Synthesis, biological evaluation, and molecular docking studies of 2,6-dinitro-4-(trifluoromethyl)phenoxysalicylaldoxime derivatives as novel antitubulin agents

Article abstract of DOI:10.1016/j.bmc.2012.03.057

A series of 2,6-dinitro-4-(trifluoromethyl)phenoxysalicylaldoxime derivatives (1h-20h) have been designed and synthesized, and their biological activities were also evaluated as potential antiproliferation and tubulin polymerization inhibitors. Among all the compounds, 2h showed the most potent activity in vitro, which inhibited the growth of MCF-7, Hep-G2 and A549 cell lines with IC₅₀ values of 0.70 ± 0.05, 0.68 ± 0.02 and 0.86 ± 0.05 μM, respectively. Compound 2h also exhibited significant tubulin polymerization inhibitory activity (IC₅₀ = 3.06 ± 0.05 μM). The result of flow cytometry (FCM) demonstrated that compound 2h induced cell apoptosis. Docking simulation was performed to insert compound 2h into the crystal structure of tubulin at colchicine binding site to determine the probable binding model. Based on the preliminary results, compound 2h with potent inhibitory activity in tumor growth may be a potential anticancer agent.

Full text of DOI:10.1016/j.bmc.2012.03.057

Bioorganic & Medicinal Chemistry 20 (2012) 3233–3241
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, biological evaluation, and molecular docking studies
of 2,6-dinitro-4-(trifluoromethyl)phenoxysalicylaldoxime derivatives
as novel antitubulin agents
Ting-Ting Zhao a, , Xiang Lu a, , Xian-Hui Yang a, Li-Ming Wang a, Xi Li a, Zhong-Chang Wang a,
a,
Hai-Bin Gong b, , Hai-Liang Zhu
⇑
⇑
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 2,6-dinitro-4-(trifluoromethyl)phenoxysalicylaldoxime derivatives (1h–20h) have been
Received 23 February 2012
Revised 23 March 2012
Accepted 24 March 2012
Available online 1 April 2012
designed and synthesized, and their biological activities were also evaluated as potential antiproliferation
and tubulin polymerization inhibitors. Among all the compounds, 2h showed the most potent activity in
vitro, which inhibited the growth of MCF-7, Hep-G2 and A549 cell lines with IC50 values of 0.70 0.05,
0.68 0.02 and 0.86 0.05
tion inhibitory activity (IC50 = 3.06 0.05
l
M, respectively. Compound 2h also exhibited significant tubulin polymeriza-
M). The result of flow cytometry (FCM) demonstrated that
l
Keywords:
compound 2h induced cell apoptosis. Docking simulation was performed to insert compound 2h into
the crystal structure of tubulin at colchicine binding site to determine the probable binding model. Based
on the preliminary results, compound 2h with potent inhibitory activity in tumor growth may be a
potential anticancer agent.
2⁄ 6-Dinitro-4-
(trifluoromethyl)phenoxysalicylaldoxime
derivatives
Antitubulin polymerization
Structure–activity relationship
Molecular docking
Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
1. Introduction
Among these structures (A, C, D, E, and compound 1h), Schiff’s
base (or oxime) is a promising skeleton that has shown the good
In conjunction with the spread of cardiovascular disease, cancer
is today amongst the worldwide health threats.1 Much work pro-
vided insights into the mechanisms of cancer and the antimitotic
agents were widely used to treat human cancers.2–4 Antimitotic
agents cause mitotic arrest in eukaryotic cells by interfering with
the polymerization–depolymerization process of tubulin dimmers
of microtubule. Actually, blocking cell division by affecting the mi-
totic spindle has historically been a successful area of research for
the advancement of cancer drugs.5,6
Classic antimitotic agents, many of which are tubulin-binding
agents, such as colchicines6–10 taxanes9 and vinca alkaloids,11 inhi-
bit tubulin polymerization or interfere with microtubule disassem-
bly, disrupting the dynamic equilibrium and then leading to cell
cycle arrest or cell apoptosis. Apart from the above antimitotic
agents, several other new compounds (Figs. 1 and 2), esp. com-
pound A sharing the similar part with 2,6-dinitro-4-(trifluoro-
methyl)phenoxysalicylaldoxime derivatives, were found to inhibit
the polymerization of tubulin by binding to the colchicine
site.10,12–14
antitubulin activity. Schiff’s base (esp. oxime) displays a variety of
biological activities, such as anti-cancer,15,16 anti-inflammatory,17
anti-tuberculosis,18 anti-fungal,19 activities, and as herbicide.20
Their broad biological properties are largely due to the exocyclic
–RC@N– bond. The introduction of substituents to the terminal
bonds of Schiff’s base remains an area of pharmacological interest
to activate Schiff’s base; by the way, the introduction of O or N is
more significant, such as Zorubicin14 (Fig. 1C).
The incorporation of 1,3-dinitro-5-(trifluoromethyl)benzene
could contribute to the binding of target compounds with tubulin.
For instance, trifluoromethyl possess lipophilicify to penetrate the
cell membrane. Trifluoromethyl substituents on phenyl ring are of
particular interest. The isosteric substitution of hydrogen by the
strong electron-withdrawing effect of CF3 groups may increase
the lipophilicity and thus enhance the rate of cell penetration,
which is a very important feature in drug delivery in vivo, referring
to prokaryotic as well as eukaryotic cells.21–23 The higher polariz-
ability due to the C–F bond may give rise to new possibilities for
binding to the receptor. Trifluoromethyl substitution can also
influence pharmacokinetic and pharmacodynamic properties of
the molecule.24 Furthermore, two nitro substituents of compounds
could form hydrogen bond with the amino hydrogen of tubulin,
which led to enhance antiproliferation and inhibition of tubulin
polymerization activities.25,26
⇑
Corresponding authors. Tel.: +86 25 8359 2572; fax: +86 25 8359 2672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
These two authors equally contributed to this paper.
0968-0896/$ - see front matter Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.03.057

3234
T.-T. Zhao et al. / Bioorg. Med. Chem. 20 (2012) 3233–3241
N
HC
O
O
O
H3CO
H3CO
OH
O
OCH3
OH
S
OCH3
B
A
O
O
HO
O
F
OH
CH3
O
H2N
O
OH
OH
O
O
O
N
N
N
N
S
Cl
OH
N
O
HN
D
C
H
C
N
O
Cl
O
O
Cl
N
C
Cl
H2
C
O2N
NO2
C
N
H2
Cl
N
CF3
compound 1h
E
Figure 1. Chemical structures of antimitotic agents and lead tubulin inhibitors.
R3
H
N
C
N
R2
HC
O
O
O
O
R4
R1
O
OH
S
NO2
O2N
O
CF3
Figure 2. Similar part of compound A and 2,6-dinitro-4-(trifluoromethyl)phenoxysalicylaldoxime derivatives.
