Discovery of 3-phenylquinolinylchalcone derivatives as potent and selective anticancer agents against breast cancers

Article abstract of DOI:10.1016/j.ejmech.2015.04.054

A number of 3-phenylquinolinylchalcone derivatives were synthesized and evaluated in vitro for their antiproliferative activities against three breast cancer cell lines (MCF-7, MDA-MB-231, and SKBR-3), and a non-cancer normal epithelial cell line (H184B5F

Full text of DOI:10.1016/j.ejmech.2015.04.054

European Journal of Medicinal Chemistry 97 (2015) 306e319
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Discovery of 3-phenylquinolinylchalcone derivatives as potent and
selective anticancer agents against breast cancers
,
,
,
Chih-Hua Tseng a b, Cherng-Chyi Tzeng b c, Chih-Yao Hsu b c, Chih-Mei Cheng d,
Chia-Ning Yang e **, Yeh-Long Chen b
a School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung City 807, Taiwan
,
, c, *
b Research Center for Natural Products & Drug Development, Kaohsiung Medical University, Kaohsiung City 807, Taiwan
c Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung City 807, Taiwan
d Department of Biomedical Science and Environmental Biology, College of Life Science, Kaohsiung Medical University, Kaohsiung City 807, Taiwan
e Department of Life Science, National University of Kaohsiung, 700 Kaohsiung University Rd, Kaohsiung, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
A number of 3-phenylquinolinylchalcone derivatives were synthesized and evaluated in vitro for their
antiproliferative activities against three breast cancer cell lines (MCF-7, MDA-MB-231, and SKBR-3), and a
non-cancer normal epithelial cell line (H184B5F5/M10). Among them, (E)-3-[3-(4-methoxyphenyl)qui-
nolin-2-yl]-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (7) was active against the growth of MCF-7,
Received 10 March 2015
Received in revised form
23 April 2015
Accepted 26 April 2015
Available online 30 April 2015
MDA-MB-231, and SKBR-3 with IC50 values of 1.05, 0.75, and 0.78 mM respectively without significant
cytotoxicity to the normal H184B5F5/M10 cell line and therefore, was selected as a new lead for further
mechanism studies. Results indicated that compound 7 inhibited the polymerization of tubulins, induced
G2/M cell cycle arrest via modulation of the cyclin B1, cdk1 and CDC25. Compound 7 ultimately induced
cell apoptosis by the increase of apoptotic protein Bax and the decrease of anti-apoptotic protein Bcl-2. In
addition, PARP was cleaved while caspase-3 and -8 activities were induced after the treatment of
compound 7 for 24 h in a concentration-dependent manner. Thus, compound 7 induces cell cycle arrest
at G2/M phase via cleavage of PARP, induces caspase-3 and -8 activities and consequently to cause the
cell death. Further study on the structure optimization of 7 is ongoing.
Keywords:
3-Phenylquinolinylchalcone derivatives
Anticancer agents
Cell cycle
Apoptosis
Breast cancers
© 2015 Elsevier Masson SAS. All rights reserved.
1. Introduction
which the cis double bond was replaced with quinoline to arrest the
cis-conformation [7]. Among them, 6-fluoro-2,3-bis{4-[2-(piperidin-
Combretastatins-A4 (CA-4) is an effective antimitotic agent pos-
sessing potent anticancer activity against a panel of cancer cells,
including multi-drug resistant cancer cell lines [1e6]. However, only
the cis-form exhibits strong antitubulin activity, but the cis-form
tends to isomerize to its inactive trans-isomer. Thus, a number of CA-
4 analogs were designed and synthesized in which the ethylene
bridge was replaced with various heterocyclic rings with objections
to overcome the spontaneous isomerization of cis-form to trans-
isomer. Recently, we have synthesized certain CA-4 analogs in
1-yl)ethoxy]phenyl}quinoline (1) which possesses the aminoalkyl
side chain, was one of the most active derivative against the growth
of Hep3B, H1299, and MDA-MB-231 with IC50 values of 0.71, 1.46,
and 0.72
certain 3-phenylquinolinylchalcone derivatives whose structures
can be considered as CA-4 analogs in which a -unsaturated
mM respectively. Furthermore, we have also synthesized
a,b
carbonyl moiety is inserted between quinoline and the C-2 phenyl
group [8]. They can also be considered as chalcone derivatives [9e17]
in which an aryl moiety was replaced with quinoline nucleus.
Among these 3-phenylquinolinylchalcone derivatives, (E)-3-[3-(4-
methoxyphenyl)quinolin-2-yl]-1-phenylprop-2-en-1-one (2) was
active against the growth of H1299 and SKBR-3 with IC50 values of
* Corresponding author. Department of Medicinal and Applied Chemistry,
Kaohsiung Medical University, Kaohsiung City 807, Taiwan.
** Corresponding author. Department of Biomedical Science and Environmental
Biology, College of Life Science, Kaohsiung Medical University, Kaohsiung City 807,
Taiwan.
1.41 and 0.70
mM respectively which was more active than the
positive topotecan [8]. The present report intends to establish
the antiproliferative structureeactivity relationships (SAR) of
3-phenylquinolinylchalcone derivatives and to identify more po-
tential anticancer drug candidates by the introduction of various
E-mail addresses: cnyang@nuk.edu.tw (C.-N. Yang), yeloch@kmu.edu.tw
(Y.-L. Chen).
http://dx.doi.org/10.1016/j.ejmech.2015.04.054
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

C.-H. Tseng et al. / European Journal of Medicinal Chemistry 97 (2015) 306e319
307
substituents, such as trimethoxy group and aminoalkyl side chains,
on the lead compound 2 (Fig. 1). Introduction of aminoalkyl side
chains on the furoquinoline pharmacophore of anticancer CIL-102
[18e21] to improve anticancer profiles, i.e., compound 3, has also
followed by the addition of 4 under basic conditions afforded com-
pound 10. Preparation of compounds 3, 5, and 6 were previously
described [8].
Pfitzinger reaction of 4-methoxyphenylacetone with 5-
fluoroisatin (11) and 5-methoxyisatin (12) afforded 6-fluoro-3-(4-
methoxyphenyl)-2-methylquinoline-4-carboxylic acid (13) and its
6-methoxy analog 14 respectively as described in Scheme 2. Com-
pounds 13 and 14 respectively, were subjected to thermal decar-
boxylation followed by oxidation with selenium oxide to afford
their respective aldehydes 17 and 18. The desired quinoli-
nylchalcones 19aeg and 20aeg were synthesized by a base cata-
lyzed ClaiseneSchmidt condensation reaction of 17 and 18
respectively with appropriately substituted acetophenones. This
method is attractive since it specifically generates solely (E)-form
isomer. From 1H NMR spectra, all quinolinylchalcones were found
to be trans based on the coupling constant between the vinylic
system of the propenone moiety (J ¼ 14.8e15.2 Hz) [8].
Compound 7 was further converted to its epoxy-chalcone de-
rivative 21 by the treatment of H2O2 in alkaline solution, cyclo-
propane derivative 22 by Corey-Chaykovsky cyclopropanation
(TMSOI/NaOH(aq) in TBAB as phase transfer catalyst), and saturated
dihydro derivative 23 by hydrogenation (H2, Pd/C) (Scheme 3).
These compounds were also evaluated for their antiproliferative
activities.
been previously
reported [22]. These substituted 3-
phenylquinolinylchalcone derivatives were evaluated in vitro
against three breast cancer cell lines (MCF-7, MDA-MB-231, and
SKBR-3) which are common malignancies in the world, and espe-
cially are the leading cause of cancer deaths in Asian countries
including Taiwan [23e27]. The normal mammary epithelial cell
(H184B5F5/M10) was also evaluated to identify potential anticancer
drug candidates which inhibit only the growth of cancer cells but not
normal mammary cells.
2. Chemistry
ClaiseneSchmidt condensation reaction of 3-(4-methoxyphenyl)
quinolin-2-carbaldehyde (4) and 3,4,5-trimethoxyacetophenones
gave
(E)-3-[3-(4-methoxyphenyl)quinolin-2-yl]-1-(3,4,5-
trimethoxyphenyl)prop-2-en-1-one (7) as outlined in Scheme 1.
Compounds 8 and 9 were synthesized by the treatment of 4 with 4-
[2-(piperidin-1-yl)ethoxy]acetophenone [28] and 4-[2-(pyrrolidin-
1-yl)ethoxy]acetophenone
respectively.
Reaction
of
4-
hydroxyacetophenone and 3-chloro-N,N-dimethylpropanamine
Fig. 1. Structures of CA-4, 6-fluoro-2,3-bis{4-[2-(piperidin-1-yl)ethoxy]phenyl}quinoline (1), (E)-3-[3-(4-methoxyphenyl)quinolin-2-yl]-1-phenylprop-2-en-1-one (2), chalcone,
CIL-102, and 3.
Scheme 1. Reagents and conditions: (i) substituted acetophenone, KOH, EtOH, rt, 2hr.

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C.-H. Tseng et al. / European Journal of Medicinal Chemistry 97 (2015) 306e319
Scheme 2. Reagents and conditions: (i) 4-methoxyphenylacetone, KOH, EtOH, 80 ꢀC, 48hr; (ii) Dowtherm A, 280 ꢀC, 4hr; (iii) SeO2, 100 ꢀC, 2hr; (iv) substituted acetophenone, KOH,
EtOH, rt, 2hr.
3. Results and discussion
3.1. Inhibition of cell proliferation
All the synthesized 3-phenylquinolinylchalcone derivatives
were evaluated in vitro against three breast cancer cells (MCF-7,
MDA-MB-231, and SKBR-3), and a non-cancer normal epithelial cell
line (H184B5F5/M10) using XTT (sodium 30-[1-(phenylamino-
carbonyl)-3,4-tetrazolium}-bis(4-methoxy-6-nitro)benzene
sul-
fonic acid hydrate) assay [29]. The concentration that inhibited the
growth of 50% of cells (IC50) was determined from the linear
portion of the curve by calculating the concentration of tested
agent that reduced absorbance in treated cells, compared to control
cells, by 50%. The IC50 results of these 3-phenylquinolinylchalcone
derivatives are summarized in Table 1. Although our first
lead compound, (E)-3-[3-(4-methoxyphenyl)quinolin-2-yl]-1-
phenylprop-2-en-1-one (2) [8] was active against the growth of
MDA-MB-231 and SKBR-3 with IC50 values of 0.86 and 0.70 mM
respectively, it was also exhibited a weak cytotoxicity to the non-
cancer normal epithelial cell line (H184B5F5/M10) with an IC50
values of 8.56 mM. Introduction of the methoxy i.e. compound 5 or
hydroxyl substituent i.e. compound 6 on the para-position of C-2
aryl moiety decreased the antiproliferative activity against all the
cancer cells tested. However, aminoalkoxy derivatives 8, 9, and 10
exhibited comparable or even more potent antiproliferative activ-
ities than that of the parent compound 2 against all cell lines tested.
Although 3,4,5-trimethoxy derivative 7 was more active than 2
against the growth of MCF-7 (1.05 v.s. 4.04
against the normal mammary epithelial cell (H184B5F5/M10) with
an IC50 value of >10 M.
mM), it was not cytotoxic
m
For the 6-fluoro substituted derivatives, 19aeg, the anti-
proliferative activity decreased in an order of 19e, 19f,
19g > 19a > 19b, 19c > 19d indicated that the introduction of
Scheme 3. Reagents and conditions: (i) H2O2, MeOH, K2CO3, rt, 3hr; (ii) TMSOI, TBAB,
NaOH; (iii) H2, Pd/C, 3hr.

