Synthesis, biological evaluation, and molecular modeling of cinnamic acyl sulfonamide derivatives as novel antitubulin agents

Article abstract of DOI:10.1016/j.bmc.2011.06.088

A series of novel cinnamic acyl sulfonamide derivatives (9a-16e) have been designed and synthesized and their biological activities were also evaluated as potential tubulin polymerization inhibitors. Among all the compounds, 10c showed the most potent growth inhibitory activity against B16-F10 cancer cell line in vitro, with an IC₅₀ value of 0.8 μg/mL. Docking simulation was performed to insert compound 10c into the crystal structure of tubulin at colchicine binding site to determine the probable binding model. Based on the preliminary results, compound 10c with potent inhibitory activity in tumor growth may be a potential anticancer agent.

Full text of DOI:10.1016/j.bmc.2011.06.088

Bioorganic & Medicinal Chemistry 19 (2011) 4730–4738
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, biological evaluation, and molecular modeling of cinnamic acyl
sulfonamide derivatives as novel antitubulin agents
⇑
Yin Luo, Ke-Ming Qiu, Xiang Lu, Kai Liu, Jie Fu, 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 cinnamic acyl sulfonamide derivatives (9a–16e) have been designed and synthesized
and their biological activities were also evaluated as potential tubulin polymerization inhibitors. Among
all the compounds, 10c showed the most potent growth inhibitory activity against B16-F10 cancer cell
Received 9 June 2011
Revised 29 June 2011
Accepted 30 June 2011
Available online 7 July 2011
line in vitro, with an IC50 value of 0.8 lg/mL. Docking simulation was performed to insert compound
10c into the crystal structure of tubulin at colchicine binding site to determine the probable binding
model. Based on the preliminary results, compound 10c with potent inhibitory activity in tumor growth
may be a potential anticancer agent.
Keywords:
Cinnamic acid
Sulfonamide
Ó 2011 Elsevier Ltd. All rights reserved.
Antiproliferative activity
Molecular docking
Antitubulin polymerization
1. Introduction
agents, such as E7010, CA-4, and HMN-214 (Fig. 1). They bound
tubulins through the same site as colchicine.9 Among these struc-
With the changes in the living environment, cancer has become
the second major cause of death in developed countries.1 Over
1 million cases of cancer occur in the United States annually and
cancer-related deaths are estimated to reach 12 million worldwide,
by the year 2015.2 The inducement of cancer is complex involving
many molecular mechanisms. Out of control in cell replication is
one of the most important mechanisms that cause cancer. During
the process of cell replication, microtubules, key cytoskeletal
filaments and involved in numerous cellular functions, play an
important role. During cell mitosis, the microtubules extend from
the cell centrosome to form mitotic spindle and fasten to the kine-
tochore of chromosomes. Then, the kinetochores are gathered
around the equatorial plate.1 Microtubules are in dynamic equilib-
rium with tubulin dimers.3 Disruption of the dynamic equilibrium
will lead to cell cycle arrest or cell apoptosis. Given their significant
roles in the growth and function of cells, microtubules or tubulins
are among the most important molecular targets for cancer chemo-
therapeutic agents.
tures (MDL-27048, compound 8, and compound 9), chalcone is a
promising skeleton that has shown the good antitubulin activity.
Chalcones, precursors of flavonoids and isoflavones, are abundant
in edible plants and display a variety of biological activities, such
as anti-cancer,10–12 anti-inflammatory,13,14 anti-tuberculosis,15
and anti-fungal activities.16 Their broad biological properties are
largely due to the a, b-unsaturated ketone moiety. The introduction
of substituents on the two aromatic rings remains an area of
pharmacological interest to screen active chalcones,17 such as
MDL-27048 (Fig. 1).18
As shown in Figure 2, chalcone and cinnamon amide own similar
part of the active structure. Cinnamoyl moiety was found in a variety
of biologically active substances.19,20 Antitumor activities of various
cinnamic acid derivatives were also explored by our research
group.21 In addition, many sulfonamides displayed potent biological
activities and low toxicities in previous reports. Among them, some
compounds showed their anticancer effects by inhibiting tubulin to
form microtubulin, such as E7010 and HMN-241.22,23 They are now
undergoing human clinical trial against various tumor types.24,25
The amide bond is not only active binding site in many active com-
pounds but also the long-term research object in our laboratory.26
Various cinnamic acid derivatives were synthesized and evaluated
their antitumor activities in our previous work.25 It encouraged us
to continuously screen new cinnamic acyl sulfonamides as potential
antitubulin agents. The two combined substructures, cinnamic acid
and sulfonamide, might exhibit synergistic effect in anticancer
activities. The objectives of present work are (1) to synthesize new
A number of small molecules were found to bind tubulins, inter-
fering with microtubule polymerization or depolymerization, and
then induce cell cycle arrest, leading to cell death.4–7 Colchicine
(Fig. 1) is the first drug that is well known to bind tubulin, and its
binding site on tubulin has been characterized recently.8 Several
other new compounds have also been synthesized as antitubulin
⇑
Corresponding author. Tel./fax: +86 25 83592672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.088

Y. Luo et al. / Bioorg. Med. Chem. 19 (2011) 4730–4738
4731
H
N
H3CO
H3CO
N
H3CO
H3CO
NHCOCH3
NH
OH
OCH3
SO2
OCH3
O
OH
OCH3
1 Colchicine
OCH3
H3CO
3 E7010
2 CombretastainA-4 (CA-4)
O
N
OCH3
O
COCH3
N
SO2
OCH3
N
H3CO
5 MDL-27048
4 HMN-214
OCH3
OCH3
F
OCH3
OCH3
F
F
O2
S
O2
S
F
F
N
H
N
H
N
F
6
7
CH3
O
O
CH3
OCH3
OCH3
H3CO
H3CO
N
OCH3
OCH3
8
9 C19
Figure 1. Chemical structures of antimitotic agents and lead tubulin inhibitors.
2. Results and discussion
H
N
2.1. Chemistry
R
O
O
The synthetic route for the novel cinnamic acyl sulfonamide deriv-
atives 9a–16e is outlined in Scheme 1. These compounds were syn-
thesized from cinnamic acids 9–16 and active sulfonamides a–e.
