Synthesis and biological evaluation of novel chalcone derivatives as a new class of microtubule destabilizing agents

Article abstract of DOI:10.1016/j.ejmech.2017.03.031

A series of novel chalcone derivatives were designed and synthesized as potential antitumor agents. Structures of target molecules were confirmed by ¹H NMR, ¹³C NMR and HR-MS, and evaluated for their in?vitro anti-proliferative activ

Full text of DOI:10.1016/j.ejmech.2017.03.031

European Journal of Medicinal Chemistry 132 (2017) 11e25
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis and biological evaluation of novel chalcone derivatives as a
new class of microtubule destabilizing agents
,
b
,
,
b
,
,
, b, *
Xiaochao Huang a 1, Rizhen Huang a 1, Lingxue Li a b, Shaohua Gou a
,
Hengshan Wang c
,
**
a Pharmaceutical Research Center and School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, China
b Jiangsu Province Hi-Tech Key Laboratory for Biomedical Research, Southeast University, Nanjing 211189, China
c State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources (Ministry of Education of China), School of Chemistry and
Pharmaceutical Sciences of Guangxi Normal University, Guilin 541004, 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 were designed and synthesized as potential antitumor agents.
Structures of target molecules were confirmed by 1H NMR, 13C NMR and HR-MS, and evaluated for their
in vitro anti-proliferative activities using MTT assay. Among them, compound 12k displayed potent ac-
tivity against the test tumor cell lines including multidrug resistant human cancer lines, with the IC50
Received 13 November 2016
Received in revised form
14 February 2017
Accepted 15 March 2017
Available online 18 March 2017
values ranged from 3.75 to 8.42 mM. In addition, compound 12k was found to induce apoptosis in NCI-
H460 cells via the mitochondrial pathway, including an increase of the ROS level, loss of mitochondrial
membrane potential, release of cytochrome c, down-regulation of Bcl-2, up-regulation of Bax, activation
of caspase-9 and caspase-3, respectively. Moreover, the cell cycle analysis indicated that 12k effectively
caused cell cycle arrest at G2/M phase. The results of tubulin polymerization assay displayed that 12k
could inhibit tubulin polymerization in vitro. Furthermore, molecular docking study indicated that 12k
can be binding to the colchicine site of tubulin.
Keywords:
Chalcone
Tubulin
Molecular modeling
Anti-tumor activity
Apoptosis
© 2017 Published by Elsevier Masson SAS.
1. Introduction
[11e13]. Anti-mitotic agents including taxanes and vinca alkaloids
have been widely used for the treatment of a variety of cancers in
Microtubules, as key components of the cytoskeleton, are
cytoskeletal filaments consisting of -tubulin heterodimers and
the past decade [14,15]. However, the clinical use of these com-
pounds was always limited by the high toxicity, the development of
drug resistance, side effects, poor solubility, low oral bioavailability
and complex synthesis [15,16]. Therefore, scientists are eager to
develop and discover novel effective anti-mitotic agents for over-
coming the above mentioned drawbacks.
Natural products and their derivatives provide a diverse source
of new medicinal leads and they play a major role in drug devel-
opment, especially in the area of cancer therapy [17]. Chalcones are
naturally occurring moieties of flavonoid and isoflavonoid com-
pounds, which are an important pharmacophore of many natural
products including curcumin, flavokawain, millepachine, and xan-
thohumol [18e21]. In addition, many researchers reported that
chalcone and its analogues also exhibited wide range of biological
activities, such as anti-oxidant, anti-filarial, anti-bacterial, anti-
fungal, anti-mitotic, anti-tumor, anti-inflammatory and inhibition
of enzymes activities [22e29]. Millepachine, as a novel chalcone
with a 2,2-dimethylbenzopyran motif (1a, Fig. 1) which was first
isolated from the Millettia pachycarpa by Chen and co-workers in
a, b
involved in a series of cellular processes including regulation of
motility, cell signaling, maintenance of cell shape, cell proliferation
and intracellular transport [1e4]. Since microtubules play an
important role in the life cycle of the cell, they have been consid-
ered as a major target for the development of novel anticancer
agents in recent years [5e7]. In recent decades, numbers of com-
pounds as anti-mitotic agents derived from natural sources or ob-
tained by chemical synthesis have been reported [8e10]. In general,
these compounds were divided into three major types such as the
taxane site for microtubule stabilizing agents, the vinca site, and
the colchicine site for microtubule polymerization inhibitors
* Corresponding author. Jiangsu Province Hi-Tech Key Laboratory for Biomedical
Research, Southeast University, Nanjing 211189, China.
** Corresponding author.
E-mail addresses: sgou@seu.edu.cn (S. Gou), whengshan@163.com (H. Wang).
Co-first author: These authors contributed equally to this work.
1
http://dx.doi.org/10.1016/j.ejmech.2017.03.031
0223-5234/© 2017 Published by Elsevier Masson SAS.

12
X. Huang et al. / European Journal of Medicinal Chemistry 132 (2017) 11e25
Fig. 1. Structures of natural products and chalcone analogues as potent inhibitors of tubulin polymerization.
2013, has been known to exert potent cytotoxicity in vitro against a
variety of cancer cells and potent in vivo antitumor activity [30]. In
order to improve the antitumor activity of millepachine, during the
last a few years, Chen and co-workers have explored the B-ring of
millepachine with different substituted groups, such as introduc-
tion of hydroxyl group on the B-ring of millepachine or replace-
ment of methoxyl with diethyl amine, to result in improvement on
their antitumor activity [21,31]. In previous work, a number of
millepachine derivatives were designed and synthesized as po-
tential anticancer agents [15,21,30e33]. Among them, compound
2a (Fig. 1) and 2b displayed excellent antitumor activities toward
various human cancer cell lines including multidrug resistant ones
in vitro and in vivo, and strongly inhibited tubulin polymerization
by binding to the colchicine site of tubulin. However, pharmaco-
kinetic studies indicated that low oral bioavailability of these
compounds has limited further study. In addition, compounds 3a
and 3b (Fig. 1), as a combretastatin A-4 analogous chalcone, were
reported to have potent antitumor activities against a panel of
cancer cell lines and markedly inhibit the polymerization of tubulin
[34,35]. These encouraging results prompted us to further design
and synthesize a new class of chalcone derivatives as potential
anticancer agents.
In an effort to discover more effective compounds that target the
tubulin-microtubule system as a tubulin de-polymerization inhib-
itor, we have designed and synthesized a new series of chalcone
derivatives and evaluated their antitumor activities in the present
study. Among all the compounds, compound 12k displayed the
most potent antitumor activity against the tested cancer cell lines
including multidrug resistant phenotype, and effectively induced
cell cycle arrest in G2/M phase. Moreover, molecular docking
analysis was made to examine whether compound 12k could
inhibit the polymerization of tubulin by binding to the colchicine
binding site.
Treatment of compound 1 with 3-chloro-3-methyl-1-butyne in the
presence of DBU and catalytic amounts of CuCl2$2H2O in CH3CN
afforded the intermediate 2 (yield, 76.1%). The resulting compound
2 was cyclized by heating in pyridine to produce the key interme-
diate 3 (yield, 73.3%). Subsequently, Claisen-Schmidt condensation
of 3 or 8 with 3-hydroxy-4-methoxybenzaldehyde in the presence
of KOH in CH3OH led to the production of the intermediate 4 (yield:
65.2%) or 9 (yield, 54.8%), which was upon etherification with a-
bromoethyl acetate, K2CO3 and KI in the presence of DMF to give
ester 5 (yield, 93.3%) or 10 (yield, 90.9%) followed by its hydrolysis
with aluminum hydroxide in the presence of THF/H2O to generate
the acid 6 (yield, 95.0%) or 11 (yield, 91.9%). Finally, target com-
pounds 7a-7l (yields, 52.3%e95.2%) and 12a-12l (yields, 43.1%e
95.6%) were achieved by the formation of amide bond between 6 or
11 and anilines, respectively, in the presence of HOBT/EDCI. The
structures of these target compounds were confirmed by 1H NMR,
13C NMR and high resolution mass spectra (HR-MS).
2.2. Biology activity
2.2.1. Cytotoxicity test
The in vitro inhibitory effect of these synthesized chalcone de-
rivatives was evaluated by MTT assay against HepG-2, NCI-H460,
MGC-803, SK-OV-3 and T-24 cancer cell lines together with HL-
7702 normal cell line, and compound 1a (Millepachine) was cho-
sen as reference. The IC50 values obtained in the performed in vitro
inhibition assays are summarized in Table 1.
As shown in Table 1, the newly synthesized chalcone derivatives
are potent anticancer agents, with IC50 values mostly in the
micromole level. Among all compounds, it was notable that com-
pound 12k exhibited the best antitumor activity against HepG-2,
NCI-H460, SK-OV-3 and T-24 cancer cells and had low cytoxicity
on normal human cell line (HL-7702) compared with compound 1a.
The results revealed that the analogues 7b and 7c, obtained by
inserting a methoxy or methyl moiety in the position 4 of the lateral
benzene group, led to significant increase in potency compared
with other analogues substituted by halogen. Compound 7b, with
2. Results and discussion
2.1. Chemistry
IC50 value of 6.73 mM against MGC-803 cells, was about 3-fold more
The general procedures for the synthesis of chalcone derivatives
are described in Schemes 1 and 2. Compounds 1e4 and 9 were
synthesized according to the reported procedures [15,21,35].
active than derivatives 7d and 7e, and undoubtedly emerged as one
of the most active compounds within this subset. 7c containing a
methyl group in the 4-position had an IC50 of 8.23 mM in the same

X. Huang et al. / European Journal of Medicinal Chemistry 132 (2017) 11e25
13
Scheme 1. Synthetic pathway to prepare target compounds 7a-7l: Reagents and conditions: (a) 3-Chloro-3-methylbut-1-yne, CuCl2$2H2O, DBU, CH3CN, 0 ꢀC, 12 h. (b) Pyridine,
120 ꢀC, 12 h (c) KOH (50% w/v aqueous solution), 3-hydroxy-4-methoxybenzaldehyde, CH3OH, 0 ꢀC, 12 h (d)
H2O, rt, 2 h (f) HOBT, EDCI, DMF, rt, overnight.
a-bromoethyl acetate, K2CO3, KI, DMF, rt, overnight. (e) LiOH$H2O, THF/
Scheme 2. Synthetic pathway to prepare target compounds 12a-12k: Reagents and conditions: (a) KOH (50% w/v aqueous solution), 3-hydroxy-4-methoxybenzaldehyde, CH3OH,
ꢀC, 12 h (b)
-bromoethyl acetate, K2CO3, KI, DMF, rt, overnight. (c) LiOH$H2O, THF/H2O, rt, 2 h (d) HOBT, EDCI, DMF, rt, overnight.
0
a
cell line. A beneficial effect was also observed with the modifica-
tions of the C-2 position. The derivative 7h was 4-fold more active
than 7j against NCI-H460. The presence of a weakly electron-
withdrawing and bulky group in position C-2 and C-4 seemed to
be associated with a general increase in activity within 7 series.
Furthermore, in the second series of compounds, 12b owning a
methoxy substituent in the para-position of the lateral benzene
ring also had significant activity against the selected cancer cell
lines, with an IC50 range from 7.07 to 9.82 mM. It was important to
note that compounds 12j-12l, possessing a halogen substituent in
the C-2 position of the benzene ring, showed reverse effects as
compared with 7j-7l, indicating that the electron-withdrawing
group might be suitable for this position. Moreover, the modifica-
tions of 4-position of the lateral phenyl ring with a halogen or
hydroxy moiety caused loss of potency, for example, compound 12b
exhibited better cytotoxicity activities than 12d-12g against the test