C.-H. Tseng et al. / European Journal of Medicinal Chemistry 97 (2015) 306e319
309
Table 1
cell cycle arrest in the G2/M phase. In the presence of compound
7 at 1 M and 5 M for 12 h, 28% and 36% respectively of the cells
Antiproliferative activities of 3-phenylquinolinylchalcones (IC50, mM).
m
m
were also arrested in G2/M phase followed by apoptosis as indi-
cated by a concentration- and time-dependent increase of cells in
the sub G1 phase, suggesting that compound 7 affects tubulin
polymerization. Significant morphological changes were also
observed by the incubation of MDA-MB-231 cells with increasing
concentrations of compound 7 for 24 h as shown in Fig. 3.
Compd
Cell lines
MCF-7
MDA-MB-231
SKBR-3
H184B5F5/M10
2
5
6
7
8
9
10
19a
19b
19c
19d
19e
19f
19g
20a
20b
20c
20d
20e
20f
20g
21
4.04 0.18
7.38 0.12
6.71 0.07
1.05 0.22
0.85 0.11
0.74 0.06
0.81 0.16
4.33 0.03
9.72 2.41
6.75 0.25
>10
0.78 0.09
0.76 0.09
0.71 0.06
7.53 0.29
>10
0.86 0.07
7.54 0.05
6.35 0.09
0.75 0.05
0.68 0.02
0.76 0.03
0.70 0.22
0.76 0.11
8.03 0.15
8.38 0.37
>10
0.74 0.13
0.64 0.09
0.65 0.05
7.92 0.09
8.76 0.42
6.88 0.12
>10
0.70 0.14
9.28 0.04
3.88 0.11
0.78 0.07
0.83 0.04
0.91 0.07
0.80 0.05
0.91 0.07
8.80 0.11
8.31 0.27
7.66 0.05
0.78 0.14
0.80 0.14
0.73 0.11
9.36 0.16
10.22 0.15
9.35 0.25
7.62 0.03
0.37 0.03
0.82 0.12
0.41 0.12
>10
8.56 0.32
>10
5.69 0.13
>10
4.66 0.16
3.63 0.12
7.67 0.11
10.10 0.85
>10
3.3. Compound 7 caused disruption of microtubule networks
G2/M cell cycle arrest was extensively reported to occur in
apoptosis induced by microtubule-interfering agents [30e32]. Due
to the similar pharmacological mechanisms of compound 7 and
CIL-102, we hypothesized that compound 7 might also inhibit
tubulin polymerization. To assure this hypothesis, microtubule or-
ganization and mitosis in response to compounds 7 and 23 (as a
negative control) treated cells were initially examined by immu-
nofluorescent microscopy. After the treatment of compound 7, cells
were found to exhibit the filament-like structure and reduce
microtubule extent in the cytoplasm (Fig. 4). The effect of com-
pound 7 on the dynamics of microtubule assembly was also
examined at the cellular level. As shown in Fig. 5, the inhibited
polymerization of tubulin was observed in MDA-MB-231 cells
9.87 0.15
>10
7.13 0.17
2.08 0.26
7.39 0.38
10.04 0.61
>10
8.35 0.17
>10
8.41 0.13
6.80 0.18
8.82 0.71
>10
6.71 0.06
>10
0.77 0.05
0.76 0.11
1.02 0.07
>10
0.66 0.04
0.72 0.03
0.67 0.15
9.05 0.88
>10
22
23
Topo
Taxol
CIL-102
>10
>10
>10
>10
8.91 3.03
0.10 0.05b
ND
>10
>10
>10
treated with compound 7 or CIL-102 (10 mM) for 24 h compared
with the control cells. Compound 7 showed a decrease in the
polymer form of tubulin in a dose dependent manner.
5.97 1.03
<0.1
>10
0.11a
0.41a
NDc
0.31 0.07
ND
0.95 0.16
a
Ref. [30].
Ref. [31].
No data.
b
c
3.4. Compound 7 induced apoptosis through regulation of Bcl-2
proteins and caspase activation
aminoalkoxy side chains increased while methoxy or hydroxyl
group decreased antiproliferative activity especially trimethoxy
group which was inactive against all cell lines tested. The same
structureeactivity relationship (SAR) was observed for the 6-
methoxy substituted derivatives, 20aeg, in which the anti-
proliferative activity decreased in an order of aminoalkoxy de-
rivatives 20e, 20f, 20g > unsubstituted derivative 20a > methoxy
20b or hydroxyl derivatives 20c > trimethoxy derivative 20d. Less
antiproliferative activity of 20a than that of 2 indicated the sub-
stitution at C-6 position is unfavorable especially the electron-
donating group such as OMe.
In order to assure compound 7-mediated inhibition of cell
growth is due to the induction of apoptosis, we analyzed the effect
of compound 7 on the expression of the Bcl-2 family, the anti-
apoptotic Bcl-2 and the proapoptotic Bad and Bax. The proteins of
the Bcl-2 family play a major role in controlling apoptosis through
the regulation of mitochondrial processes and the release of
mitochondrial proapoptotic molecules important for the cell death
pathway [33]. As shown in Fig. 6, compound 7 did not significantly
affect the expression of the apoptotic protein Bad, however, it
increased the apoptotic protein Bax and decreased the anti-
apoptotic protein Bcl-2. To determine whether compound 7 pref-
erentially kills cancer cells by apoptosis, procaspase-3, procaspase-
8, and PARP were evaluated in compound 7-treated MDA-MB-231
cells by western blotting. Caspases are the family of cysteine pro-
teases and they mediate apoptotic pathway in mammalian [34].
PARP, a nuclear poly (ADP-ribose) polymerase, is involved in DNA
repair predominantly in response to environmental stress, and is
important for the maintenance of cell viability [35]. Our results
have shown that PARP was cleaved while caspase-3 and -8 activ-
ities were induced after the treatment of compound 7 for 24 h in a
concentration-dependent manner (Fig. 6). Thus, compound 7 in-
duces cell cycle arrest at G2/M phase via cleavage of PARP, induces
caspase-3 and -8 activities, and consequently caused the cell death.
Among these newly synthesized 3-phenylquinolinylchalcone
derivatives,
(E)-3-(3-(4-methoxyphenyl)quinolin-2-yl)-1-(3,4,5-
trimethoxyphenyl)prop-2-en-1-one (7) was active against the
growth of MCF-7, MDA-MB-231, and SKBR-3 with IC50 values of
1.05, 0.75, and 0.78 mM respectively without significant cytotoxicity
to the normal H184B5F5/M10cell line. Thus, compound 7 was
selected as a lead compound and was further converted to its
ethylene oxide derivative 21, cyclopropane derivative 22, and
saturated dihydro derivative 23 for antiproliferative evaluation. Our
results indicated compounds 21e23 were inactive, implied that the
unsaturated ketone is crucial for antiproliferative activities. Com-
pound 7 was then selected for further mechanism studies due to its
potent inhibition against the growth of cancer cells but was non-
cytotoxic to the normal H184B5F5/M10 cell line.
3.5. Modulation of the expression of cell cycle-regulatory proteins
3.2. Compound 7 induced G2/M cell cycle arrest and morphological
changes
Molecular analysis of human cancers has revealed that cell cycle
regulators are frequently mutated in most common malignancies
[36]. Cdc25 is sequestered in the cytoplasm by 14-3-3 proteins,
which prevents activation of cyclin B1/CDK1 by Cdc25 and results
in G2/M arrest [37]. Western blot analysis showed that the treat-
ment of cells with compound 7 resulted in a significant dose-
dependent decrease in the protein levels of CDC25, cdk1, and
cyclin B1 (Fig. 7). These results suggested that compound 7
Cell cycle analysis of compound 7 was performed with MDA-
MB-231 cells. CIL-102, a known inhibitor of tubulin polymeriza-
tion, was used as a positive control. Compound 7 induced cell cycle
arrest in a concentration- and time-dependent manners as shown
in Fig. 2 and Table 2. The presence of 10 mM of CIL-102 for 12 h led to

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C.-H. Tseng et al. / European Journal of Medicinal Chemistry 97 (2015) 306e319
Fig. 2. Flow cytometric analysis of MDA-MB-231 cells. MDA-MB-231 cells were treated with DMSO (A), compound 7 at 1 mM (B), 5 mM (C) or 10 mM (D) or CIL-102 at 10 mM (E) for
the indicated times the cells and analyzed for propidium iodide-stained DNA content by flow cytometry. The percentage of cells in each cell cycle phase was quantified (Table 2).
Table 2
Effects of 7 and CIL-102 on MDA-MB-231 cell cycle progression.
Times (hr)
12
compd
Concentration (
m
M)
Cell cycle distribution (%)a
Sub G1
G1
S
G2/M
7
DMSO
1
5
10
10
DMSO
1
1.7 0.6
2.6 1.2
4.5 3.1
10.1 3.8
8.8 3.7
2.2 0.8
6.8 2.3
13.2 2.7
21.9 4.2
11.2 2.8
2.7 1.1
10.5 3.8
19.8 2.8
27.8 4.2
12.8 1.5
67.6 3.2
52.3 2.8
41.8 4.8
45.3 2.1
36.5 3.3
69.8 2.1
42.5 4.8
43.5 2.6
32.4 2.4
3.9 1.2
68.7 2.1
49.8 2.0
38.2 4.8
26.2 2.7
3.8 1.1
13.3 2.5
16.8 3.2
17.0 1.9
14.8 2.7
20.1 2.8
15.8 1.9
14.6 3.5
11.5 2.7
12.8 1.7
5.8 2.1
14.8 1.9
13.8 2.7
17.9 2.5
14.7 2.6
3.2 1.8
17.4 1.8
28.3 1.7
36.7 3.3
29.8 1.5
34.6 1.7
14.4 2.9
36.1 3.6
31.8 3.5
32.9 2.6
79.1 4.7
13.8 1.9
25.9 4.1
24.1 3.6
31.3 1.8
80.2 3.9
CIL-102
7
24
36
5
10
10
DMSO
1
5
10
10
CIL-102
7
CIL-102
regulated G2/M cell cycle arrest via modulation of the cyclin B1,
cdk1 and CDC25.
The docking scores in the GTP site column suggest that GTP
(Libdock score ¼ 155.3) is superior to the studied compounds
(Libdock scores ranging from 119.7 to 124.2) when filling in the
GTP binding pocket. In contrast, the docking scores in the
colchicine binding site column shows that the studied com-
pounds (Libdock scores ranging from 117.9 to 134.0) can fit better
into colchicine binding site than colchicine does (Libdock
3.6. Molecular docking of 3-phenylquinolinylchalcone derivatives
Listed in Table 3 are the docking scores of the studied com-
pounds accommodated in the GTP and colchicine binding sites.