Compounds 9–16 were prepared according to the procedure
reported by Davis et al. with some modifications.27 Aromatic
aldehydes and malonic acid were dissolved in a mixture of pyridine
and piperidine and refluxed for 24 h, and cinnamic acids were
obtainedwithyieldsof75–85%. Then,cinnamicacids9–16, active sul-
fonamides a–e, 4-dimethyaminopyridine (DMAP) and 1-[3-(dimet-
hyamino)-propyl]-3-ethylcarbodiimide hydrochloride (EDCI) were
A
B
Figure 2. Similar part of chalcone and cinnamon amide.
cinnamic acyl sulfonamides; (2) to evaluate their anticancer and
antitubulin activities; (3) to explore the preliminary mechanism of
their role in cell division cycle and (4) to investigate the inhibitor
interaction with tubulin by docking study.

4732
Y. Luo et al. / Bioorg. Med. Chem. 19 (2011) 4730–4738
R1
R1
CHO
+
COOH
HOOC
COOH
a
R2
R2
1-8
9-16
R3
R1
R3
R1
O
O
COOH
+
O
S
b
N
S
O
H
R2
NH2
O
R2
9-16
a-e
9a-16e
Scheme 1. General synthesis of cinnamic acid sulfonamide derivatives (9a–16e). Reagents and conditions: (a) piperidine, pyridine, 80–90 °C, 24 h; (b) CH2Cl2, EDCI, DMAP,
overnight.
Table 1
Structure of compounds 9a–16e
R3
R1
A
O
B
O
S
N
O
H
R2
Compounds
9a
H
H
9b
H
H
9c
H
H
F
9d
H
H
9e
H
H
10a
H
F
10b
H
F
10c
H
F
10d
H
F
10e
H
F
R1
R2
R3
H
CH3
Cl
Br
H
CH3
F
Cl
Br
Compounds
11a
H
Cl
11b
H
Cl
11c
H
Cl
F
11d
H
Cl
11e
H
Cl
12a
H
Br
H
12b
H
Br
12c
H
Br
F
12d
H
Br
12e
H
Br
R1
R2
R3
H
CH3
Cl
Br
CH3
Cl
Br
Compounds
13a
H
OCH3
H
13b
H
OCH3
CH3
13c
H
OCH3
F
13d
H
OCH3
Cl
13e
H
OCH3
Br
14a
H
N(CH3)2
H
14b
H
N(CH3)2
CH3
14c
H
N(CH3)2
F
14d
H
N(CH3)2
Cl
14e
H
N(CH3)2
Br
R1
R2
R3
Compounds
15a
H
NO2
H
15b
H
NO2
CH3
15c
H
NO2
F
15d
H
NO2
Cl
15e
H
NO2
Br
16a
NO2
H
16b
NO2
H
16c
NO2
H
16d
NO2
H
16e
NO2
H
R1
R2
R3
H
CH3
F
Cl
Br
against B16-F10 cancer cells. The results were summarized in
Table 2. Most of the synthesized sulfonamide compounds bearing
the cinnamoyl moiety showed remarkable antiproliferative effects.
The compounds with para halogen substituted (10a, 11a, 12a)
(IC50 = 2.1
antiproliferative activities in the order of F >Cl >Br. The results
demonstrated that hydrophobic and electron-withdrawing
halogen group may have slightly improved antiproliferative activity
and other electron donating substituted 13a, 14a (IC50 = 13.2 g/mL,
2.3 g/mL) had negative effects relative to those of 9a (IC50 = 3.5 g/
mL). Compounds (15a, IC50 = 4.8 g/mL) with p-substituted group
showed slightly more potent activities than those of o-substituted
(16a, IC50 = 6.0 g/mL).
In the case of constant A ring substituents, change of substitu-
ents on B ring could also affect the activities of these compounds.
Among the compounds 9a–9e, these compounds with the halogen
atom substituent (9c, 9d, 9e) showed stronger anticancer activity
and the strength order was similar with A ring: F >Cl >Br, followed
that 9a and 9b had the lowest activities. The explanation theory of
A ring could be also applied on B ring. Among all the compounds,
lg/mL, 2.2 lg/mL, 3.9 lg/mL) exhibited significant
a
l
l
l
Figure 3. Crystal structure diagram of compound 10c. H atoms are shown as small
spheres of arbitrary radii.
l
dissolved in CH2Cl2 and refluxed to give the desired compounds 9a–
16e (Table 1).
Furthermore, the crystal structure of compound 10c was deter-
mined by single crystal X-ray diffraction analysis in Figure 3.
l
2.2. Bioactivity
To test the anticancer activities of the synthesized compounds,
we evaluated antiproliferative activities of compounds 9a–16e
10c (IC50 = 0.8 lg/mL) showed the best activity while the substitu-

Y. Luo et al. / Bioorg. Med. Chem. 19 (2011) 4730–4738
4733
Table 2
polymerization activity. This result indicated the anti-proliferative
effect was produced by direct connection of tubulin and the
compound.
Furthermore, compound 10c was performed cell cycle analysis
using flow cytometry (Fig. 4). The result showed that compound
10c strongly induced G2/M arrest in B16-F10 cells, and the effect
was observed in a dose-independent manner after treatment for
24 h with increasing dose of the compound. About 30.2% of the
cells were arrested in the G2/M phase while 49.0% of the cells were
Inhibition of B16-F10 cells proliferation and tubulin polymerization by compounds
9a–16e
Compound IC50 SD (
l
g/mL)
Compound IC50 SD (
l
g/mL)
B16-F10a
Tubulinb
B16-F10a
Tubulinb
9a
9b
9c
9d
3.5 0.2
13.8 2.2
2.1 0.3
37
152
5
3
13b
13c
12.2
5.1 0.43
5.3 0.3
4
146
45
52
53
26
37
10
14
34
50
5
7
3
8
2
4
5
3
5
1
4.5 0.4 13d
2.2 0.15 14.8 0.3 13e
5.8 0.54
2.3 0.17
4.3 0.65
2.3 0.18
2.8 0.12
4.2 0.29
4.8 0.5
9e
3.9 0.3
2.1 0.1
4.8 0.14
0.8 0.14
2.6 0.12
63
4.5 0.5 14b
40 14c
2.4 0.4 14d
4.5 0.2 14e
1
14a
found to be in the G2/M phase in the presence of 1 and 5 lg/mL
10a
10b
10c
10d
10e
11a
11b
11c
11d
11e
12a
12b
12c
12d
12e
13a
10c, respectively. These findings indicated a continuing impair-
ment of cell division and confirmed compound 10c was a potent
antitubulin agent.
