Synthesis, biological evaluation and molecular docking studies of flavone and isoflavone derivatives as a novel class of KSP (kinesin spindle protein) inhibitors

Article abstract of DOI:10.1016/j.ejmech.2013.09.042

The kinesin spindle protein (KSP) is involved in the formation of bipolar mitotic spindle during cell division and it becomes a new target to overcome the neurotoxicity of MTs inhibitors. A series of flavone and isoflavone derivatives (1a-7c) have been designed, synthesized and evaluated as potential KSP inhibitors. Among them, 2c displayed the most potent inhibitory activity in vitro, which inhibited the growth of MCF-7 and Hela cell lines with IC ₅₀ values of 4.8 and 4.3 μM, respectively, and also exhibited significant KSP inhibitory activity (IC₅₀ = 0.023 μM). The new compounds can induce irregular monoastral spindles, the characteristic phenotype for KSP inhibiting agents. Docking simulation was further performed to determine the probable binding model.

Full text of DOI:10.1016/j.ejmech.2013.09.042

European Journal of Medicinal Chemistry 70 (2013) 427e433
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, biological evaluation and molecular docking studies of
flavone and isoflavone derivatives as a novel class of KSP (kinesin
spindle protein) inhibitors
,
,b,
Jing-Jun Dong a 1, Qing-Shan Li a 1, Zhi-Peng Liu a, Shu-Fu Wang a, Meng-Yue Zhao a,
Yong-Hua Yang a , Xiao-Ming Wang , Hai-Liang Zhu
,
a,
a,
*
*
*
a State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China
b School of Medical Engineering, Hefei University of Technology, Hefei 230009, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
The kinesin spindle protein (KSP) is involved in the formation of bipolar mitotic spindle during cell
division and it becomes a new target to overcome the neurotoxicity of MTs inhibitors. A series of flavone
and isoflavone derivatives (1ae7c) have been designed, synthesized and evaluated as potential KSP
inhibitors. Among them, 2c displayed the most potent inhibitory activity in vitro, which inhibited the
Received 4 August 2013
Received in revised form
18 September 2013
Accepted 20 September 2013
Available online 2 October 2013
growth of MCF-7 and Hela cell lines with IC50 values of 4.8 and 4.3
mM, respectively, and also exhibited
significant KSP inhibitory activity (IC50 ¼ 0.023
m
M). The new compounds can induce irregular mono-
astral spindles, the characteristic phenotype for KSP inhibiting agents. Docking simulation was further
performed to determine the probable binding model.
Keywords:
KSP
Flavone
Ó 2013 Elsevier Masson SAS. All rights reserved.
Isoflavone
Antimitotic
Kinesin inhibitor
Monoastral spindles
1. Introduction
critical role in centrosome separation and the formation and
maintenance of the bipolar spindle [4]. Inhibition of the KSP in
The studies of mitotic inhibitors have been carried out and some
antimitotic agents, such as colchicines, taxanes, vinblastine and
some synthetic agents, show anticancer activity by binding to the b-
proliferating tumor cells leads to the failure of the centrosome
separation and consequently irregular formation of a monopolar
spindle, ultimately, resulting in cell death [5]. Since KSP is absent
from adult central/peripheral nervous system neurons, which are
postmitotic, KSP inhibitors are not expected to cause the disruption
of the neuronal tubulin network [6,7]. And because of the over-
expression of KSP in proliferating human solid tumors, such as
breast, lung, ovarian, colon and bladder cancers, and leukemias [8e
10], the inhibitors might work as selective agents for the treatment
of malignant tumors.
Accordingly, significant efforts have been made in developing
KSP inhibitors over the past decade since monastrol (Fig. 1), the
first identification of the KSP inhibitor that can cause mitotic
arrest with monopolar spindles, was reported by Mayer et al. [6]
A number of KSP inhibitors have been reported, namely MK-0731
[11], ARRY-520 [12], ispinesib (SB715992) (Fig. 1), and several
other small molecules [13e15]. These reported inhibitors are
suggested to bind to an induced-fit pocket generating by helices
tubulin protein (a component of the microtubule) [1]. However,
because microtubules are also important in other nonproliferating
cells and required for such processes as cell signaling and vesicular
transport, the inhibition of microtubules leads to severe neuro-
toxicity and peripheral neuropathy [2,3]. Moreover, these com-
pounds encounter the acquired resistance through a variety of
mechanisms after the long-term use. Therefore, more specific and
potential agents are needed to investigate and overcome the
observed neuropathic side effects and drug resistance.
