Design, synthesis and biological evaluation of novel chalcone derivatives as antitubulin agents

Article abstract of DOI:10.1016/j.bmc.2012.03.055

A series of novel chalcone derivatives have been designed and synthesized, and their biological activities were also evaluated as potential inhibitors of tubulin. These compounds were assayed for growth-inhibitory activity against MCF-7 and A549 cell line

Full text of DOI:10.1016/j.bmc.2012.03.055

Bioorganic & Medicinal Chemistry 20 (2012) 3212–3218
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of novel chalcone derivatives
as antitubulin agents
Hui Zhang a, Jia-Jia Liu a, Jian Sun a, Xian-Hui Yang a, Ting-Ting Zhao a, Xiang Lu a, Hai-Bin Gong b,
,
⇑
Hai-Liang Zhu a,
⇑
a State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China
b Xuzhou Central Hospital, Xuzhou 221009, 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 novel chalcone derivatives have been designed and synthesized, and their biological activities
were also evaluated as potential inhibitors of tubulin. These compounds were assayed for growth-inhib-
itory activity against MCF-7 and A549 cell lines in vitro. Compound 3d showed the most potent antipro-
Received 2 March 2012
Revised 26 March 2012
Accepted 26 March 2012
Available online 1 April 2012
liferative activity against MCF-7 and A549 cell lines with IC50 values of 0.03 and 0.95
lg/mL and exhibited
the most potent tubulin inhibitory activity with IC50 of 1.42 g/mL. Docking simulation was performed to
l
insert compound 3d into the crystal structure of tubulin at colchicines binding site to determine the
probable binding model. Based on the preliminary results, compound 3d with potent inhibitory activity
in tumor growth may be a potential anticancer agent.
Keywords:
Chalcone
Antiproliferative activity
Molecular docking
Tubulin inhibitory activity
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
C@O and C@C rotates with a relatively broad angle, which may in-
crease binding energy of chalcone with the receptor. In order to at-
Dynamic mitotic-spindle microtubules are among the most suc-
cessful targets for anticancer therapy.1 Nowadays, anticancer
agents that interfere with microtubulin function are in widespread
use in man, such as colchicine and combretastatin A-4 (CA-4).2 Col-
chicine is the first compound that binds to tubulin which is the di-
meric subunit of microtubules and exerts its growth-inhibitory
effects through the inhibition of microtubule dynamics, resulting
in the growth arrest of tumor cells at the G2-M phase.3 CA-4 pos-
sessed the most potent and interesting antitumor activity. And
early work showed it inhibited tubulin polymerization and the
proliferation of murine and human cancer cells.4,5
Chalcones, precursors of flavonoids and isoflavonoid, are an
important pharmacophore of various natural products6 and display
a variety of biological activities, such as anti-cancer,7–9 anti-inflam-
matory,10,11 anti-tuberculosis,12 and anti-fungal activities.13 In the
last few years, the development of chalcones as tubulin-binding
agents has been drawn wide attention.14 Recent studies have
shown that some compounds containing the chalcone skeleton
are believed to be a result of binding to tubulin and preventing it
from polymerising into microtubules.15,16 Structural modifications
in chalcone scaffold lead to the improvement of their bioavailabil-
ity and some synthetic chalcone derivatives show selective cyto-
toxicity against MCF-7 cells.17 However, the single bond between
tempt a better binding condition and improve biological activity,
we introduce nitro group and trifluoromethyl group to the chal-
cone derivatives.
According to reports, the nitro group is a unique functional
group with a diversity of chemical and biological actions. Its very
strong electron attracting ability creates localized or regional elec-
tron deficient sites within molecules. When such compounds inter-
act with living systems these electrophilic sites may then react
with a variety of intra and extracellular biological nucleophiles,
such as proteins, amino acids and enzymes. There are numerous
drugs which exert their primary pharmacological action because
of the presence of an aromatic nitro group. The structural types
and functions are quite diverse and include antineoplastic, antibi-
otic and antiparasitic drugs.18 In all cases, the nitro group attract-
ing functionality is usually necessary for the specific biological
effect desired.19,20 On the other hand, trifluoromethyl group is con-
firmed to promote the penetration of ligands while moving
through the cell membrane.21,22 Moreover, it can provide high
in vivo stability.23
The combined substructures (chalcone, dinitrobenzene and
trifluoromethyl group) without wrecking their original effective
characteristics, might exhibit synergistic effect to improve
antitubulin and anticancer activities. Thus, we designed and syn-
thesized chalcone compounds (3a–3t) (Table 1) bearing dinitro-
benzotrifluoride, then evaluated their anticancer and antitubulin
activities.