8
2.8 0.26 14.7 0.7 15a
2.2 0.12 15.3 0.7 15b
To gain better understanding on the potency of compound 10c
and guide further SAR studies, we proceeded to examine the inter-
action of compound 10c with tubulin (PDB code: 1SA0). The molec-
ular docking was performed by simulation of compound 10c into
the colchicine binding site in tubulin. All docking runs were ap-
plied the Lamarckian genetic algorithm of Auto-Dock 4.0.28 The
binding model of compound 10c and tubulin was depicted in Fig-
ure 5. The amino acid residue which had interaction with tubulin
was labeled. In the binding mode, compound 10c was nicely bound
to the colchicine binding site of tubulin via hydrophobic interac-
tion and binding was stabilized by a hydrogen bond. The calculated
binding energies were used as the parameters 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 selected pose of 10c had an estimated binding free en-
ergy of À12.46 kcal/mol (binding free energy of control compounds
colchicine and CA-4 are À8.86 kcal/mol and À7.62 kcal/mol,
respectively). The oxygen atom of the sulfonamide system formed
one hydrogen bond with the amino hydrogen of LYS352 (bond
length: LYS352 N–HÁ Á ÁO = 2.092 Å; bond angle: LYS352 N–
HÁ Á ÁO = 142.18°). This residue (LYS352) was also found to be in-
volved in the binding of other tubulin inhibitors.29 3D model of
the interaction between compound 10c and the colchicine binding
site was depicted in Figure 6. The model was similar with the mod-
els between colchicine, CA-4 and the colchicine binding site.15
13.5
6
151 12
6.3 0.3
134 14
163 12
4.6 0.4
30
1
15c
2.2 0.13
2.0 0.17
2.5 0.13
2.8 0.25 10.8 0.4 16a
8.9 0.2 15d
9.3 0.5 15e
13.7
13.9
3
2
6.0 0.12
10.3 0.74 102 13
6.0 0.24
6.6 0.57
74
9
3.9 0.12
74
137 14
3.8 0.2 16d
55
53
8
16b
16c
12.1
3
69
78
134 16
1.3 0.4
0.7 0.2
5
3
1.2 0.1
5.2 0.27
5.1 0.35
13.2 2.7
1
6
9
16e
12.6
2
Colchicine
CA-4
0.5 0.04
0.4 0.15
160
a
Inhibition of the growth of tumor cell lines.
Inhibition of tubulin polymerization.
b
ents in the A ring and B ring were both F and F atom was p-substi-
tuted in the A ring. The results showed that electron-withdrawing
groups could enhance the activity of compounds. It could be a
promising lead for the further development of novel tubulin inhi-
bition agents.
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 9c, 10c, 12c,
showed strong inhibitory effect and their 50% tubulin polymeriza-
tion inhibition about 4.5
tively. Compound 10c displayed the most potent anti-tubulin
lg/mL, 2.4 lg/mL, 3.8 lg/mL, respec-
Figure 4. Effects of compound 10c on cell cycle progression of B16-F10 cells were determined by flow cytometry analysis. B16-F10 cells were treated with different
concentrations of 10c for 24 h. The percentage of cells in each cycle phase was indicated.

4734
Y. Luo et al. / Bioorg. Med. Chem. 19 (2011) 4730–4738
3. Conclusion
In this paper, a series of novel antitubulin polymerization inhib-
itors (9a–16e) containing cinnamoyl skeleton and sulfonamide
moiety had been synthesized and evaluated their biological activ-
ities. These compounds exhibited potent tubulin polymerization
inhibitory activities and antiproliferative activities against B16-
F10 mouse melanoma cell line. Compound 10c showed the most
potent inhibition activity with an IC50 value of 0.8
tubulin polymerization activity with an IC50 of 2.4
l
l
g/mL and anti-
g/mL. Molecu-
lar docking was further performed to study the inhibitor-tubulin
protein interactions. After analysis of the binding model of com-
pound 10c with tubulin, it was found that several interactions with
the protein residues in the colchicine binding site might play a cru-
cial role in its antitubulin polymerization and antiproliferative
activities. The information of this work might be helpful for the de-
sign and synthesis of tubulin polymerization inhibitors with stron-
ger activities.
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 DPX
300 model Spectrometer in CDCl3 and chemical shifts were re-
ported in ppm (d). ESI-MS spectra were recorded on a Mariner Sys-
tem 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 GF254) and visualized in
UV light (254 nm). Column chromatography was performed using
silica gel (200–300 mesh) eluting with ethyl acetate and petroleum
ether.
Figure 5. Binding mode of compound 10c (colored by atom: carbons: green;
oxygens: red) with tubulin (entry 1SA0 in the Protein Data Bank). The dotted lines
show the hydrogen bonds.
Comparing these models, it was found that the hydrophobic pock-
ets of colchicine binding site were occupied by compounds, and the
difference was the arrangement on the enzyme surface. In the 10c
binding model, more details revealed that there were some key
roles of the interaction of 10c and tubulin (Fig. 6). The fluorophenyl
moiety of B ring occupied a pocket bounded by ASN258, LEU255,
and LYS352. It should be noted that the static interaction between
F atom and VAL238 might played an important role. The fluoro-
phenyl moiety of A ring was embedded in a hydrophobic pocket
constructed by the side chains of residue of LYS352. Furthermore,
the binding of compound 10c to tubulin was stabilized by hydro-
gen bond with LYS352. Overall, these results suggested that com-
pound 10c could be well inserted into tubulin, similar with
colchicine and CA-4.30
4.2. General procedure for synthesis of cinnamic acids
A mixture of aromatic aldehydes (3.2 mmol), malonic acid
(3.87 mmol), piperidine (0.387 mmol) was dissolved in pyridine
and stirred on 80–90 °C for 24 h. The pyridine was removed at
the vacuum. The reaction mixture was poured into water and
washed with HCl. And the precipitate was filtered and washed
with hexane for three times, and dried under vacuum to afford
the cinnamic acids (Scheme 1).
Figure 6. Comparison of compound 10c binding model with tubulin complex. Left: 3D model of the interaction between compound 10c and the colchicine binding site. The
protein is represented by molecular surface. 10c is depicted by sticks and balls. Right: 3D model of interaction between compound 10c and the colchicine binding site. The
protein is represented by molecular surface. 10c is depicted by molecular surface, sticks and balls.

Y. Luo et al. / Bioorg. Med. Chem. 19 (2011) 4730–4738
4735
4.3. General procedure for synthesis of cinnamic
acylsulfonamides
Calcd for C15H11ClFNO3S: C, 53.02; H, 3.26; N, 4.12. Found: C,
53.17; H, 3.24; N, 4.09.
