Discovery of 2-[2-(5-nitrofuran-2-yl)vinyl]quinoline derivatives as a novel type of antimetastatic agents

Article abstract of DOI:10.1016/j.bmc.2014.11.015

A number of 2-furanylvinylquinoline derivatives were synthesized and evaluated for antiproliferative activities against the growth of four cancer cell lines including non-small cell lung cancer (A549 and H1299), breast cancer (MCF-7 and MDA-MB-231) and normal diploid embryonic lung cell line (MRC-5). Among them, (E)-6-methoxy-3-(4-methoxyphenyl)-2-[2-(5-nitrofuran-2-yl)vinyl]quinoline (10c) was found low cytotoxic in all cancer cells and normal cell. The aim of this study was to investigate the anti-invasive and anti-metastatic activity of compound 10c in H1299 human lung cancer cells. In this study, compound 10c inhibited the migration and invasion of cells in a concentration-dependent manner by wound healing assay and transwell invasion assay. Furthermore, the inhibition of both phosphorylation of Akt and ERK by compound 10c may be critical for cell migration and this may result in the down-regulation of several factors associated with cellular migration, including β-catenin transcription factor, Bcl-2 and COX-2. Interestingly, the treatment of compound 10c did not affect the expression level but reduced the activities of the MMP-2 and -9. The phosphorylation level of stress-activated kinase p38 was significantly increased following compound 10c treatment. To sum up, compound 10c had potential to suppress the migration and invasion of H1299 cancer cells in vitro and it could serve as a promising drug for the treatment of cancer metastasis.

Full text of DOI:10.1016/j.bmc.2014.11.015

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of 2-[2-(5-nitrofuran-2-yl)vinyl]quinoline derivatives
as a novel type of antimetastatic agents
Chih-Hua Tseng a, Cherng-Chyi Tzeng b, Chien-Chih Chiu c, Chih-Yao Hsu b, Chon-Kit Chou c,
Yeh-Long Chen b,
⇑
a School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung City 807, Taiwan
b Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung City 807, Taiwan
c Department of Biotechnology, College of Life Science, Kaohsiung Medical University, Kaohsiung City 807, 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 2-furanylvinylquinoline derivatives were synthesized and evaluated for antiproliferative
activities against the growth of four cancer cell lines including non-small cell lung cancer (A549 and
H1299), breast cancer (MCF-7 and MDA-MB-231) and normal diploid embryonic lung cell line (MRC-
5). Among them, (E)-6-methoxy-3-(4-methoxyphenyl)-2-[2-(5-nitrofuran-2-yl)vinyl]quinoline (10c)
was found low cytotoxic in all cancer cells and normal cell. The aim of this study was to investigate
the anti-invasive and anti-metastatic activity of compound 10c in H1299 human lung cancer cells. In this
study, compound 10c inhibited the migration and invasion of cells in a concentration-dependent manner
by wound healing assay and transwell invasion assay. Furthermore, the inhibition of both phosphoryla-
tion of Akt and ERK by compound 10c may be critical for cell migration and this may result in the down-
regulation of several factors associated with cellular migration, including b-catenin transcription factor,
Bcl-2 and COX-2. Interestingly, the treatment of compound 10c did not affect the expression level but
reduced the activities of the MMP-2 and -9. The phosphorylation level of stress-activated kinase p38
was significantly increased following compound 10c treatment. To sum up, compound 10c had potential
to suppress the migration and invasion of H1299 cancer cells in vitro and it could serve as a promising
drug for the treatment of cancer metastasis.
Received 28 August 2014
Revised 10 November 2014
Accepted 11 November 2014
Available online xxxx
Keywords:
b-Catenin
MAPK p38
Akt
Non-small cell lung cancer
Metastasis
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
growth, invasion and metastasis; are frequently over-expressed in
malignant tumors; and have been associated with an aggressive
Lung cancer is one of leading cause of death worldwide. The
human non-small cell lung cancer (NSCLC) accounts for 80–85%
of lung cancer cases. More than one of three patients with NSCLC
is surgically unavailable.1 Therefore, chemotherapy is still the
major treatment for clinical NSCLC.2–4 However, although the great
improvement of current therapies for lung cancers, both poor prog-
nosis at the advanced stage of NSCLC and chemoresistance caused
to low survival rate of NSCLC patients.4
Tumor metastasis occurs by a complex series of events including
cell adhesion, invasion, proliferation and vessel formation.5 Degra-
dation of basement membranes and stromal extracellular matrix
(ECM) is crucial for invasion and metastasis of malignant cells.
The matrix metalloproteinases (MMPs) are a family of zinc-depen-
dent proteinases involved in the degradation of the extracellular
matrix. The MMPs have been implicated in the processes of tumor
malignant phenotype and adverse prognosis in patients with
cancer.6
Quinoline ring is found in a wide variety of pharmacologically
active compounds and is frequently condensed with various het-
erocycles.7–16 Recently, a number of condensed quinoline deriva-
tives have been synthesized and evaluated for their top
I
inhibitory activities. Camptothecin (CPT), an alkaloid isolated from
Camptotheca acuminata, and its derivatives such as topotecan and
irinotecan are prototypical topo I inhibitors and currently used as
anticancer drugs. However, the chemically unstable lactone ring
limited their clinical utility. To improve chemical stability and anti-
cancer potency of CPT, we have designed and synthesized certain
quinoline derivatives for anticancer evaluation. Among them, 2,3-
diarylquinoline (1),12 6-arylindeno[1,2-c]quinoline (2),13 and 3-
arylquinolinylchalcone (3)14 were found to be more potent than
CPT against the growth of human cancer cell lines. (E)-2-[2-(5-
Nitrofuran-2-yl)vinyl]quinolin-8-ol (4)15 is another example of
quinoline derivative which exhibited potent antiproliferative activ-
⇑
Corresponding author. Tel.: +886 7 3121101x2684; fax: +886 7 3125339.
