by a Grant (No. J1103512) from National
Natural Science Foundation of China and Major Projects on Control
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