14
X. Huang et al. / European Journal of Medicinal Chemistry 132 (2017) 11e25
Table 1
Effects of chalcones derivatives 7 and 12 against different cell lines.
Compd.
IC50 (m
M)a
HepG-2
NCI-H460
MGC-803
SK-OV-3
T24
HL-7702
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
1a
20.62 1.22
8.99 1.20
9.32 1.16
22.74 1.08
15.38 1.15
14.09 0.89
15.62 1.27
7.35 0.79
9.44 0.78
27.80 1.02
11.82 1.17
19.12 1.25
10.14 1.32
7.66 0.71
8.97 0.97
10.46 1.05
20.53 1.52
21.23 1.29
>50
17.57 0.94
8.56 0.99
8.77 1.04
20.46 0.88
12.88 1.10
14.24 1.12
17.98 0.97
5.48 1.14
7.96 0.77
20.04 1.09
12.95 1.01
16.97 1.13
11.28 0.94
7.07 0.81
7.69 1.07
10.27 0.64
20.12 1.55
19.18 1.36
>50
18.36 1.08
6.73 1.14
8.23 1.00
17.22 0.98
19.35 1.07
13.49 0.48
20.85 1.04
6.72 0.95
10.34 0.83
32.72 1.23
14.18 1.19
20.56 1.06
14.55 0.75
8.52 0.89
10.34 1.32
12.90 1.43
26.30 1.38
20.92 1.20
36.63 1.13
14.93 1.18
28.35 1.31
14.55 1.46
8.42 1.23
8.09 1.05
10.98 1.21
20.78 1.23
9.02 0.96
9.58 1.11
18.96 1.07
20.01 1.12
13.86 0.86
14.47 1.09
8.24 0.86
10.02 0.84
30.28 1.21
14.56 1.08
18.71 1.24
10.67 1.36
9.82 0.93
10.28 1.29
13.05 1.33
27.15 1.49
22.47 1.35
>50
21.09 0.86
9.51 0.92
10.13 1.38
19.87 1.24
17.12 1.22
17.64 1.01
18.34 0.98
7.84 1.21
9.86 0.96
23.25 1.15
15.43 0.43
17.49 0.91
12.58 0.51
9.36 0.72
11.56 0.94
11.61 1.31
21.73 1.26
24.65 0.66
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
25.05 2.13
13.57 1.30
26.27 1.21
15.22 1.19
4.06 1.21
7.58 0.86
4.54 0.43
12.09 1.27
22.86 0.98
10.32 0.58
3.75 1.07
6.71 1.36
7.65 0.72
16.18 0.65
28.43 0.76
16.75 1.20
6.28 0.79
9.51 1.21
9.85 1.08
17.45 1.52
27.11 0.88
19.06 0.90
5.80 1.63
10.74 0.25
11.05 1.03
a
Each data represents mean S.D. from three different experiments performed in triplicate.
human cancer cell lines. In addition, the inhibition activities of
compounds 7a-7l and 12a-12l against HL-7702 normal cells were
also measured. The results are listed in Table 1. Interestingly, all
target compounds displayed lower cytotoxicity on human normal
liver cell line HL-7702 than compound 1a. The results indicated that
the cytotoxicity of most of compounds against cancer cells was
much higher than HL-7702 normal cells, making them have good
selectivity against cancer cells.
and 1.31 for resistant HeLa cells) than paclitaxel (158.6 for resistant
A549 cells and 186.4 for resistant HeLa cells), respectively.
2.2.3. In vitro tubulin polymerization inhibition activity
It is well-known that microtubule-targeting agents can be
divided into two types: microtubule stabilizing agents (e.g., pacli-
taxel) and microtubule destabilizing agents (e.g., colchicine and CA-
4) [36]. In order to further confirm which type of microtubule-
targeting agents compounds 7h and 12k are, the in vitro tubulin
polymerization inhibition activities of compounds 7h and 12k were
investigated. Compounds 7h and 12k were employed at respective
2.2.2. Antitumor activity of compound 12k against drug-resistant
cancer cell lines
15
mM for microtubule dynamics assays with paclitaxel and CA-4 as
Paclitaxel is used as first-line chemotherapy agent for human
non-small-cell lung cancer (A549) and human epithelial cervical
cancer (HeLa), while inherent or acquired resistance becomes a
major problem. According to the above biological result, we further
investigated the sensitivity of compound 12k against paclitaxel-
resistant and the corresponding sensitive cancer cells (A549 and
HeLa). As shown in Table 2, the IC50 value of paclitaxel for A549 and
positive controls, respectively. As illustrated in Fig. 2, paclitaxel
(10
(10
m
m
M) was found to stimulate tubulin polymerization, while CA-4
M) almost completely inhibited tubulin polymerization as
expected. For compounds 7h and 12k, an obvious inhibition of
polymerization was observed. These data indicated that both 7h
and 12k can effectively inhibit tubulin polymerization and act as
potential microtubule destabilizing agents for cancer therapy.
HeLa resistant cancer cell lines were increased to 1.51 and 1.53 mM,
respectively, compared with those for the corresponding sensitive
cancer cells. Interestingly, the activity of compound 12k did not
markedly vary for these two paclitaxel-resistant cancer cells
compared with the sensitive ones, with the IC50 value of compound
12k against paclitaxel-resistant cancer lines A549 and HeLa were
2.2.4. Molecular docking
To determine the possible binding mode of the chalcone de-
rivatives, compounds 7 and 12 were chosen to perform a molecular
docking to ensure that the chalcone derivatives have the same
binding site as colchicine and compound 4. The binding modes of
7h,12k, colchicine and 4 in the colchicine binding site of tubulin are
increased to 6.25 and 5.95 mM, respectively. Moreover, compound
12k had a much lower resistance index (1.20 for resistant A549 cells
Table 2
In vitro growth inhibitory effect of compound 12k on paclitaxel-resistant cancer cell lines A549 and HeLa.
Compd.
IC50
(
m
M)
resistant factora
IC50
(
m
M)
resistant factora
A549
A549/resistant
HeLa
HeLa/resistant
12k
paclitaxel
5.21 0.45
9.52 2.12b
6.25 0.75
1.51 0.53
1.20
158.6
4.53 0.62
8.21 3.27b
5.95 1.21
1.53 0.31
1.31
186.4
a
Resistance index ¼ IC50(resistant)/IC50(sensitive).
b
IC50 unit for paclitaxel is nM.

X. Huang et al. / European Journal of Medicinal Chemistry 132 (2017) 11e25
15
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
paclitaxel (10 μM)
CA-4 (10 μM)
Control
7h (15 μM)
12k (15 μM)
0
10
20
30
40
Time (min)
Fig. 2. Effects of compounds 7h and 12k on microtubule dynamics. Polymerization of tubulin at 37 ꢀC in the presence of paclitaxel (10
compound 12k (15
m
M), CA-4 (10
m
M), compound 7h (15
m
M) and
m
M) was monitored continuously by recording the absorbance at 340 nm over 40 min. The reaction was initiated by the addition of tubulin to a final con-
centration of 3.0 mg/mL.
depicted in Fig. 3 and the docking scores obtained are summarized
in Table 3. The Surflex docking scores are 12.53 for 7h and 12.31 for
12k, where higher scores indicate greater binding affinity. Biolog-
ical activity studies suggested that compounds 7h and 12k poten-
tially inhibited tubulin polymerization, hence, the docking detail of
these two compounds were examined to compare with those of
1SA0-colchicine and compound 4 to evaluate binding modes.
Fig. 3A shows that the interacting mode of compound 7h with
chalcone moiety in the binding site is surrounded by Cys241,
Leu248, Lys 254, Leu255, Lys352 and Leu354. In particular, com-
pound 7h forms three hydrogen bonds with the polar amino acids
Asn101, suggesting a probable strong electrostatic interaction with
the protein. In addition, the hydrophobic moiety of the compound
7h is well embedded in a pocket interacting with several hydro-
phobic residues making compound 7h bind tightly to tubulin. Not
surprisingly, the accommodation of compound 12k in the binding
site is also similar to colchicine (Fig. 3B). In this case, docking
simulations showed that the 3,4,5-trimethoxy-phenyl ring of
compound 12k like colchicine can also be accommodated in the
same hydrophobic groove, adopting an energetically stable
conformation. Moreover, the methoxy group, the carbonyl oxygen
atom of 3,4,5-trimethoxy-phenyl moiety and the oxygen atom of
amide in compound 12k as acceptors establish three hydrogen
bonds with Ser178, Tyr224 and Asn249, respectively, which is
consistent with the observation that colchicine stabilizes the
tubulin heterodimer and further confirms that this moiety is also
crucial for binding (Fig. 3B). Similar interactions with Ser178 or
Tyr224 can be observed in the binding mode of compound 4. It is
interesting to note that the crucial electrostatic interactions be-
tween the methoxy group of the 3,4,5-trimethoxy-phenyl unit and
represents damaged cells, the Q2 region represents necrotic cells
and later period apoptotic cells, the Q3 area represents early
apoptotic cells, and the Q4 area represents the normal cells,
respectively. It was found that compound 12k could induce
apoptosis in NCI-H460 cells. Apoptosis ratios (including the early
and late apoptosis ratios cells) were showed after treatment with
12k for 24 h at the concentration of 5 and 10 mM, respectively. The
apoptosis of NCI-H460 cells treatment with the compound
increased gradually in a dose-dependent manner. The apoptosis
ratios of compound 12k at two concentrations were found to be
15.35% and 66.4%, respectively, while that of control was only 1.44%.
The results evidently manifested that compound 12k effectively
induced apoptosis in NCI-H460 cells in comparison with the
control.
2.2.6. Morphological characterization of cell apoptosis of NCI-
H460 cells by Hoechst 33258
To further determine whether the observed cell death induced
by compound 12k was due to apoptosis, untreated cells were used
as a negative control and NCI-H460 cells treated with different
concentrations of compound 12k for 24 h were stained with
Hoechst 33258. As shown in Fig. 5, the control cells displayed weak
blue fluorescence, but it was worth noting that the nuclei of NCI-
H460 cells appeared hyper-condensed (brightly stained) after
treatment with compound 12k at 5 or 10 mM for 24 h. These results
indicated that compound 12k was able to induce apoptosis in a
dose-dependent manner.
2.2.7. Cell cycle analysis
As most anti-mitotic agents display the ability to disrupt the
regulated cell cycle distribution, the effect of a 48 h treatment with
different concentrations of compound 12k on cell cycle progression
was also evaluated by flow cytometry in NCI-H460 cells. Untreated
cells were used as a negative control and NCI-H460 cells were
residues Ser178, Lys254 and Asn101 of the neighboring a-subunit
are observed in the binding pocket, demonstrating a plausible
competitive mechanism of action at the colchicine site.
2.2.5. Compound 12k induces apoptosis in NCI-H460
treated with compound 12k at 5 and 10 mM for 48 h, respectively.
Among all the target compounds, compound 12k was found to
exhibit the best antitumor activity against the NCI-H460 cancer
cells, hence, the apoptosis ratios induced by 12k in NCI-H460
cancer cells were determined by flow cytometry. NCI-H460 cells
were co-stained with Annexin-V FITC and PI, and the apoptotic cells
were observed by flow cytometric analysis. In Fig. 4, the Q1 area
The cells were harvested, and the cell cycle phases were examined
by flow cytometry. As shown in Fig. 6, G1 period cells gradually
decreased and S period cells did not change significantly, while G2
phase cells markedly increased compared with the control group.
At 5
mM of compound 12k, 11.88% of G2 phase was arrested, while
the concentration of compound 12k was increased to 10
mM, 72.44%

16
X. Huang et al. / European Journal of Medicinal Chemistry 132 (2017) 11e25
Fig. 3. Molecular modeling of 7h, 12k, 4 and 1SA0-colchicine in complex with tubulin. Illustrated are the proposed binding mode and interaction between tubulin and selected
compounds, (A) compound 7h, (B) compound 12k, (C) 4, (D) 1SA0-colchicine. The compounds and important amino acids in the binding pockets are shown in stick model, whereas
tubulin is depicted in the ribbon model. The Mg2þ-ion is shown as a purple sphere. (For interpretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)