⇑
Corresponding authors. Tel.: +86 25 8359 2572; fax: +86 25 8359 2672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.03.055

H. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 3212–3218
3213
Table 1
Chemical structures of 3a–3t
with electron-withdrawing substituents (3i–3t) in the A-ring. A
comparison of the para substituents on the A-ring demonstrated
that an electron-donating group (3a–3h) have slightly improved
antiproliferative activity and the potency order is CH3 > OCH3,
whereas, F (3i–3l), Cl (3m–3p) and Br (3q–3t) substituent had min-
imal effects compared with CH3 (3a–3d) and OCH3 (3e–3h). In the
case of constant A-ring substituents, change of substituents on B-
ring could also affect the activities of these compounds. We found
that compounds with a halogen atom on the 5-position of salicyl-
aldehyde displayed higher anticancer activity than compounds
with two halogen atoms on the 3- and 5-position of salicylalde-
hyde. Among compounds with a halogen atom on the 5-position
of salicylaldehyde, the strength order is Cl > Br. Thus, the com-
pound 3d with para-Me group in the A-ring and Cl in the B-ring
showed the best 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,
compound 3d showed the most potent anti-tubulin polymeriza-
tion activity and 50% tubulin polymerization inhibition about
R2
O
B
R3
NO2
O
A
O2N
R1
CF3
Compound
R1
R2
R3
3a
3b
3c
3d
3e
3f
3g
3h
3i
CH3
CH3
CH3
CH3
OCH3
OCH3
OCH3
OCH3
F
F
F
F
Cl
Cl
Cl
Cl
Br
Br
Br
Br
Br
Cl
H
Br
Cl
Br
Cl
Br
Cl
Br
Cl
Br
Cl
Br
Cl
Br
Cl
Br
Cl
Br
Cl
Br
Cl
H
1.42
IC50 of 1.70
l
g/mL, respectively (the positive control colchicine with an
g/mL for tubulin). This result indicated the anti-prolif-
Br
Cl
H
l
erative effect was produced by direct connection of tubulin and the
compound.
Furthermore, compound 3d was further assayed for its effect on
cell cycle using flow cytometry (Fig. 1). As shown in Figure 1 and
67.93% of these cells were arrested at G2/M after treatment with
a 5 lg/mL concentration of 3d for 24 h. These findings indicated
a continuing impairment of cell division and confirmed compound
3d was a potent antitubulin agent.
To gain better understanding on the potency of the studied
compounds and guide further SAR studies, we proceeded to exam-
ine the interaction of compound 3d with tubulin (PDB code: 1SA0).
The molecular docking was performed by simulation of compound
3d into the colchicine binding site of tubulin. All docking runs were
applied LigandFit Dock protocol of Discovery Studio 3.1. The bind-
ing modes of compound 3d and tubulin were depicted in Figure 2.
All the amino acid residues which had interactions with tubulin
were exhibited in Figure 2b. In the binding mode, compound 3d
is nicely bound to the colchicine binding site of tubulin via hydro-
phobic interactions and binding is stabilized by one hydrogen bond
H
Br
Cl
H
3j
3k
3l
H
3m
3n
3o
3p
3q
3r
3s
3t
Br
Cl
H
H
Br
Cl
H
H
2. Results and discussion
2.1. Chemistry
The synthesis of compounds (3a–3t) is followed the general
pathway outlined in Scheme 1. They are prepared in two steps.
Firstly, the chalcones (1a–1t) were obtained by the substituted
salicylaldehydes and the substituted acetophenone, using 40%
potassium hydroxide as catalyst in ethanol. Then, chalcones (1a–
and one
p-cation interactions. The oxygen atom on B-ring of 3d
formed one hydrogen bond with the amino hydrogen of Asn
C:101 (bond length: Asn C:101 N–HÁ Á ÁO = 1.604 Å; bond angle:
Asn C:101 N–HÁ Á ÁO = 145.3°). Overall, these results of the molecu-
lar docking study showed that the compound 3d could act syner-
gistically to interact with the colchicine binding site of tubulin,
suggested that compound 3d was a potential inhibitor of tubulin.