C.-H. Tseng et al. / European Journal of Medicinal Chemistry 97 (2015) 306e319
311
Fig. 3. Induction of morphological change in MDA-MB-231 cells. Cells were treated with DMSO or compound 7 (1e10 m
M) for 24 h at 37 ꢀC and photographed under a microscope.
Fig. 4. Microtubule effect of compound 7. The confocal laser scanning micrograph shows a merged image double-labeled with DAPI and
were incubated with DMSO (A), compound 7 at 1 M (B), 5 M (C) 10 M (D), CIL-102 at 1 M (E) or 23 at 10 M (F) for 24 h. The cellular microtubule network was analyzed by an
Olympus FV10i laser confocal system using monoclonal anti- -tubulin antibody, FITC -conjugated mouse anti-human antibody, and DAPI as described in Section 5. Scale bar, 10 m.
a-tubulin antibodies. MDA-MB-231 cells
m
m
m
m
m
a
m
score ¼ 98.9). Furthermore, the correlation coefficient is positive
(0.876) when the docking scores and pIC50 values (in H1299
column, Table 3) in colchicine binding site are plotted in Fig. 8(a)
for linear relationship, whereas the correlation coefficient is
negative (ꢁ0.737) for the GTP site docking scores against the
pIC50 values (in H1299 column, Table 3) plotted in Fig. 8(b).
Accordingly, we conclude that the studied compounds are more
likely to bind to the colchicine binding site. Shown in Fig. 9aee
are docking results of compounds 7 and 20d, respectively.
Leu255 provides aliphatic-
p interaction with the quinoline
moiety in compound 7. Such an interaction is also found in
compound 20d, but is on the 3-(4-methoxyphenyl) moiety due to
the docking position shift caused by the 6-methoxy group
attached on the quinoline. In compound 7, the positively charged
Fig. 5. Effect of 7 on tubulin polymerization in MDA-MB-231 cells. Cytosolic (soluble
tubulin) and cytoskeletal (polymerized tubulin) tubulin fractions were extracted from
Hep3B cells treated with compound 7 (1e10
mM) for 24 h. CIL-102 (10 mM) was
amino acid residue Lys352 provides cation-
p interaction on the
included as controls. Cells were lysed to separate cytosolic and cytoskeletal fractions as
described in Materials and methods. Both polymerized tubulin and unpolymerized
ones were loaded on SDS-PAGE. After electrophoresis and transfer to PVDF membrane,
3,4,5-trimethoxyphenyl moiety. Although such an interaction is
not present in compound 20d docking result, with a refined
simulation where the structural flexibility are conferred upon the
amino acid residues surrounding the docked compound,
a-tubulin was visualized by Western blot analysis. Western blot data presented are
representative of those obtained in at least three separate experiments.

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C.-H. Tseng et al. / European Journal of Medicinal Chemistry 97 (2015) 306e319
Fig. 6. Effects of apoptosis-associated proteins levels in MDA-MB-231 cells. Cells were
cultured with DMSO or 1e10 M of 7 for 24 h. Then, the cells were harvested and lysed
m
for the detection of protein expression with specific antibody by Western blot analysis.
Western blot data presented are representative of those obtained in at least three
separate experiments.
Fig. 8. The correlations of activity (pIC50) versus docking score (Libdockscore) at col-
chicines binding site (a) and GTP site (b).
compound 20d can be docked into colchicine binding site in the
better manners so that the cation-
p interaction may show.
Because compound 21 is racemic mixture (labeled as 21x and
21y), we also investigated the preference of (2S, 3R)-21x or (2R,
3S)-21y form to the colchicine binding site. As shown in Figs. S1
and S2, compared to compound 21x, compound 21y has more
interaction when binding to colchicine site, in accordance with
the docking scores 123.1 and 118.0 for 21y and 21x, respectively.
Fig. 7. Effect of 7 on the expression of cell cycle regulators. MDA-MB-231 cells were
incubated in the vehicle (DMSO) or 7 (1e10 mM) for 24 h. Then, the cells were har-
vested and lysed for the detection of protein expression with specific antibody by
4. Conclusion
Western blot analysis. The data are representative of three independent experiments.
A
number of 3-phenylquinolinylchalcone derivatives were
synthesized and evaluated in vitro for their antiproliferative activ-
ities. Among them, compound 7 was active against the growth of
MCF-7, MDA-MB-231, and SKBR-3 with IC50 values of 1.05, 0.75, and
Table 3
0.78 mM respectively without significant cytotoxicity to the normal
The tubulin docking results of GTP binding site and CN binding site.
cells and therefore, was selected as a new lead for further mecha-
nism studies. Results indicated that compound 7 inhibited the
polymerization of tubulins, induced G2/M cell cycle arrest via
modulation of the cyclin B1, cdk1 and CDC25. Compound 7 ulti-
mately induced cell apoptosis by the increase of apoptotic protein
Bax and the decrease of anti-apoptotic protein Bcl-2. In addition,
PARP was cleaved while caspase-3 and -8 activities were induced
after the treatment of compound 7 for 24 h in a concentration-
dependent manner (Fig. 10).
Name
Libdockscore
GTP site
pIC50
CN site
MRC-5
H1299
7
119.6
116.5
122.2
124.2
155.3
e
133.9
129.5
118.0
123.1
e
5.12
5.32
5.16
5.16
e
6.12
6.02
5.10
5.10
e
20d
21x
21y
GTP
CN
98.9
e
e

C.-H. Tseng et al. / European Journal of Medicinal Chemistry 97 (2015) 306e319
313
Fig. 9. (a) The tubulin structure (PDB ID: 1SA0). The GTP, colchicine, and GDP were labeled by green, yellow, and blue, respectively. (b and d) The docking pose of 7 and 20d. (c and e)
The docking pose of 7 and 20d were showed by 2D diagram.