These previous researches encouraged us to integrate 1,3-dini-
performed to understand tubulin–inhibitor interaction. The dock-
ing results confirmed that hydrogen bonding may contribute to
the potent biological activities.
tro-5-(trifluoromethyl)benzene with Schiff’s base (oxime) to screen
new 2,6-dinitro-4-(trifluoromethyl)phenoxysalicylaldoxime as po-
tential antitubulin agents. The two combined substructures might
exhibit synergistic effect in anticancer activities. Herein we report
the synthesis and bioactivities of a series of novel 2,6-dinitro-4-(tri-
2. Results and discussion
2.1. Chemistry
fluoromethyl)phenoxysalicylaldoxime
derivatives
possessing
Schiff’s base to mimic the reported antimitotic agents. Their anti-
proliferation and inhibition of tubulin polymerization activities
were evaluated. Molecular modeling studies were consequently
The general reaction pathway for the synthesis of 2,6-dinitro-4-
(trifluoromethyl)phenoxysalicylaldoxime derivatives (1h–20h) is

T.-T. Zhao et al. / Bioorg. Med. Chem. 20 (2012) 3233–3241
3235
outlined in Scheme 1A–C. Firstly, the different substituted O-ben-
Cl
Cl
Cl
zylhydroxylamine hydrochloride (compound d) were coupled with
substituted salicylaldehyde to generate salicylaldoxime deriva-
tives, using triethylamine (TEA) as the deacid reagent, in a high
yield of 85–95%. Secondly, the different substituted salicyl-
aldoxime were treated with 2-chloro-1,3-dinitro-5-(trifluoro-
methyl)benzene (compound f) to afford the desired compounds
1h–20h (Table 1), using K2CO3 as the deacid reagent, with a yield
of 60–90%. By the way, the general synthetic procedure of com-
pounds d and f was prepared according to Scheme 1A–C. All of
the target compounds 1h–20h were reported for the first time,
and gave satisfactory analytical and spectroscopic data. 1H NMR
and ESI-MS spectra were consistent with the assigned structures.
NO2
NO2
O2N
vi
v
CF3
CF3
CF3
f
e
Scheme 1B. General synthesis of 2-chloro-1,3-dinitro-5-(trifluoromethyl)benzene
(compounds f). Reagents and conditions: (v) HNO3, H2SO4/SO3, 50–70 °C, 2 h; (vi)
HNO3, H2SO4/SO3, 100–115 °C, 2 h.
2.2. Crystal structure of compound
blockade. For instance, meta-substituent in the A-ring of
compounds (13h, 17h etc.) would block the binding of nitro in
the C-ring (Table 1) with the colchicine binding site of tubulin.
However, as for the B-ring (Table 1), inferred from Table 3, com-
pounds (2h, 3h) with ortho and para electron-withdrawing substit-
uents (halogen) showed more potent activities than those (1h)
without them. Among the compounds of halogen substituents,
the stronger and more electron-withdrawing effect, the higher
antiproliferative activity and the strength order is F > Cl > H. That
might be due to the high lipophilicity and metabolic stability of
fluorine substituent. In other words, the presence of fluorine often
leads to increased lipid solubility, thereby enhancing rates of
absorption and transport of drugs in vivo.27 Among all the com-
pounds, 2h displayed the best antiproliferative activity.
Among these compounds, a crystal structure of compound 4h
was determined by X-ray diffraction analysis. The crystal data
are presented in Table 2 and Figure 3 give perspective views of
4h with the atomic labeling system.
2.3. Bioactivity
2.3.1. Antiproliferative activities
The synthesized compounds were evaluated to test the antipro-
liferative activities against MCF-7, Hep-G2 and A549 cells. The
results were summarized in Table 3. As shown in Table 3, these
2,6-dinitro-4-(trifluoromethyl)phenoxysalicylaldoxime derivatives
bearing the Schiff moiety exhibited remarkable antiproliferative ef-
fects. Among them, compound 2h displayed the most potent inhib-
To examine whether the compounds interact with tubulin and
inhibit tubulin polymerization in vitro, we performed the tubulin
assembly assay. As shown in Table 3, some of these compounds
showed strong inhibitory effect. Among them, compound 2h
itory activity (IC50 = 0.70 0.05
for Hep-G2 and IC50 = 0.86 0.05
positive control combretastainA (CA-4) (IC50 = 0.41 0.04
MCF-7, IC50 = 0.19 0.04 M for Hep-G2, IC50 = 0.09 0.01
A549, respectively) and colchicine (IC50 = 0.53 0.07
l
M for MCF-7, IC50 = 0.68 0.02
M for A549), comparable to the
M for
M for
lM
l
l
l
(IC50 = 3.06 0.05
anti-tubulin polymerization activity, comparable to the positive
control (CA-4) (IC50 = 0.70 0.20 M for tubulin) and colchicine
(IC50 = 1.72 0.18 M for tubulin). The results indicated the anti-
lM for tubulin) displayed the most potent
l
l
l
M for MCF-
M for A549,
l
7, IC50 = 0.23 0.02
respectively).
l
M for Hep-G2, IC50 = 0.75 0.08
l
proliferative effect was produced by direct connection of tubulin
Subsequently, SAR (structure–activity relationships) studies
were inferred from Table 3. In general, compounds without elec-
tron-withdrawing substituents showed more potent activities than
those with them (halogen) in the A-ring (Table 1). Among the com-
pounds of halogen substituents, the stronger and more electron-
withdrawing effect, the lower antiproliferative activity. Therefore,
Cl (5h, 13h) and Br (9h, 17h) group substituents had minimal ef-
fects compared with 1h. Moreover, the position of substituents in
the A-ring also influenced the activities owing to stereospecific
and the compound.
2.3.2. Apoptosis assay
As representative of these 2,6-dinitro-4-(trifluoromethyl)phe-
noxysalicylaldoxime derivatives, compound 2h has been under
investigations in vitro experiment. We detected the mechanism
of compound 2h inhibition effect by flow cytometry (FCM) (
Fig. 4), and found that the compound could induce the apoptosis
of activated MCF-7 cells in a dose dependent manner. As shown
O
O
i
ii
O
O
+
H2N OH
N
OH
N
O
O
O
CH2
O
a
O
b
NH
NH
iii
O
+
R3
R3
iv
O
CH2
R4
CH2
O
R4
H2N
NH2 HCl
d
c
Scheme 1A. General synthesis of O-benzylhydroxylamine hydrochloride (compounds d). Reagents and conditions: (i) hydroxylamine, pyridine, 80 °C, overnight; (ii) Benzyl
chloride, NaHCO3, Bu4NHSO4, CH2Cl2/H2O, reflux, 7 h; (iii) hydrazine hydrate, ethanol, rt, 1.5 h; (iv) CH2Cl2, HCl(g), rt, 1 h.