1t), 4-chloro-3,5-dinitro-a,a,a-trifluorotoluene (2) and potassium
tert-butoxide were dissolved in DMF and reacted to obtain the
desired compounds (3a–3t) at the room temperature. All the
synthetic compounds were characterized by 1H NMR, elemental
analysis and mass spectrum, which were in full accordance with
their depicted structures.
3. Conclusion
In this study, a series of novel antitubulin polymerization inhib-
itors (3a–3t) containing chalcone skeleton had been synthesized
and evaluated their biological activities. These compounds exhib-
ited potent tubulin polymerization inhibitory activities and anti-
proliferative activities against MCF-7 and A549 cells. Among all
of the compounds, compound 3d showed the most potent inhibi-
tion activity which inhibited the growth of MCF-7 and A549 cell
2.2. Bioactivity and molecular modeling
To test the anticancer activities of the synthesized compounds,
we evaluated antiproliferative activities of compounds 3a–3t
against MCF-7 and A549 cells. The results were summarized in Ta-
ble 2. These chalcone compounds showed remarkable antiprolifer-
ative effects. Among them, compound 3d displayed the most
lines with IC50 values of 0.03 and 0.95
lg/mL and inhibited the
potent inhibitory activity (IC50 = 0.03
lg/mL for MCF-7 and
polymerization of tubulin with IC50 of 1.42
lg/mL. Molecular dock-
IC50 = 0.95 g/mL for A549), comparable to the positive control col-
l
ing was further performed to study the inhibitor-tubulin protein
chicine (IC50 = 0.51
lg/mL for MCF-7, IC50 = 0.74 lg/mL for A549,
interactions. After analysis of the binding model of compound 3d
respectively).
with tubulin, it was found that one hydrogen bond and one p-cat-
Structure-activity relationships in these chalcone derivatives
demonstrated that compounds with para electron-donating
substituents (3a–3h) showed more potent activities than those
ion interaction with the protein residues in the colchicine binding
site might play a crucial role in its antitubulin polymerization and
antiproliferative activities. In summary, compound 3d shows

3214
H. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 3212–3218
R2
O
O
OHC
OH
R2
a
R3
HO
+
R1
R3
R1
1
a-1
t
R2
R2
Cl
O
O
O2N
NO2
b
+
R3
NO2
HO
R3
O
CF3
O2N
R1
R1
CF3
2
1
a
-1
t
3a-3t
Scheme 1. The synthetic routes of compounds 3a–3t. Reagents and conditions: (a) ethanol, NaOH, rt, 5 h; (b) DMF, potassium tert-butoxide, rt, 4 h.
Table 2
Inhibition (IC50) of MCF-7 and A549 cells proliferation and inhibition of tubulin
polymerization by compounds 3a–3t
4. Experiments
Compound
IC50 SD (
A549a
lg/mL)
4.1. Materials and measurements
MCF-7a
Tubulinb
All chemicals and reagents used in current study were of analyt-
ical grade. All the 1H NMR spectra were recorded on a Bruker
DPX300 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 sil-
ica gel (200–300 mesh) eluting with ethyl acetate and petroleum
ether.
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
1.44 0.06
0.70 0.02
0.55 0.01
0.03 0.002
3.53 0.11
2.69 0.09
2.32 0.12
1.83 0.07
8.06 0.23
5.02 0.15
6.01 0.22
5.17 0.24
6.76 0.17
4.54 0.17
4.74 0.13
3.21 0.21
2.87 0.12
2.60 0.09
2.17 0.15
1.41 0.08
0.51 0.06
0.43 0.04
10.12 0.51
10.05 0.23
6.85 0.42
0.95 0.04
13.16 0.48
12.55 0.61
12.65 0.34
11.52 0.51
120.22 9.1
29.35 1.6
26.34 1.3
23.82 1.1
19.43 0.9
17.23 1.0
16.50 0.71
11.69 0.52
16.82 0.62
14.50 0.67
13.73 0.54
10.86 0.41
0.74 0.08
0.53 0.04
5.68 0.12
4.87 0.21
6.41 0.10
1.42 0.06
6.02 0.22
6.56 0.31
4.08 0.13
4.83 0.15
15.31 0.65
9.62 0.34
12.31 0.52
11.66 0.35
12.6 0.51
6.34 0.23
7.14 0.31
4.68 0.21
10.01 0.46
9.46 0.32
6.25 0.24
4.62 0.15
1.70 0.12
0.81 0.08
4.2. General synthetic procedure of chalcones (1a–1t)
Equimolar portions of the appropriately substituted salicylalde-
hydes (2 mmol, 1 equiv) and substituted acetophenone (2 mmol,
1 equiv) were dissolved in approximately 20 mL of ethanol. The
mixture was allowed to stir for several minutes at 0 °C to let dis-
solve. Then, a 0.5 mL aliquot of a 40% aqueous sodium hydroxide
solution was slowly added dropwise to the reaction flask via a
self-equalizing addition funnel. The reaction solution was allowed
to stir at room temperature for approximately 4–6 h. The mixture
was adjusted to pH 5.0 with dilute hydrochloric acid until the reac-
tion was complete. The reaction was monitored by TLC. Most com-
monly, a precipitate formed and was then collected by suction
filtration.