314
C.-H. Tseng et al. / European Journal of Medicinal Chemistry 97 (2015) 306e319
Fig. 10. A proposed model illustrates the molecular mechanism and the overall possible signaling pathways for 7-induced G2/M arrest and apoptosis in MDA-MB-231 cells.
5. Experimental
50.0e50.4 ꢀC (EtOH). 1H NMR (400 MHz, CDCl3): 1.44e1.47 (m, 2H,
piperidinyl-H), 1.58e1.63 (m, 4H, piperidinyl-H), 2.51e2.52 (m, 4H,
piperidinyl-H), 2.79 (t, 2H, J ¼ 6.0 Hz, OCH2CH2), 3.86 (s, 3H, OMe),
4.17 (t, 2H, J ¼ 6.0 Hz, OCH2CH2), 6.95e7.03 (m, 4H, 30 and 300-H),
7.33e7.36 (m, 2H, 20-H), 7.52e7.56 (m, 1H, 6-H), 7.70e7.74 (m, 1H,
7-H), 7.79 (d, 1H, J ¼ 8.0 Hz, 5-H), 7.99 (d, 1H, J ¼ 15.2 Hz, 2-CH]
CHCO), 8.06 (s, 1H, 4-H), 8.09e8.13 (m, 2H, 200-H), 8.18 (d, 1H,
J ¼ 8.4 Hz, 8-H), 8.38 (d, 1H, J ¼ 15.2 Hz, 2-CH]CHCO). 13C NMR
(100 MHz, CDCl3): 24.00, 25.75 (2C), 54.94 (2C), 55.26, 57.60, 66.06,
114.07 (2C), 114.29 (2C), 127.24, 127.25, 127.37, 128.08, 129.47,
129.62, 130.38, 130.81, 130.94 (2C), 131.01 (2C), 136.08, 136.78,
140.38, 147.07, 151.60, 159.43, 162.74, 188.38. ESI (m/z) 493.09
[Mþ1]þ. Anal. calcd for C32H32N2O3$0.25H2O: C 77.18, H 6.60, N
5.62; found C 76.99, H 6.76, N 5.35. Purity: 98.99% (tr ¼ 6.10 min).
5.1. General
Melting points were determined on a Electrothermal IA9100
melting point apparatus and are uncorrected. Nuclear magnetic
resonance (1H and 13C) spectra were recorded on a Varian-Unity-
400 spectrometer. Chemical shifts were expressed in parts per
million (d) with tetramethylsilane (TMS) as an internal standard.
Thin-layer chromatography (TLC) was performed on silica gel 60 F-
254 plates purchased from E. Merck and Co.. ESI-MS were recorded
on a Bruker Daltonics Apex II 30e. The elemental analyses were
performed in the Instrument Center of National Science Council at
National Cheng-Kung University and National Taiwan University
using Heraeus CHNeO Rapid EA, and all values are within 0.4% of
the theoretical compositions. Purity analysis of compound was
detected by high performance liquid chromatography (HPLC) sys-
tem. The HPLC (Hitachi) consisted of an L-2100 pump, L-2200
autosampler, L-2420 UVevis detector and the analysis was carried
5.1.3. (E)-3-[3-(4-Methoxyphenyl)quinolin-2-yl]-1-{4-[2-
(pyrrolidin-1-yl)-ethoxy]phenyl}-prop-2-en-1-one (9)
Compound 9 was obtained from 4 and 4-[2-(piperidin-1-yl)
ethoxy]acetophenone [28] as described for 7 in 63% yield. Mp.
106.6e107.7 ꢀC (EtOH). 1H NMR (400 MHz, CDCl3): 2.16 (br s, 4H,
pyrrolidinyl-H), 3.42 (br s, 4H, pyrrolidinyl-H), 3.52 (t, 2H,
J ¼ 5.6 Hz, OCH2CH2), 3.89 (s, 3H, OMe), 4.62 (t, 2H, J ¼ 5.6 Hz,
OCH2CH2), 6.99e7.06 (m, 4H, 300-H and 30-H), 7.35e7.38 (m, 2H, 20-
H), 7.55e7.59 (m, 1H, 6-H), 7.73e7.77 (m, 1H, 7-H), 7.83 (d, 1H,
J ¼ 8.0 Hz, 5-H), 8.00 (d, 1H, J ¼ 15.2 Hz, 2-CH]CHCO), 8.11e8.14
(m, 3H, 4-H and 200-H), 8.16 (d, 1H, J ¼ 8.4 Hz, 8-H), 8.39 (d, 1H,
J ¼ 15.2 Hz, 2-CH]CHCO). 13C NMR (100 MHz, CDCl3): 23.20 (2C),
53.76, 54.38 (2C), 55.38, 67.71, 114.15 (2C), 114.36 (2C), 127.06,
127.42, 127.44, 128.20, 129.54, 129.76, 130.42, 131.02 (2C), 131.21
(2C), 131.96, 136.17, 136.92, 140.87, 147.14, 151.53, 159.54, 161.05,
out using the Mightysil RP-18 GP column (250 ꢂ 4.6 mm i.d., 5
mm),
and the temperature was set at room temperature and the mobile
phase was composed of methanol and 0.02 M NaHCO3 (93:7). The
flow rate was 1 mL/min and the wavelength of detection was set at
282 nm.
5.1.1. (E)-3-[3-(4-Methoxyphenyl)quinolin-2-yl]-1-(3,4,5-
trimethoxyphenyl)prop-2-en-1-one (7)
Compound
4
(0.53 g, 2.0 mmol) and 30,40,5’-trimethox-
yacetophenone (0.42 g, 2.0 mmol) were stirred at 0 ꢀC for 15 min.
Aqueous solution of KOH (6 equiv) was added and the mixture was
stirred at room temperature for 2 h (TLC monitoring). After the
reaction reached completion, the resulting mixture was added 1 M
188.42.
ESI
(m/z)
479.05
[Mþ1]þ.
Anal.
calcd
for
C31H30N2O3$1.5H2O: C 73.64, H 6.58, N 5.54; found C 73.54, H 6.21,
HCl until pH
3
resulted and extracted with ethyl acetate
N 5.44. Purity: 99.01% (tr ¼ 5.77 min).
(50 mL ꢂ 3). The organic layer was collected, dried over MgSO4 and
concentrated in vacuo. The crude product was purified and crys-
tallized with EtOH to give 7 in yield 90%. Mp. 170.6e171.2 ꢀC. 1H
NMR (400 MHz, CDCl3): 3.89 (s, 3H, OMe), 3.95 (s, 6H, OMe ꢂ 2),
3.96 (s, 3H, OMe), 7.02e7.06 (m, 2H, 30-H), 7.36e7.39 (m, 4H, 20-H
and 200-H), 7.57e7.61 (m, 1H, 6-H), 7.74e7.78 (m, 1H, 7-H), 7.85 (d,
1H, J ¼ 8.0 Hz, 5-H), 8.03 (d, 1H, J ¼ 15.2 Hz, 2-CH]CHCO), 8.12 (s,
1H, 4-H), 8.11 (d,1H, J ¼ 8.4 Hz, 8-H), 8.31 (d,1H, J ¼ 15.2 Hz, 2-CH]
CHCO). 13C NMR (100 MHz, CDCl3): 55.37, 56.40 (2C), 60.96, 106.35
(2C), 114.18 (2C), 127.03, 127.50 (2C), 128.24, 129.54, 129.81, 130.44,
131.03 (2C), 133.21, 136.24, 136.99, 141.34, 142.71, 147.17, 151.59,
153.12 (2C), 159.60, 188.96. ESI (m/z) 455.97 [Mþ1]þ. Anal. calcd for
5.1.4. (E)-1-{4-[3-(Dimethylamino)propoxy]phenyl}-3-[3-(4-
methoxyphenyl)quinolin-2-yl]prop-2-en-1-one (10)
A mixture of 4-hydroxyacetophenone (3.0 mmol), 3-chloro-N,N-
dimethylpropanamine HCl (3.0 mmol), K2CO3 (1.0 mmol) in 30 mL
of EtOH was refluxed for 8 h (TLC monitoring). The solvent was
removed in vacuo and the residue was reacted with compound 4 as
described for 7 in 58% yield. Mp. 79.1e80.2 ꢀC (EtOH). 1H NMR
(400 MHz, CDCl3): 2.35e2.38 (m, 2H, OCH2CH2CH2), 2.78 (s, 6H,
NMe2), 3.14 (t, 2H, J ¼ 8.0 Hz, OCH2CH2CH2), 3.89 (s, 3H, OMe), 4.17
(t, 2H, J ¼ 5.6 Hz, OCH2CH2CH2), 6.95e6.97 (m, 2H, 300-H), 7.02e7.04
(m, 2H, 30-H), 7.35e7.37 (m, 2H, 20-H), 7.55e7.59 (m, 1H, 6-H),
7.73e7.76 (m, 1H, 7-H), 7.83 (d, 1H, J ¼ 8.0 Hz, 5-H), 8.00 (d, 1H,
J ¼ 15.2 Hz, 2-CH]CHCO), 8.10e8.12 (m, 3H, 4-H and 200-H), 8.19 (d,
1H, J ¼ 8.4 Hz, 8-H), 8.39 (d, 1H, J ¼ 15.2 Hz, 2-CH]CHCO). 13C NMR
(100 MHz, CDCl3): 24.89, 43.44 (2C), 55.36, 55.73, 65.07, 114.13 (2C),
114.17 (2C), 127.16, 127.38, 127.43, 128.17, 129.48, 129.74, 130.42,
131.00 (2C), 131.13 (2C), 131.32, 136.14, 136.89, 140.64, 147.12, 151.56,
159.51, 162.17, 188.45. ESI (m/z) 467.02 [Mþ1]þ. Anal. calcd for
C
28H25NO5: C 73.82, H 5.53, N 3.08 found C 73.74, H 5.52, N 3.01.
Purity: 98.47% (as determined by RP-HPLC, tr ¼ 5.31 min).
5.1.2. (E)-3-[3-(4-Methoxyphenyl)quinolin-2-yl]-1-{4-[2-
(piperidin-1-yl)-ethoxy]phenyl}-prop- 2-en-1-one (8)
Compound 8 was obtained from 4 and 40-[2-(pyrrolidin-1-yl)
ethoxy]acetophenone [28] as described for 7 in 46% yield. Mp.