4.3.7. (E)-N-(4-Bromophenylsulfonyl)-3-(4-fluorophenyl)
acrylamide (10e)
To a round-bottom flask (500 mL) that contained a solution of
aryl sulfonamide (6 mmol), 4-dimethyaminopyridine (DMAP,
13 mmol), and 1-[3-(dimethyamino)-propyl]-3-ethylcarbodiimide
hydrochloride (EDCI, 13 mmol) in CH2Cl2 (150 mL) was added the
synthesized cinnamic acid (6 mmol) at room temperature. The
resulting mixture was stirred at room temperature for 12 h, then
cooled to 5 °C, and acidified to pH 1 with addition of HCl aqueous
solution (10%), which was followed by extraction with CH2Cl2/
MeOH (9:1, 3 Â 100 mL). The combined organic layers were washed
with H2O and brine, dried over Na2SO4, and concentrated in vacuo.
The residue was subjected to silica gel chromatography or crystal-
lization if necessary to afford the compounds (9a–16e) (Scheme 1).
Yield 88%;mp. 194–195 °C. 1H NMR (300 MHz,CDCl3): d 6.30
(2d, J = 15.54 Hz, 1H), 7.07 (t, J = 8.51 Hz, 2H, ArH), 7.46–7.50 (m,
2H, ArH), 7.63 (s, 1H), 7.70 (d, J = 8.97 Hz, 2H, ArH), 7.98 (d,
J = 8.76 Hz, 2H, ArH), 8.42 (s, 1H, NH). ESI-MS: 383.96
(C15H12BrFNO3S, [M+H]+).Anal. Calcd for C15H11BrFNO3S: C,
46.89; H, 2.89; N, 3.65. Found: C, 46.63; H, 2.86; N, 3.62.
4.3.8. (E)-3-(4-Chlorophenyl)-N-(phenylsulfonyl)acrylamide
(11a)
Yield 88%; mp. 206–210 °C. 1H NMR (300 MHz, CDCl3): d 6.38
(d, J = 15.54 Hz, 1H), 7.34–7.42 (m, 4H, ArH), 7.54–7.66 (m, 4H,
ArH), 8.11(t, J = 4.29 Hz, 2H), 8.23 (s, 1H, NH). ESI-MS: 322.02
(C15H13ClNO3S, [M+H]+). Anal. Calcd for C15H12ClNO3S: C, 55.99;
H, 3.76; N, 4.35. Found: C, 56.25; H, 3.73; N, 4.32.
4.3.1. N-(4-Fluorophenylsulfonyl)cinnamamide (9c)
Yield 85%; mp. 139 °C. 1H NMR (300 MHz, CDCl3): d 6.40 (d,
J = 15.75 Hz, 1H), 7.20–7.26 (t, J = 9.42 Hz, 2H, ArH), 7.38 (d,
J = 6.96 Hz, 3H), 7.47–7.49 (t, J = 3.93 Hz, 2H, ArH), 7.70 (d,
J = 15.72 Hz, 1H, ArH), 8.14–8.19 (m, 2H, ArH), 8.44 (s, 1H, NH).
ESI-MS: 306.05(C15H13FNO3S, [M+H]+). Anal. Calcd for
C15H12FNO3S: C, 59.01; H, 3.96; N, 4.59. Found: C, 59.34; H, 4.00;
N, 4.57.
4.3.9. (E)-3-(4-Chlorophenyl)-N-(4-fluorophenylsulfonyl)
acrylamide (11c)
Yield 82%; mp. 213–215 °C. 1H NMR (300 MHz, CDCl3): d 6.36
(d, J = 15.54 Hz, 1H), 7.23 (t, J = 9.42 Hz, 2H, ArH), 7.34–7.43 (m,
4H, ArH), 7.63 (d, J = 15.54 Hz, 1H), 8.12–8.17 (m, 2H, ArH), 8.32
(s, 1H, NH). ESI-MS: 340.01 (C15H12ClFNO3S, [M+H]+). Anal. Calcd
for C15H11ClFNO3S: C, 53.02; H, 3.26; N, 4.12. Found: C, 53.32; H,
3.24; N, 4.15.
4.3.2. N-(4-Bromophenylsulfonyl)cinnamamide (9e)
Yield 90%; mp. 166–167 °C. 1H NMR (300 MHz, CDCl3): d 6.39
(d, J = 15.57 Hz, 1H), 7.35-7.42 (m, 3H), 7.44–7.50 (m, 2H, ArH),
7.67–7.72 (dd, J1 = 3.3 Hz, J2 = 12.06 Hz, 3H, ArH), 8.00 (d,
J = 8.79 Hz, 2H, ArH), 8.49 (s, 1H, NH). ESI-MS: 365.97
(C15H13BrNO3S, [M+H]+). Anal. Calcd for C15H12BrNO3S: C, 49.19;
H, 3.30; N, 3.82. Found: C, 49.35; H, 3.84; N, 3.82.
4.3.10. (E)-N-(4-Bromophenylsulfonyl)-3-(4-chlorophenyl)
acrylamide (11e)
Yield 88%; mp. 192–196 °C 1H NMR (300 MHz, CDCl3): d 6.35 (d,
J = 15.75 Hz, 1H), 7.34–7.43 (m, 4H, ArH), 7.61–7.71 (m, 3H), 7.98
(dd, J1 = 1.83 Hz, J2 = 6.75 Hz, 2H, ArH), 8.29 (s, 1H, NH). ESI-MS:
399.93 (C15H12BrClNO3S, [M+H]+). Anal. Calcd for C15H11BrClNO3S:
C, 44.96; H, 2.77; N, 3.50. Found: C, 45.14; H, 2.80; N, 3.53.
4.3.3. (E)-3-(4-Fluorophenyl)-N-(phenylsulfonyl)acrylamide
(10a)
Yield 82%; mp. 165–166 °C. 1H NMR (300 MHz, CDCl3): d 6.34
(d, J = 15.54 Hz, 1H), 7.04–7.10 (m, 2H, ArH), 7.45–7.50 (m, 2H),
7.54–7.57 (m, 2H, ArH), 7.59–7.68 (m, 2H, ArH), 8.10–8.17 (m,
3H). ESI-MS: 306.05 (C15H13FNO3S, [M+H]+). Anal. Calcd for
4.3.11. (E)-3-(4-Bromophenyl)-N-(phenylsulfonyl)acrylamide
(12a)
Yield 86%;mp. 222–224 °C. 1H NMR (300 MHz, CDCl3): d 6.39 (d,
J = 15.72 Hz, 1H), 7.34 (d, J = 8.61 Hz, 2H, ArH), 7.51–7.69 (m, 6H),
7.96 (s, 1H, NH), 8.12 (d, J = 7.32 Hz, 2H, ArH). ESI-MS: 365.97
(C15H13BrNO3S, [M+H]+). Anal.Calcd for C15H12BrNO3S: C, 49.19;
H, 3.50; N, 3.94. Found: C, 49.35; H, 3.52; N, 3.97.