X. Huang et al. / European Journal of Medicinal Chemistry 132 (2017) 11e25
17
G2 phase arrest was observed. These results showed that com-
3. Conclusion
pound 12k markedly arrested the G2 phase of the cell cycle in a
dose-dependent manner.
In this investigation, a class of novel chalcone analogues were
designed and synthesized, and their cell growth inhibition activ-
ities against HepG-2, NCI-H460, MGC-803, SK-OV-3 and T24 cancer
cell lines were investigated using MTT assay. The in vitro antitumor
activities result showed that among these compounds, 12k
exhibited excellent anti-proliferative activities against five human
cancer cell lines including multidrug resistant human tumor cell
lines. The apoptosis-inducing activity of 12k in NCI-H460 cells was
investigated by Hoechst 33258 staining, JC-1 mitochondrial mem-
brane potential staining, ROS generation assay and flow cytometry.
In addition, molecular mechanism studies indicated that com-
pound 12k was found to induce apoptosis in NCI-H460 cells via the
mitochondrial pathway, and the cell cycle analysis suggested that
compound 12k significantly induced cell cycle arrest in G2/M
phase. Moreover, the results of tubulin polymerization assay dis-
played that the novel chalcone analogues could inhibit tubulin
polymerization, and molecular docking analysis indicated that 12k
can be binding to the colchicine site of tubulin. Taken together,
these results suggested that compound 12k is a promising anti-
tumor agent to inhibit tubulin polymerization.
2.2.8. Compound 12k triggers ROS generation
Comprehensive literature indicated that intracellular ROS plays
a vital role in the regulation of cell apoptosis [37e39]. Thus, we
further investigated whether ROS stimulated by compound 12k
induced apoptosis in NCI-H460 cells. Cells were treated with
compound 12k (5 and 10 mM) for 24 h using the fluorescent probe
2,7-dichlorofluorescein diacetate (DCF-DA) analysis by flow
cytometry with untreated cells as a negative control. As illustrated
in Fig. 7, the cells treated with different concentrations of com-
pound 12k exhibited ROS generation level increased in a dose-
dependent manner compared with control cells. In short, these
data hinted that compound 12k caused oxidative imbalance in NCI-
H460 cells.
2.2.9. Compound 12k triggers mitochondrial pathway dependent
apoptosis
The loss of mitochondrial membrane potential (MMP) is regar-
ded as an important factor in the apoptotic pathway [40e42]. In
order to further evaluate the apoptosis-inducing effect of com-
pound 12k, MMP changes were detected using the fluorescent
probe JC-1. NCI-H460 cells were treated with compound 12k (5,
4. Experimental
All chemicals and solvents were of analytical reagent grade and
provided by Energy Chemical, Shanghai of China and used without
further purification, unless noted specifically. Column chromatog-
raphy was performed using silica gel (200e300 mesh). 1H NMR and
13C NMR spectra were recorded in CDCl3 or DMSO-d6 with a Bruker
300 or 400 MHz spectrometer. Mass spectra were measured on an
Agilent 6224 TOF LC/MS instrument.
10
mM) for 24 h using untreated cells as a negative control. As
shown in Fig. 8, compound 12k induced a dose-dependent manner
increasing in the proportion of cells compared with the control
cells, suggesting that compound 12k treatment induced mito-
chondrial membrane permeability in NCI-H460 cells. Taken
together, these results clearly demonstrated that mitochondrial
function is extremely impaired in compound 12k caused apoptosis
in NCI-H460 cells.
4.1. General procedure for the preparation of compounds 7a-7l and
12a-12k
2.2.10. Compound 12k induces apoptosis via the regulation of
apoptosis-related protein expression
Synthesis of compound 2. To a solution of compound 1 (2.32 g,
20.0 mmol) in dry CH3CN (150 mL), DBU (3.0 mL, 30.0 mmol),
CuCl2$2H2O (10 mg, 0.6 mmol), and 3-chloro-3-methyl-1-butyne
(3.06 g, 30.0 mmol) were added at 0 ꢀC. The mixture was stirred
at the same temperature for 12 h, then 1 N HCl (aq) was added to
adjust pH ¼ 2. After the solvent was removed under reduced
pressure, the residue was poured into water (50 mL) to yield yellow
precipitate that was collected by filtration and washed with pe-
troleum ether to produce compound 2 as a yellow solid (yield, 3.5 g,
76.1%) which was used directly without further purification. 1H
It is generally accepted that anticancer agents initiated apoptotic
signaling through two major pathways [43e45]. One is the death
receptor (extrinsic) pathway involving death receptor, and the
other is the mitochondrial (intrinsic) pathway. In order to investi-
gate whether compound 12k induced apoptosis via the mito-
chondrial signaling pathway, some of major protein markers
involved in mitochondria-mediated apoptosis were also analyzed
by western blot method. Initially, the level of cytochrome c released
from the mitochondria to cytosol was evaluated as shown in Fig. 9.
Compared with the control, the level of cytochrome c in cytosol
increased in a dose-dependent manner after treatment with com-
pound 12k for 48 h. In addition, it has been confirmed that the
release of cytochrome c can stimulate apoptosis via the mito-
chondrial pathway by activation of the downstream caspase-9 and
caspase-3 [46,47]. Secondly, the roles of caspase-9 and caspase-3 in
the cellular response to compound 12k were further investigated.
As illustrated in Fig. 9, western blot analysis displayed that treat-
ment of NCI-H460 cells with compound 12k effectively induced
cleavage of caspase-9 and caspase-3, respectively. Since the Bcl-2
family proteins are critical regulators of the mitochondrial
apoptotic pathway, the anti-apoptotic protein Bcl-2 and the pro-
apoptotic protein Bax are important regulators of this progress
[48,49]. Finally, to confirm the mechanism of compound 12k
induced in NCI-H460 cells apoptosis, the expression of Bax and Bcl-
2 proteins was investigated. Western blot analysis exhibited that
the protein expression level of Bax effectively increased, while the
protein expression level of Bcl-2 significantly decreased in a dose-
dependent manner compared with control (Fig. 9).
NMR (300 MHz, CDCl3)
d
7.75 (d, J ¼ 8.8 Hz, 1H, ArH), 7.18 (s, 1H,
ArH), 6.59 (d, J ¼ 8.8 Hz, 1H, ArH), 3.83 (s, 3H, -OCH3), 2.67 (s, 1H,
-C^CH), 2.57 (s, 3H, -CH3),1.74 (s, 6H, 2 ꢁ -CH3). HR-MS (m/z) (ESI):
calcd for C14H18O3 [MþHþ]: 233.11777; found: 233.11902.
Synthesis of compound 3. A solution of compound 2 (2.32 g,
10.0 mmol) in dry pyridine (50 mL) was stirred at 120 ꢀC, which was
monitored by TLC. After completion of reaction for 12 h, the
mixture was concentrated under reduced pressure, the resulting
crude product was purified by chromatography on silica gel eluted
with petroleum ether/ethyl acetate (V:V ¼ 50:1) to offer compound
3 as a yellow oil (yield: 1.7 g, 73.3%). 1H NMR (300 MHz, CDCl3)
d
7.74 (d, J ¼ 8.9 Hz, 1H, ArH), 6.67 (d, J ¼ 10.0 Hz, 1H, ArH), 6.47 (d,
J ¼ 8.9 Hz, 1H, Ph-CH]CH-C(CH3)2), 5.61 (d, J ¼ 10.0 Hz, 1H, Ph-
CH]CH-C(CH3)2), 3.86 (s, 3H, -OCH3), 2.61 (s, 3H, -CH3), 1.49 (s, 6H,
2 ꢁ -CH3). HR-MS (m/z) (ESI): calcd for C14H18O3 [MþHþ]:
233.11777; found: 233.11738.
Synthesis of compound 4. A solution of 50% KOH (15 mL, aq) was
added dropwise to a stirred solution of compound 3 (1.5 g,
6.46 mmol) and 3-hydroxy-4-methoxybenzaldehyde (0.98 g,

18
X. Huang et al. / European Journal of Medicinal Chemistry 132 (2017) 11e25
Table 3
Docking scores (kcal/mol) for all studied compounds.
Compd.
Score
Crash
Polar
D-Score
PMF Score
G-Score
Chem-Score
CScore
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
4
9.71
ꢂ1.83
ꢂ1.21
ꢂ0.83
ꢂ1.63
ꢂ1.72
ꢂ1.03
ꢂ1.57
ꢂ1.72
ꢂ1.59
ꢂ2.54
ꢂ2.01
ꢂ2.12
ꢂ2.44
ꢂ2.86
ꢂ2.06
ꢂ0.80
ꢂ3.16
ꢂ1.51
ꢂ2.03
ꢂ4.44
ꢂ2.04
ꢂ2.66
ꢂ1.44
ꢂ1.65
0.61
2.03
0.00
1.23
0.51
0.32
0.00
2.01
1.70
0.73
1.99
2.49
0.96
0.57
1.22
2.05
2.77
2.02
2.44
1.05
2.34
1.44
1.14
1.43
2.50
3.09
1.95
ꢂ174.40
ꢂ172.50
ꢂ158.68
ꢂ179.63
ꢂ171.05
ꢂ174.90
ꢂ160.68
ꢂ192.69
ꢂ167.89
ꢂ171.21
ꢂ169.44
ꢂ176.91
ꢂ167.74
ꢂ181.67
ꢂ165.12
ꢂ150.24
ꢂ170.86
ꢂ167.71
ꢂ152.99
ꢂ199.87
ꢂ162.47
ꢂ179.89
ꢂ181.13
ꢂ173.01
ꢂ120.99
ꢂ145.65
ꢂ3.78
ꢂ18.61
ꢂ9.53
ꢂ8.42
ꢂ4.01
ꢂ7.01
ꢂ4.79
3.20
7.65
9.13
ꢂ1.46
ꢂ1.21
2.94
ꢂ312.00
ꢂ323.81
ꢂ299.31
ꢂ336.89
ꢂ350.42
ꢂ325.39
ꢂ304.02
ꢂ372.87
ꢂ322.27
ꢂ292.28
ꢂ296.49
ꢂ300.44
ꢂ300.44
ꢂ308.39
ꢂ296.47
ꢂ253.22
ꢂ287.92
ꢂ264.87
ꢂ260.31
ꢂ285.07
ꢂ269.44
ꢂ312.04
ꢂ326.22
ꢂ289.46
ꢂ181.69
ꢂ229.93
ꢂ34.86
ꢂ32.49
ꢂ32.66
ꢂ32.72
ꢂ33.15
ꢂ33.77
ꢂ30.13
ꢂ33.69
ꢂ31.50
ꢂ33.27
ꢂ31.04
ꢂ28.30
ꢂ31.63
ꢂ28.82
ꢂ33.16
ꢂ25.60
ꢂ28.38
ꢂ28.11
ꢂ26.46
ꢂ37.64
ꢂ31.07
ꢂ31.52
ꢂ25.04
ꢂ27.49
ꢂ228.86
ꢂ21.31
5
5
4
4
5
5
3
4
3
3
2
4
4
4
5
2
3
3
4
4
5
5
3
3
3
2
11.70
10.97
9.35
9.64
10.09
9.68
12.53
10.39
9.20
10.02
9.89
10.53
11.13
10.61
10.95
9.07
8.95
ꢂ32.29
ꢂ24.89
ꢂ6.81
ꢂ6.95
ꢂ10.91
16.05
ꢂ19.06
ꢂ17.67
5.59
9.10
7.88
10.64
9.08
10.39
12.31
11.65
8.6
7.56
ꢂ23.37
colchicine
9.73
ꢂ1.34
2.87
Fig. 4. Apoptosis ratio detection of compound 12k by Annexin V/PI assay. (a) Untreated NCI-H460 cells as control for 24 h, (b) treated NCI-H460 cells at 5
NCI-H460 cells at 10 M for 24 h.
m
M for 24 h, (c) treated
m
6.46 mmol) in methanol (30 mL) at 0 ꢀC. The resulting mixture was
stirred at the same temperature for 12 h and monitored by TLC.
After completion of reaction, the mixture was poured into ice-
water and adjusted to pH ¼ 2 with 4 N HCl. The precipitated was
filtered, washed with water and dried to offer the crude product
which was purified by chromatography on silica gel eluted with
petroleum ether/ethyl acetate (V:V ¼ 3:1, 2:1) to give the desired
compound as a yellow solid (yield: 1.5 g, 65.2%). 1H NMR (300 MHz,
5.0 mmol) in DMF (15 mL), a-bromoethyl acetate (1.09 g, 6.5 mmol),
K2CO3 (695 mg, 10.0 mmol) and KI (85 mg, 0.5 mmol) were added.
The mixture was stirred overnight at room temperature. Then the
mixture was diluted with water (250 mL) and extracted with
CH2Cl2 (2 ꢁ 150 mL). The organic phase was dried over anhydrous
Na2SO4 and concentrated under reduced pressure. The residue was
purified by chromatography on silica gel eluted with petroleum
ether/ethyl acetate (V:V ¼ 1.5:1) to offer the desired product as a
CDCl3)
d
7.70 (d, J ¼ 8.8 Hz,1H, ArH), 7.66e7.53 (m, 2H, ArH), 7.21 (d,
yellow oil (yield: 2.1 g, 93.3%). 1H NMR (300 MHz, CDCl3)
d 7.69 (d,
J ¼ 1.7 Hz, 1H, ArH), 7.11e7.08 (m, 1H, ArH), 6.86 (d, J ¼ 8.3 Hz, 1H,
Ph-CO-CH]CH-), 6.69 (d, J ¼ 10.0 Hz, 1H, Ph-CO-CH]CH-), 6.51 (d,
J ¼ 8.8 Hz, 1H, Ph-CH]CH-C(CH3)2), 5.68 (s, 1H, -OH), 5.63 (d,
J ¼ 10.0 Hz, 1H, Ph-CH]CH-C(CH3)2), 3.93 (s, 3H, -OCH3), 3.88 (s,
3H, -OCH3), 1.51 (s, 6H, 2 ꢁ -CH3). HR-MS (m/z) (ESI): calcd for
J ¼ 8.8 Hz, 1H, ArH), 7.64e7.50 (m, 2H, ArH), 7.23 (s, 1H, ArH), 7.09
(d, J ¼ 1.5 Hz,1H, ArH), 6.91 (d, J ¼ 8.4 Hz,1H, Ph-CO-CH]CH-), 6.69
(d, J ¼ 10.0 Hz,1H, Ph-CO-CH]CH-), 6.51 (d, J ¼ 8.9 Hz,1H, Ph-CH]
CH-C(CH3)2), 5.62 (d, J ¼ 10.0 Hz, 1H, Ph-CH]CH-C(CH3)2), 4.69 (s,
2H, -O-CH2-COOCH2CH3), 4.26 (q, J ¼ 7.1 Hz, 2H, -CH2CH3), 3.91 (s,
3H, -OCH3), 3.87 (s, 3H, -OCH3), 1.50 (s, 6H, 2 ꢁ -CH3), 1.28 (t,
J ¼ 7.2 Hz, 3H, -CH2CH3). HR-MS (m/z) (ESI): calcd for C26H28O7
C
14H22O5 [MþHþ]: 367.15455; found: 367.15061.
Synthesis of compound 5. To a solution of compound 4 (1.83 g,