E-mail address: yeloch@kmu.edu.tw (Y.-L. Chen).
http://dx.doi.org/10.1016/j.bmc.2014.11.015
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Tseng, C.-H.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.11.015

2
C.-H. Tseng et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
ities with an IC50 value of 0.35 and 0.14
l
M, respectively, against
the growth of LNCaP and PC3 cancer cells. In continuation of our
search for potential anticancer drug candidates, the present study
describes the synthesis of 3-aryl-2-[2-(5-nitrofuran-2-yl)vin
yl]quinoline derivatives (Target compounds, Fig. 1) whose struc-
tures resemble compound 1 by the replacement of the 2-phenyl
group with a 2-vinylfuran ring. The target compounds can also
be considered as hybrids of compounds 3 and 4. These newly syn-
thesized compounds were evaluated for their antiproliferative and
antimetastatic activities.
2
Scheme 1. Reagents and conditions: (i) (5-nitrofuran-2-yl)methylene diacetate,
Ac2O, 150 °C, 3 h.
portion of the curve by calculating the concentration of agent that
reduced absorbance in treated cells, compared to control cells, by
50%. The results of IC50 values are summarized in Table 1. Com-
pound 4 was more active than compounds 6a–6c against all the
cancer cells tested indicated 8-hydroxy group is favorable for the
cell cytotoxicity. Introduction of 4-methoxyphenyl group at C-3
position of 6b and 6c decreased antiproliferative activities in which
10b and 10c were inactive against all the cancer cells tested. How-
ever, compound 10a was highly cytotoxic which implied that the
substitution at C-6 position of quinoline ring was unfavorable
especially the methoxy group. The same structure–activity rela-
tionship (SAR) was observed for the tetracyclic indenoquinoline
in which the antiproliferative activities decreased in an order 11a
(R = H) >11b (R = F) >11c (R = OMe). Although compounds 11a–
11c exhibited potent inhibitory activities on all the cells tested,
their methyl precursors 9a–9c were inactive, indicated that the
conjugated nitrofuran moiety is crucial. Among these newly syn-
thesized quinoline derivatives, compound 11a was the most active,
approximately equal potent to the positive daunorubicin.
2. Chemistry
Reaction of 2-methylquinoline (5a) and (5-nitrofuran-2-
yl)methylene diacetate in acetic anhydride gave (E)-2-(2-(5-nitro-
furan-2-yl)vinyl)quinoline (6a) in 81% yield as described in
Scheme 1. Accordingly, compounds 6b and 6c were prepared from
their respective 2-methylquinoline precursors 5b and 5c by the
same reaction conditions. Thermal decarboxylation of 3-(4-
methoxyphenyl)-2-methylquinoline-4-carboxylic acid (7a)14 gave
3-(4-methoxyphenyl)-2-methylquinoline (8a)14 which was then
reacted with 5-nitro-2-furaldehyde diacetate in acetic anhydride
to
afford
(E)-3-(4-methoxyphenyl)-2-(2-(5-nitrofuran-2-
yl)vinyl)quinoline (10a) as described in Scheme 2. Compounds
10b and 10c were prepared from their respective 2-methylquino-
line precursors 8b and 8c16 by the same reaction conditions. Treat-
ment of 7a with POCl3 afforded 6-methyl-11H-indeno[1,2-
c]quinolin-11-one (9a), which was then reacted 5-nitro-2-furalde-
hyde diacetate in acetic anhydride to give (E)-9-methoxy-6-[2-(5-
nitrofuran-2-yl)vinyl]-11H-indeno[1,2-c]quinolin-11-one
(11a).
Although compound 11a can be further developed as cytotoxic
anticancer drug candidates, the inactiveness of 10c against all the
cells attracted our attention due to its structure similarity to com-
bretastatin A4 (CA-4), a known anti-angiogenesis agent. Therefore,
compound 10c was selected for antimetastatic evaluations and the
possible signaling pathways in vitro.
Accordingly, compounds 11b and 11c were prepared from their
respective methyl precursors 9b and 9c which in turn were pre-
pared from their respective 4-carboxylic acid precursors 7b and
7c with POCl3.
3. Results and discussion
3.2. The effect of compound 10c on proliferation of NSCLC cells
3.1. Antiproliferative activity
To further examine the effect of compound 10c on cell growth,
H1299 cells were treated with compound 10c at various concen-
All the newly synthesized compounds were evaluated in vitro
against four cancer cell lines (H1299, A549, MCF-7 and MDA-MB-
231) using XTT [2,3-bis(2-methyloxy-4-nitro-5-sulfophenyl)-2H-
tetrazolium-5-carboxanilide] assay. The normal human fetal lung
fibroblast cell line (MRC-5) was also evaluated since a potential
anticancer drug candidate should selectively affect only tumor
cells and not somatic cells. The concentration that inhibited the
growth of 50% of cells (IC50) was determined from the linear
trations (0–200
sentative experiment shown in Figure 2A, compound 10c at
concentrations lower than 100 M had no cytotoxic effect (>90%
cell survival) while at 200 M it reduced cell viability. There was
lM) for 24 h by XTT assay. As results from a repre-
l
l
no significant change in morphology observed between the vehicle
control and 10c-treated (Fig. 2B). It has to be noted that the cell
migration and invasion assay were performed with a noncytotoxic
OMe
OMe
OR
RO
OR
OR
O
O
N
RO
N
OMe
OMe
O
N
O
N
OH
1
OR
OH
Combretastatin A-4 (CA-4)
Camptothecin (CPT)
2
OMe
OMe
R
O
N
O
NO2
Ar
N
NO2
N
OH
O
Target compounds
4
3
Figure 1. Structures of Camptothecin (CPT), Combrestatin A-4 (CA-4), 2,3-diarylquinoline (1), 6-arylindeno[1,2-c]quinoline (2), 3-arylquinolinylchalcone (3), (E)-2-[2-(5-
nitrofuran-2-yl)vinyl]quinolin-8-ol (4) and target compounds.