C15H12FNO3S: C, 59.01; H, 3.96; N, 4.59. Found: C, 59.23; H, 4.03;
N, 4.56.
4.3.4. (E)-3-(4-Fluorophenyl)-N-tosylacrylamide (10b)
Yield 86%; mp. 193–194 °C. 1H NMR (300 MHz,CDCl3): d 2.45(s,
3H), 6.37(d, J = 15.54 Hz, 1H), 7.07 (t, J = 8.60 Hz, 2H, ArH), 7.37 (d,
J = 8.04 Hz, 2H, ArH), 7.46–7.51 (m, 2H, ArH), 7.67 (d, J = 15.57 Hz,
1H), 8.01 (d, J = 8.43 Hz, 2H, ArH), 8.56 (s, 1H, NH). ESI-MS: 320.07
(C16H15FNO3S, [M+H]+). Anal. Calcd for C16H14FNO3S: C, 60.18; H,
4.42; N, 4.39.C, 60.25; H, 4.58; N, 4.46. Found: C, 60.26; H, 4.45;
N, 4.60.
4.3.12. (E)-3-(4-Bromophenyl)-N-tosylacrylamide (12b)
Yield 90%; mp. 218–220 °C. 1H NMR (300 MHz, CDCl3): d 2.44 (s,
3H), 6.41 (d, J = 15.72 Hz, 1H), 7.32–7.37 (m, 4H, ArH), 7.51 (d,
J = 8.4 Hz, 2H, ArH), 7.61 (d, J = 15.54 Hz, 1H), 8.00 (d, J = 8.4 Hz,
2H, ArH), 8.30 (s, 1H, NH). ESI-MS: 379.99 (C16H15BrNO3S,
[M+H]+). Anal. Calcd for C16H14BrNO3S: C, 50.54; H, 3.71; N, 3.68.
Found: C, 50.68; H, 3.73; N, 3.64.
4.3.5. (E)-3-(4-Fluorophenyl)-N-(4-fluorophenylsulfonyl)
acrylamide (10c)
4.3.13. (E)-3-(4-Bromophenyl)-N-(4-fluorophenylsulfonyl)
acrylamide (12c)
Yield 80%; mp. 185–186 °C. 1H NMR (300 MHz,CDCl3):d 6.33 (d,
J = 15.54 Hz, 1H), 7.09 (t, J = 8.60 Hz, 2H, ArH), 7.25 (t, J = 8.69 Hz,
2H, ArH), 7.47–7.52 (m, 2H, ArH), 7.68 (d, J = 15.72 Hz, 1H), 8.15–
8.20 (m, 2H, ArH), 8.36 (s, 1H, NH). ESI-MS: 324.04 (C15H12F2NO3S,
[M+H]+). Anal. Calcd for C15H11F2NO3S: C, 55.72; H, 3.43; N, 4.33.
Found: C, 55.65; H, 3.45; N, 4.31.
Yield 86%; mp. 212–213 °C. 1H NMR (300 MHz, CDCl3): d 6.36
(d, J = 15.72 Hz, 1H), 7.19–7.23 (m, 2H, ArH), 7.34 (d, J = 8.61 Hz,
2H, ArH), 7.52 (dd, J1 = 1.65 Hz, J2 = 6.6Hz, 2H, ArH), 7.62 (d,
J = 15.72 Hz, 1H), 8.11–8.17 (m, 2H, ArH), 8.20 (s, 1H, NH). ESI-
MS: 383.96 (C15H12BrFNO3S, [M+H]+). Anal. Calcd for
C15H11BrFNO3S: C, 46.89; H, 2.89; N, 3.65. Found: C, 46.64; H,
2.86; N, 3.62.
4.3.6. (E)-N-(4-Chlorophenylsulfonyl)-3-(4-fluorophenyl)
acrylamide (10d)
4.3.14. (E)-3-(4-Bromophenyl)-N-(4-chlorophenylsulfonyl)
acrylamide (12d)
Yield 84%; mp. 198–200 °C. 1H NMR (300 MHz, CDCl3): d 6.31
(d, J = 15.75 Hz, 1H), 7.09 (t, J = 8.51 Hz, 2H, ArH), 7.47–7.57 (m,
4H, ArH), 7.67 (d, J = 15.54 Hz, 1H), 8.08 (d, J = 8.43 Hz, 2H, ArH),
8.16 (s, 1H, NH). ESI-MS: 340.01 (C15H12ClFNO3S, [M+H]+). Anal.
Yield 92%; mp. 204–206 °C. 1H NMR (300 MHz, CDCl3): d 6.36
(d, J = 15.72 Hz, 1H), 7.33 (d, J = 8.43 Hz, 2H, ArH), 7.51–7.55 (m,

4736
Y. Luo et al. / Bioorg. Med. Chem. 19 (2011) 4730–4738
4H, ArH), 7.62 (d, J = 15.72 Hz, 1H), 8.00 (d, J = 8.79 Hz, 2H, ArH),
8.21 (s, 1H, NH). ESI-MS: 399.93 (C15H12BrClNO3S, [M+H]+). Anal.
Calcd for C15H11BrClNO3S: C, 44.96; H, 2.77; N, 3.50. Found: C,
44.72; H, 2.74; N, 3.47.
J = 6.54 Hz, 2H, ArH), 8.12 (s, 1H, NH). ESI-MS: 331.10
(C17H19N2O3S, [M+H]+). Anal. Calcd for C17H18N2O3S: C, 61.80; H,
5.49; N, 8.48. Found: C, 62.02; H, 5.51; N, 8.45.
4.3.22. (E)-3-(4-(Dimethylamino)phenyl)-N-tosylacrylamide
(14b)
4.3.15. (E)-3-(4-Bromophenyl)-N-(4-bromophenylsulfonyl)
acrylamide (12e)
Yield 85%; mp. 218–220 °C. 1H NMR (300 MHz, CDCl3): d 2.43(s,
3H), 3.03 (s, 6H, N(CH3)2), 6.18 (d, J = 15.36 Hz, 1H), 6.68 (d,
J = 8.79 Hz, 2H, ArH), 7.32–7.39 (m, 4H, ArH), 7.61 (d,
J = 15.36 Hz, 1H), 7.93 (s, 1H, NH), 7.99 (d, J = 8.40 Hz, 2H, ArH).