The kinesin spindle protein (KSP, also known as HsEg5) has
recently gained significant attention as a novel biological target for
anticancer therapy. KSP, a member of the kinesin-5 family, plays a
* Corresponding authors. Tel./fax: þ86 25 83592672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
1
a2 and a3 and loop L5 of the KSP [7,16]. Of these compounds,
Both authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.09.042

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J.-J. Dong et al. / European Journal of Medicinal Chemistry 70 (2013) 427e433
2.2. Crystal structure of compound 4a and 4c
Among these compounds, the crystal structure of compound 4a
and 4c was determined by X-ray diffraction analysis. The crystal-
lographic data for the structural analysis have been deposited with
the Cambridge Crystallographic Data Centre Nos. 960224 for 4a and
Nos. 960225 for 4c. The perspective views of 4a and 4c with the
atomic labeling system were presented in Fig. 2.
2.3. Bioassay
2.3.1. Antiproliferative activity
To test the anticancer activities of the synthesized compounds,
we evaluated antiproliferative activities of compounds 1ae7c
against MCF-7 (human breast cancer cell lines) and Hela cells. The
results were summarized in Table 1. To our delight, a number of
flavone and isoflavone derivatives showed remarkable effects on
anticancer activities. Among them, compound 2c displayed the
Fig. 1. Chemical structures of flavone, isoflavone and KSP inhibitors.
most potent inhibitory activity (IC50 ¼ 4.8
IC50 ¼ 4.3 M for Hela), comparable to the positive control ispinesib
(IC50 ¼ 1.3 M for MCF-7 and IC50 ¼ 1.0 M for Hela, respectively).
mM for MCF-7 and
m
m
ispinesib was the first KSP inhibitor in phase 2 clinical trials [17].
Encouraged by these findings, we have tried our best to discover
further KSP inhibitors.
Flavones or isoflavones (Fig. 1), such as genistein, have been
reported to have many biological activities including anti-
proliferative, [18] anti-inflammatory [19], anti-estrogenic [20] and
anti-oxidant effects [21]. Many researchers also found that some of
m
2.3.2. KSP inhibitory activity
The series of flavone and isoflavone derivatives 1ae7c were
evaluated for their ability to inhibit the KSP activity and the IC50
values of the compounds were summarized in Table 1. It was
observed that all the synthesized compounds showed obviously
inhibitory activity for KSP with IC50 values between 0.023 and
them could inhibit the activation of nuclear transcription factor
kB
(NF- B), a sequence-specific transcription factor that can maintain
k
0.170 mM. In particular, compound 2c had demonstrated the most
the balance between cell survival and programmed cell death [22].
However, few reports have been dedicated for studying its KSP
inhibitory activity. Many drugs containing nitrobenzotrifluoride
possess anticancer activity because of their strong electron
attracting ability. Upon interaction with the tumor cells, the elec-
trophilic groups (CF3, NO2) of these drugs get tightly bind to nu-
cleophiles present in and around the cells. Additionally, by
introduction of the electron-withdrawing group CF3, the lip-
ophilicity of the compounds might become better for its penetra-
tion through the cell membrane [23]. Taking these factors into
consideration, we tried to combine these advantages together to
find new KSP inhibitors with enhanced activity.
Herein, we are reporting in the present work the design, syn-
thesis and biological evaluation of new compounds, which contain
flavone or isoflavone and nitrobenzotrifluoride simultaneously, as a
novel class of KSP inhibitors. Biological evaluation indicated that
some of the synthesized compounds were potent KSP inhibitors
preliminarily. Mitotic phenotype helped us to seek their role in
inducing cell death further, with the findings that these compounds
could induce the monoastral spindles. Docking simulations were
also performed using the X-ray crystallographic structure of the
KSP in complex with an inhibitor to explore the binding modes of
these compounds.
significant KSP inhibitory activity.
As shown in Table 1, structureeactivity relationships in com-
pounds 1ae7c demonstrated that compounds bearing disubsti-
tuted nitro-compounds showed better KSP inhibitory activity (2b,
2c, 3a, 5c, 6ae7c) than those with monosubstitution (1ae2a, 3b,
4ae4c, 5b). At the same time, perhaps for the same reason, the
substitution of chlorine had better activity (6c, 7c), while a loss of
activity was observed when it was replaced by a hydrogen atom
(6a, 7a). Comparison of the inhibitory activity data also showed that
for the compounds possessing the same molecular mass, the IC50
value of isoflavone derivatives (6b, 6c) is higher than that observed
for flavone derivatives (2b, 2c). Furthermore, influence of the hy-
droxyl group at C-5 was found to be critical in the determination of
the enzyme activity.
The above results indicated that the flavone and isoflavone core
along with the NO2 substitution might have played an important
role in the KSP inhibitory activity. Molecular docking of all the
compounds of this series had demonstrated the point and this was
conformed our estimate.