X. Huang et al. / European Journal of Medicinal Chemistry 132 (2017) 11e25
19
Fig. 5. Hoechst 33258 staining of compound 12k in NCI-H460 cells. (a) Untreated NCI-H460 cells were used as control at for 24 h and (b, c) NCI-H460 cells were treated with
compound 12k at 5 and 10 M for 24 h, respectively.
m
Fig. 6. Investigation of cell cycle distribution by flow cytometric analysis. (a) Untreated NCI-H460 cells as a control. (b) and (c) NCI-H460 cells were treated with increasing
concentrations of compound 12k (5 and 10 M) for 48 h, respectively.
m
31.8 mmol) was added and stirred at room temperature for 2 h.
After the reaction mixture was adjusted pH ¼ 2 with 1 N HCl so-
lution, 150 mL of water was added. The solution was extracted with
CH2Cl2 (2 ꢁ 100 mL), then the organic phase was dried over
anhydrous Na2SO4 and concentrated under reduced pressure to
obtain the target product as a yellow solid (yield: 3.2 g, 95.0%). 1H
NMR (300 MHz, CDCl3)
d
7.69 (d, J ¼ 8.8 Hz, 1H, ArH), 7.64e7.49 (m,
2H, ArH), 7.22 (s, 1H, ArH), 7.12 (s, 1H, ArH), 6.88 (d, J ¼ 8.3 Hz, 1H,
Ph-CO-CH]CH-), 6.67 (d, J ¼ 10.1 Hz, 1H, Ph-CO-CH]CH-), 6.50 (d,
J ¼ 8.8 Hz, 1H, Ph-CH]CH-C(CH3)2), 5.61 (d, J ¼ 10.0 Hz, 1H, Ph-
CH]CH-C(CH3)2), 4.66 (s, 2H, -O-CH2-COOH), 3.87 (s, 6H, 2 ꢁ -
OCH3), 1.48 (s, 6H, 2 ꢁ -CH3). HR-MS (m/z) (ESI): calcd for C24H24O7
[MþHþ]: 425.16003; found: 425.14932.
Synthesis of compound 9. A solution of 50% KOH (15 mL, aq) was
added dropwise to a stirred solution of compound 8 (1.5 g,
7.14 mmol) and 3-hydroxy-4-methoxybenzaldehyde (1.09 g,
7.14 mmol) in methanol (25 mL) at 0 ꢀC. The resulting mixture was
stirred at the same temperature for 12 h and monitored by TLC.
After completion of reaction, the mixture was poured into ice-
water and adjusted to pH ¼ 2 with 4 N HCl. The deposit was
filtered, washed with water and dried to offer the crude product,
which was purified by chromatography on silica gel eluted with
petroleum ether/ethyl acetate (V:V ¼ 4:1, 3:1, 2:1) to give the
desired compound as a yellow solid (yield: 2.3 g, 54.8%). 1H NMR
Fig. 7. ROS generation assay of compound 12k in NCI-H460 cells after treatment with
5 and 10 M for 24 h.
m
[MþHþ]: 453.19133; found:453.18403.
Synthesis of compound 6. To a solution of compound 5 (3.6 g,
7.96 mmol) in THF/H2O (50/10 mL) lithium hydroxide (1.33 g,
(300 MHz, DMSO-d6)
d 9.13 (s, 1H, ArH), 7.73e7.59 (m, 2H, ArH),
7.40 (s, 2H, ArH), 7.37 (s, 1H, -OH), 7.30 (d, J ¼ 8.4 Hz, 1H, Ph-CO-

20
X. Huang et al. / European Journal of Medicinal Chemistry 132 (2017) 11e25
Fig. 8. JC-1 mitochondrial membrane potential staining of compound 12k in NCI-H460 cells. (a) Untreated cells as control for 24 h and (b, c) treated cells with compound 12k at 5
and 10 M for 24 h, respectively.
m
Fig. 9. Western blot analysis of Cyt c, Bax, Bcl-2, caspase-9 and caspase-3 after treatment of NCI-H460 cells with compound 12k at the indicated concentrations and for the
indicated times. -actin antibody was used as reference control.
b
CH]CH-), 7.00 (d, J ¼ 8.3 Hz,1H, Ph-CO-CH]CH-), 3.90 (s, 6H, 2 ꢁ -
OCH3), 3.84 (s, 3H, -OCH3), 3.76 (s, 3H, -OCH3). HR-MS (m/z) (ESI):
calcd for C19H20O6 [MþHþ]: 345.13381; found: 345.13371.
Synthesis of compound 10. To a solution of compound 9 (1.75 g,
extracted with CH2Cl2 (2 ꢁ 100 mL), the organic phase was dried
over anhydrous Na2SO4 and concentrated under reduced pressure
to obtain the target product as a yellow solid(yield: 1.7 g, 91.9%). 1H
NMR (300 MHz, DMSO-d6)
d 12.97 (s, 1H, -COOH), 7.76 (d,
5.1 mmol) in DMF (15 mL),
a
-bromoethyl acetate (1.02 g, 6.1 mmol),
J ¼ 15.5 Hz,1H, ArH), 7.67 (d, J ¼ 15.5 Hz,1H, ArH), 7.54e7.43 (m, 2H,
ArH, Ph-CO-CH]CH-, overlapped signals), 7.39 (s, 2H, ArH), 7.07 (d,
J ¼ 8.4 Hz, 1H, Ph-CO-CH]CH-), 4.78 (s, 2H, -O-CH2-COOH), 3.90 (s,
6H, 2 ꢁ -OCH3), 3.85 (s, 3H, -OCH3), 3.77 (s, 3H, -OCH3). HR-MS (m/
z) (ESI): calcd for C21H22O8 [M-Hþ]: 401.12364; found: 401.12445.
Synthesis of compounds 7a-7l. To a solution of compound 6
(150 mg, 0.353 mmol), EDCI (104 mg, 0.530 mmol) and HOBT
(72 mg, 0.530 mmol) in dry DMF (3 mL), anilines (0.353 mmol) was
added. After the mixture was stirred overnight at room tempera-
ture, it was diluted with water (250 mL) and extracted with CH2Cl2
(100 mL). The organic phase was dried over anhydrous Na2SO4 and
concentrated under reduced pressure. The residue was purified by
chromatography on silica gel eluted with petroleum ether/ethyl
acetate (V:V ¼ 6:1, 3:1) to offer the desired products as yellow
solids.
K2CO3 (1.4 g, 10.2 mmol) and KI (85 mg, 0.51 mmol) were added.
The mixture was stirred overnight at room temperature, then it was
diluted with water (300 mL) and extracted with CH2Cl2
(2 ꢁ 150 mL). The organic phase was dried over anhydrous Na2SO4
and concentrated under reduced pressure. The residue was purified
by chromatography on silica gel eluted with petroleum ether/ethyl
acetate (V:V ¼ 1.5:1) to offer the desired product as a yellow solid
(yield: 2.0 g, 90.9%). 1H NMR (300 MHz, CDCl3)
d
7.80 (d, J ¼ 15.5 Hz,
1H, ArH), 7.41e7.38 (m, 2H, ArH), 7.34 (s, 2H, ArH), 7.25 (d,
J ¼ 8.6 Hz, 1H, Ph-CO-CH]CH-), 7.02 (d, J ¼ 8.3 Hz, 1H, Ph-CO-CH]
CH-), 4.81 (s, 2H, -O-CH2-COOCH2CH3), 4.41e4.33 (m, 2H,
-CH2CH3), 4.03 (d, J ¼ 6.5 Hz, 12H, 4 ꢁ -OCH3), 1.38 (t, J ¼ 7.1 Hz, 3H,
-CH2CH3). HR-MS (m/z) (ESI): calcd for C23H26O8 [MþHþ]:
431.17059; found: 431.16823.
Synthesis of compound 11. To a solution of compound 10 (2.0 g,
4.65 mmol) in THF/H2O (30/5 mL) lithium hydroxide (979 mg,
23.3 mmol) was added and stirred at room temperature for 2 h. The
reaction mixture was adjusted pH ¼ 2 with 1 N HCl solution, then
150 mL of water was added. The resulting aqueous solution was
Synthesis of compounds 12a-12k. To a solution of compound 11
(130 mg, 0.323 mmol), EDCI (93 mg, 0.485 mmol) and HOBT
(65 mg, 0.485 mmol) in dry DMF (3 mL), anilines (0.323 mmol) was
added in reaction. The mixture was stirred overnight at room
temperature, then it was diluted with water (300 mL) and extracted