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C.-H. Tseng et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
2
2
Scheme 2. Reagents and conditions: (i) Dowtherm A, 280 °C 4 h; (ii) POCl3, 150 °C, 48 h; (iii) (5-nitrofuran-2-yl)methylene diacetate, Ac2O, 150 °C, 30 h.
Table 1
Antiproliferative activity of 2-furanylvinylquinoline derivatives (IC50
, lM)
Compound
Cell lines
Lung cancer
Breast cancer
Normal cell
MRC-5
H1299
A549
MCF-7
MDA-MB-231
4
6a
6b
6c
9a
9b
9c
10a
10b
10c
11a
11b
11c
Daunorubicin
0.09 0.03
0.62 0.09
0.84 0.10
0.14 0.02
21.35 2.36
31.55 1.59
33.69 2.54
0.43 0.12
9.63 0.30
86.72 9.23
0.08 0.02
0.46 0.06
0.89 0.16
0.096 0.007
0.76 0.04
5.06 0.16
8.69 1.15
0.77 0.17
61.59 6.78
71.14 3.25
75.89 9.15
0.99 0.06
51.58 6.87
78.69 4.57
0.07 0.03
0.74 0.08
1.32 0.25
0.08 0.04
0.53 0.03
1.46 0.12
4.93 0.27
0.87 0.08
32.59 2.68
39.65 3.96
41.98 3.89
0.67 0.16
36.32 3.99
80.99 27.05
0.62 0.08
0.58 0.05
0.87 0.16
0.65 0.07
0.37 0.12
0.75 0.05
0.74 0.08
0.31 0.07
7.30 0.57
7.50 0.48
0.88 0.11
65.38 6.58
43.21 5.74
78.35 6.91
0.78 0.26
50.81 3.72
81.56 8.92
0.093 0.008
0.80 0.19
6.07 1.06
0.078 0.007
0.19 0.06
41.98 3.89
52.14 5.29
62.34 7.93
0.65 0.43
87.12 14.29
88.08 5.58
0.095 0.006
0.093 0.008
0.93 0.13
0.091 0.009
Figure 2. Effect of compound 10c on proliferation of NSCLC cells. 5 ꢀ 103 cells were seeded onto a 96-well plate, and cells were treated with the indicated doses of compound
10c for 24 h, respectively. (A) Cell proliferation was determined using the XTT assay described in materials and methods. (B) The cellular morphology of H1299 following
compound 10c treatment. Magnification: 200ꢀ. *p <0.05 and **p <0.001 for compound 10c treatments against vehicle, respectively.
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C.-H. Tseng et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Figure 3. Effect of compound 10c on migration of NSCLC cells. Compound 10c inhibits cell migration of NSCLC tumor H1299 cells. (A) 5 ꢀ 105 cells were seeded onto a 12-
well plate and cells were scraped to create a clean 1-mm wide wound area within the confluent culture. Cells were treated with the indicated doses of vehicle, 0.2, 0.5, 1 and
2 l
M of compound 10c for 16 h. Afterwards, the wound gaps were photographed using an inverted phase-contrast microscopy. (B) The quantification analysis. *p <0.05 and
**p <0.001 for compound 10c treatments against vehicle, respectively.
Figure 4. Effect of compound 10c on invasion of H1299 cells. (A) The Boyden’s transwell invasion assay showed that a noncytotoxic dose of compound 10c dramatically
inhibited the cellular invasion (14 h). (B) Quantitative analysis. *p <0.05 and **p <0.001, for compound 10c treatments against vehicle respectively.
concentration of 10c. Thus, compound 10c at concentrations lower
than 10 M were applied in the following experiments.
compound 10c concentrations used at vehicle, 0.2, 0.5, 1, and 2 lM
was 81.28 3.2%, 68.97 2.8%, 66.02 3.2% and 56.64 1.5%,
l
respectively. Compound 10c attenuates migration of H1299 lung
3.3. Compound 10c attenuates migration of H1299 lung cancer
cells
cancer cells (IC50 value of 2.71 0.35 lM). These results indicate
that noncytotoxic dose of compound 10c inhibited the migration
of H1299 lung cancer cells.