2.4. Mitotic phenotype
The compounds exhibiting antiproliferating activities in tumor
cells and inhibition of KSP in vitro were tested for the ability to
induce monoastral spindles. Monoastral spindles, the typical
phenotype of cells treated with known KSP inhibitors, is formed by
the failure of separating the spindle MTs in the nucleation due to
loss of KSP activity. The mitotic phenotype of Hela cells and the
presence of bipolar and monoastral spindles treated with com-
pound 2c, 6c and 4a were shown in Figs. 3 and 4. The Hela cells
2. Results and discussion
2.1. Chemistry
The synthesis of flavone and isoflavone derivatives followed the
general reaction pathway outlined in Scheme 1. Compounds 1ae7c
were synthesized by coupling substituted dinitro-compounds with
equimolar (series A) or double mole quantities (series B) of flavones
or isoflavones in DMF using t-BuOK as catalyst. All the compounds
1ae7c were reported for the first time. All of the synthetic com-
pounds gave satisfactory analytical and spectroscopic data. 1H NMR
and ESI-MS spectra were consistent with the assigned structures.
treated with compound 2c at 1
monoastral spindles compared with that treated at 5
at 5 M, compounds 2c, 6c and 4a were capable of inducing a
mM gave less obvious phenotype of
mM. Moreover,
m
conspicuous cell morphological change (Fig. 3). And in Fig. 4, we
detected that the number of the monoastral spindles appears to
increase as the concentration of the compound gets bigger.

J.-J. Dong et al. / European Journal of Medicinal Chemistry 70 (2013) 427e433
429
Scheme 1. General synthesis of compounds 1ae7c. Reagents and conditions: (A). 1.0 eq. 8e10, 1.0 eq. t-BuOK, DMF, r t. (B). 2.0 eq. 8e10, 2.0 eq. t-BuOK, DMF, r t.
2.5. Molecular docking study
in the binding pocket, which were stabilized by Pi-Cation bond,
hydrogen bond, and charge interaction that shown in 2D diagram.
Besides, the model suggests that extensive hydrophobic interactions
are formed between 2c and residues Thr112, Pro121, Trp127, Leu132,
Ala133, Ile136, Pro137 and Ala218 of the KSP. Most significantly, the
interaction between F atom of the compound 2c and ADP can form a
KSP-ADP-2c ternary complex, which cannot bind to ATP quickly and
subsequently cannot undergo further ATP-driven conformational
changes. Overall, these results of the molecular docking, along with
the enzyme assay data and mitotic phenotype, suggested that
compound 2c is a potential inhibitor of KSP.
To gain better understanding on the potency of the synthesized
compounds and guide further SAR studies, we proceeded to
examine the interaction of the compounds with KSP crystal struc-
ture (PDB code: 2Q2Y). The molecular docking was performed by
inserting the compounds into the MKR binding site of KSP. All
docking runs were applied by Discovery Studio 3.5. The binding
model of compound 2c and KSP was depicted in Fig. 5. The amino
acid residues which interacted with KSP were labeled. Docking re-
sults revealed that NO2 group of the compound 2c played vital roles

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J.-J. Dong et al. / European Journal of Medicinal Chemistry 70 (2013) 427e433
Fig. 2. The molecular structure of compound 4a and 4c showing 30% probability displacement ellipsoids for non-hydrogen atoms.
3. Conclusion
glass-backed silica gel sheets (Silica Gel 60 GF254) and visualized in
UV light (254 nm). Separation of the compounds by column chro-
matography was carried out with silica gel 60 (200e300 mesh
ASTM, E. Merck). The quantity of silica gel used was 50e100 times
the weight charged on the column. Melting points were deter-
mined on an XT4 MP apparatus (Taike Corp., Beijing, China). All the
Proton nuclear magnetic resonance (1H NMR) spectra were recor-
ded on a Bruker PX500 or DPX300 model Spectrometer in DMSO-d6
at 25 ꢀC with TMS and chemical shifts were reported in parts per
million (d). The solvent signals allotted as internal stands.
Elemental analyses were performed on a CHNeO-Rapid instru-
ment. All the compounds gave satisfactory chemical analyses
(ꢁ0.4%).
In this study, a series of novel KSP inhibitors (1ae7c) bearing
flavone or isoflavone skeleton had been synthesized and evaluated
their biological activities. These compounds exhibited potent
antiproliferative activities against MCF-7 and Hela cells and KSP
inhibitory activities. Among all of the compounds, 2c showed the
most potent inhibition activity which inhibited the growth of MCF-
7 and Hela cell lines with IC50 values of 4.8 and 4.3
mM and inhibited
the KSP with IC50 of 0.023 M. Otherwise, the antiproliferative
m
properties of the synthesized compounds could be explained by
their ability to inhibit KSP activity through the mitotic phenotype.
Furthermore, after analysis of the docking results, it was found that
many interactions between compound 2c with the protein residues
in the binding site might play a crucial role in its KSP inhibitory
activities. The information of this work might be helpful for the
design and synthesis of KSP inhibitors with stronger activities.