C.-H. Tseng et al. / European Journal of Medicinal Chemistry 97 (2015) 306e319
315
C30H30N2O3$0.5H2O: C 75.76, H 6.57, N 5.89; found C 75.88, H 6.93,
N 6.28. Purity: 96.27% (tr ¼ 6.16 min).
monitoring) and then cooled to room temperature. The reaction
mixture was filtered through celite to remove the black residue.
Evaporation of the solvent afforded a residue which was dissolved
in CH2Cl2 (200 mL), washed with brine (100 mL), H2O (100 mL),
saturated sodium bicarbonate solution (100 mL) and dried
(MgSO4). The crude product was recrystallized with EtOH to give
0.62 g (73%) of 17 as a yellow solid. mp. 116.4e117.2 ꢀC (EtOH). 1H
NMR (400 MHz, CDCl3): 3.89 (s, 3H, OMe), 7.01e7.05 (m, 2H, 30-H),
7.34e7.38 (m, 2H, 20-H), 7.49 (dd, 1H, J ¼ 8.4, 2.8 Hz, 5-H), 7.58 (ddd,
1H, J ¼ 9.2, 8.4, 2.8 Hz, 7-H), 8.13 (s, 1H, 4-H), 8.33 (dd, 1H, J ¼ 9.2,
5.6 Hz, 8-H), 10.24 (s, 1H, CHO). 13C NMR (100 MHz, CDCl3): 55.37,
110.43, 114.08 (2C), 121.02, 128.65, 129.94, 130.78 (2C), 133.33,
136.32, 137.72, 144.06, 149.43, 159.88, 162.23, 192.23. Anal. calcd for
5.1.5. 6-Fluoro-3-(4-methoxyphenyl)-2-methylquinoline-4-
carboxylic acid (13)
A
mixture of 5-fluoroisatin (11, 6.60 g, 40 mmol), 4-
methoxyphenylacetone (7.88 g, 48 mmol) and KOH (6.74 g,
120 mmol) in EtOH (200 mL) was refluxed for 48 h (TLC moni-
toring). Evaporation of the solvent afforded a residue which was
dissolved in H2O (50 mL), and the solution was washed twice with
Et2O (30 mL). The aqueous phase was acidified to pH 1 with 37% HCl
in ice bath, and the precipitate was collected, washed with H2O and
recrystallized with EtOH to give 13 (11.58 g, 93%) as a white solid.
Mp. 269 ꢀC (Dec) (EtOH). 1H NMR (400 MHz, DMSO-d6): 2.45 (s, 3H,
2-Me), 3.83 (s, 3H, OMe), 7.05e7.08 (m, 2H, 30-H), 7.29e7.32 (m, 2H,
20-H), 7.45 (dd, 1H, J ¼ 9.6, 2.8 Hz, 5-H), 7.73 (ddd, 1H, J ¼ 9.2, 8.4,
2.8 Hz, 7-H), 8.12 (dd, 1H, J ¼ 9.2, 5.6 Hz, 8-H). 13C NMR (100 MHz,
DMSO-d6): 24.43, 55.15, 108.07, 113.82 (2C), 119.78, 122.49, 128.17,
130.49 (2C), 131.27, 131.59, 139.63, 143.39, 157.60, 159.03, 159.92,
167.79. Anal. calcd for C18H14FNO3: C 69.45, H 4.53, N 4.50; found C
69.45, H 4.59, N 4.57.
C17H12FNO2: C 72.59, H 4.30, N 4.98; found C 72.82, H 4.45, N 4.76.
5.1.10. 6-Methoxy-3-(4-methoxyphenyl)quinoline-2-carbaldehyde
(18)
Compound 18 was obtained from 16 as described for 17 in 70%
yield. Mp. 100.4e102.4 ꢀC. 1H NMR (400 MHz, CDCl3): 3.89 (s, 3H,
OMe), 3.97 (s, 3H, 6-OMe), 7.01e7.05 (m, 2H, 30-H), 7.10 (d, 1H,
J ¼ 2.8 Hz, 5-H), 7.35e7.39 (m, 2H, 20-H), 7.45 (dd, 1H, J ¼ 9.2, 2.8 Hz,
7-H), 8.05 (s, 1H, 4-H), 8.20 (d, 1H, J ¼ 9.2 Hz, 8-H), 10.22 (s, 1H,
CHO). 13C NMR (100 MHz, CDCl3): 55.35, 55.70, 104.30, 113.93 (2C),
123.81, 129.28, 130.57, 130.77 (2C), 132.09, 136.25, 136.86, 143.23,
147.59, 159.66, 160.12, 192.37. Anal. calcd for C18H15NO3: C 73.69, H
5.16, N 4.91; found C 73.63, H 4.91, N 4.70.
5.1.6. 6-Methoxy-3-(4-methoxyphenyl)-2-methylquinoline-4-
carboxylic acid (14)
Compound 14 was obtained from 12 as described for 13 in 84%
yield. Mp. 278.1 ꢀC (Dec) (EtOH). 1H NMR (400 MHz, DMSO-d6):
2.40 (s, 3H, 2-Me), 3.82 (s, 3H, OMe), 3.87 (s, 3H, 6-OMe), 7.03e7.06
(m, 3H, 30-H and 5-H), 7.27e7.29 (m, 1H, 20-H), 7.45 (d, 1H,
J ¼ 8.0 Hz, 7-H), 7.95 (d, 1H, J ¼ 8.4 Hz, 8-H). 13C NMR (100 MHz,
DMSO-d6): 24.16, 55.19, 55.52, 102.71, 113.81 (2C), 121.98, 122.80,
129.21, 130.22, 130.60 (2C), 139.38, 142.29, 155.06, 157.53, 158.95,
168.38. Anal. calcd for C19H17NO4$0.2H2O: C 69.80, H 5.36, N 4.28;
found C 69.78, H 5.44, N 4.16.
5.1.11. General procedure for the preparation of quinolinyl-
chalcones, 19aeg and 20aeg
Compound 17 (2.0 mmol) and appropriate acetophenone
(2.0 mmol) in EtOH (50 mL) were stirred for 15 min, followed by the
addition of an aqueous solution of KOH (6 equiv) at 0 ꢀC for 1 h. The
mixture was stirred at room temperature overnight. After the re-
action reached completion (TLC monitoring), the resulting mixture
was concentrated in vacuo, diluted with water (50 mL), acidified
with 1 M HCl to pH ¼ 3, extracted with CH2Cl2 (50 mL ꢂ 3). The
organic layer was dried over MgSO4 and concentrated in vacuo. The
crude product was purified by flash chromatography on silica gel
(CH2Cl2/MeOH 50/1) and recrystallized with EtOH to give
quinolinyl-chalcones 19aeg. Compounds 20aeg were prepared
from 18 (2.0 mmol) and appropriate acetophenone (2.0 mmol)
under the same reaction procedures.
5.1.7. 6-Fluoro-3-(4-methoxyphenyl)-2-methylquinoline (15)
A mixture of 13 (1.55 g, 5.0 mmol) in 10 mL dowtherm was
heated in 280 ꢀC for 4 h (TLC monitoring). The mixture was cooled
and then hexane (50 mL) was added and stirred at room temper-
ature for 2 h. The resulting precipitate was collected, washed with
H2O, and then purified by flash chromatography on silica gel
(hexane/CH2Cl2 1/1) and recrystallized from EtOH to give 0.98 g
(73%) of 15 as a brown solid. mp. 87.9e88.7 ꢀC (EtOH). 1H NMR
(400 MHz, CDCl3): 2.66 (s, 3H, 2-Me), 3.89 (s, 3H, OMe), 6.99e7.03
(m, 2H, 30-H), 7.31e7.34 (m, 2H, 20-H), 7.39 (dd,1H, J ¼ 9.2, 2.8 Hz, 5-
H), 7.44 (ddd, 1H, J ¼ 8.8, 8.4, 2.8 Hz, 7-H), 7.88 (s, 1H, 4-H), 8.05 (dd,
1H, J ¼ 9.6, 5.6 Hz, 8-H). 13C NMR (100 MHz, CDCl3): 24.48, 55.34,
110.29, 113.92 (2C), 119.23, 127.47, 130.28 (2C), 130.80, 131.81,
135.33, 136.20, 143.95, 157.04, 159.28, 160.18. Anal. calcd for
5.1.12. (E)-3-[6-Fluoro-3-(4-methoxyphenyl)quinolin-2-yl]-1-
phenylprop-2-en-1-one (19a)
Yield 90%; Mp.146.1e147.6 ꢀC (EtOH). 1H NMR (400 MHz,CDCl3):
3.89 (s, 3H, OMe), 7.02e7.06 (m, 2H, 30-H), 7.34e7.38 (m, 2H, 20-H),
7.49 (dd, 1H, J ¼ 8.4, 2.8 Hz, 5-H), 7.49e7.62 (m, 4H, 7-H, 300-H and
400-H), 7.99 (d, 1H, J ¼ 15.2 Hz, 2-CH]CHCO), 8.04 (s, 1H, 4-H),
8.10e8.13 (m, 2H, 200-H), 8.19 (dd, 1H, J ¼ 9.2, 5.2 Hz, 8-H), 8.36 (d,
1H, J ¼ 15.2 Hz, 2-CH]CHCO). 13C NMR (100 MHz, CDCl3): 55.38,
110.32, 114.24 (2C), 121.25, 127.28, 128.61 (2C), 128.74 (2C), 128.86,
130.03, 131.00 (2C), 132.24, 133.04, 136.20, 136.94, 137.86, 140.98,
144.33, 150.95, 159.71, 161.08, 190.25. ESI (m/z) 383.94 [Mþ1]þ.
Anal. calcd for C25H18FNO2: C 78.30, H 4.74, N 3.65; found C 78.32, H
4.76, N 3.58. Purity: 99.34% (tr ¼ 6.02 min).
C
17H14FNO: C 76.39, H 5.28, N 5.24; found: C 76.25, H 5.38, N 5.18.
5.1.8. 6-Methoxy-3-(4-methoxyphenyl)-2-methylquinoline (16)
Compound 16 was obtained from 14 as described for 15 in 70%
yield. Mp. 208.1e209.5 ꢀC. 1H NMR (400 MHz, CDCl3): 2.63 (s, 3H, 2-
Me), 3.88 (s, 3H, OMe), 3.92 (s, 3H, 6-OMe), 6.98e7.02 (m, 2H, 30-H),
7.04 (d, 1H, J ¼ 2.8 Hz, 5-H), 7.31e7.35 (m, 3H, 20-H and 7-H), 7.84 (s,
1H, 4-H), 7.95 (d, 1H, J ¼ 9.2 Hz, 8-H). 13C NMR (100 MHz, CDCl3):
24.22, 55.32, 55.48, 104.94, 113.81 (2C), 121.72, 127.77, 129.78,
130.31 (2C), 132.38, 135.02, 135.60, 142.95, 154.92, 157.38, 159.08.
Anal. calcd for C18H17NO2$0.4H2O: C 75.45, H 6.26, N 4.89; found C
75.63, H 6.21, N 4.82.
5.1.13. (E)-3-[6-Fluoro-3-(4-methoxyphenyl)quinolin-2-yl]-1-(4-
methoxyphenyl)prop-2-en-1-one (19b)
Yield 87%; Mp. 155.3e157.6 ꢀC (EtOH). 1H NMR (400 MHz,
CDCl3): 3.89 (s, 3H, OMe), 3.90 (s, 3H, OMe), 6.98e7.06 (m, 4H, 300-H
and 30-H), 7.34e7.38 (m, 2H, 20-H), 7.44 (dd, 1H, J ¼ 8.8, 2.4 Hz, 5-H),
7.52 (ddd, 1H, J ¼ 9.6, 8.8, 3.2 Hz, 7-H), 7.98 (d, 1H, J ¼ 14.8 Hz, 2-
CH]CHCO), 8.04 (s, 1H, 4-H), 8.11e8.15 (m, 2H, 200-H), 8.19 (dd,
5.1.9. 6-Fluoro-3-(4-methoxyphenyl)quinoline-2-carbaldehyde
(17)
A mixture 15 (0.80 g, 3.0 mmol) and selenium dioxide (0.66 g,
6.0 mmol) in 1,4-dioxane (50 mL) was heated at 100 ꢀC for 2 h (TLC
1H, J ¼ 9.2, 5.2 Hz, 8-H), 8.36 (d, 1H, J ¼ 14.8 Hz, 2-CH]CHCO). 13
C