X. Huang et al. / European Journal of Medicinal Chemistry 132 (2017) 11e25
21
with CH2Cl2 (100 mL). The organic phase was dried over anhydrous
Na2SO4 and concentrated under reduced pressure. The residue was
purified by chromatography on silica gel eluted with petroleum
ether/ethyl acetate (V:V ¼ 8:1, 5:1) to offer the desired products as
yellow solids.
116.83, 115.23, 112.18, 110.49, 103.56, 70.29, 56.14, 55.82, 28.10. HR-
MS (m/z) (ESI): calcd for
C
30H28O6NCl [MþHþ]:534.16834;
found:534.15679.
Compound 7f: yield, 93.1%; 1H NMR (400 MHz, CDCl3)
d 8.86 (s,
1H, -CO-NH-), 7.71 (d, J ¼ 8.8 Hz, 1H, ArH), 7.61 (s, 2H, ArH),
7.59e7.56 (m, 2H, ArH), 7.31e7.24 (m, 2H, ArH), 7.05 (t, J ¼ 8.6 Hz,
2H, ArH), 6.97 (d, J ¼ 8.4 Hz, 1H, Ph-CO-CH]CH-), 6.70 (d,
J ¼ 10.0 Hz, 1H, Ph-CO-CH]CH-), 6.52 (d, J ¼ 8.8 Hz, 1H, Ph-CH]
CH-C(CH3)2), 5.63 (d, J ¼ 10.0 Hz, 1H, Ph-CH]CH-C(CH3)2), 4.65 (s,
2H, -O-CH2-CO-NH-), 3.97 (s, 3H, -OCH3), 3.88 (s, 3H, -OCH3), 1.50
Compound 7a: yield, 95.2%; 1H NMR (400 MHz, CDCl3)
d 8.87 (s,
1H, -CO-NH-), 7.71 (d, J ¼ 8.8 Hz, 1H, ArH), 7.62e7.60 (m, 4H, ArH),
7.35 (t, J ¼ 7.8 Hz, 2H, ArH), 7.30e7.24 (m, 2H, ArH), 7.14 (t,
J ¼ 7.4 Hz,1H, ArH), 6.96 (d, J ¼ 8.4 Hz,1H, Ph-CO-CH]CH-), 6.70 (d,
J ¼ 10.0 Hz, 1H, Ph-CO-CH]CH-), 6.51 (d, J ¼ 8.9 Hz, 1H, Ph-CH]
CH-C(CH3)2), 5.63 (d, J ¼ 10.0 Hz, 1H, Ph-CH]CH-C(CH3)2), 4.66 (s,
2H, -O-CH2-CO-NH-), 3.97 (s, 3H, -OCH3), 3.87 (s, 3H, -OCH3), 1.50
(s, 6H, 2 ꢁ -CH3). 13C NMR (100 MHz, CDCl3)
d 189.62, 166.36,
160.79, 158.48, 158.36, 153.66, 151.39, 147.42, 140.31, 133.27. 133.24,
131.72, 129.36, 128.59, 126.32, 124.84, 121.66, 121.61, 121.58, 116.83,
115.87, 115.65, 115.11, 112.16, 110.49, 103.56, 70.21, 56.13, 55.82,
28.09. HR-MS (m/z) (ESI): calcd for C30H28O6NF [MþHþ]:518.19789;
found:518.18735.
(s, 6H, 2 ꢁ -CH3). 13C NMR (100 MHz, CDCl3)
d 189.62, 166.39,
158.47, 153.66, 151.45, 147.49, 140.37, 137.22, 131.71, 129.31, 129.10,
128.61, 126.27, 124.85, 124.68, 121.63, 119.87, 116.82, 115.05, 112.13,
110.49, 103.55, 70.28, 56.11, 55.81, 28.10. HR-MS (m/z) (ESI): calcd
for C33H29O6N [MþHþ]: 500.20731; found: 500.19839.
Compound 7g: yield, 85.9%; 1H NMR (400 MHz, CDCl3)
d 8.76 (s,
Compound 7b: yield, 91.5%; 1H NMR (400 MHz, CDCl3)
d
8.74 (s,
1H, -CO-NH-), 7.71 (d, J ¼ 8.8 Hz, 1H, ArH), 7.61 (s, 2H, ArH), 7.41 (d,
J ¼ 8.8 Hz, 2H, ArH), 7.30e7.21 (m, 2H, ArH), 6.96 (d, J ¼ 8.4 Hz, 1H,
ArH), 6.83 (d, J ¼ 8.8 Hz, 2H, ArH, Ph-CO-CH]CH-, overlapped
signals), 6.69 (d, J ¼ 10.0 Hz, 1H, Ph-CO-CH]CH-), 6.52 (d,
J ¼ 8.9 Hz, 1H, Ph-CH]CH-C(CH3)2), 5.63 (d, J ¼ 10.0 Hz, 1H, Ph-
CH]CH-C(CH3)2), 4.64 (s, 2H, -O-CH2-CO-NH-), 3.95 (s, 3H, -OCH3),
3.88 (s, 3H, -OCH3),1.50 (s, 6H, 2 ꢁ -CH3). 13C NMR (100 MHz, CDCl3)
1H, -CO-NH-), 7.71 (d, J ¼ 8.8 Hz, 1H, ArH), 7.61 (s, 2H, ArH),
7.55e7.48 (m, 2H, ArH), 7.30e7.25 (m, 1H, ArH), 7.24 (d, J ¼ 1.9 Hz,
1H, ArH), 6.96 (d, J ¼ 8.4 Hz, 1H, ArH), 6.97e6.88 (m, 2H, ArH, Ph-
CO-CH]CH-, overlapped signals), 6.70 (d, J ¼ 10.0 Hz, 1H, Ph-CO-
CH]CH-), 6.51 (d, J ¼ 8.9 Hz, 1H, Ph-CH]CH-C(CH3)2), 5.63 (d,
J ¼ 10.0 Hz, 1H, Ph-CH]CH-C(CH3)2), 4.65 (s, 2H, -O-CH2-CO-NH-),
3.96 (s, 3H, -OCH3), 3.88 (s, 3H, -OCH3), 3.80 (s, 3H, -OCH3), 1.50 (s,
d
190.00, 166.45, 158.55, 153.75, 153.43, 151.44, 147.44, 140.70,
6H, 2 ꢁ -CH3). 13C NMR (100 MHz, CDCl3)
d
189.64, 166.10, 158.46,
131.73, 129.72, 129.24, 128.64, 126.20, 124.75, 122.15, 121.54, 116.78,
115.86, 115.02, 112.12, 110.51, 103.58, 70.08, 56.11, 55.82, 28.10. HR-
156.68, 153.66, 151.43, 147.49, 140.40, 131.70, 130.33, 129.30, 128.61,
126.25, 124.78, 121.63, 116.81, 114.92, 114.25, 112.10, 110.49, 103.54,
70.18, 56.09, 55.81, 55.50, 28.10. HR-MS (m/z) (ESI): calcd for
MS (m/z) (ESI): calcd for
C
30H29O7N [Mþ Naþ]:538.18417;
found:538.1773.
C
31H31O7N [MþHþ]: 530.21788; found: 530.21028.
Compound 7h: yield, 89.0%; 1H NMR (400 MHz, CDCl3)
d 9.25 (s,
Compound 7c: yield, 94.5%; 1H NMR (400 MHz, CDCl3)
d
8.79 (s,
1H, -CO-NH-), 8.41 (d, J ¼ 8.0 Hz, 1H, ArH), 7.71 (d, J ¼ 8.8 Hz, 1H,
ArH), 7.61 (s, 2H), 7.29e7.26 (m, 1H, ArH), 7.23 (d, J ¼ 1.9 Hz, 1H,
ArH), 7.10e7.06 (m, 1H, ArH), 7.00e6.89 (m, 3H, ArH, Ph-CO-CH]
CH-, overlapped signals), 6.69 (d, J ¼ 10.0 Hz, 1H, Ph-CO-CH]CH-),
6.51 (d, J ¼ 8.9 Hz, 1H, Ph-CH]CH-C(CH3)2), 5.62 (d, J ¼ 10.0 Hz, 1H,
Ph-CH]CH-C(CH3)2), 4.65 (s, 2H, -O-CH2-CO-NH-), 3.95 (s, 3H,
-OCH3), 3.90 (s, 3H, -OCH3), 3.87 (s, 3H, -OCH3), 1.49 (s, 6H, 2 ꢁ -
1H, -CO-NH-), 7.71 (d, J ¼ 8.8 Hz, 1H, ArH), 7.61 (s, 2H, ArH),
7.49e7.47 (m, 2H, ArH), 7.31e7.22 (m, 2H, ArH), 7.15 (d, J ¼ 8.2 Hz,
2H, ArH), 6.96 (d, J ¼ 8.4 Hz, 1H, Ph-CO-CH]CH-), 6.70 (d,
J ¼ 10.0 Hz, 1H, Ph-CO-CH]CH-), 6.51 (d, J ¼ 8.9 Hz, 1H, Ph-CH]
CH-C(CH3)2), 5.63 (d, J ¼ 10.0 Hz, 1H, Ph-CH]CH-C(CH3)2), 4.65 (s,
2H, -O-CH2-CO-NH-), 3.97 (s, 3H, -OCH3), 3.88 (s, 3H, -OCH3), 2.33
(s, 3H, -CH3), 1.50 (s, 6H, 2 ꢁ -CH3). 13C NMR (100 MHz, CDCl3)
CH3). 13C NMR (100 MHz, CDCl3)
d 189.70, 166.05, 158.43, 153.64,
d
189.65, 166.20, 158.46, 153.66, 151.45, 147.51, 140.39, 134.65,
151.58, 148.43, 147.28, 140.63, 131.68, 129.00, 128.64, 126.96, 126.06,
124.59, 124.32, 121.69, 121.10, 120.09, 116.80, 113.98, 112.10, 110.49,
110.24, 103.52, 69.48, 56.06, 55.88, 55.80, 28.06. HR-MS (m/z) (ESI):
calcd for C31H31O7N [MþHþ]: 530.21788; found: 530.20973.
134.32, 131.71, 129.58, 129.31, 128.61, 126.26, 124.83, 121.65, 119.92,
116.81, 115.00, 112.10, 110.49, 103.54, 70.26, 56.10, 55.81, 28.10,
20.91. HR-MS (m/z) (ESI): calcd for C31H31O6N [MþHþ]: 514.22296;
found: 514.21420.
Compound 7i: yield, 82.5%; 1H NMR (400 MHz, CDCl3)
d 8.71 (s,
Compound 7d: yield, 90.6%; 1H NMR (400 MHz, CDCl3)
d
8.82 (s,
1H, -CO-NH-), 8.05 (d, J ¼ 8.0 Hz, 1H, ArH), 7.72 (d, J ¼ 8.8 Hz, 1H,
ArH), 7.62 (s, 2H, ArH), 7.29e7.19 (m, 4H, ArH), 7.08 (t, J ¼ 7.4 Hz, 1H,
ArH), 6.95 (d, J ¼ 8.4 Hz, 1H, Ph-CO-CH]CH-), 6.70 (d, J ¼ 10.0 Hz,
1H, Ph-CO-CH]CH-), 6.51 (d, J ¼ 8.9 Hz, 1H, Ph-CH]CH-C(CH3)2),
5.63 (d, J ¼ 10.0 Hz, 1H, Ph-CH]CH-C(CH3)2), 4.67 (s, 2H, -O-CH2-
CO-NH-), 3.92 (s, 3H, -OCH3), 3.88 (s, 3H, -OCH3), 2.32 (s, 3H, -CH3),
1H, -CO-NH-), 7.63 (d, J ¼ 8.8 Hz, 1H, ArH), 7.53 (s, 2H, ArH),
7.45e7.42 (m, 2H, ArH), 7.40e7.37 (m, 2H, ArH), 7.23e7.21 (m, 1H,
ArH), 7.15 (d, J ¼ 1.9 Hz,1H, ArH), 6.89 (d, J ¼ 8.4 Hz,1H, Ph-CO-CH]
CH-), 6.62 (d, J ¼ 10.0 Hz, 1H, Ph-CO-CH]CH-), 6.44 (d, J ¼ 8.9 Hz,
1H, Ph-CH]CH-C(CH3)2), 5.56 (d, J ¼ 10.0 Hz, 1H, Ph-CH]CH-
C(CH3)2), 4.57 (s, 2H, -O-CH2-CO-NH-), 3.89 (s, 3H, -OCH3), 3.80 (s,
3H, -OCH3), 1.42 (s, 6H, 2 ꢁ -CH3). 13C NMR (100 MHz, CDCl3)
1.51 (s, 6H, 2 ꢁ -CH3). 13C NMR (100 MHz, CDCl3)
d 189.65, 165.94,
158.46, 153.66, 151.22, 147.07, 140.50, 135.18, 131.70, 130.50, 129.07,
128.61, 128.17, 126.91, 126.15, 125.08, 124.52, 121.91, 121.66, 116.83,
113.29, 111.89, 110.50, 103.55, 69.08, 55.91, 55.81, 28.10, 17.34. HR-
MS (m/z) (ESI): calcd for C31H31O6N [MþHþ]: 514.22296; found:
514.21447.
d
189.62, 166.48, 158.49, 153.66, 151.38, 147.38, 140.28, 136.32,
132.08, 131.72, 129.38, 128.59, 126.34, 124.89, 121.60, 121.38, 117.27,
116.83, 115.24, 112.18, 110.49, 103.56, 70.30, 56.14, 55.82, 28.10. HR-
MS (m/z) (ESI): calcd for C30H28O6NBr [MþNaþ]:602.09772; found:
602.08574.
Compound 7j: yield, 54.5%; 1H NMR (400 MHz, CDCl3)
d 9.28 (s,
Compound 7e: yield, 95.2%; 1H NMR (400 MHz, CDCl3)
d
8.89 (s,
1H, -CO-NH-), 8.43 (d, J ¼ 8.3 Hz, 1H, ArH), 7.72 (d, J ¼ 8.8 Hz, 1H,
ArH), 7.61 (d, J ¼ 0.8 Hz, 2H, ArH), 7.58e7.56 (m, 1H, ArH), 7.39e7.20
(m, 4H, ArH), 7.04e7.00 (m, 1H, ArH), 6.96 (d, J ¼ 8.4 Hz, 1H, Ph-CO-
CH]CH-), 6.70 (d, J ¼ 10.0 Hz, 1H, Ph-CO-CH]CH-), 6.52 (d,
J ¼ 8.9 Hz, 1H, Ph-CH]CH-C(CH3)2), 5.63 (d, J ¼ 10.0 Hz, 1H, Ph-
CH]CH-C(CH3)2), 4.68 (s, 2H, -O-CH2-CO-NH-), 3.93 (s, 3H, -OCH3),
3.88 (s, 3H, -OCH3),1.50 (s, 6H, 2 ꢁ -CH3). 13C NMR (100 MHz, CDCl3)
1H, -CO-NH-), 7.71 (d, J ¼ 8.8 Hz, 1H, ArH), 7.61 (s, 2H, ArH), 7.57 (d,
J ¼ 8.7 Hz, 2H, ArH), 7.33e7.23 (m, 4H, ArH), 6.97 (d, J ¼ 8.4 Hz, 1H,
Ph-CO-CH]CH-), 6.70 (d, J ¼ 10.0 Hz, 1H, Ph-CO-CH]CH-), 6.52 (d,
J ¼ 8.8 Hz, 1H, Ph-CH]CH-C(CH3)2), 5.63 (d, J ¼ 10.0 Hz, 1H, Ph-
CH]CH-C(CH3)2), 4.65 (s, 2H, -O-CH2-CO-NH-), 3.97 (s, 3H, -OCH3),
3.88 (s, 3H, -OCH3),1.50 (s, 6H, 2 ꢁ -CH3). 13C NMR (100 MHz, CDCl3)
d
189.61, 166.46, 158.49, 153.66, 151.38, 147.40, 140.27, 135.81,
d
189.71, 166.36, 158.44, 153.64, 151.50, 147.01, 140.59, 135.19,
131.72, 129.66, 129.39, 129.13, 128.59, 126.35, 124.89, 121.61, 121.05,
132.49, 131.70, 128.93, 128.61, 128.40, 126.09, 125.60, 124.70, 121.89,