3r
3s
3t
Colchicine
CA-4
a
Inhibition of the growth of tumor cell lines.
Inhibition of tubulin polymerization.
b
promising biological activity. Also detailed investigations are
continuing to study the mechanisms of the inhibitory activity
reported here.

H. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 3212–3218
3215
Figure 1. Effects of compound 3d on cell cycle progression of MCF-7 cells were determined by flow cytometry analysis. MCF-7 cells were treated with 3d for 24 h. The
percentage of cells in each cycle phase was indicated.
Figure 2. (a) Compound 3d (colored by atom: carbons: gray; nitrogen: blue; oxygen: red; sulfur: yellow) is bond into tubulin (entry 1SA0 in the Protein Data Bank). The
dotted lines show the hydrogen bond and the yellow line show the p-cation interactions. (b) 2D ligand interaction diagram of compound 3d 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.

3216
H. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 3212–3218
4.3. General procedure for synthesis of target compounds (3a–3t)
557.0 (C23H14Cl2F3N2O7, [M+H]+). Anal. Calcd for C23H13Cl2F3N2O7:
C, 49.57; H, 2.35; N, 5.03. Found: C, 49.65; H, 2.33; N, 5.10
A mixture of chalcones (1a–1t) (1 mmol), 4-chloro-3,5-dini-
tro-
a
,
a
,
a
-trifluorotoluene (2) (1 mmol) and potassium tert-
4.3.7. (E)-3-(3-Bromo-2-(2,6-dinitro-4-(trifluoromethyl)phen
oxy)phenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (3g)
butoxide (0.75 mmol) was dissolved in DMF and stirred at the
room temperature for 4 h. The products were extracted with
ethyl acetate and water. The extract was dried over anhydrous
Na2SO4, filtered and evaporated. The residue was purified by col-
umn chromatography using petroleum ether and ethyl acetate
(3:1).
Yellow solid, yield 72.5%, mp: 172 °C. 1H NMR (300 MHz, CDCl3)
d: 3.03 (s, 3H), 7.29 (d, J = 7.59 Hz, 1H), 7.56–7.59 (m, 2H), 7.84 (d,
J = 2.58 Hz, 1H), 8.07 (d, J = 2.37 Hz, 2H), 8.46 (s, 1H), 8.54 (s, 4H).
MS (ESI): 566.9 (C23H15BrF3N2O7, [M+H]+). Anal. Calcd for
C23H14BrF3N2O7: C, 48.70; H, 2.49; N, 4.94. Found: C, 48.65; H,
2.48; N, 4.89.
4.3.1. (E)-3-(3,5-Dibromo-2-(2,6-dinitro-4-(trifluoromethyl)phen-
oxy)phenyl)-1-p-tolylprop-2-en-1-one (3a)
4.3.8. (E)-3-(3-Chloro-2-(2,6-dinitro-4-(trifluoromethyl)phenoxy)
phenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (3h)
Yellow solid, yield 72.9%, mp: 145–147 °C. 1H NMR (300 MHz,
CDCl3) d: 2.42 (d, J = 11.0 Hz, 3H), 7.29 (t, J = 9.06 Hz, 2H), 7.55 (t,
J = 16.38 Hz, 1H), 7.67 (d, J = 2.19 Hz, 1H), 7.78 (d, J = 2.19 Hz,
1H), 7.83 (s, 1H), 7.91 (t, J = 6.77 Hz, 2H), 8.27 (s, 2H). MS (ESI):
628.9 (C23H14Br2F3N2O6, [M+H]+). Anal. Calcd for C23H13Br2F3N2O6:
C, 43.84; H, 2.08, N, 4.45. Found: C, 43.78; H, 2.11; N, 4.48.