4.2. General procedure for the synthesis of flavone and isoflavone
derivatives (1ae7c)
The synthesis of flavone and isoflavone derivatives followed the
general reaction pathway outlined in Scheme 1. The substituted
dinitro-compounds (series A: 1 mmol; series B: 2 mmol) mixed
with flavones or isoflavones (1 mmol) through by using t-BuOK
(series A: 1 mmol; series B: 2 mmol) as catalyst in 20e30 ml DMF
for 3e4 h at room temperature. The reaction was monitored by TLC.
4. Experiments
4.1. Materials and measurements
All chemicals and reagents used in current study were analytical
grade. Thin layer chromatography (TLC) was performed on the
Table 1
Antiproliferative and inhibitory activities of compounds 1ae7c (IC50, mM).
Compounds
MCF-7
Hela
KSP
1a
1b
1c
2a
2b
2c
3a
3b
4a
4b
4c
5b
5c
14.9 ꢁ 1.24
15.6 ꢁ 1.38
12.7 ꢁ 1.09
13.2 ꢁ 1.20
5.2 ꢁ 0.48
4.8 ꢁ 0.45
5.1 ꢁ 0.56
12.9 ꢁ 1.33
13.6 ꢁ 1.42
19.8 ꢁ 1.76
15.4 ꢁ 1.20
15.7 ꢁ 1.34
12.6 ꢁ 1.32
9.9 ꢁ 0.90
11.3 ꢁ 1.09
9.8 ꢁ 0.86
8.9 ꢁ 0.75
8.2 ꢁ 0.72
1.3 ꢁ 0.10
13.9 ꢁ 1.37
14.8 ꢁ 1.36
14.2 ꢁ 1.51
12.8 ꢁ 1.27
4.9 ꢁ 0.44
4.3 ꢁ 0.39
5.3 ꢁ 0.52
15.3 ꢁ 1.39
17.8 ꢁ 1.65
17.6 ꢁ 1.37
18.9 ꢁ 1.82
16.9 ꢁ 1.26
11.2 ꢁ 1.17
10.2 ꢁ 1.20
10.8 ꢁ 1.06
9.6 ꢁ 0.80
9.0 ꢁ 0.92
7.8 ꢁ 0.67
1.0 ꢁ 0.08
0.134 ꢁ 0.024
0.122 ꢁ 0.013
0.123 ꢁ 0.032
0.098 ꢁ 0.010
0.027 ꢁ 0.0018
0.023 ꢁ 0.0014
0.038 ꢁ 0.0030
0.091 ꢁ 0.0087
0.168 ꢁ 0.034
0.170 ꢁ 0.031
0.157 ꢁ 0.037
0.149 ꢁ 0.029
0.076 ꢁ 0.0043
0.067 ꢁ 0.0035
0.078 ꢁ 0.0058
0.063 ꢁ 0.0046
0.059 ꢁ 0.0039
0.052 ꢁ 0.0050
0.005 ꢁ 0.0002
6a
6b
6c
7a
7c
Fig. 3. Mitotic phenotype of Hela cells with flavone and isoflavone derivatives 2c-1
(2c: 1 mM), 2c-2 (2c: 5 mM), 6c (5 mM) and 4a (5 mM). Microtubules are colored in green,
and DNA in blue. (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)
Ispinesib

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431
4.2.3. 7-(3-Chloro-2,6-dinitro-4-(trifluoromethyl)phenoxy)-5-
hydroxy-2-phenyl-4H-chromen-4-one (1c)
Yellow powder, yield: 82%. Mp: 232e234 ꢀC. 1H NMR (300 MHz,
CDCl3,
d
ppm): 6.32 (d, J ¼ 2.34 Hz,1H); 6.57 (d, J ¼ 2.37 Hz,1H); 6.74
(s, 1H); 7.54e7.59 (m, 3H); 7.87e7.90 (m, 2H); 8.64 (s, 1H); 12.92 (s,
1H). MS (ESI): 523.8 ([M þ H]þ). Anal. Calcd for C22H10ClF3N2O8: C,
50.55; H, 1.93; N, 5.36%; Found: C, 50.67; H, 1.87; N, 5.25%.
4.2.4. 7-(2,6-Dinitro-4-(trifluoromethyl)phenoxy)-5-hydroxy-2-(4-
hydroxyphenyl)-4H-chromen-4-one (2a)
Yellow powder, yield: 84%. Mp: 258e260 ꢀC. 1H NMR (300 MHz,
DMSO-d6,
d
ppm): 6.64 (d, J ¼ 2.34 Hz, 1H); 7.01e7.12 (m, 2H); 8.10
(d, J ¼ 8.82 Hz, 2H); 8.97 (d, J ¼ 8.13 Hz, 4H); 12.98 (s, 1H). MS (ESI):
505.3 ([M þ H]þ). Anal. Calcd for C22H11F3N2O9: C, 52.39; H, 2.20; N,
5.55%; Found: C, 52.21; H, 2.18; N, 5.63%.