22
X. Huang et al. / European Journal of Medicinal Chemistry 132 (2017) 11e25
121.70, 116.82, 113.71, 113.67, 111.99, 110.48, 103.53, 69.18, 55.93,
CDCl3) d 189.00, 166.53, 153.19, 151.95, 147.37, 143.81, 142.61, 136.23,
55.81, 28.09. HR-MS (m/z) (ESI): calcd for
C
30H28O6NBr
133.54, 132.11, 128.53, 125.55, 121.42, 120.43, 117.38, 115.45, 112.17,
[Mþ Naþ]:602.09772; found: 602.08456.
106.18, 70.32, 61.00, 56.49, 56.18. HR-MS (m/z) (ESI): calcd for
Compound 7k: yield, 52.3%; 1H NMR (400 MHz, CDCl3)
d
9.34 (s,
C
27H26O7NBr [MþNaþ]: 580.07669; found:580.06663.
1H, -CO-NH-), 8.46 (d, J ¼ 7.7 Hz, 1H, ArH), 7.72 (d, J ¼ 8.8 Hz, 1H,
ArH), 7.59 (d, J ¼ 16.2 Hz, 2H, ArH), 7.40 (d, J ¼ 7.7 Hz, 1H, ArH),
7.32e7.26 (m, 2H, ArH), 7.22 (s, 1H, ArH), 7.16e6.92 (m, 3H, ArH, Ph-
CO-CH]CH-, overlapped signals), 6.70 (d, J ¼ 10.0 Hz, 1H, Ph-CO-
CH]CH-), 6.52 (d, J ¼ 8.8 Hz, 1H, Ph-CH]CH-C(CH3)2), 5.63 (d,
J ¼ 10.0 Hz, 1H, Ph-CH]CH-C(CH3)2), 4.68 (s, 2H, -O-CH2-CO-NH-),
3.91 (d, J ¼ 19.9 Hz, 6H, 2 ꢁ -OCH3), 1.50 (s, 6H, 2 ꢁ -CH3). 13C NMR
Compound 12e: yield, 90.5%; 1H NMR (400 MHz, CDCl3)
d 8.88
(s, 1H, -CO-NH-), 7.77e7.33 (m, 1H, ArH), 7.64e7.52 (m, 2H, ArH),
7.41e7.24 (m, 7H, ArH, Ph-CO-CH]CH-, Ph-CO-CH]CH-, over-
lapped signals), 7.00e6.97 (m, 1H, ArH), 4.70 (s, 2H, -O-CH2-CO-
NH-), 3.99e3.95 (m, 12H, 4 ꢁ -OCH3). 13C NMR (100 MHz, CDCl3)
d
188.92, 166.47, 153.18, 151.97, 147.38, 143.75, 142.62, 135.75,
133.53, 129.72, 129.13, 128.51, 125.50, 121.07, 120.41, 115.50, 112.15,
(100 MHz, CDCl3)
d
189.69, 166.34, 158.43, 153.63, 151.50, 147.05,
106.19, 70.31, 60.97, 56.46, 56.15. HR-MS (m/z) (ESI): calcd for
140.56, 134.08, 131.69, 129.21, 128.94, 128.60, 127.75, 126.09, 125.05,
124.70, 123.25, 121.69, 12154, 116.81, 113.77, 111.97, 110.48, 103.52,
69.26, 55.92, 55.80, 28.08. HR-MS (m/z) (ESI): calcd for C30H28O6NCl
[MþHþ]:534.16834; found:534.15718.
C
27H26O7NCl [MþNaþ]:534.12955; found:534.11949.
Compound 12f: yield, 89.0%; 1H NMR (400 MHz, CDCl3)
d
8.81 (s,
1H, -CO-NH-), 7.73 (d, J ¼ 15.5 Hz, 1H, ArH), 7.58e7.54 (m, 2H, ArH),
7.36e7.32 (m, 2H, ArH, Ph-CO-CH]CH-, overlapped signals), 7.27
(s, 1H, ArH), 7.25 (s, 2H, ArH), 7.03 (t, J ¼ 8.6 Hz, 2H, ArH), 6.97 (d,
J ¼ 8.4 Hz, 1H, Ph-CO-CH]CH-), 4.68 (s, 2H, -O-CH2-CO-NH-), 3.96
(s, 3H, -OCH3), 3.93 (d, J ¼ 2.1 Hz, 9H, 3 ꢁ -OCH3). 13C NMR
Compound 7l: yield, 65.3%; 1H NMR (400 MHz, CDCl3)
d 9.15 (s,
1H, -CO-NH-), 8.42e8.37 (m, 1H, ArH), 7.72 (d, J ¼ 8.8 Hz, 1H, ArH),
7.61 (s, 2H, ArH), 7.31e7.24 (m, 2H, ArH), 7.17e7.07 (m, 3H, ArH),
6.96 (d, J ¼ 8.4 Hz, 1H, Ph-CO-CH]CH-), 6.70 (d, J ¼ 10.0 Hz, 1H, Ph-
CO-CH]CH-), 6.52 (d, J ¼ 8.9 Hz, 1H, Ph-CH]CH-C(CH3)2), 5.63 (d,
J ¼ 10.0 Hz, 1H, Ph-CH]CH-C(CH3)2), 4.69 (s, 2H, -O-CH2-CO-NH-),
(100 MHz, CDCl3)
d 188.94, 166.40, 160.81, 158.39, 153.18, 151.97,
147.39, 143.82, 142.58, 133.54, 133.19, 133.17, 128.48, 125.48, 121.71,
121.63, 120.37, 115.89, 115.67, 115.35, 112.13, 106.16, 70.22, 60.98,
3.96 (s, 3H, -OCH3), 3.88 (s, 3H, -OCH3), 1.51 (s, 6H, 2 ꢁ -CH3). 13
C
56.47, 56.16. HR-MS (m/z) (ESI): calcd for
C27H26O7NF
NMR (100 MHz, CDCl3)
d 189.66, 166.60, 158.45, 153.65, 151.64,
[MþNaþ]:518.15910; found:518.14944.
147.42, 140.47, 131.71, 129.07, 128.60, 126.16, 125.86, 125.76, 124.91,
124.82, 124.75, 124.63, 121.68. 121.67, 116.82, 115.05, 114.86, 114.80,
112.01, 110.48, 103.54, 70.13, 56.00, 55.81, 28.09. HR-MS (m/z) (ESI):
calcd for C30H28O6NF [MþHþ]:518.19789; found:518.18747.
Compound 12g: yield, 87.6%; 1H NMR (400 MHz, DMSO-d6)
9.78 (s, 1H, -CO-NH-), 9.26 (s, 1H, -OH), 7.84e7.66 (m, 2H, ArH),
d
7.60e7.49 (m, 2H, ArH), 7.44e7.34 (m, 4H, ArH), 7.10 (d, J ¼ 8.4 Hz,
1H, Ph-CO-CH]CH-), 6.71 (d, J ¼ 8.7 Hz, 2H, ArH, Ph-CO-CH]CH-,
overlapped signals), 4.74 (s, 2H, -O-CH2-CO-NH-), 3.88 (s, 9H, 3 ꢁ -
Compound 12a: yield, 93.5%; 1H NMR (400 MHz, CDCl3)
d 8.83
(s,1H, -CO-NH-), 7.75 (d, J ¼ 15.5 Hz,1H, ArH), 7.61 (d, J ¼ 8.0 Hz, 2H,
ArH), 7.38e7.33 (m, 4H, ArH), 7.29 (d, J ¼ 1.5 Hz, 1H, Ph-CO-CH]
CH-), 7.27 (s, 2H, ArH), 7.15 (t, J ¼ 7.4 Hz,1H, ArH), 6.98 (d, J ¼ 8.4 Hz,
1H, Ph-CO-CH]CH-), 4.70 (s, 2H, -O-CH2-CO-NH-), 3.98 (s, 3H,
-OCH3), 3.94 (d, J ¼ 2.1 Hz, 9H, 3 ꢁ -OCH3). 13C NMR (100 MHz,
OCH3), 3.76 (s, 3H, -OCH3). 13C NMR (100 MHz, DMSO-d6)
d 188.35,
166.25,154.22, 153.36,152.07,148.03,144.46,142.39,133.73,130.33,
128.00, 124.98, 121.85, 120.36, 115.61, 114.65, 112.75, 106.65, 69.15,
60.67, 56.69, 56.30. HR-MS (m/z) (ESI): calcd for C27H27O8N
[MþNaþ]:516.16344; found:516.15421.
CDCl3)
d
188.95, 166.43, 153.18, 152.02, 147.47, 143.85, 142.55, 137.13,
Compound 12h: yield, 82.3%; 1H NMR (400 MHz, CDCl3)
d 9.14
133.57, 129.12, 128.45, 125.52, 124.76, 120.33, 119.90, 115.23, 112.09,
(s,1H, -CO-NH-), 8.32 (d, J ¼ 8.0 Hz,1H, ArH), 7.68 (d, J ¼ 15.6 Hz,1H,
ArH), 7.29e7.24 (m, 2H, ArH), 7.21 (d, J ¼ 1.9 Hz,1H, Ph-CO-CH]CH-
), 7.19 (s, 2H, ArH), 7.04e7.02 (m, 1H, ArH), 6.93e6.89 (m, 2H, ArH),
6.84 (d, J ¼ 8.1 Hz, 1H, Ph-CO-CH]CH-), 4.63 (s, 2H, -O-CH2-CO-
NH-), 3.90 (s, 3H, -OCH3), 3.87 (d, J ¼ 3.3 Hz, 9H, 3 ꢁ -OCH3), 3.83 (s,
106.14, 70.31, 60.99, 56.47, 56.15. HR-MS (m/z) (ESI): calcd for
C
27H27O7N [MþNaþ]:500.16852; found:500.15859.
Compound 12b: yield, 95.6%; 1H NMR (400 MHz, CDCl3)
d 8.70
(s, 1H, -CO-NH-), 7.75 (d, J ¼ 15.6 Hz, 1H, ArH), 7.52e7.50 (m, 2H,
ArH), 7.37e7.33 (m, 2H, ArH), 7.29 (d, J ¼ 1.9 Hz, 1H, Ph-CO-CH]
CH-), 7.27 (s, 2H, ArH), 6.98 (d, J ¼ 8.4 Hz, 1H, Ph-CO-CH]CH-),
6.90e6.88 (m, 2H, ArH), 4.70 (s, 2H, -O-CH2-CO-NH-), 3.98 (s, 3H,
3H, -OCH3). 13C NMR (100 MHz, CDCl3)
d 189.07, 166.17, 153.18,
152.15, 148.45, 147.31, 144.13, 142.51, 133.63, 128.17, 126.84, 125.31,
124.45, 121.13, 120.15, 114.24, 112.05, 110.28, 106.12, 69.61, 60.99,
56.47, 56.09, 55.89. HR-MS (m/z) (ESI): calcd for C28H29O8N
[MþNaþ]:530.17909; found:530.16653.
-OCH3), 3.95 (d, J ¼ 3.2 Hz, 9H, 3 ꢁ -OCH3), 3.80 (s, 3H, -OCH3). 13
C
NMR (100 MHz, CDCl3)
d 188.95, 166.16, 156.74, 153.18, 152.00,
147.48, 143.89, 142.55, 133.58, 130.21, 128.43, 125.47, 121.69, 120.30,
115.04, 114.27, 112.06, 106.13, 70.20, 60.99, 56.47, 56.13, 55.50. HR-
Compound 12i: yield, 75.5%; 1H NMR (400 MHz, CDCl3)
d 8.68 (s,
1H, -CO-NH-), 8.02 (d, J ¼ 8.0 Hz, 1H, ArH), 7.76 (d, J ¼ 15.5 Hz, 1H,
ArH), 7.38e7.32 (m, 2H, ArH), 7.27 (s, 2H), 7.26e7.20 (m, 3H, ArH,
Ph-CO-CH]CH-, overlapped signals), 7.10 (t, J ¼ 7.5 Hz, 1H, ArH),
6.97 (d, J ¼ 8.4 Hz, 1H, Ph-CO-CH]CH-), 4.73 (s, 2H, -O-CH2-CO-
NH-), 3.95 (d, J ¼ 3.3 Hz, 12H, 4 ꢁ -OCH3), 2.32 (s, 3H, -CH3). 13C
MS (m/z) (ESI): calcd for
C
28H29O8N [MþNaþ]:530.17909;
found:530.16858.
Compound 12c: yield, 94.5%; 1H NMR (400 MHz, CDCl3)
d 8.74 (s,
1H, -CO-NH-), 7.75 (d, J ¼ 15.6 Hz, 1H, ArH), 7.49e7.47 (m, 2H, ArH),
7.37e7.33 (m, 2H, ArH), 7.29 (d, J ¼ 1.9 Hz, 1H, Ph-CO-CH]CH-),
7.27 (s, 2H, ArH), 7.16 (d, J ¼ 8.2 Hz, 2H, ArH), 6.98 (d, J ¼ 8.4 Hz, 1H,
Ph-CO-CH]CH-), 4.70 (s, 2H, -O-CH2-CO-NH-), 3.98 (s, 3H, -OCH3),
3.94 (d, J ¼ 3.1 Hz, 9H, 3 ꢁ -OCH3), 2.33 (s, 3H, -CH3). 13C NMR
NMR (100 MHz, CDCl3)
d 188.99, 166.09, 153.18, 151.77, 147.11,
144.02, 142.56, 135.05, 133.59, 130.54, 12837, 128.22, 126.93, 125.24,
125.20, 122.08, 120.1, 113.45, 111.86, 106.15, 69.14, 60.99, 56.47,
55.96, 17.35. HR-MS (m/z) (ESI): calcd for
C
28H29O7N
(100 MHz, CDCl3)
d 188.97, 166.27, 153.18, 152.02, 147.49, 143.89 (s),
[MþNaþ]:514.18417; found:514.17411.
142.55, 134.55, 134.45, 133.58, 129.61, 128.44, 125.52, 120.31, 119.97,
115.10, 112.06, 106.13, 70.28, 60.99, 56.47, 56.13, 20.91. HR-MS (m/z)
(ESI): calcd for C28H29O7N [MþNaþ]:514.18417; found:514.17419.
Compound 12j: yield, 53.5%; 1H NMR (400 MHz, CDCl3)
d 9.26 (s,
1H, -CO-NH-), 8.43 (d, J ¼ 7.5 Hz, 1H, ArH), 7.76 (d, J ¼ 15.5 Hz, 1H,
ArH), 7.62e7.53 (m, 1H, ArH), 7.39e7.32 (m, 3H, ArH, Ph-CO-CH]
CH-, overlapped signals), 7.28 (s, 1H, ArH), 7.27 (s, 2H, ArH),
7.04e7.01 (m, 1H, ArH), 6.98 (d, J ¼ 8.3 Hz, 1H, Ph-CO-CH]CH-),
4.73 (s, 2H, -O-CH2-CO-NH-), 3.95e3.94 (m, 12H, 4 ꢁ -OCH3). 13C
Compound 12d: yield, 91.2%; 1H NMR (400 MHz, CDCl3)
d 8.80
(s,1H, -CO-NH-), 7.67 (d, J ¼ 15.6 Hz,1H, ArH), 7.45 (d, J ¼ 8.9 Hz, 2H,
ArH), 7.39 (d, J ¼ 8.9 Hz, 2H, ArH), 7.31e7.24 (m, 2H, ArH), 7.21 (d,
J ¼ 1.4 Hz, 1H, Ph-CO-CH]CH-), 7.19 (s, 2H, ArH), 6.92 (d, J ¼ 8.4 Hz,
1H, Ph-CO-CH]CH-), 4.62 (s, 2H, -O-CH2-CO-NH-), 3.91 (s, 3H,
-OCH3), 3.87 (d, J ¼ 4.0 Hz, 9H, 3 ꢁ -OCH3). 13C NMR (100 MHz,
NMR (100 MHz, CDCl3)
d 188.98, 166.43, 153.18, 152.11, 147.08,
144.06, 142.53, 135.12, 133.63, 132.50, 128.45, 128.08, 125.68,
125.43, 121.91, 120.13, 114.10, 113.67, 111.99, 106.13, 69.37, 60.99,