Yellow solid, yield 67.2%, mp: 165 °C. 1H NMR (300 MHz, CDCl3)
d: 3.11 (s, 3H), 7.32 (d, J = 6.87 Hz, 1H), 7.60 (d, J = 7.21 Hz, 1H),
7.89 (d, J = 11.23 Hz, 2H), 8.14 (d, J = 4.62 Hz, 2H), 8.63 (s, 3H),
8.71 (d, J = 7.72 Hz, 2H). MS (ESI): 523.0 (C23H15ClF3N2O7,
[M+H]+). Anal. Calcd for C23H14ClF3N2O7: C, 52.84; H, 2.70; N,
5.36. Found: C, 52.76; H, 2.74; N, 5.29.
4.3.2. (E)-3-(3,5-Dichloro-2-(2,6-dinitro-4-(trifluoromethyl)phen
oxy)phenyl)-1-p-tolylprop-2-en-1-one (3b)
4.3.9. (E)-3-(3,5-Dibromo-2-(2,6-dinitro-4-(trifluoromethyl)phen
oxy)phenyl)-1-(4-fluorophenyl)prop-2-en-1-one (3i)
Yellow solid, yield 81.9%, mp: 170 °C. 1H NMR (300 MHz, CDCl3)
d: 2.44 (s, 3H), 7.31 (d, J = 7.86 Hz, 2H), 7.36 (d, J = 2.4 Hz, 1H), 7.57
(d, J = 15.75 Hz, 1H), 7.64 (d, J = 2.4 Hz, 1H), 7.92 (t, J = 8.15 Hz, 3H),
8.30 (s, 2H). MS (ESI): 541.2 (C23H14Cl2F3N2O6, [M+H]+). Anal. Calcd
for C23H13Cl2F3N2O6: C, 51.04; H, 2.42; N, 5.18. Found: C, 51.07; H,
2.39; N, 5.16.
Yellow solid, yield 51.7%, mp: 157 °C. 1H NMR (300 MHz, CDCl3)
d: 7.18 (t, J = 8.51 Hz, 2H), 7.52 (d, J = 15.72 Hz, 2H), 7.68 (d,
J = 2.04 Hz, 1H), 7.79 (d, J = 2.01 Hz, 2H), 7.88 (d, J = 15.72 Hz,
1H), 8.28 (s, 2H). MS (ESI): 632.9 (C22H11Br2F4N2O6, [M+H]+). Anal.
Calcd for: C22H10Br2F4N2O6: C, 41.67; H, 1.59; N, 4.42. Found: C,
41.55; H, 1.63; N, 4.38.
4.3.3. (E)-3-(3-Bromo-2-(2,6-dinitro-4-(trifluoromethyl)phenoxy)
phenyl)-1-(p-tolyl)prop-2-en-1-one (3c)
4.3.10. (E)-3-(3,5-Dichloro-2-(2,6-dinitro-4-(trifluoromethyl)phen
oxy)phenyl)-1-(4-fluorophenyl)prop-2-en-1-one (3j)
Yellow solid, yield 77.2%, mp: 150 °C. 1H NMR (300 MHz,
CDCl3) d: 2.45 (s, 3H), 6.51 (d, J = 8.76 Hz, 1H), 7.24 (t,
J = 6.86 Hz, 1H), 7.32 (d, J = 7.5 Hz, 2H), 7.61 (s, 1H), 7.77 (d,
J = 2.25 Hz, 1H), 7.95 (d, J = 7.86 Hz, 2H), 8.07 (d, J = 15.18, 1H),
8.50 (s, 2H). MS (ESI): 551.0 (C23H15BrF3N2O6, [M+H]+). Anal.
Calcd for C23H14BrF3N2O6: C, 50.11; H, 2.56; N, 5.08. Found: C,
50.07; H, 2.54; N, 5.11.