316
C.-H. Tseng et al. / European Journal of Medicinal Chemistry 97 (2015) 306e319
NMR (100 MHz, CDCl3): 55.39, 55.49, 110.32, 113.84 (2C), 114.20
(2C), 120.19, 127.26, 128.81, 130.10, 130.90, 131.00 (2C), 131.12 (2C),
132.19, 136.19, 136.90, 140.21, 144.32, 151.17, 159.69, 161.02, 163.60,
(2C), 114.39 (2C), 120.19, 127.25, 128.82, 130.11, 130.87, 130.99 (2C),
131.12 (2C), 132.21, 136.18, 136.91, 140.27, 144.34, 151.17, 159.71,
161.04, 162.47, 188.45. ESI (m/z) 497.06 [Mþ1]þ. Anal. calcd for
188.48. ESI
(m/z) 435.95 [Mþ1]þ.
Anal. calcd
for
C31H29FN2O3$0.2H2O: C 74.44, H 5.92, N 5.60; found C 74.26, H 6.03,
C
26H20FNO3$0.8H2O: C 72.68, H 5.11, N 3.26; found C 72.40, H 5.05,
N 5.37. Purity: 97.74% (tr ¼ 6.18 min).
N 3.06. Purity: 97.26% (tr ¼ 5.03 min).
5.1.18. (E)-1-{4-[3-(Dimethylamino)propoxy]phenyl}-3-[6-fluoro-
3-(4-methoxyphenyl)-quinolin-2-yl]prop-2-en-1-one (19g)
5.1.14. (E)-3-[6-Fluoro-3-(4-methoxyphenyl)quinolin-2-yl]-1-(4-
hydroxyphenyl)prop-2-en-1-one (19c)
Yield 62%; Mp. 103.5e104.6 ꢀC (EtOH). 1H NMR (400 MHz,
CDCl3): 2.03e2.10 (m, 2H, OCH2CH2CH2N), 2.36 (s, 6H, NMe2), 2.60
(t, 2H, J ¼ 7.2 Hz, OCH2CH2CH2N), 3.89 (s, 3H, OMe), 4.12 (t, 2H,
J ¼ 5.6 Hz, OCH2CH2CH2N), 6.96e7.05 (m, 4H, 30-H and 300-H),
7.34e7.38 (m, 2H, 20-H), 7.44 (dd, 1H, J ¼ 8.8, 2.8 Hz, 5-H), 7.52 (ddd,
1H, J ¼ 9.2, 8.8, 2.4 Hz, 7-H), 7.97 (d, 1H, J ¼ 15.2 Hz, 2-CH]CHCO),
8.04 (s, 1H, 4-H), 8.10e8.14 (m, 2H, 200-H), 8.18 (dd, 1H, J ¼ 9.2,
5.2 Hz, 8-H), 8.36 (d, 1H, J ¼ 15.2 Hz, 2-CH]CHCO). 13C NMR
(100 MHz, CDCl3): 26.88, 45.05 (2C), 55.37, 56.08, 66.21, 110.3,
114.22 (2C), 114.28 (2C), 120.17, 127.27, 128.82, 130.09, 130.86,
130.99 (2C), 131.11 (2C), 132.17, 136.18, 136.88, 140.17, 144.31, 151.17,
159.68, 161.01, 162.95, 188.45. ESI (m/z) 485.03 [Mþ1]þ. Anal. calcd
for C30H29FN2O3$0.2H2O: C 73.81, H 6.07, N 5.74; found C 73.65, H
6.11, N 5.69. Purity: 95.56% (tr ¼ 6.29 min).
Yield 65%; Mp. 231.7e232.3 ꢀC (EtOH). 1H NMR (400 MHz,
DMSO-d6): 3.87 (s, 3H, OMe), 6.91e6.95 (m, 2H, 300-H), 7.12e7.16
(m, 2H, 30-H), 7.42e7.46 (m, 2H, 20-H), 7.71e7.77 (m, 2H, 7-H and 2-
CH]CHCO), 7.84 (dd, 1H, J ¼ 9.6, 2.8 Hz, 5-H), 7.95e7.99 (m, 2H, 200-
H), 8.19e8.28 (m, 2H, 8-H and 2-CH]CHCO), 8.35 (s, 1H, 4-H), 10.52
(s, 1H, OH). 13C NMR (100 MHz, DMSO-d6): 55.28, 110.79, 114.18
(2C), 115.60 (2C), 120.45, 127.04, 128.56, 128.76, 129.68, 131.06 (2C),
131.18 (2C), 131.99, 136.24, 136.60, 139.07, 143.77, 150.38, 159.30,
160.31, 162.49, 187.24. ESI (m/z) 399.93 [Mþ1]þ. Anal. calcd for
C
25H18FNO3: C 75.18, H 4.54, N 3.51; found C 74.96, H 4.74, N 3.42.
Purity: 99.52% (tr ¼ 3.15 min).
5.1.15. (E)-3-[6-Fluoro-3-(4-methoxyphenyl)quinolin-2-yl]-1-
(3,4,5-trimethoxyphenyl)-prop-2-en-1-one (19d)
Yield 86%; . Mp. 200.3e200.7 ꢀC (EtOH). 1H NMR (400 MHz,
CDCl3): 3.89 (s, 3H, OMe), 3.95 (s, 3H, OMe), 3.96 (s, 6H, OMe ꢂ 2),
7.00e7.08 (m, 2H, 30-H), 7.35e7.37 (m, 4H, 20-H and 200-H), 7.45 (dd,
1H, J ¼ 8.8, 2.4 Hz, 5-H), 7.49e7.55 (m, 1H, 7-H), 8.00 (d, 1H,
J ¼ 15.2 Hz, 2-CH]CHCO), 8.06 (s, 1H, 4-H), 8.20 (dd, 1H, J ¼ 9.2,
5.2 Hz, 8-H), 8.27 (d, 1H, J ¼ 14.8 Hz, 2-CH]CHCO). 13C NMR
(100 MHz, CDCl3): 55.38, 56.41 (2C), 60.98, 106.37 (2C), 110.36,
114.25 (2C), 120.28, 126.95, 128.88, 130.05, 130.99 (2C), 132.19,
133.16, 136.27, 136.94, 141.04, 142.77, 144.32, 151.01, 153.14 (2C),
159.75, 161.10, 188.90. ESI (m/z) 473.98 [Mþ1]þ. Anal. calcd for
5.1.19. (E)-3-[6-Methoxy-3-(4-methoxyphenyl)quinolin-2-yl]-1-
phenylprop-2-en-1-one (20a)
Yield 87%; Mp. 130.2e130.7 ꢀC (EtOH). 1H NMR (400 MHz,
CDCl3): 3.89 (s, 3H, OMe), 3.95 (s, 3H, 6-OMe), 7.01e7.05 (m, 2H, 30-
H), 7.07 (d,1H, J ¼ 2.8 Hz, 5-H), 7.34e7.38 (m, 2H, 20-H), 7.40 (dd,1H,
J ¼ 9.2, 2.8 Hz, 7-H), 7.48e7.61 (m, 3H, 300-H and 400-H), 7.98 (s, 1H,
4-H), 8.00 (d, 1H, J ¼ 15.2 Hz, 2-CH]CHCO), 8.08 (d, 1H, J ¼ 9.2 Hz,
8-H), 8.10e8.13 (m, 2H, 200-H), 8.19 (d,1H, J ¼ 15.2 Hz, 2-CH]CHCO).
13C NMR (100 MHz, CDCl3): 55.36, 55.59, 104.47, 114.12 (2C), 123.03,
126.23, 128.55 (2C), 128.71 (2C), 129.43, 130.60, 131.01 (2C), 131.15,
132.87, 135.56, 136.63, 138.02, 141.50, 143.47, 148.93, 158.64, 159.51,
190.34. ESI (m/z) 395.96 [Mþ1]þ. Anal. calcd for C26H21NO3: C 78.97,
H 5.35, N 3.54; found C 78.84, H 5.23, N 3.52. Purity: 99.00%
(tr ¼ 5.79 min).
C
28H24FNO5$0.2H2O: C 70.49, H 5.15, N 2.94; found C 70.64, H 5.17,
N 2.88. Purity: 98.13% (tr ¼ 5.27 min).
5.1.16. (E)-3-[6-Fluoro-3-(4-methoxyphenyl)quinolin-2-yl]-1-{4-
[2-(piperidin-1-yl)ethoxy]-phenyl}prop-2-en-1-one (19e)
Yield 55%; Mp. 198.6e199.2 ꢀC (EtOH). 1H NMR (400 MHz,
CDCl3): 1.43e1.49 (m, 2H, piperidinyl-H), 1.60e1.65 (m, 4H,
piperidinyl-H), 2.53 (br s, 4H, piperidinyl-H), 2.81 (t, 2H, J ¼ 6.0 Hz,
OCH2CH2N), 3.87 (s, 3H, OMe), 4.20 (t, 2H, J ¼ 6.0 Hz, OCH2CH2N),
6.97e7.05 (m, 4H, 30-H and 300-H), 7.34e7.37 (m, 2H, 20-H), 7.43 (dd,
1H, J ¼ 8.8, 2.8 Hz, 5-H), 7.51 (ddd, 1H, J ¼ 9.2, 8.8, 2.4 Hz, 7-H), 7.93
(d, 1H, J ¼ 15.2 Hz, 2-CH]CHCO), 8.04 (s, 1H, 4-H), 8.10e8.14 (m,
2H, 200-H), 8.19 (dd, 1H, J ¼ 9.2, 5.2 Hz, 8-H), 8.36 (d, 1H, J ¼ 15.2 Hz,
2-CH]CHCO). 13C NMR (100 MHz, CDCl3): 24.10, 25.87 (2C), 55.06
(2C), 55.37, 57.70, 66.19, 110.32, 114.16 (2C), 114.38 (2C), 120.16,
127.24, 128.79, 130.07, 130.86, 130.98 (2C), 131.08 (2C), 132.17,
136.17, 136.88, 140.16, 144.30, 151.16, 159.67, 160.99, 162.87, 188.42.
ESI (m/z) 511.03 [Mþ1]þ. Anal. calcd for C32H31FN2O3$0.5H2O: C:
73.97, H: 6.21, N: 5.39; found C: 73.82, H: 6.48, N: 5.03. Purity:
98.57% (tr ¼ 6.06 min).
5.1.20. (E)-3-[6-Methoxy-3-(4-methoxyphenyl)quinolin-2-yl]-1-(4-
methoxyphenyl)prop-2-en-1-one (20b)
Yield 82%; Mp. 147.6e148.7 ꢀC (EtOH). 1H NMR (400 MHz,
CDCl3): 3.89 (s, 3H, OMe), 3.90 (s, 3H, OMe), 3.96 (s, 3H, 6-OMe),
6.97e7.00 (m, 2H, 300-H), 7.01e7.05 (m, 2H, 30-H), 7.07 (d, 1H,
J ¼ 2.8 Hz, 5-H), 7.34e7.39 (m, 2H, 20-H), 7.40 (dd, 1H, J ¼ 9.2, 2.8 Hz,
7-H), 7.98 (s, 1H, 4-H), 7.99 (d, 1H, J ¼ 14.8 Hz, 2-CH]CHCO), 8.08
(d, 1H, J ¼ 9.2 Hz, 8-H), 8.12e8.16 (m, 2H, 200-H), 8.34 (d, 1H,
J ¼ 14.8 Hz, 2-CH]CHCO). 13C NMR (100 MHz, CDCl3): 55.60, 55.69,
55.83, 104.76, 114.01 (2C), 114.36 (2C), 123.19, 126.47, 129.61, 130.92,
131.25 (2C), 131.31 (2C), 131.37, 135.80, 136.84, 140.95, 143.72,
149.43, 158.82, 159.74, 163.72, 188.84. ESI (m/z) 426.00 [Mþ1]þ.
Anal. calcd for C27H23NO4$0.2H2O: C 75.58, H 5.50, N 3.26; found C
75.32, H 5.52, N 3.17. Purity: 98.86% (tr ¼ 6.02 min).
5.1.21. (E)-1-(4-Hydroxyphenyl)-3-[6-methoxy-3-(4-
5.1.17. (E)-3-[6-Fluoro-3-(4-methoxyphenyl)quinolin-2-yl]-1-{4-
[2-(pyrrolidin-1-yl)ethoxy]-phenyl}prop-2-en-1-one (19f)
methoxyphenyl)quinolin-2-yl]prop-2-en-1-one (20c)
Yield 68%; Mp. 223.4e223.8 ꢀC (EtOH). 1H NMR (400 MHz,
DMSO-d6): 3.83 (s, 3H, OMe), 3.89 (s, 3H, 6-OMe), 6.87e6.90 (m,
2H, 300-H), 7.08e7.11 (m, 2H, 30-H), 7.36e7.40 (m, 3H, 20-H and 5-H),
7.44 (dd, 1H, J ¼ 9.2, 2.8 Hz, 7-H), 7.70 (d, 1H, J ¼ 14.8 Hz, 2-CH]
CHCO), 7.91e7.95 (m, 2H, 200-H), 8.01 (d, 1H, J ¼ 9.2 Hz, 8-H), 8.17 (s,
1H, 4-H), 8.18 (d, 1H, J ¼ 14.8 Hz, 2-CH]CHCO), 10.46 (s, 1H, OH).
13C NMR (100 MHz, DMSO-d6): 55.24, 55.62, 105.31, 114.12 (2C),
115.56 (2C), 123.06, 125.84, 128.90, 129.16, 130.18, 130.62, 131.01
(2C), 131.07 (2C), 135.65, 135.98, 139.52, 142.75, 148.09, 158.17,
159.15, 162.36, 187.30. ESI (m/z) 411.96 [Mþ1]þ. Anal. calcd for
Yield 57%; . Mp. 88.0e88.9 ꢀC (EtOH). 1H NMR (400 MHz, CDCl3):
1.89e1.93 (m, 4H, pyrrolidinyl-H), 2.84 (br s, 4H, pyrrolidinyl-H),
3.08 (t, 2H, J ¼ 6.0 Hz, OCH2CH2N), 3.89 (s, 3H, OMe), 4.30 (t, 2H,
J ¼ 6.0 Hz, OCH2CH2N), 6.99e7.05 (m, 4H, 30-H and 300-H), 7.34e7.39
(m, 2H, 20-H), 7.44 (dd, 1H, J ¼ 8.8, 2.8 Hz, 5-H), 7.52 (ddd, 1H,
J ¼ 9.2, 8.4, 2.4 Hz, 7-H), 7.97 (d, 1H, J ¼ 14.8 Hz, 2-CH]CHCO), 8.04
(s, 1H, 4-H), 8.10e8.14 (m, 2H, 200-H), 8.19 (dd, 1H, J ¼ 9.2, 5.2 Hz, 8-
H), 8.35 (d, 1H, J ¼ 14.8 Hz, 2-CH]CHCO). 13C NMR (100 MHz,
CDCl3): 23.42 (2C), 54.59, 54.62 (2C), 55.39, 66.49, 110.31, 114.24