Fig. 4. The presence of bipolar spindles and monoastral spindles were scored in four
different concentrations of each of the indicated inhibitors in Hela cells, and the % of
monoastral spindles were determined and plotted as bar graphs.
4.2.5. 7-(2,4-Dinitro-6-(trifluoromethyl)phenoxy)-2-(4-(2,4-
dinitro-6-(trifluoromethyl)phenoxy)phenyl)-5-hydroxy-4H-
chromen-4-one (2b)
The products are extracted with ethyl acetate and saturated salt
water, dried over Na2SO4, filtered and evaporated. The residue is
purified by column chromatography using petroleum ether and
ethyl acetate.
Yellow powder, yield: 76%. Mp: 237 ꢀC. 1H NMR (300 MHz,
CDCl3,
d
ppm): 6.23 (d, J ¼ 2.28 Hz, 1H); 6.52 (d, J ¼ 2.31 Hz, 1H);
6.67 (s, 1H); 7.01 (d, J ¼ 8.79 Hz, 2H); 7.87 (d, J ¼ 8.82 Hz, 2H); 8.92
(d, J ¼ 2.49 Hz, 2H); 9.11 (dd, J1 ¼ 2.67 Hz, J2 ¼ 17.34 Hz, 2H); 12.82
(s,1H). MS (ESI): 739.0 ([M þ H]þ). Anal. Calcd for C29H12F6N4O13: C,
47.17; H, 1.64; N, 7.59%; Found: C, 47.22; H, 1.58; N, 7.67%.
4.2.1. 7-(2,6-Dinitro-4-(trifluoromethyl)phenoxy)-5-hydroxy-2-
phenyl-4H-chromen-4-one (1a)
White powder, yield: 80%. Mp: 176e178 ꢀC. 1H NMR (500 MHz,
CDCl3,
d
ppm): 6.26 (d, J ¼ 2.10 Hz,1H); 6.56 (d, J ¼ 2.15 Hz,1H); 6.73
4.2.6. 7-(3-Chloro-2,6-dinitro-4-(trifluoromethyl)phenoxy)-2-(4-
(3-chloro-2,6-dinitro-4-(trifluoromethyl)phenoxy)phenyl)-5-
hydroxy-4H-chromen-4-one (2c)
(s, 1H); 7.51e7.57 (m, 3H); 7.87 (d, J ¼ 7.45 Hz, 2H); 8.55 (s, 2H);
12.90 (s, 1H). MS (ESI): 489.1 ([M þ H]þ). Anal. Calcd for
Yellow powder, yield: 78%. Mp: 253e255 ꢀC. 1H NMR (300 MHz,
C
22H11F3N2O8: C, 54.11; H, 2.27; N, 5.74%; Found: C, 54.23; H, 2.19;
N, 5.64%.
DMSO-d6,
d
ppm): 6.74 (d, J ¼ 2.40 Hz,1H); 7.11e7.16 (m, 2H); 7.39 (d,
J¼ 9.03Hz, 2H);8.11(d,J¼ 9.03Hz,2H);8.87(d, J¼ 7.26Hz, 2H);12.99
(s,1H). MS(ESI):807.0([Mþ H]þ). Anal. Calcd forC29H10Cl2F6N4O13: C,
43.14; H, 1.25; N, 6.94%; Found: C, 43.26; H, 1.34; N, 6.85%.
4.2.2. 7-(2,4-Dinitro-6-(trifluoromethyl)phenoxy)-5-hydroxy-2-
phenyl-4H-chromen-4-one (1b)
White powder, yield: 75%. Mp: 158e160 ꢀC. 1H NMR (500 MHz,
CDCl3,
d
ppm): 5.93 (s, 1H); 6.24 (d, J ¼ 2.15 Hz, 1H); 6.51 (d,
4.2.7. 6,7-Bis(2,6-dinitro-4-(trifluoromethyl)phenoxy)-5-hydroxy-
2-phenyl-4H-chromen-4-one (3a)
J ¼ 2.15 Hz, 1H); 7.50e7.58 (m, 3H); 7.86 (d, J ¼ 7.30 Hz, 2H); 8.91 (d,
J ¼ 2.50 Hz,1H); 9.13 (d, J ¼ 2.55 Hz,1H). MS (ESI): 489.1 ([M þ H]þ).
Anal. Calcd for C22H11F3N2O8: C, 54.11; H, 2.27; N, 5.74%; Found: C,
54.02; H, 2.18; N, 5.68%.
Yellow powder, yield: 78%. Mp: 189e190 ꢀC. 1H NMR (300 MHz,
CDCl3, d ppm): 6.55e6.74 (m, 3H); 7.46e7.49 (m,4H); 7.78e7.81 (m,
2H); 8.31e8.52 (m, 2H); 13.02 (m, 1H). MS (ESI): 739.0 ([M þ H]þ).