ESI-MS: 345.12 (C18H21N2O3S, [M+H]+). Anal. Calcd for
Yield 93%; mp. 213–214 °C. 1H NMR (300 MHz, CDCl3): d 6.29
(d, J = 15.57 Hz, 1H), 7.54–7.69 (m, 5H), 8.10 (t, J = 10.61 Hz, 4H,
ArH), 8.23 (s, 1H, NH). ESI-MS: 443.88 (C15H12Br2NO3S, [M+H]+).
Anal. Calcd for C15H11Br2NO3S: C, 40.47; H, 2.49; N, 3.15.C, 40.22;
H, 2.37; N, 3.02. Found: C, 40.72; H, 2.46; N, 3.04.
C18H20N2O3S: C, 62.77; H, 5.85; N, 8.13. Found: C, 62.93; H, 5.89;
N, 8.16.
4.3.16. (E)-3-(4-Methoxyphenyl)-N-(phenylsulfonyl)acrylamide
(13a)
4.3.23. (E)-3-(4-(Dimethylamino)phenyl)-N-(4-fluorophenyl-
sulfonyl)acrylamide (14c)
Yield 80%; mp. 173–175 °C. 1H NMR (300 MHz,CDCl3): d 3.85 (s,
3H), 6.30 (d, J = 15.54 Hz, 1H), 6.90 (d, J = 8.76 Hz, 2H, ArH), 7.45 (d,
J = 8.97 Hz, 2H, ArH), 7.57 (t, J = 7.50 Hz, 2H, ArH), 7.66 (t,
J = 7.77 Hz, 2H), 8.13 (t, J = 2.93 Hz, 2H, ArH), 8.27 (s, 1H, NH).
ESI-MS: 318.07 (C16H16NO4S, [M+H]+). Anal. Calcd for
Yield 77%; mp. 207–209 °C 1H NMR (300 MHz,CDC3): d 3.04 (s,
6H, N(CH3)2), 6.15 (d, J = 15.47 Hz, 1H), 6.67 (d, J = 8.57 Hz, 2H,
ArH), 7.32 (d, J = 7.78 Hz, 2H, ArH), 7.34-7.37 (m, 2H, ArH), 7.62
(d, J = 15.48 Hz, 1H), 7.73 (t, J = 4.56 Hz, 2H, ArH), 8.09 (s, 1H,
NH). ESI-MS: 349.09 (C17H18FN2O3S, [M+H]+). Anal. Calcd for
C16H15NO4S:C, 60.55; H, 4.76; N, 4.41. Found: C, 60.61; H, 4.73;
N, 4.43.
C17H17FN2O3S: C, 58.61; H, 4.92; N, 8.04. Found: C, 58.73; H,
4.95; N, 8.01.
4.3.17. (E)-3-(4-Methoxyphenyl)-N-tosylacrylamide (13b)
Yield 86%; mp. 195–197 °C. 1H NMR (300 MHz, CDCl3): d 2.43 (s,
3H), 3.83 (s, 3H), 6.27 (d, J = 15.54 Hz, 1H), 6.88 (d, J = 8.79 Hz, 2H,
ArH), 7.34 (d, J = 8.04 Hz, 2H, ArH), 7.43 (d, J = 8.79 Hz, 2H, ArH),
7.62 (d, J = 15.54 Hz, 1H), 7.99 (d, J = 8.22 Hz, 2H, ArH), 8.30 (s,
1H, NH). ESI-MS: 332.09 (C17H18NO4S, [M+H]+). Anal. Calcd for
4.3.24. (E)-N-(4-Chlorophenylsulfonyl)-3-(4-(dimethylamino)
phenyl)acrylamide (14d)
Yield 81%; mp. 220–222 °C. 1H NMR (300 MHz,CDCl3): d 3.06 (s,
6H, N(CH3)2), 6.37 (d, J = 15.54 Hz, 1H), 6.67 (d, J = 8.85 Hz, 2H,
ArH), 7.41 (d, J = 15.52 Hz, 1H), 7.64 (d, J = 8.94 Hz, 2H, ArH), 7.78
(d, J = 7.84 Hz, 2H, ArH), 7.84 (d, J = 7.73 Hz, 2H, ArH), 8.04(s, 1H,
NH). ESI-MS: 365.06 (C17H18ClN2O3S, [M+H]+). Anal. Calcd for
C17H17NO4S: C, 61.61; H, 5.17; N, 4.23. Found: C, 61.83; H, 5.15;
N, 4.25.
C17H17ClN2O3S: C, 55.96; H, 4.70; N, 7.48. Found: C, 55.86; H,
4.3.18. (E)-N-(4-Fluorophenylsulfonyl)-3-(4-methoxyphenyl)
acrylamide (13c)
4.67; N, 7.51.
Yield 78%; mp. 175–177 °C. 1H NMR (300 MHz, CDCl3): d 3.83 (s,
3H), 6.26 (d, J = 15.54 Hz, 1H), 6.87 (d, J = 8.76 Hz, 2H, ArH), 7.18–
7.26 (m, 2H, ArH), 7.43 (d, J = 8.79 Hz, 2H, ArH), 7.65(d,
J = 15.54 Hz, 1H), 8.13–8.18 (m, 2H, ArH), 8.50 (s, 1H, NH). ESI-
MS: 336.06 (C16H15FNO4S, [M+H]+). Anal. Calcd for C16H14FNO4S:
C, 57.30; H, 4.21; N, 4.18. Found: C, 57.43; H, 4.24; N, 4.22.
4.3.25. (E)-N-(4-Bromophenylsulfonyl)-3-(4-(dimethylamino)
phenyl)acrylamide (14e)
Yield 84%; mp. 226–228 °C. 1H NMR (300 MHz, CDCl3): d 3.04 (s,
6H, N(CH3)2), 6.54 (d, J = 15.57 Hz, 1H), 6.67 (d, J = 8.86 Hz, 2H,
ArH), 7.40 (d, J = 15.57 Hz, 1H), 7.70 (d, J = 6.45 Hz, 2H, ArH),
7.86–7.90 (m, 4H, ArH), 8.13 (s, 1H, NH). ESI-MS: 409.01
(C17H18BrN2O3S, [M+H]+). Anal. Calcd for C17H17BrN2O3S: C,
49.89; H, 4.19; N, 6.84. Found: C, 49.76; H, 4.23; N, 6.81.