Yellow solid, yield 70.3%, mp: 163 °C. 1H NMR (500 MHz, CDCl3)
d: 7.18 (t, J = 8.52 Hz, 3H), 7.25 (s, 1H), 7.38 (d, J = 2.15 Hz, 1H), 7.49
(s, 1H), 7.67 (d, J = 15.7 Hz, 1H), 7.93 (d, J = 15.9 Hz, 1H), 8.06 (m,
2H). MS (ESI): 544.9 (C22H11Cl2F4N2O6, [M+H]+). Anal. Calcd for
C22H10Cl2F4N2O6: C, 48.46; H, 1.85; N, 5.14. Found: C, 48.38; H,
1.81; N, 5.11.
4.3.11. (E)-3-(3-Bromo-2-(2,6-dinitro-4-(trifluoromethyl)phen
oxy)phenyl)-1-(4-fluorophenyl)prop-2-en-1-one (3k)
4.3.4. (E)-3-(3-Chloro-2-(2,6-dinitro-4-(trifluoromethyl)phenoxy)
phenyl)-1-(p-tolyl)prop-2-en-1-one (3d)
Yellow solid, yield 65.5%, mp: 157 °C. 1H NMR (300 MHz, CDCl3)
d: 6.44 (d, J = 8.79 Hz, 1H), 7.18 (t, J = 8.15 Hz, 2H), 7.37 (d,
J = 8.76 Hz, 1H), 7.58 (d, J = 15.72 Hz, 1H), 7.91 (s, 1H), 8.03–8.10
(m, 3H), 8.50 (s, 2H). MS (ESI): 554.9 (C22H12BrF4N2O6, [M+H]+).
Anal. Calcd for: C22H11BrF4N2O6: C, 47.59; H, 2.00; N, 5.05. Found:
C, 47.48; H, 1.99; N, 5.10.
Yellow solid, yield 80.1%, mp: 145 °C. 1H NMR (300 MHz, CDCl3)
d: 2.44 (s, 3H), 6.51 (d, J = 8.76 Hz, 1H), 7.24 (t, J = 6.86 Hz, 1H), 7.32
(d, J = 7.5 Hz, 2H), 7.61 (s, 1H), 7.77 (d, J = 2.25 Hz, 1H),7.95 (d,
J = 7.86 Hz, 2H),8.07 (d, J = 15.18, 1H), 8.50 (s, 2H). MS (ESI):
507.0 (C23H15ClF3N2O6, [M+H]+). Anal. Calcd for C23H14ClF3N2O6:
C, 54.51; H, 2.78; N, 5.53. Found: C, 54.43; H, 2.82; N, 5.48.
4.3.12. (E)-3-(3-Chloro-2-(2,6-dinitro-4-(trifluoromethyl)phenoxy)
phenyl)-1-(4-fluorophenyl)prop-2-en-1-one (3l)
4.3.5. (E)-3-(3,5-Dibromo-2-(2,6-dinitro-4-(trifluoromethyl)phen
oxy)phenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (3e)
Yellow solid, yield 67.9%, mp: 190 °C. 1H NMR (300 MHz, CDCl3)
d: 7.38 (d, J = 2.37 Hz, 1H), 7.52 (d, J = 15.9 Hz, 1H), 7.64 (t,
J = 10.34 Hz, 4H), 7.92 (t, J = 12.62 Hz, 3H), 8.30 (s, 2H). MS (ESI):
511.0 (C22H12ClF4N2O6, [M+H]+). Anal. Calcd for: C22H11ClF4N2O6:
C, 51.73; H, 2.17; N, 5.48. Found: C, 51.67; H, 2.21; N, 5.41.
Yellow solid, yield 76.1%, mp: 163 °C. 1H NMR (300 MHz, CDCl3)
d: 3.88 (t, J = 8.25 Hz, 3H), 6.96 (t, J = 13.64 Hz, 2H), 7.53 (d,
J = 15.75 Hz,1H), 7.67 (d, J = 1.10 Hz,1H), 7.78 (d, J = 2.19 Hz,1H),
7.85 (d, J = 15.75 Hz, 1H), 8.02 (d, J = 8.79 Hz, 2H), 8.27 (s, 2H).