Fig. 5. 2D diagram of binding model between KSP and compound 2c.

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J.-J. Dong et al. / European Journal of Medicinal Chemistry 70 (2013) 427e433
Anal. Calcd for C29H12F6N4O13: C, 47.17; H, 1.64; N, 7.59%; Found: C,
47.23; H, 1.58; N, 7.72%.
4.2.15. 7-(2,4-Dinitro-6-(trifluoromethyl)phenoxy)-3-(4-(2,4-
dinitro-6-(trifluoromethyl)phenoxy)phenyl)-5-hydroxy-4H-
chromen-4-one (6b)
4.2.8. 7-(2,4-Dinitro-6-(trifluoromethyl)phenoxy)-5,6-dihydroxy-
2-phenyl-4H-chromen-4-one (3b)
Yellow powder, yield: 75%. Mp: 212e213 ꢀC. 1H NMR (300 MHz,
DMSO-d6,
d
ppm): 6.61 (d, J ¼ 2.34 Hz, 1H); 6.90 (d, J ¼ 2.34 Hz, 1H);
Yellow powder, yield: 83%. Mp: 236e238 ꢀC. 1H NMR (300 MHz,
7.11 (d, J ¼ 8.82 Hz, 2H); 7.57 (d, J ¼ 8.88 Hz, 2H); 8.58 (s, 1H); 8.92
(dd, J1 ¼ 2.67 Hz, J2 ¼ 7.11 Hz, 2H); 9.22 (dd, J1 ¼ 2.70 Hz,
J2 ¼ 10.71 Hz, 2H); 12.95 (s, 1H). MS (ESI): 739.0 ([M þ H]þ). Anal.
Calcd for C29H12F6N4O13: C, 47.17; H, 1.64; N, 7.59%; Found: C, 47.32;
H, 1.60; N, 7.68%.
DMSO-d6,
d ppm): 6.68 (s, 1H); 7.03 (s, 1H); 7.58e7.64 (m, 3H);
8.08e8.11 (m, 2H); 8.75 (d, J ¼ 2.67 Hz,1H); 9.03 (d, J ¼ 2.67 Hz,1H);
11.79 (s, 1H); 13.26 (s, 1H). MS (ESI): 505.0 ([M þ H]þ). Anal. Calcd
for C22H11F3N2O9: C, 52.39; H, 2.20; N, 5.55%; Found: C, 52.46; H,
2.13; N, 5.72%.
4.2.16. 7-(3-Chloro-2,6-dinitro-4-(trifluoromethyl)phenoxy)-3-(4-
(3-chloro-2,6-dinitro-4-(trifluoromethyl)phenoxy)phenyl)-5-
hydroxy-4H-chromen-4-one (6c)
4.2.9. 7-(2,6-Dinitro-4-(trifluoromethyl)phenoxy)-3-(4-
methoxyphenyl)-4H-chromen-4-one (4a)
White powder, yield: 85%. Mp: 188e190 ꢀC. 1H NMR (500 MHz,
Yellow powder, yield: 73%. Mp: 236e238 ꢀC. 1H NMR (300 MHz,
CDCl3,
d
ppm): 3.84 (s, 3H); 6.93e6.98 (m, 4H); 7.48 (d, J ¼ 8.55 Hz,
DMSO-d6,
d
ppm): 6.32 (d, J ¼ 2.40 Hz, 1H); 6.51 (d, J ¼ 2.37 Hz, 1H);
2H); 7.93 (s, 1H); 8.32 (d, J ¼ 8.70 Hz, 1H); 8.54 (s, 2H). MS (ESI):
503.1 ([M þ H]þ). Anal. Calcd for C23H13F3N2O8: C, 54.99; H, 2.61; N,
5.58%; Found: C, 54.70; H, 2.68; N, 5.66%.
7.02 (d, J ¼ 8.82 Hz, 2H); 7.53 (d, J ¼ 8.82 Hz, 2H); 7.96 (s, 1H); 8.49e
8.64 (m, 2H); 12.85 (s, 1H). MS (ESI): 807.0 ([M þ H]þ). Anal. Calcd
for C29H10Cl2F6N4O13: C, 43.14; H, 1.25; N, 6.94%; Found: C, 43.26; H,
1.31; N, 6.87%.