3236
T.-T. Zhao et al. / Bioorg. Med. Chem. 20 (2012) 3233–3241
R3
H
C
R3
R2
N
R2
NH2 HCl
O
O
O
vii
OH
OH
R4
1g-20g
R4
R1
R1
d
H
R3
C
N
R2
Cl
O
H
C
R3
R2
N
O2N
NO2
O
O
viii
R4
R1
O2N
NO2
OH
R4
R1
CF3
f
1g-20g
1h-20h
CF3
Scheme 1C. General synthesis of compounds 1h–20h. Reagents and conditions: (vii) TEA, ethanol, rt, 0.5 h; (viii) 2-chloro-1,3-dinitro-5-(trifluoromethyl)benzene, K2CO3,
DMF, 50–60 °C, 1 h.
Table 2
Crystal data for compound 4h
Table 1
Structures of compounds 1h–20h
Crystal parameters
Compound 4h
C21H12Cl2F3N3O6
R3
H
Formula
C
N
R2
Crystal size (mm)
0.2 Â 0.2 Â 0.1
MW (g molÀ1
)
530.24
O
A
Crystal system
(°)
b(°)
(°)
Monoclinic
90.00
114.087(3)
90.00
B
a
O
R4
c
a(Å)
b(Å)
12.530(5)
14.445(5)
13.827(5)
2284.9(15)
R
O2N
1
NO2
c(Å)
V(Å3)
C
Z
30
h limits(°)
hkl limits
F(000)
2.27 6 h 6 23.90
À14 6 h 6 14, À16 6 k 6 16, À15 6 l 6 15
1980
CF3
Data/restraints/parameters
Absorption coefficient (mmÀ1
Reflections collected
Independent reflections
R1 wR2 [I>2&Gs(I)]
R1 wR2 (all data)
GOF
3534/0/316
1.177
14072
3534[Rint = 0.0475]
0.1157/0.3195
0.1661/0.3455
2.006
)
Compound 1h
2h
3h
4h
5h
6h
7h
8h
9h
10h
R1
R2
R3
R4
H
H
H
H
H
H
F
H
H
Cl
H
H
H
Cl
Cl
H
Cl
H
H
H
Cl
F
H
H
H
Br
H
H
H
Br
F
Cl
Cl
H
Cl
Cl
Cl
H
H
H
Compound 11h 12h 13h 14h 15h 16h 17h 18h 19h 20h
R1
R2
R3
R4
H
H
Cl
Cl
H
Cl
Cl
F
Cl
Cl
Cl
H
Cl
Cl
Cl
Cl
Br
Br
H
Br
Br
F
Br
Br
Cl
H
Br
Br
Cl
Cl
Br
Cl
H
Br
Cl
Cl
H
H
H
H
in Figure 4, MCF-7 cells were treated with 0, 0.01, 0.05, 0.25 lM of
compound 2h for 24 h. The compound increased the percentage of
apoptosis by Annexin V-FITC/PI. The result indicated that com-
pound 2h could induce apoptosis of MCF-7 cells.
2.3.3. Binding model of compounds 2b into the colchicines
binding site of tubulin
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 2h with tubulin (PDB code: 1SA0).
The molecular docking was performed by simulation of compound
2h into the colchicine binding site of tubulin. All docking runs were
Figure 3. Crystal structure diagrams of compound 4h.

T.-T. Zhao et al. / Bioorg. Med. Chem. 20 (2012) 3233–3241
3237
Table 3
Inhibition (IC50) of MCF-7, Hep-G2 and A549 cells proliferation and inhibition of
tubulin polymerization by compounds 1h–20h
Compound
IC50 SD (lM)
MCF-7a
Hep-G2a
A549a
Tubulinb
1h
2h
3h
4h
5h
6h
7h
8h
1.33 0.09
0.70 0.05
1.1 0.07
1.33 0.17
0.68 0.02
1.17 0.12
1.02 0.09
1.79 0.07
1.02 0.06
1.51 0.14
1.28 0.08
1.18 0.15
0.89 0.12
1.24 0.02
1.07 0.05
1.12 0.07
0.82 0.18
1.12 0.24
0.94 0.11
1.48 0.13
1.10 0.08
2.45 0.10
1.05 0.10
0.19 0.04
0.23 0.02
1.47 0.15
0.86 0.05
1.43 0.11
1.61 0.07
3.41 0.28
2.57 0.06
5.03 0.33
3.44 0.29
2.24 0.25
1.28 0.03
3.43 0.32
2.06 0.19
1.45 0.15
1.23 0.02
2.16 0.15
1.42 0.04
2.15 0. 26
1.56 0.05
4.12 0.43
6.19 0.05
0.09 0.01
0.75 0.08
13.98 0.93
3.06 0.05
6.92 0.53
4.08 0.44
30.89 2.16
7.07 0.78
9.05 0.81
7.54 0.63
15.36 0.97
6.90 0.79
13.24 1.02
7.06 0.63
9.02 0.93
3.65 0.18
16.12 1.07
6.18 0.70
18.01 1.63
4.53 0.08
18.09 1.51
15.66 1.24
0.70 0.20
1.72 0.18
0.94 0.11
1.22 0.13
1.11 0.08
3.95 0.28
1.14 0.13
1.15 0.05
1.12 0.07
2.84 0.25
1.34 0.08
1.21 0.12
0.80 0.09
1.96 0.03
1.03 0.09
1.83 0.21
0.88 0.07
2.32 0.14
2.09 0.16
0.41 0.04
0.53 0.07
9h
10h
11h
12h
13h
14h
15h
16h
17h
18h
19h
20h
CA-4
Colchicine
Figure 5A. The 3D model of the interaction between compound 2h and colchicine
bonding site. The protein is represented by molecular surface.
activities against MCF-7, Hep-G2 and A549 cells and tubulin poly-
merization inhibitory activity. Among them, compound 2h demon-
strated the most potent activity which inhibited the growth of
MCF-7, Hep-G2 and A549 cells with IC50 values of 0.70 0.05,
a
Inhibition of the growth of tumor cell lines.
Inhibition of tubulin polymerization.
b
0.68 0.02 and 0.86 0.05
of tubulin with IC50 of 3.06 0.05
l
M and inhibited the polymerization
M. The result of flow cytometry
l
applied LigandFit Dock protocol of Discovery Studio 3.1. The bind-
ing modes of compound 2h and tubulin were depicted in Figure 5.
All the amino acid residues which had interactions with tubulin
were exhibited in Figure 5B. In the binding mode, compound 2h
was nicely bound to the colchicine binding site of tubulin by three
hydrogen bonds. The oxygen atom of the hydroxyimino formed
one hydrogen bond with the amino hydrogen of Asn C: 101
(N–HÁ Á ÁO = 2.188 Å; 135.9°), the oxygen atom of the nitro groups
on C-ring of 2h formed another two hydrogen bonds with the ami-
no hydrogen of Gly C: 144 (N–HÁ Á ÁO = 1.732 Å; 147.5°) and Lys
D254 (N–HÁ Á ÁO = 2.154 Å; 116.3°), respectively. The 3D model of
the interaction between compound 2h and colchicin bonding site
was showed in Figure 5A, which revealed that the molecule was
well embedded in the active pocket, suggested that compound
2h is a potential inhibitor of tubulin.