MS (ESI):644.9 (C23H14Br2F3N2O7, [M+H]+). Anal. Calcd for
C
23H13Br2F3N2O7: C, 42.75; H, 2.03; N, 4.34. Found: C, 42. 66; H,
4.3.13. (E)-1-(4-Chlorophenyl)-3-(3,5-dibromo-2-(2,6-dinitro-4-
(trifluoromethyl)phenoxy)phenyl)prop-2-en-1-one (3m)
Yellow solid, yield 72.7%, mp: 163 °C. 1H NMR (300 MHz, CDCl3)
d: 7.47–7.53 (m, 3H), 7.68 (d, J = 2.4 Hz, 1H), 7.80 (d, J = 2.37 Hz,
1H), 7.90 (d, J = 15.72 Hz, 1H), 7.93–7.98 (m, 2H), 8.28 (d,
J = 0.57 Hz, 2H). MS (ESI): 648.8 (C22H11Br2ClF3N2O6, [M+H]+). Anal.
Calcd for: C22H10Br2ClF3N2O6: C, 40.62; H, 1.55; N, 4.31. Found: C,
40.67; H, 1.59; N, 4.37.
1.99; N, 4.30.
4.3.6. (E)-3-(3,5-Dichloro-2-(2,6-dinitro-4-(trifluoromethyl)phen
oxy)phenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (3f)
Yellow solid, yield 80.2%, mp: 166 °C. 1H NMR (300 MHz, CDCl3)
d: 3.83 (t, J = 8.23 Hz, 3H), 7.16 (d, J = 2.30 Hz, 2H), 7.54 (d,
J = 9.75 Hz, 2H), 8.11 (d, J = 6.75 Hz, 2H), 8.85 (m, 4H). MS (ESI):

H. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 3212–3218
3217
4.3.14. (E)-1-(4-Chlorophenyl)-3-(3,5-dichloro-2-(2,6-dinitro-4-
(trifluoromethyl)phenoxy)phenyl)prop-2-en-1-one (3n)
Yellow solid, yield 76.2%, mp: 165 °C. 1H NMR (300 MHz, CDCl3)
d: 7.50 (t, J = 7.97 Hz, 3H), 7.68 (d, J = 2.22 Hz, 1H), 7.80 (d,
J = 2.22 Hz, 1H), 7.90 (d, J = 15.72 Hz, 1H), 7.96 (d, J = 8.61 Hz,
2H), 8.28 (s, 2H). MS (ESI): 560.9 (C22H11Cl3F3N2O6, [M+H]+). Anal.
Calcd for C22H10Cl3F3N2O6: C, 47.04; H, 1.79; N, 4.99. Found: C,
47.11; H, 1.85; N, 4.98.
grown to log phase in RPMI 1640 medium supplemented with
10% fetal bovine serum. After diluting to 2 Â 104 cells mLÀ1 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 six wells with colchicines and CA-4
co-assayed as positive references. After 48 h exposure period,
40
zol-2-yl)-2,5-diphenyltetrazolium bromide)) was added to each
well. Four hours later, 100 L extraction solution (10% SDS–5% iso-
l
L of PBS containing 2.5 mg mLÀ1 of MTT (3-(4,5-dimethylthia-
4.3.15. (E)-3-(3-Bromo-2-(2,6-dinitro-4-(trifluoromethyl)phen
oxy)phenyl)-1-(4-chlorophenyl)prop-2-en-1-one (3o)
l
Yellow solid, yield 80.1%, mp: 168 °C. 1H NMR (300 MHz, CDCl3)
d: 6.45 (d, J = 8.79 Hz, 1H), 7.31–7.40 (m, 1H), 7.47–7.50 (m, 2H),
7.55 (d, J = 15.93 Hz, 1H), 7.90 (d, J = 2.19 Hz, 1H), 7.97–7.80 (m,
2H), 8.05 (d, J = 15.75 Hz, 1H), 8.50 (s, 2H). MS (ESI): 570.9
(C22H12BrClF3N2O6, [M+H]+). Anal. Calcd for C22H11BrClF3N2O6: C,
46.22; H, 1.94; N, 4.90. Found: C, 46.31; H, 1.91; N, 4.86.
butyl alcohol–0.01 M HCl) was added. After an overnight incuba-
tion at 37 °C, the optical density was measured at a wavelength
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 summa-
rized in Table 2.