4.2.10. 7-(2,4-Dinitro-6-(trifluoromethyl)phenoxy)-3-(4-
methoxyphenyl)-4H-chromen-4-one (4b)
4.2.17. 7-(2,6-Dinitro-4-(trifluoromethyl)phenoxy)-2-(4-(2,6-
dinitro-4-(trifluoromethyl)phenoxy)phenyl)-5-hydroxychroman-4-
one (7a)
White powder, yield: 83%. Mp: 184e186 ꢀC. 1H NMR (500 MHz,
CDCl3,
d ppm): 3.84 (s, 3H); 6.88e6.98 (m, 4H); 7.47e7.49 (m, 2H);
7.93 (s, 1H); 8.31 (d, J ¼ 3.80 Hz, 1H); 8.92 (d, J ¼ 2.50 Hz, 1H); 9.11
Yellow powder, yield: 85%. Mp: 160e162 ꢀC. 1H NMR (300 MHz,
(d, J ¼ 2.70 Hz, 1H). MS (ESI): 503.1 ([M þ H]þ). Anal. Calcd for
CDCl3,
d
ppm): 2.87 (dd, J1 ¼ 3.00 Hz, J2 ¼ 17.22 Hz, 1H); 3.12 (dd,
C
23H13F3N2O8: C, 54.99; H, 2.61; N, 5.58%; Found: C, 54.77; H, 2.63;
J1 ¼13.38 Hz, J2 ¼ 17.22 Hz, 1H); 5.45 (dd, J1 ¼ 2.88 Hz, J2 ¼ 13.38 Hz,
1H); 6.04 (dd, J1 ¼ 2.40 Hz, J2 ¼ 5.76 Hz, 2H); 6.97 (d, J ¼ 8.73 Hz,
2H); 7.43 (d, J ¼ 8.76 Hz, 2H); 8.47 (s, 2H); 8.53 (s, 2H); 11.97 (s, 1H).
MS (ESI): 741.1 ([M þ H]þ). Anal. Calcd for C29H14F6N4O13: C, 47.04;
H, 1.91; N, 7.57%; Found: C, 47.15; H, 1.87; N, 7.62%.
N, 5.69%.
4.2.11. 7-(3-Chloro-2,6-dinitro-4-(trifluoromethyl)phenoxy)-3-(4-
methoxyphenyl)-4H-chromen-4-one (4c)
White powder, yield: 78%. Mp: 168e170 ꢀC. 1H NMR (500 MHz,
CDCl3,
d
ppm): 3.77 (s, 3H); 6.90e6.94 (m, 4H); 7.41 (d, J ¼ 8.65 Hz,
4.2.18. 7-(3-Chloro-2,6-dinitro-4-(trifluoromethyl)phenoxy)-2-(4-
(3-chloro-2,6-dinitro-4-(trifluoromethyl)phenoxy)phenyl)-5-
hydroxychroman-4-one (7c)
2H); 7.87 (s, 1H); 8.25 (d, J ¼ 8.85 Hz, 1H); 8.55 (s, 1H). MS (ESI):
537.0 ([M þ H]þ). Anal. Calcd for C23H12ClF3N2O8: C, 51.46; H, 2.25;
N, 5.22%; Found: C, 51.35; H, 2.22; N, 5.29%.
Yellow powder, yield: 72%. Mp: 213e214 ꢀC. 1H NMR (300 MHz,
CDCl3,
d
ppm): 2.84 (d, J ¼ 3.03 Hz, 1H); 3.11 (dd, J1 ¼ 13.32 Hz,
4.2.12. 7-(2,4-Dinitro-6-(trifluoromethyl)phenoxy)-3-(4-
hydroxyphenyl)-4H-chromen-4-one (5b)
J2 ¼ 17.25 Hz, 1H); 5.43e5.48 (m, 1H); 6.06 (dd, J1 ¼ 2.43 Hz,
J2 ¼ 13.11 Hz, 2H); 7.00 (d, J ¼ 8.76 Hz, 2H); 7.43 (d, J ¼ 8.70 Hz, 2H);
8.58 (d, J ¼ 23.22 Hz, 2H); 11.95 (s, 1H). MS (ESI): 809.0 ([M þ H]þ).
Anal. Calcd for C29H12Cl2F6N4O13: C, 43.04; H, 1.49; N, 6.92%; Found:
C, 43.13; H, 1.52; N, 6.87%.
White powder, yield: 76%. Mp: 192e194 ꢀC. 1H NMR (500 MHz,
CDCl3,
d
ppm): 6.79 (s, 1H); 6.86 (d, J ¼ 2.80 Hz, 3H); 7.50 (d,
J ¼ 8.60 Hz, 2H); 7.96e7.98 (m, 2H); 8.80 (d, J ¼ 2.15 Hz,1H); 9.02 (d,
J ¼ 2.35 Hz, 1H). MS (ESI): 489.1 ([M þ H]þ). Anal. Calcd for
C
22H11F3N2O8: C, 54.11; H, 2.27; N, 5.74%; Found: C, 54.32; H, 2.36;
4.3. Crystal structure determination
N, 5.68%.