(FCM) demonstrated that compound 2h induced cell apoptosis.
3. Conclusion
Figure 5B. The molecular docking of compound 2h into tubulin. Compound 2h
(colored by atom: carbons: gray; nitrogen: blue; oxygen: red; chlorine: green) was
nicely bound to the tubulin via three hydrogen bonds. (N–HÁ Á ÁO = 2.188 Å; 135.9°.
N–HÁ Á ÁO = 1.732 Å; 147.5°. N–HÁ Á ÁO = 2.154 Å; 116.3°.)
In our present work, a series of novel antitubulin polymeriza-
tion inhibitors (1h–20h) have been firstly synthesized and evalu-
ated. These compounds exhibited potent antiproliferative
Figure 4. MCF-7 cells were cultured with various concentrations of compound 2h for 24 h. Cells were stained by Annexin V-FITC/PI and apoptosis was analyzed by flow
cytometry. Inhibition included early and late apoptosis.

3238
T.-T. Zhao et al. / Bioorg. Med. Chem. 20 (2012) 3233–3241
Molecular docking showed that compound 2h bound to the colchi-
cines binding site by three hydrogen bond which might play a cru-
cial role in its antitubulin polymerization and antiproliferative
activities. This study might be helpful for the design and synthesis
of tubulin polymerization inhibitors with stronger activities.
anhydrous Na2SO4, filtered and evaporated. Finally the precipitate
was filtered and recrystallized from ethanol to yield 1h–20h.
4.3. Spectral properties of 2,6-dinitro-4-(trifluoromethyl)
phenoxysalicylaldoxime derivatives
4.3.1. 2-(2, 6-Dinitro-4-(trifluoromethyl) phenoxy)
4. Experiments
benzaldehyde O-benzyl oxime (1h)
Yellow solid, yield 75%, mp: 92–94 °C. IR (KBr, m
, cmÀ1): 1544 (s,
C@N); 1319 (s, NO2); 1144 (s, C–O–C). 13C NMR (300 MHz, CDCl3,
ppm): d 160.7; 153.8 (CH@N); 144.6; 137.3; 132.8; 131.1; 128.8;
127.8; 126.8; 126.7; 126.6; 123.3; 122.4; 119.7; 76.9 (CH2). 1H
NMR (CDCl3, 300 MHz): 5.23 (s, 2H); 6.97 (d, J = 8.22 Hz, 1H);
7.21 (t, J = 7.59 Hz, 1H); 7.32–7.45 (m, 6H); 7.81 (t, J = 3.84 Hz,
1H); 8.51 (s, 1H); 8.93 (s, 2H). ESI-MS: 461.35 (C21H15F3N3O6
[M+H]+). Anal. Calcd for C21H14F3N3O6: C, 54.67; H, 3.06; N, 9.11.
Found: C, 54.74; H, 3.09; N, 9.13.
4.1. Materials and measurements
All chemicals and reagents used in the current study were of
analytical grade. Melting points (uncorrected) were determined
on an XT4 MP apparatus (Taike Corp., Beijing, China). All the 1H
NMR spectra were recorded on a Bruker DPX300 model Spectrom-
eter in DMSO-d6 and chemical shifts were reported in ppm (d). FT-
IR spectra (KBr) were run on a Nexus 870 FT-IR spectrophotometer.
ESI-MS spectra were recorded on a Mariner System 5304 Mass
spectrometer. Elemental analyses were performed on a CHN-O-Ra-
pid instrument. TLC was performed on the glassbacked silica gel
sheets (Silica Gel 60 GF254) and visualized in UV light (254 nm).
4.3.2. 2-(2,6-Dinitro-4-(trifluoromethyl)phenoxy)benzaldehyde
O-(2-fluorobenzyl) oxime (2h)
Yellow solid, yield 78%, mp: 98–100 °C. IR (KBr, m
, cmÀ1): 1543
(s, C@N); 1320 (s, NO2); 1143 (s, C–O–C). 13C NMR (300 MHz,
CDCl3, ppm): d 162.5; 159.2; 154.2 (CH@N); 144.6; 139.7; 132.8;
131.1; 130.7; 130.6; 129.8; 128.3; 127.8; 126.8; 126.7; 124.6;
124.4; 123.9; 123.3; 122.3; 119.6; 115.4; 115.1; 70.1 (CH2). 1H
NMR (CDCl3, 300 MHz): 5.35 (d, J = 8.40 Hz, 2H); 6.50 (d,
J = 8.22 Hz, 1H); 7.43 (t, J = 9.135 Hz, 1H); 7.17(t, J = 7.05 Hz, 2H);
7.23–7.27 (m, 1H); 7.28–7.36 (m, 1H); 7.48–7.52 (m, 1H); 7.94–
7.97 (m, 1H); 8.45 (s, 2H); 8.58 (s, 1H). ESI-MS: 479.34
(C21H14F4N3O6 [M+H]+). Anal. Calcd for C21H13F4N3O6: C, 52.62;
H, 2.73; N, 8.77. Found: C, 52.52; H, 2.70; N, 8.73.
4.2. Synthesis
4.2.1. General synthetic procedure of O-benzylhydroxylamine
hydrochloride (d)
A mixture of substituted isobenzofuran-1,3-dione and hydrox-
ylamine was dissolved in pyridine and stirred on 80 °C overnight.
The reaction was monitored by TLC. The mixture was poured into
water and the product was precipitated. Then it was washed with
trichlormethane and dried over anhydrous MgSO4, filtered and
evaporated to gain compound a. After reaction of compound a with
NaHCO3, the mixture was basified to pH 8–9, added Bu4NHSO4 and
Benzyl chloride, and refluxed 7 h. The product was extracted with
CH2Cl2/H2O, dried over anhydrous MgSO4, filtered and evaporated
to gain compound b. Then compound b was added into ethanol.
After cooling down to 0 °C, hydrazine hydrate was dropped. The
mixture was reacted and stirred for 1.5 h to get compound c. At
last, compound c was dissolved in CH2Cl2, and dry HCl(g) was
vented. After reacting 1 h, HCl(g) was blowed with N2 and com-
pound d was yielded.