To investigate if compound 10c treatments could influence the
migration of cancer cells, the wound-healing assays were per-
formed. Figure 3A showed that the migration of H1299 lung cancer
cells was significantly inhibited by compound 10c in a dose-depen-
dent manner at 16-h post-treatment. Figure 3B showed the quan-
titative analysis on the inhibition of migration ability by compound
10c treatments compared with the vehicle control. The calculated
denuded zone (indicating the migratory ability of H1299 cells) for
3.4. Compound 10c attenuates invasion of H1299 lung cancer
cells
We further tested the effect of compound 10c on cellular inva-
sion of H1299 cells. The Boyden’s chamber transwell invasion assay
was performed. Figure 4A showed that the invasion ability of
H1299 lung cancer cells was significantly inhibited by compound
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C.-H. Tseng et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5
19.13 0.7%, 47.83 1.4%, and 76.65 4.2% when cells were treated
with 0.2, 0.5, 1 and 2 M of compound 10c, respectively. Compound
10c attenuates invasion of H1299 lung cancer cells (IC50 value of
1.08 0.12 M).
l
l
3.5. Compound 10c significantly inhibits MMPs activities
Because the degradation of extracellular matrix by MMPs is a
critical step important for tumor invasion,17 the gelatin zymogra-
phy was performed to investigate the activity of MMP-2 and -9
in cell treated with various concentrations of compound 10c. As
shown by gelatinolytic activity data, compound 10c inhibited the
activity of MMP-2 and -9 in a dose-dependent manner compared
with the control group (Fig. 5A). Quantification analysis indicated
that MMP-9 activity reduced by 63.85 1.6%, 31.48 0.4% and
18.77 0.1% and MMP-2 activity reduced by 75.47 10.3%,
63.69 8.0% and 56.29 0.7% when cells were treated with 1, 2,
5 lM of 10c, respectively (Fig. 5B). Furthermore, we found that
compound 10c more significantly attenuates MMP-9 activity than
MMP-2.
3.6. Compound 10c attenuates activation of Akt signaling
Many studies have reported that the protein level and activity
of MMP-2 and -9 are regulated by various transcription factors
including several members of MAPK superfamily including ERK1/
2, JNK1/2, P38, and Akt.18–21 To further figure out the mechanism
underlying the 10c-mediated down-regulation of MMP-2/9, we
examined the phosphorylation status (activation states) of MAPKs
and Akt in H1299 cells treated with 10c at different concentrations
Figure 5. Effect of compound 10c on activities of MMP-2 and MMP-9. After H1299
cells were treated with or without different concentrations of compound 10c for
24 h, the activities of (A) MMP-2 and MMP-9 were detected by gelatin zymography
(n = 3). (B) Quantitative analysis. Representative results are shown here. *p <0.05
and **p <0.001 for compound 10c treatments against vehicle, respectively.
Figure 6. Modulation of migration-associated proteins by compound 10c. Cells
were subject to compound 10c treatment with the dose course of vehicle control,
0.2, 0.5, 1,
2 and 5 lM, respectively. The result of Western blot showed the
modulation of cell migration associated signaling pathways, and the regulation of
cellular migration associated transcription factors and their downstream genes. b-
actin was measured as an internal control. Each blot is representative of three
independent experiments.
10c in a dose-dependent manner at 14-h post-treatment. Figure 4B
showed the quantitative analysis on the inhibition of invasion abil-
ity indicated that invasion was reduced by 19.13 2.1%,
Figure 7. Proposed model of compound 10c-induced cell migration inhibition in
H1299 lung cancer cells.
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C.-H. Tseng et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
for 24 h. Results from Western blotting analyses showed that com-
pound 10c (1–5 M) significantly reduced protein levels of p-Akt,
(m, 2H, Ar-H), 8.41 (d, 1H, J = 8.4 Hz, 4-H). 13C NMR (100 MHz,
DMSO-d6): 114.11, 115.36, 119.72, 120.87, 126.89, 127.45,
127.86, 128.88, 130.15, 133.10, 136.90, 147.60, 151.34, 153.76,
155.11. Anal. Calcd for C15H10N2O3: C 67.67, H 3.79, N 10.52;
found: C 67.79, H 3.80, N 10.52.
l
p-ERK, b-catenin transcription factor, Bcl-2 and COX-2. On the con-
trary, 10c causes the prophorylation levels of p-38 in examined
cells (Fig. 6). It is reported that activation of Akt and MAPKs plays
a critical role in the regulation of MMPs activities. The overexpres-
sion of cyclooxygenase-2 (COX-2) is seen in many malignancies
including lung cancer. In non-small cell lung cancer (NSCLC),
COX-2 is overexpressed in most adenocarcinomas and squamous
cell carcinomas. There is also sufficient evidence from animal stud-
ies to indicate that selective COX-2 inhibitors are effective in the
prevention of carcinogenesis and induce apoptosis in a range of
cancer cells.22–24 In order to demonstrate whether apoptosis of
H1299 cells induced by 10c is due to COX-2 regulation, we ana-
lyzed the amount of COX-2 in H1299 cells treated with 10c. Fig-
ure 6 showed that, compared to the control cells, the expression
level of COX-2 was decreased in H1299 cells treated with 10c for
24 h.
5.1.2. (E)-6-Fluoro-2-[2-(5-nitrofuran-2-yl)vinyl]quinoline (6b)
From 5b as described for the preparation of 6a: 75% yield. Mp
183–185 °C. 1H NMR (400 MHz, DMSO-d6): 7.21 (d, 1H, J = 4.0 Hz,
furanyl-H), 7.56 (d, 1H, J = 16.0 Hz, @CH), 7.67–7.81 (m, 4H, Ar-,
furanyl-H), 7.95 (d, 1H, J = 8.4 Hz, 3-H), 8.06 (dd, 1H, J = 9.6,
5.6 Hz, 8-H), 8.38 (d, 1H, J = 8.4 Hz, 4-H). 13C NMR (100 MHz,
DMSO-d6): 110.99 (J = 22.0 Hz), 114.11, 115.33, 119.71, 120.20
(J = 25.7 Hz), 121.56, 128.11 (J = 10.6 Hz), 131.70 (J = 9.1 Hz),
132.82, 136.43 (J = 5.4 Hz), 144.80, 151.33, 153.36 (J = 2.3 Hz),
155.02, 159.86 (J = 244.9 Hz). ESI-HRMS calcd,
C15H10FN2O3
[M+H]+ 285.0675, observed 285.0673.