4.3.19. (E)-N-(4-Chlorophenylsulfonyl)-3-(4-methoxyphenyl)
acrylamide (13d)
Yield 84%; mp. 183–185 °C. 1H NMR (300 MHz,CDCl3): d 3.83 (s,
3H), 6.24 (d, J = 15.54 Hz, 1H), 6.89 (d, J = 8.76 Hz, 2H, ArH), 7.43 (d,
J = 8.61 Hz, 2H, ArH), 7.52 (d, J = 8.58 Hz, 2H, ArH), 7.64 (d,
J = 15.54 Hz, 1H), 8.06 (d, J = 8.61 Hz, 2H, ArH), 8.43 (s, 1H, NH).
ESI-MS: 352.03 (C16H15ClNO4S, [M+H]+). Anal. Calcd for
4.3.26. (E)-N-(4-Fluorophenylsulfonyl)-3-(2-nitrophenyl)
acrylamide (15c)
Yield 75%; mp. 165–167 °C. 1H NMR (300 MHz, CDCl3): d 6.31
(d, J = 15.57 Hz, 1H), 7.21–7.27 (m, 2H, ArH), 7.54–7.57 (m, 2H,
ArH), 7.65 (t, J = 6.86 Hz, 1H, ArH), 8.04 (t, J = 4.85, 2H, ArH), 8.10
(s, 1H, NH), 8.12–8.17 (m, 2H). ESI-MS: 351.04 (C15H12FN2O5S,
[M+H]+). Anal. Calcd for C15H11FN2O5S: C, 51.43; H, 3.16; N, 8.00.
Found: C, 51.21; H, 3.13; N, 7.96.
C16H14ClNO4S: C, 54.62; H, 4.01; N, 3.98. Found: C, 54.74; H,
4.03; N, 4.02.
4.3.20. (E)-N-(4-Bromophenylsulfonyl)-3-(4-methoxyphenyl)
acrylamide (13e)
4.3.27. (E)-N-(4-Chlorophenylsulfonyl)-3-(2-
Yield 88%; mp. 188–190 °C. 1H NMR (300 MHz,CDCl3): d 3.83 (s,
3H), 6.23 (d, J = 15.54 Hz, 1H), 6.89 (d, J = 8.79 Hz, 2H, ArH), 7.43 (d,
J = 8.79 Hz, 2H, ArH), 7.62 (s,1H), 7.69 (d, J = 8.79 Hz, 2H, ArH), 7.98
(d, J = 8.76 Hz, 2H, ArH), 8.22 (s, 1H, NH). ESI-MS: 395.98
(C16H15BrNO4S, [M+H]+). Anal. Calcd for C16H14BrNO4S: C, 48.50;
H, 3.56; N, 3.53. Found: C, 48.43; H, 3.60; N, 3.57.
nitrophenyl)acrylamide (15d)
Yield 86%; mp. 178–180 °C. 1H NMR (300 MHz, CDCl3): d 6.36
(d, J = 15.57 Hz, 1H), 7.53–7.70 (m, 6H, ArH), 8.03–8.14 (m, 4H),
8.64 (s, 1H, NH). ESI-MS: 367.01 (C15H12ClN2O5S, [M+H]+). Anal.
Calcd for C15H11ClN2O5S: C, 49.12; H, 3.02; N, 7.64. Found: C,
50.16; H, 3.06; N, 7.67.
4.3.21. (E)-3-(4-(Dimethylamino)phenyl)-N-(phenylsulfonyl)
acrylamide (14a)
4.3.28. (E)-N-(4-Bromophenylsulfonyl)-3-(2-nitrophenyl)
acrylamide (15e)
Yield 75%; mp. 202–204 °C. 1H NMR (300 MHz, CDCl3): d 3.05 (s,
6H, N(CH3)2), 6.20 (d, J = 15.54 Hz, 1H), 6.66 (d, J = 8.67 Hz, 2H,
ArH), 7.32 (d, J = 8.43 Hz, 2H, ArH), 7.54 (d, J = 8.56 Hz, 2H, ArH),
7.62 (d, J = 15.54 Hz, 1H), 7.76 (d, J = 15.54 Hz, 1H, ArH), 8.01(d,
Yield 85%; mp. 175–176 °C. 1H NMR (300 MHz, CDCl3): d 6.29
(d, J = 15.54 Hz, 1H), 7.53–7.60 (m, 2H, ArH), 7.65 (d, J = 7.32 Hz,
1H, ArH), 7.72 (d, J = 8.79 Hz, 2H, ArH), 8.00 (d, J = 8.61 Hz, 2H,
ArH), 8.05–8.12 (m, 2H), 8.49 (s, 1H, NH). ESI-MS: 410.96

Y. Luo et al. / Bioorg. Med. Chem. 19 (2011) 4730–4738
4737
(C15H12BrN2O5S, [M+H]+). Anal. Calcd for C15H11BrN2O5S: C, 43.81;
Table 3
Crystallographical and experimental data for compound 10c
H, 2.70; N, 6.81. Found: C, 43.56; H, 2.68; N, 6.84.
Compounds
10c
Empirical formula
Formula weight
Crystal system
Space group
a (Å)
b (Å)
c (Å)
a (°)
C
15H11F2NO3S
4.3.29. (E)-3-(4-Nitrophenyl)-N-(phenylsulfonyl)acrylamide
(16a)
323.31
Monoclinic
P21/c
14.450(7)
10.908(5)
9.586(4)
90
92.800(4)
90
1509.2(12)
17
1.760
2.34–28.42
799
8178/3563 [R(int) = 0.0239]
3563/0/203
0.730
Yield 78%; mp. 225–227 °C. 1H NMR (300 MHz, CDCl3): d 6.52
(d, J = 15.54 Hz, 1H), 7.35 (d, J = 8.73 Hz, 2H, ArH), 7.66 (d,
J = 7.88 Hz, 2H, ArH), 7.73 (d, J = 8.54 Hz, 2H, ArH), 7.82 (d,
J = 15.54 Hz, 1H), 8.01 (d, J = 4.52 Hz, 2H, ArH), 8.21 (s, 1H, NH).
ESI-MS: 333.05(C15H13N2O5S, [M+H]+). Anal. Calcd for
b (°)
c (°)
V (Å)
C15H12N2O5S: C, 54.21; H, 3.64; N, 8.43. Found: C, 54.33; H, 3.67;
N, 8.45.