4.3.3. (E)-2-(2,6-Dinitro-4-(trifluoromethyl)
phenoxy)benzaldehyde O-(2-chloro- benzyl) oxime (3h)
Yellow solid, yield 68%, mp: 141–142 °C. IR (KBr, m
, cmÀ1): 1553
(s, C@N); 1320 (s, NO2); 1155 (s, C–O–C). 13C NMR (300 MHz,
CDCl3, ppm): d 160.4; 152.8 (CH@N); 140.1; 137.9; 135.2; 133.5;
131.3; 128.9; 128.1; 127.3; 125.7; 121.7; 121.0; 120.4; 73.5
(CH2). 1H NMR (CDCl3, 300 MHz): 6.50–6.53 (m, 1H); 7.14–7.23
(m, 1H); 7.23–7.27 (m, 1H); 7.27–7.29 (m, 2H); 7.30–7.32 (m,
2H); 7.39–7.42 (m, 1H); 7.51–7.54 (m, 1H); 7.87–7.97 (m, 1H);
8.46 (m, 2H); 8.63 (m, 1H). ESI-MS: 495.79 (C21H14ClF3N3O6
[M+H]+). Anal. Calcd for C21H13ClF3N3O6: C, 50.87; H, 2.64; N,
8.48. Found: C, 50.78; H, 2.69; N, 8.63.
4.2.2. General synthetic procedure of 2-chloro-1,3-dinitro-5-
(trifluoromethyl) benzene (f)
A mixture of nitric acid and sulfur trioxide was poured into
1-chloro-4-(trifluoromethyl)benzene. And the reaction was run at
50–70 °C for 3 h. The product was extracted with ethyl acetate
and saturated NaHCO3, filtered and recrystallized from ethanol to
gain compound e. Similarly, the same mixture was poured into
compound e. Then, the reaction was run at 100–115 °C for 2 h to
synthsis 2-chloro-1,3-dinitro-5-(trifluoromethyl)benzene (com-
pound f).
4.3.4. (E)-2-(2,6-Dinitro-4-(trifluoromethyl)
phenoxy)benzaldehyde O-(2,4-dichlo-robenzyl) oxime (4h)
Light yellow solid, yield 67%, mp: 142–143 °C. IR (KBr, m
, cmÀ1):
1545 (s, C@N); 1321 (s, NO2); 1144 (s, C–O–C). 13C NMR (300 MHz,
CDCl3, ppm): d 154.2 (CH@N); 144.6; 134.0; 133.8; 132.8; 131.3;
130.4; 128.4; 127.9; 127.1; 126.8; 126.7; 124.6; 123.3; 122.1;
119.6; 112.8; 73.4 (CH2). 1H NMR (CDCl3, 300 MHz): 5.35 (d,
J = 7.02 Hz, 2H); 6.50–6.53 (m, 1H); 7.14–7.23 (m, 1H); 7.24–7.27
(m, 1H); 7.27–7.30 (m, 1H); 7.42 (d, J = 2.19 Hz, 1H); 7.46 (d,
J = 8.25 Hz, 1H); 7.91–7.94 (m, 1H); 8.46 (s, 2H); 8.62 (s, 1H). ESI-
MS: 530.24 (C21H13Cl2F3N3O6 [M+H]+). Anal. Calcd for
4.2.3. General synthetic procedure of target compounds (1h–
20h)
A mixture of substituted O-benzylhydroxylamine hydrochloride
(1 equiv), TEA (1 equiv) and substituted salicylaldehyde (1 equiv)
was dissolved in absolute ethanol and stirred for 0.5 h. The reac-
tion was monitored by TLC. The precipitate was filtered and recrys-
tallized from ethanol to gain salicylaldoxime (compounds 1g–20g).
Then K2CO3 (2 equiv), substituted salicylaldoxime (1 equiv) and
2-chloro-1,3-dinitro-5-(trifluoromethyl)benzene (1 equiv) were
mixed and dissolved in DMF and stirred on 50–60 °C for 1 h. The
solvent was evaporated until no longer liquid outflow. The prod-
ucts were extracted with ethyl acetate. The extract was washed
successively with saturated NaCl solution, and then dried over
C21H12Cl2F3N3O6: C, 47.57; H, 2.28; N, 7.92. Found: C, 47.65; H,
2.31; N, 7.90.
4.3.5. 5-Chloro-2-(2,6-dinitro-4-(trifluoromethyl)
phenoxy)benzaldehyde O-benzyl oxime (5h)
Yellow solid, yield 89%, mp: 143–146 °C. IR (KBr,
m
, cmÀ1): 1549
(s, C@N); 1318 (s, NO2); 1149 (s, C–O–C). 13C NMR (300 MHz,
CDCl3, ppm): d 161.3; 154.8 (CH@N); 143.2; 137.6; 137.2; 133.8;
131.2; 129.1; 127.0; 124.6; 121.7; 120.8; 120.6; 118.4; 110.8;
75.4 (CH2). 1H NMR (CDCl3, 300 MHz): 5.25 (s, 2H); 7.08 (d,

T.-T. Zhao et al. / Bioorg. Med. Chem. 20 (2012) 3233–3241
3239
J = 9.03 Hz, 1H); 7.37–7.45 (m, 6H); 7.76 (d, J = 2.55 Hz, 1H); 8.47
(s, 1H); 8.94 (s, 2H); ESI-MS: 495.79 (C21H14ClF3N3O6 [M+H]+).
Anal. Calcd for C21H13ClF3N3O6: C, 50.87; H, 2.64; N, 8.48. Found:
C, 50.79; H, 2.64; N, 8.43.
(t, J = 7.31 Hz, 1H); 8.09 (d, J = 2.55 Hz, 1H); 8.48 (d, J = 3.12 Hz,
3H). ESI-MS: 558.23 (C21H13BrF4N3O6 [M+H]+). Anal. Calcd for
C21H12BrF4N3O6: C, 45.18; H, 2.17; N, 7.53. Found: C, 45.28; H,
2,15; N, 7.57.
4.3.6. 5-Chloro-2-(2,6-dinitro-4-(trifluoromethyl)
phenoxy)benzaldehyde O-(2-fluorobenzyl) oxime (6h)
4.3.11. (E)-5-Bromo-2-(2,6-dinitro-4-(trifluoromethyl)
phenoxy)benzaldehyde O-(2-chlorobenzyl) oxime (11h)
Yellow solid, yield 78%, mp: 122–126 °C. IR (KBr,
m
, cmÀ1): 1537
Yellow solid, yield 68%, mp: 108–109 °C. IR (KBr, m
, cmÀ1): 1546
(s, C@N); 1323 (s, NO2); 1156 (s, C–O–C). 13C NMR (300 MHz,
CDCl3, ppm): d 156.5; 154.7; 153.8 (CH@N); 142.9; 138.8; 132.2;
129.1; 127.5; 127.4; 127.3; 126.1; 125.9; 124.6; 123.9; 123.2;
121.5; 120.1; 114.9; 68.8 (CH2). 1H NMR (CDCl3, 300 MHz): 5.29
(s, 2H); 7.08 (d, J = 8.97 Hz, 1H); 7.21–7.27 (m, 2H); 7.39–7.45
(m, 2H); 5.21 (t, J = 7.59 Hz, 1H); 7.76 (d, J = 2.76 Hz, 1H); 8.46 (s,
1H); 8.94 (s, 2H). ESI-MS: 513.78 (C21H13ClF4N3O [M+H]+). Anal.