5.1.3. (E)-6-Methoxy-2-[2-(5-nitrofuran-2-yl)vinyl]quinoline
(6c)
4. Conclusion
From 5c as described for the preparation of 6a: 83% yield. Mp
250–252 °C. 1H NMR (400 MHz, DMSO-d6): 4.01 (s, 3H, OMe),
7.26 (d, 1H, J = 3.6 Hz, furanyl-H), 7.66 (d, 1H, J = 16.4 Hz, @CH),
7.72–7.78 (m, 3H, Ar-H), 8.15 (d, 1H, J = 9.2 Hz, 8-H), 8.17 (d, 1H,
J = 16.4 Hz, @CH), 8.50 (d, 1H, J = 9.2 Hz, 3-H), 8.98 (d, 1H,
J = 9.2 Hz, 4-H). 13C NMR (100 MHz, DMSO-d6): 56.60, 107.54,
111.40, 115.11, 118.15, 120.09, 122.85, 123.09, 127.47, 127.97,
130.49, 134.11, 144.42, 148.41, 153.39, 160.54. Anal. Calcd for
A number of 2-[2-(5-nitrofuran-2-yl)vinyl]quinoline derivatives
were synthesized and evaluated for antiproliferative activities
against the growth of six cancer cell lines. Among them, 10c was
found low cytotoxic in all cancer cells and normal cell. Compound
10c has anti-metastatic activities and has the potential to be devel-
oped into an anti-metastatic agent for lung cancer. The methoxy
group substituted at C-6 position of the quinoline ring is crucial
for anti-metastatic activity. Methoxy groups have also been found
in the structure of CA-4. The possible signal pathways for 10c to
inhibit migration and invasion in H1299 cells may be through
down-regulation of MAPKs and Akt signaling pathways and inhibi-
tion of MMP-2 and -9 (Fig. 7). The in vivo efficacy studies with 10c
should be done in animal models in future investigations.
C16H12N2O4: C 64.86, H 4.08, N 9.46; found: C 64.62, H 4.03, N 9.27.
5.1.4. 6-Methyl-11H-indeno[1,2-c]quinolin-11-one (9a)
A solution of 3-(4-methoxyphenyl)-2-methylquinoline-4-car-
boxylic acid (7a)14 (2.93 g, 10 mmol) in POCl3 (30 mL) was heated
at 150 °C for 48 h (TLC monitoring). After cooling, the mixture was
poured into ice water (150 mL). The resulting precipitate that sep-
arated was collected by filtration. The filtered cake was suspended
in 5% NaHCO3 solution (200 mL) with vigorous stirring for 1 h. The
resulting precipitate was collected, washed with H2O, and purified
by FC (CH2Cl2 as eluent) to give 9a (0.58 g, 21%) as a red solid. Mp
179–180 °C. 1H NMR (400 MHz, CDCl3): 2.93 (s, 3H, 6-Me), 3.87 (s,
3H, 9-OMe), 6.94 (dd, 1H, J = 8.4, 2.4 Hz, 8-H), 7.22 (d, 1H,
J = 2.4 Hz, 10-H), 7.50–7.63 (m, 3H, Ar-H), 7.94 (d, 1H, J = 8.4 Hz,
1-H), 8.73–8.75 (m, 1H, 4-H). 13C NMR (100 MHz, CDCl3): 24.68,
55.76, 111.10, 119.21, 122.29, 123.77, 124.19, 128.62, 128.77,
129.51, 133.36, 135.62, 135.65, 137.61, 149.02, 153.54, 160.87,
195.37. Anal. Calcd for C18H13NO2ꢁ0.25H2O: C 77.26, H 4.87, N
5.01; found: C 77.38, H 4.85, N 4.89.
5. Experimental
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 Gemini
200 spectrometer or Varian-Unity-400 spectrometer. Chemical
shifts were expressed in parts per million (d) with tetramethylsil-
ane (TMS) as an internal standard. Thin-layer chromatography
was performed on silica gel 60 F-254 plates purchased from E.
Merck and Co. High-resolution mass spectroscopic (HRMS) data
were provided by the Joint Center for High Valued Instruments at
National Sun Yat-sen University using Bruker APEX II (ESI) mass
spectrometer. The elemental analyses were performed in the
Instrument Center of National Science Council at National Cheng-
Kung University and National Taiwan University using Heraeus
CHN-O Rapid EA, and all values are within 0.4% of the theoretical
compositions.