4.3.16. (E)-3-(3-Chloro-2-(2,6-dinitro-4-(trifluoromethyl)phen
oxy)phenyl)-1-(4-chlorophenyl)prop-2-en-1-one (3p)
4.5. Effects on tubulin polymerization
Yellow solid, yield 77.2%, mp: 172 °C. 1H NMR (300 MHz, CDCl3)
d: 6.51 (d, J = 8.79 Hz, 1H), 7.23–7.27 (m, 1H), 7.48 (t, J = 4.31 Hz,
2H), 7.56 (t, J = 7.97 Hz, 1H), 7.76 (d, J = 2.55 Hz,1H), 7.98 (t,
J = 2.66 Hz, 2H), 7.99–8.09 (m, 1H), 8.50(s, 2H). MS (ESI): 526.9
(C22H12Cl2F3N2O6, [M+H]+). Anal. Calcd for C22H11Cl2F3N2O6: C,
50.12; H, 2.10; N, 5.31. Found: C, 50.19; H, 2.06; N, 5.28.
Bovine brain tubulin was purified as described previously.25 To
evaluate the effect of the compounds on tubulin assembly in vi-
tro,26 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 turbid metrically. The IC50 was
defined as the compound concentration that inhibited the extent
of assembly by 50% after 20 min incubation.
4.3.17. (E)-1-(4-Bromophenyl)-3-(3,5-dibromo-2-(2,6-dinitro-4-
(trifluoromethyl)phenoxy)phenyl)prop-2-en-1-one (3q)
Yellow solid, yield 75.4%, mp: 185 °C. 1H NMR (300 MHz, CDCl3)
d: 7.49 (d, J = 15.72 Hz, 1H), 7.65 (d, J = 8.61 Hz, 3H), 7.80 (s, 1H),
7.90 (t, J = 7.86 Hz, 3H), 8.28 (s, 2H). MS (ESI): 692.8
(C22H11Br3F3N2O6, [M+H]+). Anal. Calcd for C22H10Br3F3N2O6: C,
38.02; H, 1.45, N, 4.03. Found: C, 38.11; H, 1.47; N, 4.01.
4.6. Docking simulations
Molecular docking of compound 3d into the three-dimensional
X-ray structure of tubulin (PDB code: 1SA0) was carried out using
LigandFit Dock protocol of Discovery Studio 3.1. For protein prep-
aration, the hydrogen atoms were added, and the water and impu-
rities were removed. The whole tubulin was defined as a receptor
and the site sphere was selected based on the ligand binding loca-
tion of colchicine, then the colchicine molecule was removed and
3d was placed during the molecular docking procedure. Types of
interactions of the docked protein with ligand were analyzed after
the end of molecular docking.
4.3.18. (E)-1-(4-Bromophenyl)-3-(3,5-dichloro-2-(2,6-dinitro-4-
(trifluoromethyl)phenoxy)phenyl)prop-2-en-1-one (3r)
Yellow solid, yield 69.6%, mp: 165 °C. 1H NMR (300 MHz, CDCl3)
d: 7.37 (d, J = 2.37 Hz, 1H), 7.52 (d, J = 15.72 Hz, 1H), 7.61–7.67 (m,
3H), 7.92 (t, J = 12.53 Hz, 3H), 8.31 (s, 2H). MS (ESI): 604.9
(C22H11BrCl2F3N2O6, [M+H]+). Anal. Calcd for C22H10BrCl2F3N2O6:
C, 43.59; H, 1.66; N, 4.62. Found: C, 43.48; H, 1.65; N, 4.58.
4.3.19. (E)-3-(3-Bromo-2-(2,6-dinitro-4-(trifluoromethyl)phenoxy)
phenyl)-1-(4-bromophenyl)prop-2-en-1-one (3s)
References and notes
Yellow solid, yield 71.9%, mp: 158 °C. 1H NMR (300 MHz, CDCl3)
d: 6.45 (d, J = 8.79 Hz, 1H), 7.38–7.41 (m, 1H), 7.54 (d, J = 15.93 Hz,
1H), 7.64 (t, J = 9.96 Hz, 2H), 7.89–7.92 (m, 3H), 8.06 (d,
J = 15.72 Hz, 1H), 8.50 (s, 2H). MS (ESI): 614.9 (C22H12Br2F3N2O6,
[M+H]+). Anal. Calcd for C22H11Br2F3N2O6: C, 42.89; H, 1.80; N,
4.55. Found: C, 42.98; H, 1.83; N, 4.49.
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Yellow solid, yield 76.2%, mp: 165 °C. 1H NMR (300 MHz, CDCl3)
d: 6.51 (d, J = 8.79 Hz, 1H), 7.16–7.22 (m, 3H), 7.58 (d, J = 15.72 Hz,
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H. Zhang et al. / Bioorg. Med. Chem. 20 (2012) 3212–3218
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