Calcd for C21H12ClF4N3O6: C, 49.09; H, 2.35; N, 8.18. Found: C,
49.18; H, 2.33; N, 8.03.
(s, C@N); 1323 (s, NO2); 11474 (s, C–O–C). 13C NMR (300 MHz,
CDCl3, ppm): d 145.8 (CH@N); 142.5; 134.7; 133.2; 132.0; 130.0;
129.4; 129.2; 127.6; 126.9; 126.7; 126.4; 126.2; 126.1; 126.0;
123.5; 123.3; 119.7; 76.5 (CH2). 1H NMR (CDCl3, 300 MHz): 5.39
(s, 2H); 6.39 (d, J = 8.85 Hz, 1H); 7.26–7.30 (m, 2H); 7.32–7.36
(m, 1H); 7.4005 (t, J = 4.58 Hz, 1H); 7.49 (d, J = 6.39 Hz, 1H); 8.08
(d, J = 2.40 Hz, 1H); 8.47 (s, 2H); 8.53 (s, 1H). ESI-MS: 574.69
(C21H13BrClF3N3O6 [M+H]+). Anal. Calcd for C21H12BrClF3N3O6: C,
43.89; H, 2.10; N, 7.31. Found: C, 43.78; H, 2.15; N, 7.38.
4.3.7. 5-Chloro-2-(2,6-dinitro-4-(trifluoromethyl)
phenoxy)benzaldehyde O-(2-chlorobenzyl) oxime (7h)
4.3.12. (Z)-5-Bromo-2-(2,6-dinitro-4-(trifluoromethyl)phenoxy)
benzaldehyde O-(2,4-dichlorobenzyl) oxime (12h)
Yellow solid, yield 87%, mp: 159–160 °C. IR (KBr,
m
, cmÀ1): 1546
Yellow solid, yield 73%, mp: 117–125 °C. IR (KBr, m
, cmÀ1): 1546
(s, C@N); 1319 (s, NO2); 1151 (s, C–O–C). 13C NMR (300 MHz,
CDCl3, ppm): d 160.2; 153.6 (CH@N); 140.3; 138.8; 134.3; 133.8;
130.4; 127.9; 127.1; 125.9; 121.6; 120.9; 120.5; 117.8; 110.5;
77.6 (CH2). 1H NMR (CDCl3, 300 MHz): 5.39 (s, 2H); 6.45 (d,
J = 8.79 Hz, 1H); 7.18–7.21 (m, 1H); 7.26–7.32 (m, 2H); 3.73 (t,
J = 4.58 Hz, 1H); 7.50 (t, J = 6.17 Hz, 1H); 7.93 (d, J = 2.55 Hz, 1H);
8.47 (s, 2H); 8.53 (s, 1H). ESI-MS: 530.24 (C21H13Cl2F3N3O6
[M+H]+). Anal. Calcd for C21H12Cl2F3N3O6: C, 47.57; H, 2.28; N,
7.92. Found: C, 47.49; H, 2.33; N, 7.84.
(s, C@N); 1318 (s, NO2); 1145 (s, C–O–C). 13C NMR (300 MHz,
CDCl3, ppm): d 158.7; 152.7 (CH@N); 142.7; 138.7; 136.7; 136.3;
134.6; 133.8; 131.6; 130.3; 127.3; 127.1; 125.9; 123.3; 122.1;
118.4; 111.0; 71.5 (CH2). 1H NMR (CDCl3, 300 MHz): 5.33 (s, 2H);
6.39 (d, J = 8.79 Hz, 1H); 7.28 (d, J = 10.05 Hz, 1H); 7.33–7.37 (m,
1H); 7.43 (t, J = 7.68 Hz, 2H); 8.05 (d, J = 3.12 Hz, 1H); 8.44 (d,
J = 11.88 Hz, 3H). ESI-MS: 609.13 (C21H12BrCl2F3N3O6 [M+H]+).
Anal. Calcd for C21H11BrCl2F3N3O6: C, 41.41; H, 1.82; N, 6.90.
Found: C, 41.34; H, 1.78; N, 6.97.
4.3.8. 5-Chloro-2-(2,6-dinitro-4-(trifluoromethyl)
4.3.13. 3,5-Dichloro-2-(2,6-dinitro-4-(trifluoromethyl)phenoxy)
phenoxy)benzaldehyde O-(2,4-d ichlorobenzyl) oxime (8h)
benzaldehyde O-benzyl oxime (13h)
Yellow solid, yield 75%, mp: 166–167 °C. IR (KBr,
m
, cmÀ1): 1543
Yellow solid, yield 79%, mp: 139–145 °C. IR (KBr, m
, cmÀ1): 1545
(s, C@N); 1320 (s, NO2); 1151 (s, C–O–C). 13C NMR (300 MHz,
CDCl3, ppm): d 158.5; 153.8 (CH@N); 141.3; 139.8; 135.3; 134.6;
133.8; 130.9; 130.6; 129.7; 128.6; 127.3; 127.1; 125.4; 123.9;
122.3; 121.5; 120.1; 72.5 (CH2). 1H NMR (CDCl3, 300 MHz): 6.45
(d, J = 8.97 Hz, 1H); 7.18–7.22 (m, 1H); 7.26–7.30 (m, 3H); 7.39–
7.42 (m, 1H); 7.49–7.61(m, 1H); 7.93 (d, J = 0.15 Hz, 1H); 8.46 (s,
2H); 8.53 (s, 1H). ESI-MS: 564.68 (C21H12Cl3F3N3O6 [M+H]+). Anal.
Calcd for C21H11Cl3F3N3O6: C,44.67; H, 1.96; N, 7.44. Found:
C,44.59; H, 1.93; N, 7.44.
(s, C@N); 1317 (s, NO2); 1143 (s, C–O–C). 13C NMR (300 MHz,
CDCl3, ppm): d 161.6; 152.8 (CH@N); 143.2; 139.8; 137.3; 133.8;
130.4; 128.9; 127.1; 125.9; 121.4; 120.9; 120.1; 117.8; 110.4;
76.6 (CH2). 1H NMR (CDCl3, 300 MHz): 5.28 (d, J = 10.62 Hz, 2H);