5.1.5. 2-Fluoro-6-methyl-11H-indeno[1,2-c]quinolin-11-one
(9b)
From 7b16 as described for the preparation of 9a: 16% yield as a
red solid. Mp 239–240 °C. 1H NMR (400 MHz, TFA-d): 3.00 (s, 3H,
6-Me), 3.76 (s, 3H, 9-OMe), 7.03 (dd, 1H, J = 8.4, 2.4 Hz, 8-H),
7.28 (d, 1H, J = 2.4 Hz, 10-H), 7.50 (d, 1H, J = 8.4 Hz, 7-H), 7.55–
7.60 (m, 1H, 3-H), 7.93 (dd, 1H, J = 9.2, 4.4 Hz, 4-H), 8.45 (dd, 1H,
J = 8.0, 2.4 Hz, 1-H). 13C NMR (100 MHz, TFA-d): 16.89, 53.38,
106.62 (J = 25.7 Hz), 111.28, 119.64, 120.49 (J = 9.9 Hz), 122.77
(J = 12.1 Hz), 123.68, 124.25 (J = 28.0 Hz), 129.99, 132.39, 134.86,
137.79 (J = 7.6 Hz), 138.36, 149.01, 160.90, 162.25 (J = 258.4 Hz),
190.66. Anal. Calcd for C18H12FNO2ꢁ0.5H2O: C 71.51, H 4.34, N
4.63; found: C 71.85, H 4.07, N4.55.
5.1.1. (E)-2-[2-(5-Nitrofuran-2-yl)vinyl]quinoline (6a)
A mixture of 2-methylquinoline (5a, 0.14 g, 1 mmol), (5-nitro-
furan-2-yl)methylene diacetate (0.72 g, 3 mmol) and acetic anhy-
dride (30 mL) was heated at 150 °C for 30 h (TLC monitoring).
After cooling, the solvent was removed in vacuo to provide the
crude product, which was purified by flash column chromatogra-
phy (FC, silica gel use CH2Cl2 as eluent) to give 6a (0.22 g, 81%)
as a yellow solid. Mp 169–170 °C. 1H NMR (400 MHz, DMSO-d6):
7.22 (d, 1H, J = 4.0 Hz, furanyl-H), 7.57–7.63 (m, 2H), 7.77–7.83
(m, 3H, Ar-, furanyl-H), 7.92 (d, 1H, J = 8.4 Hz, 3-H), 7.97–8.03
5.1.6. 2-Methoxy-6-methyl-11H-indeno[1,2-c]quinolin-11-one
(9c)
From 7c16 as described for the preparation of 9a: 28% yield as a
red solid. Mp 241–242 °C. 1H NMR (400 MHz, TFA-d): 2.94 (s, 3H,
Please cite this article in press as: Tseng, C.-H.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.11.015

C.-H. Tseng et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
7
6-Me), 3.76 (s, 3H, 2-OMe), 3.87 (s, 3H, 9-OMe), 7.02 (dd, 1H,
J = 8.8, 2.8 Hz, 8-H), 7.27 (d, 1H, J = 2.4 Hz, 10-H), 7.45–7.49 (m,
2H, Ar-H), 7.76 (d, 1H, J = 9.6 Hz, 4-H), 8.12 (d, 1H, J = 2.4 Hz, 1-
H). 13C NMR (100 MHz, TFA-d): 16.32, 53.38, 53.53, 99.14,
118.96, 119.51, 124.46, 123.72, 127.93 (2C), 130.54, 132.54,
134.73, 136.15, 137.47, 144.67, 150.50, 160.98, 191.79. Anal. Calcd
for C19H15NO3ꢁ0.5H2O: C 72.59, H 5.14, N 4.46; found: C 72.15, H
4.98, N 4.36.
139.81, 142.86, 149.98, 159.11, 160.97, 190.66. Anal. Calcd for
C23H14N2O5: C 69.34, H 3.54, N 7.03; found: C 69.04, H 3.57, N 6.94.
5.1.11. (E)-2-Fluoro-9-methoxy-6-[2-(5-nitrofuran-2-yl)vinyl]-
11H-indeno[1,2-c] quinolin-11-one (11b)
From 9b as described for the preparation of 6a: 28% yield as a
yellow solid. Mp 258–260 °C. 1H NMR (400 MHz, TFA-d): 4.06 (s,
3H, 9-OMe), 7.16 (d, 1H, J = 4.0 Hz, furanyl-H), 7.35 (dd, 1H,
J = 8.4, 2.4 Hz, 8-H), 7.59 (d, 1H, J = 2.4 Hz, 10-H), 7.62 (d, 1H,
J = 4.0 Hz, furanyl-H), 7.84 (d, 1H, J = 16.0 Hz, @CH), 7.87–7.92
(m, 2H, Ar-H), 8.03 (d, 1H, J = 16.0 Hz, @CH), 8.33 (dd, 1H, J = 9.2,
4.4 Hz, 4-H), 8.78 (dd, 1H, J = 8.4, 2.8 Hz, 1-H). 13C NMR
(100 MHz, TFA-d): 53.38, 106.69 (J = 25.0 Hz), 111.28, 112.19,
116.65, 119.88, 120.80 (J = 9.8 Hz), 123.36 (J = 12.2 Hz), 124.13,
125.23 (J = 28.1 Hz), 126.58, 129.46, 132.75, 135.74, 137.94,
138.82 (J = 6.8 Hz), 142.60, 149.82, 150.99, 158.40, 161.19, 162.48
(J = 257.7 Hz), 190.02. Anal. Calcd for C23H13FN2O5: C 66.35, H
3.15, N 6.73; found: C 66.01, H 3.24, N 6.62.