C.-H. Tseng et al. / European Journal of Medicinal Chemistry 97 (2015) 306e319
317
C26H21NO4$0.2H2O: C 75.24, H 5.20, N 3.37; found C 74.88, H 5.13, N
3.42. Purity: 99.27% (tr ¼ 3.27 min).
114.18 (2C), 122.88, 126.15, 129.28, 130.59, 130.86, 130.94 (2C),
130.98 (2C), 131.02, 135.47, 136.49, 140.58, 143.38, 149.07, 158.48,
159.42, 162.90, 188.46. ESI (m/z) 497.06 [Mþ1]þ. Anal. calcd for
5.1.22. (E)-3-[6-Methoxy-3-(4-methoxyphenyl)quinolin-2-yl]-1-
(3,4,5-trimethoxyphenyl)-prop-2-en-1-one (20d)
C31H32N2O4$0.2H2O: C 74.44, H 6.53, N 5.60; found C 74.35, H 6.52,
N 5.56. Purity: 97.05% (tr ¼ 5.98 min).
Yield 92%; Mp. 179.4e179.5 ꢀC (EtOH). 1H NMR (400 MHz,
CDCl3): 3.89 (s, 3H, OMe), 3.95 (s, 6H, OMe), 3.96 (s, 3H, OMe), 3.96
(s, 3H, 6-OMe), 7.02e7.05 (m, 2H, 30-H), 7.08 (d, 1H, J ¼ 2.8 Hz, 5-H),
7.35e7.38 (m, 4H, 20-H and 200-H), 7.41 (dd, 1H, J ¼ 9.2, 2.8 Hz, 7-H),
8.00 (s, 1H, 4-H), 8.01 (d, 1H, J ¼ 15.2 Hz, 2-CH]CHCO), 8.09 (d, 1H,
J ¼ 9.2 Hz, 8-H), 8.26 (d, 1H, J ¼ 15.2 Hz, 2-CH]CHCO). 13C NMR
(400 MHz, CDCl3): 55.35, 55.60, 56.36 (2C), 60.95, 104.50, 106.23
(2C), 114.11 (2C), 123.10, 125.85, 129.44, 130.56, 131.00 (2C), 131.03,
133.32, 135.67, 136.64, 141.54, 142.53, 143.41, 148.96, 153.07 (2C),
158.68, 159.52, 188.96. ESI (m/z) 486.00 [Mþ1]þ. Anal. calcd for
5.1.26. {3-[3-(4-Methoxyphenyl)quinolin-2-yl]oxiran-2-yl}(3,4,5-
trimethoxyphenyl)methan-one (21)
A suspension of 7 (0.45 g, 1 mmol) in MeOH (30 mL) is treated
successively with K2CO3 (0.33 g, 3 mmol) and 35% aqueous
hydrogen peroxide solution (1 mL, 10 mmol), the mixture was
stirred at room temperature for 3 h (TLC monitoring). The solvent
was removed under reduced pressure and the resulting residue
dissolved in CH2Cl2 (50 mL), and washed with H2O (40 mL). The
organic phase was separated, washed with brine and dried by
MgSO4, then the solvent removed under reduced pressure and
recrystallized with EtOH to give compound 21 (0.42 g, 90%). Mp.
151.9e152.9 ꢀC (EtOH). 1H NMR (400 MHz, CDCl3): 3.86 (s, 3H,
OMe), 3.90 (s, 6H, OMe), 3.93 (s, 3H, OMe), 4.26 (d, 1H, J ¼ 2.0 Hz),
5.29 (d, 1H, J ¼ 2.0 Hz), 6.99e7.02 (m, 2H, 20-H), 7.46e7.79 (m, 4H,
200-and 20-H), 7.58e7.62 (m, 1H, 6-H), 7.73e7.77 (m, 1H, 7-H), 7.87
(d, 1H, J ¼ 8.4 Hz, 5-H), 8.13e8.16 (m, 2H, 4- and 8-H). 13C NMR
C
29H27NO6: C 71.74, H 5.61, N 2.88; found C 71.53, H 5.28, N 2.78.
Purity: 98.53% (tr ¼ 5.19 min).
5.1.23. (E)-3-[6-Methoxy-3-(4-methoxyphenyl)quinolin-2-yl]-1-
{4-[2-(piperi-din-1-yl)eth-oxy]phenyl}prop-2-en-1-one (20e)
Yield 60%; Mp. 98.2e99.1 ꢀC (EtOH). 1H NMR (400 MHz, CDCl3):
1.42e1.49 (m, 2H, piperidinyl-H), 1.58e1.66 (m, 4H, piperidinyl-H),
2.54 (m, 4H, piperidinyl-H), 2.81 (t, 2H, J ¼ 6.0 Hz, OCH2CH2N), 3.89
(s, 3H, OMe), 3.95 (s, 3H, 6-OMe), 4.20 (t, 2H, J ¼ 6.0 Hz, OCH2CH2N),
6.97e7.00 (m, 2H, 300-H), 7.01e7.05 (m, 2H, 30-H), 7.07 (d, 1H,
J ¼ 2.8 Hz, 5-H), 7.34e7.38 (m, 2H, 20-H), 7.40 (dd, 1H, J ¼ 9.2, 2.8 Hz,
7-H), 7.97 (s, 1H, 4-H), 7.98 (d, 1H, J ¼ 15.2 Hz, 2-CH]CHCO), 8.08
(d, 1H, J ¼ 9.2 Hz, 8-H), 8.10e8.14 (m, 2H, 200-H), 8.34 (d, 1H,
J ¼ 15.2 Hz, 2-CH]CHCO). 13C NMR (100 MHz, CDCl3): 24.12, 25.87
(2C), 55.06 (2C), 55.38, 55.60, 57.72, 66.20, 104.56, 114.15 (2C),
114.37 (2C), 122.93, 126.31, 129.38, 130.72, 131.25 (2C), 131.06 (2C),
131.16, 135.56, 136.61, 140.69, 143.51, 149.24, 158.61, 159.54, 162.77,
(100 MHz, CDCl3)
d 55.36, 56.31 (2C), 58.18, 59.01, 60.97, 106.06
(2C), 114.26, 114.30 (2C), 127.49, 127.56, 127.90, 129.30, 129.52,
129.77, 130.40, 130.79 (2C), 136.01, 136.78, 143.29, 146.92, 151.41,
153.20, 159.66, 192.68. ESI (m/z) 471.96 [Mþ1]þ. Anal. calcd for
C28H25NO6: C 71.32, H 5.34, N 2.97; found C 71.30, H 5.43, N 2.76.
Purity: 97.53% (tr ¼ 4.49 min).
5.1.27. {2-[3-(4-Methoxyphenyl)quinolin-2-yl]cyclopropyl}(3,4,5-
trimethoxyphenyl)methan-one (22)
A mixture of compound 7 (0.45 g, 1 mmol), trimethylsulfoxo-
nium iodide (0.26 g, 2 mmol) and tetrabutylammonium bromide
(0.06 g, 0.2 mmol) in CH2Cl2 (10 mL) was stirred at room temper-
ature for 15 min. 50% aq NaOH (10 mL) solution was added drop-
wise and the reaction mixture was refluxed at 80 ꢀC for 4 h (TLC
monitoring). The reaction mixture was cooled, CH2Cl2 (50 mL) was
added and washed twice with H2O (50 mL). The organic layer was
dried over MgSO4 and concentrated in vacuo. The crude product
was purified by flash chromatography on silica gel (CH2Cl2/MeOH
100/1) and recrystallized with EtOH to give 22 (0.42 g, 89%) as a
gray solid. Mp.: 148.6e150.3 ꢀC (EtOH). 1H NMR (400 MHz, CDCl3):
1.86e1.91 and 1.99e2.04 (two m, 2H, cyclopropyl-CH2), 2.93e2.97
(m, 1H, 2-CHCHCO), 3.59e3.64 (m, 1H, 2-CHCHCO), 3.84 (s, 3H,
OMe), 3.89 (s, 6H, OMe), 3.91 (s, 3H, OMe), 6.92e6.96 (m, 2H, 20-H),
7.33 (s, 2H, 200-H), 7.34e7.38 (m, 2H, 20-H), 7.47e7.51 (m, 1H, 6-H),
7.66e7.70 (m, 1H, 7-H), 7.79 (d, 1H, J ¼ 8.0 Hz, 5-H), 7.96 (s, 1H, 4-H),
8.04 (d, 1H, J ¼ 8.4 Hz, 8-H). 13C NMR (100 MHz, CDCl3): 20.87,
29.02, 29.44, 55.26, 56.26 (2C), 60.88, 105.74 (2C), 113.98 (2C),
126.02, 126.87, 127.24, 128.54, 129.18, 130.59 (2C), 130.99, 132.95,
135.25, 135.85, 142.39,146.83,152.97 (2C),157.45,159.22,197.37. ESI
(m/z) 470.01 [Mþ1]þ. Anal. calcd for C29H27NO5: C 74.18, H 5.80, N
2.98; found C 74.12, H 5.66, N 2.77. Purity: 99.45% (tr ¼ 4.45 min).
188.96.
ESI (m/z) 523.06 [Mþ1]þ.
Anal. calcd for
C
33H34N2O4$0.2H2O: C: 75.32, H: 6.59, N: 5.32; found C: 75.04, H:
6.56, N: 5.10. Purity: 97.93% (tr ¼ 6.27 min).
5.1.24. (E)-3-[6-Methoxy-3-(4-methoxyphenyl)quinolin-2-yl]-1-
{4-[2-(pyrrolidin-1-yl)eth-oxy]phenyl}prop-2-en-1-one (20f)
Yield 58%; Mp. 66.7e67.1 ꢀC (EtOH). 1H NMR (400 MHz, CDCl3):
1.80e1.85 (m, 4H, pyrrolidinyl-H), 2.66e2.67 (m, 4H, pyrrolidinyl-
H), 2.96 (t, 2H, J ¼ 6.0 Hz, OCH2CH2N), 3.89 (s, 3H, OMe), 3.96 (s,
3H, 6-OMe), 4.21 (t, 2H, J ¼ 6.0 Hz, OCH2CH2N), 6.98e7.07 (m, 4H,
30-H and 300-H), 7.07 (d, 1H, J ¼ 2.8 Hz, 5-H), 7.34e7.38 (m, 2H, 20-H),
7.40 (dd, 1H, J ¼ 9.2, 2.8 Hz, 7-H), 7.97 (s, 1H, 4-H), 7.98 (d, 1H,
J ¼ 15.2 Hz, 2-CH]CHCO), 8.08 (d,1H, J ¼ 9.2 Hz, 8-H), 8.11e8.14 (m,
2H, 200-H), 8.34 (d, 1H, J ¼ 15.2 Hz, 2-CH]CHCO). 13C NMR
(100 MHz, CDCl3): 23.46 (2C), 54.70 (2C), 54.82, 55.36, 55.58, 67.17,
104.51, 114.12 (2C), 114.33 (2C), 122.94, 126.24, 129.36, 130.68,
131.01 (2C), 131.05 (2C), 131.09, 131.13, 135.55, 136.59, 140.69,
143.48, 149.19, 158.57, 159.50, 162.73, 188.58. ESI (m/z) 509.02
[Mþ1]þ. Anal. calcd for C32H32N2O4$0.4H2O: C 74.51, H 6.41, N 5.43;
found C 74.58, H 6.59, N 5.10. Purity: 97.86% (tr ¼ 6.11 min).
5.1.25. (E)-1-{4-[3-(Dimethylamino)propoxy]phenyl}-3-[6-
methoxy-3-(4-methoxyphenyl)-quinolin-2-yl]prop-2-en-1-one
(20g)
5.1.28. 3-[3-(4-Methoxyphenyl)quinolin-2-yl]-1-(3,4,5-
trimethoxyphenyl)propan-1-one (23)
Yield 53%; Mp. 49.7e51.8 ꢀC (EtOH). 1H NMR (400 MHz, CDCl3):
1.97e2.03 (m, 2H, OCH2CH2CH2N), 2.28 (s, 6H, NMe2), 2.50 (t, 2H,
J ¼ 7.2 Hz, OCH2CH2CH2N), 3.87 (s, 3H, OMe), 3.93 (s, 3H, 6-OMe),
4.09 (t, 2H, J ¼ 6.4 Hz, OCH2CH2CH2N), 6.95e7.03 (m, 4H, 30-H and
300-H), 7.05 (d, 1H, J ¼ 2.8 Hz, 5-H), 7.33e7.36 (m, 2H, 20-H), 7.39 (dd,
1H, J ¼ 9.2, 2.8 Hz, 7-H), 7.96 (s, 1H, 4-H), 7.99 (d, 1H, J ¼ 15.2 Hz, 2-
CH]CHCO), 8.07 (d, 1H, J ¼ 9.2 Hz, 8-H), 8.09e8.13 (m, 2H, 200-H),
8.33 (d, 1H, J ¼ 15.2 Hz, 2-CH]CHCO). 13C NMR (100 MHz, CDCl3):
27.16, 45.28 (2C), 55.27, 55.49, 56.08, 66.28, 104.45, 114.04 (2C),
A solution of 7 (0.91 g, 2 mmol) in acetic acid (30 mL) was hy-
drogenated for 2 h (TLC monitoring) under H2 with Pd/C (120 mg).
The reaction mixture was filtered with celite and the filtrate
concentrated in vacuo to give a residual solid, and recrystallized
from EtOH to give 23 (0.85 g, 93%) as a white solid. Mp.:
128.1e129.6 ꢀC (EtOH). 1H NMR (CDCl3): 3.94 (t, 2H, J ¼ 6.8 Hz, 2-
CH2CH2CO), 3.52 (t, 2H, J ¼ 6.8 Hz, 2-CH2CH2CO), 3.88 (s, 3H,
OMe), 3.90 (s, 6H, OMe), 3.92 (s, 3H, OMe), 7.00e7.03 (m, 2H, 30-H),
7.28 (s, 2H, 200-H), 7.38e7.41 (m, 2H, 20-H), 7.46e7.50 (m, 1H, 6-H),