6.38 (d, J = 8.76 Hz, 1H); 7.26–7.44 (m, 5H); 8.08 (d, J = 2.40 Hz,
1H); 8.47 (d, J = 6.06 Hz, 3H). ESI-MS: 530.24 (C21H13Cl2F3N3O6
[M+H]+). Anal. Calcd for C21H12Cl2F3N3O6: C, 47.57; H, 2.28; N,
7.92. Found: C, 47.50; H, 2.31; N, 7.89.
4.3.14. 3,5-Dichloro-2-(2,6-dinitro-4-(trifluoromethyl)phenoxy)
4.3.9. 5-Bromo-2-(2,6-dinitro-4-(trifluoromethyl)
phenoxy)benzaldehyde O-benzyl oxime (9h)
benzaldehyde O-(2-fluorobenzyl) oxime (14h)
Yellow solid, yield 83%, mp: 111–112 °C. IR (KBr, m
, cmÀ1): 1547
Yellow solid, yield 72%, mp: 130–139 °C. IR (KBr,
m
, cmÀ1): 1536
(s, C@N); 1319 (s, NO2); 1142 (s, C–O–C). 13C NMR (300 MHz,
CDCl3, ppm): d 159.4; 152.7 (CH@N); 147.4; 141.3; 139.8; 132.4;
130.7; 129.2; 128.9; 128.7; 127.6; 127.3; 127.0; 125.9; 124.5;
122.3; 122.1; 115.7; 69.8 (CH2). 1H NMR (CDCl3, 300 MHz): 5.28
(t, J = 10.10 Hz, 2H); 7.06–7.13 (m, 1H); 7.17 (d, J = 7.50 Hz, 1H);
7.30–7.37 (m, 2H); 7.44 (t, J = 7.40 Hz, 1H); 7.83 (d, J = 2.55 Hz,
1H); 8.29 (s, 2H); 8.39 (s, 1H). ESI-MS: 548.23 (C21H11Cl3F4N3O6
[M+H]+). Anal. Calcd for C21H11Cl2F4N3O6: C, 46.01; H, 2.026; N,
7.66. Found: C, 46.11; H, 2.01; N, 7.66
(s, C@N); 1323 (s, NO2); 1151 (s, C–O–C). 13C NMR (300 MHz,
CDCl3, ppm): d 158.9; 152.8 (CH@N); 141.3; 139.8; 137.3; 137.2;
130.7; 128.6; 127.5; 127.3; 127.0; 125.7; 122.3; 122.1; 118.4;
111.3; 77.6 (CH2). 1H NMR (CDCl3, 300 MHz): 7.26 (d,
J = 19.92 Hz, 2H); 6.70 (d, J = 8.76 Hz, 1H); 7.33–7.44 (m, 5H);
7.52–7.56 (m, 1H); 7.89 (d, J = 2.58 Hz, 1H); 8.46 (s, 1H); 8.93 (s,
2H). ESI-MS: 540.24 (C21H14BrF3N3O6 [M+H]+). Anal. Calcd for
C21H13BrF3N3O6: C, 46.69; H, 2.43; N, 7.78. Found: C, 46.77; H,
2.46; N, 7.73.
4.3.15. (E)-3,5-Dichloro-2-(2,6-dinitro-4-(trifluoromethyl)
phenoxy)benzaldehyde O-(2-chlorobenzyl) oxime (15h)
4.3.10. 5-Bromo-2-(2,6-dinitro-4-(trifluoromethyl)
phenoxy)benzaldehyde O-(2- fluorobenzyl) oxime (10h)
Yellow solid, yield 69%, mp: 154–156 °C. IR (KBr, m
, cmÀ1): 1545
Yellow solid, yield 82%, mp: 121–128 °C. IR (KBr,
m
, cmÀ1):
(s, C@N); 1318 (s, NO2); 1150 (s, C–O–C). 13C NMR (300 MHz,
CDCl3, ppm): d 153.8 (CH@N); 147.4; 143.3; 139.8; 138.2; 132.4;
130.7; 129.0; 128.9; 128.5; 127.3; 127.0; 125.9; 122.9; 122.1;
70.9 (CH2). 1H NMR (CDCl3, 300 MHz): 5.37 (s, 2H); 7.26–7.30
(m, 1H); 7.33 (d, J = 2.55 Hz, 1H); 7.39–7.42 (m, 1H); 7.44–7.51
(m, 1H); 7.83 (d, J = 2.55 Hz, 1H); 8.30 (s, 2H); 8.44 (s, 1H). ESI-
MS: 564.68 (C21H12Cl3F3N3O6 [M+H]+). Anal. Calcd for
1536 (s, C@N); 1322 (s, NO2); 1156 (s, C–O–C). 13C NMR
(300 MHz, CDCl3, ppm): d 159.7; 153.7 (CH@N); 141.3; 138.9;
137.1; 130.3; 129.2; 128.7; 127.9; 127.3; 125.8; 124.5; 122.7;
122.1; 118.4; 115.7; 111.4; 70.8 (CH2). 1H NMR (CDCl3,
300 MHz): 5.35 (t, J = 10.52 Hz, 2H); 6.39 (d, J = 8.76 Hz, 1H);
7.07 (m, 1H); 7.19 (d, J = 7.50 Hz, 1H); 7.32–7.37 (m, 2H); 7.48

3240
T.-T. Zhao et al. / Bioorg. Med. Chem. 20 (2012) 3233–3241
C21H11Cl3F3N3O6: C, 44.67; H, 1.96; N, 7.44. Found: C, 44.75; H,
1.99; N, 7.42.
4.4. Crystal structure determination
Crystal structure determination of compound 4h were carried
out on a Nonius CAD4 diffractometer equipped with graphite-
monochromated Mo Ka (k = 0.71073 Å) radiation (Fig. 3). The
structure was solved by direct methods and refined on F2 by full-
matrix least-squares methods using SHELX-97.28 All non-hydrogen
atoms of compound 4h were refined with anisotropic thermal
parameters. All hydrogen atoms were placed in geometrically ide-
alized positions and constrained to ride on their parent atoms. The
crystal data, data collection and refinement parameters for the
compound 4h are listed in Table 2.
4.3.16. (E)-3,5-Dichloro-2-(2,6-dinitro-4-(trifluoromethyl)
phenoxy)benzaldehyde O-(2,4-dichlorobenzyl) oxime (16h)
Yellow solid, yield 75%, mp: 170–171 °C. IR (KBr, m
, cmÀ1): 1543
(s, C@N); 1319 (s, NO2); 1151 (s, C–O–C). 13C NMR (300 MHz,
CDCl3, ppm): d 154.8 (CH@N); 147.7; 142.3; 139.8; 136.4; 134.6;
133.7; 132.4; 130.6; 129.9; 128.0; 127.3; 127.1; 127.0; 125.9;
122.6; 122.1; 70.5 (CH2). 1H NMR (CDCl3, 300 MHz): 5.30 (d,
J = 9.69 Hz, 2H); 7.26–7.30 (m, 1H); 7.33 (d, J = 2.55 Hz, 1H);
7.39–7.44 (m, 1H); 7.46–7.51 (m, 1H); 7.80 (d, J = 2.55 Hz, 1H);