X. Huang et al. / European Journal of Medicinal Chemistry 132 (2017) 11e25
23
56.48, 55.98. HR-MS (m/z) (ESI): calcd for C27H26O7NBr
[MþNaþ]:580.07669; found:580.06677.
ether)-N,N,N0,N'-tetraacetic acid (EGTA), 1 mM GTP, and 10.2% glyc-
erol. Test compounds were added in different concentrations, and
then all components except the purified tubulin were warmed to
37 ꢀC. The reaction was initiated by the addition of tubulin to a final
concentration of 3.0 mg/mL. Paclitaxel was used as positive controls
under the same conditions. The optical density was measured for
40 min at 1 min intervals in BioTek's Synergy 4 multifunction
Compound 12k: yield, 43.1%; 1H NMR (400 MHz, CDCl3)
d 9.31
(s,1H, -CO-NH-), 8.46 (d, J ¼ 8.3 Hz,1H, ArH), 7.76 (d, J ¼ 15.6 Hz,1H,
ArH), 7.42e7.39 (m, 1H, ArH), 7.37e7.31 (m, 3H, ArH), 7.29e7.27 (m,
4H, ArH, Ph-CO-CH]CH-, overlapped signals), 7.09 (t, J ¼ 7.7 Hz,
1H), 6.97 (d, J ¼ 8.4 Hz, 1H, Ph-CO-CH]CH-), 4.73 (s, 2H, -O-CH2-
CO-NH-), 3.95 (s, 6H, 2 ꢁ -OCH3, overlapped signals), 3.95 (d,
microplate spectrophotometer with
a temperature controlled
J ¼ 1.9 Hz, 6H, 2 ꢁ -OCH3). 13C NMR (100 MHz, CDCl3)
d
188.98,
cuvette holder. Assays were performed according to the manufac-
turer's instructions and under conditions similar to those employed
for the tubulin polymerization assays described above [8,51].
166.40, 153.18, 152.11, 147.10, 144.05, 142.53, 134.02, 133.62, 129.22,
128.09, 127.80, 125.42, 125.14, 123.24, 121.57, 120.12, 114.12, 111.96,
106.13, 69.42, 60.99, 56.47, 55.97. HR-MS (m/z) (ESI): calcd for
C
27H26O7NCl [MþNaþ]:534.12955; found:534.11866.
4.5. Molecular docking
Compound 12l: yield, 63.3%; 1H NMR (400 MHz, CDCl3)
d
9.11 (s,
1H, -CO-NH-), 7.41e7.36 (m, 1H, ArH), 7.75 (d, J ¼ 15.5 Hz, 1H, ArH),
7.38e7.33 (m, 2H, ArH), 7.30 (d, J ¼ 1.7 Hz, 1H, Ph-CO-CH]CH-),
7.28 (s, 2H, ArH), 7.17e7.08 (m, 3H, ArH), 6.97 (d, J ¼ 8.4 Hz, 1H, Ph-
CO-CH]CH-), 4.73 (s, 2H, -O-CH2-CO-NH-), 3.97 (s, 3H, -OCH3),
3.95 (d, J ¼ 4.1 Hz, 9H, 3 ꢁ -OCH3). 13C NMR (100 MHz, CDCl3)
All the docking studies were carried out using Sybyl-X 2.0 on a
windows workstation. The crystal structure of the tubulin in
complex with colchicine was retrieved from the RCSB Protein Data
Bank (PDB: 1SA0.pdb) [52]. The synthetic chalcone analogues were
selected for the docking studies. The 3D structures of these selected
compounds were first built using Sybyl-X 2.0 sketch followed by
energy minimization using the MMFF94 force field and Gasteiger-
Marsili charges. We employed Powell's method for optimizing the
geometry with a distance dependent dielectric constant and a
termination energy gradient of 0.005 kcal/mol. All the selected
compounds were automatically docked into the colchicine binding
pocket of tubulin by an empirical scoring function and a patented
search engine in the Surflex docking program. The polar hydrogen
atoms were added and also the automated docking manner was
applied in the present work. Other parameters were established by
default to estimate the binding affinity characterized by the
Surflex-Dock scores in the software. Surflex-Dock total scores,
which were expressed in -log10 (Kd) units to represent binding
affinities, were applied to evaluate the ligand-receptor interactions
of newly designed molecules. A higher score represents stronger
binding affinity. The optimal binding pose of the docked com-
pounds was selected based on the Surflex scores and visual in-
spection of the docked complexes.
d
188.96, 166.63, 153.84, 153.18, 152.22, 151.41, 147.43, 143.94,
142.54, 133.61, 128.22, 125.77, 125.67, 125.58, 124.92, 124.84, 124.66,
124.62, 121.73, 120.21, 115.07, 114.88, 111.99, 106.14, 70.21, 60.98,
56.46, 56.03. HR-MS (m/z) (ESI): calcd for
C27H26O7NF
[MþNaþ]:518.15910; found:518.14879.
4.2. Cell culture and maintenance
All human cancer cell lines and human normal liver cell line HL-
7702 in this study were purchased from China Life Science Collage
(Shanghai, PRC). Culture medium Dulbecco's modified Eagle me-
dium (DMEM), fetal bovine serum (FBS), phosphate buffered saline
(PBS, pH ¼ 7.2), and Antibiotice-Antimycotic came from KeyGen
Biotech Company (China). Cell lines were grown in the supple-
mented with 10% FBS, 100 units/ml of penicillin and 100 g/mL of
streptomycin in a humidified atmosphere of 5% CO2 at 37 ꢀC.
4.3. Cytotoxicity assay
The anticancer activity of the all compounds were investigated
in five human cancer cell lines (HepG-2, NCI-H460, MGC-803, SK-
OV-3 and T24) and human normal liver cell line (HL-7702).
Among all compounds, compound 12k was further evaluated on
paclitaxel resistance cancer cell lines (A549 and HeLa). About
1 ꢁ 105 cells/mL cells, which were in the logarithmic phase, were
grown in each well of 96-well plates and incubated for 12 h of 5%
CO2 at 37 ꢀC. All compounds at five different concentrations were
then added to the test well and the cells were incubated at 37 ꢀC in
a 5% CO2 atmosphere for 48 h, respectively. An enzyme labeling
instrument was used to read absorbance with 570/630 nm double
wavelength measurement. Cytotoxicity was evaluated on the per-
centage of cell survival compared with control group. The final IC50
values were calculated by the Bliss method (n ¼ 5). All of the tests
were repeated in triplicate.
4.6. Flow cytometry analysis of cell cycle distribution
The NCI-H460 cells were grown on 6-well plates and treated
with compound 12k (5, 10 mM), and maintained with the proper
culture medium in 5% CO2 at 37 ꢀC for 24 h. After completion of
incubation, cells were harvested and washed twice with ice-cold
PBS, fixed with ice-cold 70% ethanol at ꢂ20 ꢀC for overnight. The
cells were treated with 100 m
g/mL RNase A for 30 min at 37 ꢀC after
washed with twice ice-cold PBS, and finally stained with 1 mg/mL
propidium iodide (PI) in the dark at 4 ꢀC for 1 h. Analysis was
performed with the system software (Cell Quest; BD Biosciences).
4.7. Apoptosis analysis
Apoptosis was examined by flow cytometry analysis of annexin
V/PI staining. NCI-H460 cells were seeded in each well of 96-well
plates at the density of 1 ꢁ 105 cells/mL of the DMEM medium
with 10% FBS to the final volume of 2 mL. The plates were incubated
for overnight and treated with different concentrations of the test
compound 12k for 24 h. Briefly, cells were harvested and washed
with three times ice-cold PBS, and then suspended cells in the
annexin-binding buffer at a concentration of 5 ꢁ 105 cells/ml. cells
4.4. Tubulin polymerization assay in vitro assays
Tubulin polymerization assay was monitored by the change in
optical density at 340 nm using a modification of methods described
by Jordan et al. [50] Purified brain tubulin polymerization kit was
purchased from Cytoskeleton (BK006P, Denver, CO). The final buffer
concentrations for tubulin polymerization contained 80.0 mM
were then incubated with 5 mL of annexin V-FITC and 5 mL of PI for
30 min at room temperature in the dark. The cells were analyzed by
system software (Cell Quest; BD Biosciences).
piperazine-N,N0-bis(2-ethanesulfonic
(pH ¼ 6.9), 2.0 mM MgCl2, 0.5 mM ethylene glycol bis(
acid)sequisodium
salt
b-aminoethyl

24
X. Huang et al. / European Journal of Medicinal Chemistry 132 (2017) 11e25
4.8. Mitochondrial membrane potential assay
the Central Universities (Project 2242016K30020) for supplying
basic facilities to our key laboratory. We also want to express our
gratitude to the Priority Academic Program Development of Jiangsu
Higher Education Institutions for the construction of fundamental
facilities (Project 1107047002). Huang is grateful to the Innovation
Program of Jiangsu Province postgraduate education (Project
KYLX16_0263). The research was also supported by the Scientific
Research Foundation of Graduated School of Southeast University
(YBJJ1677).
The mitochondrial membrane potential was measured by flow
cytometry using the JC-1 fluorescent probe (Beyotime, Haimen,
China, Molecular Probe), as previously described [53]. NCI-
H460 cells were treated with different concentrations of the test
compound 12k for 24 h. After for 24 h, the JC-1 fluorescent probe
was added 20 min after replacing with fresh medium. Cells were
harvested at 2000 rpm and washed twice with ice-cold PBS and the
lost of mitochondrial membrane potential were investigated by
flow cytometry. The emission fluorescence for JC-1 was monitored
at 530 and 590 nm, under the excitation wavelength at 488 nm,
respectively.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2017.03.031.
4.9. ROS assay
References
The production of ROS was examined by flow cytometry using
DCFH-DA (Molecular Probe, Beyotime, Haimen, China), as previ-
ously described [53]. NCI-H460 cells were grown into six-well
plates and subjected to various treatments. On the following
treatment, cells were collected at 2000 rpm and washed twice with
ice-cold PBS, and then resuspend cells in 10 mM DCFH-DA dis-
solved in cell free medium at 37 ꢀC for 20 min in dark, and then
washed twice with ice-cold PBS. Cellular fluorescence was analyzed
by flow cytometry at an excitation of 485 nm and an emission of
538 nm.
[1] J. Seligmann, C. Twelves, Tubulin: an example of targeted chemotherapy,
Future Med. Chem. 5 (2013) 339e352.
[2] R.A. Stanton, K.M. Gernert, J.H. Nettles, R. Aneja, Drugs that target dynamic
microtubules:
a new molecular perspective, Med. Res. Rev. 31 (2011)
443e481.
[3] Y.V. Voitovich, E.S. Shegravina, N.S. Sitnikov, V.I. Faerman, V.V. Fokin,
H.G. Schmalz, S. Combes, D. Allegro, P. Barbier, I.P. Beletskaya,
E.V. Svirshchevskaya, A.Y. Fedorov, Synthesis and biological evaluation of
furanoallocolchicinoids, J. Med. Chem. 58 (2015) 692e704.
[4] M. Kavallaris, Microtubules and resistance to tubulin-binding agents, Nat. Rev.
Cancer 10 (2010) 194e204.
[5] R. Romagnoli, P.G. Baraldi, M.K. Salvador, D. Preti, M.A. Tabrizi, A. Brancale,
X.H. Fu, J. Li, S.Z. Zhang, E. Hamel, R. Bortolozzi, E. Porcú, G. Basso, G. Viola,
Discovery and optimization of a series of 2-Aryl-4-Amino-5-(30,40,50-trime-
thoxybenzoyl) thiazoles as novel anticancer agents, J. Med. Chem. 55 (2012)
5433e5445.
4.10. Western blot analysis
[6] J.R. McIntosh, E. Grishchuk, R.R. West, Chromosome-microtubule interactions
during mitosis, Annu. Rev. Cell Dev. Biol. 18 (2002) 193e219.
[7] R. Heald, E. Nogales, Microtubule dynamics, J. Cell Sci. 115 (2002) 3e4.
[8] P.B. Schiff, J. Fant, S.B. Horwitz, Promotion of microtubule assembly in vitro by
taxol, Nature 277 (1979) 665e667.
[9] R. Kaur, G. Kaur, R.K. Gill, R. Soni, J. Bariwal, Recent developments in tubulin
polymerization inhibitors: an overview, Eur. J. Med. Chem. 87 (2014) 89e124.
[10] Q.X. Yue, X. Liu, D.A. Guo, Microtubule-binding natural products for cancer
therapy, Planta Med. 76 (2010) 1037e1043.
Western blot analysis was performed as described previously
[52]. NCI-H460 cells were treated with different concentrations of
the test compound 12k for 48 h. After that time, cells were har-
vested, centrifuged, and washed twice with ice-cold PBS. The pellet
was then resuspended in lysis buffer. After the cells were lysed on
ice for 20 min, lysates were centrifuged at 20000 g at 4 ꢀC for 5 min.
The protein concentration in the supernatant was analyzed using
the BCA protein assay reagents (Imgenex, USA). Equal amounts of
protein per line were was separated on 12% SDS polyacrylamide gel
electrophoresis and transferred to PVDF Hybond-P membrane (GE
Healthcare). Membranes were incubated with 5% skim milk in Tris-
buffered saline with Tween 20 (TBST) buffer for 1 h and then the
membranes being gently rotated overnight at 4 ꢀC. Membranes
were then incubated with primary antibodies against Bcl-2, Bax,
[11] M.A. Jordan, L. Wilson, Microtubules as a target for anticancer drugs, Nat. Rev.
Cancer 4 (2004) 253e265.
[12] B. Gigant, C. Wang, R.B. Ravelli, F. Roussi, M.O. Steinmetz, P.A. Curmi, A. Sobel,
M. Knossow, Structural basis for the regulation of tubulin by vinblastine,
Nature 435 (2005) 519e522.
€
[13] J. Lowe, H. Li, K.H. Downing, E. Nogales, Refined structure of ab-tubulin at 3.5
Å resolution, J. Mol. Biol. 313 (2001) 1045e1057.
[14] M.A. Jordan, L. Wilson, Microtubules as a target for anticancer drugs, Nat. Rev.
Cancer 4 (2004) 253e265.
[15] G.C. Wang, C.Y. Li, L. He, K. Lei, F. Wang, Y.Z. Pu, Z. Yang, D. Cao, L. Ma,
J.Y. Chen, Y. Sang, X.L. Liang, M.L. Xiang, A.H. Peng, Y.Q. Wei, L.J. Chen, Design,
synthesis and biological evaluation of a series of pyrano chalcone derivatives
containing indole moiety as novel anti-tubulin agents, Bioorg. Med. Chem. 22
(2014) 2060e2079.
[16] M. Alami, S. Messaoudi, B. Treguier, A. Hamze, O. Provot, J.F. Peyrat, J.R. De
Losada, J.M. Liu, J. Bignon, J. Wdzieczak-Bakala, S. Thoret, J. Dubois, J.D. Brion,
Iso combretastatins A versus combretastatins A: the forgotten iso CA-4 isomer
as a highly promising cytotoxic and antitubulin agent, J. Med. Chem. 52 (2009)
4538e4542.
Cyt c, caspase-9, caspase-3, or b
-actin for overnight at 4 ꢀC. Mem-
branes were next incubated with peroxidase labeled secondary
antibodies for 2 h. Then all membranes were washed with TBST
three times for 15 min and the protein blots were investigated with
chemiluminescence reagent (Thermo Fischer Scientifics Ltd.). The
X-ray films were developed with developer and fixed with fixer
solution.
[17] D.J. Newman, G.M. Cragg, K.M. Snader, Natural products as sources of new
drugs over the period 1981-2002, J. Nat. Prod. 66 (2003) 1022e1037.
[18] B. Zhang, D. Duan, C. Ge, J. Yao, Y. Liu, X. Li, J. Fang, Synthesis of xanthohumol
analogues and discovery of potent thioredoxin reductase inhibitor as poten-
tial anticancer agent, J. Med. Chem. 58 (2015) 1795e1805.
[19] W. Cai, B. Zhang, D. Duan, J. Wu, J. Fang, Curcumin targeting the thioredoxin
system elevates oxidative stress in HeLa cells, Toxicol. Appl. Pharmacol. 262
(2012) 341e348.
4.11. Statistical analysis
All statistical analysis was performed with SPSS Version 10. Data
was analyzed by one-way ANOVA. Mean separations were per-
formed using the least significant difference method. Each experi-
ment was replicated thrice, and all experiments yielded similar
results. Measurements from all the replicates were combined, and
treatment effects were analyzed.
[20] N.K. Sahu, S.S. Balbhadra, J. Choudhary, D.V. Kohli, Exploring pharmacological
significance of chalcone scaffold:
209e225.
a review, Curr. Med. Chem. 19 (2012)
[21] Z. Yang, W. Wu, J. Wang, L. Liu, L. Li, J. Yang, G. Wang, D. Cao, R. Zhang,
M. Tang, J. Wen, J. Zhu, W. Xiang, F. Wang, L. Ma, M. Xiang, J. You, L.J. Chen,
Synthesis and biological evaluation of novel millepachine derivatives as a new
class of tubulin polymerization inhibitors, J. Med. Chem. 57 (2014)
7977e7989.
[22] J. Chen, J. Yan, J. Hu, Y. Pang, L. Huang, X. Li, Synthesis, biological evaluation
and mechanism study of chalcone analogues as novel anti-cancer agents, RSC
Adv. 5 (2015) 68128e68135.
Acknowledgments
We are grateful to the National Natural Science Foundation of
China (Grant No. 21571033) for financial aid to this work. The au-
thors would also like to thank the Fundamental Research Funds for