5.1.7. (E)-3-(4-Methoxyphenyl)-2-[2-(5-nitrofuran-2-
yl)vinyl]quinoline (10a)
From 8a14 as described for the preparation of 6a: 78% yield as a
yellow solid. Mp 185–187 °C. 1H NMR (400 MHz, CDCl3): 3.92 (s,
3H, 9-OMe), 6.64 (d, 1H, J = 3.6 Hz, furanyl-H), 7.05–7.09 (m, 2H,
Ar-H), 7.34 (d, 1H, J = 3.6 Hz, furanyl-H), 7.37–7.41 (m, 2H, Ar-H),
7.52–7.56 (m, 1H, Ar-H), 7.58 (d, 1H, J = 15.6 Hz, @CH), 7.71–7.75
(m, 1H, Ar-H), 7.81 (d, 1H, J = 8.0 Hz, Ar-H), 7.85 (d, 1H,
J = 15.6 Hz, @CH), 8.07 (s, 1H, 4-H), 8.11 (dd, 1H, J = 8.4, 0.8 Hz,
Ar-H). 13C NMR (100 MHz, CDCl3): 55.43, 112.46, 113.92, 114.21
(2C), 119.97, 127.08, 127.46, 127.85, 129.27, 129.79, 130.53,
130.98 (2C), 131.09, 135.39, 137.01 (2C), 147.19, 151.56, 155.77,
159.63. Anal. Calcd for C22H16N2O4ꢁ0.25H2O: C 70.10, H 4.42, N
7.43; found: C 70.05, H 4.31, N 7.30.
5.1.12. (E)-2,9-Dimethoxy-6-[2-(5-nitrofuran-2-yl)vinyl]-11H-
indeno[1,2-c]quinolin-11-one (11c)
From 9c as described for the preparation of 6a: 26% yield as a
yellow solid. Mp 246–247 °C. 1H NMR (400 MHz, TFA-d): 3.77, (s,
3H, 2-OMe), 3.89 (s, 3H, 9-OMe), 6.82 (d, 1H, J = 4.0 Hz, furanyl-
H), 7.06 (dd, 1H, J = 8.4, 2.4 Hz, 8-H), 7.28 (d, 1H, J = 2.4 Hz, 10-
H), 7.33 (d, 1H, J = 4.0 Hz, furanyl-H), 7.45 (d, 1H, J = 16.0 Hz,
@CH), 7.51 (dd, 1H, J = 9.6, 2.8 Hz, 3-H), 7.57 (d, 1H, J = 8.4 Hz, 7-
H), 7.72 (d, 1H, J = 16.0 Hz, @CH), 7.87 (d, 1H, J = 9.6 Hz, 4-H),
8.16 (d, 1H, J = 2.8 Hz, 1-H). 13C NMR (100 MHz, TFA-d): 53.38,
53.69, 99.08, 110.96, 112.38, 115.70, 117.04, 119.32, 119.72,
123.96, 124.50, 124.91 (2C), 129.22, 129.96, 132.91, 135.76,
136.77, 137.15, 138.67, 150.22, 160.81, 160.52, 191.14. Anal. Calcd
for C24H16N2O6: C 67.29, H 3.76, N 6.54; found: C 67.24, H 3.79, N
6.41.
5.1.8. (E)-6-Fluoro-3-(4-methoxyphenyl)-2-[2-(5-nitrofuran-2-
yl)vinyl]quinoline (10b)
From 8b16 as described for the preparation of 6a: 73% yield as a
yellow solid. Mp 194–195 °C. 1H NMR (400 MHz, CDCl3): 3.92 (s,
3H, 9-OMe), 6.64 (d, 1H, J = 3.6 Hz, furanyl-H), 7.05–7.09 (m, 2H,
Ar-H), 7.34 (d, 1H, J = 3.6 Hz, furanyl-H), 7.36–7.43 (m, 3H, Ar-H),
7.47–7.52 (m, 1H, Ar-H), 7.55 (d, 1H, J = 15.6 Hz, @CH), 7.82 (d,
1H, J = 15.6 Hz, @CH), 8.00 (s, 1H, 4-H), 8.10 (dd, 1H, J = 9.2,
5.2 Hz, 8-H). 13C NMR (100 MHz, CDCl3): 55.42, 110.33
(J = 22.0 Hz), 112.45, 113.86, 114.27 (2C), 119.89, 120.19
(J = 26.5 Hz), 128.42 (J = 10.6 Hz), 130.13, 130.76, 130.92 (2C),
131.84 (J = 9.1 Hz), 136.08, 136.26 (J = 6.1 Hz), 144.33, 150.99,
152.26 (J = 2.3 Hz), 155.65, 159.78, 160.84 (J = 247.8 Hz). Anal.
Calcd for C22H15FN2O4: C 67.69, H 3.87, N 7.18; found: C 67.35, H
3.71, N 7.11.
5.2. Biological testing
5.2.1. Reagents
Dulbecco’s modified Eagle’s medium (DMEM), F-12 medium,
fetal bovine serum (FBS), penicillin G and streptomycin and phos-
phate-buffered saline (PBS) were obtained from Gibco BRL (Gai-
thersburg, MD, USA). Dimethyl sulphoxide (DMSO) and 6-
diamidino-2-phenylindole (DAPI) were purchased from Sigma–
Aldrich (St. Louis, MO, USA). Antibodies against Bcl-2, COX-2, survi-
vin, phospho-Akt (Ser473), phospho-ERK, ERK, phospho-p38, p38,
phospho-JNK and b-actin were obtained from Santa Cruz Biotech-
nology (Santa Cruz, CA, USA). Antibodies against MMP-2, MMP-9,
Sp1 and b-catenin were purchased from Anaspec (San Jose, CA,
USA). Anti-mouse and anti-rabbit IgG peroxidase-conjugated sec-
ondary antibodies were purchased from Pierce (Rockford, IL, USA).