8.29 (s, 2H); 8.44 (s, 1H). ESI-MS: 599.13 (C21H11Cl4F3N3O6
[M+H]+). Anal. Calcd for C21H10Cl4F3N3O6: C, 42.10; H, 1.68; N,
7.01. Found: C, 42.03; H, 1.69; N, 7.61.
4.5. Antiproliferation assay
The antiproliferative activities of the prepared compounds
against MCF-7, Hep-G2 and A549 cells were evaluated as described
in the literature29 with some modifications. Target tumor cell lines
were grown to log phase in RPMI 1640 medium supplemented
with 10% fetal bovine serum. After diluting to 2 Â 104 cells mLÀ1
4.3.17. 3,5-Dibromo-2-(2,6-dinitro-4-(trifluoromethyl)
phenoxy)benzaldehyde O-benzyl oxime (17h)
Yellow solid, yield 79%, mp: 123–124 °C. IR (KBr, m
, cmÀ1): 1544
(s, C@N); 1315 (s, NO2); 1141 (s, C–O–C). 13C NMR (300 MHz,
CDCl3, ppm): d 153.8 (CH@N); 147.7; 144.6; 142.5; 136.7; 133.7;
129.2; 128.9; 128.1; 126.9; 126.6; 126.1; 126.0; 119.8; 112.1;
76.9 (CH2). 1H NMR (CDCl3, 300 MHz): 5.12 (s, 2H); 7.28–7.37
(m, 5H); 7.90 (d, J = 2.19 Hz, 1H); 8.07 (d, J = 2.4 Hz, 1H); 8.41 (s,
1H); 8.83 (s, 2H). ESI-MS: 619.14 (C21H13Br2F3N3O6 [M+H]+). Anal.
Calcd for C21H12Br2F3N3O6: C, 40.74; H, 1.95; N, 6.79. Found: C,
40.83; H, 1.99; N, 6.76.
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-
4.3.18. 3,5-Dibromo-2-(2,6-dinitro-4-(trifluoromethyl)
l
phenoxy)benzaldehyde O-(2-fluorobenzyl) oxime (18h)
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 3.
Yellow solid, yield 88%, mp: 94–96 °C. IR (KBr, m
, cmÀ1): 1548 (s,
C@N); 1316 (s, NO2); 1144 (s, C–O–C). 13C NMR (300 MHz, CDCl3,
ppm): d 162.5; 159.2 (CH@N); 147.3; 142.5; 130.6; 130.5; 130.0;
129.9; 128.0; 126.9; 126.7; 126.2; 126.1; 126.0; 124.0; 123.9;
123.8; 119.8; 119.7; 115.5; 115.2; 112.1; 70.6 (CH2). 1H NMR
(CDCl3, 300 MHz): 5.23 (d, J = 37.32 Hz, 2H); 7.15–7.23 (m, 2H);
7.36–7.43 (m, 2H); 7.95 (d, J = 2.37 Hz, 1H); 8.07 (d, J = 2.40 Hz,
1H); 8.41 (s, 1H); 8.83 (s, 2H). ESI-MS: 637.13 (C21H12Br2F4N3O6
[M+H]+). Anal. Calcd for C21H11Br2F4N3O6: C, 39.59; H, 1.74; N,
6.60. Found: C, 39.51; H,1.74; N, 6.63.
4.6. Effects on tubulin polymerization
Bovine brain tubulin was purified as described previously.30 To
evaluate the effect of the compounds on tubulin assembly in vi-
tro,31 varying concentrations were preincubated with 10
lM tubu-
4.3.19. 3,5-Dibromo-2-(2,6-dinitro-4-(trifluoromethyl)
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.
phenoxy)benzaldehyde O-(2-chlorobenzyl) oxime (19h)
Yellow solid, yield 75%, mp: 112–114 °C. IR (KBr, m
, cmÀ1): 1550
(m, C@N); 1339 (m, NO2); 1167 (s, C–O–C). 13C NMR (300 MHz,
CDCl3, ppm): d 163.7; 153.6 (CH@N); 141.4, 137.8; 136.3; 133.8;
132.7; 128.6; 127.5; 125.9; 121.6; 120.9; 120.4; 117.6; 110.6;
77.5(CH2). 1H NMR (CDCl3, 300 MHz): 5.35 (d, J = 9.51 Hz, 2H);
6.45 (d, J = 8.76 Hz, 1H); 7.19–7.23 (m, 1H); 7.28 (t, J = 5.04 Hz,
1H); 7.43 (t, J = 3.93 Hz, 2H); 7.90 (d, J = 2.73 Hz, 1H); 8.47 (s,
2H); 8.52 (s, 1H). ESI-MS: 653.58 (C21H12Br2ClF3N3O6 [M+H]+).
Anal. Calcd for C21H11Br2ClF3N3O6: C, 38.59; H, 1.70; N, 6.43.
Found: C, 38.65; H, 1.56; N, 6.37.
4.7. Apoptosis assay
MCF-7 cells were cultured with various concentrations of com-
pounds for 24 h. Then, the cells were harvested and stained with
both Annexin V-FITC (fluorescein isothiocyanate) and PI (propidi-
um iodide) for 20 min. The samples were analyzed by flow cytom-
eter (FCM).
4.3.20. (E)-3,5-Dibromo-2-(2,6-dinitro-4-(trifluoromethyl)
phenoxy)benzaldehyde O-(2,4-dichlorobenzyl) oxime (20h)
Yellow solid, yield 66%, mp: 157–159 °C. IR (KBr,
m
, cmÀ1):
4.8. Docking simulations
1547 (s, C@N); 1323 (s, NO2); 1145 (s, C–O–C). 13C NMR
(300 MHz, CDCl3, ppm): d 161.5; 152.8 (CH@N); 142.7; 138.3;
137.3; 133.7; 130.4; 127.9; 127.1; 125.6; 121.9; 120.8; 120.1;
119.8; 111.8; 76.3 (CH2). 1H NMR1H NMR(CDCl3, 300 MHz):
7.24 (s, 1H); 7.36 (t, J = 9.61 Hz, 1H); 7.41–7.44 (m, 2H); 7.64 (t,
J = 2.28 Hz, 2H); 7.97 (d, J = 2.19 Hz, 1H); 8.27 (s, 2H); 8.41 (s,
1H). ESI-MS: 688.03 (C21H11Br2Cl2F3N3O6 [M+H]+). Anal. Calcd
for C21H10Br2Cl2F3N3O6: C, 36.66; H, 1.46; N, 6.11. Found: C,
36.73; H, 1.48; N, 6.07.
The crystal structure of tubulin (PDB code: 1SA0)10,32 was ob-
tained from the RCSB Protein Data Bank (http://www.rcsb.org/
pdb/home/home.do). The molecular docking procedure was per-
formed by using LigandFit protocol within Discovery Studio 3.1.
For ligand preparation, the 3D structures of 2h were generated
and minimized using Discovery Studio 3.1. For protein preparation,
the hydrogen atoms were added, and the water and impurities
were removed. The whole tubulin was defined as a receptor and

T.-T. Zhao et al. / Bioorg. Med. Chem. 20 (2012) 3233–3241
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