Crystal structure determination of compound 4a and 4c were
4.2.13. 7-(3-Chloro-2,6-dinitro-4-(trifluoromethyl)phenoxy)-3-(4-
(3-chloro-2,6-dinitro-4-(trifluoromethyl)phenoxy)phenyl)-4H-
chromen-4-one (5c)
carried out on a Nonius CAD4 diffractometer equipped with graph-
ꢀ
itemonochromated MoKa (k ¼ 0.71073 A) radiation. The structure
was solved by direct methods and refined on F2 by full-matrix least-
squares methods using SHELX-97. All non-hydrogen atoms of com-
pound 4a and 4c were refined with anisotropic thermal parameters.
All hydrogen atoms were placed in geometrically idealized positions
and constrained to ride on their parent atoms.
Yellow powder, yield: 77%. Mp: 251e253 ꢀC. 1H NMR
(300 MHz, CDCl3,
d ppm): 6.99e7.02 (m, 4H); 7.56 (d,
J ¼ 8.64 Hz, 2H); 7.98 (s, 1H); 8.32 (d, J ¼ 8.70 Hz, 1H); 8.54 (s,
1H); 8.64 (s, 1H). MS (ESI): 791.0 ([M þ H]þ). Anal. Calcd for
C
29H10Cl2F6N4O12: C, 44.02; H, 1.27; N, 7.08%; Found: C, 44.32; H,
1.35; N, 7.26%.
4.4. Antiproliferation activity
4.2.14. 7-(2,6-Dinitro-4-(trifluoromethyl)phenoxy)-3-(4-(2,6-
dinitro-4-(trifluoromethyl)phenoxy)phenyl)-5-hydroxy-4H-
chromen-4-one (6a)
The antiproliferative activities of the prepared compounds
against MCF-7 and Hela cell lines were evaluated as described
elsewhere with some modifications [24]. Target tumor cell lines
were grown to log phase in RPMI 1640 medium supplemented with
10% fetal bovine serum. After diluting to 2 ꢂ 104 cells/mL with the
White powder, yield: 83%. Mp: 221e222 ꢀC. 1H NMR (300 MHz,
DMSO-d6,
d
ppm): 6.67 (d, J ¼ 2.40 Hz, 1H); 6.96 (d, J ¼ 2.40 Hz, 1H);
7.16 (dd, J1 ¼ 2.07 Hz, J2 ¼ 6.84 Hz, 2H); 7.55e7.58 (m, 2H); 8.59 (s,
1H); 8.95 (d, J ¼ 8.10 Hz, 4H); 12.97 (s, 1H). MS (ESI): 739.0
([M þ H]þ). Anal. Calcd for C29H12F6N4O13: C, 47.17; H, 1.64; N,
7.59%; Found: C, 47.28; H, 1.54; N, 7.72%.
complete medium,100 mL of the obtained cell suspensionwas 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

J.-J. Dong et al. / European Journal of Medicinal Chemistry 70 (2013) 427e433
433
were added to six wells with ispinesib as positive references. After
48 h exposure period, 40 L of PBS containing 2.5 mg/mL of MTT (3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-razolium bromide) was
added to each well. Four hours later, 100 L extraction solution (10%
SDS-5% isobutyl alcohol ꢃ0.01 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.
protein data bank (http://www.pdb.org). The molecular docking
procedure was performed by using CDOCKER protocol for recep-
toreligand interactions section of Discovery Studio 3.5 (Accelrys
Software Inc, San Diego, CA) [26]. All bound water and ligands were
eliminated from the protein and the polar hydrogen was added. The
whole KSP complex was defined as a receptor and the site sphere
was selected based on the ligand binding location of MKR, then the
MKR molecule was removed and the tested compound was placed
during the molecular docking procedure. Types of interactions of
the docked protein with ligand were analyzed after the end of
molecular docking.
m
m
4.5. KSP inhibitory assay
The microtubule-stimulated KSP ATPase reaction was per-
formed in a reaction buffer [20 mM PIPES-K (pH 6.8), 25 mM KCl,
2 mM MgCl2,1 mM EGTA-KOH (pH 8.0)] containing 38 nM bacteria-
expressed KSP motor domain (1e369) fused to a histidine tag at the
carboxyl terminus and 350 nM microtubules in 96-well half-area
plates (Corning). Each chemical compound in DMSO at different
concentrations was diluted 12.5-fold with the chemical dilution
buffer [10 mM Tris-OAc (pH 7.4), 0.04% (v/v) NP-40]. After pre-
Acknowledgments
This work was supported by Major Projects on Control and
Rectification of Water Body Pollution (No. 2011ZX07204-001-004),
and was supported by ‘PCSIRT’ (IRT1020).
Appendix A. Supplementary data
incubation of 9.7
chemical solution at 25 ꢀC for 30 min, the ATPase reaction was
initiated by the addition of 1.5 L of 0.3 mM ATP, followed by in-
cubation at 25 ꢀC for 15 min. The reaction was terminated by the
addition of 15 L Kinase-Glo Plus reagent (Promega). The ATP
mL of the enzyme solution with 3.8 mL of each
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.09.042.
m
m
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