5.1.9. (E)-6-Methoxy-3-(4-methoxyphenyl)-2-[2-(5-nitrofuran-
2-yl)vinyl]quinoline (10c)
From 8c16 as described for the preparation of 6a: 75% yield as a
yellow solid. Mp 217–218 °C. 1H NMR (400 MHz, CDCl3): 3.92 (s,
3H, 6-OMe), 3.95 (s, 3H, 9-OMe), 6.60 (d, 1H, J = 3.6 Hz, furanyl-
H), 7.04–7.08 (m, 3H, Ar-H), 7.34 (d, 1H, J = 3.6 Hz, furanyl-H),
7.33–7.39 (m, 3H, Ar-H), 7.55 (d, 1H, J = 15.6 Hz, @CH), 7.78 (d,
1H, J = 15.6 Hz, @CH), 7.94 (s, 1H, 4-H), 8.00 (d, 1H, J = 8.8 Hz, 8-
H). 13C NMR (100 MHz, CDCl3): 55.41, 55.60, 104.60, 112.04,
114.01, 114.17 (2C), 118.88, 122.95, 128.96, 130.71, 130.82,
130.95 (2C), 131.28 (2C), 135.69 (2C), 143.48, 149.08, 156.07,
158.38, 159.60. Anal. Calcd for C23H18N2O5ꢁ0.25H2O: C 67.88, H
4.59, N 6.89; found: C 67.87, H 4.23, N 6.81.
5.2.2. Cell culture
Non-small cell lung cancer (A549 and H1299), breast cancer
(MCF-7 and MDA-MB-231) and normal diploid embryonic lung cell
line (MRC-5) were obtained from the American Type Culture Col-
lection (ATCC) (Manassas, VA, USA). Cells were maintained in
DMEM: F-12 medium (1:1 ratio) and supplemented with 8% FBS,
2 mM glutamine and antibiotics (100 units/ml penicillin and
5.1.10. (E)-9-Methoxy-6-[2-(5-nitrofuran-2-yl)vinyl]-11H-
indeno[1,2-c]quinolin-11-one (11a)
From 9a as described for the preparation of 6a: 34% yield as a
yellow solid. Mp 270–271 °C. 1H NMR (400 MHz, TFA-d): 4.06 (s,
3H, 9-OMe), 7.16 (d, 1H, J = 3.6 Hz, furanyl-H), 7.36 (dd, 1H,
J = 8.8, 2.4 Hz, 8-H), 7.59 (d, 1H, J = 2.4 Hz, 10-H), 7.62 (d, 1H,
J = 3.6 Hz, furanyl-H), 7.86 (d, 1H, J = 16.0 Hz, @CH), 7.88 (d, 1H,
J = 8.8 Hz, 7-H), 8.00–8.04 (m, 1H, Ar-H), 8.06 (d, 1H, J = 16.0 Hz,
@CH), 8.12–8.17 (m, 1H, Ar-H), 8.25 (d, 1H, J = 8.8 Hz, Ar-H), 9.17
(d, 1H, J = 8.4 Hz, Ar-H). 13C NMR (100 MHz, TFA-d): 53.41,
111.13, 112.26, 116.53, 116.85, 117.83, 120.16, 122.20, 122.91,
123.97, 126.50, 130.12, 130.78, 132.82, 134.79, 136.92, 138.77,
100 lg/ml streptomycin) at 37 °C in a humidified atmosphere of
5% CO2.25
5.2.3. Antiproliferative assay
Cells (5 ꢀ 103 cells/well) were treated as indicated for 72 h in
medium containing 10% FBS. (The medium was then changed
(100
lL), and the cells were incubated for another day. Fifty micro-
liters of serum-free medium containing XTT (1 mg/mL) and phen-
Please cite this article in press as: Tseng, C.-H.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.11.015

8
C.-H. Tseng et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
azine methosulfate (PMS) (10 lM) was added to the cells which
Acknowledgments
were then incubated at 37 °C for 4 h. Color was measured spectro-
photometrically 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 con-
trol cells and used to determine the percent of control growth as
(Abtreated/Abcontrol) ꢀ 100, where Ab represents the mean absor-
bance (n = 3). The concentration that killed 50% of cells (IC50) was
determined from the linear portion of the curve by calculating
the concentration of agent that reduced absorbance in treated cells,
compared to control cells, by 50%.26
Financial support of this work by grants from the Kaohsiung
Medical University Research Foundation (KMU-Q103028) and
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) are gratefully acknowledged. We also
thank the National Center for High-Performance Computing for pro-
viding computer resources and chemical database services.
Supplementary data
5.2.4. Gelatin zymography
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.11.015.
MMP-2 and MMP-9 activities were assessed by gelatin-zymog-
raphy as previously described.27 In brief, 3 ꢀ 105 H1299 cells were
seeded onto 12-well plates and cultured with indicated concentra-
tions of compound 10c for 24 h, respectively, after which aliquots
of culture medium were harvested for gelatin zymography analy-
sis. The samples were resolved by 10% SDS polyacrylamide gels
containing 1% gelatin. Afterwards, gels were washed with distilled
water and incubated with reaction buffer (40 mM Tris–HCl, pH 8.0,
10 mM CaCl2, 0.02% NaN3) at 37 °C for overnight. Gels were stained
with Coomassie brilliant blue R-250 and destained with methanol-
acetic acid–water (50/75/875, v/v/v). The gelatinase activity of
MMP-2 and MMP-9 was detected as the unstained gelatin-degra-
dation zones within the gel.
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The Western blot assay was performed as described previ-
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Please cite this article in press as: Tseng, C.-H.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.11.015