318
C.-H. Tseng et al. / European Journal of Medicinal Chemistry 97 (2015) 306e319
7.62e7.66 (m, 1H, 7-H), 7.78 (d, 1H, J ¼ 8.0 Hz, 5-H), 7.92 (d, 1H,
J ¼ 8.4 Hz, 8-H), 7.94 (s, 1H, 4-H). 113C NMR (CDCl3): 30.69, 36.51,
55.34, 56.26 (2C), 60.94, 105.56 (2C), 113.95 (2C), 126.07, 126.83,
127.39, 128.47, 129.06, 129.47, 130.46 (2C), 131.80, 132.67, 135.34,
136.22, 142.22, 146.81, 152.96, 159.16, 159.20, 198.61. ESI (m/z)
457.98 [Mþ1]þ. Anal. calcd for C28H27NO5: C 73.50, H 5.95, N 3.06;
found C 73.60, H 6.01, N 2.97. Purity: 99.39% (tr ¼ 4.48 min).
for 5 min at room temperature in the dark. Removed the excess
DAPI solution and washed with PBS twice. Mounted the samples
before analyzing under a fluorescence microscopy.
5.2.4. Immunoblot analysis
After treatment, cells were collected and washed twice with
cold PBS. The cells were then lysed in lysis buffer (50 mM TriseHCl,
pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 2 mM EDTA, 1 mM EGTA,
5.2. Pharmacological methods
1 mM NaVO3, 10 mM NaF, 1 mM DTT, 1 mM PMSF, 25
mg/mL
aprotinin, and 25 mg/mL leupeptin) and kept on ice for 30 min. The
lysates were then centrifuged at 12,000 g at 4 ꢀC for 20 min; the
5.2.1. Antiproliferative assay
supernatants were stored at ꢁ70 ꢀC until use. The protein con-
Cancer cells (H460, A549, H1299, MCF-7, MDA-MB-231, SKBR-3),
normal lung cell (MRC-5) and normal mammary epithelial cell
(H184B5F5/M10) were purchased from Bioresources Collection and
Research Center, Taiwan. Cell lines were maintained in the same
standard medium and grown as a monolayer in DMEM (Gibco, USA)
and supplemented with 10% fetal bovine serum (FBS) and antibi-
centration was determined by the Bradford method. 20 mg protein
were separated by 8e12% SDS-PAGE and transferred onto a PVDF
membrane using a glycine transfer buffer (192 mM glycine, 25 mM
TriseHCl, pH 8.8, and 20% methanol [v/v]). After blocking with 5%
non-fat dried milk, the membrane was incubated for 2 h with
primary antibodies, followed by 30 min with secondary antibodies
in milk containing Tris-buffered saline (TBS) and 0.5% Tween. Anti-
human-PARP, pro-caspase 8, caspase 8 and Bad antibodies were
used at a 1:1000 dilution as the primary antibodies, while horse-
radish peroxidase-conjugated horse anti-rabbit IgG (Vector Labo-
ratories, Burlingame, CA, USA) was used at a 1:5000 dilution as the
secondary antibody. The membrane was then exposed to X-ray
film. Protein bands were detected using the enhanced chem-
iluminescence blotting detection system (Amersham, USA).
otics i.e. 100 IU/mL penicillin, 0.1 mg/mL streptomycin and 0.25 mg/
mL amphotercin. Culture was maintained at 37 ꢀC with 5% CO2 in a
humidified atmosphere.
Cells (5 ꢂ 103 cells/well) were treated as indicated for 72 h in
medium containing 10% FBS. Cell viability was quantitated with the
use of sodium 30-[1-(phenylamino-carbonyl)- 3,4-tetrazolium}-
bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate (XTT) color-
imetric assay (Biological Industries, Beit-Haemek, Israel). XTT la-
beling reagent (1 mg/mL) was mixed with electron-coupling
reagent, following the manufacturer's instructions, and 50
mL of
the mixture was added directly to the cells. The plates were further
incubated at 37 ꢀC for 4 h. Color was measured spectrophotomet-
rically in a microtiter plate reader at 492 nm and used as a relative
measure of viable cell number. The number of viable cells following
treatment was compared to solvent and untreated control cells and
5.3. Molecular modeling
Libdoc was performed by Discovery Studio 3.5 (Accelrys, San
Diego, CA, USA) [38e40]. Ligand conformations were generated
and adjusted by Solvent Accessible Solvent Area (SASA) methods;
polar and non-polar probes were employed to place grid in the
protein binding site. The hot spots would be pointed out in the
binding site by this step. In libdock, “docking” means that confor-
mations of ligands would be aligned to hot spots by the triplets
method. In the scoring step, Broyden-Fletcher-Goldfarb-Shanno
(BFGS) pose optimization was achieved and shown on the top of
simple pair-wise scores [41]. These results were also called by
Libdock scores. In our study, the tubulin structure was retrieved
from Protein Data Bank (PDB code: 1SA0) where the GTP site and
colchicine binding site are labeled in Fig. 9.
used to determine the percent of control growth as (Abtreated
/
Abcontrol) ꢂ 100, where Ab represents the mean absorbance (n ¼ 3).
The concentration that killed 50% of cells (GI50) was determined
from the linear portion of the curve by calculating the concentra-
tion of agent that reduced absorbance in treated cells, compared to
control cells, by 50% [29].
5.2.2. Cell cycle analysis
MDA-MB-231 cells treated with DMSO, compound 7 at different
concentration (1, 5, and 10 mM) or CIL-102 (10 mM) for 12, 24 and 36 h
were harvested, rinsed in PBS, resuspended and fixed in 70% ethanol
and store cells at ꢁ20 ꢀC in fixation buffer until ready for analysis.
Then the pellets were suspended in 1 mL of propidium iodide (PI)
Acknowledgments
solution containing 20 mg/mL of PI, 0.2 mg/mL RNase, and 0.1% (v/v)
Financial support of this work by grants from the Ministry of
Science and Technology, the Republic of China (NSC 101-2320-B-
037-039-MY3; NSC 101-2320-B-037-046-MY3; NSC 102-2320-B-
037-005-MY2) and Kaohsiung Medical University (KMU-
TP103H06; KMU-TP103H07; KMU-TP103H08) are gratefully
acknowledged. We also thank the National Center for High-
Performance Computing for providing computer resources and
chemical database services.
Trition X-100. Cell samples were incubated at room temperature in
the dark for at least 30 min and analyzed by a flow cytometer
(Coulter Epics). Data recording was made using Epics software and
cell cycle data were analyzed using Multicycle software (coulter).
5.2.3. Immunofluorescence analysis
MDA-MB-231 Cells were seeded on cover glasses in 12-well
plates with compound 7 (1e10
mM), CIL-102 (10 mM) or 23
(10 M) treatment for 24 h. After incubation, cells were washed
m
Appendix A. Supplementary data
with 1ꢂ PBS twice and fixed in 4% paraformaldehyde for 1 h. Then,
cells were washed with PBS containing 0.1 M Glycine for 5 min and
permibilized with solution containing 2% FBS and 0.4% TritonX-100
in PBS at room temperature for 15 min. After permibilization, cells
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.04.054.
were stained with
b-tubulin monoclonal antibody (Santa Cruza
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mouse IgG antibody (Origene, 1:200 diluted) at room temperature
for 1 h. Finally, washed with PBS and stained with DAPI (0.1 mg/ml)
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