Z
Dcalcd/g cm-3
4.3.30. (E)-3-(4-Nitrophenyl)-N-tosylacrylamide (16b)
h range (°)
F(000)
Reflections collected/unique
Data/restraints/parameters
Absorption coefficient (mmÀ1
Yield 92%; mp. 189–190 °C. 1H NMR (300 MHz, CDCl3): d 2.45
(s,3H), 6.55 (d, J = 15.72 Hz, 1H), 7.37 (d, J = 8.22 Hz, 2H, ArH),
7.63 (d, J = 8.61 Hz,2H, ArH), 7.71 (d, J = 15.72 Hz, 1H), 8.00 (d,
J = 8.40 Hz, 2H, ArH), 8.24 (d, J = 8.58, 3H). ESI-MS: 347.06
(C16H15N2O5S, M+H]+). Anal. Calcd for C16H14N2O5S: C, 55.48; H,
4.07; N, 8.09. Found: C, 55.53; H, 4.05; N, 8.12.
)
R1; wR2 [I >2
r
(I)]
0.0456/0.1033
0.0759/0.1203
1.024
R1; wR2 (all data)
GOF
4.3.31. (E)-N-(4-Fluorophenylsulfonyl)-3-(4-nitrophenyl)
acrylamide (16c)
Yield 75%; mp. 139–141 °C. 1H NMR (300 MHz, DMSO-D6): d
6.75 (d, J = 15.90 Hz, 1H), 7.49 (t, J = 8.87 Hz, 2H, ArH), 7.68 (d,
J = 16.08 Hz, 1H), 7.85 (d, J = 8.94 Hz, 2H, ArH), 8.03–8.08 (m, 2H,
ArH), 8.27 (d, J = 8.76 Hz, 2H), 12.52 (s, 1H, NH). ESI-MS: 351.04
(C15H12FN2O5S, [M+H]+). Anal. Calcd for C15H11FN2O5S: C, 51.43;
H, 3.16; N, 8.00. Found: C, 51.72; H, 3.35; N, 8.28.
4.5. Antiproliferation assay
The antiproliferative activities of the prepared compounds
against B16-F10 mouse melanoma cell line were evaluated as de-
scribed elsewhere with some modifications.31 Target tumor cell
lines were grown to log phase in RPMI 1640 medium supple-
mented with 10% fetal bovine serum. After diluting to 2 Â 104
cells mLÀ1 with the complete medium, 100
lL of the obtained cell
4.3.32. (E)-N-(4-Chlorophenylsulfonyl)-3-(4-nitrophenyl)
acrylamide (16d)
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 before the cytotoxicity assessments. Tested samples at
pre-set concentrations were added to six wells with colchicine
and CA-4 coassayed as positive references. After 48 h exposure per-
Yield 81%; mp. 198–200 °C. 1H NMR (300 MHz, CDCl3): d 6.48
(d, J = 15.54 Hz, 1H), 7.55 (d, J = 8.76 Hz, 2H, ArH), 7.62 (t,
J = 6.59 Hz, 2H, ArH), 7.73 (d, J = 15.54 Hz, 1H), 8.08 (d,
J = 8.78 Hz, 2H, ArH), 8.11 (s, 1H, NH), 8.25 (d, J = 8.79 Hz, 2H, Ar-
H).ESI-MS: 367.01 (C15H12ClN2O5S, [M+H]+). Anal. Calcd for
iod, 40
thiazol-2-yl)-2,5-diphenyltetrazolium bromide)) was added to
each well. Four hours later, 100 L extraction solution (10% SDS-
l
L of PBS containing 2.5 mg mLÀ1 of MTT (3-(4,5-dimethyl-
C15H11ClN2O5S: C, 49.12; H, 3.02; N, 7.64. Found: C, 49.25; H,
l
3.04; N, 7.60.
5% isobutyl alcohol-0.01 M HCl) was added. After an overnight
incubation at 37 °C, the optical density was measured at a wave-
length of 570 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 2.
4.3.33. (E)-N-(4-Bromophenylsulfonyl)-3-(4-nitrophenyl)
acrylamide (16e)
Yield 86%; mp. 209–212 °C. 1H NMR (300 MHz,CDCl3): d 6.48(d,
J = 15.75 Hz,1H), 7.63 (d, J = 8.79 Hz, 2H, ArH), 7.72 (d, J = 8.58 Hz,
2H, ArH), 7.75 (s, 1H), 7.98 (d, J = 8.79 Hz, 2H, ArH), 8.25 (d,
J = 8.76, 2H, ArH), 8.28 (s, 1H, NH). ESI-MS: 410.96 (C15H12BrN2O5S,
[M+H]+). Anal. Calcd for C15H11BrN2O5S: C, 43.81; H, 2.70; N, 6.81.
Found: C, 43.73; H, 2.67; N, 6.85.
4.6. Effects on tubulin polymerization
Bovine brain tubulin was purified as described previously.32 To
evaluate the effect of the compounds on tubulin assembly in vi-
4.4. Crystal structure determination
tro,33 varying concentrations were preincubated with 10
lM tubu-
lin in glutamate buffer at 30 °C and then cooled to 0 °C. After
addition of GTP, the mixtures were transferred to 0 °C cuvettes in
a recording spectrophotometer and warmed up to 30 °C and the
assembly of tubulin was observed turbidimetrically. The IC50 was
defined as the compound concentration that inhibited the extent
of assembly by 50% after 20 min incubation.
Crystal structure determination of compound 10c were carried
out on a Nonius CAD4 diffractometer equipped with graphite-
monochromated Mo K
a (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 10c are listed in Table 3.
4.7. Docking simulations
Molecular docking of compound 10c into the 3D X-ray structure
of tubulin (PDB code: 1SA0) was carried out using the Auto-Dock
software (version 4.0) as implemented through the graphic user
interface Auto-Dock Tool Kit (ADT 1. 4.6).34

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Y. Luo et al. / Bioorg. Med. Chem. 19 (2011) 4730–4738
7. Romagnoli, R.; Baraldi, P. G.; Carrion, M. D.; Cruz-Lopez, O.; Cara, C. L.; Basso,
The graphical user interface ADT was employed to set up the
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enzymes: all hydrogens were added, Gasteiger charges were calcu-
lated and nonpolar hydrogens were merged to carbon atoms. For
macromolecules, generated pdbqt files were saved.
The 3D structures of ligand molecules were built, optimized
(PM3) level, and saved in Mol2 format with the aid of the molecu-
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the docking input files. In all docking
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a grid box size of
42 Â 45 Â 43 points in x, y, and z directions was built, the maps
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This work was supported by Jiangsu National Science Founda-
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the Central Universities (No. 1092020804 & 1106020824).
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