X. Huang et al. / European Journal of Medicinal Chemistry 132 (2017) 11e25
25
[23] D. Kumar, N.M. Kumar, K. Akamatsu, E. Kusaka, H. Harada, T. Ito, Synthesis and
biological evaluation of indolyl chalcones as antitumor agents, Bioorg. Med.
Chem. Lett. 20 (2010) 3916e3919.
bridging skeleton and aryl moiety, J. Med. Chem. 59 (2016), 3439e345.
[37] J. Wang, J. Yi, Cancer cell killing via ROS: to increase or decrease, that is the
question, Cancer Biol. Ther. 7 (2008) 1875e1884.
[24] M.L. Edwards, D.M. Stemerick, P.S. Sunkara, Chalcones: a new class of anti-
mitotic agents, J. Med. Chem. 133 (1990) 1948e1954.
[25] S. Ducki, D. Rennison, M. Woo, A. Kendall, J.F.D. Chabert, A.T. McGown,
N.J. Lawrence, Combretastatin-like chalcones as inhibitors of microtubule
polymerization. Part 1: synthesis and biological evaluation of anti-vascular
activity, Bioorg. Med. Chem. 17 (2009) 7698e7710.
[26] S.P. Bahekar, S.V. Hande, N.R. Agrawal, H.S. Chandak, P.S. Bhoj, K. Goswami,
M.V.R. Reddy, Sulfonamide chalcones: synthesis and in vitro exploration for
therapeutic potential against Brugia malayi, Eur. J. Med. Chem. 124 (2016)
262e269.
[27] K.H. Jeon, Lee, E.K.Y. Jun, J.E. Eom, S.Y. Kwak, Y. Na, Y. Kwon, Neuroprotective
effect of synthetic chalcone derivatives as competitive dual inhibitors against
m-calpain and cathepsin B through the downregulation of tau phosphoryla-
tion and insoluble Ab peptide formation, Eur. J. Med. Chem. 121 (2016)
433e444.
[38] Y.L. Chu, C.T. Ho, J.G. Chung, R. Raghu, Y.C. Lo, L.Y. Sheen, Allicin induces anti-
human liver cancer cells through the p53 gene modulating apoptosis and
autophagy, J. Agric. Food Chem. 61 (2013) 9839e9848.
[39] S.S. Zhang, S.P. Nie, D.F. Huang, Y.L. Feng, M.Y. Xie, A novel polysaccharide
from ganoderma atrum exerts antitumor activity by activating mitochondria-
mediated apoptotic pathway and boosting the immune system, J. Agric. Food
Chem. 62 (2014) 1581e1589.
[40] J.D. Ly, D.R. Grubb, A. Lawen, The mitochondrial membrane potential (Djm) in
apoptosis: an update, Apoptosis 8 (2003) 115e128.
[41] D.R. Green, G. Kroemer, The pathophysiology of mitochondrial cell death,
Science 305 (2004) 626e629.
[42] X.C. Huang, L. Jin, M. Wang, D. Liang, Z.F. Chen, Y. Zhang, Y.M. Pan, H.S. Wang,
Design, synthesis and in vitro evaluation of novel dehydroabietic acid de-
rivatives containing a dipeptide moiety as potential anticancer agents, Eur. J.
Med. Chem. 89 (2015) 370e385.
[28] T. Kraege, K. Stefan, K. Juvale, T. Ross, T. Willmes, M. Wiese, The combination
of quinazoline and chalcone moieties leads to novel potent heterodimeric
modulators of breast cancer resistance protein (BCRP/ABCG2), Eur. J. Med.
Chem. 117 (2016) 212e229.
[29] A. Hammuda, R. Shalaby, S. Rovida, Edmondson, D.E.C. Binda, A. Khalil, Design
and synthesis of novel chalcones as potent selective monoamine oxidase-B
inhibitors, Eur. J. Med. Chem. 114 (2016) 162e169.
[30] W. Wu, H. Ye, L. Wan, X. Han, G. Wang, J. Hu, M. Tang, X. Duan, Y. Fan, S. He,
L. Huang, H. Pei, X. Wang, X. Li, C. Xie, R. Zhang, Z. Yuan, Y. Mao, Y. Wei,
L.J. Chen, Millepachine, a novel chalcone, induces G2/M arrest by inhibiting
CDK1 activity and causing apoptosis via ROS-mitochondrial apoptotic
pathway in human hepatocarcinoma cells in vitro and in vivo, Carcinogenesis
34 (2013) 1636e1643.
[31] G. Wang, W. Wu, F. Peng, D. Cao, Z. Yang, L. Ma, N. Qiu, H. Ye, X. Han, J. Chen,
J. Qiu, Y. Sang, X. Liang, Y. Ran, A. Peng, Y. Wei, L.J. Chen, Design, synthesis, and
structure-activity relationship studies of novel millepachine derivatives as
potent antiproliferative agents, Eur. J. Med. Chem. 54 (2012) 793e803.
[32] G. Wang, F. Peng, D. Cao, Z. Yang, X. Han, J. Liu, W. Wu, L. He, L. Ma, J. Chen,
Y. Sang, M. Xiang, A. Peng, Y. Wei, L.J. Chen, Design, synthesis and biological
evaluation of millepachine derivatives as a new class of tubulin polymeriza-
tion inhibitors, Bioorg. Med. Chem. 21 (2013) 6844e6854.
[33] B.J. An, S. Zhang, J. Yan, L. Huang, X.S. Li, Synthesis, in vitro and in vivo eval-
uation of new hybrids of millepachine and phenstatin as potent tubulin
polymerization inhibitors, Org. Biomol. Chem. 15 (2017) 852e862.
[34] S. Ducki, R. Forrest, J.A. Hadfield, A. Kendall, N.J. Lawrence, A.T. McGown,
D. Rennison, Potent antimitotic and cell growth inhibitory properties of
substituted chalcones, Bioorg. Med. Chem. Lett. 8 (1998) 1051e1056.
[35] R. Schobert, B. Biersack, A. Dietrich, S. Knauer, M. Zoldakova, A. Fruehauf,
T. Mueller, Pt(II) complexes of a combretastatin A-4 analogous chalcone: ef-
fects of conjugation on cytotoxicity, tumor specificity, and long-term tumor
growth suppression, J. Med. Chem. 52 (2009) 241e246.
[43] A. Thorburn, Death receptor-induced cell killing, Cell. Signal. 16 (2004)
139e144.
[44] E. Er, L. Oliver, P.F. Cartron, P. Juin, S. Manon, F.M. Vallette, Mitochondria as the
target of the pro-apoptotic protein Bax, Biochim. Biophys. Acta-Bioenergetics
1757 (2006) 1301e1311.
[45] X.C. Huang, R.Z. Huang, Z.X. Liao, Y.M. Pan, S.H. Gou, H.S. Wang, Synthesis and
pharmacological evaluation of dehydroabietic acid thiourea derivatives con-
taining bisphosphonate moiety as an inducer of apoptosis, Eur. J. Med. Chem.
108 (2016) 381e391.
[46] X. Jiang, X. Wang, Cytochrome C-mediated apoptosis, Annu. Rev. Biochem. 73
(2004) 87e106.
[47] J.S. Bose, V. Gangan, R. Prakash, S.K. Jain, S.K. Manna, A dihydrobenzofuran
lignan induces cell death by modulating mitochondrial pathway and G2/M
cell cycle arrest, J. Med. Chem. 52 (2009) 3184e3190.
[48] S.S. Zhang, S.P. Nie, D.F. Huang, Y.L. Feng, M.Y. Xie, A novel polysaccharide
from ganoderma atrum exerts antitumor activity by activating mitochondria-
mediated apoptotic pathway and boosting the immune system, J. Agric. Food
Chem. 62 (2014) 1581e1589.
[49] T. Kuwana, D.D. Newmeyer, Bcl-2-family proteins and the role of mitochon-
dria in apoptosis, Curr. Opin. Cell Biol. 15 (2003) 691e699.
[50] M.A. Jordan, L. Wilson, Microtubules as a target for anticancer drugs, Nat. Rev.
Cancer 4 (2004) 253e265.
[51] A.V. Schofield, C. Gamell, R. Suryadinata, B. Sarcevic, O. Bernard, Tubulin
polymerization promoting protein
1 (Tppp1) phosphorylation by rho-
associated coiled-coil kinase (rock) and cyclin-dependent kinase 1 (Cdk1)
inhibits microtubule dynamics to increase cell proliferation, J. Biol. Chem. 288
(2013) 7907e7917.
[52] Y.J. Qin, Y.J. Li, A.Q. Jiang, M.R. Yang, Q.Z. Zhu, H. Dong, H.L. Zhu, Design,
synthesis and biological evaluation of novel pyrazoline containing derivatives
as potential tubulin assembling inhibitors, Eur. J. Med. Chem. 94 (2015)
447e457.
[36] V. Chaudhary, J.B. Venghateri, H.P.S. Dhaked, A.S. Bhoyar, S.K. Guchhait,
D. Panda, Novel Combretastatin-2-aminoimidazole analogues as potent
tubulin assembly inhibitors: exploration of unique pharmacophoric impact of
[53] J.Y. Zhang, T. Yi, J. Liu, Z. Zhao, H.B. Chen, Quercetin induces apoptosis via the
mitochondrial pathway in KB and KBv200 cells, J. Agric. Food Chem. 61 (2013)
2188e2195.